The Lipid Handbook with CD-ROM, Third Edition [3 ed.] 0849396883, 9780849396885

Citation preview

The Lipid Handbook with CD-ROM Third Edition

The Lipid Handbook with CD-ROM Third Edition Edited by

Frank D. Gunstone John L. Harwood Albert J. Dijkstra

Boca Raton London New York

CRC Press is an imprint of the Taylor & Francis Group, an informa business

CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2007 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-10: 0-8493-9688-3 (Hardcover) International Standard Book Number-13: 978-0-8493-9688-5 (Hardcover) This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data The lipid handbook with CD-ROM / [edited by] Frank D. Gunstone, John L. Harwood, Albert J. Dijkstra. -- 3rd ed. p. ; cm. Includes bibliographical references and index. ISBN-13: 978-0-8493-9688-5 (alk. paper) ISBN-10: 0-8493-9688-3 (alk. paper) 1. Lipids--Handbooks, manuals, etc. I. Gunstone, F. D. II. Harwood, John L. III. Dijkstra, Albert J. [DNLM: 1. Lipids. QU 85 L7633 2007] OP751.L547 2007 572’57--dc22 Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

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CONTENTS

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix Editors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii

1

Fatty Acid and Lipid Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 C.M. Scrimgeour and J.L. Harwood 1.1 Fatty acid structure (CMS). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.2 Lipid structure (JLH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

2

Occurrence and Characterisation of Oils and Fats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 F.D. Gunstone and J.L. Harwood 2.1 Introduction (FDG) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 2.2 Major oils from plant sources (FDG). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 2.3 Minor oils from plant sources (FDG) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 2.4 Milk fats, animal depot fats and fish oils (FDG). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 2.5 Waxes (JLH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .108 2.6 Egg lipids (JLH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .115 2.7 Milk lipids (JLH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .117 2.8 Liver and other tissue lipids (JLH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .119 2.9 Cereal lipids (JLH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .121 2.10 Leaf lipids (JLH). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .123 2.11 Algal lipids (JLH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .125 2.12 Fungal lipids (JLH). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .128 2.13 Bacterial lipids (JLH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .134 2.14 Lipids of viruses (JLH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .141

3

Production and Refining of Oils and Fats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .143 A.J. Dijkstra and J.C. Segers 3.1 Introduction (AJD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .143 3.2 Production of animal oils and fats (AJD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .147 3.3 Production of vegetable oils and fats (AJD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .155 3.4 Degumming of oils and fats (AJD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .177 3.5 Alkali refining of oils and fats (AJD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .191 3.6 Soapstock and by-product treatments (AJD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .204 3.7 Bleaching of oils and fats (AJD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .212 3.8 Dewaxing of oils (AJD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .231 3.9 Vacuum stripping of oils and fats (AJD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .235 3.10 HACCP for oils and fats supply chains (JCS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .251

4

Modification Processes and Food Uses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .263 A.J. Dijkstra 4.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .263 4.2 Hydrogenation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .264 v

Contents

4.3 4.4 4.5 4.6

Interesterification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .285 Fractionation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .300 Food grade emulsifiers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .315 Food uses of oils and fats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .333

5

Synthesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .355 M.S.F. Lie Ken Jie, J.L. Harwood and F.D. Gunstone (with W.H. Cheung and C.N.W. Lam) 5.1 Unsaturated fatty acid synthesis via acetylene (MSFLKJ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .355 5.2 Fatty acid synthesis by the Wittig reaction (MSFLKJ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .359 5.3 Isotopically labelled fatty acids (MSFLKJ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .363 5.4 Synthesis of acylglycerols (MSFLKJ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .368 5.5 Fullerene lipids (MSFLKJ). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .375 5.6 Glycerophospholipids (JLH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .386 5.7 Sphingolipids (JLH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403 5.8 Glycosylglycerides (JLH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .406 5.9 Bulk separation procedures (FDG). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .410

6

Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .415 A. J. Dijkstra, W.W. Christie and G.Knothe 6.1 Introduction (AJD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .415 6.2 Requirements stemming from quality control and process investigation (AJD) . . . . . . . . . . . . . . . . . . . .420 6.3 Some selected analytical methods (AJD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .423 6.4 Chromatographic analysis of lipids (WWC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .426 6.5 Nuclear Magnetic Resonance Spectroscopy (GK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .455

7

Physical Properties: Structural and Physical Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .471 I. Foubert, K. Dewettinck, D. Van de Walle, A.J. Dijkstra and P.J. Quinn 7.1 Introduction (IF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .471 7.2 Crystallisation and melting (IF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .472 7.3 Phase behaviour (KD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .491 7.4 Lipid/water interactions (DVdW) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .495 7.5 Interaction between lipids and proteins (PJQ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .503 7.6 Biological membranes (PJQ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .509

8

Chemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .535 G. Knothe, J.A. Kenar and F.D. Gunstone 8.1 Autoxidation and photo-oxidation (GK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .535 8.2 Enzymatic oxidation (GK). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .542 8.3 Epoxidation, hydroxylation and oxidative fission (GK). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .546 8.4 Halogenation and halohydrins (GK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .551 8.5 Oxymercuration (JAK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .552 8.6 Metathesis (JAK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .554 8.7 Stereomutation (JAK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .555 8.8 Double-bond migration and cyclisation (JAK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .557 8.9 Cyclisation (GK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .559 8.10 Dimerisation (JAK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .564 8.11 Chain branching and extension (GK). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .566 8.12 Hydrolysis, alcoholysis, esterification and interesterification (GK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .570 8.13 Acid Chlorides, Anhydrides and Ketene Dimers (JAK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .576 8.14 Peroxy acids and related compounds (JAK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .577 8.15 Nitrogen-containing compounds (JAK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .579 8.16 Other reactions of the carboxyl group (JAK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .583 8.17 Oleochemical carbonates (JAK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .585 8.18 Guerbet compounds (GK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .587

9

Nonfood Uses of Oils and Fats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .591 F.D. Gunstone, J. Alander, S.Z. Erhan, B.K. Sharma, T.A. McKeon and J.-T. Lin 9.1 Introduction (FDG) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .591 9.2 Basic oleochemicals (FDG) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .592 9.3 Surfactants (FDG) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .597 9.4 Cosmetics and personal care products (JA). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .604 9.5 Lubricants (SZE and BKS). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .610 9.6 Biofuels (FDG) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .625 9.7 Surface coatings and inks (FDG) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .629 9.8 Castor oil products (TAMcK and J-TL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .632 vi

Contents

10

Lipid Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .637 J.L. Harwood 10.1 Fatty acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .637 10.2 Glycerophospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .668 10.3 Glyceride metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .680 10.4 Glycosylglycerides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .686 10.5 Sphingolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .689 10.6 Lipids as signalling molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .694 10.7 Sterol esters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .698 10.8 Control mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .699

11

Medical and Agricultural Aspects of Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .703 J.L. Harwood, M. Evans, D.P. Ramji, D.J. Murphy and P.F. Dodds 11.1 Human dietary requirements (JLH). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .703 11.2 Lipids and cardiovascular disease (ME) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .710 11.3 Clinical aspects of lipids with emphasis on cardiovascular disease and dyslipaemia (DPR) . . . . . . . . . .721 11.4 Skin lipids and medical implications (JLH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .742 11.5 Sphingolipidoses (JLH). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .746 11.6 Other disorders of lipid metabolism (JLH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .749 11.7 Pulmonary surfactant (lung surfactant) (JLH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .751 11.8 Agricultural aspects (DJM, JLH and PFD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .756

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 783

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i Dictionary Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Name Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 617

vii

PREFACE

The Lipid Handbook was first published in 1984, with a second edition in 1994. We now present the third edition of this successful book, with Albert Dijkstra replacing Fred Padley as a member of the editorial team. The decision to revise this book was made late in 2004 and most of the writing was completed during 2005. We planned the book to take account of the many changes in lipid science and technology that have occurred in the past 10 years, but we sought to maintain the approach and organisation of material used in the earlier editions. Compared to the second edition, some chapters have been combined — “Fatty acid structure” with “Lipid structure” (Chapter 1), “Separation and isolation” with “Analytical methods” (Chapter 6), along with the two chapters on “Physical properties” (Chapter 7). Other chapters have been divided — The former chapter on “Processing” now appears as separate chapters devoted to “Production and refining of oils and fats” (Chapter 3) and to “Modification processes and food uses” (Chapter 4). One new chapter —“Nonfood uses” (Chapter 9) has been introduced. All chapters have been rewritten (often by a new author) and we have sought to present information on the basis of thinking and practice in the present day. One interesting change is that the processing sections refer to patents now easily accessible through espacenet.com or uspto.gov. In addition, the Dictionary section has been extended on the basis of the latest Taylor & Francis Group databases. This contains a wealth of information covering chemical structures, physical properties, and references to hundreds of lipid and lipid-related molecules, only some

of which can be detailed in the text. We are grateful to Taylor & Francis for allowing us to include this information and we thank Fiona Macdonald for assistance in selecting and organising it. In order to make our task manageable in the time scale agreed between the publishers and the editors and to present authoritative coverage of our topics, we have secured the assistance of several contributors from Europe, Hong Kong and the United States. Only one contributor (P. J. Quinn) and two of the editors (F. D. Gunstone and J. L. Harwood) were involved with the previous edition and almost the entire text now has different authors. This brings fresh minds to the volume. By bringing a wide range of information into a single volume, we hope that the book will be useful to all who work in the lipid field as scientists or technologists, in industrial or academic laboratories, as newcomers, or as those who already know their way around the field. Lipid science is of increasing interest for metabolic, nutritional, and environmental reasons and we offer this revised and updated volume as a contribution to that growth. For 20 years the book has provided assistance to a generation of those working with lipids and we offer LH-3 (our acronym for this work) to the next generation. The third edition is also available on a CD-ROM (included with the book). This will provide a compact form of the so-called “Handbook” and will be easily searchable, thereby providing easy access to material hidden in tables and figures and in the extensive list of references, which now come with full titles.

F. D. Gunstone J. L. Harwood A. J. Dijkstra

ix

EDITORS

Frank D. Gunstone, Ph.D., is professor emeritus of the University of St. Andrews (Scotland) and holds an honorary appointment at the Scottish Crop Research Institute (Invergowrie, Dundee, Scotland). He received his Ph.D. from the University of Liverpool (England) in 1946 for studies with the late Professor T. P. Hilditch, and subsequently, there followed an academic career in two Scottish Universities: Glasgow (1946 to 1954) and St. Andrews (1954 to 1989). He continues to be professionally active and has spent over 60 years studying fatty acids and lipids with many publications to his credit. Since his retirement in 1989, Dr. Gunstone has written or edited several books. He has given many invited lectures and has received distinguished awards in the United States (1973, 1999, 2005, and 2006), Britain (1962 and 1963), France (1990), Germany (1998), and Malaysia (2004). For many years he has been the editor of Lipid Technology, an activity that gives him continued contact with lipid scientists of many differing interests.

currently editor of four journals, including executive editor of Progress in Lipid Research. Dr. Harwood has published nearly 500 scientific papers and communications, plus authoring three books (including Lipid Biochemistry) and editing 14 others. He has given many plenary and named lectures, received his D.Sc. in 1979 and is in receipt of personal prizes. He also has awards for his publications and those of his students. He is an honorary visiting scientist at the Malaysian Palm Oil Board (Kuala Lumpur), Centre d’Etudes Nucléaires (Grenoble), and the Hungarian Academy of Sciences (Szeged). Albert J. Dijkstra, Ph.D., specialised in gas kinetics with Professor A. F. Trotman-Dickenson at University College of Wales, Aberystwyth, before defending his Ph.D. thesis at Leyden University in 1965. He joined ICI, first at the Petrochemical & Polymer Laboratory in Runcorn, Cheshire, then at the ICI Holland Rozenburg Works, The Netherlands, and finally at the ICI Europa headquarters in Everberg, Belgium. He became involved in edible oils and fats in 1978 when he joined the Vandemoortele Group in Izegem, Belgium, as its R&D director. Dr. Dijkstra is the inventor in a dozen patents and has published numerous articles on edible oil processing. He was the first nonAmerican to receive the American Oil Chemists’ Society (AOCS) Chang Award (1997) and the first to receive the EuroFedLipid Technology Award (2002). Although officially retired, he continues to be active in the field of edible oils and fats as author and scientific consultant.

John L. Harwood, Ph.D., is head of the School of Biosciences at Cardiff University (Wales, United Kingdom). He received his Ph.D. from the University of Birmingham in 1969, with studies on the metabolism of inositol lipids with Professor J. N. Hawthorne and, subsequently, learned about plant fatty acid synthesis at the University of California with Professor P. K. Stumpf. Following a tenure at the University of Leeds, he moved to Cardiff where he was promoted via reader to professor in 1984. He is

xi

CONTRIBUTORS

J. Alander

AarhusKarlshamn, Sweden AB

W. W. Christie

The Scottish Crop Research Institute Invergowrie Dundee, Scotland

K. Dewettinck

Laboratory of Food Technology and Engineering Ghent University Ghent, Belgium

A. J. Dijkstra

Scientific Consultant St. Eutrope-de-Born, France

P. F. Dodds

Department of Biology Imperial College at Wye Wye, Ashford, U.K.

F. D. Gunstone

The Scottish Crop Research Institute Invergowrie Dundee, Scotland

J. L. Harwood

Cardiff School of Biosciences Cardiff University Cardiff, Wales

J. A. Kenar

USDA, ARS, NCAUR Peoria, Illinois USA

G. Knothe

USDA, ARS, NCAUR Peoria, Illinois USA

M. S. F. Lie Ken Jie

M. Evans

Jiann-Tsyh Lin

Llandough Hospital Llandough, Cardiff, Wales

I. Foubert

Laboratory of Food Technology and Engineering Ghent University Ghent, Belgium

School of Applied Sciences University of Glamorgan Pontypridd, Wales

P. J. Quinn

S. Z. Erhan

USDA, ARS, NCAUR Peoria, Illinois USA

D. J. Murphy

Hong Kong University Hong Kong, China

Department of Life Sciences King’s College London, UK

D. P. Ramji

School of Biosciences Cardiff University Cardiff, Wales

C. M. Scrimgeour

The Scottish Crop Research Institute Invergowrie Dundee, Scotland

J. C. Segers

Jacques Segers Consultancy Nieuwerkerk aan den IJssel, The Netherlands

B. K. Sharma USDA, ARS, WRRC Albany, California USA

Department of Chemical Engineering Pennsylvania State University University Park, Pennsylvania USA

D. Van De Walle

T. A. McKeon

USDA, ARS, WRRC Albany, California USA

xiii

Laboratory of Food Technology and Engineering Ghent University Ghent, Belgium

1 FATTY ACID AND LIPID STRUCTURE

C. M. Scrimgeour and J. L. Harwood

1.1

Fatty acid structure

1.1.1

Introduction and nomenclature of fatty acids

substituents. However, the terms cis and trans (abbreviated c and t) are widely used to describe double bond geometry, as with only two types of substituents there is no ambiguity that requires the systematic Z/E convention (Figure 1.1). However, a recent proposal for systematic naming for use in lipidomic and bioinformatic databases requires the use of Z or E (Fahy et al., 2005a, 2005b). Systematic names for fatty acids are cumbersome in general use and both shorthand alternatives and trivial names are widely used. Trivial names seldom convey any structural information, often reflecting a common or early source of the acid. The shorthand names use two numbers separated by a colon for the chain length and number of double bonds, respectively. Octadecenoic acid with 18 carbons and 1 double bond is, therefore, 18:1. The position of double bonds is indicated in a number of ways — explicitly, defining the position and configuration or locating double bonds relative to the methyl or carboxyl ends of the chain. In the biomedical literature, it is common to number the chain from the methyl end rather than the systematic numbering from the carboxyl end, to emphasise the biosynthetic relationship of different double bond patterns. Numbering from the methyl end is written n-x or ωx, where x is the double bond carbon nearest the methyl end. If there is more than one double bond, a cis configuration, methylene-interrupted pattern is implied. Although the n-x notation is recommended, both n-x and ωx are widely used in the current biomedical literature and wider nutritional contexts. The ∆ notation is used to make it explicit that the numbering is from the carboxyl end. Other substituents may also be included in the shorthand notation; for example 12-OH 18:1 9c for ricinoleic acid (12-hydroxy-9-cis-octadecenoic acid). The order and style used for shorthand names varies widely in the literature.

Fatty acids are aliphatic, usually straight chain, monocarboxylic acids. The broadest definition includes all chain lengths, but most natural fatty acids have even chain lengths between C4 and C22, with C18 the most common. Natural fatty acid structures reflect their common biosynthesis — the chain is built in two-carbon units and cis double bonds are inserted at specific positions relative to the carboxyl carbon. Over 1000 fatty acids are known with different chain lengths, positions, configurations and types of unsaturation, and a range of additional substituents along the aliphatic chain. However, only around 20 fatty acids occur widely in nature; of these, palmitic, oleic, and linoleic acids make up ~80% of commodity oils and fats. Figure 1.1 shows the basic structure of fatty acids and a number of the functional groups found in fatty acids. A list of many of the known structures, sources, and trivial names is available online (Adlof and Gunstone, 2003). Table 1.1 illustrates the naming of some commonly encountered fatty acids (additional examples are found in the following sections). Fatty acids are named systematically as carboxylic acid derivatives, numbering the chain from the carboxyl carbon (IUPAC-IUB, 1976). Systematic names for the series of saturated acids from C1 to C32 are given in Table 1.2. The -anoic ending of the saturated acid is changed to -enoic, -adienoic, -atrienoic, -atetraenoic, -apentaenoic, and -ahexaenoic to indicate the presence of one to six double bonds, respectively. Carbon–carbon double bond configuration is shown systematically by Z or E, which is assigned following priority rules for the 1

1.1

Fatty acid structure

or

COOH

CH3(CH2)16 COOH

Fatty acid with saturated alkyl chain R

R′

R

R

Methylene interrupted double bonds

R′

H

R

R

H

H

H

R′

trans (E)

cis (Z)

Conjugated double bonds

Double bond configuration OH

. Acetylene

Allene

Methyl branch

Hydroxyl

O O Cyclopropane

FIGURE 1.1 TABLE 1.1

Furan

Fatty acid structure and some functional groups found in fatty acids. Structure, systematic, trivial, and shorthand names of some common fatty acids

Structure CH3(CH2)l0COOH CH3(CH2)12COOH CH3(CH2)14COOH CH3(CH2)5CH=CH(CH2)7COOH CH3(CH2)16COOH CH3(CH2)7CH=CH(CH2)7COOH CH3(CH2)5CH=CH(CH2)9COOH CH3(CH2)5CH=CH(CH2)9COOH CH3(CH2)3(CH2CH=CH)2(CH2)7COOH CH3(CH2CH=CH)3(CH2)7COOH CH3(CH2)3(CH2CH=CH)3(CH2)4COOH CH3(CH2)18COOH CH3(CH2)3(CH2CH=CH)4(CH2)3COOH CH3(CH2CH=CH)5(CH2)3COOH CH3(CH2)20COOH CH3(CH2)7CH=CH(CH2)11COOH CH3(CH2CH=CH)6(CH2)2COOH CH3(CH2)22COOH CH3(CH2)7CH=CH(CH2)13COOH a

Epoxide

Cyclopropene

Trivial Name/ Abbreviation

Systematic Name Dodecanoic Tetradecanoic Hexadecanoic Z-9-hexadecenoic Octadecanoic Z-9-octadecenoic Z-11-octadecenoic E-11-octadecenoic Z,Z- 9,12-octadecadienoic Z,Z,Z- 9,12,15-octadecatrienoic Z,Z,Z- 6, 9,12-octadecatrienoic eicosanoica Z,Z,Z,Z- 5,8,11,14-eicosatetraenoica Z,Z,Z,Z,Z- 5,8,11,14,17eicosapentaenoica docosanoic Z-13-docosenoic Z,Z,Z,Z,Z,Z- 4,7,10,13,16,19docosahexaenoic tetracosanoic Z-15-tetracosenoic

Shorthand Name

lauric myristic palmitic palmitoleic stearic oleic cis-vaccenic vaccenic linoleic (LA) α-linolenic (ALA) γ-linolenic (GLA) arachidic arachidonic (ARA) EPA

12:0 14:0 16:0 16:1 9c 18:0 18:1 9c 18:1 11c 18:1 11t 18:2 9c,12c 18:3 9c,12c,15c 18:3 6c,9c,12c 20:0 20:4 5c,8c,11c,14c 20:5 5c,8c,11c,14c,17c

behenic erucic DHA

22:0 22:1 13c 22:6 4c,7c, 10c,13c,16c,19c 24:0 24:1 15c

lignoceric nervonic

n- or ω

7 9 7 7 6 3 6 6 3

9 3

9

Icosa- replaced eicosa- in systematic nomenclature in 1975, but the latter is still widely used in the current literature.

The following sections describe classes of naturally occurring fatty acids, emphasising acids that are nutritionally and biologically important, are components of commodity oils and fats, or are oleochemical precursors. The structures of many fatty acids are contained in the dictionary section of this book. Up to date information on fatty acid occurrence in seed oils can be found online (Aitzetmuller et al., 2003) and this is the source of much of the data in Section 1.1.2. Further information on fatty acid structure is available online at http://www.lipidlibrary.co.uk/ and http://www.cyberlipid.org/. The structures of naturally occurring fatty acids are most easily

rationalised by considering their biosynthesis; a few basic processes build and extend the chain and insert double bonds, producing the common families of fatty acids. We do not consider the details of these biochemical processes here (see Section 10.1), but the reader should be aware of the result of the various enzyme processes that build and modify fatty acids. Saturated fatty acids are built from two carbon units, initially derived from acetate, added to the carboxyl end of the molecule, usually until there are 18 carbons in the chain. Double bonds are introduced by desaturase enzymes at specific positions relative to the carboxyl group. Elongases further 2

Fatty Acid and Lipid Structure

TABLE 1.2

Systematic, trivial, and shorthand names and melting points of saturated fatty acids

Systematic Name

Trivial Name

Shorthand Name

methanoic ethanoic propanoic butanoic pentanoic hexanoic heptanoic octanoic nonanoic decanoic undecanoic dodecanoic tridecanoic tetradecanoic pentadecanoic hexadecanoic heptadecanoic octadecanoic nonadecanoic eicosanoic heneicosanoic docosanoic tricosanoic tetracosanoic pentacosanoic hexacosanoic heptacosanoic octacosanoic nonacosanoic triacontanoic hentriacontanoic dotriacontanoic

formic acetic propionic butyric valeric caproic enanthic caprylic pelargonic capric

1:0 2:0 3:0 4:0 5:0 6:0 7:0 8:0 9:0 10:0 11:0 12:0 13:0 14:0 15:0 16:0 17:0 18:0 19:0 20:0 21:0 22:0 23:0 24:0 25:0 26:0 27:0 28:0 29:0 30:0 31:0 32:0

a

lauric myristic palmitic margaric stearic arachidic behenic lignoceric cerotic carboceric montanic melissic lacceric

Melting Pointa (°C) 8.4 16.6 –20.8 –5.3 –34.5 –3.2 –7.5 16.5 12.5 31.6 29.3 44.8 41.8 54.4 52.5 62.9 61.3 70.1 69.4 76.1 75.2 80.0 79.6 84.2 83.5 87.8 87.6 90.9 90.4 93.6 93.2 96.0

Data from The Lipid Handbook, 2nd Edition (1994), Chapman & Hall, London. With permission.

References

extend the chain in two carbon units from the carboxyl end. These processes produce most of the fatty acids of commercial importance in commodity oils and fats, and which are considered to be of most value in food and nutrition. A great diversity of fatty acid structures is produced by variations on the basic process. The start, particularly, of the chain elongation process may be derived from acids other than acetate, resulting in odd or branched chains. Enzymes closely related to the desaturases may introduce functional groups other than double bonds, but usually with similar positional patterns. The result is a great variety of fatty acid structures, often restricted to a few related plant genera in which the altered enzymes have evolved. Additional structural variety is introduced by subsequent modification of fatty acids, e.g., oxidation at or near the carboxyl or methyl end. The Euphorbiacae and Compositae (Asteracae) are particularly adept at producing many and varied fatty acid structures. Fatty acids may be modified further, producing other groups of natural products, such as polyacetylenes, ecosanoids, and oxylipins. The following sections illustrate these various structures, but are not exhaustive.

Adlof, R.O. and Gunstone, F.D. (2003) Common (non-systematic) names for fatty acids. http://www.aocs.org/member/ division/analytic/fanames.asp Aitzetmuller, K. et al. (2003) A new database for seed oil fatty acids — the database SOFA, Eur. J. Lipid Sci. Technol., 105, 92–103. http://www.bagkf.de/sofa/ Fahy, E. et al. (2005a) A comprehensive classification system for lipids. J. Lipid Res., 46, 839–861. Fahy, E. et al. (2005b) A comprehensive classification system for lipids. Eur. J. Lipid Sci. Technol., 107, 337–364. IUPAC-IUB (1976) Nomenclature of Lipids, World Wide Web version, prepared by G.P. Moss. http://www.chem. qmul.ac.uk/iupac/lipid/

1.1.2 1.1.2.1

Fatty acids Saturated acids

Saturated fatty acids form a homologous series of monocarboxylic acids (CnH2n+1COOH). Table 1.2 lists the saturated acids from C1 to C32 with their systematic and trivial names and melting points. Naturally occurring saturated acids are mainly of even chain length between C4 3

1.1

Fatty acid structure

and C24. Fats rich in saturated acids are high melting and are characteristic of many tropical species. Odd chain acids are usually minor or trace components of plant and animal lipids, but some are more abundant in bacterial lipids. Short chain acids, particularly butyric (4:0), are found mainly in ruminant milk fats. Medium chain fatty acids (8:0, 10:0, 12:0, and 14:0) occur together in coconut and palm kernel oils, both tropical commodity oils. In both of these oils, lauric acid (12:0) predominates (45 to 55%), with 14:0 next most abundant. A number of Lauracae and Myristacae species contain in excess of 80% of 12:0 or 14:0, respectively. Cuphea, a temperate genus, has species rich in individual medium chain acids, e.g., C. pulcherrima >90% 8:0, C. koehneana >90% 10:0, and C. calophylla ~85% 12:0. These include some of the highest levels of single fatty acids in seed oils. Palmitic acid (16:0) is the most abundant and widespread natural saturated acid, present in plants, animals, and microorganisms. Levels of 20 to 30% are common in animal lipids, 10 to 40% in seed oils. Palm oil is a rich commodity oil source and contains over 40% of palmitic acid. Stearic acid (18:0) is also ubiquitous, usually at low levels, but is abundant in cocoa butter (~34%) and some animal fats, e.g., lard (5 to 24%) and beef tallow (6 to 40%). A few tropical plant species contain 50 to 60+% of 18:0, e.g., Shorea, Garcinia, Allanblackia, and Palaquium. Arachidic acid (20:0) is 20 to 30% of the seed oils of some tropical Sapindaceae species, but is usually a minor component of plant and animal lipids. Groundnut oil is the only commodity oil with significant amounts (~1%). Saturated acids are often most easily obtained by hydrogenation of more readily available unsaturated acids, e.g., docosanoic acid (22:0) could be obtained by hydrogenation of erucic acid (22:1). Chain shortening and chain extension reactions give access to odd or even chain lengths not readily found in natural sources. Saturated acids with 10 or more carbons are solids, and melting points increase with chain length (see Table 1.2). Melting points alternate between odd and even chain length, with odd chain lengths having a lower melting point than the preceding even chain acid. Polymorphism occurs, where one or more lower melting, metastable forms exist. 1.1.2.2

The most common monoene is oleic acid (18:1 9c). Oleic acid (1) is found in most plant and animal lipids and is the major fatty acid in olive oil (70 to 75%) and several nut oils, e.g., macadamia, pistachio, pecan, almond, and hazelnut (filbert) contain 50 to over 70%. High oleic varieties of sunflower and safflower contain 75 to 80% oleic acid. COOH Oleic acid (1)

Cis-vaccenic acid (18:1 11c, n-7) is common in bacterial lipids and a minor component of plant and animal lipids, co-occurring with the more abundant oleic acid. Cis-vaccenic is relatively abundant in sea buckthorn pulp, which is also rich in its n-7 biosynthetic precursor 16:1 9c. Petroselinic acid (18:1 6c) makes up over 50% of seed oil fatty acids of Umbelliferae species, such as carrot, parsley, and coriander, and is also found in the Araliaceae, Garryaceae, and Geraniaceae species. The biosynthesis of petroselinic acid involves a ∆4 desaturase acting on palmitic acid (16:0) followed by two carbon chain elongation (Cahoon et al., 1994). Palmitoleic acid (16:1 9c, n-7) is a ubiquitous minor component in animal lipids; somewhat more abundant in fish oils. A few plant oils are richer sources, e.g., nuts such as macadamia (20 to 30%) and the pulp of sea buckthorn (25 to 40%). C20 monoenes (11c and 13c) are present in brassica seed oils and the 9c and 11c isomers are found in fish oils. 20:1 5c is >60% of meadowfoam (Limnanthes alba) seed oil fatty acids. Erucic acid (22:1 13c, n-9) is up to 50% of Cruciferae oils, e.g. rape, mustard, crambe and over 70% in some Tropaeolum species. Nervonic acid (24:1 15c, n-9) occurs at 15 to 20% in Lunaria annua seed oil, along with higher levels of erucic acid. Some monoenes are used as or have potential use as oleochemicals. Erucic acid, as the amide, is used as an antislip agent for polythene film. 20:1 5c from meadowfoam oil can be used to prepare estolide lubricants and other novel materials. ω-Olefins, such as 10-undecenoic acid available from pyrolysis of castor oil, are useful oleochemical intermediates. Cis-monoenes with 18 or less carbons are liquids at room temperature or low-melting solids; higher homologues are low-melting solids. Trans-monoenes are higher melting, closer to the corresponding saturated acids. Double bond position also influences the melting point; both cis- and trans-C18 monoenes are higher melting when the double bond is at even positions than at odd positions; a pattern most distinct for double bonds between C4 and C14. The solid acids may exist as a number of polymorphs, with different melting points, resulting from subtly different packing in the crystal (Table 1.3).

Monoenoic acids

Straight-chain, cis-monoenoic acids with an even number of carbons are common constituents of many lipids and commodity oils. Trans- monoenes are rare components of natural oils and fats (see Section 1.2.6). The cis (Z) double bond is usually inserted by a ∆9-desaturase enzyme into preformed saturated acids; this may be followed by twocarbon chain extension at the carboxyl end. Starting with 16:0, this results in n-7 monoenes, while desaturation of 18:0 leads to the n-9 family. Monoenes may also result from desaturation at the ∆4 or ∆5 positions since oils with unsaturation at these positions occur in a few plant genera.

1.1.2.3

Methylene-interrupted polyunsaturated acids

Most unsaturated fatty acids with two or more double bonds show a characteristic methylene-interrupted pattern

4

Fatty Acid and Lipid Structure

fatty acids found in most plants, animals, and commodity oils and fats. Linoleic acid (18:2 n-6, 2) is present in most plant oils and is abundant (>50%) in corn, sunflower, and soybean oils, and exceeds 70% in safflower oil. γ-linolenic acid (18:3 n-6, 3) is usually a minor component of animal lipids, but is relatively abundant in some plant oils, e.g., evening primrose (~10%), borage (~20%), blackcurrant (~15%), and echium (~25%). Other n-6 acids, dihomo-γ-linolenic acid (20:3 n-6) and arachidonic acid (20:4 n-6) are present in animal tissues, but do not usually accumulate at significant levels in storage fats. These two C20 acids are the precursors of the PG1 and PG2 prostaglandin families, respectively. Some fungi, e.g., Mortierella species produce up to 50% arachidonic acid in storage lipids and are a commercial source of this acid (Ratledge, 2004).

TABLE 1.3 Trivial names and melting points of some monoene fatty acids Fatty Acid 16:1 9c (n-7) 16:1 9t (n-7) 18:1 9c (n-9) 18:1 9t (n-9) 18:1 6c 18:1 11c (n-7) 18:1 11t (n-7) 20:1 5c (n-16) 20:1 11c (n-9) 22:1 13c (n-9) 24:1 15c (n-9) a

Trivial Name palmitoleic palmitelaidic oleic elaidic petroselinic cis-vaccenic vaccenic gondoic erucic nervonic

Melting Pointa (°C) 0.5 32 16.2, (13.3) 45.5 31, (29) 15.5 44.1 27 25 33.5 (–52, –7, 2, 14) 45, 41

Data from The Lipid Handbook, 2nd Edition (1994), Chapman & Hall, London. With permission. Also references in Section 1.1.3. Polymorph melting points in parentheses.

of unsaturation, with one CH2 between cis double bonds. This pattern results from the operation of a few specific desaturases and chain-elongation enzymes. Plants generally insert double bonds at the ∆9, ∆12, and ∆15 positions in C18 fatty acids, giving n-9, n-6, and n-3 compounds, respectively. Animals can also insert double bonds at the ∆9 position, but not at ∆12 or ∆15; instead, further double bonds are introduced between the carboxyl group and the ∆9 position by ∆5 and ∆6 desaturase enzymes and the chain can then be extended in two carbon units at the carboxyl end of the molecule. The resulting n-6 and n-3 polyenes are shown in Figure 1.2. The step leading to DHA appears to be the result of a ∆4 desaturase, but is usually the net result of two elongations, a ∆6 desaturase and subsequent two-carbon chain shortening. Leonard et al. (2004) have reviewed the biosynthesis of long chain polyenes. Along with a few saturates (mainly 16:0 and 18:0, but also 10:0 to 14:0) and oleic acid, the n-6 and n-3 polyenes make up the n–9 18:1 9c oleic

COOH Linoleic acid (2) COOH γ-linolenic acid (3)

α-linolenic acid (18:3 n-3, 4) is ubiquitous in plant leaf lipids and is present in several commodity seed oils: 8 to 10% in soybean and canola, >50% in linseed oil, and 65 to 75% of perilla oil. The seed oils of many Labiatae species are >50% α-linolenic acid. In plant leaves, chloroplast lipids contain up to 50% α-linolenic acid accompanied, in some species, by its C16 homologue, 16:3 7c, 10c,13c (Mongrand et al., 1998). Stearidonic acid (18:4 n-3, 5) is a minor component of animal lipids and fish oils and is found in some seed oils, e.g., blackcurrant (up to 5%) and echium (~7%). The n-3 long-chain, polyunsaturated fatty

n–6 ∆12 D

n–3

18:2 9c, 12c linoleic

18:3 9c, 12c, 15c ∆15 D

∆6 D

α–linolenic ∆6 D

18:3 6c, 9c, 12c

18:4 6c, 9c, 12c, 15c stearidonic

γ-linolenic E

E

20:3 8c, 11c, 14c

20:4 8c, 11c, 14c, 17c

∆5 D

∆5 D

20:4 5c, 8c, 11c, 14c arachidonic

20:5 5c, 8c, 11c, 14c, 17c EPA E, E, ∆6 D, –2C 22:6 4c, 7c, 10c, 13c, 16c, 19c DHA

FIGURE 1.2

Biosynthesis of n-6 and n-3 polyenes (D = desaturase, E = elongase, -2C = two-carbon chain shortening).

5

1.1

Fatty acid structure

acids (LC-PUFA) 20:5 (EPA, 6) and 22:6 (DHA, 7) are important nutritionally and are mainly obtained from oily fish and fish oils where they are present at levels from 5 to 20%. EPA is the precursor of the PG3 prostaglandin series. Attempts are being made to produce EPA and DHA in plant lipids by the incorporation of appropriate enzymes because of the desire to have new sources of these important acids. Two types of microorganisms, a dinoflagellate Crypthecodinium cohnii and marine protist Schizochytrium species, are commercial single-cell oil sources of DHA (Ratledge, 2004). α-linolenic acid

∆5t double bond occur in Thalictrum species (see Section 1.2.6). COOH Pinolenic acid (8)

Sponges and some other marine invertebrates contain a wide range of fatty acids with 5c,9c double bonds, with chain lengths (both odd and even) ranging from C16 to C34, known as demospongic acids. Additional double bonds are usually n-7 or n-9 and methyl branching may also be present (Dembitsky et al., 2003).

COOH

1.1.2.5

COOH Stearidonic acid (5)

COOH EPA (6) COOH DHA (7)

While the n-3 and n-6 polyenes are the most widely occurring and of prime biological and nutritional interest, a large number of other methylene-interrupted polyenes are known, produced by the same desaturation and elongation steps, but starting with fatty acids of different chain length and initial unsaturation. For example, animals deprived of linoleic or linolenic acids can use oleic acid as substrate for the ∆6 desaturase and subsequent steps, resulting in an n9 polyene series. The accumulation of 20:3 n-9 (Mead’s acid) in animals is considered to be a symptom of essential fatty acid (i.e., linoleic acid) deficiency. The presence of two or more cis double bonds results in a large lowering of the melting point compared to saturates of the same chain length and these polyenes are all liquid at room temperature. Linoleic acid melts at −5°C. 1.1.2.4

Conjugated acids

Fatty acids with two or more conjugated double bonds are found in some plants and animals. Ruminant fats contain small amounts (~1%) of “conjugated linoleic acid” (CLA), resulting from bio-hydrogenation of linoleic and α-linolenic acids in the rumen, which gives mainly the 18:2 9c,11t isomer (rumenic acid, 9). The only reported long chain, conjugated diene from a plant is 18:2 10t,12t (~10%), which occurs in Chilopsis linearis along with the more abundant conjugated triene 18:3 9t,11t,13c. Estolides in stillingia oil (Sapium sebiferum) and Sebastiana species contain 10:2 2t,4c linked to a short chain allenic hydroxy acid (Spitzer et al., 1997; Figure 1.3). Conjugated dienes (and higher polyenes) are prepared chemically from methylene-interrupted fatty acids by alkaline isomerisation. Under controlled conditions, linoleic acid produces a mixture containing only the 9c11t and 10t12c CLA isomers (Sæbø, 2001). These isomers have potential uses in modifying body composition and as anticancer agents.

(4)

COOH Rumenic acid (9)

Conjugated trienes and tetraenes are found in several plant species. They are produced biologically from methylene-interrupted polyenes by a conjugase enzyme similar to ∆12 desaturase, which shifts an existing double bond into conjugation with a new double bond (Dyer et al., 2002). Table 1.4 gives the structure, common name, source, and melting point of the known conjugated trienes and tetraenes from plants. Conjugated trienes and tetraenes containing cis double bonds readily isomerise to the all trans form on heating or on exposure to light. Tung oil, containing >60% α-eleostearic acid (10), oxidises and

Bis- and polymethylene-interrupted acids

Fatty acids with bis- or polymethylene-interrupted double bonds, or a mixture of methylene and polymethylene separated unsaturation, occur in some plant species and marine organisms. Often these have a double bond inserted at the ∆5 position in addition to one or more double bonds in more usual positions. Bis-methylene-interrupted acids with a ∆5c double bond are common in gymnosperms (conifers), a typical example being pinolenic acid (18:3 5c,9c,12c) (8), occurring at levels of 25 to 30% in a number of pine and larch species (Wolff et al. 2001). Among angiosperms, Limnanthes alba (meadowfoam) seed oil contains the polymethylene-interrupted 22:2 5c,13c (~20%) and other ∆5 acids. Bis-methylene-interrupted acids with a

O O R

O

R′

O

. O

O O

FIGURE 1.3 18:2, 18:3.

6

O

Estolide from stilingia oil. R, R’ 16:0, 18:0, 18:1,

Fatty Acid and Lipid Structure

tion of commodity oils may result in low levels of trans isomers, particularly of polyenes. The undesirable nutritional properties of trans acids have led to alternative ways of producing hardened fats, such as interesterification or blending with fully saturated fats, and to the use of milder deodorisation procedures.

polymerises readily and is used as a drying agent in paints and varnishes. Along with CLA, there has been recent interest in the biological and nutritional properties of conjugated polyenes. COOH

α-eleostearic acid (10)

1.1.2.6

1.1.2.7

Trans acids

Fatty acids with acetylenic and allenic unsaturation are rare. The two types of unsaturation are isomeric and can be interconverted. In the allenic function, the double bonds are rigidly held at right angles and introduce a twist in the molecule, resulting in optical activity when they are asymmetrically substituted. The estolide oil in stillingia oil contains the allenic hydroxy acid 8-hydroxy-5,6-octadienoic acid (Spitzer et al., 1997; Figure 1.3). The (R,E) form of 2,4,5-tetradecatrienoic acid is an insect sex pheromone. Fatty acids with a 5,6 allene are found in the seed oils of a few Labiatae species: laballenic acid (18:2 5,6; 11) is up to 25% of Phlomis tuberosa and some Leucas species; lamenallenic acid (18:3 5,6,16t) is up to 10% in Lamium purpureum.

Monoenes and methylene-interrupted polyenes are predominantly cis. A few trans monoenes and dienes with typical double bond positions are known, e.g., 18:1 9t in Butyrospermum parkii (12.5%) and Dolichos lablab (15%), co-occurring with 18:1 9c, and 18:2 9t12t, (~15%) in Chilopsis linearis, associated with conjugated acids. Thalictrum (and some other Ranunculaceae species) contain several acids with a ∆5t bond, 16:1 5t (~2%), 18:1 5t (~20%), 18:2 5t,9c (~6%), and 18:3 5t,9c,12c (~45%). A similar pattern with ∆3t unsaturation is seen in some Aster species. 16:1 3t occurs widely in leaves associated with chloroplast lipids. Vaccenic acid, 18:1 11t, is the most abundant trans monoene in ruminant lipids, which contain a complex mixture of both cis and trans positional isomers resulting from biohydrogenation of linoleic and linolenic acids. Conjugated acids usually contain one or more trans double bonds (see Section 1.2.5). Trans isomers, mainly monoenes, are produced during catalytic partial hydrogenation, and can be present in substantial amounts in hardened fats, generally as a mixture of positional isomers. Heat treatment during deodorisaTABLE 1.4

Acetylenic and allenic acids

.

COOH

Laballenic acid (11)

Fatty acids containing an acetylenic group are tariric acid (18:1 6a, 12), up to 85% of some Picramnia species and crepenynic acid (18:2 9c,12a, 13) 50 to 75% of some

Common name, source, and melting point of some conjugated fatty acids

Fatty Acid

Common Name

10:2 2t,4c

Source

Melting Pointa (°C)

Sapium sebiferum (stillingia oil) (~5 to 10%)

18:2 18:2 18:2 18:2 18:2 18:2 18:2

8t,10t 9t,11t 9c,11c 9c,11t 10t,12t 10t,12c 10c,12c

CLA CLA CLA CLA CLA CLA CLA

18:3 18:3 18:3 18:3 18:3 18:3 18:3 18:3

8t,10t,12t 8t,10t,12c 8c,10t,12c 9t,11t,13t 9c,11t,13tb 9t,11t,13c 9c,11c,13t 9c,11t,13c

β-calendic calendic jacaric β-eleostearic α-eleostearic catalpic – punicic

Calendula officinalis (tr) Calendula officinalis (60%) Jacaranda mimosifolia (36%) Aleurites fordii (11%) Aleurites fordii (Tung oil), Parinarium spp., Momordica sp. (>60%) Catalpa spp. (~40%) – Punica granatum (~70%), Momordica balsamina (~60%)

78 40 44 72 49 32 62 45

α-parinaric β-parinaric

Parinarium laurinum (>50%), Impatiens spp. (>20%) –

86 96

18:4 9c,11t,13t,15cc 18:4 9t,11t,13t,15t a b

c

ruminant fats Chilopsis linearis (~10%)

56 54 43 20 56 23 39

Data from The Lipid Handbook, 2nd Edition (1994), Chapman & Hall, London. With permission. Occurs also as the 18-hydroxy (kamlolenic acid, Mallotus philippinensis (70%)) and 4-oxo (licanic acid, Licania rigida (80%)) derivatives. Occurs also as the 4-oxo derivative (Chrysobalanus icaco (18%)).

7

1.1

Fatty acid structure

Crepis species. In C. alpina, the acetylenic bond is introduced by a ∆12-desaturase-like enzyme (Lee et al., 1998). Crepenynic acid is the starting point for the biosynthesis of a large number of fatty acid-derived acetylenic and polyacetylenic secondary natural products (e.g., matricaria ester). Stearolic acid (18:1 9a), the acetylenic analogue of oleic acid (from which it is easily prepared), is not often found in nature, other than as a minor component. However, it is more abundant in some Pyrularia species, P. edulis containing over 50%. Tariric acid (12)

are, however, abundant in the surface waxes of plant leaves. Fatty acids with a mid-chain methyl branch are characteristic of some bacteria. For example, 10-R-methyloctadecanoic acid (tuberculostearic acid) (14) is the major normal chain length fatty acid in Mycobacterium tuberculosis, the causative agent of tuberculosis, and is found in a number of other actinomycetes. The biosynthesis involves methylation of oleic acid, the methyl carbon being derived from the C-1 pool. C16 to C24 mid-chain methyl branched acids are also found in Mycobacterium species. H

COOH

COOH Tuberculostearic acid

COOH

(14)

Polymethyl fatty acids include those of isoprenoid origin, derived from partial metabolism of the phytyl chain from dietary chlorophyll. Phytanic (15) and pristanic acids (16) are the most common examples and are minor components of fish oils. A different pattern is seen in fatty acids from bird uropygial glands where the methyl groups are found on alternate, usually even, carbons, with two to four methyl groups present, e.g., (17) found in the preen gland wax of the graylag goose. Dimycocerosate esters, found in mycobacteria, contain a range of polyketidederived polymethyl fatty acids. These also have the methyls on alternate even carbons (Onwueme et al., 2005).

Crepenynic acid (13)

1.1.2.8

Branched chain acids

Straight chain fatty acids are the norm, but a wide variety of branched chain structures are known, mainly from bacterial and some animal sources. These acids are usually saturated or monoenes and the alkyl branch is a methyl group. Acids with a methyl group on the n-2 or n-3 carbon (iso and anteiso, respectively; Figure 1.4) are common in bacteria; their occurrence and distribution being strong taxonomic indicators. The biosynthesis of these acids involves the normal two-carbon chain extension, but instead of starting with a two-carbon acetate-derived unit, they start with 2-methyl propionic acid (from valine) or 2-methyl butanoic acid (from leucine), respectively. The resulting iso and anteiso acids, thus, have an even and odd total number of carbons, but α-oxidation may subsequently shorten the chain resulting in both odd and even carbon iso and anteiso acids. The shorthand nomenclature for these acids can be confusing, as the total number of carbons is shown, while the systematic name uses the number of carbons in the longest alkyl chain. For example, 15-methyl hexadecanoic acid is iso-17:0. Iso and anteiso acids found in animal fats, particularly ruminant fats, are mostly derived from bacteria in the diet or digestive system. However, some specific acids are of animal origin: 18-methyleicosanoic acid is the major thioester-bound fatty acid on the surface of wool and mammalian hair fibres, producing a continuous hydrophobic layer (Jones and Rivett, 1997). Iso and anteiso acids are rarely found in plant oils, apart from 14-methylhexadecanoic acid, which is found as a taxonomically useful minor component (~1%) in the Pinacae family. These acids

iso

FIGURE 1.4

CH3

COOH Phytanic acid (15)

COOH Pristanic acid (16)

COOH (17)

1.1.2.9

Cyclic fatty acids

Cyclic fatty acids, with a carbon ring along or at the end of the alkyl chain, occur naturally in some bacteria and plants. In addition, a variety of carbocyclic structures are formed from methylene-interrupted polyenes during heating, for example, during deep frying. The sources, synthesis, and biological properties of cyclic fatty acids have been reviewed by Sebedio and Grandgirard (1989). Fatty acids with a mid-chain cyclopropane group are found mainly in bacteria, with cis-9,10-methylenehexadecanoic (9,10 cpa 17:0); cis-9,10-methyleneoctadecanoic acid (9,10 cpa 19:0; dihydrosterculic acid); and cis-10,11-methyleneoctadecanoic acid (10,11 cpa 19:0; lactobacillic acid, 18) most common. They are found in diverse bacterial species, both aerobic and anaerobic, and in both Gram-negative

anteiso

Iso and anteiso branched-chain structures.

8

Fatty Acid and Lipid Structure

and Gram-positive species. Depending on culture conditions, they may be up to 35% of the membrane lipids.

various chain lengths. The most abundant is usually the C16 hydnocarpic acid (22), but in Oncoba and Caloncoba species the C18 chaulmoogric acid (23) predominates (~70%). Gorlic acid (24), C18 with a ∆6 double bond, is usually 10 to 20% of these oils. Related homologues from C6 to C20 are often found at low levels. Arum maculatum seed oil contains ~20% of 13-phenyltridecanoic acid (25)

COOH Lactobacillic acid (18)

Biosynthesis of the cyclopropane ring involves addition of a methylene group, derived from S-adenosylmethione (the “C1 pool”), to an existing double bond, for example, lactobacillic acid is derived from cis-vaccenic acid, the most abundant monoene in many bacteria. The cyclopropane acids that have been found in protozoa, slime moulds, and invertebrates are most likely derived from bacteria in their diet. The distribution and biosynthesis of cyclopropane acids in bacteria has been reviewed by Grogan and Cronan (1997). Cyclopropane acids are often found at low levels (~1%) in plant oils containing cyclopropene acids (see below). Litchi chinensis, however, contains ~40% dihydrosterculic acid (9,10 cpa 19:0) along with small amounts of shorter chain homologues. Cyclopropene acids are found in plant oils of the Malvalaceae, Sterculiaceae, Bombacaea, Tiliaceae, and Sapicidacaea families. These are mainly sterculic acid (9,10methyleneoctadec-9-enoic acid; 9,10 cpe 19:1; 19) and malvalic acid (8,9-methyleneheptadec-8-enoic acid; 8,9 cpe 18:0; 20). Sterculic acid is usually the more abundant (>50% in Sterculia foetida oil) accompanied by smaller amounts of malvalic acid. 2-hydroxysterculic acid may also occur in these oils, probably an intermediate in the biosynthesis of malvalic acid by α-oxidation of sterculic acid. 9,10-methyleneoctadec9-en-17-ynoate (sterculynic acid) occurs in Sterculia alata (~8%). The biosynthesis of the cyclopropene ring is not fully understood, but is thought to proceed from oleic acid to the cyclopropane, produced by the same mechanism as in bacteria, followed by further desaturation. Long chain cyclopropane and cyclopropylidene fatty acids have been found in sponges, for example, (21) from the Amphimedon species (Nemoto et al., 1997). Their biosynthesis is unknown.

COOH Hydnocarpic acid (22)

COOH Chaulmoorgic acid (23)

COOH Gorlic acid (24)

COOH

13-phenyltridecanoic acid (25)

Bacteria isolated from the extreme environment of hot springs produce fatty acids with a terminal cyclohexyl group. In strains of the acidophilic and thermophilic Bacillus acidocardarius, 11-cyclohexylundecanoic acid and 13-cyclohexyltridecanoic acid (26) account for 70 to over 90% of the fatty acids in the bacteria (Oshima and Ariga, 1975). One of the most unusual fatty acid structures reported to date is a terminal concatenated cyclobutane or ladderane, containing up to five cis-fused four membered rings (e.g. 27). These occur as glycerol and methyl esters in the unusually dense membranes of anammox bacteria (Damste et al., 2002).

COOH

COOH

Sterculic acid (19)

13-cyclohexyltridecanoic acid

COOH Malvalic acid

(26)

(20)

COOH

COOH (21)

(27)

Fatty acids with terminal rings are thought to be produced by incorporating a cyclic acid rather than acetate at the start of the chain, although the biosynthetic origin of the cyclic acid has not always been unequivocally established. Up to 80% of the seed oils of Hydnocarpus species and other genera of the Flacourtiaceae are terminal cyclopentenyl acids of

1.1.2.10

Fatty acids with oxygen-containing functional groups

Most fatty acids contain only double bonds, but a number of fatty acids and their metabolites have oxygen-containing functional groups, most commonly a hydroxyl or epoxide. 9

1.1

Fatty acid structure

Some of these are introduced by enzyme-mediated oxidation of methylene-interrupted fatty acids, e.g., by lipoxygenase or the initial stages of fatty acid catabolism, the latter giving hydroxyl groups near the carboxyl or methyl end of the chain. Autoxidation, occurring in the absence of enzymes also gives oxygen-containing products (hydroxy, keto, epoxy, etc.) with less positional specificity. In a few plant oils, hydroxy and epoxy groups are introduced in mid-chain positions by enzymes with the same positional specificity as desaturases. Castor oil, rich in ricinoleic acid (12-OH 18:1 9c), is the only commodity oil containing a fatty acid with a functional group other than double bonds. Oils containing vernolic acid (an epoxy acid) have been investigated as oleochemical precursors. Ricinoleic acid (R-12-hydroxy-9-cis-octadecenoic acid; 12-OH 18:1 9c; 28) is 80 to 90% of castor oil (from Ricinus communis). It occurs at similar levels in Hiptage species and is found in a number of other species. In Azima tetracantha, Argyreia cuneata, and Anogeissus latifolia, it occurs at levels of 10 to 25% along with lower amounts of the cyclopropene, which contain malvalic and sterculic acids (see Section 1.2.9). The sclerotia of the ergot fungus (Claviceps purpurea) contain up to 50% ricinoleic acid (see below). Isoricinoleic acid (R-9-hydroxy-12-cis-octadecenoic acid; 9-OH 18:1 12c) is over 70% of the Wrightia species. Lesquerolic acid (R-14-hydroxy-11-cis-eicosenoic acid; 29), the C20 homologue of ricinoleic acid, occurs in Lesquerella species (50 to 70%). It is produced from ricinoleic acid by an elongase specific for hydroxy acids (Moon et al., 2001). Related acids found in Lesquerella species include densipolic acid (12-OH 18:2 9c, 15c) and auricolic acid (14-OH 20:2 11c, 17c). Hydroxy (and keto) acids are also found with conjugated double bonds (see Table 1.4). These include kamlolenic acid (18-OH 18:3 9c, 11t, 13t) in Mallotus philippinensis (70%) and coriolic acid (13-OH 18:2 9t,11c) in Coriaria species (~70%).

Cutin, a cross-linked polyester constituent of plant cuticle, contains a number of C16 and C18 mono, di, and trihydroxy fatty acids. The C16 acids, derived from palmitic acid contain a terminal hydroxyl group and a mid-chain hydroxyl between C7 and C10. The predominant C18 acids, derived from oleic acid, are 18-hydroxyoleic, 9,10,18-trihydroxystearic and 9,10-epoxy-18-hydroxystearic acids. The primary hydroxyls are mainly ester linked, while the midchain hydroxyls are only partially esterified. Polyhydroxy acids are not usually found in seed oils; however, 9,10,18trihydroxy-12-cis-octadecenoic acid occurs as ~14% of Chamaepeuce afra oil. 2-hydroxy or α-hydroxy acids occur in sphingolipids, skin lipids, wool wax, bacterial cell wall lipids, and in a few seed oils. In some Thymus species 2-hydroxylinolenic occurs up to ~13%, along with linolenic acid and its C17 homologue (17:3 8c,11c,14c). The hydroxy acid is probably an intermediate in the biosynthesis of the C17 acid (see also hydroxysterculic acid, Section 1.2.9). Salvia nilotica oil contains α-hydroxy oleic, linoleic, and linolenic acids along with traces of C17 acids. 3-hydroxy or β-hydroxy fatty acids are found in bacterial lipids, both medium to normal chain-length saturates and in mycolic acids. Mycolic acids are very long chain compounds, typically C60 to C90, branched at C2, with unsaturation or cyclopropane groups along the long chain in addition to the 3-hydroxy group. Vernolic acid (12-epoxy-9-cis-octadecenoic acid, 30) is the most widespread epoxy acid in plant oils occurring in a number of Compositae, Malvaceae and Euphorbiaceae species. It makes up 60 to 80% of Vernonia oils and is over 90% of Bernardia pulchella oil. (+)-vernolic acid with the 12S,13R configuration is the most usual form, but the other optical isomer (–)-vernolic acid, has been isolated from some seed oils of the Malvaceae. In Crepis palaestina and Vernonia galamensis, the epoxide group is introduced by a ∆12-desaturase-like enzyme (Lee et al., 1998). However, in Euphorbia lagascae, the epoxygenase is a cytochrome P450 acting on linoleic acid (Cahoon et al., 2002).

OH COOH Ricinoleic acid (28)

O COOH OH

Vernolic acid

COOH

(30)

Lesquerolic acid (29)

Other epoxy acids include coronoric acid (9,10-epoxy12-cis-octadecenoic acid), which occurs in a number of mainly Compositae species and is ~15% of Chrysanthemum coronarium oil. It is also found in sunflower and other oils after prolonged storage of the seeds. 9,10-epoxyoctadecanoic acid is found at low levels in Tragopogon porrifolius oil, and alchornoic acid (14,15-epoxy-11-cis-eicosanoic acid), the C20 homologue of vernolic acid, occurs in Alchornea cordifolia (~50%). A number of oxygen-containing fatty acid derivatives are produced from methylene-interrupted fatty acids following

A hydroxyl group along the acyl chain can be esterified to other fatty acids, forming an estolide. In castor oil, ricinoleic acid is present only in simple triacylglycerols, but in the ergot fungus Claviceps purpurea, ricinoleic acid is extensively esterified with both nonhydroxy acids and other molecules of ricinoleic acid in polyestolide groups (Batrakov and Tolkachev, 1997). Seed oils of Lesquerella and related species rich in lesquerolic acid contain estolides (Hayes et al., 1995).

10

Fatty Acid and Lipid Structure

R2

O

R1

COOH PGE1

lipoxygenases R2

R2

R1

R1

HO 20:3 n–6

OOH

HOO

OH

HO COOH PGF1α

jasmonates, leukotrienes (a)

HO

OH

O COOH

C OOH PGE2 HO

arachidonic acid 20:4 n–6 cyclooxygenase

OH

HO COOH

O

PGF2α

COOH HO

O OOH

OH

O

cyclic endoperoxide

C OOH PGE3 HO

prostaglandins, prostacyclins, thromboxane (b)

OH

20:5 n–3 HO

FIGURE 1.5 Formation of hydroperoxides and cyclic endoperoxides catalysed by (a) lipoxygenase and (b) cyclooxygenase enzymes.

COOH PGF3α HO

the formation of a hydroperoxide or cyclic endoperoxide catalysed by lipoxygenase and cyclooxygenase enzymes, respectively (Figure 1.5). Subsequent cyclisation and modification leads to physiologically active products, such as eicosanoids (in mammals) and jasmonates and divinyl ether fatty acids (in plants), and also to furanoid fatty acids. Although the enzyme-catalysed, oxygen addition is stereo and regiospecific, the range of starting acids and subsequent modifications results in many different products; only a few representative structures are shown here. Eicosanoids are biologically active C20 fatty acid metabolites acting as short-lived hormones or mediators, and include prostaglandins, thromboxanes, and leukotrienes. The PG1, PG2, and PG3 families of prostaglandins are derived from dihomo-g-linolenic acid (20:3 n-6), arachidonic acid (20:4 n-6), and eicosapentaenoic acid (EPA, 20:5 n-3), respectively, via their cyclic endoperoxides (Figure 1.6). Christie (2005) has recently reviewed ecosanoid structure and function. Among other functions, prostaglandins are involved in the inflammatory response, platelet aggregation, vasodilation, and smooth muscle function. Jasmonates are produced in plants following lipoxygenase catalysed conversion of 16:3 n-3, 18:2 n-6, and

FIGURE 1.6

OH

The PG1, PG2, and PG3 families of prostaglandins.

18:3 n-3 to conjugated hydroperoxides, which are then converted to a range of metabolites. The most widely studied is (–)- jasmonic acid (31), which is derived from 13-hydroperoxylinolenic acid. The cyclised product is chain shortened to C12 by β-oxidation. Jasmonates have hormone properties, regulating plant growth and development and are involved in leaf senescence and in defence against pathogens and in wound signalling (Farmer et al., 2003). O

COOH Jasmonic acid (31)

Divinyl ether synthase in plant leaves and roots converts hydroperoxides generated by lipoxygenase to divinyl ethers. In the potato, the 9-hydroperoxides of linoleic and linolenic acids lead to colneleic (32) and colnelenic acids

11

1.1

Fatty acid structure

e.g., (36) from the Parmelia species. A number of toxic ωfluoro fatty acids have been isolated from the South African plant Dichapetalum toxicarium. The origin of the fluorine is fluoroacetic acid, which can accumulate in the leaves of Dichapetalum species. The most abundant is 18-fluoro-oleic acid.

(33), respectively. Structurally similar compounds are derived from the 13-hydroperoxides in Ranunculus leaves, e.g., etherolenic acid (34) (Hamberg, 1998). These compounds are thought to be plant defence compounds protecting against pathogen attacks. O

COOH

Colneleic acid COOMe

(32) Br

O

COOH

(36)

Colnelenic acid (33) O

Sulfur-containing fatty acids have been reported at trace levels (,90% α-linolenic acid in chloroplastic 26

Fatty Acid and Lipid Structure

TABLE 1.11

Some bacterial sn-O-glycosyldiacylglycerols

Glyceride

Structure of Glycoside Moiety

Monoglucosyldiacylglycerol Diglucosyldiacylglycerol Diglucosyldiacylglycerol Dimannosyldiacylglycerol Galactofuranosyldiacylglycerol Galactosylglucosyldiacylglycerol Glucosylgalactosylglucosyldiacylglycerol

Occurrence Pneumococcus, Mycoplasma Staphylococcus Mycoplasma, Streptococcus Microccus lysodeikticus Mycoplasma, Bacteroides Lactobacillus Lactobacillus

α-D-Glucopyranoside β-D-Glucopyranosyl(1→6)-O-β-D-glucopyranoside α-D-Glucopyranosyl(1→2)-O-β-D-glucopyranoside α-D-Mannopyranosyl(1→3)-O-D-mannopyranoside α-D-Galactofuranoside α-D-Galactopyranosyl(1→2)-O-α-D-glucopyranoside α-D-Glucopyranosyl(1→6)-O-α-Dgalactopyranosyl(1→2)-O-α-D-glucopyranoside

Adapted from Kates (1972).

A number of gluco- and galactoglycerolipids have been isolated in small quantities from animal tissue. Their structures are given in Murray and Narasimhan (1990). The majority of galactoglycerolipids contain a single galactose residue, which is linked in a β-glycosidic link between the C-1 of galactose and the C-3 of glycerol. The glucoglycerolipids constitute a large number of compounds with up to eight glucose residues linked α(1→6). Alkylacyl and diacyl lipids as well as sulfated forms have been reported (Slomiany et al., 1987).

TABLE 1.12 danica

Derivative MonochloroDichloroPentachloroHexachloro-

References

Position of substitution in 1,14dicosane-disulfate 13 11,15 3,3,11,13,16 3,3,11,13,15,16

Position of substitution in 1,15-tetracosanedisulfate 14 2,12,14,16,17 2,2,12,14,16,17

Chlorosulpholipids are found in some fungi (e.g., caldariomycin) and certain algae. Ochromonas danica contains particularly high amounts of chlorosulpholipids, where they represent almost half of the total membrane lipids. An entire family of compounds can be found with two sulfate ester functions and from one to six chlorines (Haines, 1973), as shown in Table 1.12. Some other structures (e.g., trichloro derivatives) have been reported but not characterized (Haines, 1973). An unusual glycolipid sulfate ester (36) has been reported in extremely halophilic bacteria by Kates and coworkers (cf. Kates, 1972), and a glycolipid sulfate (2,3,6,6′-tetraacetyl-α-α-tetrahalose-2′-sulfate) in Mycobacterium tuberculosis (Goren, 1970). For a review of sulfated glycolipids in Archaebacteria, see Kates (1990), and for mycobacterial sulphoglycolipids, see Goren (1990). Sulfated glycolipids and sterols are minor components of animal tissues (see Murray and Narasimhan, 1990). Lactosyl sulfatide and seminolipid are examples of the former (Ishizuka, 1997). A number of novel taurine-containing lipids have been isolated from the ciliated protozoan, Tetrahymena. These have the structures shown in (37).

Harwood, J.L. (1980) Plant acyl lipids: structure, distribution and analysis. In Biochemistry of Plants, vol. 4, P.K. Stumpf and E. E. Conn, Eds., New York: Academic Press, pp. 1–55. Harwood, J.L. and Okanenko, A.A. (2003) Sulphoquinovosyl diacylglycerol — the sulpholipid of higher plants. In Sulfur in Plants, Y.P. Abrol and A. Ahmad, Eds., Dordrecht: Kluwer, pp. 189–219. Kates, M. (1972) Techniques in Lipidology, 2nd ed., Amsterdam: Elsevier. Kates, M. (1990) Glyco-, phosphoglyco- and sulfoglycoglycerolipids from bacteria. In Handbook of Lipid Research, vol.6, M. Kates, Ed., New York: Plenum, pp. 1– 122. Murray, R.K and Narasimhan, R. (1990) Glycerolipids in animal tissues. In Handbook of Lipid Research, vol. 6, M. Kates, Ed., New York: Plenum, pp. 321–361. Slomiany, B.L., Murty, V.L.N., Liau, Y.N., and Slomiany, A. (1987) Animal glycoglycerolipids. Prog. Lipid Res., 26, 29– 51.

1.2.6

Structures of the sulfolipids of Ochromonas

Sulfur–containing lipids

In addition to cerebroside sulfates, other sulfur-containing sphingolipids (Section 1.2.4) and diacylsulpho-quinovosylglycerol and sulfated gluco- or galactogly-cerolipids (Section 1.2.5), various other sulfur-containing lipids have been reported. These include alkyl sulfates (Mayers et al., 1969) in microorganisms (35).

–O SO 3

– 3-Gal(1

6)Man(1

2)Glc(1

1') –OCH2 ROCH

OSO3–

ROCH2

H3C[CH2]7CH[CH2]12CH2OSO3–

(36) Glycolipid sulfate ester

(35) (1,14S)-Docosanediol-1,14-disulfate

27

1.2 Lipid structure

development. Small quantities of such diols are found in mammalian and fish liver, mammalian adipose tissue, egg yolk, corn seed, and yeast. Diesters of butane-1,3-diol and butane-1,4-diol are produced by various yeasts (Ratledge and Wilkinson, 1988) and mixed acyl and alk-1-enyl derivatives of these and other simple diols (e.g., ethylene glycol) have been reported (Batrakov et al., 1974). Extracellular acyl esters of arabinitol, xylitol, or mannitol have been reported (Stodola et al., 1967) and an acylated diol phospholipid has been isolated from the yeast Lipomyces starkeyi, after growth on propane-1,2-diol (Suzuki and Hasegawa, 1974).

CH3(CH2)24COO R1 CONHCH2CH2SO3H R2

R3

(37) Taurine-containing lipids Lipid Taurolipid A Taurolipid B Taurolipid C 7-Acyltaurolipid A

R1 OH OH OH CH3(CH2)14COO

R2 H OH OH H

R3 H H OH H

H2COCOR1

An alkaline-stable, taurine-containing lipid, lipo-taurine (2-(7,13-dihydroxy-2-trans-octadecenoylamino)ethanesulphonic acid) was also detected and probably plays a role as a metabolic intermediate. Other related compounds, such as 2-(octadecanoylamino)ethanesulphonic acid, were also identified. The isolation, characterization, and biochemistry of the taurolipids have been reviewed (Kaya, 1992).

CH2 H2COCOR2

(38) Diacylpropane-1,3-diol

References Batrakov, S.G. et al. (1974) Identification of threo-butane-2,3diol phospholipid from Actinomyces olivaceus., Biochim. Biophys. Acta, 337, 29– 40. Ratledge, C. and Wilkinson, S.G. (1988) An overview of microbial lipids. In Microbial Lipids, vol. 1, C. Ratledge and S.G. Wilkinson, Eds., Academic Press, London, pp. 3–22. Stodola, F.H. et al. (1967) Extracellular lipids of yeasts. Bacterial Rev , 31, 194–213. Suzuki, T. and Hasegawa, K. (1974) Diol lipids in the phospholipid fraction of Lipomyces starkeyi grown in the medium containing 1,2-propanediol. Agric. Biol. Chem., 38, 613–620.

References Goren, M.B. (1970) Sulfolipid I of Mycobacterium tuberculosis, strain H37Rv. II Structural studies. Biochim. Biophys. Acta, 210, 127– 138. Goren, M.B. (1990) Mycobacterial fatty acid esters of sugars and sulfosugars. In Handbook of Lipid Research, vol. 6, M. Kates, Ed., Plenum, New York, pp. 363– 461. Haines, T.H. (1973). Halogenated sulphatides. In The Biochemistry of Lipids, T.W. Goodwin, Ed., MTP International Reviews of Science, Biochemistry Series 1, vol. 4, Butterworths, London, pp. 271–286. Ishizuka, I (1997) Chemistry and functional distribution of sulfoglycolipids, Prog. Lipid Res, 36, 245–319. Kates, M. (1986) Techniques of Lipidology,2 nd ed. Elsevier, Amsterdam. Kates, M. (1990) Glyco-, phosphoglyco-, and sulfoglycoglycerolipids of bacteria. In Handbook of Lipid Research, vol. 6 (M. Kates, Ed.), Plenum, New York, pp. 1–122. Kaya, K. (1992) Chemistry and biochemistry of taurolipids. Prog. Lipid Res., 31, 87–108. Lederer, E. (1967). Glycolipids of mycobacteria and related microorganisms. Chem. Phys. Lipids, 1, 294–315. Mayers, G.M. et al. (1969). Microbial sulfolipids. III. Disulfate of (+) -1, 14-docosanediol in Ochromonas danica. Biochemistry, 8, 2981–2986. Murray, R.K. and Narasimhan, R. (1990) Glycerolipids of animal tissues. In Handbook of Lipid Research, vol. 6, M. Kates, Ed., Plenum, New York, pp. 321–361.

1.2.8

Other esters

A wide variety of other lipid esters have been reported. Wax esters are a typical example. Although the term “wax” should, strictly speaking, only be used for esters of long-chain fatty acids with long-chain primary alcohols, common usage, unfortunately, often equates “wax” with an entire mixture of lipids of which the true waxes are but a part. Ester waxes are found in animals and plants where they form part of the water-repellent surface coating (i.e., skin surface of animals and the leaf cuticle (cf. Section 1.2.11)). The general formula for a simple wax is shown in (39). The preen glands of birds, in addition, contain esters of normal alcohols with mono- or multibranched fatty acids (Odham, 1967). H3C[CH2]xCOO[CH2]yCH3

1.2.7

Diol lipids

(39) A simple wax

Complex waxes are compounds where either the fatty acid or the alcohol component or both has a complex structure. For example, the waxes of Mycobacterium spp. are diesters of phthiocerols (C33-C35 branched-chain diols)

Only recently has the presence of diol lipids, such as diacylpropane-1,3-diol (38), been confirmed for a wide variety of tissues. This is probably because techniques for the elucidation of their structures are a recent 28

Fatty Acid and Lipid Structure

with mycocerosic acids (C29-C32 branched-chain acids) (Asselineau, 1966; see Barry et al., 1998).

C22H45 CH2OCOCHCH[CH2]17CH CH[CH2]17CH3 OH O

H HO

OH

H

H

OH

RCOO

H

H O

H

OH

OH H H O OH O H OH O CCHCH[CH2]17CH CH[CH2]17CH3 C22H45

(40) Cholesterol ester FIGURE 1.15

Cord factor from Mycobacterium smegmatis.

CH2OCOR

Esters are found in most of the commonly occurring sterols (40), including those from plant tissues (Mudd, 1980). The fatty acid constituents usually reflect those of the acylglycerols from the same source. Likewise, esters are found in vitamin alcohols, such as vitamin A, the D vitamins, and vitamin E. Examples are shown in (41) to (43). Carotenoid esters have been reported from a few plant sources (Hitchcock and Nichols, 1971). Flower pigments, for example, are known to contain saturated fatty acid esters of carotenoid alcohols. These compounds are also found in green algae. Where dihydroxy alcohols are involved, then both substitutents are usually esterified, e.g., (44). In addition, acyl esters of terpenoid alcohols have been reported. For example, Dunphy and Allcock (1971) showed that 30 to 60% of the total monoterpenoid alcohol content of rose petals occurred as acyl esters with geranyl stearate (45) predominating. An important example of a carbohydrate ester is the so-called cord factor from Mycobacteria spp. This contains an ester of the disaccharide, trehalose, with two molecules of a complex acid, mycolic acid. The latter is a general term embracing a whole series of fatty acids containing 60 to 90 carbons. They are hydroxy fatty acids that differ in their degree of unsaturation and chain branching (see Section 1.1.2.10). In the example given in Figure 1.15, the mycolic acid is the 60-carbon compound found in Mycobacterium smegmatis. Various other esters have been reported in different bacteria. For example, propionibacteria contain diacyl myo-inositol mannosides in which the mannose is glycosidically linked to position 2 of myo-inositol. The 1 and 6 positions of inositol are esterified with fatty acids. Other bacteria and yeasts contain esters of glucose and certain other sugars (Lederer, 1967; Weete, 1980). Not only are simple carbohydrate esters found in Nature, but fatty acyl derivatives of amino acids have also been reported. These include serratamic acid (N-(D-3-hydroxydecanoyl)-L-serine), siolipin A (46), and siolipin B. The latter is the ornithine analogue of siolipin A.

(41) Acyl retinol

CH3 RCOO

(42) Acyl cholecaldiferol CH3 RCOO H3C

O CH3

H 3

(43) Acyl-α-tocopherol

OCO[CH2]14CH3

H3C[CH2]14COO

(44) Luteol dipalmitate

CH2OCO[CH2]16CH3

(45) Geranyl stearate

Animal skin-surface lipids have two types of diester waxes. In the first, a hydroxy acid has its hydroxyl group esterified to a normal fatty acid and its carboxyl group to a fatty alcohol. The second wax type consists of an alkane α, βdiol in which both hydroxyls are esterified with fatty acids (Nicolaides et al. 1970). 29

1.2 Lipid structure

Mycosides from mycobacteria are glycosides of methylated sugars with a long-chain, highly branched hydroxylated hydrocarbon terminated by a phenol group. The hydroxy groups of the long-chain glycol are esterified with mycocerosic and palmitic acids (Kates, 1972). Acylated steryl glycosides of plants usually contain Dglucose attached via a β-glycosidic linkage to the 3-position of sterols, such as sitosterol, stigmasterol, campesterol, and cholesterol (see Figure 1.17). The 6-position of the glucose is esterified with fatty acids, such as palmitic, stearic, oleic, linoleic, and linolenic (Mudd and Garcia, 1975). In some tissues, such as potato tubers, acylated sterol glycosides are major components (around 20% total) (Mudd, 1980). Although glucose is the major esterified sugar, galactose, mannose, xylose, and gentiobiose have been found in isolated cases and an α-glycosidic link also reported (Harwood, 1980). Nitrogen-fixing cyanobacteria produce heterocyst cells containing characteristic glycolipids (see Murata and Nishida, 1987). The chemical structures of major components of Anabaena cylindrica are shown in (47) and (48). Glycerol ester glycolipids (49) and (50) are also present (Murata and Nishida, 1987). The heterocyst lipids of Nodularia harveyana (a marine cyanobacterium) have been purified

[CH2]4NH2 R1CONHCHCOOR2 R1COOH = normal, branched and β-hydroxy-branched acids R2OH = long-chain polyalcohols

(46) Siolipin A

The peptidolipids that occur in mycobacteria and Nocardia spp. are N-acyl oligopeptides. They often occur as glycoside derivatives (Kates, 1972).

References Asselineau, J. (1966) The Bacterial Lipids, Hermann, Paris. Barry, C.E. et al. (1998) Mycolic acids: structure, biosynthesis and physiological functions. Prog. Lipid. Res. 37, 143–179. Dunphy, P.J. and Allcock, C. (1971) quoted by Hitchcock and Nichols (1971). Hitchcock, C. and Nichols, B.W. (1971) Plant Lipid Biochemistry, Academic Press, London. Kates, M. (1986) Techniques in Lipidology, 2 nd ed., Elsevier, Amsterdam. Lederer, E. (1967) Glycolipids of mycobacteria and related microorganisms. Chem. Phys. Lipids, 1, 294–315. Mudd, J.B. (1980) Sterol interconversions. In The Biochemistry of Plants, P.K. Stumpf and E.E. Conn, Eds., vol. 4. Academic Press, New York, pp. 509–534. Nicolaides, F., Fu, H.C., and Ansari, M.N.A. (1970) Diester waxes in surface lipids of animal skin. Lipids, 5, 299–307. Odham, G. (1967) Fatty acids in the feather waxes of some water birds. Fette Seifen Anstrichm. 69, 164–172. Weete, J.D. (1980) Lipid Biochemistry of Fungi and Other Organisms, Plenum, New York.

HO

O

CH3 OCHCH2COOCHCH2COOH

O HO

OH

OH

C7H15

C7H15

(a) CH2OH O HO

1.2.9

O

HO

CH3

Glycosides

CH2OH O

Several types of glycosides can be identified — those of hydroxy fatty acids, aromatic glycols and sterols. Glycolipids of some microorganisms, particularly yeasts, are extracellular products. There has been increasing interest in several of these compounds as biosurfactants (Solaiman, 2005). Emulsan, a polyanionic heteropolysaccharide having acyl chains esterified to the sugar moieties, is produced by Acinetobacter calcoaceticus. Often the lipids contain a mono- or disaccharide glycosidically linked to a hydroxy acid. Examples would be a rhamnolipid from Pseudomonas aeruginosa, a sophorolipid from Candida bombicola (see Figure 1.16) and cellobiolipids from Ustilago maydis (see Ratledge and Wilkinson, 1988). Emulsan is used as a degreasing agent and detergent. Rhamnolipid is used for oil recovery, in the printing industry and in a multitude of applications as a detergent in the agrochemical, food and cosmetic industry, and as a component of germicidal solutions for food and medical uses (Solaiman, 2005).

HO

OCH[CH2]15COOH

OH

CH3 O

OH OH (b)

FIGURE 1.16 (a) Rhamnolipid of Pseudomonas aeruginosa; (b) sophorolipid (yeasts).

RCOOCH2 O HO

O

OH OH

FIGURE 1.17

30

Acylated steryl glucoside (ASG).

Fatty Acid and Lipid Structure

recently and fully characterized as 1-(O-α-D-glucopyranosyl)-3R, 25R-hexacosanediol, 1-(O-α-D-glucopyranosyl)-3S, 25R-hexa-cosanediol, and 1-(O-α-D-glucopyranosyl)-3keto-25R-hexacosanol (Soriente et al., 1992).

Ratledge, C. and Wilkinson, S..G. Eds., (1988) Microbial Lipids, vol. 1, Academic Press, London. Solaiman, D.K.Y. (2005) Applications of microbial biosurfactants. INFORM 16, 408–410. Soriente, A., Sodano, G., Gambacorta, A., and Trincone, A. (1992) Structure of “heterocyst glycolipids” from the marine cyanobacterium, Nodularia harveyana. Tetrahedron, 48, 5375–5384.

Glycosidic glycolipids HOCH2

HOCH2 O HO

OH

O

HO OH

and

O

OH

(90%)

1.2.10

O CH2

OH

OH

OH

The occurrence of lipid-soluble arsenic compounds in marine organisms was first reported over 30 years ago (Lunde 1973). At the present time, more than 100 naturally occurring arsenolipids have been reported, as reviewed recently (Dembitsky and Levitsky, 2004). They are found in a wide variety of organisms ranging from lichens, fungi, and plants, to freshwater and marine algae, and invertebrates, fishes, and animals. The primary analytical technique used for separation, identification, and quantification of arsenolipids is LC coupled to various types of mass spectrometry and a full discussion is given in Dembitsky and Levitsky (2004). Many species of bacteria seem to be active in metabolising arsenic compounds and, in particular, have been shown to be capable of producing methylated derivatives, such as trimethylarsine. These include soil organisms, such as Flavobacterium or Pseudomonas spp. (Shariatpanahi et al., 1981) as well as microorganisms from the deep sea (1000 to 3500 m) where they seem important for the metabolism of arsenic compounds eventually to simpler metabolites and inorganic arsenic (Hanaoka et al., 1997). Other notable microorganisms with high contents of arsenic, including arsenobetaine and arsenocholine compounds, are various halophytes (Oremland and Stolz, 2003). Some examples of arsenolipids found in freshwater and marine algae are given in Figure 1.18. Marine brown algae have been well studied (Dembitsky and Levitsky, 2004), but green and red algae have also been noted to actively metabolise arsenic compounds. From freshwater environments, Chlorella spp. and Chlamydomonas reinhardtii have been studied. Most algal species seem well capable of methylating arsenic as part of the conversion to compounds isolated in the polar lipid fraction. A variety of arsenolipids were identified and these could accumulate in the range of 1.5 to 33.8 µg/g dry weight. Arsenolipids have been identified in marine invertebrates (Benson, 1989), including jellyfish, crustacea, worms and molluscs. Freshwater molluscs, crustacea, and earthworms have also been studied. The major arsenic compound in marine fish and animals is arsenobetaine, first detected in lobsters (Edmonds et al., 1977). Other animals studied include the sperm whale. In plants, arsenic-containing lipids have been identified in species ranging from higher plants through ferns and lichens.

(10%)

(47) 3,25-Dihydroxyhexacosanyl α-D-glycopyranoside HOCH2

HOCH2

O HO

OH

O

HO O

OH

and

OH

O CH2 OH

(90%)

HO

HO OH

(10%)

(48) 3,25,27-Trihydroxyoctacosanyl α-D-glycopyranoside

Glycosyl ester glycolipids HOCH2 O HO

O

OH

O C OH

OH

(49) α-D-Glucopyranosyl 25-hydroxyhexacosanate HOCH2 O HO

OH

O O C

OH

Arsenolipids

HO OH

(50) α-D-Glucopyranosyl 25,27-dihydroxyoctacosanate

References Harwood, J.L. (1980) Plant acyl lipids: structure, distribution and analysis. In Biochemistry of Plants, Vol. 4, P.K. Stumpf and E.E. Conn, Eds., Academic Press, New York, pp. 1–55. Kates, M. (1986) Techniques in Lipidology, 2nd ed. Elsevier, Amsterdam. Mudd, J.B. (1980) Sterol interconversions. In The Biochemistry of Plants, P.K. Stumpf and E.E. Conn, Ed., vol. 4, Academic Press, New York, pp. 509–534. Mudd, J.B. and Garcia, R.E. (1975) Biosynthesis of glycolipids. In Recent Advances in the Chemistry and Biochemistry of Plant Lipids, T. Galliard and E. I. Mercer, Eds., Academic Press, New York, pp. 161–201. Murata, N. and Nishida, I. (1987) Lipids of blue-green algae (cyanobacteria). In The Biochemistry of Plants, vol. 9, P.K. Stumpf and E. E. Conn, Eds., Academic Press, New York, pp. 315–347.

31

1.2 Lipid structure

Shariatpanahi, M. et al. (1981) Biotransformation of the pesticide sodium arsenate. J. Environ. Sci. Health, 16, 35–47.

O H3C–As–CH2 O CH3

OR

18.R = Me OH

19.R = OH

OH OH 20.R =

OH

P O O O

21.R =

OH

22.R =

OH O

26. R = OH

OSO3H OH

OH

SO3H OH

P O O

OCOR1 OCOR

23.R =

SO3– NH3+

24.R =

27. R = OH

OH P O O O

+

O O O

OH OH

OH OH

O 29. R =

OH

28. R =

SO3–

NH3

O

OH OH

OH O

O

O

30. R =

N H

31. R =

Waxes

Plant wax is the general term used to describe the lipid components of the cuticle that covers the outer surface of aerial plant tissues or is associated with the suberin matrix of underground or wound tissues. The components of plant cuticular waxes have been reviewed by Kolattukudy (1980, 1987) and Walton (1990). Major components include hydrocarbons, very long-chain fatty acids, alcohols and monoesters (Table 1.13 and Table 1.14). Surface waxes are exposed to the environment and, therefore, are chemically rather stable. Thus, there is an absence of functional groups, which might be susceptible to attack by atmospheric agents. Furthermore, the very long carbon chains of most wax components reduces their volatility. In addition, many of compounds present in surface waxes are rather stable metabolically and are not readily susceptible to microbial degradation (Kolattukudy, 1976). Certain general structural features of natural waxes have been described by Kolattukudy (1976), and these are summarized in Table 1.13. However, it must also be stressed that the structure and composition of surface waxes vary considerably from organism to organism. Thus, with regard to Table 1.13, the longer aliphatic chains are more abundant in plant waxes than in animal surface waxes, whereas bird waxes may contain appreciable amounts of chains of less than 16 carbons. With regard to branching, methyl branches are the most common, but, in birds, ethyl and propyl branches are found. Although polyunsaturated carbon chains are nearly always absent from surface waxes, in insects substantial proportions of di-unsaturated hydrocarbons have been found. In this case, autoxidation may be reduced by the simultaneous presence of cuticular phenolics. So far as the general composition of surface waxes is concerned, very long chain hydrocarbons are common in insects and plants, but rare in animals. Higher plant waxes contain the most complex

OH

OH 25. R =

1.2.11

OH

OH OH

FIGURE 1.18 Arsenolipids isolated from freshwater and marine algae. (From Dembitsky, V.M. and Levitsky D.O. (2004) Arsenolipids. Prog. Lipid Res., 43, 403–448. With permission.)

Some plants are hyper-accumulators and may take up and accumulate more than 1 mmol As g-1 dry weight (Brooks et al., 1977). Details of all these reports of different arsenolipids in various species and their possible metabolism are given in Dembtisky and Levitsky (2004).

References Benson, A.A. (1989) In Marine Biogenic Lipids, Fats and Oils, R.G. Actaman, Ed., vol. 1, Boca Raton, FL: CRC Press, pp. 243– 250. Brooks R.R. et al. (1977) Detection of nickeliferous rocks by analysis of herbarium specimens of indicator plants. J. Geochem. Explor. 7, 49–57. Dembitsky, V.M. and Levitsky D.O. (2004) Arsenolipids. Prog. Lipid Res., 43, 403–448. Edmonds, J.S. et al. (1977) Isolation, crystal-structure and synthesis of arsenobetaine, arsenical constituent of western rock lobster Panulirus longipes cygnus George. Tetrahedron Lett. 18, 1543–1546. Hanoaka, K. et al. (1997) Arsenobetaine-decomposing ability of marine microorganisms occurring in particles collected at depths of 1100 and 3500 metres. Appl. Organomet. Chem. 11, 265–271. Lunde, G. (1973) Synthesis of fat and water-soluble arseno organic compounds in marine and limnetic algae. Acta Chem. Scand. 27, 1586–1594. Oremland, R.S. and Stolz, J.F. (2003) The ecology of arsenic. Science, 300, 939–944.

TABLE 1.13 Chain length Branching Unsaturation

Functional types

32

General structural features of natural waxes Very long chains (up to C62) are common Branched carbon chains common, with methyl branches frequent Polyunsaturated chains nearly always absent; double bonds, when present, at different position from those of internal lipids Saturated hydrocarbons, olefins, wax esters, aldehydes, ketones, primary and secondary alcohols, and terpenoids can be present; the bulk of the surface lipid is distinctly different from the major internal lipids of the same organism

Fatty Acid and Lipid Structure

TABLE 1.14 Major classes of plant aliphatic wax components Wax Class n-Alkanes Secondary alcohols

Chain-Length Range in Plants C21-C35 C21-C35

Ketones Fatty alcohols

C21-C35 C22-C34

Fatty acids

C16-C34

Aldehydes

C21-C35

Wax esters

C32-C64

Major Arabidopsis Components C29,C31,C27 C29,C31,C27

Notes for Plants in General Common; usually C29, C31 About as common as ketones Not as common as alkanes Common, even chains predominate Very common; even-chain saturated usually Usually minor; not as common as alcohols Common

C29 C28,C30, C26

% in Arabidopsis Stems 38 10 30 12

C30, C28

3

C30, C28

6

-

1

Source: See Kolattukudy, P.E. (1980) In Biochemistry of Plants, vol. 4 (P.K. Stumpf and E. E. Conn, Eds.), Academic Press, New York, pp. 571–645; Kunst, L. and Samuels, A.L. (2003) Prog. Lipid. Res. 42, 51–80.

mixture of components, while insects and birds have the simplest. The wax associated with suberin has also been examined, and very long-chain fatty acids, alcohols and terpenes have all been found. These are all typical components of cuticular wax, but certain differences have been noted. The hydrocarbons in suberin have a broader chain-length distribution with a predominance of shorter carbon chains and more even-numbered carbon chains than cuticular wax. Suberin-associated wax also contains a high proportion of free fatty acids. Free and esterified alkan-2-ols are also present (Kolattukudy, 1980). Further details of the wax components of other organisms will be found in Section 2.5.

Kunst, L. and Samuels, A.L. (2003) Biosynthesis and secretion of plant cuticular wax. Prog. Lipid. Res. 42, 51–80. Walton, T.J. (1990) Waxes, cutin and suberin. In Methods in Plant Biochemistry, vol. 4, J.L. Harwood and J.R. Boyer, Eds., Academic Press, London, pp. 105–158.

1.2.12

In many organisms the outer envelope or covering consists of polymers of carbohydrate or amino acids. In plants, however, the covering (cuticle) consists of a hydroxy fatty acid polymer called cutin. The underground parts and healed wound surfaces of plants are covered with an analogous material, suberin. Both cutin and suberin are embedded in or associated with a complex mixture of lipids, which is termed wax (see Section 1.2.11). The structure and composition of cutin and suberin are reviewed by Kolattukudy (1980, 1987) and by Walton (1990). Cutin contains C16 and C18 families of acids. The former is predominate in rapidly growing plants, while both are present in the thicker cuticle of slower- growing plants. The C16 family is based on palmitic acid, while the C18 family is based on oleic acid (Table 1.15) (see also Kolattukudy, 1980). In the cutin structure, a polyester intramolecular structure exists where crosslinking is mainly influenced by the availability of secondary hydroxyl groups. Thus, cutins that contain large amounts of epoxy, oxo, and ω-hydroxy

References Hamilton, R.J. Ed., (1996) Waxes: chemistry, molecular biology and functions. The Oily Press, Dundee, Scotland. Kolattukudy, P.E. Ed., (1976) Chemistry and Biochemistry of Natural Waxes, Elsevier, New York. Kolattukudy, P.E. (1980) Cutin, suberin and waxes. In Biochemistry of Plants, vol. 4, P.K. Stumpf and E.E. Conn, Eds., Academic Press, New York, pp. 571– 645. Kolattukudy, P.E. (1987) Lipid-derived defensive polymers and waxes and their role in plant-microbial interaction. In Biochemistry of Plants, vol. 9, P.K. Stumpf and E.E. Conn, Eds., Academic Press, New York, pp. 291–314. TABLE 1.15

Cutin and suberin

The major components of cutin, the cutin acids

C16 Family H3C[CH2]14COOH HOCH2[CH2]14COOH HOCH2[CH2]XCHOH[CH2]yCOOH (x + y = 13; y = 5-8)

C18 Familya H3C[CH2]7CH = CH[CH2]7COOH HOCH2[CH2]7CH = CH[CH2]7COOH

HOCH2[CH2]7CH–CH[CH2]7COOH O HOCH2[CH2]7CHOHCHOH[CH2]7COOH

∆12 unsaturated analogues also occur. Note: For further details, see Harwood (1980) and Kolattukudy (1977).

a

33

1.2 Lipid structure

TABLE 1.16

Typical cutin monomers and their ability to form polyesters Cutin Monomers (Acids) Capable of Cross-Linking a Polyester Polymer

HO[CH2]6CHOH[CH2]8COOH HOOC[CH2]5CHOH[CH2]8COOH OHC[CH2]6CHOH[CH2]7COOHa HO[CH2]8CHOHCHOH[CH2]7COOHa HO[CH2]5CHOHCHOHCH2CHOHCHOH[CH2]7COOH

10,16-dihydroxyhexadecanoic acid (and other positional isomers) 7-hydroxyhexadecanedioic acid (and other positional isomers) 9-hydroxy-16-oxohexadecanoic acid (and other positional isomers) 9,10,18-trihydroxyoctadecanoic acid (and its ∆12 analogue) 9,10,12,13,18-pentahydroxyoctadecanoic acid

a

Cutin Monomers (Acids) Capable of Forming Only a Linear Polyester Monobasic α, ω-Dibasic ω-Hydroxymonobasic namely HO[CH2]6CO[CH2]8COOHa

16:0, 18:0, 18:1(9), 18:2 (9,12) 16-hydroxy-10-oxohexadecanoic acid (and other positional isomers)

O HO[CH2]8CH – CH[CH2]7COOHa

9,10-epoxy-18-hydroxyoctadecanoic acid (and its ∆ 12 analogue)

Major components of cutin. Source: Adapted from Deas and Holloway (1977). a

TABLE 1.17

Polymeric form of dihydroxyhexadecanoic acid and related C18 acids in four plant cutins Total Monomers (%)

Polymeric Form Tomato Cutin-O[CH2]15COO-Cutin Cutin-OOC[CH2]5CHOH[CH2]8COO-Cutin

Cutin

O

Cutin-OOC[CH2]5CH[CH2]8COO-Cutin HO[CH2]6CHOH[CH2]8COO-Cutin Cutin-O[CH2]6CHOH[CH2]8COO-Cutin

Cutin-O[CH2]6CH[CH2]8COO-Cutin

O

Lemon

7 6 5

18 5

14 2 1

2 48

5 50

3 38

2 25

36

24

30

5

3 2

3 1

4 2

1 51

Cutin

HO[CH2]6CH[CH2]8COO-Cutin

O

Rosehip

Cutin

O

Cutin

Blackcurrant leaf

5 1 4

[CH2]6CO[CH2]8COO

Cutin

Source: Adapted from Deas and Holloway (1977).

monomers must be predominantly linear (Table 1.16) (Deas and Holloway, 1977). Esterification appears to occur chiefly through the primary hydroxy groups of the monomers. A significant portion (up to 40%) of the monomers is also cross-linked through secondary hydroxyl groups (Table 1.16 and Table 1.17). Considerable diversity is evident when cutins from different sources are compared in detail. It is of interest, though, that the cutin composition of delicate tissues, such as spinach leaves, is essentially similar to that of much more substantial membranes. The wax part of the epidermal layer (see Section 1.2.11) is also usually similar between species, but with differences in detail (Harwood. 1980; Kolattukudy, 1980). The major aliphatic components of suberins are ωhydroxy acids and dicarboxylic acids. Octadec-9-enedioic

acid is the usual dicarboxylic acid, and 18-hydroxyoleic, the major hydroxy fatty acid. The proportion of very longchain fatty acids (>C20) is usually much greater in the ωhydroxy acid fraction than in the dicarboxylic acid fractions. Among the α,ω-diols, fatty alcohols and fatty acids, which are often found as significant components of suberin, long chains are common. Kolattukudy has suggested some basic rules for the classification of hydroxy acid phytopolymers as cutin or suberin (Table 1.18). However, these rules must be regarded only as a guide, since the examination of individual plant species has provided exceptions. Indeed, it should be noted that, apart from species or varietal differences, environmental conditions may cause large changes in surface lipids. Thus, light, temperature, and age have all been found to affect leaf cuticular components (Harwood. 1980).

34

Fatty Acid and Lipid Structure

TABLE 1.18 suberin

Biochemistry of Plants, vol. 9, P.K. Stumpf and E.E. Conn, Eds., Academic Press, New York, pp. 291–314. Walton, T.J. (1990) Waxes, cutin and suberin. In Methods in Plant Biochemistry, vol. 4, J.L. Harwood and J.R. Boyer, Eds., Academic Press, London, pp. 105–158.

Compositional differences between cutin and

Monomer Dicarboxylic acids In-chain substituted acids Phenolics Very long-chain (C20-C26) acids Very long-chain alcohols

Cutin Minor Major Low Rare and minor Rare and minor

Suberin Major Minor High Common and substantial Common and substantial

1.2.13

A full discussion of the varied lipid structures found in bacterial cell walls is beyond the scope of this book, but the reader will find detailed accounts in Rogers et al. (1980) and Goldfine (1982). More recent detailed updates on the biochemistry and distribution of the cell-wall lipids of mycobacteria and other actinomycetes and of Gram-negative bacteria will be found in Brennan (1988) and Wilkinson (I988, 1996), respectively. The structure, biosynthesis, and physiological functions of mycolic acids are reviewed in Barry et al. (1998). The unique lipid-rich cell walls of mycobacteria contribute to their resilience and contain many compounds known to increase pathogenicity. The dimycocerosate esters (also called phthiocerol diesters) are particularly important and have been reviewed recently (Onwueme et al., 2005). Gram-negative bacteria have a cell envelope containing two membranes, with the outer membrane having lipopolysaccharide in its outer leaflet. Lipopolysaccharide is complex and consists of four parts. On the outside is the O-antigen, which is a polysaccharide of variable structure. This is attached to a core polysaccharide, which is in two parts, an outer core and a backbone. The backbone is connected to a glycolipid, called lipid A, through a short “link” usually composed of 3-deoxy-D-manno-octulosonic acid (KDO). These structures are shown in Figure 1.19. The role of lipid A as bacterial endotoxin and further details of different structures are given in Raetz and Whitfield (2002).

Source: Kolattukudy, P.E. (1975) In Recent Advances in the Chemistry and Biochemistry of Plant Lipids (T. Galliard and E.I. Mercer, Eds.), Academic Press, New York, pp. 203–246. With permission.

References Deas, A.H.B. and Holloway. P.J. (1977) The intracellular structure of some plant cutins. In Lipids and Lipid Polymers in Higher Plants, M. Tevini and H.K. Lichtenthaler, Eds., Springer-Verlag, Berlin. pp. 293–299. Harwood, J.L. (1980) Plant acyl lipids: structure, distribution and analysis. In Biochemistry of Plants. vol. 4, P.K. Stumpf and E.E. Conn, Eds., Academic Press, New York. pp. 1–55. Kolattukudy, P.E. (1975) Biochemistry of cutin, suberin and waxes on the lipid barriers of plants. In Recent Advances in the Chemistry and Biochemistry of Plant Lipids, T. Galliard and E.I. Mercer, Eds., Academic Press, New York, pp. 203–246. Kolattukudy. P.E. (1977) Biosynthesis and degradation of lipid polymers. In Lipids and Lipid Polymers in Higher Plants, M. Tevini and H.K. Lichtenthaler, Eds., Springer-Verlag, Berlin. pp. 271–292. Kolattukudy, P.E. (1980) Cutin, suberin and waxes. In Biochemistry of Plants, vol. 4, P.K. Stumpf and E.E. Conn, Eds., Academic Press, New York, pp. 571–645. Kolattukudy, P.E. (1987) Lipid-derived defensive polymers and waxes and their role in plant-microbe interaction. In GlcN

Gal

Glc

Gal

Glc

Abe Man

Hep

Hep EtN

Gal n

Rha

Bacterial-wall lipids

P

P

Outer core

O-antigen

(KDO)3 P

lipid A

EtN

Backbone R-core

(a) Lipopolysaccharide M P

CH2O M0 O

P

O

O

CH2 O

EtN

P

KDO O KDO KDO

NH

M O

O

M0

M

O P

NH M0

(b) Lipid A–KDO link region

FIGURE 1.19 Generalized structures of lipopolysaccharide and lipid A. Abbreviations: Abe, abequose; Man, mannose, Rha, rhamnose; Gal, galactose; Glc, glucose; Hep, heptose; KDO, 3-deoxy-D-manno-octulsonic acid; P , phosphate; EtN, ethanolamine; M, myristate; M0, β-hydroxymyristate. (From Harwood and Russell (1984). With permission.).

35

1.2 Lipid structure

O–

OCH2

H

CHOCOR2 D-alanine O CH O or CH2OPO K OCH2 D-glucose O– n n = 28–35

CH2CHCH2OPO O

CH2OCOR1

O

D-glucose or D-alanine

(a) Teichoic acid (general formula)

(b) Lipoteichoic acid (from Streptococcus lactis) CH2OCOR1 CHOCOR2

D-mannose

OCH2

O

n = 52–75 Succinic acid n (c) Lipomannan (from Micrococcus lysodeikticus)

FIGURE 1.20 Structures of some anionic polymers in bacteria. Abbreviations: K, kojibiose (6,O,β-D-glucosyl-D-glucose); R1 and R2, fatty acids. (From Harwood and Russell (1984) With permission.)

The cell walls and membranes of most Gram-positive bacteria contain a series of highly anionic polymers. Quantitatively, one of the most important of these is teichoic acid, which can be covalently linked to a glycolipid to give a lipoteichoic acid (Figure 1.20). An alternative type of anionic polymer. which is found in Gram-positive bacteria such as Micrococcus lysodeikticus, is succinylated lipomannan (Figure 1.20). Like teichoic acid, the lipomannan is embedded in the membrane by linkage to a diacylglycerol moiety.

S.G. Wilkinson, Eds., Academic Press, London. pp. 203–285. Goldfine, H. (1982) Lipids of prokaryotes — structure and distribution. Curr. Top. Membr. Transp, 17, 1–43. Harwood, J.L. and Russell, N.J. (1984) Lipids in Plants and Microbes, Allen and Unwin, Hemel Hempstead, U.K. Onwueme, K.C. et al. (2005) The dimycocerosate ester polyketide virulence factors of mycobacteria. Prog. Lipid. Res. 44, 259–302. Raetz, C.R.H. and Whitfield, C. (2002) Lipopolysaccharide endotoxins. Annu. Rev. Biochem. 71, 635–700. Rogers, H.J. et al. (1980) Microbial Cell Walls and Membranes, Chapman & Hall, London. Wilkinson, S.G. (1988) Gram-negative bacteria. In Microbial Lipids. vol. 1, C. Ratledge and S.G. Wilkinson, Eds., Academic Press, London, pp. 299–488. Wilkinson, S.G. (1996) Bacterial lipopolysaccharides — themes and variations. Prog. Lipid Res. 35, 283–343.

References Barry, C.E. et al. (1998) Mycolic acids: structure, biosynthesis and physiological function. Prog. Lipid. Res. 37, 181–207. Brennan, P.J. (1988) Mycobacterium and other actinomycetes. In Microbial Lipids. vol. 1, C. Ratledge and

36

2 OCCURRENCE AND CHARACTERISATION OF OILS AND FATS

F. D. Gunstone and J. L. Harwood

2.1

Introduction

dramatic example relates to the attempts — which must surely be ultimately successful — to grow plants that generate long chain PUFA (polyunsaturated fatty acids), such as eicosapentaenoic (EPA) and docosahexaenoic (DHA) in their seed lipids. • For a long time, fats were considered to be useful only as a source of calories, but now, in addition to the recognition of essential fatty acids and to the important minor components present in oils and fats, it is recognised that many fatty acid derivatives and lipids act as signalling molecules in the complex interactions that make up life in both animals and plants. • Gunstone (2005) has calculated the annual fatty acid production during 2004 and 2005 on the basis of the production in that year of 17 commodity oils and fats. Of the 136.4 million tonnes produced, he calculated levels for the following acids: lauric (3.4 million tonnes, 2.5% of total production), myristic (2.6, 1.9%), palmitic (27.4, 20.0%), stearic (7.2, 5.3%), oleic (47.8, 35.1%), linoleic (37.5, 27.5%), linolenic (4.5, 3.3%), and other (6.0, 4.4%). Other figures relate to oils and fats used for food purposes and show the changes resulting from industrial hydrogenation. Attention is drawn to the serious consequence of hydrogenation for the level of linolenic acid, which is the major source of omega-3 PUFA.

The first chapter in this book was concerned with the wide range of fatty acids that occur naturally and with the various natural lipids of which the acids are major constituents. This chapter is devoted to information about the natural occurrence of the lipids covering the important materials (mainly triacylglycerols) that furnish our food lipids and are the basis of the (growing) oleochemical industry and also the less common lipids, such as those occurring in leaves, in algae, etc. These may seem to be mature topics, but they are being developed in many new and important ways. The following are typical: • Oils and fats are being produced in ever-increasing quantities. In Chapter 3 of the second edition of this book (published in 1994), it was forecast that the average annual supply of oils and fats (from 17 commodity sources) in the 5-year period of 2003 to 2007 would be 104 metric tonnes. In the harvest year 2004–2005, the supply was 136 million tonnes, with soybean oil and palm oil predominating, each at levels of 33 million tonnes. • We now recognise a close relation between health and disease on the one hand and dietary intake of lipids on the other. Our increasing ability to modify lipid composition through seed breeding with or without genetic modification is leading to extensive changes in fatty acid composition, driven in large part by nutritional influences. These new products are considered to be healthier fats. This will be illustrated in several ways in the following section. The most

References Gunstone, F.D., Fatty acid production for human consumption, Inform, 16, 736–737, 2005.

37

2.2 Major oils from plant sources

2.2

Major oils from plant sources

2.2.1

Introduction

Akoh, C.C. and Min, D.B. Eds. Food Lipids — Chemistry, Nutrition and Biotechnology, 2nd ed., Marcel Dekker, New York, 2002. Ching Kuang Chow Ed. Fatty Acids in Foods and Their Health Applications, Marcel Dekker, New York, 2000. Firestone, D. Ed. Official Methods and Recommended Practices of the American Oil Chemists’ Society: Physical and Chemical Characteristics of Oils, Fats and Waxes, AOCS Press, Champaign, IL, 1997, reprinted as a book and disc, 1999. Gunstone, F.D. Ed. Vegetable Oils in Food Technology — Composition, Properties and Uses, Blackwell Publishing, Oxford, U.K., 2002. Gunstone, F.D. Ed. Modified Foods For Use in Lipids, Woodhead Publishing Ltd., Cambridge, U.K., 2006. Murphy, D.J., Ed. Plant Lipids — Biology, Utilisation and Manipulation, Blackwell Publishing, Oxford, U.K., 2005. O’Brien, R.D., Fats and Oils — Formulating and Processing for Applications, 2nd ed., CRC Press, Boca Raton, FL, 2004. Rossell, J.B. and Pritchard, J.L.R., Ed. Analysis of Oilseeds, Fats, and Fatty Foods, Elsevier Applied Science, London, 1991. Seed oil fatty acids database (SOFA) www.bagkf.de/SOFA Shahidi, F. Ed. Bailey’s Industrial Oil and Fat Products, 6th ed., Wiley-Interscience, Hoboken, New Jersey, USA, 2005. Ucciani, E., Nouveau Dictionnaire des Huiles Végétales, Compositions en acides gras, Lavoisier Tec & Doc, Paris, 1995. Websites (general) www.codexalimentarius.net www.cyberlipid.org www.fosfa.org www.ncaur.usda.gov/currentres www.usda.gov/nas www.margarine.org www.nal.usda.gov/fnic www.oilworld.de www.gafta.com

This discussion on vegetable fats is divided arbitrarily into major and minor oils, so it is necessary to consult Section 2.3 for a long list of minor oils. The oils in each section are presented in alphabetical order. The major oils are discussed where possible in the following terms: production; harvest yields; trade (exports and imports); major components (fatty acids and triacylglycerols); minor components (phospholipids, sterols, tocols, other); major uses; and sources of information. Information is tabulated where appropriate and data for several oils may be collated in a single table. Tables at the end of this section contain information on many different oils. They include: Table 2.43a: Past, present, and future production of oils and fats. Table 2.43b: Past and present production of oils and fats. Table 2.44: Predicted total (million tonnes) and per capita consumption (kg per annum) on a global basis and for selected countries/regions throughout the century. Table 2.45: Production, consumption, imports, and exports of 17 oils and fats (million tonnes) by country/region for the calendar years 2000 to 2004 by country/region. Table 2.46a and Table 2.46b: Range of fatty acid composition for some major oils taken from Codex Alimentarius. Table 2.47: Sterols (mg/100 g oil) in a range of crude vegetable oils. Table 2.48: Content of tocols in selected vegetable oils, animal fats, and nuts and berries. Table 2.49: Some physical and chemical properties of major vegetable oils adapted from Firestone (1997).

2.2.2

Castor oil (Ricinus communis)

Castor oil is unique among commodity oils in that it is rich in a hydroxy acid (ricinoleic, 12-hydroxyoleic) and is used only for industrial and cosmetic purposes. The distinct physical and chemical properties of the oil depend on the unusual chemical nature of this acid. The hydroxyl group provides additional functionality and polarity in a mid-chain position. Compared with common vegetable oils, castor oil is more viscous, less soluble in hexane, more soluble in ethanol, and is optically active. It can be converted to a range of interesting and useful materials (see Section 9.8). The castor plant is grown mainly in India, China, and Brazil (Table 2.1). Extraction by pressing and with solvent furnishes castor oil and residual meal. The latter contains a mildly toxic alkaloid (ricinine), an extremely poisonous protein (ricin), and a heat-stable allergen. Castor oil contains about 90% ricinoleic acid and small amounts of palmitic, stearic, oleic, linoleic, and 9,10dihydroxystearic acids. Most of the triacylglycerols are triricinolein or glycerol esters with two ricinoleic and one other acyl chain. In contrast to some other (less common) hydroxy acid-containing oils, the hydroxyl groups in castor oil remain free and are not themselves acylated. Ricinoleic

Useful general information is available in the following sources: Rossell and Pritchard, 1991; Ucciani, 1995; Ching Kuang Chow, 2000; Gunstone, 2002 and 2006; Akoh and Min, 2002; O’Brien, 2004; Shahidi, 2005; and Murphy, 2005. Aitzetmüller and his colleagues have prepared a valuable database on seed oil fatty acids (www.bagkf.de/ SOFA) and have described this in Aitzetmüller et al (2003a and 2003b). An older database is also available (www.ncaur.usda.gov/nc/ncdb).

References Aitzetmüller, K., and Matthäus, B., Potential uses of the seed oil fatty acids database ‘SOFA’, Lipid Technol. Newsl., 9, 123–127, 2003a. Aitzetmüller, K. et al. A new database for seed oil fatty acids — the database SOFA, Eur. J. Lipid Sci. Technol.105, 92–103, 2003b.

38

Occurrence and Characterisation of Oils and Fats

dibasic acid, when reacted appropriately, produces a nylon (polyamide) and efficient lubricants (esters). Splitting ricinoleic acid with steam yields C7 and C11 products. This splitting process has been much improved by the development of a continuous steam-cracking process. Heptanal is used in perfumes and 10-undecenoic acid shows antifungal properties and can be converted, via 11-amino-undecanoic acid, to a polyamide (Rilsan). A new plasticiser made from fully hydrogenated castor oil and acetic acid is particularly effective with PVC and, unlike the presently used phthalates, shows no hormone disrupting effects. It is metabolised like other vegetable oils and is fully biodegradable (Anon., 2005). For further information, see Table 2.43 to Table 2.49.

TABLE 2.1 Major countries/regions involved in the production (from indigenous or imported seed), consumption, export, and import of castor oil in 2004/05 (1000 tonnes) Total (kt) Production Consumption

522 519

Exports Imports Seed yield (t/ ha)

258 260 0.99

Countries/Regions India 316, China 106, Brazil 70 China 146, EU-25 110, India 87, Brazil 65, US 39, Japan 25, Thailand 20 India 224 EU-25 110, China 45, US 39, Japan 24 India 1.07, China 0.96, Brazil 0.91

Source: Adapted from Oil World Annual 2005, ISTA Mielke GmbH, Hamburg, 2005.

acid is produced in nature by hydroxylation of oleic acid, probably present in a phosphatidylcholine molecule. The oil contains some sterols and some tocols, but since it is not used for food purposes these are not considered to be very important. Castor oil differs from other commodity oils and fats in that it contains high levels of ricinoleic acid (12hydroxyoleic acid) and the oil or castor acids is a starting point for several useful chemicals (Caupin, 1997). Sulfation converts the secondary hydroxyl group (>CHOH) to a sulfate (>CHOSO2OH) with improved surfactant properties. Apart from soap, this is the earliest anionic surfactant (1874) and is still used in textile processing, leather treatment, and as an additive for cutting oils and hydraulic fluids. The sulfated hydrogenated oil has the consistency of an ointment and gives adjustable viscosity to water-based formulations with excellent skin compatibility. Castor oil has been converted to estolides by acylation of the free hydroxyl groups with oleic acid at 175 to 250°C in the absence of any catalyst (Isbell and Cermak, 2002). Reaction with other acids has been achieved using tetrabutyl titanate as catalyst (Kulkarni and Sawant, 2003). Dehydration of castor oil and of castor acids gives products rich in diene acids (mainly 9,11- and 9,12-18:2), some of which have conjugated unsaturation. These products are valuable alternatives to drying oils, such as tung oil, which contain conjugated trienoic acids (see Section 2.3.109). Hydrogenated castor oil and hydrogenated castor acids, with higher melting points than the nonhydrogenated material, are used in cosmetics, coatings, and greases. Greases prepared from tallow are much improved when salts of 12-hydroxystearic acid are added. Castor oil reacts with isocyanates to give polyurethanes, which are frequently used for wood preservation and have been developed as encapsulating materials. Splitting ricinoleic acid with caustic soda gives C8 and C10 products. At 180 to 200°C with a 1:1 caustic/castor ratio, the major products are 2-octanone and 10-hydroxydecanoic acid. At 250 to 275°C and a 2:1 ratio, the products are 2-octanol and sebacic (decanedioic) acid. The

References Anon., Danisco’s plasticizer base on vegetable oil gets EU approval, Lipid Technol., 17, 51–52, 2005. Caupin, H-J., Products from castor oil in Gunstone, F.D. and Padley, F.B. Eds. Lipid Technologies and Applications, Marcel Dekker, New York, 1997, pp. 787–795. Isbell, T.A. and Cermak, S.C., Synthesis of triglyceride estolides from lesquerella and castor oils, J. Amer. Oil Chem. Soc., 79, 1227–1233, 2002. Kulkarni, M.G. and Sawant, A.B., Some physical properties of castor oil esters and hydrogenated castor oil esters, Eur. J. Lipid Sci. Technol., 105, 214–218, 2003.

2.2.3

Cocoa butter (Theobroma cacao)

The commercial exploitation of cacao or cocoa beans was probably first practised by the Aztecs. The Spanish transferred the bean from Mexico to Europe in the 16th century, where it was consumed as a drink. Chocolate was developed only in the 19th century. The plant is an evergreen tree growing to 5 to 10 metres. The fruit is a large pod approximately 15 to 20 cm long and 7 cm in diameter containing 25 to 50 seeds embedded in a soft sweet edible pulp (Nickless, website). Production figures for cocoa butter are not included in the statistics generally cited for oil and fat production, but according to information cited in www.gobi.co.uk, world consumption of cocoa butter was over 700 kt in 2003 and is growing at a rate around 2% a year. Europe is the largest consuming region accounting for 60% of world consumption and Germany, the U.S., and France are the main importing countries. Cocoa is grown mainly in West Africa (Ghana, Ivory Coast, Nigeria), Malaysia, Brazil, Central America, India, and Sri Lanka. The composition of cocoa butter from these different sources varies somewhat as shown in Table 2.2 for cocoa butter from Ghana, Ivory Coast, Brazil, and Malaysia. Small differences in fatty acid composition are reflected in the iodine value, but more significantly in the triacylglycerol composition and, consequently, in the melting profile. The average content 39

2.2 Major oils from plant sources

behaviour gives it properties that are significant in chocolate. At ambient temperature, it is hard and brittle giving chocolate its characteristic snap, but also it has a steep melting curve with complete melting at mouth temperature. This gives a cooling sensation and a smooth creamy texture. For example, the content of solids falls from 45 to 1% between 30 and 35°C. The hardness of cocoa butter is related to its solid fat content at 20 and 25°C. This melting behaviour is related in turn to the chemical composition of cocoa butter. The fat is rich in palmitic (24 to 30%), stearic (30 to 36%), and oleic acids (32 to 39%) and its major triacylglycerols are of the kind SOS, where S represents saturated acyl chains in the 1 and 3 positions and O represents an oleyl chain in the 2 position. There are three major components: POP, POSt, and StOSt (P = palmitic acid and St = stearic acid). These triacylglycerols have 50, 52, or 54 carbon atoms in their 3 acyl chains and the levels of these can be determined by high temperature gas chromatography (GC) with the ratio of these being used to detect adulteration of cocoa butter. (Triacylglycerol molecular species are detailed in Table 2.2.) Cocoa butter has a high content of saturated acids that raises health concerns, but it has been argued that much of this is the noncholesterolemic stearic acid. Chocolate is also a rich source of flavonoids, which are considered to be powerful antioxidants (Beckett, 1999 and 2000). Triacylglycerol analysis of cocoa butter is generally carried out by capillary GC and the results of an interlaboratory study have been reported (Buchgruber et al., 2003). Seventeen triacylglycerol species were recognised, including POSt (39.8%), StOSt (28.0%), POP (15.6%), PLSt (3.2%), StLSt/StOO (2.9%), POO (1.9%), PLP (1.8%), OOO (1.5 %), StOA (1.0%), and seven others (total 3.3%). The crystal structure of cocoa butter has been studied extensively because of its importance in understanding the nature of chocolate (Section 4.6.5). The solid fat has been identified in six crystalline forms designated I to VI. Some crystals show double chain length (D) and some triple chain length (T) (Sato et al 1989). The six forms have the following melting points (°C) and D/T structure: I (17.3, D), II (23.3, D), III (25.5, D), IV (27.3, D), V (33.8, T), and VI (36.3, T). Form V is the one preferred for chocolate. This crystalline form gives good molding characteristics and has a stable gloss and favourable snap at room temperature. It is desirable to promote the formation of form V and to inhibit its conversion to form VI. Form V is usually obtained as a result of extensive tempering (putting molten chocolate through a series of cooling and heating processes), which have been found to optimise production of the appropriate polymorph. Alternatively, molten chocolate can be seeded with cocoa butter already crystallised in form V. Transition from form V to the more stable form VI leads to the appearance of white crystals of fat on the surface of the chocolate. This phenomenon (“bloom”) is promoted

TABLE 2.2 Composition and properties of cocoa butter from different countries

Iodine value Melting point °C Diacylglycerols (%) Free acid (%)

Ghana

Ivory Coast

Brazil

Malaysia

35.8 32.2 1.9 1.53

36.3 32.0 2.1 2.28

40.7 32.0 2.0 1.24

34.2 34.3 1.8 1.21

23.7 32.9 37.4 4.0 1.0

24.8 37.1 33.2 2.6 1.1

Component acids (%) Palmitic Stearic Oleic Linoleic Arachidic

24.8 37.1 33.1 2.6 1.1

25.4 35.0 34.1 3.3 1.0

Component triacylglycerols (%) Trisaturated Monounsaturated POP POSt StOSt Diunsaturated Polyunsaturated

0.7 84.0 15.3 40.1 27.5 14.0 1.3

0.6 82.6 15.2 39.0 27.1 15.5 1.3

trace 71.9 13.6 33.7 23.8 24.1 4.0

1.3 87.5 15.1 40.4 31.0 10.9 0.3

Solid content (pulsed NMR) — after tempering for 40 hours at 26°C 20°C 25°C 30°C 35°C

(%) (%) (%) (%)

76.0 69.6 45.0 1.1

75.1 66.7 42.8 0.0

62.6 53.3 23.3 1.0

82.6 77.1 57.7 2.6

Source: Adapted from Shukla, V.J.S., Inform, 8, 152–162, 1997. The original paper contains more details along with information on cocoa butter from India, Nigeria, and Sri Lanka.

of the important SOS triacylglycerols (S = saturated, O = oleic) varies between 87% in Malaysian and 72% in Brazilian cocoa butter, with the African samples midway between these extremes (Shukla, 1995 and 1997, see also Kurvinen et al, 2002). There is, however, some evidence that the cocoa butters of different geographical origins are becoming more alike. Harvested pods are broken open and left in heaps on the ground for about a week during which time the sugars ferment. The beans are then sun dried and are ready for transportation and storage. To recover the important components, the beans are roasted at ~150°C, shells are separated from the cocoa nib, and the latter is ground to produce cocoa mass. When this is pressed, it yields cocoa butter and cocoa powder still containing some fat (10 to 24%). Typically, 100 g of beans produce 40 g of cocoa butter by pressing, expelling, or solvent extraction; 40 g of cocoa powder; and 20 g of waste material (shell, moisture, dirt, etc.). Increasingly the beans are processed in the country where they grow and cocoa liquor, cocoa powder, and cocoa butter (usually in 25 kg parcels) are exported to the chocolate-producing countries. Cocoa butter carries a premium price and is sometimes adulterated (Crews 2002). Cocoa butter is a solid fat melting at 32 to 35°C (Table 2.2). It is in high demand because its characteristic melting 40

Occurrence and Characterisation of Oils and Fats

by fluctuations in temperature during storage and by migration of liquid oils from nut centres. This change is undesirable because it detracts from the appearance of the chocolate and may be mistaken for microbiological contamination. Bloom can be inhibited by the addition of a small amount of 2-oleo 1,3-dibehenin (BOB), milk fat, or other form V stabilisers to the cocoa butter. This phenomenon is discussed in more detail by Padley, 1997; Smith, 2001; Timms, 2003; and Longchampt and Hartel, 2004. Minor components include phospholipids (0.050.13%), tocopherols (~200 ppm, — mainly γ-tocopherol), sterols, 4-methylsterols, and triterpene alcohols. Cocoa butter is also used in cosmetics (Section 9.4). For further information, see Table 2.43 to Table 2.49. For information on cocoa butter replacers and cocoa butter substitutes, see the appropriate minor vegetable oils and the section on chocolate.

because of their high content of lauric acid. Some other lauric oils occur among the minor oils (Section 2.3). This oil has been reviewed by Pantzaris and Yusof Basiron, 2002; O’Brien, 2004; Canapi et al., 2005; and Gervajio, 2005. Coconuts grow in coastal regions between 20º N and 20º S of the equator. The trees bear fruit after 5 or 6 years and for up to 60 years thereafter. The shell is split open and allowed to dry. The “meat” on the inside of the shell is called copra and is the source of coconut oil in a yield of ~65%. The oil is extracted by pressing, usually followed by solvent extraction. As indicated in Table 2.3, coconut oil is produced mainly in the Philippines, Indonesia, and India and exported from the first two countries to EU-25 and U.S., in particular. Both lauric oils are used for a similar range of food and nonfood purposes. They are used to make soaps and other surface-active products and in the production of spreads and other food products. They are also the source of the C8 and C10 acids required to make MCT (medium chain triglycerides). These liquid products are used as lubricants in food-making equipment and, because they are easily metabolised, they appear in food preparations for invalids and athletes. Both oils can be fractionated into oleins and stearins, and hydrogenated to modify their properties and extend their range of uses. Palm kernel stearin is used as a chocolate substitute fat. Coconut stearin is somewhat softer and is used as a confectionery filling fat. The two lauric oils differ slightly from one another mainly in that coconut oil is the richer in the 6:0 to 10:0 acids and palm kernel oil is the richer in unsaturated C18 acids (Table 2.4). This is reflected in the triacylglycerol composition usually expressed in terms of carbon number (the sum of the carbon atoms in the three acyl groups and ignoring the three glycerol carbon atoms). The C36 triacylglycerols, dominant in both oils, will be mainly, but not

References Beckett, S.T., Ed. Industrial Chocolate Manufacture and Use, Blackwell Science, Oxford, 1999. Beckett, S.T., The Science of Chocolate, The Royal Society of Chemistry, Cambridge, 2000. Buchgruber, M. et al. Capillary GC: a robust method to characterise the triglyceride profile of cocoa butter — results of an intercomparison study, Eur. J. Lipid Sci. Technol., 105, 754–760, 2003. Crews, C., Authenticity of cocoa butter, in Oils and Fats Authentication, Jee, M. Ed. Blackwell Publishing, Oxford, U.K., 2002. Kurvinen, J.-P. et al. Rapid MS method for analysis of cocoa butter TAG, J. Amer. Oil Chem. Soc.,79, 621–626, 2002. Longchampt, P.L. and Hartel, R.W., Fat bloom in chocolate and compound coatings, Eur. J. Lipid Sci. Technol., 106, 241–274, 2004. Nickless, H., Cocoa butter quality, Guest contribution: www. britanniafood.com Padley, F.B., Chocolate and confectionery fats, in Lipid Technologies and Applications, Gunstone, F.D. and Padley, F. B. Eds. Marcel Dekker, New York, 1997, pp. 391–432. Sato, K. et al., Polymorphism of POP and SOS. I Occurrence and polymorphic transformation, J. Am. Oil Chem. Soc., 66, 664–674, 1989. Shukla, V.J.S., Chocolate — the chemistry of pleasure, Inform, 8, 152–162, 1997. Shukla, V.J.S., Cocoa butter — properties and quality, Lipid Technol., 7, 54–57, 1995. Smith, K.W., Cocoa butter and cocoa butter equivalents, in Structured and Modified Lipids, Gunstone, F.D. Ed. Marcel Dekker, New York, 2001. Timms, R.E., Confectionery Fats Handbook — Properties, Production, and Application, The Oily Press, Bridgwater, England, 2003.

2.2.4

TABLE 2.3 Major countries/regions involved in the production (from indigenous or imported seed), consumption (food and nonfood uses), export, and import of coconut oil in 2004/05 (million tonnes)

Coconut oil (Cocos nuciferus)

Coconut oil and palm kernel oil differ from other commodity oils and are known collectively as lauric oils

Total (mt)

Countries/Regions

Production

3.01

Consumption

2.99

Exports

1.86

Imports

1.87

Seed yield (t/ha)

0.52

Philippines 1.27, Indonesia 0.74, India 0.40, Mexico 0.11 EU-25 0.69, India 0.43, US 0.36, Philippines 0.28, Indonesia 0.20, China 0.12, Mexico 0.12 Philippines 0.98, Indonesia 0.56, Malaysia 0.13 EU-25 0.72, US 0.38, Malaysia 0.17, China 0.12 Philippines 0.90, Indonesia 0.46, India 0.35, Mexico 1.36

Source: Adapted from Oil World Annual 2005, ISTA Mielke GmbH, Hamburg, 2005.

41

2.2 Major oils from plant sources

TABLE 2.4

Fatty acid composition (% weight) of coconut oil and palm kernel oil Coconut oil

Palm kernel oil

Mean (b)

Range (b)

Range (c)

Mean (d)

Range (d)

0.4 7.3 6.6 47.8 18.1 8.9 2.7 6.4 1.6

0–0.6 4.6–9.4 5.5–7.8 45.1–50.3 16.8–20.6 7.7–10.2 2.5–3.5 5.4–8.1 1.0–2.1

0–0.7 4.6–10.0 5.0–8.0 45.1–53.2 16.8–21.0 7.5–10.2 2.0–4.0 5.0–10.0 1.2–2.5 0–0.2 0–0.2 0–9.2 6.3–10.6

0.2 3.3 3.5 47.8 16.3 8.5 2.4 15.4 2.4

0–0.8 2.1–4.7 2.6–4.5 43.6–53.2 15.3–17.2 7.1–10.0 1.3–3.0 11.9–19.3 1.4–3.3

0–0.8 2.4–6.2 2.6–5.0 45.0–55.0 14.0–18.0 6.5–10.0 1.0–3.0 12.0–19.0 1.0–3.5

17.5 26.4

14.1–21.0 24.0–28.3

14.1–21.0

6:0 8:0 10:0 12:0 14:0 16:0 18:0 18:1 18:2 18.3 20:0 20.1 IV (a) SMP (ºC)

0.1

0–0.2

8.5 24.1

6.3–10.6 23.0–25.0

Range (c)

Source: Adapted from Pantzaris, T.P. and Yusof Basiron, in Gunstone, F.D. (Ed.) Vegetable Oils in Food Technology — Composition, Properties and Uses, Blackwell Publishing, Oxford, pp 157–202, 2002. (a) Iodine value calculated from fatty acid composition (b) Leatherhead Food Research Association (LFRA) survey, 35 samples (c) Codex Alimentarius values (d) LFRA survey, 71 samples Values in the original references cited as trace and as not detected have been replaced by 0 in this table.

entirely, trilaurin because of the very high level of this acid. Careful study of Table 2.5 shows small differences in triacylglycerol composition between the two oils reflecting the differences in fatty acid composition referred above. Interesting results reported by Caro et al. (2004) show that the sn-2 position is enriched in lauric acid and the two unsaturated C18 acids, and that the remaining saturated acids are enriched at the sn-1/3 positions. Other work cited by Caro et al. (Table 2.6) shows that the 1 and 3 positions differ in their fatty acids with 6:0, 8:0, and 10:0 occurring particularly at sn-3. Coconut oil is highly saturated with an iodine value between 7 and 10 and this is probably associated with the low levels of tocols in the oil. These have been cited at a mean level of 10 ppm (ranging between 0 and 44 ppm) with α-tocopherol and α-tocotrienol the major members. Among the sterols (mean 836 ppm, range 470 to 1140 ppm) β-sitosterol, ∆ 5 -avenasterol, stigmasterol, and campesterol predominate and account typically for 46, 27, 13, and 9% of total sterols, respectively. Polycyclic aromatic hydrocarbons (PAH) are usually present at levels of 150 ppb in crude vegetable oils and β1. A different order is observed in relative selectivity of phosphatidylserine compared with phosphatidylethanolamine in which the order is α2 > β2 > α1 > β1. The involvement of Drs2 protein in the transbilayer movement and distribution of phospholipids in the plasma membrane of the S. cerevisiae end4∆ mutant in which both growth and the internalization step of endocytosis are blocked at a restrictive temperature of >34°C has been investigated (Pohl et al., 2005). It is known that several proteins, such as ATP-binding cassette multidrug transporters Pdr5 and Ste6, accumulate in the plasma membrane of the yeast after incubation under nonpermissive conditions and it was thought that this may have consequences for the organization and dynamics of the plasma-membrane lipid phase. It is known that the Ste6 protein transports synthetic alkyl phospholipids, but not whether the protein-mediated transbilayer movement and the transbilayer asymmetry of lipids in the plasma membrane of the S. cerevisiae end4∆ mutant are altered. The transbilayer movement of fluorescently labelled analogues of choline and serine phosphatides has been measured in an S. cerevisiae end4∆drs2∆ strain, to determine whether TABLE 7.4 Relative phospholipid specificities for activation of different isoforms of recombinant ATPaseII Relative Specificity PS/PC PS/PE

α1

α2

β3

β4

53.0 2.7

68.0 10.9

25.0 1.3

32.5 3.5

PS, phosphatidylserine; PC, phosphatidylcholine; PE, phosphatidylethanolamine. Source: Data from Ding, J., et al. (2000), J. Biol. Chem., 275, 23378–23386.

519

7.6 Biological membranes

internalization of analogues by transbilayer movement depended on a functional drs2 gene in endocytosis-deficient yeast cells. It has been shown that exposure of endogenous aminophospholipids to the exoplasmic leaflet of the mutant cells is altered with respect to wild-type cells. There is also a family of membrane P-glycoproteins involved in transmembrane phospholipid movement. The translocase responsible for nonspecific movement of phospholipid from the cytoplasmic leaflet to the cell surface has been shown to be identical to the multidrug resistance protein-1. The proteins appear to translocate particular phospholipids on different cell types (Sugawara et al., 2005). Sphingolipids, for example, are preferentially transported across the plasma membrane of LLC-PK1 kidney epithelial cells transfected with MRP1 cDNA, but phosphatidylcholine was the preferred phospholipid translocated by MRP3 in bile canicular cells where the protein predominates in the plasma membrane of fibroblasts of transgenic mice. The loss of membrane phospholipid asymmetry is known to be an important signalling mechanism as it results in the appearance of specific phospholipids on opposite sides of the membrane, a situation that triggers a variety of cellular responses (Zwaal et al., 2005). A family of membrane proteins referred to as phospholipid scramblases (PLSCR) have been implicated in dissipating phospholipid asymmetry in a process that depends on elevated cytoplasmic calcium concentration. Four genes have been identified that code for phospholipid scramblases in human and mouse and all have been conserved through evolution. The amino acid sequence deduced for one of the human phospholipid scramblases, HuPLSCR1, indicates that the scramblase is a Type-2 membrane protein of molecular weight 35 kDa (318 amino acid residues) with a transmembrane helical domain located towards the C-terminus (Wiedmer et al., 2000). The N-terminal cytoplasmic domain was found to possess a putative phosphorylation site and a calciumbinding segment. There is evidence that regulation of scramblase activity may be mediated by phosphorylation in the presence of PKCδ, the action of which results in the surface exposure of phosphatidylserine in apoptosing cells (Frasch et al., 2000). Regulation of scramblase activity and its localization in the nucleus may also occur via palmitolylation at cysteine thiol residues of the protein (Ben-Efraim et al., 2004).

7.6.4

the use of membrane lipid analogues that incorporate para-magnetic, fluorescent, or other probes and it is important to recognise that it is the motion of the probe that is observed and not the native molecules. It is not unknown for probe molecules to exert a considerable perturbation on the motion and structure of the surrounding lipids or to behave in a manner distinctly different from the lipids they are designed to mimic. Another problem that also has to be addressed is the need to introduce the probe into the membrane without disturbing the structure. The most common method is to incubate cells or membrane suspensions in a medium into which a small aliquot of lipid probe has been injected from a concentrated solution in organic solvent, such as ethanol. Alternatively the probe may be codispersed with unlabelled phospholipid or mild detergent. The strategy of choice often depends on whether the probe increases or decreases the hydropathy of the lipid analogue compared to the native lipids, which comprise the membrane. Possibly the least perturbing method is the use of phospholipid transfer proteins to perform the task, but this method relies on close physical resemblance between the analog and the phospholipid for which the protein has affinity. Nevertheless, with these restrictions in mind the general picture that has emerged is that lipid molecules are relatively mobile within the membrane and constraints on this motion occur by interactions with the proteins and other membrane constituents. 7.6.4.1 Motion of lipids in membranes The two methods that have been exploited to greatest effect to examine lateral diffusion of lipids in the plane of biological membranes and from one leaflet of the membrane lipid bilayer to the other are electron paramagnetic resonance and fluorescence probe techniques. A range of probe molecules have been synthesized as analogs of membrane lipids. One example is the synthesis of Lucifer yellow derivatives of phospholipids and cholesterol, which have been introduced into human erythrocyte ghost membranes and living melanoma cells to measure lateral diffusion rates. The rates of diffusion were then compared with diffusion in phospholipid bilayer membranes. Lateral diffusion rates were obtained using a spot fluorescence photo-bleaching recovery method. The method involves bleaching of probe molecules in a defined surface area of membrane using an intense pulse of laser light and monitoring recovery of fluorescence as unbleached probe molecules from the surrounding area of membrane diffuse into the bleached domain. It was found that Lucifer yellow derivatives of cholesterol and phosphatidylethanolamine diffused rapidly with a diffusion coefficient >1 µm2 s–1 in lipid dispersions at temperatures greater than the gel to liquid-crystalline phase transition temperature, but the diffusion rate decreased dramatically for probes in gel

Membrane lipid dynamics

With the notable exception of lipids in the purple membrane of Halobacterium, which adopt more or less a crystal structure, the lipid matrix of biological membranes is said to be fluid. The term fluidity is not a precise quantitative parameter, but it implies that the molecules of the structure exhibit motion with respect to one another. The measurement of this motion is often performed by 520

Physical Properties: Structural and Physical Characteristics

phase lipid ( T > TC

T > TH (a)

(b)

T > TH

TC > T

Bilayer-forming lipid (d)

Non-bilayer-forming lipid (c)

FIGURE 7.38 Illustration of the lipid phase separation model for low temperature to cell membranes. The scheme shows the consequences of cooling from the growth temperature (a) to a temperature below the hexagonal-II to liquid-crystalline phase transition temperature of the nonbilayer forming lipids (b) and subsequently below the gel to liquid-crystalline phase transition of all the membrane lipids (c). The effect of reheating to the growth temperature is shown in (d). (Data from Quinn, P.J. (1985), Cryobiology, 22, 128–146.)

cultured at 28oC and 38oC that occur at 2o and 4oC, respectively. This observation clearly shows that it is the high melting point lipids that phase separate from the membrane proteins in the intact biological membrane. The question remains as to which lipids have higher melting points and which lipids have low melting points. This has important implications with regard to the ability of the membrane to restore a random distribution of components after thermally induced phase separations. It is well known, for example, that the phase separations of the type observed in Synococcus result in irreversible changes and loss in viability of the cells, which are unable to repair their leaky membranes. It is seen that the lipid composition of Synococcus is relatively simple, and appreciable proportions of the membrane lipids undergo phase transitions over a relatively narrow range of temperatures. With more complex mixtures, typical of many biological membranes of higher organisms, the transition endotherms observed on heating membranes previously cooled to low temperatures exhibit transitions that are invariably broad and extend over tens of degrees. Attempts have been made to resolve these broad transitions in erythrocyte membranes and lipid mixtures simulating the membrane lipid composition into components contributed to by each of the major lipid classes present in the membrane. The results of such experiments using human erythrocyte phospholipids have shown that the phase separation of the sphingolipids dominated the higher temperature phase behaviour of the outer leaflet of the membrane, and the phosphatidylethanolamines appear to phase separate initially in the inner leaflet upon cooling the membrane. 524

Physical Properties: Structural and Physical Characteristics

at higher temperatures causes a phase separation of nonbilayer lipids into stable aggregates of cylindrical inverted micelles. Interpretation of the effects of temperatures greater than 45oC is based on phase conditions that result in a release of the constraints imposed by interaction of the major nonbilayer-forming lipid of the membrane, monogalactosyldiacylglycerol, with other membrane components and its segregation into domains of nonbilayer lipid structure. Gross phase separations of this type require that the shift in thermal stability of the stacked membrane is relatively large because three-dimensional aggregates of lipid are not observed if the chloroplast membrane is destacked by manipulation of the ionic environment before heat treatment. It has been suggested that a shift in the phase of the nonbilayer lipid, monogalactosyldiacylglycerol, underlies these structural changes and that the functional role of this nonbilayer lipid may be to package the light-harvesting chlorophyll a/b-protein complexes together with the photosystem-II core protein complex into an efficient functional unit localised within the grana stack. All membranes contain molecular species of lipid that at physiological temperatures do not form bilayer structures. These include phosphatidylethanolamine, monoglycosyldiacylglycerol, and cardiolipin. While it is generally assumed that such lipids are constrained into a bilayer arrangement by interaction with other membrane constituents, their phase separation to form inverted micelles is an event that is thought to be associated with membrane fusion. Recent NMR evidence, however, has identified lipids undergoing isotropic motion in intact cell membranes that are said to be associated with raft domains (Ferretti et al., 2003). Motion of this type may be a feature of the boundary region separating fluid from liquidordered domains where packing faults and molecular mismatches are expected to occur.

Non-bilayer lipid structures 100 nm

FIGURE 7.39 An electron micrograph of a freeze-fracture replica prepared from bean chloroplasts heated for 5 min at 55°C before thermal quenching.

that the higher melting point lipids present in gel-phase domains will be rich in the hexagonal-II-forming lipids. This assumption is based on the fact that, with equivalent hydrocarbon substituents, lipids that tend to form hexagonal-II structures have liquid-crystalline to gel-phase transitions at temperatures that are considerably higher than those corresponding to bilayer-forming lipids. Where phase separations between lipid classes of this type are created, the changes are not likely to be reversed on reheating to temperatures above the hexagonal-II to lamellar-phase transition temperature. In practice, it would be improbable for there to be a single temperature where all the nonlamellar lipids would co-exist together with the bilayer-forming lipids in the fluid phase, allowing the original distribution of the lipids and the proteins to be restored. Damage to the membrane in these circumstances would be expected to result when the membrane is reheated to temperatures where the domains of the phase-separated hexagonal-II-rich lipids tend to form nonbilayer structures. The creation of nonbilayer structures, such as inverted lipid micelles, unless suppressed or dealt with by normal homeostatic mechanisms operating within the membrane, would serve to destroy the permeability barrier properties of the membrane. If this breakdown of membrane barrier is of sufficient duration to permit loss of essential components or irreversible alterations in the intracellular compartmentation, loss of cell viability will result. Phase separations of the constituents of biological membranes can also be driven by exposure of membranes to salts or pH that screen charges on the acidic phospholipids as well as to high temperatures. The structural changes in thylakoid membranes of higher plant chloroplasts subjected to thermal stress is illustrated in Figure 7.39. Chloroplasts maintain a normal morphology during a brief exposure (5 min) to temperatures up to 35oC. Incubation at temperatures of between 35o and 45oC causes complete destacking of the grana and incubation

7.6.4.4

Domain creation by lipid–lipid interactions

Great emphasis has more recently been placed on lipid phase separations that are brought about by the association between particular membrane lipids to create lateral domains within the bilayer. The phase separation in these instances is driven by the order created in the bilayer by the formation of complexes. Complexes have been described between phosphatidylcholine and diacylglycerol, phosphatidylcholine and α-tocopherol, phosphatidylethanolamine and glucosylceramide, and choline phosphatides and cholesterol. The ordered phase in each case depends subtly on the molecular species of lipid involved in the complex and the extent of order created within the phase-separated domains again depends on the molecules involved. Diacylglycerol accumulates transiently in cell membranes as a consequence of phospholipase C-type enzymes activated by a variety of hormones, growth factors, and neurotransmitters. The amount of diacylglycerol formed 525

7.6 Biological membranes

can reach 2 mol% in some physiological situations and the resulting change in membrane lipid composition represents a molecular signal transducing mechanism responding to the interaction of agonists with their respective cell surface receptors. Structural changes in the lipid bilayer have been detected in phospholipids containing diacylglycerol and these physical perturbations may be responsible for the molecular signal, such as activation of protein kinase C or to enhance membrane fusion processes. Diacylglycerols when codispersed with disaturated phosphatidylcholines in molar ratios of up to 30 mol% create lateral phase separated domains of a stoichiometric 1:1 complex of diacylglycerol and phosphatidylcholine within bilayers of pure phospholipid (Quinn et al., 1995). It may be expected that physiological proportions of diacylglycerol may result in lateral phase separations in small domains within membranes. The domains created by the demixing process tend to destabilize the phospholipid bilayer. The presence of much higher, and unphysiological, proportions of diacylglycerols (greater than 30 mol%) is known to induce nonbilayer phases such as hexagonal-II (HII) and cubic phases in disaturated phosphatidylcholines. The transient nature of lamellar phase complexes between diacylglycerol and membrane phospholipids is necessary in the process of switching off the signal generated by the formation of diacylglycerol. This is accomplished by diacylglycerol kinase, which converts the substrate into phosphatidic acid. Control is presumably exercised by the molecular species of diacylglycerol that are generated from the parent phospholipid. Studies on the effect of acyl chain length of diacylglycerol on activation of protein kinase C has shown that diacylglycerol with shorter hydrocarbon chains (C6 to C10) are more effective than molecular species containing longer acyl chains. The miscibility of the short-chain diacylglycerols with the phospholipid in liquid-crystalline bilayers could be the explanation for the high activity of the short-chain diacylglycerol. By contrast, longer chain diacylglycerols form complexes with the phospholipids, which exist in gel phase at temperatures higher than the fluid phase transition of the phospholipid and are unable to activate protein kinase C. Complex formation has been characterised in binary mixtures of both dipalmitoylphosphatidylcholine/ dipalmitoylglycerol and dimyristoylphosphatidylcholine/ dimyristoylglycerol. In terms of the effect of diacylglycerol on domain formation in membranes it is clear that this is highly dependent on the molecular species of diacylglycerol and the degree of unsaturation of the polar lipids comprising the membrane lipid bilayer matrix. Interaction and complex formation between saturated molecular species may generate liquid-ordered domains, whereas interaction between unsaturated molecular species tends to destabilize the bilayer in favour of the formation of inverted phases

that result in repackaging of intrinsic proteins or membrane fusion. Diacylglycerol generation from proteolipid complexes containing polyphosphoinositol lipids by intranuclear phospholipase C is a recognised signalling pathway in the control of gene expression (Martelli et al., 2003). These complexes have been localized to interchromatin granule clusters by immunostaining with monoclonal antibodies in preparations that have been extracted with cold TritonX100. On this basis it is said that the complexes do not originate from the membrane bilayer lipid matrix of the nuclear envelope; however, the possibility that they represent detergent-resistant membrane domains of the envelope cannot be discounted on the present evidence. The consequence of the presence of ceramide in membranes is dominated by its tendency to self assemble into rigid bilayer structures especially in the presence of sphingomyelin and cholesterol. This has led to the suggestion that ceramides play an important role in membrane signaling processes, membrane fusion, and permeability of solutes through bilayers (Goni et al., 2005). Evidence that is consistent with these functions is that ceramides are recovered in significant amounts in detergent-resistant membrane fractions even from cells in an unstimulated state. Ceramide domains are more hydrophobic than the parent sphingolipid domains, promoting membrane fusion. In relatively minor proportions (3 to 6 mol%), ceramide synergises with cholesterol in stabilising ordered lipid domains. Ceramide alone is able to promote order in the lipid chains of phosphatidylcholine, even with monounsaturation in the sn-2 chain, and it partitions into liquid-ordered domains with an affinity markedly higher than that of other (fatty acid-matched) sphingolipids (Wang, 2003). The generation of ceramide in membranes takes place at different subcellular sites. De novo biosynthesis occurs at the endoplasmic reticulum, whereas at the plasma membrane ceramide is derived by hydrolysis of sphingomyelin and complex sphingoglycolipids. The hydrolytic enzymes responsible for production of ceramide at the plasma membrane are designated by their pH optima into acid and neutral sphingomyelinases. Cellular modulators of ceramide production include diacylglycerol and other protein kinase C activators and serine proteases. Degradation of ceramide by ceramidases is enhanced by a variety of agents including cytokines, cell differentiating agents, death receptor ligands, cancer chemotherapeutic agents, and ionizing radiation (Jaffrezou et al., 2002). It has been proposed that the cellular response to ceramide elevation or depletion depends directly on where the ceramide is located (Blitterswijk et al., 2003). According to this view ceramide in the Golgi influences biosynthetic pathways in formation of sphingolipids and their transport in vesicles to the plasma membrane where they promote domain formation. Clustering of receptors on the cell surface, promoted by rafts, induces endocytosis in 526

Physical Properties: Structural and Physical Characteristics

response to transient ceramide formation. Finally, changes in membrane permeability associated with the action of ceramidases on mitochondrial and plasma membrane are said to mediate apoptotic events. Ceramides are known to be one of the main mediators of apoptosis in cells. Increasing the levels of ceramide accumulating during inhibition of ceramidases, which convert ceramide to sphingosine and free fatty acid, results in cell death. Likewise, inhibition of ceramide production by blocking the de novo synthesis pathway or inhibiting neutral sphingomyelinase activity slows down apoptosis in response to a variety of factors including chemotherapeutic agents, tumor necrosis factor-ß, angiotensin-II, and B-cell activation (El Bawab et al., 2002). Perhaps the most studied of the complexes is that between choline phospholipids and cholesterol. Cholesterol interacts with lipids containing long-chain (>C14) saturated fatty acids in a manner dominated by van der Waals interactions. In order for van der Waals’ forces to operate, the sterol and lipid must fit closely. This steric constraint requires of the sterol that it have the 3-OH head group, the sterol rings must be planar, the hydrocarbon tail must be of the appropriate length, and it should be the natural enantiomer (Miao et al., 2002). On the phospholipid side, the primary requirement is for long, saturated lipid chains; in particular, cis double bonds are poorly tolerated in that part of the fatty acid chain (approximately the first 12 carbons) that interacts with the planar sterol rings. Glycerolipids, made in the endoplasmic reticulum, often have 1 to 4 double bonds in this region of their sn-2 acyl chain, whereas sphingolipids (made in the Golgi) are either fully saturated or have a double bond at the C15 position where it is accommodated by the flexible hydrocarbon tail of cholesterol. However, this requirement for saturation applies strictly only to the fatty acid in the sn-1 position for a glycerolipid; a single double bond, even in a relatively superficial position abutting the sterol rings of cholesterol, is tolerated nearly as well as a fully saturated chain in this position. It is the presence of multiple double bonds in one chain, or unsaturation in both chains, that strongly prevents condensation. The range of membrane lipids that can condense with cholesterol and partition into a liquid-ordered (LO) phase is much greater than is commonly assumed. This includes, in particular, the glycerolipids of the inner leaflet of the plasma membrane, and of internal membranes within the cell, provided they meet the condition of saturation in the sn-1 chain and no more than one double bond in the sn-2 chain. The fatty acid attached at sn-1 to glycerolipids is normally fully saturated except in certain tissues, such as brain and spermatozoa, and sphingolipids have a linear hydrocarbon chain at this position as part of the sphingosine base. Interactions between cholesterol and phospholipids are also believed to take place through hydrogen bonds. Sphingomyelin contributes both hydrogen bond acceptors (esteric O) and donors (-OH and -NH) to the membrane

surface, whereas phosphatidylcholine has only acceptors (esteric O). The principal hydrogen bond site appears to be with the -NH group of the sphingomyelin, which can be detected indirectly by effects, such as the accessibility of 3-OH group of cholesterol to cholesterol oxidase, the extractability of cholesterol from the membrane, or the effect of cholesterol on interfacial elasticity. The multiple hydrogen-bonding propensity of the sphingomyelin molecule results in the formation of relatively large hydrogen-bonded clusters (4 to 8 molecules) of sphingomyelin in the membrane (Mombelli et al., 2003). Cholesterol can only form a single hydrogen bond and so acts as a terminator of the hydrogen bonding networks. The gain to the membrane of the single hydrogen bond formed with cholesterol is at the expense of larger clusters of hydrogenbonded sphingolipids, and so makes a relatively minor contribution to membrane properties. The condensing effect of cholesterol on phospholipids to create a liquid-ordered phase can also be viewed from the simple action of the sterol in spacing out the phospholipid molecules at the bilayer interface. The polar groups of the phospholipids tend to repel each other via charge–charge interactions and this effect is reduced by the interposition of uncharged cholesterol molecules. Computer modeling of the interacting molecules shows that cholesterol makes closer contacts with the sn-1 rather than sn-2 fatty acid chain of glycerolipids, which explains why unsaturation is allowed in the sn-2 chain. Moreover, such studies show that the main interaction is not that of cholesterol with the lipid chains, but of the tightly packed lipid chains with each other (Rog and Pasenkiewicz-Gierula, 2001). Cholesterol acts as a hydrophobic spacer that allows the saturated lipids to pack tightly and thereby interact more with each other along their entire length. Presumably constraint upon the flexibility of the first C12 atoms of the fatty acid chains by the presence of cholesterol promotes van der Waals’ interactions between the chains. The small -OH head group of cholesterol is probably of key importance in allowing room for tighter packing of the bulky phosphocholine head group, which can orient on the bilayer surface to prevent exposure of the hydrophobic region of the bilayer to water. One of the material advances in understanding the role of liquid-ordered phases in cell biology has been the isolation of these phase-separated domains from living cells. This is achieved by exploiting the relatively low solubility of the liquid-ordered phase in weak nonionic detergents. The fraction of membrane that survives solubility can be separated by density gradient centrifugation from the remaining solubilized membrane components. The isolation of detergent-resistant membrane has been modeled using giant unilamellar vesicles containing a lipid composition in which liquid-ordered phase is created in a matrix of fluid lipid. An example is shown in Figure 7.40. Giant unilamellar vesicles formed from a mixture of phosphatidycholine, sphingomyelin, and cholesterol (mole ratio 527

7.6 Biological membranes

(a)

(b)

produces a marked effect on membrane characteristics as its mol% in a monolayer or bilayer of saturated lipids rises above 20 mol% to a maximum of 50 mol% after which it forms a separate cholesterol-only phase. Normal homeostatic processes operate to ensure that the level of cholesterol in the plasma membrane is strictly regulated and can only be manipulated experimentally by removing cholesterol (typically with methyl ß-cyclodextrin or cholesterol oxidase). However, red blood cells have only one membrane, which is exposed continuously to a rich supply of cholesterol in the blood plasma. In membrane rafts isolated from both human and goat red blood cells (ruminant cells have twice as much sphingolipid as human red cells), there is a strict 1:1 stoichiometry between cholesterol and sphingomyelin, although in these cells the cholesterol level is higher in the solubilised (disordered) membrane than in the rafts (Koumanov et al., 2005). This suggests that lipid-ordered microdomains impose an absolute equimolar stoichiometry between cholesterol and the saturated lipid, and as readily exclude excess sterol as they include it when needed. Although the condensing effect of cholesterol on lipids becomes marked at >20 mol% cholesterol, other effects occur at concentrations too low (60%) of 2-ethylhexanol is to serve in the production of plasticizers, such as diethylhexyl phthalate (DEHP; dioctyl phthalate) and diethylhexyl adipate (Bahrmann et al., 2001). Other uses of 2-ethylhexanol include the production of 2-ethylhexyl acrylate, which in turn is used in coating materials, adhesives, and inks, 2-ethylhexylnitrate used as cetane improver additives for diesel fuel, and 2-ethylhexylphosphates as an additive for lubricating oils (Bahrmann et al. 2001). An alternative synthesis of the corresponding acid, 2-ethylhexanoic acid, is achieved by hydrogenating the δ-lactone (depicted in Figure 8.61) in presence of a watersoluble rhodium-phosphine catalyst (Behr and Brehme 2002). The δ−lactone is obtained from a reaction of butadiene with carbon dioxide.

Properties and Applications

CH3

Their wide liquidity range documented by melting points (especially) and boiling points is one reason why Guerbet compounds have significant practical applications. For example, 1-octanol has a melting point of −16.7°C and a boiling point of 194.4°C. The Guerbet alcohol with the same number of carbon atoms, 2-ethylhexanol, has a melting point of −70°C and a boiling point of 184.6°C.

H 2C O

FIGURE 8.61

588

O

Chemical Properties

References

Carlini, C. et al. Guerbet condensation of methanol with npropanol to isobutyl alcohol over heterogeneous bifunctional catalysts based on Mg-Al mixed oxides partially substituted by different metal components, J. Molec. Catal. A: Chem., 232, 13–20, 2005. Gast, L.E. et al. Reactions of unsaturated fatty alcohols. VI. Guerbet reaction of soybean and linseed alcohols, J. Am. Oil Chem. Soc., 35, 703–707, 1958. Guerbet, M., Action de l’alcool amylique de fermentation sur dérivé sodé, Comptes rendus, 128, 511–513, 1899. Kenar, J.A. et al. Synthesis and characterization of dialkyl carbonates prepared from mid-, long-chain, and Guerbet alcohols, J. Am. Oil Chem. Soc., 81, 285–291, 2004. Kenar, J.A. et al. Physical properties of oleochemical carbonates, J. Am. Oil Chem. Soc., 82, 201–205, 2005. Knothe, G. and Carlson, K.D., Synthesis, mass spectrometry, and nuclear magnetic resonance characterization of di-Guerbet esters, J. Am. Oil Chem. Soc., 75, 1861–1866, 1998. Knothe, G., Characterization of esters of fatty acids and dicarboxylic acids with Guerbet alcohols, J. Am. Oil Chem. Soc., 78, 537–540, 2001. Knothe, G., Synthesis, applications, and characterization of Guerbet compounds and their derivatives, Lipid Technol., 14, 101–104, 2002. Krause, H.-J. and Syldatk, A., Neue Tenside aus gemischten Guerbet-Alkoholen, Fette, Seifen, Anstrichm., 87, 386–390, 1985. Markownikoff, W. and Zuboff, P., Ueber die Condensation höherer Alkohole: Tricaprylalkohol, Chem. Ber., 34, 3246–3249, 1901. Miller, R.E., The Guerbet reaction. I. The reaction of amines under guerbet conditions, J. Org. Chem., 25, 2126–2128, 1960. O’Lenick, A.J., Guerbet chemistry, J. Surfact. Deterg., 4, 311–315, 2001. Sulzbacher, M., The Guerbet reaction of cetyl alcohol, J. Appl. Chem., 5, 637–641, 1955. Ueda, W. et al. A low-pressure Guerbet reaction over magnesium oxide catalyst, J. Chem. Soc., Chem. Commun., 1558–1559, 1990. Veibel, S. and Nielsen, J.I., On the mechanism of the Guerbet reaction, Tetrahedron 23, 1723–1733, 1967.

Bahrmann, H. et al. 2-Ethylhexanol, in Ullmann’s Encyclopedia of Industrial Chemistry, 6th ed., Wiley-VCH, Weinheim, Germany; online version, 2001. Behr, A. and Brehme, V.A., Homogeneous and heterogeneous catalyzed three-step synthesis of 2-ethylheptanoic acid from carbon dioxide, butadiene and hydrogen, J. Molec. Catal. A, 187, 69–80, 2002. Behr, A. and Döring, N., Synthesis of branched fatty acids by catalytic oxidation of alcohols. (Herstellung verzweigter Fettsäuren durch katalytische Oxidation von Alkoholen.), Fat Sci. Technol., 94, 13–18, 1992. Burk, P.L. et al. The rhodium-promoted Guerbet reaction. Part I. Higher alcohols from lower alcohols, J. Mol. Catal., 33, 1–14, 1985a. Burk, P.L. et al. The rhodium-promoted Guerbet reaction. Part II. Secondary alcohols and methanol as substrates, J. Mol. Catal., 33, 15–21, 1985b. Carlini, C. et al. Selective synthesis of isobutanol by means of the Guerbet reaction. Part 1. Methanol/n-propanol condensation by using copper based catalytic systems, J. Mol. Catal. A: Chem., 184, 273–280, 2002. Carlini, C. et al. Selective synthesis of isobutanol by means of the Guerbet reaction. Part 3. Methanol/n-propanol condensation by using bifunctional catalytic systems based on nickel, rhodium, and ruthenium species with basic components, J. Molec. Catal. A: Chem., 206, 409–418, 2003a. Carlini, C. et al. Selective synthesis of isobutanol by means of the guerbet condensation of methanol with n-propanol in the presence of heterogeneous and homogeneous palladium-based catalytic systems, J. Molec. Catal. A: Chem., 204–205, 721–728, 2003b. Carlini, C. et al. Selective synthesis of isobutanol by means of the Guerbet reaction. Part 2. Reaction of methanol/ethanol and methanol/ethanol/n-propanol mixtures over copper based / MeONa catalytic systems, J. Molec. Catal. A: Chem., 200, 137–146, 2003c. Carlini, C. et al. Guerbet condensation of methanol with npropanol to isobutyl alcohol over heterogeneous copper chromite/Mg-Al mixed oxides catalysts, J. Molec. Catal. A: Chem., 220, 215–220, 2004.

589

9 NONFOOD USES OF OILS AND FATS

F.D. Gunstone, J. Alander, S.Z. Erhan, B.K. Sharma, T.A. McKeon and J.-T. Lin

9.1

Introduction

levels of burdensome stocks, to raise prices by removal of excess oil, or to meet targets for the reduction of carbon dioxide production. In contrast to fossil fuels, oils and fats represent a renewable resource produced by agricultural systems from carbon dioxide and water with sunlight providing the necessary energy. But some numbers should be applied to this issue to maintain a sense of perspective. Annual production of mineral oil, at 3.5 billion tonnes, is almost 30 times that of oils and fats, and the greater part of these latter are essential for food purposes. For example, biodiesel can never replace the demand for conventional fossil fuel — it can only diminish it marginally (Dumelin, 2005). Other environmental reasons are based on the fact that oils and fats are biodegraded more quickly than petrochemical products and, therefore, disappear more easily from the environment when used or spilled. Finally, when fully degraded, these materials liberate carbon dioxide trapped only months earlier and, therefore, do not add to total carbon dioxide, one of the greenhouse gases responsible for global warming. This contrasts with petrochemical products, which are oxidised to carbon dioxide trapped millennia earlier. Environmental issues are more complex than is generally appreciated. It must not be forgotten that the growing, harvesting, and transporting of oilseeds and their products are achieved only at some environmental cost. Hirsinger (2001) has reviewed the relation between oleochemicals and the environment and Urata et al. (2001) have described the contribution of surfactants and lipids to “Green Chemistry.” Yanagawa (2001) has discussed sustainable growth of the Asian-Pacific surfactant and detergent industries and Williams (2005) has reported on European detergent rules.

Annual production of commodity oils and fats is now (2004) around 130 million tonnes and the general consensus is that ~ 80% is used for human food, ~ 6% for animal feed, and ~ 14% for the oleochemical industry though these ratios may change through the increasing use of oils and fats for oleochemical purposes, especially biodiesel. This chapter is devoted to the major uses of that last 14% (18 million tonnes). Before mineral oil and gas were used to supply many of our needs, oils and fats were widely used as illuminants and lubricants. Illumination was provided by a wick burning in olive oil in Mediterranean countries in biblical times or in the early days of the railways using rapeseed oil (then a high-erucic variety known as colza oil). At one time, lighthouses used seal oil or whale oil in their lamps. In a book written by T. P. Hilditch in 1927, an entire chapter was devoted to “the use of fats in candles and illuminants.” There are suggestions that the axles of early chariots were greased with a mixture of animal fat and lime producing calcium soaps. More importantly, soap made from fat and alkali (wood ash) has been used for centuries. Other uses are based on oxidative drying (hardening) of oil films to form coatings and have been exploited by artists, decorators, and producers of linoleum. With the development of fossil fuels and the inventiveness of chemists in the petrochemical industry, oils and fats ceased to be used as illuminants or as lubricants. Today there is a limited return to the use of oils and fats for some of these purposes, mainly on environmental grounds. Oil and gas supplies are not known with certainty, but they are finite and will not last indefinitely. Oils and fats can sometimes be used in place of fossil fuels and are even being burnt in electrical power stations to reduce the 591

9.2

Basic oleochemicals

References

The use of oils and fats as oleochemicals depends either on the physical properties of fatty acids and esters or on chemical properties relating to the carboxyl group, to olefinic centres, or to the whole molecule (as in its combustion). Linseed and castor oil are classed as industrial oils. Other oils used for nonfood purposes include significant proportions of the lauric oils (coconut and palm kernel), palm oil and palm stearin, tallow, rapeseed oil (both the high-erucic and the low-erucic oil), and lesser amounts of soybean and other oils. This listing may change when larger volumes of biodiesel are prepared. USDA figures for 2003/04 show the proportion of the commodity food oils used for industrial purposes in EU-15 as rapeseed oil (39%), palm kernel oil (29%), coconut oil (17%), palm oil (10%), soybean oil (7%), sunflower oil (6%), with olive oil, groundnut oil, and cottonseed oil at 1% or less. In the U.S. about 3% of total usage of soybean oil is for industrial purposes. Another important oleochemical feedstock is tall oil (a name based on tallolja, the Swedish word for pine oil). Tall oil fatty acids are by-products of the wood pulp industry and result when pine wood chips are digested, under pressure, with an aqueous mixture of sodium hydroxide and sodium sulfide during which the acids are converted to their sodium salts. Tall oil is the cheapest source of fatty acids rich in oleic and linoleic acids. In volume terms, surface-active compounds dominate among oleochemicals — a proportion of 90% has been reported, but this share is falling with increasing use as a fuel or by production of biodiesel. Surfactant molecules are mainly saturated or monounsaturated and find different uses according to their chain length. Three categories are recognised: C12 and C14 compounds from the lauric oils competing with identical products from the petrochemical industry, C16 and C18 compounds mainly from tallow and palm stearin, and C20 and C22 compounds from fish oils, high-erucic rapeseed oil, and crambe oil. These compounds may be used as acids or salts (soaps), as esters of methanol, other alkanols, or glycerol, as long-chain alcohols, or as nitrogen-containing compounds. Other oleochemical uses exploit the high unsaturation of oils, such as linseed and soybean oil, while castor oil is a unique source of several important chemicals. Several aspects of this topic have been covered in the sixth edition of Bailey’s Industrial Oil and Fat Products with chapters on soaps (Burke, 2005), detergents and detergency (Lynn, 2005), glycerine (Schroeder, 2005), biodiesel (Reaney et al., 2005), lubricants, hydraulic fluids, and inks (Erhan, 2005), polymers and plastics (Narine and Kong, 2005), paints (Lin, 2005), leather and textiles (Kronich and Kamath, 2005), and pharmaceutical and cosmetic uses (Hernandez, 2005). Hauthal and Wagner (2004) have reviewed household cleaning, care, and maintenance products.

Burke, M.R., Soaps, in Bailey’s Industrial Oil and Fat Products, 6th ed., Shahidi, F., Ed., Wiley Interscience, New York, 2005, Chap. 3. Dumelin, E.E., Biodiesel — a blessing in disguise? Eur. J. Lipid Sci. Technol., 107, 63–64, 2005. Erhan, S.Z., Vegetable oils as lubricants, hydraulic fluids, and inks, in Bailey’s Industrial Oil and Fat Products, 6th ed., Shahidi, F., Ed., Wiley Interscience, New York, 2005, Chap. 7. Hauthal, H.G. and Wagner, G. Eds., Household Cleansing, Care, and Maintenance Products, Ziolkowsky GmbH publishing, Augsberg, Germany, 2004. Translation of the original German volume published in 2003. Hernandez, E., Pharmaceutical and cosmetic use of lipids, in Bailey’s Industrial Oil and Fat Products, 6th ed., Shahidi, F., Ed., Wiley Interscience, New York, 2005, Chap. 12. Hilditch, T.P., The Industrial Chemistry of Fats and Waxes, Baillière, Tindall and Cox, London, 1927. Hirsinger, F., Oleochemicals and the environment, in Oleochemical Manufacture and Applications, Gunstone, F.D. and Hamilton, R.J., Eds., Sheffield Academic Press, Sheffield, U.K., 2001, Chap 10. Kronick, P. and Kamath, Y.K., Leather and textile uses of fats and oils, in Bailey’s Industrial Oil and Fat Products, 6th ed., Shahidi, F., Ed., Wiley Interscience, New York, 2005, Chap. 10. Lin, K.F., Paints, varnishes, and related products, in Bailey’s Industrial Oil and Fat Products, 6th ed., Shahidi, F., Ed., Wiley Interscience, New York, 2005, Chap. 9. Lynn, Jr., J.L., Detergents and detergency, in Bailey’s Industrial Oil and Fat Products, 6th ed., Shahidi, F., Ed., Wiley Interscience, New York, 2005, Chap. 4. Narine, S.S. and Kong Xiaohua, Vegetable oils in production of polymers and plastics, in Bailey’s Industrial Oil and Fat Products, 6th ed., Shahidi, F., Ed., Wiley Interscience, New York, 2005, Chap. 8. Reaney, M.J.T., Vegetable oils as biodiesel, in Bailey’s Industrial Oil and Fat Products, 6th ed., Shahidi, F., Ed., Wiley Interscience, New York, 2005, Chap. 6. Schroeder, K., Glycerine, in Bailey’s Industrial Oil and Fat Products, 6th ed., Shahidi, F., Ed., Wiley Interscience, New York, 2005, Chap. 5. Urata, K. and Takaishi, N., A perspective on the contribution of surfactants and lipids toward “Green Chemistry:” present status and future potential, J. Surfact. Deterg., 4, 191–200, 2001. Williams, M., Europe tightens detergent rules, Oils Fats Int., 21(2), 20–21, 2005. Yanagawa, Y., Perspectives on the sustainable growth of AsianPacific surfactant and detergent industries towards the new millennium, J. Oleo Sci., 50, 281–393, 2001.

9.2

Basic oleochemicals

The basic oleochemicals are fatty acids, methyl esters, alcohols, amines, and glycerol. Traditionally these have been produced mainly in North America, Western Europe, and Japan from local or imported oils and fats. But this is changing, and countries in South East Asia, particularly Malaysia, have become major producers of 592

Nonfood Uses of Oils and Fats

basic oleochemicals using their increasing indigenous supplies of raw material. This is shown in Table 9.1 containing projected figures up to the year 2010. Over the 10-year period, the production of oleochemicals is expected to rise by one-third from 5.76 to 7.75 million tonnes. Although this rise is apparent in all regions, market share will fall in North America and Western Europe, but rise in Asia. It is predicted that by 2010 Asian production will equal production in Western Europe and North America combined. This statement refers particularly to Malaysia and possibly underestimates the contribution likely to be made by China by 2010 (MPOB, 2005). The materials used in the oleochemical industry and the processes by which oleochemicals are produced are summarised in Table 9.2 and Table 9.3. It is interesting to note in Table 9.3 the wide range of products that contain oleochemicals.

TABLE 9.3 Materials, processes, and products of the oleochemical industry Raw materials

Unit operations to produce basic oleochemicals Basic oleochemicals Operations to produce downstream products from the basic oleochemicals Oleochemical derivatives

End-use markets

TABLE 9.1 Estimates for 2000, 2005, and 2010 of basic oleochemicals (million tonnes) by region and by commodity (percentage figures are given in parentheses) 2000 World total By region Western Europe North America Asia Other By commodity Fatty acids Methyl esters Alcohols Amines Glycerol

5.76 1.76 1.36 2.27 0.37 3.05 0.66 1.44 0.57 0.75

2005 6.69

(31%) (24%) (39%) (6%)

1.87 1.52 2.79 0.51

2010 7.75

(28%) (23%) (42%) (7%)

3.50 0.73 1.73 0.62 0.86

1.96 1.66 3.54 0.59

(25%) (21%) (46%) (8%)

Source: Adapted from Gunstone, F.D., The Chemistry of Oils and Fats, Blackwell Publishing, Oxford, 2004.

9.2.1

4.00 0.80 2.07 0.70 1.00

TABLE 9.2 Basic oleochemicals and downstream products produced from triacylglycerols

Fatty acids

Methyl esters Alcohols Amines Glycerol

Fatty acids

Fat hydrolysis gives fatty acids and glycerol. The former are used in large quantities to make soaps and also as intermediates to produce methyl esters, amides, amines, and other important nitrogen-containing compounds, acid chlorides, anhydrides, ketene dimers, and peroxy acids and esters (Table 9.2). As there is often confusion about the weight relationships between fats and fatty acids, it is worth noting that hydrolysis of glycerol trioleate (100 g) involves reaction with water (6.1 g) to produce oleic acid (95.7 g) and glycerol (10.4 g). The contribution of water is often overlooked and it is erroneously argued that because the reaction produces 10 g of glycerol there will only be 90 g of fatty acids. The theoretical yield of free acid is close to 96%. The conversion of oils and fats to soaps (saponification) is carried out by a long-established process involving treatment with aqueous alkali at around 100ºC. Glycerol is obtained as a valuable by-product. The sodium and potassium salts are conventional soaps. Salts with other metals are used to promote polymerisation of drying oils, as components of greases and lubricants, and are incorporated into animal feeds for ruminants. Fats can also be hydrolysed by water itself in a fatsplitting process to yield free acids. This is probably a

Note: For more recent data on glycerol see Tables 9.4 and Table 9.5 and MPOB (2005). Source: Adapted from Gunstone, F.D., The Chemistry of Oils and Fats, Blackwell Publishing, Oxford, 2004.

Basic Oleochemicals

Tall oil, lauric oils, palm oil, highand low-erucic rapeseed oil, soybean oil, sunflower oil Splitting, distillation, fractionation, hydrogenation, methylation, hydrophilisation Fatty acids, methyl esters, fatty alcohols, fatty amines, glycerol Amidation, dimerisation, epoxidation, ethoxylation, quaternisation, sulfation, sulfonation, saponification, transesterification Amides, dimer and trimer acids, epoxidised oils and esters, ethoxylates and propoxylates, sulfates, sulfonates, esters, soaps, salts Building auxiliaries, candles, cleaning agents, cosmetics, detergents, flotation agents, food emulsifiers, inks, insecticides, leather treatment, lubricants, paints, pesticides, pharmaceuticals, plastics, soaps, textiles, tyres

Downstream Derivatives Esters, metal salts (soaps), amides and amines, ketene dimers, anhydrides, acid chlorides, peroxy acids, and esters Acids, other esters, alcohols, α-sulfonates Ethylene oxide adducts, sulfates, Guerbet alcohols and acids Various cationic surfactants – Section 9.3.6 Polyglycerol, mono- and diacylglycerols and their acetates, tartates, lactates, etc.

Note: Some of the compounds are not discussed in this chapter, but are described in Chapter 7, which is devoted to physical properties. Source: Adapted from Gunstone, F.D., The Chemistry of Oils and Fats, Blackwell Publishing, Oxford, 2004.

593

9.2

Basic oleochemicals

homogeneous reaction between fat and a small amount of water dissolved in the fat. The procedure is usually carried out in a continuous, high pressure (20 to 60 bar), uncatalysed, counter-current process at 250ºC, though lower temperatures are desirable for highly unsaturated oils. Under these vigorous conditions, both the fatty acids and the glycerol will be discoloured and may have to be distilled. Splitting capacity in Malaysia has risen from 0.68 million tonnes in 1996 to 1.77 million tonnes in 2004 (Lim, 2005). This is in addition to the production of 0.47 million tonnes of soap noodles. Further information is found in Malaysian Oil Palm Statistics (MPOB, 2005). Toilet soaps are made from tallow (mainly in North and South America) or from vegetable oil (mainly palm oil) elsewhere in the world. The traditional process involves saponification (alkaline hydrolysis of vegetable oils or animal fats), but soaps are made increasingly by neutralisation of distilled fatty acids. There is a growing trade in soap noodles (sodium salts of fatty acids) that are converted elsewhere to coloured, scented, wrapped bars of toilet soap. This product is now in competition with liquid soaps (Table 9.7). Hydrolysis promoted by lipases, such as those from Rhizomucor miehei and Candida rugosa, takes about 20 hours at 20ºC or 6 hours at 45ºC, but gives cleaner products with less waste than the fat splitting process. Despite this and the saving in energy costs, it is not yet widely used on an industrial scale.

9.2.2

Claims have been made for the use of calcium carbonate as an environmentally acceptable catalyst (Suppes et al., 2001). Just as hydrolysis can occur without a catalyst at an elevated temperature, methanolysis is also possible without a catalyst at temperatures up to 200ºC, but this process is not used on an industrial scale. Candida antarctica lipase has been used to promote the methylation of waste fatty acids. Conversions of around 95% are achieved at 30ºC in 24 hours and under appropriate conditions the lipase maintains it activity through 45 cycles. Enzymatic methanolysis has also been examined with a range of vegetable oils and waste edible oil using a fixed bed reactor. Glycerolysis is another alcoholysis process employed on a commercial scale to convert triacylglycerols to mixtures of monoacylglycerols and diacylglycerols by reaction with glycerol in the presence of a basic catalyst when the following equilibrium is established: triacylglycerol + glycerol ] monoacylglycerol + diacylglycerol The composition of the product mix depends on the amount of glycerol dissolved in the fat phase. It can also be modified by the use of appropriate solvents. Concentrates of monoacylglycerol (90 to 95%), produced by molecular distillation, are widely used as emulsifiers. A similar process has been described using enzymes as catalysts, but this has not been used on an industrial scale.

Fatty esters

9.2.3

Esters can be made by esterification of acids by reaction with alcohols or from existing esters, including triacylglycerols, by reaction with alcohols (a process known as alcoholysis), acids (acidolysis), or other esters (interesterification). Alcoholysis is more widely practised than acidolysis and includes the important reactions of esters (especially triacylglycerols) with methanol (methanolysis) or with glycerol (glycerolysis). On an industrial scale ester production of methyl esters is most commonly undertaken by methanolysis of triacylglycerols (natural oils and fats) in the presence of an acidic, alkaline, or enzymatic catalyst. Large-scale methanolysis is used to make methyl esters for use as biofuel, as solvent, or as an intermediate in the production of alcohols. They can also be hydrolysed to acids. Oils low in free acid can be converted directly to methyl esters with an alkaline catalyst. Glycerol is also produced in this reaction and is recovered as a second marketable product. In a continuous process for the conversion of vegetable oils to methyl esters, conversion is >98% using sodium hydroxide as the catalyst. Under optimum conditions the reaction requires 6 to 8 minutes and may even take place during passage through the reaction plant.

Fatty alcohols

Long-chain alcohols (RCH2OH) with structures similar to the better-known acids (RCOOH) occur naturally in the free state and, more commonly, as esters. These last include wax esters made from long-chain alcohols and long-chain acids and occur in jojoba and other vegetable and animal waxes. Many insect pheromones are fatty alcohol acetates. Long-chain alcohols are important oleochemicals produced on a commercial scale by hydrogenolysis of acids, methyl esters, or triacylglycerols. This is a catalytic process and, depending on the choice of catalyst, olefinic double bonds may also be reduced or be left unchanged. These processes are generally applied to natural mixtures and the products, therefore, are mixtures varying in chain length. C8 to C14 alcohols are produced from lauric oils (coconut and palm kernel), C16 and C18 compounds from tallow, lard, palm oil, or palm stearin, and C22 alcohols from erucic acid-rich oils. Individual alcohols can be obtained by fractional distillation of the mixed products. Dodecanol and similar alcohols are also produced by the petrochemical industry through oligomerisation of ethene (ethylene). Commercial production of long-chain alcohols is now around 2 million tonnes annually of which twothirds or more is fat-based. About 75% of these fatty 594

Nonfood Uses of Oils and Fats

alcohols are used as alcohol sulfates, alcohol ethoxylates, or alcohol ethoxylate sulfates (see Section 9.3.3 to Section 9.3.5). Lim (2005) reports that since 2001 new production capacity for C12 to C18 alcohols has been 0.78 million tones, while capacity of only 0.24 million tonnes has been closed. An increase in the capacity to produce alcohols of 135,000 tonnes in Malaysia and 60,000 tonnes in Indonesia was reported for 2005. Malaysian exports of fatty alcohols rose from 288,000 tonnes in 2003 to 328,000 tonnes in 2004 (MPOB, 2005). Although commodity oils and fats are the starting point for most of these processes, the glycerol esters themselves are not generally used directly since, among other reasons, the valuable glycerol would be lost. More usually, hydrogenolysis is carried out on acids, methyl esters, or on wax esters made in situ from acids and alcohols. A wide range of minor products may also be formed during this reaction, including esters, aldehydes, alkanes, ethers, and acetals. In the methyl ester route, acid-free esters are first made from the natural oils by methanolysis (with release and recovery of glycerol) and then subjected to hydrogenolysis using pure hydrogen (>99.9%) and a copper chromite catalyst, usually in a fixed bed reactor, at 250 to 300 bar and 210°C. The volatile mixture of hydrogen and methanol can be separated and each component recycled. The alcohol product is stripped of methanol and the long-chain alcohols are used as such or are fractionated by distillation into individual components. This procedure can be adapted to produce olefinic alcohols with a copper-zinc catalyst free of chromium. Nickel catalysts activated with chromium, iron, or preferably rhodium can also be used for reactions at 200 to 230°C and 200 bar. Arguments have been presented for the acid route using some new technologies. This involves: (1) conversion of oil to acids and fractionation of these, some of which may be sold as acids; (2) preparation of methyl esters from acids using a resin bed as catalyst; and (3) reduction of esters to alcohols using a fixed bed catalyst (40 bar, 200 to 250°C, chromium-free catalyst). The combined procedures can be used flexibly to produce acids, methyl esters, and alcohols as required. In the wax ester route, the starting materials are fatty acids (distilled or fractionated) and some pre-made, longchain alcohols. At an appropriate temperature the acids and alcohols react without a catalyst to produce wax esters. These and pure hydrogen are then passed to the fixed-bed hydrogenation reactor charged with catalyst where hydrogenolysis takes place. Thereafter, hydrogen is separated from the alcohols. Some of these are returned to make more wax ester and the balance is distilled. This procedure has the advantage that it is not necessary to use or to recover methanol. These commercial processes are limited by the rates of hydrogen transfer between the gas, liquid, and solid

phases. Laboratory scale reactions, conducted under supercritical conditions with propane, are 5 to 10 times quicker than the conventional reaction. A copper-based catalyst free of chromium at 150 bar and 240 to 250°C is employed (van den Hark et al., 1999).

9.2.4

Fatty amines

Fatty amines, produced at a level of around 500 kt per annum, are the starting point for several types of nitrogencontaining compounds used as surfactants. Acids are first converted to nitriles — probably via amides — by reaction with ammonia at 280 to 360°C in the presence of alumina, thoria, titanium oxide, zinc oxide, manganese acetate, bauxite, or cobalt salts as catalysts. RCOOH → RCONH2 → RCN → RCH2NH2 → [RCH2]2NH Hydrogenation of nitrile occurs with nickel or cobalt as catalysts. Double bonds may be reduced at the same time, but conditions are usually selected to minimise this. Some conversion of cis to trans isomers may also occur. The major product is usually the primary amine (RCH2NH2), but this may be accompanied by secondary ([RCH2]2NH) and tertiary amines ([RCH2]3N). The formation of secondary and tertiary amines can be promoted by adjusting the reaction conditions. Aldimine (RCH = NH), the first product in the conversion of nitriles to primary amines, reacts with more hydrogen to form primary amine, with preformed primary amine to form secondary amine, or with preformed secondary amine to give tertiary amine. RC ≡ N → RCH = NH → RCH2NH2 reaction with hydrogen RC ≡ N → RCH = NH → [RCH2]2NH reaction with primary amine RC ≡ N → RCH = NH → [RCH2]3N reaction with secondary amine Tertiary amines with two methyl groups (RCH2NMe2) are made from primary amines by reductive alkylation with formaldehyde (methanal), from N,N-dimethylalkyl amides by catalytic reduction, or from fatty alcohols by catalysed reaction with dimethylamine. RCH2NH2 + 2CH2O → RCH2NMe2 RCONMe2 → RCH2NMe2 RCH2OH + Me2NH → RCH2NMe2 Still other nitrogen-containing, surface-active compounds may be made from carboxylic acids, alcohols, and amines (see Section 9.3.7 and Table 9.9 and Table 9.10).

595

9.2

Basic oleochemicals

9.2.5

Glycerol

In 2003, the annual production of glycerol (930 kt) came from countries with significant oleochemical industries including the U.S., Europe, Japan, and Southeast Asia. Significant importers were the U.S. (37% of its glycerol requirement) and Japan (50% of its glycerol requirement), while Southeast Asia was the major exporting region. Four ASEAN (Association of Southeast Asian Nations) countries (Malaysia, Indonesia, Philippines, Thailand, and Singapore) alone exported around 164,000 tonnes of glycerol in 2003. Table 9.4 clearly shows that ASEAN countries are now important producers of glycerol and have become the dominant exporter of this commodity. Sources of glycerol by oleochemical products are detailed in Table 9.5. Between 1999 and 2008, glycerol production is expected to rise 38% (from 804 to 1110 kt). Changes in the supply levels from various oleochemical processes over this 10-year period are soaps (–58kt), fatty acids (+88 kt), biodiesel (+293 kt), fatty alcohols (+32 kt), and petrochemical glycerol (–50 kt). These figures demonstrate the growing importance of biodiesel production and the continuing demand for fatty acids and fatty alcohols as sources of glycerol. The change through increased biodiesel production is mainly in Europe, but is expected to become more apparent in North America and elsewhere. The market for fatty acids continues to increase and new plants are being established in China and other developing countries. These will add to local supplies of glycerol and affect import requirements. In 2003, 41% of the European glycerol supply came from biodiesel production and that share is expected to increase. Following changes in taxation rules in Germany for a 95:5 blend of diesel with biodiesel, usage of biodiesel is expected to increase rapidly leading eventually to the production of 140 kt of glycerol in Germany, in addition to part of the 65 kt produced in 2003 for the existing pure biodiesel market. The biodiesel market in the rest of Europe will also move ahead strongly based mainly on locally produced rapeseed oil. European biodiesel capacity in Europe in 2004 was 2.2 million tonnes. New projects for large biodiesel plants continue to be announced

Oils and fats are mainly triacylglycerols and are generally used for dietary consumption in this form. However, in the oleochemical industry, oils and fats are used mainly to manufacture acids, soaps, methyl esters, alcohols, or nitrogen-containing derivatives and the production of these compounds will almost always involve the liberation of glycerol (1,2,3-propanetriol) at a level of around 10% of the oil or fat. This is a useful and valuable by-product and its economic value is an important part of the profitability of the oleochemical industry. Glycerol is also a product of the petrochemical industry where it is made from propene via epichlorohydrin (1-chloro2,3-epoxypropane). The increasing supply of glycerol from the oleochemical industry, the high price of propene, and the demand for epichlorohydrin for other purposes have together made the petrochemical supply route less important. It is now about 5% or less of total supply compared with 25% 20 years ago (Gunstone and Heming, 2004). Glycerol is available in several grades varying in purity and the requirements of the industries to which it is sold. Refined material is at least 86.5% pure and generally greater than 99.5%. Its value lies in its physical properties: it is hygroscopic, colourless, odourless, viscous, sweet-tasting, low boiling, nontoxic, emollient, a good solvent, and water-soluble. It is also easily biodegradable. Its major uses include oral care products, food and food emulsifiers, tobacco products, polyurethanes, prescription drugs, over-the-counter medicines, and cosmetics. Attempts are being made to develop new uses by conversion to other valuable compounds, such as glycidol (2,3-epoxypropanol), glycerol carbonate, and polyglycerols and their esters (Barrault et al., 2005, and Stepan website). In some of its uses, glycerol (produced at an annual level of around 1 million tonnes) competes with other polyols, such as pentaerythitol and trimethylolpropane (together 0.4 million tonnes), sorbitol (1.1 million tonnes.), propylene glycol (1.5 million tonnes), and ethylene glycol (7.5 million tonnes). The figures in parentheses represent annual production levels in 2003. TABLE 9.4

Production, consumption, exports, and imports of glycerol (kilotonnes) in 2003 Production

World U.S. Europe China Japan ASEANb Rest of world

Consumption

Exports

Imports

930a

936

251

248

142 315 20 45 197 211

201 325 65 85 33 227

24 25 2 2 164 34

75 35 45 43 – 50

Imports From:

SE Asia, Europe, S. America SE Asia Malaysia and Indonesia SE Asia

Details of sources by oleochemical products are given in Table 9.5. Malaysia, Indonesia, Philippines, Thailand, and Singapore. Source: Gunstone, F.D. and Heming, M.P.D., Glycerol – an important product of the oleochemical industry, Lipid Technol., 16, 177–179, 2004. a

b

596

Nonfood Uses of Oils and Fats

Typically emulsifiers have HLB values of 5 to 6, wetting agents of 7 to 9, and detergents of 13 to 15. Surfactants are produced both by the petrochemical and oleochemical industries, though only the latter will be considered in this section. This is most obvious in the production of long-chain alcohols. There are environmental and economic reasons why lipid-based molecules find favour in a time of high-priced oil and gas. Amphiphilic molecules can exist comfortably at an oil–water interface and reduce the surface tension at such interfaces. This property is fundamental in all living systems and in many foods and other manmade systems. Depending on the HLB, appropriate amphiphilic molecules influence a range of important surfactant properties, such as emulsification, deemulsification, wetting, foaming, defoaming, water-repelling, dispersing, solubilising, detergency, sanitising, lubricity, and emolliency. The simplest and oldest examples are soaps, such as sodium palmitate in which the palmitic acid chain is lipophilic and the carboxylate group is hydrophilic. The lipophilic alkyl chain varies mainly in chain length with C12 and C14 chains from the lauric oils; C16 and C18 chains from tallow, palm oil, or palm stearin; and the C20 and C22 chains from fish oils or high-erucic rapeseed oil. The alkyl chain may also have some unsaturation and may be branched. The polar head group shows greater variations through carboxylates, sulfates and sulfonates, phosphates, partial esters of polyhydric alcohols, such as glycerol and carbohydrates, polyoxyethlene derivatives of alcohols or amines, derivatives of amino acids, and many other nitrogen-containing molecules. There are four major groups of surfactants – anionic, nonionic, cationic, and zwitterionic (amphoteric) – with these terms describing the nature of the head group. Of these, anionics are used in greatest amount, but nonionics are increasing faster than any of the other groups. Some figures of usage in 2000 are given in Table 9.6. For automatic dishwashers two, three, or four surfactants may be combined in a single washing tablet. Many complex molecules have been designed and synthesised as superior surfactants. Typical of these is the amphoteric surfactant shown below with four functional groups (amino, carboxy, hydroxy, and ether groups) (Hidaka et al., 2003).

TABLE 9.5 Production of glycerol (kilotonnes) in 1999, 2003, 2004, and forecast for 2008 by the oleochemical product of which it is a by-product 1999

2003

2004

2008

Total

804

930

970

1110

Soaps Fatty acids Biodiesel Fatty alcohol Synthetic Other

198 322 57 108 75 44

180 350 160 110 80 50

170 365 210 120 50 55

140 410 350 140 25 45

Source: Gunstone, F.D. and Heming, M.P.D., Glycerol – an important product of the oleochemical industry, Lipid Technol., 16, 177–179, 2004.

although doubts have been expressed about the economic viability of some of these programmes. Nevertheless, it is quite possible that European biodiesel capacity will reach 3 million tonnes by the end of 2005, with the largest capacities being in Germany (1.1 million tonnes/year), Italy (0.6 million tonnes/year) and France (0.4 million tonnes/year) (see Section 9.6).

References Barrault, et al., Polyglycerols and their esters as an additional use of glycerol, Lipid Technol., 17, 131–135, 2005. Dumelin, E.E., Biodiesel — a blessing in disguise? Eur. J. Lipid Sci. Technol., 107, 63–64, 2005. Gunstone, F.D., The Chemistry of Oils and Fats, Blackwell Publishing, Oxford, 2004, Chaps. 8, 11. Gunstone, F.D. and Heming, M.P.D., Glycerol — an important product of the oleochemical industry, Lipid Technol., 16, 177–179, 2004. Lim, S., Oleochemicals — biodiesel and glycerine overshadow market, Oils Fats Int., 21 (1) 37–39, 2005. MPOB (Malaysian Palm Oil Board), 2004 Malaysian Oil Palm Statistics, 2005. Stepan (Stephan Company): www.stepan.com Suppes, G.J., et al., Calcium carbonate catalysed alcoholysis of fats and oils, J. Am. Oil Chem. Soc., 78, 139–145, 2001. Van den Hark, S., et al., Hydrogenation of fatty acid methyl esters to fatty alcohols at supercritical conditions, J. Am. Oil Chem. Soc., 76, 1363–1370, 1999.

9.3

Surfactants

9.3.1

Introduction

C12H25OCH2CH(OH)CH2N(CH2CH2OH) CH2CH2COONa

Surfactants are surface-active molecules as a consequence of their amphiphilic nature. This means that one part of a surfactant molecule (the alkyl chain) is lipophilic (hydrophobic) and the other part (the polar head group) is hydrophilic (lipophobic). The balance between these forces is an important property of a surfactant molecule and is defined as the hydrophilic lipophilic balance (HLB) by the equation:

A detergent is a formulation of many components (Hargreaves, 2003) in which the surfactant is responsible for washing and cleansing properties. Detergents enter widely into daily life and play an important part in keeping the human environment clean and wholesome. Their many uses include: cleaning agents for floors, surfaces, laundry, dishes, and personal care products. They are frequently used in pharmaceuticals and as lubricants, and are employed in industries devoted to food, agriculture, metal-working, textiles, and building. Appel (2000) has summarised the

HLB = 20 (molecular weight of the hydrophilic portion/ molecular weight of the whole molecule) 597

9.3

Surfactants

modern methods of detergent manufacture and Berna et al. (1998) have reviewed laundry products still used in bar form for manual washing. Rosen and Dahanayake (2000) have written a book on the industrial utilization of surfactants and Hargreaves (2003) has authored a simple but useful work on formulation containing a large number of typical recipes some of which are cited in Table 9.7. There has been a general consolidation of the surfactants industry both of suppliers and retailers (Anon., 2001). Many natural products have surfactant properties. Hill (2001) has reviewed the use of oils and fats as oleochemical raw materials. Dembitsky (2004, 2005a-e) has demonstrated the diversity of these compounds and has reported their chemical structures and biological activities. Many microorganisms also produce biosurfactants (Solaiman, 2005). Urata and Takaishi (1999, 2002) have discussed a number of synthetic routes to novel compounds capable of self-assembly. Analytical methods for the examination of surfactants have been reported by Thin Sue Tang (2001), Morelli and Szajer (2000, 2001), and Waldhoff and Spilker (2005). A useful website is The Surfactants Virtual Library (see reference list).

TABLE 9.6 Consumption of surfactants (million tonnes) in 2000 by category and usage in three countries/regions of the world (excluding 7 million tonnes of soaps)

Total By category Anionic Nonionic Cationic Amphoteric By usage Household products Cosmetics and toiletries Cleaning products Textiles Mining and petroleum Plastics and paints Agrochemicals Other

Western Europe

North America

Japan

2.61

3.34

1.19

1.32 0.97 0.25 0.07

1.95 1.04 0.27 0.09

0.49 0.56 0.10 0.04

1.41 0.12 0.21 0.16 0.30 0.16 0.08 0.15

1.32 0.19 0.28 0.31 0.37 0.60 0.12 0.15

0.32 0.13 0.10 0.12 0.11 0.06 0.04 0.31

Note: Other information is available in references: Anon., 2000 2002a,b, 2003. Source: Adapted from Gunstone, F.D., The Chemistry of Oils and Fats, Blackwell Publishing, Oxford, 2004.

TABLE 9.7

Selected typical formulations for a range of products indicating the surfactants present

Product

Formulation

% 8.0 6.0 4.0 7.0 2.0

Shampoo for dry hair

40% triethanolamine lauryl sulfate 27% ammonium lauryl sulfate 70% sodium lauryl ether sulfate Coco amido propyl betaine Coconut diethanolamide and citric acid, salt, perfume, colour, preservative, and water

Foam bath

27% sodium lauryl ether sulfate 30% lauryl betaine and salt, citric acid, perfume, colour, preservative, and water

60.0 5.0

Shower gel

30% sodium lauryl ether sulfate Coconut diethanolamide 30% alkylamido propyl betaine Cocoamine oxide and salt, lactic acid, perfume, colour, preservative, and water

40.0 2.0 5.0 2.0

Liquid soap

27% sodium lauryl ether sulfate Monoethanolamine lauryl sulfate 30% lauryl betaine and salt, citric acid, perfume, colour, preservative, and water

20.0 10.0 7.0

Toothpaste

Glycerol Sodium lauryl sulfate and sodium carbomethoxycellulose, sodium monofluorophosphate, dicalcium phosphate dihydrate, flavour, preservative, and water

25.0 1.5

Moisturising cream

Caprylic/capric triacylglycerols Octyl cocoate Cetyl esters Cetyl/stearyl alcohol Polysorbate 60 Sorbitan stearate Glycerol and hydrolysed vegetable protein, perfume, colour, preservative, and water

13.0 3.0 3.0 3.0 3.0 2.0 3.0

Dish washing liquids (20% active)

Sodium dodecylbenzene sulfonate Coconut diethanolamide 27% sodium lauryl ether sulfonate and salt, perfume, colour, preservative, and water

23.3 2.0 13.3

Carpet shampoo

28% sodium lauryl ether sulfonate Coconut diethanolamide Isopropanol and perfume, colour, preservative, and water

35.7 3.0 10.0

Screenwash

30% sodium lauroyl sarcosinate (1.0%) Isopropanol (25.0%), colour, and water

1.0 25.0

Source: Adapted from Hargreaves, T., Chemical Formulation, RSC, Cambridge, U.K., 2003.

598

Nonfood Uses of Oils and Fats

9.3.2

Anionic surfactants from carboxylic acids

Tracy and Reierson, 2002), sulfosuccinates (maleic anhydride and sodium sulfite), ethoxy carboxylates (sodium chloroacetate), or carbonate ethoxylates (dimethyl carbonate) by reaction with the reagents indicated in parentheses (Table 9.8). These are active components in detergents used in personal care products and for washing clothes and hard surfaces (floors, walls, dishes). Surfactant, in the range of 5 to 20%, is accompanied by other materials, such as phosphate, zeolite, bleaching agent, optical brightener, fragrance, and water. The sarcosinates, taurates, and isethionates (Table 9.8) are long-chain amides of sarcosine and taurine, respectively, or are esters of isethionic acid. α-Sulfonate esters are made from methyl esters of saturated acids by reaction with sulfur trioxide. The product, after neutralisation, is a mixture of monoester salts (from RCH(SO3H)COOCH3) and di-sodium salts (from RCH(SO3H)COOH) usually in a ratio of 80:20. Further details are given in chapters written by Porter (1997) and by Roberts (2001).

As the name indicates, anionic surfactants have a negatively charged species and a counterion that is usually a metal, but may be a type of ammonium group. The anionics are used in greater volume than any other class of surfactants. Soap belongs to this category and is still the surfactant used in largest amount. In many countries, hard (tablet) soap is being replaced by a liquid soap that is not a carboxylate salt (see Table 9.7). Soap has the disadvantage that it can only be used at pH8 and above and that it forms an insoluble scum with the calcium salts present in hard water. Alternative and superior surfactants, therefore, have been developed. These are generally sulfates or sulfonates in place of carboxylates. In all of these, the alkyl chain is the most expensive component in the surfactant. Carboxylates Fatty alcohol sulfates Fatty alcohol ether sulfates α-Sulfonated esters

RCOOH ROSO3H R(OCH2CH2)nOSO3H RCH(SO3H)COOCH3

Sodium and potassium salts of fatty acids (traditional soaps) are still made by saponification of appropriate fats and also increasingly by neutralisation of carboxylic acids resulting from splitting (see Section 9.2.1). Cohen and Trujillo (1998) have reported the synthesis, characterization by GC-MS (gas chromatography-mass spectrometry), and by infrared spectroscopy, surface tension, and specific conductivity of methyl ester sufonates. The production of methyl ester sulfonates in the U.S. has been reported (Watkins, 2001). Scheibel (2004) has discussed changes in anionic surfactant technology in the laundry detergent industry.

9.3.3

9.3.4

Nonionic surfactants

Nonionic surfactants contain the usual type of lipophilic chain from a petrochemical or oleochemical source. The head group is not charged, but is polar through the presence of an appropriate collection of hydroxy, amino, or ether groups. The last come from ethylene oxide or propylene oxide products (see Section 9.3.5) and the former from glycerol, polyglycerol, low molecular weight carbohydrates, or amino acids or other amines. Typical structures include polyethylene oxide derivative of the fatty alcohol ROH (alcohol ethoxylate AEO), such as R(OCH2CH2)nOH and bis-polyethylene oxide derivatives of the fatty amine RNH2, such as

Anionic surfactants from alcohols

Anionic surfactants of various kinds can be made from fatty alcohols or fatty alcohol ethoxylates (see Section 9.2.3 and Section 9.3.4) mainly as sulfates through reaction with sulfur trioxide or chlorosulfonic acid and used as sodium, ammonium, or monoethanolamine (HOCH2CH2NH2) salts. The long-chain alcohols may also be used as phosphates (phosphorus pentoxide,

HO(CH2CH2O)n NR(OCH2CH2)mOH Ethylene oxide is itself a product of the petrochemical industry and a hazardous chemical with undesirable environmental properties. It is for this reason that there is growing interest in the acyl and alkyl derivatives of glucose and other carbohydrates where all the reactants are

TABLE 9.8 Anionic surfactants produced from fatty alcohols and their ethoxylates Name Alcohol sulfates Ethoxy sulfates Monoacylglycerol sulfates Ethoxy phosphates Sulfosuccinates Ethoxycarboxylates Carbonate ethoxylates Sarcosinates Taurates Isethionates a

Structurea ROSO3H AEOSO3H RCOOCH2CH(OH)CH2OSO3H AEOPO3H2 or (AEO)2PO2H AEOCOCH2CH(OSO3Na)COONa AEOCH2COONa ROCOO(CH2CH2O)nH RCON(CH3)CH2COOH RCONHCH2CH2SO3H RCOOCH2CH2SO3H

Typical Uses Shampoo, toothpaste Shampoo, bubble bath Electroplating Carpet cleaner, oil spill dispersants

Corrosion inhibitor Toilet bars

RO represents a fatty alcohol, RCOO an acyl group and AEO an alcohol ethoxylate R(OCH2CH2)nO.

599

9.3

Surfactants

natural products coming from renewable resources (Section 9.3.7). Bognolo (1997) has written a full account of nonionic surfactants.

9.3.5

dermatological properties (Cox et al., 1998; Hreczuch et al., 2001). The reaction is not confined to methyl esters and has been applied to compounds with two or more ester functions. Interesting products result from triacylglycerols including natural mixtures, such as the lauric oils (coconut and palmkernel) and tallow. Reaction can occur at all three ester functions to give products with the structure shown in Figure 9.1 Subsequent partial hydrolysis gives a mixture of ethoxylated triacylglycerols (unreacted material), fatty acid soaps (RCOONa), and products in which 1, 2, or 3 acyl groups have been removed. The composition of the mixture depends on the degree of hydrolysis. Even without further separation this mixture has good surfactant properties (Cox and Weerasooriya, 2000). Another development is the replacement of ethylene oxide, wholly or in part, by propylene oxide in the reaction both with alcohols and esters. The repeating unit (-CH2CH2O-) is then replaced by (-CHMeCH2O-) or there is a mixture of both units in the poly-ether chain. The resulting branched-methyl compounds have interesting modified surfactant properties, important among which is their greater ability to reduce foaming when compared with the ethylene oxide derivatives. This reaction requires a calcium aluminum complex as catalyst (Cox et al., 1998; Weerasooriya, 1999). Further information is given by Bognolo (1997) and Gunstone (2001).

Ethoxylation and propoxylation of alcohols and esters

A substantial portion of the medium and long-chain alcohols are used only after conversion to ethoxylates or propoxylates. Ethoxylation of long-chain alcohols with ethylene oxide occurs at 135°C under pressure in 30 minutes in a reaction usually catalysed by ~ 0.2% of NaOH or KOH. The product is hydrophilic by virtue of one hydroxyl group and many ether links and may be a mixture of compounds with up to 20 ethylene oxide (EO) units (Figure 9.1). Since important surfactant properties vary with the number and range of EO units, there is a drive to produce materials with different narrow ranges of EO units. For example, when dodecanol is reacted with ethylene oxide (50% wt ≡ 4.4 mol) the product is a mixture of compounds with up to 20 or more EO units and no individual compound greater than 10%. With proprietary catalysts, such as ZrSO4(OR)2 or similar aluminium compounds, the products contain only 0 to 10 EO units with those having 4 to 6 EO units each around 20% and those with 3 and 7 units ~ 10%. These narrow range ethoxylates have good stability and skin mildness in liquid dishwashing products (Di Serio et al., 1998). The ethoxylation of esters, rather than alcohols, is an interesting development in this field since products with improved surfactant properties can be obtained from esters that are less expensive starting materials than the alcohols. Using a methyl ester and an appropriate catalyst, such as a composite aluminum and calcium metal oxide at 180°C and 3 bar the product has a narrow range of molecular weights with mainly 5 to 10 ethylene oxide units (see Figure 9.1). While alcohol ethoxylates can assume a linear arrangement ester ethoxylates are necessarily bent (boomerang shape) because of the trigonal ester carbon atom. The ester products have outstanding ROH

R(OCH2CH2)nOH

RCOOMe

RCO(OCH2CH2)nOMe

CH2O(CH2CH2O)xCOR

9.3.6

Alkyl polyglycosides

The term alkyl polyglycoside is the name given to technical products made from starch (or other source of glucose) and a fatty alcohol. These are sugar ethers in contrast to the sugar esters used in olestra. The alcohol is usually a mixture of C8/10, C12/14, or C16/18 alcohols derived from appropriate fatty acid sources. All the substrates are renewable resources. The reaction between starch and alcohol is usually catalysed by acids, such as sulfuric or 4toluenesulfonic and is accompanied by extensive depolymerisation of the carbohydrate polymer so that the product is mainly, but not entirely, an alkyl glucoside. In the two-step process (see equation below) butanol is used first and in the second step the fatty alcohol mixture (ROH) of desired chain length. The product is a mixture of compounds with R groups differing in chain length and values of n lying between 1 and 5. The average value of n (degree of polymerisation) is usually 1.3 to 1.7. Products with a value of 1.3 will contain molecules with one (~60%), two (~20%), three (~10%), and four and five glucose units. Products made from alcohols having 8 to 14 carbon atoms are water-soluble and are used as surfactants, those with 16 and 18 carbon atoms are not water-soluble, but are used as emulsifying agents and in cosmetic preparations (Hill et al., 1997).

Ethylene oxide derivative based on an alcohol

Ethylene oxide derivative based on a methyl ester

Ethylene oxide derivative based on a triacylglycerol

CHO(CH2CH2O)yCOR CH2O(CH2CH2O)zCOR

FIGURE 9.1 Ethylene oxide derivatives of alcohols, methyl esters, and triacylglycerols.

600

Nonfood Uses of Oils and Fats

starch + BuOH → [glucose]nOBu

again with acrylonitrile to give a triamine RNH(CH2)3 NH(CH2)3NH2.

[glucose]nOBu + ROH → [glucose]nOR

ROH + CH2=CHCN → RO(CH2)2 CN → RO(CH2)3NH2 (ether amine)

Piispanen et al. (2004) have described the structures and structure/property relationships of surfactants derived from natural products and Dembitsky (2004, 2005a-d) has reported the structure and biological activity of a large number of natural glycoside surfactants. The acyl esters of sugars are not yet commercial surfactants, but there is considerable interest in the preparation of acylated derivatives of fructose (Jung et al., 1998), glucose, or other monosaccharides, and of sucrose. These can be prepared chemically or, more specifically, with enzymatic catalysts and studies of their synthesis, hydrolysis, biodegradation, and some surfactant properties have been reported (Allen and Tau, 1999; Baker et al., 2000a,c; Polat et al., 2001).

9.3.7

RNH2 + CH2=CHCN → RNH(CH2)2 CN → RNH(CH2)3NH2 (diamine) Ether amines and diamines react with ethylene oxide to give ethyloxylated products (Table 9.8 and Table 9.9) as in the following equations: RO(CH2)3NH2 → RO(CH2)3NH(CH2CH2O)nH (ethoxylated ether amine) RNH(CH2)3NH2 → RNH(CH2)3NH(CH2CH2O)nH (ethoxylated diamine) RNH2 → H(OCH2CH2)nN(R)(CH2CH2O)mH (ethoxylated amine)

Cationic surfactants

Cationic surfactants are nitrogen containing-compounds. They show high substantivity (i.e., strong adherence) to natural surfaces and find extensive use in fabric softening, hair conditioning, corrosion inhibition, mineral flotation, and as bactericides (Karsa, 2001). Levinson (1999) has reviewed rinse-added fabric softener technology. Alcohols and amines add to acrylonitrile and, after catalytic hydrogenation, furnish ether amines (often written as one word) and diamines as shown in the equations below (Table 9.9 and Table 9.10). The diamine can react TABLE 9.9

RCONH(CH2)2NH(CH2)2NHCOR (diamidoamine or diacylated triamine) Diamidoamines (or diacylated amines) formed from carboxylic acids and diethylene triamine (H2N(CH2)2 NH(CH2)2NH2) have the structure shown and readily cyclise to imidazolines (Figure 9.2). Further information is provided by James (1997) and by Franklin et al. (2001).

Cationic surfactants made from fatty amines or fatty alcohols

Product Name Ether amine Diamine Ethoxylated ether amine Ethoxylated diamine Ethoxylated amine Diamido amineb

Product Structure

Starting Material

RO(CH2)3NH2 RNH(CH2)3NH2 a RO(CH2)3NH(CH2CH2O)nH RNH(CH2)3NH(CH2CH2O)nH H(OCH2CH2)nN(R)(CH2CH2O)mH RCONH(CH2)2NH(CH2)2NHCOR a

ROH RNH2 RO(CH2)3NH2 RNH(CH2)3NH2 RNH2 H2N(CH2)2NH(CH2)2NH2

Reactant CH2=CHCN CH2=CHCN Ethylene oxide Ethylene oxide Ethylene oxide RCOOH

Product after catalytic hydrogenation of an intermediate nitrile. This is the name given to a diacylated triamine. The products are readily cyclised to imidazolones (Figure 9.2). Note: R represents an alkyl chain.

a

b

TABLE 9.10

Cationic surfactants made from fatty amines

Products Amines Quaternary salts (quats) Amine oxides Amido amines Imidazolines Ester amines Ether amines

Structure

Reactants

RNH2, R2NH, R3N [R2NMe2]+ X[RNMe2]+ ORCONH(CH2)3NMe2 Figure 9.2 RCOOCH2CH2NMe2 RO(CH2)3NH2

Nitriles – hydrogenation Tertiary amines and RX Tertiary amines and H2O2 Polyamines and RCOOH Polyamines and RCOOH Ethanolamines and RCOOH Fatty alcohols and acrylonitrile

Source: Adapted from Gunstone, F.D., The Chemistry of Oils and Fats, Blackwell Publishing, Oxford, 2004.

601

9.3

Surfactants

N

N

O

R

R N

N

R O

CH2CH2NHCOR

FIGURE 9.3

FIGURE 9.2 Imidazolines from RCOOH and diamine (H2N(CH2)2NH2) or triamine (H2N(CH2)2NH(CH2)2NH2).

9.3.8

O

R

OH O

CH2OH

Acetals from RCHO and glycerol.

possibility of using these compounds for drug delivery. The weak bonds can be broken when required with the help of enzymes, by reactions occurring at sewage plants, or by chemical or physical processes involving acid, alkali, ozone, heat, or ultraviolet light. These compounds are generally acetals/ketals or ortho esters (Hellberg, 2003). Cyclic acetals/ketals result when aldehydes or ketones react with polyhydric alcohols, such as glycerol (Figure 9.3), pentaerythryitol, or glucose. The products are 1,3dioxolanes (5-membered hetero ring) or 1,3-dioxanes (6-membered hetero ring). Any unreacted hydroxyl groups can be further functionalised. These compounds are made under anhydrous acidic conditions and are readily hydrolysed under aqueous acidic conditions (Hellberg et al., 2000a). Ortho-esters are made from ethyl orthoformate [HC(OEt)3], alcohols, and monomethyl polyethylene glycol (HO(C2H4O)nMe) in the presence of aluminum chloride. The product is a mixture of many compounds having the structures shown below in which x, y, and z have values 0-3 and x + y + z = 3. The products formed in largest amount have values of x, y, and z of 1,1,1 or 0,2,1, or 0,1,2. Such product mixtures are used for temporary emulsions, hard surface cleaners, textile treatment processing, etc. They are hydrolysed under mild acid or alkaline conditions and have good biodegradability (Hellberg et al., 2000b).

Gemini surfactants and cleavable surfactants

Most surfactants contain one lipophilic chain and one hydrophobic head group. Gemini or dimeric surfactants contain two of each of these linked together by a short aliphatic group or through an aromatic ring. A book devoted to this subject has been edited by Zana and Xia (2003). One example formulated below shows two quaternary groups linked through a tetramethylene spacer (Rosen and Tracy, 1998). Similar compounds with an aromatic spacer have been prepared and assessed for protection of steel fabrics against 2M hydrochloric acid. (Negm and Mohamed, 2004). R(Me)2N+(CH2)4N+(Me)2 R R3N+CH2COOC6H4OCOCH2N+R3 Unlike conventional surfactants, gemini consist of two molecules of monomeric surfactant linked through a flexible spacer. Such molecules are capable of wide variations in terms of each of their three components – lipophilic chain, head group, and spacer — and frequently show remarkable properties. Gemini surfactants generally display unusual patterns of self-assembly and some intriguing physicochemical properties. For example, ionic Gemini surfactants have critical micelle concentrations two orders of magnitude lower than their monomeric alkylsulfonate analogues and are very efficient at reducing surface tension both on their own and in combination with conventional surfactants. Gemini surfactants have much lower Krafft points and higher solubility in water compared to their monomeric counterpoints. Valivety et al. (1998) have described the synthesis of amino acid based gemini surfactants. One example is the compound formulated below and based on myristic acid (2 mols), serine (2 mols) and1,10-dihydroxydecane (1 mol).

[OR]y  H  C  [O(C2H4O)nMe]z  [OEt]x Ono et al. (2004, 2005) have described the preparation of cleavable surfactants from methyl pyruvate and from diethyl tartrate as shown below. CH3COCOOMe → CH3CH(OR)2COONaR = C8H17, C10H21, or C12H23

C13H27COOCH2CH(NH2)COO(CH2)10OCOCH(NH2) – CH2OCOC13H27

[EtO2CCH(OH)]2 → [RO2CCH(OSO3Na)]2R = C8H17, C10H21, or C12H23

decanediyl-1,10-bis-(3-O-myristoyl-L-serine)

Quaternary ammonium compounds, much used as rinse-aids in the past, have been largely replaced by ester quats. Typical structures of these two categories of compounds are shown:

Another group of surfactants have a weak bond built into the molecule. These are interesting because this feature leads to improved biodegradabilty and opens up the 602

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Dembitsky, V.M., Astonishing diversity of natural surfactants: 3. Carotenoid glycosides and isoprenoid glycolipids, Lipids, 40, 535–557, 2005b. Dembitsky, V.M., Astonishing diversity of natural surfactants: 4. Fatty acid amide glycosides, their analogues and derivatives, Lipids, 40, 641–660, 2005c. Dembitsky, V.M., Astonishing diversity of natural surfactants: 5. Biologically active glycosides of aromatic metabolites, Lipids, 40, 869–900, 2005d. Dembitsky, V.M., Astonishing diversity of natural surfactants: 6. Biologically active marine and terrestrial alkaloid glycosides, Lipids, 40, 1081–1105, 2005e. Di Serio, M., et al., Narrow-range ethoxylation of fatty alcohols promoted by a zirconium alkoxide sulfate catalyst, J. Surfact. Deterg., 1, 83–91, 1998. Franklin, R., et al., Cationic and amine-based surfactants, in Oleochemical Manufacture and Applications, Gunstone, F.D., Hamilton, R.J., Eds., Sheffield Academic Press, Sheffield, U.K., 2001, Chap 2. Gunstone, F.D., Chemical reactions of fatty acids with special reference to the carboxyl group, Eur. J. Lipid Sci. Technol., 103, 307–314, 2001. Gunstone, F.D., The Chemistry of Oils and Fats, Blackwell Publishing, Oxford, 2004, Chaps. 8, 11. Gunstone, F.D. and Heming, M.D., Glycerol – an important product of the oleochemical industry, Lipid Technol., 16, 177–179, 2004. Hargreaves, T., Chemical Formulation, RSC, Cambridge, U.K., 2003. Hellberg, P-E., Cleavable surfactants — giving an unstable advantage, Lipid Technol., 15, 101–105, 2003. Hellberg, P-E., Ortho ester-based cleavable cationic surfactants, J. Surfact. Deterg., 5, 217–227, 2002. Hellberg, P-E., et al., Cleavable surfactants, J. Surfact. Deterg., 3, 81–91, 2000a. Hellberg, P-E., et al., Nonionic cleavable ortho ester surfactants, J. Surfact. Deterg., 3, 369–379, 2000b. Hidaka, H., et al., Preparation and properties of new tetrafunctional amphoteric surfactants bearing amino, carboxyl, and hydroxyl groups and an ether bond, J. Surfact. Deterg., 6, 131136, 2003. Hill, K., et al., Alkylpolyglycosides: Technology, Properties, and Applications, VCH, Weinheim, Germany, 1997. Hill, K., Fats and oils as oleochemical raw materials, J. Oleo Sci., 59, 433–444, 2001. Hreczuch, W., et al., Direct ethoxylation of longer-chain aliphatic ester, J. Surfact. Deterg., 4, 167–173, 2001. James, A.D., Cationic surfactants, in Lipid Technologies and Applications, Gunstone, F.D., Padley, F.B., Eds., Marcel Dekker, New York, 1997, Chap 24. Jung, S., et al., Structure and surface-active property determinations of fructose mono-oleates, J. Surfact. Deterg., 1, 53–57, 1998. Karsa, D. R., Quaternary ammonium surfactants, Lipid Technol., 7, 81–86, 2001. Levinson, M.I., Rinse-added fabric softener technology at the close of the twentieth century, J. Surfact. Deterg., 2, 223–235, 1999. Morelli, J.J. and Szajer, G., Analysis of Surfactants: Part I, J. Surfact. Deterg., 3, 539–552, 2000. Morelli, J.J. and Szajer, G., Analysis of surfactants: Part II, J. Surfact. Deterg., 4, 75–83, 2001. Negm, N.A. and Mohamed, A.S., Surface and thermodynamic properties of diquaternary bola-form amphphiles containing an aromatic spacer, J. Surfact. Deterg., 7, 23–30. 2004.

quat: R2N+Me2 Xester quat: (RCOOCH2CH2)2N+Me2 XThe ester quats are stable to acids, but are easily hydrolysed by alkali to soap and the compound ((HOCH2CH2)2 N+Me2X-). They show better environmental characteristics than the quats themselves (Hellberg, 2002). Alkyl ethoxylates (R(OC2CH2)nOH) are viscous oils that are not always easy to handle, but they react with carbon dioxide to form carbonates (R(OCH 2 CH 2 ) n OCO2Na). These are solid and are easily incorporated into granular detergents. In an alkaline solution, the ethoxylates are quickly regenerated from the carbonates.

References Allen, D.K. and Tao, B.Y., Carbohydrate-alkyl ester derivatives as biosurfactants, J. Surfact. Deterg., 2, 383–390, 1999. Anon., Publications and reports, J. Surfact. Deterg., 3, 139–141, 2000. Anon., Consolidation in the surfactants industry, Inform, 12, 872–882, 2001. Anon., Publications and reports, J. Surfact. Deterg., 5, 426, 2002a. Anon., LAS decline, Inform, 13, 704–705, 2002b. Anon., Publications and reports, J. Surfact. Deterg., 6, 396, 2003. Appel, P.W., Modern methods of detergent manufacture, J. Surfact. Deterg., 3, 395–405, 2000. Baker, I.J.A., et al., Sugar fatty acid ester surfactants: structure and ultimate aerobic biodegradability, J. Surfact. Deterg., 3, 1–11, 2000a. Baker, I.J.A., et al., Sugar fatty acid ester surfactants: biodegradation pathways, J. Surfact. Deterg., 3, 13–27, 2000b. Baker, I.J.A., et al., Sugar fatty acid ester surfactants: basecatalysed hydrolysis, J. Surfact. Deterg., 3, 29–32, 2000c. Berna, J.L., et al., Laundry products in bar form, J. Surfact. Deterg., 1, 263–271, 1998. Bognolo, G., Nonionic surfactants, in Lipid Technologies and Applications, Gunstone, F.D., Padley, F.B., Eds., Marcel Dekker, New York, 1997, Chap 25. Cohen, L. and Trujillo, F., Synthesis, characterisation, and surface properties of sulfoxylated methyl esters, J. Surfact. Deterg., 1, 353341, 1998. Cox, M.F. and Weerasooriya, U., Impact of molecular structures on the performance of methyl ester ethoxylates, J. Surfact. Deterg.. 1, 11–22, 1998. Cox, M.F. and Weerasooriya, U., Enhanced propoxylation of alcohols and alcohol ethoxylates, J. Surfact. Deterg.. 2, 59–68, 1999. Cox, M.F. and Weerasooriya, U., Partially saponified triglyceride ethoxylates, J. Surfact. Deterg.. 3, 213, 2000. Cox, M.F. et al. Methyl ester propoxylates, J. Surfact. Deterg.. 1, 167–175, 1998. Dembitsky, V.M., Astonishing diversity of natural surfactants: 1. Glycosides of fatty acids and alcohols, Lipids, 39, 933–953, 2004. Dembitsky, V.M., Astonishing diversity of natural surfactants: 2. Polyester glycosidic ionophores and macrocyclic glycosides, Lipids, 40, 219–248, 2005a.

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9.4

Cosmetics and personal care products

of lipids make them suitable for controlling product consistency and to favourably influence the skin feel of the preparation. Knowledge about polymorphism and crystallisation kinetics is essential to optimise formulations based on lipids. The second aspect of lipids in cosmetic formulations being covered is the ability of essential fatty acids, tocopherols, and phytosterols to improve skin health. Lipids are important building blocks of the cell membranes; they act as antioxidants and anti-inflammatory agents and can have a fundamental effect on collagen production and break down in the skin. All of this illustrates the versatility of lipids as formulating tools in skin care and cosmetics. Lipids are important multifunctional ingredients commonly used in cosmetic and personal care products. They may function as emollients, moisturisers, emulsifiers, solubilisers, dispersing agents, texturisers and skin-feel improvers. Some lipids show interesting bioactivity (antiinflammatory and antioxidative) and influence protein synthesis and degradation in the skin. They find uses in skin-care emulsions, ointments, and balms as well as colour cosmetics and personal care products, such as shower gels, shampoos, and hair conditioners. The definition of lipids in cosmetic applications is variable and in this review the emphasis will be on acylglycerols, tocopherols, and phytosterols. The use of lipids and lipid-derived materials as texturisers will first be described, followed by examples of their bioactivity and interactions with the skin.

Ono, D., et al., Synthesis and properties of soap types of doublechain cleavable surfactants derived from pyruvate, J. Oleo Sci., 53, 89–95, 2004. Ono, D., et al., Preparation and properties of bis(sodium sulfate) types of cleavable surfactants derived from diethyl tartrate, J. Oleo Sci., 54, 51–57, 2005. Piispanen, P.S., et al., Surface properties of surfactants derived from natural products. Syntheses and structure/property relationships – Part 1, solubility and emulsification, Part 2, foaming, dispersion and wetting, J. Surfact. Deterg., 7, 147–159, 161–167, 2004. Polat, T. and Linhardt, R.J., Syntheses and applications of sucrose-based esters, J. Surfact. Deterg., 4, 415, 2001. Porter, M.R., Anionic detergents, in Lipid Technologies and Applications, Gunstone, F.D., Padley, F.B., Eds., Marcel Dekker, New York, 1997, Chap 23. Roberts, D.W., Manufacture of anionic surfactants, in Oleochemical Manufacture and Applications, Gunstone, F.D., Hamilton, R.J., Eds., Sheffield Academic Press, Sheffield, U.K., 2001, Chap 3. Rosen, M.J. and Tracy, D.J., Gemini surfactants, J. Surfact. Deterg., 1, 547–554, 1998. Rosen, M.J. and Dahanayake, M., Industrial Utilisation of Surfactants, Principles and Practice, AOCS Press, Champagne, IL, 2000. Schiebel, J.J., The evolution of anionic surfactant technology to meet the requirements of the laundry detergent industry, J. Surfact. Deterg., 7, 319–328, 2004. Solaiman, D.K.Y., Applications of microbial biosurfactants, Inform, 126, 408–410, 2005. The Surfactants Virtual Library: www.surfactants.net Thin Sue Tang, Analysis of oleochemicals, in Oleochemical Manufacture and Applications, Gunstone, F.D., Hamilton, R.J., Eds., Sheffield Academic Press, Sheffield, U.K., 2001, Chap 8. Tracy, D.J. and Reierson, R.L., Commercial synthesis of monoalkyl phosphates, J. Surfact. Deterg., 5, 169–172, 2002. Urata, K. and Takaishi, N., Newer synthetic approaches to surfactants, lipids, and related compounds based on C-3 building blocks: Recent advances related to fatty chemistry, J. Surfact. Deterg., 2, 91–103, 1999. Urata, K. and Takaishi, N., Self-assembly of compounds based on a glycerin skeleton as a C-3 building block, J. Surfact. Deterg., 5, 287294, 2002. Valivety, R., et al., Application of enzymes to the synthesis of amino acid-based bola and gemini surfactants, J. Surfact. Deterg., 1, 177–185, 1998. Waldhoff, H. and Spilker, R., Eds., Handbook of Detergents, Part C: Analysis, vol. 123, Marcel Dekker, New York, 2005. Watkins, C., Methyl ester sulfonates, Inform, 12, 11531159. 2001. Weerasooriya, U., Ester alkoxylation technology, J. Surfact. Deterg., 2, 373–381, 1999. Zana, R. and Xia, J., Eds., Gemini Surfactants: Synthesis, Interfacial and Solution-Phase Behaviour and Applications, Marcel Dekker, New York, 2003.

9.4

Cosmetics and personal care products

9.4.1

Introduction

9.4.2

Texture control using solid and semisolid lipids

A cosmetic skin-care formulation is a complex mixture of ingredients with varying functionality. A majority of such products are stabilised emulsions of an emollient in water with added bioactive materials for delivering real or apparent benefits to the well-being of the skin. The formulation needs to deliver both water-soluble actives and oil-soluble ingredients to the stratum corneum and preferentially control the penetration of these substances to the epidermis and dermis. The formulation regularly has to have a shelf life of more than 24 months, putting a lot of requirements on the emulsifying and stabilising system. Finally, the formulation also needs to be aesthetically and sensorially acceptable in order to fulfill the demands of the consumer. The main component in the oil phase of skin-care emulsions is the emollient. It comprises one or more oils of differing composition and chemical structure. Many lipidbased and lipid-derived materials have been suggested for use as emollients, including esters of long-chain fatty acids with both short- and long-chain alcohols, natural and synthetic triacylglycerols, and naturally occurring hydrocarbons, such as squalene and squalane. The primary function of these emollients is to lubricate the skin, decrease the water permeability, and to act as a carrier of

This review covers two aspects of using lipids in cosmetic and skin-care formulations. The physicochemical properties 604

Nonfood Uses of Oils and Fats

the actives in the formulation. A secondary function of increasing importance is to influence the formulation texture and the sensory aspects when applying the emulsion to the skin. Important considerations when selecting emollients are their effects on emulsion structure and how that effect is translated to sensory properties on application (Wiechers et al., 2004). Physicochemical properties, such as viscosity and polarity, are strongly related to the ability of an emollient to spread onto and penetrate into the skin. By combining emollients with different characteristics in terms of spreading ability and viscosity, optimal performance on different timescales can be obtained. The ability of an emollient to interact with surfactants in liquid crystalline phases also plays a significant role in the performance (Wiechers, 2003). The microstructure and the resulting texture of cosmetic emulsions can be strongly influenced using semisolid and solid crystallising materials in the oil phase. By a careful selection of semisolid fats and waxes, the consistency of a cosmetic cream or lotion can be fine tuned to obtain optimal sensory properties and good product stability. In this context, the crystallisation behaviour of the ingredients used is strongly influencing the final result. There are three physicochemical concepts that are useful in the design of an emollient for cosmetic creams: the solids content of the oil phase, the polymorphic behaviour of the solids, and, finally, the kinetics of crystallisation.

9.4.3

In general terms, the more solids at a given temperature, the harder and more brittle the emulsion will appear. It is important to optimise the solid phase content for the different temperature ranges that the product will encounter. For example, the solid phase content at body or skin temperature (34 to 37°C) determines the sensory properties and the spreadability of the product. The solids content at 40 to 45°C will have an influence on the high temperature storage stability of the formulation. Finally, the solids content at 20 70 25°C will determine the texture and consistency of the product at room temperature. Characteristic ranges for skin-care creams are less than 50% solids at room temperature, less than 30% solids at body temperature, and 5 to 15% solids at 40°C (Figure 9.4). With these solids ranges, a product will have good stability during storage, be nicely spreadable on the skin, and have good high temperature resistance.

9.4.4

The polymorphic behaviour of the solids used in cosmetic formulations is generally not well known by the formulators. Most simple waxes (wax esters and hydrocarbonbased waxes) crystallise in orthorhombic crystals similar to the beta-prime polymorph of triacylglycerols (Small, 1984). Semisolid and solid triacylglycerol based ingredients display a more complex crystallisation behaviour including the alpha-, beta-prime and beta forms. Many hydrogenated vegetable fats, such as hydrogenated soybean oil and hydrogenated palm oil, as well as the lauric oils, coconut oil, and palm kernel oil are generally stable in the beta-prime form. Many of the exotic “butters” used in cosmetic applications — cocoa butter, shea butter,

Solid phase content

First of all, the solid phase content is directly correlated with the consistency and sensory properties of the product.

Solid phase content (%)

Polymorphism

80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0

Soft emollient Waxy emollient

5

10

15

20

25

30

35

40

45

50

Temperature (°C)

FIGURE 9.4 Solid phase content as a function of temperature for two typical emollient blends: (a) an emollient with soft, plastic consistency at room temperature and good stability at elevated temperatures, and (b) an emollient with hard, waxy texture at room temperature and almost complete melting at body temperature. Solid phase content in the mixtures was determined by low resolution pulsed NMR after tempering at 26°C for 40 hours.

605

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Cosmetics and personal care products

9.4.6

shorea butter and so on — crystallise in the triple-chain packed beta form. These butters normally require a good control of the crystallisation conditions (“tempering”) when used in higher concentrations if problems with the storage stability are to be avoided. When mixing ingredients from different groups, the resulting complex polymorphic behaviour can strongly influence the stability and sensory properties of the formulation. The best results in both aspects are obtained if the polymorphic behaviour of the system is matched; all ingredients should be either in the beta prime or in the beta modification. For further discussion of polymorphism, see Section 7.2.4.

9.4.5

The combination of polymorphic behaviour and response on cooling will determine the morphology of the crystals produced. Depending on the shape and size distribution of the crystals, the consistency can be softer or harder. Needle-like crystals, characteristic of the beta-prime polymorphs of fats and wax esters, will give a higher degree of elasticity compared to the more regularly shaped crystals associated with the beta polymorphs. Shear during cooling will also influence the shape and the size distributions of the particles. Rapid cooling can sometimes lock the particles in an unstable polymorph that will slowly transform on storage, resulting in undesired consistency changes. Both polymorphism and the solid phase content are dependent on the liquid phase used in the formulation. The solubility of the fats and waxes used will be different in different emollients and unexpected effects on product consistency and stability can be seen when the emollient composition is changed. In emulsions, the emulsion droplet sizes can also influence both solid phase content as well as polymorphic forms. If the emulsion droplets are small enough, a super-cooling of the oil phase can sometimes be observed with unstable crystal forms such as alpha being more stable. In anhydrous formulations, such as balms and ointments, as well as colour cosmetics (lipsticks, pencils, mascaras and foundations), the compatibility between the solid phases becomes very important for product performance (Matsuda and Yamaguchi, 2001). Co-crystallising solids that cover a wide melting point range can give very stable formulations with excellent consistency, stability, and skin feel.

Crystallisation kinetics

The response of a formulation to processing, especially cooling conditions, is dependent on the inherent crystallisation behaviour of the mixture of solids used in the product. Cooling conditions available in cosmetic product manufacturing frequently are not designed for rapid cooling, a fact the formulator needs to consider when selecting ingredients. Control of cooling conditions is also relevant when considering scale-up effects and product stability during storage. The most obvious influence of cooling conditions is on product consistency. In most systems, rapid cooling results in a massive burst of crystal nuclei that will have a limited potential of growth, resulting in small crystals and a harder consistency (Figure 9.5). The surface of the product is smooth and glossy if the crystals are small. Slow cooling results in fewer crystal nuclei that will grow to large sizes with time. Such systems are usually softer in consistency and spread more easily. However, uncontrolled growth can also lead to a grainy product with obvious “bloom” on the surfaces.

Penetrometer hardness (g)

500

Combined effects

Fast crystallisation Slow crystallisation

400

300

200

100

0 5

10

15

20

25

30

Solid fat content @ 22 C (%)

FIGURE 9.5 Hardness of emollient mixtures crystallised at different conditions: (a) rapidly cooled from melt to 20°C, cooling rate 10°C/minute, and (b) slowly cooled from melt to 20°C, cooling rate 0.3°C/minute. Solid phase content in the mixtures was determined by low resolution pulsed NMR at 20°C after tempering for 2 hours.

606

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9.4.7

Bioactive lipids in skin-care applications

(4 to 17%), cholesterol sulfate (1.5 to 4.5%), and triacylgly-cerols (5 to 25%) (Engblom, 1996).

The skin is the largest organ of the human body, covering about 1.5 to 2 m2, presenting a total thickness of less than 2 mm in most locations and weighing about 4 kg in adults. It is the only organ completely exposed to the environment, making the major role of the skin that of a barrier against air, microorganisms, and environmental pollutants. The outermost layer, stratum corneum or the horny layer, consists of cells named corneocytes embedded in a lipid matrix. The lipids form an intercellular lamellar sheet between the cornified cells and constitute the primary barrier of the skin. The structure and function of the stratum corneum is extensively studied and several models for the interaction between the epidermal lipids and the corneocytes have been proposed, including the “brick-andmortar” of Michaels (Michaels, Chandrasekaran, and Shaw, 1975), the “domain mosaic model” of Forslind (Forslind, 1994), and more recently the “single gel phase” model of Norlén (Norlén, 2001). A schematic cross section through human skin is shown in Figure 9.6. The outermost part of the epidermis is normally considered as the main target for cosmetic and personal care products. For optimal function, the skin requires a selection of different lipids. There are two main types of skin lipids; on the skin surface, there is the sebum, generated in the sebaceous glands and dominated by triacylglycerols, wax esters, and squalene. The epidermal lipids, which are generated in the epidermal cells, are essential for maintaining the integrity of the skin barrier by preventing the penetration of impurities, chemicals, microorganisms, and water, while also protecting against undesirable water loss through the skin. The main components of the epidermal lipids are ceramides (15 to 41%), free fatty acids (7 to 23%), cholesterol (13 to 34%), cholesterol esters

9.4.8

Lipids in skin care

Lipids are important ingredients in all skin-care categories and of special importance for dry and sensitive skin, and for antiaging and protecting skin-care formulations. Apart from acting as emollients, many lipids also function as delivery agents for various bioactive materials in the formulations. Dry-skin conditions have become a widespread problem in many parts of the world mainly due to life-style changes, including altered dietary patterns, changes in workplace conditions, and a comparatively older population. Dry skin is characterised by a reduced content of water and an altered lipid composition in the stratum corneum. A defective skin barrier results in increased water evaporation and an increased sensitivity to the environment. Thus, an ideal skin-care formulation should contain ingredients that improve barrier function and repair as well as supplement the natural epidermal lipids (Loden and Maibach, 2005; Park, 2001).

9.4.9

Essential fatty acids

Essential fatty acids are those polyunsaturated fatty acids (PUFA) that are necessary for good health, but cannot be synthesized in the body. Dry and atopic skin shows a decrease in linoleic acid content, the important precursor of ceramides essential for the barrier function of the skin (Horrobin, 2000). Both topical application and dietary intake of essential fatty acids have been shown to restore dry skin conditions as well as having therapeutic effects on skin disorders, such as atopic dermatitis, psoriasis, and

Intercellular lipid barrier

Stratum corneum Stratum granulosum Stratum spinosum

Epidermis hickness 0.05-1 mm Corneocytes form in stratum basale and migrate to the stratum corneum Dermis hickness 3-5 mm Hair follicles and growing hair Sebaceous glands produce sebum Sweat gland Fibroblasts producing structural proteins Blood vessels, nerves

FIGURE 9.6

Schematic representation of skin structure.

607

9.4

Cosmetics and personal care products

acne (Conti, 1996; Spector, 1999). Common vegetable oils with a high content of essential fatty acids, such as linoleic acid, that can be used as emollients in skin-care products, are products derived from rapeseed, sunflower, corn, soybean, arachis (groundnut, peanut), and cottonseed. Another polyunsaturated fatty acid – γ-linolenic acid – has also shown potential for treating dry skin conditions caused by atopic dermatitis. γ-Linolenic acid metabolism leads to antiinflammatory prostaglandins that can ameliorate the effects of inflammatory skin disorders (Horrobin, 2000; Ziboh, Miller, and Cho, 2000). Oils derived from evening primrose, borage, blackcurrant, and echium are known for their high content of γ-linolenic and stearidonic fatty acids (Section 2.3.1.5). Due to the high sensitivity of polyunsaturated fatty acids with respect to oxidation, the addition of an optimised antioxidant system during oil processing is recommended. The added antioxidant will protect the oil during storage, but also contribute to the protection of the formulation.

9.4.10

acid, are shown to offer significant antioxidant and membrane stabilising properties in human skin (Kitazawa et al., 1997; Shindo et al., 1994). In addition, α-tocopherol has been demonstrated in vitro to reduce the age-dependent increase of collagenase activity, potentially delaying the progression of skin aging (Ricciarelli et al., 1999). The antiinflammatory action of γ-tocopherol has been demonstrated in dietary studies (Jiang et al., 2001; Jiang et al., 2000;, Jiang and Ames, 2003). The combination of α- and γ-tocopherol found in many seed oils is interesting for the dual activity these oils can have when used as emollients in topical applications. These nontoxic and biologically active substances are present at varying quantities in vegetable oils. Typical compositions for some common oils are given in Chapter 2.

9.4.11

Sterols and triterpene alcohols

Sterols (desmethylsterols), triterpene alcohols (4,4-dimethylsterols) and their derivatives such as hormone and vitamin precursors impart various important biological functions within the body. Desmethylsterols are commonly found in vegetable oils in concentrations ranging from 0.1 to 1% (Section 2.2.19; Table 2.47). The predominant sterol in seed oils is normally beta-sitosterol, but other sterols can also be found in significant amounts in specific plants. Triterpene alcohols are normally present in much lower concentrations in seed oils. Uniquely, high concentrations of triterpene alcohols are found in shea butter, the fat extracted from the kernels of the shea tree (Vitellaria paradoxa or Butyrospermum parkii) (Peers, 1977), growing in the arid regions of subSaharan Africa.

Tocopherols

The human skin is rich in lipids, proteins and DNA, all of which are extremely sensitive to oxidation and a proper protection against oxidation is necessary for the health of the skin (Kohen, 1999; Rengarajan, 1999). Beside the obvious oxidative damage to the skin constituents, free radicals and reactive oxygen species can initiate inflammatory reactions and activate matrix metalloproteinases (Brenneisen, Sies, and Scharffetter-Kochanek, 2002). Free radicals are closely linked with aging and oxidative stress in the skin, being associated not only with decreased cell viability and DNA damage, but as a significant agent in the skin-aging process when the skin loses its elasticity and regenerative power. It, therefore, is essential to protect the skin against the effects of UV radiation and oxygen-derived free radicals and many formulation strategies to achieve this have been developed during the past decades. The lipophilic tocopherols are essential for the stabilisation of biological membranes, especially those containing large amounts of polyunsaturated fatty acids. α-Tocopherol is particularly effective in protecting against oxidative damage of cellular membranes and biomolecules, such as lipids, proteins, and nucleic acids (Kohen 1999, Nachbar and Korting, 1995; Thiele et al., 2001). The tocopherols act as antioxidants primarily by a free radical scavenging mechanism. It has been shown to be of obvious physiological advantage to deliver tocopherols topically, which can ameliorate the early phase of an oxidative stress response. For example, topical application of vitamin E is shown to reduce the appearance of fine facial lines and wrinkles as well as increasing stratum corneum hydration and enhancing its water-binding capacity (Gehring, Fluhr, and Gloor, 1998). Synergistic effects between tocopherols and other antioxidants, such as flavonoids and ascorbic

9.4.12

Antiinflammatory effects of sterols and tocopherols

Phytosterols from rapeseed oil have been shown to impart antiinflammatory and healing effects on surfactant damaged skin (Loden and Andersson, 1996). The benefits using the combination of tocopherols and phytosterols in an emollient were demonstrated in a clinical study using rapeseed oil fractions with different levels of sterols and tocopherols. It was shown that a pretreatment with rapeseed oil fractions reduced the transepidermal water loss (TEWL) and erythema in skin treated with sodium lauryl sulfate solutions. The reference materials were petrolatum, an inert occlusive emollient, borage oil, which is rich in γ-linolenic acid, and a hydrocortisone cream, a known antiinflammatory preparation. The fractionated rapeseed oil showed activity comparable to the hydrocortisone cream, while no effects were observed with petrolatum and borage oil. Phytosterols are also known for having a structural role by interacting with the lameIlar lipid layers, strengthening the lipid barrier, and improving dry skin conditions, squamation, and erythema (Chlebarov, 1989). 608

Nonfood Uses of Oils and Fats

9.4.13

Bioactivity of triterpene alcohols

lupeol and its palmitate and linoleate were inhibitors of trypsin activity, while no effect on porcine pancreatic elastase was observed. Metalloproteases (e.g., collagenase) and serine proteases (trypsin, chymotrypsin, porcine pancreatic elastase, human leukocyte elastase) are inhibited in vitro by various types of triterpenes, including lupeol and its esters. Rajic et al. (2000) showed that esterification increases the degree of inhibition of trypsin and chymotrypsin. Lupeol palmitate, lupeol linoleate, and alpha-amyrin linoleate were potent trypsin inhibitors, while free lupeol and alpha-amyrin were less efficient. Chymotrypsin was inhibited by lupeol, the other tested compounds being weaker inhibitors. These examples show that there is a potentially useful effect of the triterpene alcohols from shea butter to prevent aging effects on the skin by inhibiting the degrading activity of proteases. Triterpene derivatives are also known for stimulating collagenesis of skin fibroblasts (Laugel, 1998), indicating a second mechanism for delaying skin aging and changes due to lowered content of the structural proteins.

There have been many bioactivity studies conducted with various types of triterpene alcohols. The studies performed with these alcohols indicate that there are at least two areas where interesting bioactive effects can be expected. The antiinflammatory effects of phytosterols, including the triterpene alcohols, are well demonstrated for both the free alcohols and their esters. The second effect is associated with the synthesis and degradation of the structural proteins collagen and elastin. Many individual triterpene alcohols and their natural mixtures have been investigated for their antiinflammatory properties. Several studies show that the two amyrins as well as lupeol and butyrospermol are antiinflammatory in different types of inflammation models. For example, Akihisa et al. (1997) presented data on the antiinflammatory effect of a large number of triterpene alcohols found in Theaceae oils (Camellia and Sasanqua), including butyrospermol, lupeol, alpha- and beta-amyrin, as well as taraxasterol, psitaraxasterol, and 24-methylenedammarenol (all of them also found in high concentrations in shea butter). All of these triterpene alcohols (in the form of free alcohols) showed inhibitory activities in the same concentration range as the control substance indomethacin, when tested in an inflammation model in mice. The mechanism for the inflammatory action of lupeol and its esters was investigated by Fernandez et al. (2001). The antiinflammatory activity of lupeol was studied in models demonstrating effects on two different inflammatory pathways. Topically applied lupeol had a significant antiinflammatory effect in the TPA model (cyclooxygenase pathway), while the effect was less pronounced in the arachidonic acid-induced oedema (lipoxygenase pathway), only the highest concentration tested gave any significant effect. It was concluded that lupeol is an inhibitor of certain proinflammatory mediators, such as prostaglandin E2 (a cyclooxygenase metabolite) and cytokines, but not leukotrienes (lipoxygenase metabolites). Some of the triterpene alcohols found in shea butter are also inhibitors of protein degrading enzymes, proteases. Different types of proteases are active in the skin, degrading collagen and elastin, two of the major structural proteins contributing to the toughness and strength of the skin. The production of collagen and elastin decreases with increasing age, resulting in thinner and less elastic skin. The effects of this natural aging process can be alleviated by stimulating collagen and elastin synthesis or by inhibiting the activity of the collagenases and elastases. Proteases are also implicated in the breakdown of connective tissue in rheumatoid arthritis and the triterpene alcohols and their derivatives have been investigated as alternatives to conventional pharmaceutical products, such as hydrocortisone and indomethacin. Several studies have been conducted to evaluate the inhibitory effect of triterpene alcohols on different types of proteases. For example, Hodges et al. (2003) showed that

9.4.14

Products enriched with minor lipids

Normally the natural levels of triterpene alcohols, sterols, and tocopherols are too low for having extensive effect on topical application. Vegetable oils with naturally high contents of sterols include wheat germ oil, rapeseed oil, and soybean oil. Shea butter, avocado oil, rice bran oil, and olive oil also contribute to high levels of triterpene alcohols (4,4-dimethylsterols). The naturally occurring levels of these minor components can be increased by different processing methods including saponification followed by extraction and distillation (Clark, 1996). A nondestructive way of isolating these components is based on low-temperature solvent fractionation (Alander et al., 2005; Mellerup, Bach, and Enkelund, 2002). These concentrated fractions can be used as part of the emollient system in a cosmetic formulation to deliver the bioactive minor components to the skin.

References Akihisa, T. et al. 1997, Triterpene alcohols from camellia and sasanqua oils and their anti- inflammatory effects, Chem.Pharm.Bull.(Tokyo), 45, 2016–2023. Alander, J. et al. 2005, Fractionation process, EP 1084215, Karlshamns AB, (patent). Brenneisen, P. et al. 2002, Ultraviolet-B irradiation and matrix metalloproteinases: from induction via signaling to initial events, Ann. N.Y. Acad. Sci, 973, 31–43. Chlebarov, S., 1989, Notabene Medici, vol. 2 and 3. Clark, J.P. 1996, Tocopherols and sterols from soybeans, Lipid Technol., 8, 111–114. Conti, A., 1996, Seasonal influences on stratum corneum ceramide I fatty acids and the influence of topical essential fatty acids, Int. J. Cosmet. Sci., 18, 1–12. Engblom, J., 1996, On the phase behaviour of lipids with respect to skin barrier function, Department of Food Technology, Lund University, Lund, Sweden.

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Lubricants

protein kinase C inhibition, Free Radic. Biol. Med., 27, 729–737. Shindo, Y. et al. 1994, Enzymic and non-enzymic antioxidants in epidermis and dermis of human skin, J. Invest. Dermatol., 102, 122–124. Small, D.M., 1984, Lateral chain packing in lipids and membranes, J. Lipid Res., 25, 1490–1500. Spector, A.A., 1999, Essentiality of fatty acids, Lipids, 34, S1–S3. Thiele, J.J. et al. 2001, The antioxidant network of the stratum corneum, Curr. Probl. Dermatol., 29, 26–42. Wiechers, J.W., 2003, The EEC concept: how emollients and emulsifiers work together to create more efficacious cosmetic products, SÖFW-J., 129, 2225. Wiechers, J.W. et al. 2004, Formulating for efficacy, Int. J. Cosm. Sci., 26, 173–182. Ziboh, V.A. et al. 2000, Metabolism of polyunsaturated fatty acids by skin epidermal enzymes: generation of antiinflammatory and antiproliferative metabolites, Am. J. Clin. Nutr., 71, 361S–366S.

Fernandez, M.A. et al. 2001, New insights into the mechanism of action of the anti-inflammatory triterpene lupeol, J. Pharm. Pharmacol., 53, 1533–1539. Forslind, B., 1994, A domain mosaic model of the skin barrier, Acta Derm. Venereol., 74, 1–6. Gehring, W. et al. 1998, Influence of vitamin E acetate on stratum corneum hydration, Arzneimittelforschung., 48, 772–775. Hodges, L.D. et al. 2003, Antiprotease effect of anti-inflammatory lupeol esters, Mol. Cell Biochem, 252, 97–101. Horrobin, D.F., 2000, Essential fatty acid metabolism and its modification in atopic eczema, Am. J. Clin. Nutr., 71, 1 Suppl, 367S–372S. Jiang, Q. and Ames, B.N., 2003, Gamma-tocopherol, but not alpha-tocopherol, decreases proinflammatory eicosanoids and inflammation damage in rats, FASEB J., 17, 816–822. Jiang, Q. et al. 2001, Gamma-tocopherol, the major form of vitamin E in the U.S. diet, deserves more attention, Am. J. Clin. Nutr., 74, 714–722. Jiang, Q. et al. 2000, Gamma-tocopherol and its major metabolite, in contrast to alpha-tocopherol, inhibit cyclooxygenase activity in macrophages and epithelial cells, Proc. Natl. Acad. Sci. U.S.A., 97, 11494–11499. Kitazawa, M. et al. 1997, Interactions between vitamin E homologues and ascorbate free radicals in murine skin homogenates irradiated with ultraviolet light, Photochem. Photobio., 65, 355–365. Kohen, R., 1999, Skin antioxidants: their role in aging and in oxidative stress—new approaches for their evaluation, Biomed. Pharmacother., 53, 181–192. Laugel, C., 1998, Incorporation of triterpenic derivatives within an o/w/o multiple emulsion: structure and release studies, Int. J. Cosmet. Sci., 20, 183–191. Loden, M. and Andersson, A.C., 1996, Effect of topically applied lipids on surfactant-irritated skin, Br. J. Dermatol., 134, 215–220. Loden, M. and Maibach, H.I., 2005, Dry Skin and Moisturizers: Chemistry and Function, 2nd ed., CRC Press, Boca Raton, FL. Matsuda, H. and Yamaguchi, M., 2001, Separation and crystallization of oleaginous constituents in cosmetics —sweating and blooming, in Crystallization Processes in Fats and Lipid Systems, Garti, N. and Sato, K., Eds, Marcel Dekker, New York, pp. 485–503. Mellerup, J. et al. 2002, A process for preparing vegetable oil fractions rich in non-tocolic, high-melting, unsaponifiable matter, WO0250221, Aarhus Oliefabrik A/S, (patent). Michaels, A.S. et al. 1975, Drug permeation through human skin; theory and in vitro experimental measurement, AIChE J., 21, 985–996. Nachbar, F. and Korting, H.C., 1995, The role of vitamin E in normal and damaged skin, J. Mol. Med., 73, 7–17. Norlen, L., 2001, Skin barrier structure and function: the single gel phase model, J. Invest. Dermatol., 117, 830836. Park, W.S., 2001, Improvement of skin barrier function using lipid mixtures, SÖFW-J., 127, 9. Peers, K.E., 1977, The non-glyceride saponifiables of shea butter, J. Sci. Food Agric., 28, 1000–1009. Rajic, A. et al. 2000, Inhibition of serine proteases by antiinflammatory triterpenoids, Planta Medica, 66, 206–210. Rengarajan, H., 1999, Skin delivery of Vitamin E, J. Cosmet. Sci., 50, 249–279. Ricciarelli, R. et al. 1999, Age-dependent increase of collagenase expression can be reduced by alpha-tocopherol via

9.5

Lubricants

9.5.1

Introduction

Lubrication is the use of a material to improve the smoothness of movement of one surface over another. The material used to achieve this is called a lubricant. These are usually liquids or semiliquids, but may be solids or gases or any combination of solids, liquids, and gases. Smoothness of movement is improved by reducing friction. However, this is not always the case, and there may be situations in which it is more important to maintain steady friction than to obtain the lowest possible friction. In addition to simply lubricating the metal parts that come in contact, lubricants are expected to reduce or control friction between metal parts to save energy, reduce wear or prevent weld of metal surfaces, clean metal surfaces of dirt or sludge to prevent scratching or scoring, clean metal surfaces of water and acids to prevent corrosion, and often to prevent overheating. Annual consumption of oil-based lubricants in the U.S. is close to 10 million metric tons and valued at more than $8 billion (USD). The U.S. usage accounts for 27% of the world lubricant consumption (37.5 million metric tons). With a share of almost a third of global lubricant consumption, Asia-Pacific remains the leading lubricant region, followed by Europe and North America. More than 70% of total lubricant volume is used in motor oils for automotive engines and approximately 10% in hydraulic fluids. Other application areas, mostly industrial lubricants, are less significant. Lubricants are usually divided into four basic classes. 9.5.1.1

Liquid lubricants or oils

Liquid lubricants cover mineral oils, fatty oils, synthetics, emulsions, or even process fluids. Mineral oils are most often used as the base stock in lubricant formulation. Synthetic oils (synthetic esters, phosphate esters, silicones, and fluorocarbons) are used for lubricants, which are 610

Nonfood Uses of Oils and Fats

expected to operate in extreme conditions, i.e., high performance aircraft, missiles, and in space. Vegetable oils are used in formulating lubricants intended for the food and pharmaceutical industries, but even in these applications their use is quite limited. The advantages and disadvantages of oils stem from their ability to flow easily. Thus, on the credit side, it is very easy to pour them from a container, to feed them into a bearing by dripping, splashing or pumping, and to drain them out of a machine when no longer fit for use. Other advantages are the cooling of a bearing by carrying away heat, and cleaning it by removing debris. 9.5.1.2

lubricant will not flow at all. Similarly, the advantages and disadvantages of gas lubricants are like the extremes of oils, where the flow properties are almost too good. 9.5.1.4

The gas used in gas bearings is generally air, but any gas can be used so long as it does not attack the bearings or decompose.

9.5.2

Lubricant base oils

Lubricants are made from a base oil (80 to 100%) and suitable additive package. The additives are used to enhance the most important properties for each specific application. Most base oils originate from petroleum, including many synthetic esters and poly-alpha-olefins. Less than 2% of the base oils are the product of oleochemical and related industries. The primary area of their application has been as hydraulic fluids. The various base oils used in lubricant formulations are described below.

Greases

Greases are more than very viscous lubricating oils. Technically they are oils, which contain a thickening agent to make them semisolid. Thus, grease consists of oil constrained by microscopic thickener fibres to produce a stable and colloidal structure or gel. Greases contain three basic active ingredients: a base oil, additives, and thickener. The base oil may be mineral, synthetic, or vegetable oil. For thickeners, metal soaps and clays are mostly used apart from some nonsoap thickeners, which are inorganic (silica and bentonite clays) or organic (polyurea) materials. Metal soaps are prepared by heating fats or oils in the presence of an alkali, e.g., NaOH. Fats and oils can be animal or vegetable origin, and are produced from cattle, fish, castor bean, coconut, cottonseed, etc. The reaction products are soap, glycerol, and water. Soaps are very important in the production of greases. The most commonly used soap-type greases are calcium, lithium, aluminum, and sodium. In most cases the oil plays the most important role in determining the grease performance, but in some instances the additives and the thickener can be critical. Very often additives, which are similar to those in lubricating oils, are used. The behavior of greases is very similar to that of oils, but the former are used where the advantages of easy flow are outweighed by the disadvantages. Greases do not easily leak out of a machine or container, do not migrate away, and will form an effective seal against contaminants. 9.5.1.3

Gases

9.5.2.1

Mineral/petroleum oils

Since mineral oils are the most widely used lubricating oils, they are often the standard with which other oils are compared. Mineral oils are generally oils obtained from petroleum, although they may come from similar sources, such as oil shales and tar stands. The mineral oils used for lubrication were originally distilled fractions with suitable viscosity for lubrication. But now they are obtained through various steps of refining and extraction before being blended with specialty chemicals, called additives, to enhance existing performance characteristics. Mineral oils are mainly hydrocarbons of three basic types: paraffins, naphthenes, and compounds containing aromatic systems. Finally, there is usually a small proportion (~2%), containing aromatic ring systems. In addition to these hydrocarbons, there may be small quantities of compounds containing other elements, such as oxygen, sulfur, phosphorus, or nitrogen. Mineral base oils used for lubricants are generally molecules with 20 to 50 carbon atoms. Mineral base oils continue to be economical and provide superior performance characteristics in various applications, but they present a potential hazard because they are not readily biodegradable and are environmentally toxic. During the past few decades, the level of public awareness of environmental issues has risen considerably and materials that do not meet accepted standards of biodegradability are disapproved of by environmentalists and government bodies. It is believed that federal directives in the U.S. will be strictly imposed in the next 2 to 5 years, eventually resulting in newer regulations on the development and application of environmentally friendly base stocks.

Solids or dry lubricants

The lubricants used in solid form may be bulky solids, paint-like coatings, or loose powders interposed between two surfaces in moving contact. Depending upon the nature of the two surfaces, a wide variety of solid materials can reduce friction and prevent seizure. For example, dust, sand, or gravel on the surface of a road can cause vehicles to skid because they decrease friction between tires and the road surface. The majority of solid lubricant applications are met by only three materials: graphite, molybdenum disulfide, and PTFE (polytetrafluoroethylene). The advantages and disadvantages of solid lubricants are rather like the extremes for greases, where the 611

9.5

Lubricants

9.5.2.2

Synthetic fluids

different. Tocopherols have important antioxidant properties. They are a series of benzopyranols with 1, 2, or 3 methyl groups attached to the phenolic ring along with a C16 side chain on the pyran ring. Antioxidant activity as a result of tocopherol content is high in corn oil, soybean oil, walnut oil, and cottonseed oil (Rossell et al., 1991 and Section 8.1). Fully refined oils (refined, bleached, and deodorized) have free fatty acid contents of less than 0.1% (normally 0.01 to 0.05% generally expressed as oleic acid). The quality of crude oils largely depends on the content of free fatty acids and quality generally deteriorates as the acid content rises. Free fatty acids are produced by hydrolysis of oils catalyzed by acids or enzymes.

Many of the alternatives to mineral oils are synthetic materials manufactured from various feed stocks by chemical processes. There are several types of synthetic oil, which differ from each other in performance and properties. Widely used synthetic oils include hydrocarbons, diesters, polyol esters, phosphate ester, silicones, polyglycols, polyphenyl ethers and perfluoroalkyl polyethers. Out of these fluids, low molecular weight polyalphaolefins (PAO 2, PAO 4, essentially 20:1 and 10:1 mixtures of hydrogenated dimers and trimers of αdecene), dialkyl adipates (isodecyl, isotridecyl), or polyol esters (mostly neopentyl glycol or trimethylol propane with fatty acids) are biodegradable synthetic base oils (Rudnick, 2002). The most important are PAOs, which are branched-chain paraffins, and resemble highly refined mineral oils in their structure, properties, and performance. Their inherent oxidation resistance is good, but their boundary lubrication is not as good as that of the highly refined mineral oils. Synthetic oils offer improved performance but at a price. Most of the esters are biodegradable and offer superior thermal and oxidative stability. Prices for these niche products are higher than vegetable oils and significantly higher than petroleum base stocks. Although specialized synthetic lubricants have been successfully replacing mineral oil in various applications for many years, general-purpose synthetic lubricants have only recently been introduced on a large scale. They are generally more expensive, but have better oxidation and thermal resistance than mineral oils. Low resistance to oxidative degradation and poor low temperature behavior of vegetable base oils have triggered the development and rise in demand for biodegradable synthetic base stocks. 9.5.2.3

9.5.2.3.1 Economics and availability Until recently, mineral oil had a significant cost advantage over vegetable oils and so petroleum has been the base oil of economic choice. Recent rises in oil prices along with the low vegetable oil prices has narrowed the price difference to close to $0.05/lb (0.11/kg), and there is now more interest in vegetable oils base stocks (Table 9.11). Though most lubricants used currently originate from petroleum base stocks, vegetable oils have seen a promising increase as biodegradable fluids over the last decade. Environmental concerns as well as economics and performance issues will drive the market share for these oils and government legislation may force this issue. Today, less than 2% of the base stocks are products of the oleochemical and related industries with the primary area of their application in hydraulic fluids, which have the highest need for biodegradable lubricants (Padavich et al., 1995). This is consumed at approximately 5MMT/ year in the U.S. market. Because soybean oil provides nearly 80% of the seed oils produced annually in the U.S. and is the cheapest vegetable oil in the U.S. market, its relatively low cost and dependable supply make it one of the more important sources of lubricant base oil in the U.S. Another commonly used vegetable oil in lubricant applications is rapeseed oil due to its relatively good oxidative stability

Natural oils

These include vegetable oils and animal fats. They are usually excellent boundary lubricants, but they are much less stable than mineral oils, and tend to break down to give sticky deposits. Vegetable oils are used in various industrial applications, such as emulsifiers, lubricants, plasticizers, surfactants, plastics, solvents, and resins. The natural oils are mainly triacylglycerols (98%), diacylglycerols (0.5%), free fatty acids (0.1%), sterols (0.3%), and tocopherols (0.1%). The fatty acids exist mostly as esters of glycerol. They have a carbon chain length of 12 to 24 carbon atoms. The predominant carbon chain length of the fatty acids from plants is 18 carbon atoms. They are the fully acylated derivative of glycerol. The resulting structure is abundantly present in vegetable oils, and resembles a tuning fork in shape. Vegetable oils have 6 oxygen atoms and around 60 carbon atoms per molecule, compared to an average of 30 carbon atoms in mineral base oils. The fatty acid constituents of triacylglycerol molecules may be all identical (e.g., triolein in olive oil and tripalmitin in palm oil), two different, or all

TABLE 9.11

Cost of various base oils in 2004

Base Oils

Cost ($/lba)

Cost ($/kg)

0.22–0.25 ~0.29 0.45–0.55

0.48–0.55 0.638 0.99–1.21

~1.00 ~1.25 ~0.90

~2.20 ~2.76 ~1.98

~0.25

~0.55

Vegetable oils Soybean oil Canola oil High-oleic sunflower oil Synthetic oils TMPb trioleate TMPb trioleate (high-oleic) PAO8c Mineral base oil (Group I and II) a

b c

612

Price will vary based on quantity, customer supplier relationships, and market conditions. TMP, trimethylolpropane. PAO, polyalphaolefin.

Nonfood Uses of Oils and Fats

therefore, are necessary to suppress or eliminate triacylglycerol crystallization and to improve oxidation stability. The inherent problems of poor low temperature performance and oxidation stability in vegetable oils can be partially improved by a variety of reactions at either the fatty acid carboxy groups or the hydrocarbon chain depending on end use applications. More than 90% of chemical modifications have been those occurring at the fatty acid carboxy groups, while less than 10% have involved reactions at fatty acid hydrocarbon chain. Without sacrificing favorable viscosity–temperature characteristics and lubricity, unsaturated vegetable oils can be converted into thermo-oxidatively stable products by saturation of carbon–carbon double bonds using alkylation, arylation, cyclization, hydrogenation, epoxidation, and other reactions. Chemical modifications at the carboxyl group of vegetable oils include transesterification, hydrolysis, etc. Reactions at double bond and carboxyl positions of vegetable oils are discussed by Erhan et al. (2005) and Hwang et al. (2002). With improvements in their low temperature performance and oxidation stability, they can be used in automotive and industrial lubricant applications with the additional advantage of being clean, biodegradable, nontoxic, and requiring lesser amounts of expensive additives (e.g., VI improvers are not required and lesser amounts of antiwear/antifriction additives are required).

compared to other vegetable oils, its reasonable cost, and its wide availability in Europe and North America (Whitby, 2004). Other vegetable oils used as lubricants include olive, sunflower, and castor oil. 9.5.2.3.2 Natural oil advantages Environmental concerns over the use of petroleum-based products in activities such as forestry, farming, mining, boating, and others, has led to increased interest in the use of environmentally friendly fluids. The beneficial aspects of vegetable oils as lubricants are mainly their biodegradability and nontoxicity, which are not exhibited by conventional mineral base oils (Randles et al., 1992; Battersby et al., 1989). Their volatility is low due to the high molecular weight triacylglycerol structure and they have a narrow range of viscosity change with temperature (high viscosity index, VI) and high flash point. Lower volatility results in decreased exhaust emission and high VI means the oil is a naturally multigrade oil. The high VI index of vegetable oils eliminates the need for the polymeric VI improvers used with mineral oils, resulting in high shear stability of vegetable oils. The ester linkages deliver inherent lubricity on metallic surfaces due to their adhesive property. Higher lubricity or lower friction results in more power and better fuel economy. Vegetable oils also have superior solubilizing power for contaminants and additive molecules compared to mineral base fluids. The ester structures provide improved solvency for polar deposits and sludge containing worn metals. Further, vegetable oils have higher shear stability.

9.5.2.5

Monounsaturated fatty acids are more thermally stable than polyunsaturated fats and, therefore, are highly desired components in vegetable oils. Ideally, vegetable oils having high stability and low pour points contain only monounsaturated fatty acids. Advanced plant breeding and genetic engineering has enabled the development of vegetable oils with higher concentration of oleic acid and lower linoleic and linolenic acids. The oleic content of high-oleic varieties of rapeseed and soybean oil is 75 to 85%, while that of high-oleic sunflower oil is 80 to 92% (Whitby, 2004). The oxidative stabilities of these high-oleic oils are three to six times greater than normal vegetable oils. Specialty canola oil products that Monsanto (Monsanto Company, St. Louis, MO) expects to market include oil containing medium-chain fatty acids for lubricants as well as for nutritional and high-energy food products (Schmidt et al., 2005).

9.5.2.3.3 Natural oil disadvantages Performance limitations of vegetable oil base stocks are poor oxidative stability due to bis-allylic hydrogen atoms in the fatty acyl chain, deposit-forming tendency, low temperature solidification, and low hydrolytic stability. Oxidation results in increased acidity, corrosion, and viscosity and volatility of the lubricant. The inherently narrow viscosity range limits their use in various viscosity grades, especially at lower viscosities. The polar nature of triacylglycerols contributes to air entrainment and problems of foaming. On the other hand, parameters like lubricity, antiwear protection, load carrying capacity, rust prevention, foaming, demulsibility, etc., are mostly additive-dependent. Antioxidant additives (Becker et al., 1996) have limited capability to improve on oxidative stability; therefore, other approaches are required to improve the above characteristics. The performance limitations of vegetable oil base stocks can be overcome by genetic modification, chemical modification, processing changes, and development in additive technology. 9.5.2.4

Genetically modified vegetable oils

9.5.3

Lubricant additives

Lubricant additives are chemicals, nearly always organic or organo-metallic, that are added to oils in quantities of a few weight percent to improve the lubricating capacity and durability of the oil. Specific purposes of various lubricant additives are discussed below. Wear and friction improvers are used to improve the wear and friction characteristics by adsorption and

Chemically modified vegetable oils

Low temperature testing shows that vegetable oils solidify at –20°C on long-term exposure. Poor oxidative stability of vegetable oils is due to bis-allylic hydrogen atoms in the fatty acyl chain. Chemical modifications, 613

9.5

Lubricants

extreme pressure (EP) lubrication. This includes adsorption or boundary additives, antiwear additives, and extreme pressure additives. The adsorption or boundary additives in current use are mostly the fatty acids and the esters and amines of these fatty acids. Common examples of antiwear additives are zinc dialkyldithiophosphate, tricresyl phosphate, dilauryl phosphate, diethyl phosphate, dibutyl phosphate, tributyl phosphate, and triparacresyl phosphate. The most commonly used EP additives are dibenzyldisulfide, phosphosulfurized isobutene, and chlorinated paraffin, sulfurchlorinated sperm oil, sulfurized derivatives of fatty acids and sulfurized sperm oil, cetyl chloride, mercaptobenzothiazole, chlorinated wax, lead naphthenates, chlorinated paraffinic oils, and molybdenum disulfide. Antioxidants improve the oxidation resistance and, thus, prevent a gradual increase in the viscosity and acidity of oil. Widely used antioxidant additives are zinc dialkyldithiophosphate, metal deactivators, phenol derivatives, amines, and organic phosphates. Sulfur-based EP and antiwear additives are also quite effective as antioxidants. Sulfur and phosphorus in elemental form or incorporated into organic compounds are also effective as antioxidants and antiwear additives. To control the corrosion of metal parts, corrosion inhibitors and rust inhibitors are used. Corrosion inhibitors are used to protect the nonferrous surfaces of bearings, seals, etc., against corrosive attack by oxidation products and additives containing reactive elements, such as sulfur, phosphorus, iodine, or chlorine. The commonly used corrosion inhibitors are benzotriazole, substituted azoles, zinc diethyldithiophosphate, zinc diethyldithiocarbamate, and trialkyl phosphites. Rust inhibitors are used to protect ferrous components against corrosion. Widely used rust inhibitors are metal sulfonates (i.e., calcium, barium, etc.), amine succinates, or other polar organic acids. Contamination control additives restrict contamination by reaction products, wear particles, and other debris. The other possible contaminants are soot from inefficient fuel combustion, unburnt fuel, breakdown products of the base oil, corrosion products, dust from the atmosphere, organic debris from microbiological decomposition of the oil, etc. Without proper control of contamination, the oil will lose its lubricating capacity, become corrosive and become unsuitable for service. Additives, which prevent the development of all these detrimental effects, are known as “detergents” or “dispersants.” Mild dispersants are typically low molecular weight polymers of methylacrylate esters, long chain alcohols, or polar vinyl compounds. Over-based dispersants are calcium, barium, or zinc salts of sulfonic, phenol, or salicylic acids. Viscosity index (VI) improvers reduce excessive decrease of lubricant viscosity at high temperatures. They are usually high molecular weight polymers that are dissolved in the oil and change shape from spheroidal to linear as the temperature increases. Unfortunately these additives are easily degraded by excessive shear rates and

oxidation. Under high shear rates, VI improvers can suffer permanent or temporary viscosity loss. Pour point depressants (PPDs) enhance lubricant characteristics by reducing the pour point through interfering with the crystallization mechanism. PPDs do not prevent wax crystallization in the oil, but they are absorbed on the wax crystals and, thus, reduce the amount of oil occluded on the crystal. Reducing the crystal volume permits lubricant flow. Typically used PPDs are maleic anhydride-styrene copolymers and polymethacrylates. These can be used in mineral as well as vegetable oils, though a higher percentage is generally required in the latter. Antifoam agents/foam inhibitors include silicones and miscellaneous organic copolymers. The most common package of additives used in oil formulations contains antiwear and extreme pressure lubrication additives, oxidation inhibitors, detergents, dispersants, viscosity improvers, pour point depressants, and foam inhibitors.

9.5.4

Physico-chemical and performance properties of bio-based lubricant base oils

The physical and chemical properties of vegetable oils are determined mainly by the fatty acid (FA) profile. Table 9.12 shows the FA composition of some vegetable oils that are being used as potential lubricant base oils for industrial applications and also chemical properties associated with C = C unsaturation. High unsaturation in the triacylglycerol molecule (and particularly high levels of 18:2 and 18:3) increases the rate of oxidation resulting in polymerization and an increase in viscosity (Brodnitz, 1968). On the other hand, high saturation increases the melting range of the oil (Hagemann et al., 1972). Therefore, suitable adjustment between low temperature properties and oxidative stability must be made when selecting a vegetable oil base stock for a particular industrial application. The fatty acids can be saturated or unsaturated resulting in a straight chain or bent chain configuration, respectively. With the increase of double bonds in the chain, ability to gain a close packed conformation is prevented and, therefore, the oil remains liquid. The higher the IV, the more unsaturated (the greater the number of double bonds) the oil and, therefore, the higher is the potential for the oil to polymerize through oxidation. Any attempt to increase the saturation content of the oil through hydrogenation will increase the melting range temperature of the oil as shown in Table 9.13 (Swern, 1970). An IV of less than 25 is required if the neat oil is to be used for long-term applications in unmodified diesel engines. Triacylglycerols in the range of IV 50 to 100 may result in decreased engine life and, in particular, will reduce the life of fuel pump and injector. Most vegetable oils are unsuitable for lubricant applications due to their high saturated or polyunsaturated 614

Nonfood Uses of Oils and Fats

TABLE 9.12

Analytical data of vegetable oils used as lubricant base oils

Vegetable Oil

16:0

Soybean oil High-oleic soybean oil Sunflower oil High-oleic sunflower oil Safflower oil High-oleic safflower oil High-linoleic safflower oil Rapeseedc Corn oil Cottonseed oil a

b c

11.1 6.2 6.1 3.5 6.4 4.6 6.7 3.0 10.6 18.0

Fatty Acid Compositiona (%) 18:0 18:1 18:2 4.8 3.0 5.3 4.4 2.5 2.2 2.6 1.0 2.0 2.0

53.6 3.7 66.4 10.4 73.2 13.2 75.2 14.0 59.8 38.0

18:3 6.3 1.7 – – – – – 10.0 0.9 1.0

131.0 85.9 124.8 80.8 135.2 83.6 121.2 99.1 119.9 109.1

Gas chromatography analysis (16:0 palmitic, 18:0 stearic, 18:1 oleic, 18:2 linoleic, 18:3 linolenic), AACC Method 58-18, 1993. Iodine value (mg I2/g), AOCS method, Cd 1-25, 1993. Rapeseed oil also contains other fatty acids, such as 1% of 20:0, 6% of 20:1, and 49% of 22:1.

(Annual Book of ASTM Standards, 2000). The viscosity of any fluid changes with temperature, increasing as temperature decreases, and decreasing as temperature rises. Viscosity may also change with alterations in shear stress or shear rate. To compare base oils with respect to viscosity variations with temperature, ASTM Method D2270 provides a means to calculate a VI. This is an arbitrary number used to characterize the variation of kinematic viscosity of a base oil with temperature. The calculation is based on kinematic viscosity measurements at 40 and 100°C. For oils of similar kinematic viscosity, the higher the viscosity index, the smaller the effect of temperature. The benefits of higher VI are:

TABLE 9.13 Melting point and iodine values of some vegetable oils Oil Coconut oil Palm kernel oil Palm oil Olive oil Castor oil Rapeseed oil Cotton seed oil Sunflower oil Soybean oil Linseed oil

Iodine Value 18 16–19 54 81 85 98 105 125 130 178

Tristearin (18:0) Triolein (18:1) Trilinolein (18:2) Trilinolenin (18:3)

0 86 173 261

Approx. Melting Point (°C) 25 24 35 –6 –18 –10 –1 –17 –16 –24 74 5 –11 –24

1. Higher viscosity at high temperature, which results in lower oil consumption and less wear. 2. Lower viscosity at low temperature, which for engine oil may result in better starting capability and lower fuel consumption during warm-up.

fatty acid content. The oxidation stability of polyunsaturated fatty acids can be improved significantly by converting them to saturated fats; this, however, will cause the low temperature behaviour of the material to deteriorate. Optimally, vegetable oils with high oxidation stability and low pour points are the high oleic varieties. The oxidation stability of such oils is three to six times greater than that of conventional vegetable oils. Such oils will provide both high thermo-oxidation stability and reasonably low temperature flow properties. Bio-based lubricants that are accepted as environmentally friendly must pass bench tests designed to evaluate the potential performance as lubricants in addition to biodegradability and toxicity. 9.5.4.1

24.2 83.6 21.4 80.3 17.9 77.5 14.6 16.0 26.7 41.0

IVb

Other viscosity measurements are used to check properties at low or high temperatures. The Cannon minirotary viscometer is used to measure low temperature properties (yield stress and apparent viscosity) as per ASTM standard method D4684-97. ASTM method D4624-93 is used to determine if the test oils have the appropriate high-temperature characteristics required for an engine. This test is aimed at the performance of lubricants in the bearing systems of an engine. The viscosities of vegetable oils, synthetic biodegradable lubricant base oils polyalphaolefin (PAO), trimethylolpropane (TMP) ester, and adipate, and mineral base oil (Erhan et al., 2000) are compared in Table 9.14. The mineral oil is a nonbiodegradable base oil mostly used for formulations of automotive lubricants. Except for natural antioxidants, these fluids do not have any additives. The vegetable oils have excellent VI. The linear fatty acids contribute to the high VI because the molecule is generally long.

Viscosity

The primary consideration for any base oil to be used in lubricant formulation is its viscosity. Viscosities of base oils are mostly reported as kinematic viscosities measured at 40 and 100°C, according to ASTM (American Society for Testing and Materials) standard method D445-95 615

9.5

Lubricants

process when the microcrystalline structures initially formed become macrocrystalline and rapidly change to a solid-like consistency. This results in a rapid viscosity increase leading to poor pumpability, lubrication, and rheological behavior. Wax crystallization at low temperature is also controlled by steric and geometrical constraints in these molecules. The fatty acid chains of triacylglycerol molecule have a “tuning fork” conformation and undergo molecular stacking during the cooling process. Another problem with vegetable oils is the manner in which they solidify. Mineral oil, which is a mixture of short- and longbranched chains, solidifies a little at a time under cold conditions, displaying a cloud point when the first solid appears in the liquid, and a pour point when the apparent viscosity of the solid/liquid mix is too high to allow immediate gravity flow. Vegetable oil molecules are mostly of the same size, so they solidify at very nearly the same temperature, giving vegetable oils a freezing point rather than a pour point. This can result in oil solidification after more than few days in cold winter weather. In vegetable oil triacylglycerols, the presence of double bonds in the fatty acyl chain influences the low temperature behaviour as shown in Table 9.13. The decreased melting temperatures of these compounds are a result of disorganization of the crystalline lattice by the presence of double bonds. It has been firmly established (de Jong et al., 1991; D’Souza et al., 1991) that presence of cis unsaturation, lower molecular weights, and diverse chemical structures of triacylglycerols favour lower temperatures of solidification. This demonstrates the contradiction of having both low temperature properties and the best possible oxidative stability in a given triacylglycerol molecule. PPDs are used to suppress formation of large crystals during solidification, although the mechanism of PPD action on triacylglycerol crystallization remains undisclosed (Bentz et al., 1969). PPDs, like polymethacrylate, allow inclusion of the PPD molecule’s branches into the growing crystal (Erhan, 2004). The effect of PPDs on pour points of vegetable oils (Table 9.15) shows that an amount of 0.4% by weight of PPD significantly reduces rates of solidification. Increased amount of PPD may slow down solidification, but further depression ceases quite rapidly. Low temperature and cold storage properties of vegetable oils, thus, do not respond appreciably to the PPD, as opposed to mineral oils (Asadauskas et al., 1999) and have

TABLE 9.14 Viscosities and pour points of vegetable oil, synthetic oil, and mineral base oils KVa at 40°C, cST

KV at 100°C, cST

VIb

Pour Points, °C

ASTM D445

ASTM D445

ASTM D2270

ASTM D97

31.5 31.6 39 33 31.9 40.3 38.3 36.9 255.5

7.6 7.7

227 226

7.7 9.1 8.4 8.3 19.5

223 217 203 212 87

–9 – –12 –18 –15 –18 –9 3 –24

139 191

Vegetable oils Soybean Sunflower High-oleic sunflower Canola Corn Rapeseed Olive Peanut Castor Synthetic oils Diisotridecyl adipate TMPc trioleate PAO2d PAO4 PAO6 PAO8

27 46.8 5.54 16.8 31 45.8

5.4 9.4 1.8 3.9 5.9 7.8

129 138 140

–51 –39 –65 –70 –68 –63

Mineral oil

65.6

8.4

97

–18

a b c d

Kinematic viscosity. Viscosity Index. Trimethylolpropane. Polyalphaolefin (kinematic viscosity 2 mm2/s at 100°C).

9.5.4.2

Low temperature properties

Pour point measurement is most commonly used to check the low temperature properties of the oils. Pour points of various base oils are shown in Table 9.14. Overall, the data shows that low temperature properties of vegetable oils are inferior to those of synthetic base oils or even mineral oil. In vegetable oils, castor oil demonstrates PP notably lower than those of soybean, high-oleic sunflower, and canola oils, suggesting that hydrogen bonding between the hydroxy groups of ricinoleic acid interferes with the crystal growth. It must be noted that some fluids still pour after quite significant durations at slightly lower temperatures than their determined PP. A good example is castor oil, which pours after more than 24 hours storage at –25°C, although its PP appeared as –24°C in triplicate runs. Increasing molecular weight of fatty acids and full saturation contribute to the increase in PP, whereas cis unsaturation favors the decrease. The relatively poor low temperature flow properties of vegetable oils arise from the appearance of waxy crystals that rapidly agglomerate resulting in the solidification of the oil. A vegetable oil is a complex mixture; therefore, the transition from liquid to solid state occurs over a wide temperature range involving several polymorphic forms (α, β′, β) (Hagemann et al., 1983; Hagemann, 1988). Wax appearance and crystallization is a slow continuous

TABLE 9.15 Effect of PPD (polyalkylmethacrylate copolymer of 8000 amu, canola oil carrier 1:1.) on pour points (°C) of vegetable oils Amount of PPD % (w/w) Vegetable Oil Soybean High-oleic sunflower Canola

616

0

0.4

1

2

–9 –12 –18

–18 –21 –30

–18 –24 –33

–18 –24 –33

Nonfood Uses of Oils and Fats

shown unsatisfactory performance when exposed to low temperatures for longer durations (Antila et al., 1966). Studies show that diluents have a significant role in lowering the pour point of the vegetable oils (Asadauskas et al., 1999). However, high dilution does not necessarily translate to proportionate depression of pour point and no synergism exists between diluents and PPD molecules. During the cooling process, the response to diluents and PPD molecules is dependent to some extent on the vegetable oil FA composition and its geometry. Pour point determinations of safflower, high-oleic safflower, and high-linoleic safflower in the presence of diluent and PPD are shown in Table 9.16. The addition of a synthetic ester as a diluent to safflower and high-linoleic safflower oils showed a larger decrease in the pour point compared to the high-oleic oil. Due to the presence of multiple unsaturation in safflower and high-linoleic safflower oil, the triacylglycerol molecules encounter significant steric-hindrance from the “zigzag” nature of the FA chain during the cooling process. The presence of diluent molecules in the system enhances this effect by lowering the viscosity and by interfering with the stacking process during cooling. The addition of PPDs further lower the pour point. High-oleic and high-linoleic oils appear to show a better response in the presence of additive molecules. In addition to exhibiting good low-temperature behavior, base oils should be stable over extended time at low temperature to qualify for any industrial and automotive applications. Although high-oleic oils exhibit good thermaloxidative behaviour and acceptable PPD response, they fail in an industry-specified, low-temperature extended storage stability test. Table 9.16 shows the cold storage stability data of selected vegetable oils in the presence of diluents and PPD (Erhan et al., 2002). Using the optimized diluent and PPD concentration, safflower and high-linoleic safflower oils showed acceptable fluidity well beyond 7 days with some TABLE 9.16

loss in optical clarity. Therefore, to meet the viscometric properties of vegetable oil-based lubricants for engine applications, PPDs, synthetic hydrocarbons, and synthetic fluids have to be used in various combinations to produce base fluids meeting SAE 30, 5W-30, and 10W-30 viscosity requirements as shown in Table 9.17. These requirements can be met using a proper selection and combination of various vegetable oils along with PPD and synthetic oil diluent. All of the blends shown in the table contain antioxidants and antiwear additives and are compared with commercially available 5W-30 oil. Similar lubricants can be prepared using combinations of soybean, castor, canola, high-oleic oils, esters, and synthetic hydrocarbons. 9.5.4.3

Oxidation stability

Oxidation is the single most important reaction of oils resulting in increased acidity, corrosion, viscosity, and volatility when used as lubricant base oils for engine oils. A number of tests are used to evaluate the oxidation stability of lubricants (Booser, 1997). The more common choices for synthetic and vegetable oil fluids are the TOST test (dry) ASTM D943, Rotary Bomb Oxidation Test (RBOT) ASTM D2272, Modified Thin Film Oxygen Uptake Test (TFOUT), Pressurized Differential Scanning Calorimetry (PDSC) ASTM method D6186-98, and Penn State thinfilm Microoxidation (TFMO) test (Cvitkovic et al., 1979). Several benchtop oxidation tests are available as screening tools for oxidative stability of vegetable oils. Evaluation of oxidation is complex and a fully acceptable protocol has yet to emerge. Estimation of peroxide value (PV) can be used as an index of oxidation if the peroxides formed are stable and do not decompose after formation, which in most cases is not true. The activation energy for the formation of peroxide is 146-272 kJ/mol (Labuza, 1971) and that of decomposition of lipid peroxide is 84184.5 kJ/mol, suggesting peroxides are less stable than

Response of diluents and PPD on pour point and cold storage stability of vegetable oils Pour Point in °Ca

Fluid

Oil + Diluentb

Diluent + PPDc

Oil + Diluentb

Diluent + PPDc

–21 –21 –21

–39 –39 –27

–48 –45 –36

7+ 7+ 1

7+ 7+ 1

High-linoleic safflower oil Safflower oil High-oleic safflower oil a b c

ASTM D97. Oil:diluent ratio of 65:35 (vol/vol). Pour point depressants (PPDs) concentration of 1%.

TABLE 9.17

SAE requirements and properties of typical environmentally friendly lubricants

Lubricant Oil SAE requirements Rapeseed + castor High-oleic sunflower oil + PPD Corn oil + synthetic ester + PPD Commercial lubricant a

Number of Days at –25°C

Neat Oil

SAE Viscosity Grade

KV at 100°C

Pumping Viscositya (cP)

30 wt 10W-30 5W-30 5W-30

9.8–12.5 cSt 9.72 10.10 10.34 10.42

60,000 (max) Not required 4651 26,287 25,664

Measured using Cannon minirotary viscometer (MRV) as per ASTM D4684-97 method.

617

9.5

Lubricants

lipids (Swern, 1970). In the active oxygen method (AOM) (AOCS Official Method Cd-12-57, 1983), test oil is heated to 100°C and the oxidation is followed by measuring the PV of heated sample at regular time intervals until PV = 100 meq/kg is reached, which gives the AOM endpoint. A large amount of sample, numerous analysis, and critical control of airflow is required. With samples that form unstable peroxides, a PV = 100 meq/kg may never be reached and such measurements have no meaning. In the AOM method, consumption of O2 may also be a measure for induction period. The Rancimat method (Oxidationsstabilität, 1994; Laubli et al., 1988) is based on the fact that the volatile acids formed during oxidation (Loury, 1972; De Man et al., 1987) can be used for automated endpoint detection. Gordon and Mursi (1994) have shown good correlation of Rancimat results at 100°C with oil stability as measured by peroxide development during storage at 20°C. In another study, Jebe et al. (1993) pointed out the advantages of the Rancimat method at higher temperature. In the Sylvester test (Wewala, 1997), the sample is heated to 100°C in a closed vessel and pressure decrease due to O2 consumption is monitored. The Oxidograph (Wewala, 1997) is an automated version of this method and the induction period is determined from the sudden decrease in the O2 pressure. Oxidative status of oil can also be obtained by integrating the light curve during a chemiluminescence reaction (Matthäus et al., 1994). The method is highly sensitive for the measurement of lipid oxidation. Matthäus et al. (1993) described a linear correlation (R2 = 0.99) between the iodimetric peroxide determination (DGF Einheitsmethoden; 1984) and the chemiluminescence method. Another official method to measure induction period is the oil stability index (OSI) (AOCS Method Cd-12b-92, 1993). OSI values generally correspond well with AOM values if PV is 100 meq/kg or greater (Laubli et al., 1986). The method is automated and much easier compared to AOM. However, lengthy experimental time, large errors associated with small changes in O2/air flow rate (Hill et al., 1995) and inability to differentiate between small changes in vegetable oil matrix are major disadvantages. PDSC is also popular for the determination of oxidative stabilities of vegetable oils (Shankwalkar et al., 1993; Kowalski, 1989; Kowalski, 1991). The TFMO method (Lee et al., 1993) is often the method of choice for studying vegetable oils because it is simple and reproducible. The test is especially effective when thermally induced volatility is low and insoluble deposit formation through polymerization is to be considered rather than rates of inhibitor depletion. In vegetable oils, unsaturation, due to C = C from oleic, linoleic, and linolenic acid moieties, provides active sites for various oxidation reactions. Saturated FAs have relatively high oxidation stability (Brodnitz, 1968), but this decreases with increasing unsaturation in the molecule. The rate of oxidation depends on the degree of unsaturation of a fatty acyl chain. In general, the rate of oxidation

TABLE 9.18

Oxidation rates of simple triacylglycerols

Triacylglycerols Tristearin (18:0) Triolein (18:1) Trilinolein 18:2) Trilinolenin (18:3)

Oxidation Low Moderate High Very high

Relative Rate 1 10 100 200

of linoleic (18:2) is 10 (or more) times greater than oleic (18:1), while linolenic (18:3) is twice as great as the linoleic fatty acyl chain (Table 9.18). Oxidation usually takes place through a radical initiated chain mechanism (Murray et al., 1982). Initiation RH ÆR• R• + O2 Æ RO2• Propagation RO2• + RH Æ RO2H + R• R• + O2 Æ RO2• Branching RO2H Æ RO• + •OH RO• + RH + O2 Æ ROH + RO2• •OH + RH + O Æ H O + RO • 2 2 2 Inhibition In H + RO2•Æ In• + RO2H Peroxide decomposition RO2H Æ RO• + •OH + inert products The free radicals generated during the initiation stage react with O2 to form peroxy free radicals and hydroperoxides (Privett et al., 1962). During this period, O2 is consumed in a zero-order process (Labuza et al., 1983), apparently leading to intermediates that are not well characterized, prior to the formation of peroxides (Privett et al., 1962). The latter undergoes further reaction to form alcohols, ketones, aldehydes, carboxylic acids (Shahidi, 1997), leading to rancidity and toxicity (Grosch, 1979). The formation of polar functionalities further accelerates the oil degradation process (Steinberg et al., 1989; Harman, 1982). These compounds have molecular weights that are similar to vegetable oils and, therefore, remain in solution. As the oxidation proceeds, the oxygenated compounds polymerize to form viscous material that, at a particular point, becomes oil insoluble leading to oil thickening and deposits. The extent of oxidation and formation of oxidation products are further complicated by the amount of unsaturation, structural differences in the various triacylglycerol molecules, and the presence of antioxidants. All these factors, together or individually, can change the specific compounds formed and the rates of their formation (Coates et al., 1986). In addition to unsaturation in the molecule, oxidative degradation and kinetics of oxidation is influenced by methylene chain length, bisallylic methylene groups, etc. The cumulative effect of various structural parameters in the triacylglycerol molecule makes oxidation a highly complex process and no simple kinetic model alone would hold good for such systems. Oxidative stabilities of various base oils using the Penn State TFMO test are compared in Table 9.19. In this test, a thin film of oil is oxidized in air high temperatures. The 618

Nonfood Uses of Oils and Fats

interaction of the sample with the reactant gas (oxygen). A film thickness of less than 1 mm is required to ensure proper oil–O2 interaction and eliminate any discrepancy in the result due to oxygen diffusion limitations (Kowalski, 1993; Adhvaryu et al., 1999). Oxygen gas (dry, 99% pure, obtained commercially) is pressurized in the module at a constant pressure of 3450 kPa and maintained throughout the length of the experiment. The sample is then heated at 10°C/min to 250°C. From the DSC thermogram, the onset temperature (OT) is determined. This represents the temperature when rapid increase in the rate of oxidation is observed in the system. This temperature is obtained from extrapolating the tangent drawn on the steepest slope of reaction exotherm. A high OT would suggest a high oxidative stability of the vegetable oil. The OT for various vegetable oils is shown in Table 9.20. The OT is influenced by the degree of poly-unsaturation present in the vegetable oils. It is generally observed that a high polyunsaturation (linoleic and linolenic acid content) decreases, while high oleic content in the FA chain increases the OT. The increase in saturated fatty acids improves the resistance to initial thermal breakdown. The activation energy requirement for such system is considerably high. This results in delaying the onset of initial oxidation process where bond scission takes place to form primary oxidation products. The percentage of oleic acid (see Table 9.12) in the different vegetable oils explains the observed trends in oxidation stability. However, the polyunsaturated and saturated fatty acid contents do not conclusively explain the relative variation of OT among the vegetable oils. The role of different structural parameters obtained using 1H and 13C NMR on the oxidation behavior of unmodified and genetically modified vegetable oils has been explained elsewhere (Adhvaryu et al., 2000). Improvements in oxidation stability are needed due to more stringent demands being placed on lubricant performance. Use of antioxidant (AO) additives along with higholeic vegetable oils improves the oxidation stability. The AO package has to be optimized for vegetable oils. Typically a mixture of AO is required. Table 9.21 shows the effects of the AO package (commercial LZ7652) optimized for vegetable oils when evaluated in the ASTM D2272 RBOT and ASTM D943 TOST methods (Lawate, 2002; Rudnick, 2002). Also in these studies, it was found that high-oleic soybean oil is oxidatively more stable than conventional soybean oil, but less than mineral oil. In TFMO

TABLE 9.19 Oxidative degradation tendencies of various base oils using TFMO (30 min at 150°C) Base Oils Soybean oil High-oleic sunflower oil Diisotridecyl adipate PAO4a Mineral oil a

Deposits (%)

Evaporation (%)

48 13 3 6 5

2 0 5 45 5

Polyalphaolefin with viscosity of ~4 cSt at 100°C.

losses due to evaporation and oxidation and the depositforming tendencies (oxypolymerization) of the test sample are determined in the test as shown in Table 9.19. A temperature of 150°C and times of 30 to 60 minutes chosen for testing were high enough to cause a quantifiable polymerization in unsaturation-free base stocks, yet not too severe to result in oxidative gelation of vegetable oils. Therefore, the side processes, such as oxidative cleavage and formation of solids, were not too substantial. It appears from the data that vegetable oils oxypolymerize considerably faster than unsaturation-free fluids. Although high-oleic sunflower oil containing only 5% of linoleic acid shows higher resistance to oxypolymerization than soybean oil, its oxidative stability is still far less than those of PAO or adipate. Oxypolymerization proceeds much faster and slows down only when side processes, especially formation of solids, become more pronounced. It has been established that methylene-interrupted polyunsaturation is the key factor causing low oxidative stability of vegetable oils (Gardner, 1989). At higher temperatures, such as 175 and 200°C, evaporation was substantially higher in oils having high oleic content. Conversely, increase in polyunsaturation resulted in low evaporation of vegetable oil. A similar trend was observed in safflower oil: higholeic, and high-linoleic. The evaporative loss was greatest in high-oleic oil at 175 and 200°C. As the polyunsaturated FA content increased (as in high-linoleic safflower oil), the percent evaporation decreased at 175°C and remained comparable to safflower oil at 200°C. The deposit-forming tendency is the inverse of the evaporation trend with least deposit in high-oleic oils and more deposit in more polyunsaturated high-linoleic oils. The presence of polyunsaturation in the FA is the primary reason of low oxidative stability, as divinyl methylene hydrogen atoms are highly susceptible to free radical attack leading to substitution with O2 molecule and consequent formation of polymeric oxy-polar compounds. These compounds are the precursors of oil insoluble deposits often encountered with high temperature oxidation of vegetable oils. PDSC is another popular approach for rapid measurement of the oxidative stability of vegetable oils (Kowalski, 1993; Shankwalkar et al., 1993). The procedure is fast, requires only a small quantity of sample, and is extremely reproducible. A small amount of sample is placed in a hermetically sealed aluminum pan with a pinhole lid for

TABLE 9.20 Oxidation stability of vegetable oils using pressurized differential scanning calorimetry Vegetable Oil Cottonseed Safflower High-oleic safflower High-linoleic safflower Sunflower High-oleic sunflower

619

OT (°C) 150 166 178 166 145 177

9.5

Lubricants

9.5.4.6

TABLE 9.21 Oxidation stability of vegetable oils and synthetic ester using the RBOT method RBOT (min) Base Oils

There are two types of biodegradability tests: primary and ultimate biodegradation (Battersby, 2000; Product Review, 1996). Primary biodegradation involves the disappearance of the parent organic compounds under specific test conditions and may or may not indicate that the substrate will biodegrade completely. The primary biodegradation test method is CEC L-33-A-93 from the Coordinating European Council (CEC). This method measures the degree of degradation by the disappearance of specific hydrocarbons bands using IR (infrared) spectroscopy. Since the method does not identify the type of products produced, conclusions regarding the extent of degradation are limited. As regulations are tightened in Europe, the method may not be acceptable for certification of the fluid as environmentally friendly. Total or ultimate biodegradation is measured by tests that result in the complete degradation to carbon dioxide and water by microbial action within 28 days. The two most widely used tests are ASTM standard method D5846 (Annual Book of ASTM Standards, 2000) and the Organization of Economic Cooperation and Development (OECD) test method OECD 301B. The OECD method is accepted worldwide and is the basis for the German chemical laws, dangerous substances legislation (UBA WGK Water hazard), and Eco-labeling (Blue Angel) (Laemmle, 2002). The choice of base oil used is critical for environmentally friendly (EF) lubricants since it dictates the biodegradability and ecotoxicity of the finished lubricant. The biodegradability of different base oils is shown in Table 9.22. The use of PAO and mineral oils in EF lubricants is thus limited due to low biodegradability. Among the base oils listed, vegetable oils possess the highest biodegradability and lowest ecotoxicity.

a

No Antioxidant

3.0 % Antioxidants

15 15 15

79 169 232

15

280

15

170

TOST, hb

Vegetable oil Canola High-oleic canola High-oleic sunflower High-oleic soybean

< 100 ~ 350 ~ 500

Synthetic oil TMP trioleate Mineral oil (200 N) a b

350

~ 2000

Min to 25 lb pressure loss, ASTM D2272. Time to TAN = 2, ASTM D943.

tests the deposit-forming tendencies and oxidative volatility of vegetable oils are significantly reduced by the addition of AO additives (Perez et al., 2002). The proper combinations of high-oleic vegetable oils and additives have oxidative stability at par with off-the-shelf 10W-30 commercial mineral oil-based lubricant. 9.5.4.4

Friction-wear properties

Tests to evaluate the friction and wear characteristics of lubricants are numerous and range from bench tests to engine and pump stand tests (Annual Book of ASTM Standards, 2000; Booser, 1997). Pump stand tests include vane- (Dennison T-50, Vickers V104C) and piston-type tests (Dennison P-46). The DIN 51354 FZG test is popular both in Europe and the U.S. There are four different ASTM bench tests (Annual Book of ASTM Standards, 2000) used to evaluate wear and load-carrying ability: ASTM standard method D2266 (Four-Ball Wear), D2783 (Four-Ball EP), D2782 (Timken test), and D3233 (Falex EP). Each lab uses its own modification of the four-ball test, such as a sequential test correlated with the pump stand tests (Perez et al., 1986). In addition, the fluids are subjected to brake, clutch, and friction disc tests (Booser, 1997). Friction and wear performance of the vegetable oil based lubricants using a commercial additive package are acceptable for industrial and automotive lubricants. 9.5.4.5

Biodegradability

9.5.4.7

Toxicity

Aquatic toxicity tests measure the extent to which a fluid will poison selected environmental species, such as algae (OECD 201), Daphnia magna (OECD 202-12), flathead TABLE 9.22

Biodegradability of various base oils % Biodegradability by

Base Oils

Hydrolytic Stability

CEC Method

OECD Method

Vegetable oils

A serious threat to the stability of vegetable oils and synthetic esters is the presence of water. The reaction can yield organic acids that further catalyze the reaction resulting in further degradation of the base fluid and corrosive wear of metal surfaces. Additives can be used to improve hydrolytic stability (Booser, 1994), but good maintenance practices are the best insurance for long life. The stability of the fluids can be evaluated using the “coke bottle” test, ASTM standard method D2619 (Annual Book of ASTM Standards, 2000).

Soybean Canola High-oleic sunflower

100 100 100

>70 >70 >70

90 90 60 >60 30

15

5

Synthetic oils TMP trioleate TMP trioleate (high-oleic) PAO8 Mineral oil (150N)

620

Nonfood Uses of Oils and Fats

alternative oilseeds in the U.S. Sunflower oil is considered a premium oil due to its light colour, mild flavour, low level of saturated fats and ability to withstand high cooking temperatures. Although the sunflower has the potential for many industrial uses, in the U.S., it is mostly used for food or feed purposes.

minnows Oncorhyuchus mykis (OECD 203-13), and bacteria (OECD 209).

9.5.5

Suitability of natural oils as lubricants

The following vegetable oils are most suitable for use in lubricant applications. More details about these oils can be found in Chapter 2. 9.5.5.1

9.5.5.4

There are two types of safflower varieties: one that produces oil that is high in monounsaturated fatty acids (oleic acid), and the other with high concentrations of polyunsaturated fatty acids (linoleic acid). The high-linoleic safflower oil contains nearly 75% linoleic acid, which is considerably higher than corn, soybean, cottonseed, peanut, or olive oils. Higholeic safflower variety may contain up to 80% oleic acid and is comparable to olive oil, and stable when heated. Regular safflower oil is considered as a drying or semidrying oil, with properties intermediate between soybean and linseed oils, and is currently used in manufacturing paints and other surface coatings. The oil is light in color and will not yellow with aging. This oil can also be used as lubricant base oil, but like most vegetable oils, is currently too expensive for this use. High-oleic safflower oil is rapidly gaining recognition as one of nature’s most valuable vegetable oils with extensive industrial applications.

Soybean oil

Soy oil, extracted through pressing or via solvent extraction, is used for a number of industrial applications. Bioengineered (high oleic and/or low linoleic) soybeans may provide highly desirable improvements for fuels and other industrial products. Current research priorities are focused on developing improved soy oil-based lubricants with improved oxidation stability and cold weather pour properties. Developments in the area of total loss lubricants, hydraulic fluids, and crankcase lubricants are showing promising results. Products, such as biodegradable grease and 2-cycle engine oils, based on soybean oil are currently available. These are environmentally friendly when used in outboard motors, lawnmowers and other small engines. Soybean oil used in crankcases must exhibit properties such as high lubricity, viscosity index, flash point and low evaporation loss. 9.5.5.2

9.5.5.5

Canola oil

Sunflower oil

Traditional sunflower oil consists of 68% linoleic acid and about 20% oleic acid. Two varieties of the oil are currently available: normal and high-oleic. The high-oleic variety has higher oxidation stability and is suitable for industrial applications. Both canola and sunflowers are popular

9.5.5.6

TABLE 9.23 Fatty acid distribution and physical properties of natural and high-oleic canola oil Fatty Acid Distribution/ Technical Properties Palmitic acid (16:0) Stearic acid (18:0) Oleic acid (18:1) Linoleic acid (18:2) Linolenic acid (18:3) Melting temperature (ºC) PDSC (minutes) at 130 °C

High-Oleic Canola Oil

Canola Oil

3.5 1.5 84.0 3.5 4.0 –5.5 71

3.5 1.5 61.5 19.5 10.5 –9.5 25

Meadowfoam oil

Meadowfoam oil contains three previously unknown longchain fatty acids and resembles high erucic acid rapeseed oil in some respects (Bosisio, 1989). It is unusually high in long-chain fatty acids (over 90% C20 to C22 fatty acids) with very high levels of monounsaturation and very low levels of polyunsaturation. These characteristics make meadowfoam oil very stable, even when heated or exposed to air and crude meadowfoam oil is more oxidatively stable than other regular vegetable oils. Meadowfoam oil has the added benefit of enhancing the properties of other oils when mixed with them. Less expensive oils can be mixed with meadowfoam oil without the loss of the qualities of either oil and it can increase the stability of the oils to which it is added. The oil can be used as a lubricant, apart from other applications like light coloured premium grade solid wax, a sulfur compound valuable to the rubber industry, or used as a detergent or plasticizer.

Canola oil is typically referred to in the industry as a penetrating oil and generally has a higher level of linolenic fatty acid than soybean oil. High α-tocopherol content (19 mg/100gm), higher levels of oleic acid and lower levels of polyunsaturated acids contribute to the oil stability as compared to soybean oil. With the rapid development of high-oleic variety oils, various technical properties of the oil can be significantly improved, thereby meeting industrial specifications. Table 9.23 presents the comparison of natural and high oleic canola oil. 9.5.5.3

Safflower oil

Lesquerella oil

Lesquerella oil contains a hydroxy fatty acid and so resembles castor oil, which is an important raw material used by industry for making lubricating greases, resins, waxes, nylons, plastics, corrosion inhibitors, coatings, and cosmetics (Smith Jr. et al., 1961, 1962). Saturated hydroxy fatty acids produced by hydrogenation could be useful in the production of greases in the form of their lithium soaps. Many of the properties may be enhanced over those of castor oil because of the increased chain length of this new crop oil.

621

9.5

Lubricants

9.5.5.7

9.5.6

Cuphea oil

Cuphea oil contains high levels of short-chain saturated fatty acids, C8, C10, C12, and C14, which are used in the production of solvents, detergents, and emulsifiers. Other uses of the seed oil include cosmetics and motor oil. 9.5.5.8

Lubricants provide a well-established and highly competitive market, but growing only at an average rate of less than 1% per year (Padavich et al., 1995). More than 70% of total lubricant volume is used as motor oils for automotive engines and approximately 10% as hydraulic fluids. Other small areas of usage include: cutting oils, two stroke engine oils, chainsaw bar oils, wire rope oils, bicycle chain oils, railroad oils, pump oils, outboard engine oils, drilling oils, and other niche markets. Nonfood uses of vegetable oils have grown very little during the past 40 years (with the exception of the burgeoning demand for biodiesel). Although some markets have expanded or new ones added, other markets have been lost to competitive petroleum products. Vegetable oils are currently being used in various industrial applications, such as emulsifiers, lubricants, plasticizers, surfactants, plastics, solvents, and resins. Research and development approaches take advantage of the natural properties of these oils for lubricant applications, namely amphiphilic character to deliver inherent lubricity, high molecular weight, low volatility, high viscosity index, good solubilizing power for additive molecules and being eco-friendly (Randles et al., 1992; Battersby et al., 1989) among others. Vegetable oils and other lipid derivatives have shown significant increase in use as biodegradable lubricants over the last decade, but still contributing less than 2% of all base oils used in the market. A major application area is industrial hydraulic fluids, which represents a 222 million gallon market in the U.S., with potential use in waterways, farms, and forests. Vegetable base oils are suitable as metal cutting oils and fluids, and avoid the hazardous mist formation from mineral oils during use. Canola-based motor oils have rapidly evolved into a competitive product as a potential substitute for mineral oil-based products. In terms of pricing, they are highly competitive with synthetic motor oils. They are also the most “environmentally friendly” of the motor oils available maintaining properties of nontoxicity and biodegradability. In terms of functionality, they have exceeded expectations by surpassing both conventional and synthetic oils in the tests conducted. There has been significant reduction in tailpipe gas emissions of nitrogen oxides, carbon monoxide, and hydrocarbons, therefore providing an easy and effective way to reduce air pollution. As crankcase oil, though vegetable oil-based lubricants have limited contact with the environment, active development work is in progress on base stocks (e.g., canola, corn, soybean oil) for use in air-cooled engines. Other significant niche market areas are cutting and drive chain oils, two stroke engine oils, chain saw bar oil, wire rope oil, marine oils and outboard engine lubricants, oil for water and underground pumps, rail flange lubricants, agricultural equipment lubricants, metal cutting oils, tractor oils, dedusting, and several others.

Jojoba oil

Perhaps the most commercially advanced of the new crops is jojoba. Jojoba “oil” is a liquid ester wax rather than the familiar triacylglycerols produced in well-known oil seeds like canola. The principal oil structures contain 40 and 42 carbon atoms. The major market for jojoba oil continues to be within the cosmetic industry. An estimated 2000 metric tonnes per annum is consumed by this industry in the form of jojoba oil, hydrogenated jojoba oil, jojoba esters, hydrolyzed jojoba oil, ethoxylated jojoba oil, and other value-added jojoba oil derivatives. In lubricant applications, jojoba oil provides a market for approximately 100 tonnes annually. In general the price of jojoba is too high for this market compared to other available lubricant oils. The molecular structure of this oil is such that it is stable even at high temperatures and pressures unlike most of other lubricants. 9.5.5.9

Tallow and yellow grease

Tallow is inedible grease derived from animal fat renderings. Products such as lubricants, soap, cosmetics, and plastics are made from tallow. Tallow is gradually being replaced by yellow grease (waste vegetable oils) from used cooking oil from fast food restaurants. It contains a mix of unsaturated and saturated fatty acids (generally in a ratio of about 2.8:1). Other application areas of yellow grease are the manufacture of soap, makeup, clothing, rubber, and detergents. 9.5.5.10

Applications

Medium-chain triglycerides

Medium-chain triglycerides (MCT) are medium-chain fatty acid esters of glycerol. Medium-chain fatty acids are fatty acids containing from 6 to 12 carbon atoms. These fatty acids are constituents of coconut and palm kernel oils and are also found in camphor tree drupes. Coconut and palm kernel oils are called lauric oils because of their high content of the lauric acid (C12). MCT used for nutritional and other commercial purposes are derived from lauric oils. They differ from other fats in that they have a slightly lower calorie content (Bach et al., 1996) and they are more rapidly absorbed and burned as energy, resembling carbohydrate more than fat (Bach et al., 1982). With mineral oil falling out of favour in use as a processing lubricant and as a mould release and polishing agent in hard candy production, MCTs may be an option. Many European countries are banning mineral oil usage in food applications, a trend that may spread to the U.S. A recent development is an increase in use of castor oil and palm oil in manufacture of biodegradable- and food industry-grade lubricants. 622

Nonfood Uses of Oils and Fats

A recent product is a soy-based transformer dielectric fluid. This product is used to insulate and cool electrical distribution products, such as transformers, and is safe for the environment and the public. The fluid is biodegradable based on testing done by the U.S. Environmental Protection Agency (USEPA). The fluid also enhances the performance and life of a utility's transformer assets. The fluid extends paper insulation life five to eight times, lowering life cycle costs. The increased insulation life also translates to extended and enhanced transformer life or the ability to carry higher loads during peak demand periods without leading to premature insulation failure. The enhanced performance allows utilities to manage their assets more profitably and forestall costly capital expenditures. The fluid also has excellent fire resistant qualities. The fluid offers an ignition fire point of 360°C and flash point of 330°C, more than twice that of petroleum-based mineral oils. The soy-based fluid has been shown to enhance the loading performance of new transformers by up to 14% or extend their insulation life five to eight times. It also has a similar positive impact on larger units already in service, such as those found in electric substations. Although the cost of the fluid is slightly higher than mineral oils, its soy-based properties contribute to long-term savings that mineral oil cannot deliver. Accelerated aging tests have shown that this fluid extends transformer life well beyond that of units with mineral oil.

9.5.7

challenges that must be overcome in order to improve their usefulness as a sustainable alternative to petroleum base oils. These challenges are of such a scope that it is unlikely that one laboratory, or even one country's scientific community, will easily overcome them alone. New applications of vegetable oil-based lubricants are constantly gaining predominance in areas where stiff regulations require expensive clean up and disposal, and environmental safety plays a major role, despite the higher costs involved. One such class of lubricants is total loss lubricants. The applications where lubricants are lost directly to the environment (railroad rails and switches, wire cables on cranes, the bars of chain saws, and other power equipment) are the most likely application areas to initiate the use of biodegradable lubricants. In these limited-life applications, the stability of the lubricant is not a factor, giving vegetable oil an advantage as the base oil. Widespread use of vegetable oils will depend on how well they perform with a wide range of factors, including temperature, pressure, metal surface, other functional fluids and existing technology. Chemically and genetically modified derivatives of vegetable oils have resulted in significant improvement in thermo-oxidative, low temperature stability, and lubrication properties, thereby increasing their use in a variety of industrial lubricant applications. Development of genetically modified vegetable oil is a lengthy process and it is currently not extensively available as an all-purpose less expensive material capable of delivering all performance qualities for nonfood uses. Nevertheless, it is evident that over the last decade there has been a significant progress in the research and development of lipid technology for innovative industrial uses. In spite of all these technological advancements, an appropriate strategy is needed to convince users of lubricants. This drive toward the use of renewable resources was given a major boost in the U.S. government with the Federal Executive Order 13010 (which set a goal that 25% of all government purchases be biobased), which has been encouraging companies to make use of renewable, bio-degradable base stocks, rather than petroleum base stocks, in many applications (Patin et al., 2002). Achieving the U.S. federal government’s goal of tripling use of bio-based products and bioenergy by 2010 could create $15 to 20 billion in new income for farmers and rural America and reduce fossil fuel emissions by an amount of up to 100 million metric tonnes of carbon (Soya and Oilseed Industry News, 2003). Another opportunity to accelerate the development of this market exists in the 2002 Farm Bill (Farm Security and Rural Investment Act, published January 11, Federal Register). Section 9002 includes language directing all federal agencies to give preference to bio-based products (lubricants is one of the categories specified in the guidelines), unless it is unreasonable to do so, based on price, availability, or performance.

Future prospects of bio-based lubricants

Bio-based lubricants are becoming increasingly important in Europe, particularly for total loss lubricants. In Western Europe, bio-lubricants were 3.1% of total lubricant consumption in 2002 (Whitby, 2004). Their interest in biodegradable lubricants started in the mid 1980s. While the strongest environmental pressures and the resulting acceptance of biodegradable lubricants began in Northern Europe, the same pressures now appear in other European Countries. The first country in Europe to introduce bio-lubricants was Sweden, in 1988, when hydraulic fluids based on rapeseed oil were introduced. The City of Gothenburg published the first “Clean Lubrication” list in 1995, comprising 14 products from 10 lubricant suppliers. The list has been further expanded every year since then. Swedish standard SS 15 54 34 was revised in 1997 to include the environmental criteria in the “Clean Lubrication” list. By 2000, around 25% of all hydraulic fluids sold in Sweden were environmentally friendly, of which 80% were synthetic esters (Whitby, 2004). Almost all forestry operations now use environmentally friendly lubricants, including greases, gear oils, and chain bar oils, because this is now mandated for all forestry operators in Sweden. Biobased lubricants have the potential to create new market opportunities for farmers while easing society’s reliance on petroleum. Vegetable oils, however, offer some 623

9.5

Lubricants

The supply side of the market along with environmental pressure groups, agricultural companies and research institutes are more enthusiastic than the demand side of the market, mainly as a result of higher costs of bio-based lubricants. Users of lubricants are reluctant to use a more expensive product unless there are compelling economic or regulatory pressures to do so. Future outlook for vegetable oil-based lubricants, therefore, will be dictated by consumer awareness and regulations that mandate use of bio-based lubricants. The most encouraging development is OEM interest in these types of fluids, which provides an optimistic future to biobased lubricants.

Bosisio, M., Meadowfoam: pretty flowers pretty possibilities oilseed soaps limnanthes alba nutlets, Agric. Res., USDA Res. Serv., 1989, 37(2), 10. Brodnitz, M.H., Autoxidation of saturated fatty acids. a review, J. Agric. Food Chem., 16, 994, 1968. Coates, J.P. and Setti, L.C., Infrared spectroscopic methods for the study of lubricant oxidation products, ASLE Trans., 29, 394, 1986. Cvitkovic, E. et al. A thin film test for measurement of the oxidation and evaporation of ester type l ubricants, ASLE Trans., 22, 395, 1979. D’Souza, V. et al. Polymorphic behavior of high-melting glycerides from hydrogenated canola oil, J. Am. Oil Chem. Soc., 68, 907, 1991. de Jong, S. et al. Crystal structures and melting points of unsaturated triacylglycerols in the β-phase, J. Am. Oil Chem. Soc., 68, 371, 1991. de Man, J.M. et al. Formation of short chain volatile organic acids in the automated aom method, J. Am. Oil Chem. Soc., 64, 993, 1987. DGF Einheitsmethoden zur Untersuchung von Fetten, Fettprodukten, Tensiden un verwandten Stoffen, Deutsche Gesellchaft für Fettwissenschaft e.V., Munster, Wissenschaftliche Verlagsgesellschaft GmbH Stuttgart, Method C-IV 6a, 1984. Erhan, S.Z. and Adhvaryu, A., Vegetable-Based Base Stocks, in Biobased Industrial Fluids and Lubricants, Erhan, S.Z. and Perez, J.M., Eds., AOCS Press, Champaign, IL, 2002, Chap.1. Erhan, S.Z. and Asadauskas, S., Lubricant basestocks from vegetable oils, Ind. Crops Prod., 11, 277, 2000. Erhan, S.Z. et al. Chemically Functionalized Vegetable Oils, in Synthetics, Mineral Oils, and Bio-Based Lubricants Chemistry and Technology, Rudnick, L.R., Ed., CRC Press, Boca Raton, FL, 2005, Chap.22. Erhan, S.Z., Vegetable oils as lubricants, hydraulic fluids, and inks, in Bailey's Industrial Oil and Fat Products, Volume 6, Industrial and Nonedible Products from Oils and Fats, 6th ed., Shahidi, F., Ed., John Wiley & Sons, New York, 2004, Chap.7. Gardner, H.W., Oxygen radical chemistry of polyunsaturated fatty acids, Free Rad. Biol. Med., 7, 65, 1989. Gordon, M.H. and Mursi, E., Comparison of oil stability based on the Metrohm Rancimat with storage at 20°C, J. Am. Oil Chem. Soc., 71, 649, 1994. Grosch, W., Moll, C. and Biermann, U., Occurrence and formation of bitter-tasting trihydroxy fatty acids in soybeans, J. Agric. Food Chem., 27, 239, 1979. Hagemann, J.W. and Rothfus, J.A., Computer modeling of theoretical structures of monoacid triglyceride alpha-forms in various subcell arrangements, J. Am. Oil Chem. Soc., 60, 1308, 1983. Hagemann, J.W., Tallent, W.H., and Kolb, K.E., Differential scanning calorimetry of single acid triglycerides: effect of chain length and unsaturation, J. Am. Chem. Soc., 49, 118, 1972. Hagemann, J.W., Thermal behavior and polymorphism of acylglycerols, in Crystallization and Polymorphism of Fats and Fatty Acids, Garti, N. and Sato, K., Eds., Marcel Dekker, New York, 1988, 9–95.

References Adhvaryu, A. et al., Application of quantitative NMR spectroscopy to oxidation kinetics of base oils using a pressurized differential scanning calorimetry technique, Energ. Fuels, 13, 493, 1999. Adhvaryu, A. et al., Oxidation kinetic studies of oils derived from unmodified and genetically modified vegetables using pressurized differential scanning calorimetry and nuclear magnetic resonance spectroscopy, Thermochem. Acta, 364, 87, 2000. American Oil Chemist’s Society Official Method Cd 12-57, Fat Stability, Active Oxygen Method, Am. Oil Chem. Soc., Champaign, IL, 1983. American Oil Chemist’s Society Official Method Cd12b-92, Oil Stability Index (OSI), Am. Oil Chem. Soc., Champaign, IL, 1993. Annual Book of ASTM Standards, Section Five: Petroleum Products, Lubricants, and Fossil Fuels, vol. 05.02, ASTM, West Conshohocken, PA, 2000. Antila, V., Fatty acid composition, solidification and melting of Finnish butter fat, Finn. J. Dairy Sci., 27, 1, 1966. Asadauskas, S. and Erhan, S.Z., Depression of pour points of vegetable oils by blending with diluents used for biodegradable lubricants, J. Am. Oil Chem. Soc., 76, 316, 1999. Bach, A.C. and Babayan, V.K., Medium-chain triglycerides: an update, Am. J. Clin. Nutr., 36, 950–62, 1982. Bach, A.C. et al., The usefulness of dietary medium-chain triglycerides in body weight control: Fact or fancy?, J. Lipid Res., 37, 708–26, 1996. Battersby, N.S., The biodegradability and microbial toxicity testing of lubricants — some recommendations, Chemosphere, 41, 1011, 2000. Battersby, N.S. et al. A correlation between the biodegradability of oil products in the CEC L-33-T-82 and modified Sturm tests, Chemosphere, 24, 1989, 1992. Becker, R. and Knorr, A., An evaluation of antioxidants for vegetable oils at elevated temperatures, Lubr. Sci., 8, 95, 1996. Bentz, A.P. and Breidenbach, B.G., Evaluation of the differential scanning calorimetric method for fat solids, J. Am. Chem. Soc., 46, 60, 1969. Booser, E.R., CRC Handbook of Lubrication and Tribology, vol. III, CRC Press, Boca Raton, FL, 1994, 282. Booser, E.R., CRC Tribology Data Handbook, CRC Press, Boca Raton, FL, 1997, 136.

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Harman, D., Free Radical in Biology, vol. 5, Academic Press, New York, 1982, 255. Hill, S.E. and Perkins, E.G., Determination of oxidation Stability of Soybean oil with the oxidative stability instrument: operation parameter effects, J. Am. Oil Chem. Soc., 72, 741, 1995. Hwang, H.S. and Erhan, S.Z., Lubricant base stocks from modified soybean oil, in Bio-Based Industrial Fluids and Lubricants, Erhan, S.Z. and Perez, J.M., Eds., AOCS Press, Champaign, IL, 2002, Chap. 2. Jebe, T.A. et al. Collaborative study of the oil stability index analysis, J. Am. Oil Chem. Soc., 70, 1055, 1993. Kowalski, B, Evaluation of activities of antioxidants in rapeseed oil matrix by pressure differential scanning calorimetry, Thermochim. Acta, 213, 135, 1993. Kowalski, B., Determination of oxidative stability of edible vegetable oils by pressure differential scanning calorimetry, Thermochim. Acta, 156, 347, 1989. Kowalski, B., Thermal-oxidative decomposition of edible oils and fats. DSC studies, Thermochim. Acta, 184, 49, 1991. Labuza, T.P. and Bergquist, S., Kinetics of oxidation of potato chips under constant temperature and sine wave temperature conditions, J. Food Sci., 48, 712, 1983. Labuza, T.P., et al., Metal-catalyzed oxidation in the presence of water in foods, J. Am. Oil Chem. Soc., 48, 527, 1971. Laemmle, P., Biodegradable Hydraulic Fluids and Utto-Lubricants, SAE 2002-01-1455, NCFP I02-20.3, 2002. Laubli, M.W. and Bruttel, P.A., Determination of the oxidative stability of fats and oils: comparison between the active oxygen method (AOCS Cd 12-57) and the Rancimat method, J. Am. Oil Chem. Soc., 63, 792­795, 1988. Laubli, M.W. and Bruttel, P.A., Determination of the oxidative stability of fats and oils: comparison between the active oxygen method (AOCS Cd 12-57) and the rancimat method, J. Am. Oil Chem. Soc., 63, 792, 1986. Lawate, S., Environmentally friendly hydraulic fluids, in Biobased Industrial Fluids and Lubricants, Erhan, S.Z. and Perez, J.M., Eds., AOCS Press, Champaign, IL, 2002, chap.3. Lee, C.J. and Klaus, E.E., Evaluation of deposit forming tendency of mineral and synthetic base oils using the Penn state microoxidation test, Lubr. Eng., 49, 441, 1993. Loury, M., Possible mechanisms of Autoxidative Rancidity, Lipids, 7, 671, 1972. Matthäus, B. et al. Fast chemiluminescence method for detection of oxidized lipids, Fat Sci. Technol., 96, 95, 1994. Matthäus, B. et al. Bestimmung von Hydroperoxiden in Fetten und Ölen durch Chemilumineszenz, Lebensmittelchemie, 47, 85, 1993. Murray, D.W. et al., Effect of basestock composition on lubricant oxidation performance, Petrol. Rev., 36 (421), 36–40, 1982. Oxidationsstabilität von Ölen und Fetten-Rancimatmethode, Metrohm AG, Herisau, Application Bulletin Metrohm Nr. 204/1 d, 1994. Padavich, R.A. and Honary, L., A market research and analysis report on the vegetable oil based industrial lubricants, SAE Tech paper 952077, p. 13, 1995. Patin, L.J. and James, D.K., Biodegradable and fire-resistant hydraulic fluids: now you can have both, Lubric. World, Nov. 26, 2002. Perez, J.M. and Boehman, A.L., Environmentally friendly fuels and lubricants, in Biobased Industrial Fluids and

Lubricants, Erhan, S.Z. and Perez, J.M., Eds., AOCS Press, Champaign, IL, 2002, Chap.6. Perez, J.M. et al. Comparative evaluation of several hydraulic fluids in operational equipment, a full scale pump test stand and the four-ball wear tester–Part III. New and used hydraulic fluids, Lubr. Engr., 52, 416, 1986. Privett, O.S. and Blank, M.L., The initial stages of autoxidation, J. Am. Oil Chem. Soc., 39, 465, 1962. Product review on biodegradable fluids and lubricants, Ind. Lub. & Tribol., 48(2), 17, 1996. Randles, S.I. and Wright, M., Environmentally considerate ester lubricants for the automotive and engineering industries, J. Syn. Lubr. 9, 145, 1992. Rossell, J.B. and Pritchard, J.L.R., Analysis of Oil Seeds, Fats and Fatty Foods, Elsevier, London, 1991. Rudnick, L.R., A comparison of synthetic and vegetable oil esters for use in environmentally friendly fluids, in Bio-Based Industrial Fluids and Lubricants, Erhan, S.Z. and Perez, J.M., Eds., AOCS Press, Champaign, IL, 2002, Chap. 4. Schmidt, M.A. et al. Biotechnological enhancement of soybean oil for lubricant applications, in Synthetics, Mineral Oils, and Bio-Based Lubricants Chemistry and Technology, Rudnick, L.R., Ed., CRC Press, Boca Raton, FL, 2005, Chap. 23. Shahidi, F., Natural Antioxidants Chemistry, Health Effects and Application, AOCS Press, Champaign, IL, 1997, 1. Shankwalkar, S. and Placek, D., Oxidation kinetics of tricresyl phosphate (TCP) using differential scanning calorimetry (DSC), Lubr. Eng., 50, 261, 1994. Smith, C.R. et al., Lesquerolic acid, a new hydroxy acid from Lesquerella seed oil, J. Org. Chem., 26, 2903, 1961. Smith, C.R. et al., Densipolic acid: a unique hydroxydienoid acid from Lesquerella densipila seed oil, J. Org. Chem., 27, 3112, 1962. Soya & Oilseed Industry News, ‘BioEnergy’ conference aims to help power U.S. energy independence, Oct. 30, 2003. Steinberg, D. et al., Beyond cholesterol: modifications of lowdensity lipoprotein that increase its atherogenicity, New Engl. J. Med., 320, 915, 1989. Swern, D., Organic Peroxides, vol. 1, Wiley/Interscience, New York, 1970, 115. Wewala, A.R., Natural Antioxidants. Chemistry, Health Effects and Applications, AOCS Press, Champaign, IL, 1997, 331. Whitby, R.D., Market share of bio-lubricants in Europe and the USA, Lipid Technol., 16(6), 125–130, 2004.

9.6

Biofuels

9.6.1

What are biofuels and why are they attracting so much interest?

For the last century and more, man has exploited fossil fuels (coal, mineral oil, and gas) to provide energy for warmth (and cooling), transport, and to drive industrial machinery. The supplies of fossil fuels are large but finite and in recent decades and particularly in the last few years there has been a revived interest in alternative forms of energy among which are two based on plant matter — bioethanol and biodiesel (usually methyl esters derived from vegetable oils and animal fats). The reasons for this 625

9.6

Biofuels

new emphasis have included the rising cost of mineral oil, concern about security of supply and about environmental issues, and pressures from oil and fat producers and from governments. Europe has led the way in the production of biodiesel. North and South America have favoured bioethanol, but here too biodiesel is becoming more important. In 2004, the U.S. produced 4 billion gallons (19 million tonnes) of bioethanol, but only 30 million gallons (0.13 million tonnes) of biodiesel. In 1973 and again in 1978 the price of crude petroleum rose very rapidly and unexpectedly. This had serious consequences for the economies of importing countries in Asia, Europe, and North America and led to a serious global economic downturn. The power of the mineral oilproducing countries, particularly in the Middle East, to control prices and supplies made the western countries concerned about security of supply and caused them to consider alternative sources of petroleum supply and alternative sources of energy. Those concerns remain and are deepened by current fluctuating prices that are frequently high and occasionally very high. Environmental issues are immediate and local leading to pollution and also long-term and global leading to global warming. In cities, particularly, our dependence on cars, vans, and lorries (trucks) to transport goods and persons leads to poor atmospheric conditions (smog) and a consequent interest in fuels that are less polluting. There is also a growing concern about global warming that many consider to be manmade and arising in large part from the production of greenhouse gases of which carbon dioxide resulting from burning of fossil fuels is of greatest concern. There is also long-term worry about the depletion of our valuable but nonrenewable petroleum supplies. Despite the increasing demand for oils and fats as food and for oleochemicals there are times of over-supply and pressures then come from producers of oils and fats to find new uses for these materials. There is also pressure from governments who, having set and accepted targets for the use of nontraditional forms of energy, are struggling to meet those targets. These alternative sources of energy are further seen as a way of avoiding the use of nuclear energy, considered by many as a less acceptable nonfossil fuel solution. One way to meet some of these problems, in small part, is to replace fossil fuels with biodiesel (usually methyl esters derived from oils and fats). These materials have the advantage that they can be used neat or in various blends with petrodiesel without engine modification and with the approval of the vehicle manufactures. They can also be distributed easily through the existing fuel supply chain. Development and limited use of biodiesel goes back over 20 years, but these activities have accelerated in the last few years and many countries, both developed and developing, have plans to produce and use biodiesel if they are not already doing so. Several EU countries are in the lead, but they are being followed by rapid growth in Asia and in

North and South America. However, biodiesel is probably more expensive to produce than petrodiesel and can only be economic with government assistance. In Europe this is provided in the form of lower taxation compared with conventional fuel. In the U.S. where fuels are less heavily taxed assistance can be provided via The Clean Air Act Amendments of 1990, the Energy policy Act of 1992, or by tax rebate. Currently this amounts to $0.01 per 1% of biodiesel from soybean oil or $0.005 from other sources. This is equivalent to 20 and 10 cents, respectively, for each litre of a 20% blend. The doubt about relative costs expressed earlier relates to the difficulty in determining the true cost of production free of taxes and subsidies and in the problem of assessing in financial terms the environmental and social benefits that result from producing and using biodiesel and the cost of global warming made worse by doing nothing. For example, in Brazil production of biodiesel from locally grown crops is seen not only as a way of reducing the environmental and economic cost of transporting fuel by road over long distances, but as a way of providing rural employment and slowing the drift of unemployed people to already overcrowded cities. Governments lend their support to the production and use of biodiesel for differing reasons including reduction in pollution, in carbon dioxide emissions, in the use of nonrenewable resources, and to encourage the use of domestically produced material. Despite the popularity of biodiesel as a way of overcoming some environmental concerns, some serious questions have been raised (Dumelin, 2005). First it has to be recognised that biodiesel can only make a small contribution to the total requirement for energy. Until we develop new energy sources, such as hydrogen or atomic fusion, we will remain dependent on fossil fuels. The annual production of mineral oils is about 30 times larger than that of commodity oils and fats. Secondly, oils and fats are needed as an important source of energy-rich food and of essential nutrients. The demand for food increases with the growth in population and in income and there remains a substantial unmet need. Why then should we divert this irreplaceable food source? The Indian government has decreed that biodiesel cannot be made from any oil or fat that could be used for food and alternative supplies are being sought, such as the nonedible oil from Jatropha curcas seeds. There are claims already that the supply/demand system is being disturbed and this is reflected in pressure on prices. However, it is worth noting that Fry (2001) has forecast that this situation will change during the present century. Production levels will continue to increase through rising yields, population will level off about mid-century, and there is a limit to how much fat we can eat with the consequence that from around 2050 onwards there will be quite large supplies available for nonfood purposes including biofuels. A third concern is that biodiesel is only economic through manipulation of taxation. When the supply of biodiesel is small, this may not matter, but as it increases the downturn in tax revenues will have to be met in another way. Perhaps this is a way of transferring a part of the cost from 626

Nonfood Uses of Oils and Fats

the customer to the taxpayer (frequently, but not always the same person). Reaney et al. (2005) also take an optimistic view. They consider that biodiesel will become the largest market for oils and fats exceeding demands for food, feed, and the other nonfood uses discussed in this chapter. This will involve developments in agriculture (mainly higher yields and new biodiesel crops), better use of by-products (meal and glycerol), and improved production technology. Although 100% biodiesel can be used in vehicles and is so used in Germany and Austria, it is more often offered as a blend with regular diesel. In the U.S. biodiesel generally contains 20% of added methyl esters, but in Europe 5% blends are more common. In addition to road vehicles, these products are being used in agricultural machinery, in boats — both for business and for pleasure — and in trains. Methyl esters can also be used in central heating systems and it is of interest that rapeseed methyl esters are used to heat the Reichstag building in Berlin, newly renovated after the union of West and East Germany into a single state in 1990. The methyl esters also serve in one route to fatty alcohols (Section 9.2.3).

9.6.2

for this purpose. It is desirable to avoid too high a content of saturated esters that may solidify at ambient temperature and high levels of polyunsaturated esters, especially those with more than two double bonds, which lead to undesirable oxidation and may cause problems during storage of the fuel or at the moment of use. Nevertheless a wide range of materials is available for use with convenience of supply being the most important. Rapeseed or canola oil (Europe), soybean oil (North and South America), palm oil (Malaysia), coconut oil (Philippines), and tallow (New Zealand) have been used or examined in the countries indicated. There is even a report of fish oil being used in Canada after removal of the valuable EPA and DHA. The fatty acid composition of some of these oils is shown in Table 9.24. About 80% of the production cost of biodiesel lies in the price of the starting oil when this is an appropriately refined vegetable oil. Attempts to reduce cost have led to the use of animal fats (tallow, chicken fat, and animal fats unfit for human consumption), vegetable oils already used for frying and cooking, free acids removed during refining, and alternative vegetable oils, such as castor, palm, jatropha (Jatropha curcas), and karanja (Pongamnia pinnata) oils from India and Africa, babassu oil, jojoba oil (a wax that would yield methyl esters and fatty alcohols on methanolysis), and fatty acids recovered from refining processes (Table 9.25). The quality of these less common starting materials needs to be controlled and specifications for each

From what material is biodiesel made?

Good quality biodiesel must meet certain criteria (see below) and this may limit the range of oils and fats that can be used TABLE 9.24

Fatty acid composition of selected oils and fats

Palmitic Stearic Oleic Linoleic Linolenic

Rapeseed

Sunflower

Soybean

Palm

Tallow

4–5 1–2 55–63 20–31 9–10

3–6 1–6 14–43 44–69

2–11 2–6 22–31 49–53 2–10

32–46 4–6.3 37–53 6–12

25–37 14–29 26–50 1–3

Source: Adapted from Knothe, G. and Dunn, R.O., in Oleochemical Manufacture and Applications, Gunstone, F D., Hamilton, R.J., Eds., Sheffield Academic Press, Sheffield, U.K., 2001, Chap. 5.

TABLE 9.25

Fuel-related properties of selected oils and fats and of the methyl esters derived from them IV

CN

35 61 94–120 117–143 110–143 35–48

42 37.6 37.9 37.1

HG

Viscosity

CP

PP

FP

–3.9 –3.9 7.2

–31.7 –12.2 –15.0

–15.0

–33.0

246 254 274 201 52

–2 2 0 12

–9 –1 -4 9

Oils and Fats Palm Rapeseed Soybean Sunflower Tallow Petrodiesel

47

39709 39623 39575 40054 45343

37.0 32.6 37.1 51.1 2.7

(37.8) (37.8) (37.8) (40) (37.8)

Derived Methyl Esters Rapeseed Soybean Sunflower Tallow

54.4 46.2 46.6

40449 39800 39800 39949

6.7 4.1 4.2 4.1

(40) (40) (40) (40)

84 171 96

Note: Iodine value, cetane number, gross heat of combustion (kJ/kg), kinematic viscosity min2/s) at the temperature indicated (°C), cloud point (°C), pour point (°C), flash point (°C). Source: Adapted from Knothe, G. and Dunn, R.O., in Oleochemical Manufacture and Applications, Gunstone, F D., Hamilton, R.J., Eds., Sheffield Academic Press, Sheffield, U.K., 2001, Chap. 5.

627

9.6

Biofuels

would be required. Since large-scale production plants are generally preferred, it is necessary to secure a source of oil or fat available continually and in appropriate quantity.

9.6.3

purification), methanol (for reuse), and biodiesel. Plants vary in size from 10 to 200 kt/year. The reaction is an equilibrium process involving two phases and interesterification may be carried out in two or three stages. At the end of each stage glycerol is removed and fresh methanol and a catalyst is charged to the reactor. Reaction proceeds at about 60°C with an overall residence time of about 2 hours. Plants are now available that are flexible enough to handle a range of different feed stocks (Meyer and Koerbitz, 2004). Information is available on the websites of companies, such as Ballestra, Bayer, Crown Iron Works, De Smet, Lurgi Life Science, and Westphalia available through Google.

How is biodiesel manufactured?

Despite the desire to use cheap starting materials for biodiesel production, the final product must meet defined specifications and this may be achieved more easily with good quality oil. The cost of upgrading a poor product may take up the saving in a cheaper starting material. For biodiesel production, crude oils should be degummed (removal of phospholipids) and neutralised (removal of free acids), but bleaching and deodorisation is not generally required. In general, oils and fats (triacylglycerols) are converted to methyl esters by reaction with methanol in the presence of an acidic, basic, or enzymatic catalyst (Section 9.2.2). Reaction with a basic catalyst (NaOH, KOH, or preferably NaOMe) is the preferred route, but is only appropriate with oils with low levels of free acid. The other product of this reaction is glycerol (Section 9.2.5) and the economic value of this by-product is an important part of the economics of the whole process. Since many other oleochemical processes are dependent on the economic value of glycerol, there is concern about the over supply of this by-product with the consequence of this for prices (Section 9.2.5). It is important that the methanolysis reaction proceed as far as possible as partial glycerol esters as well as methanol, glycerol, and free acids are considered to be undesirable impurities in biodiesel. Walker (2004) has reported that one tonne of rapeseed can be expected to produce crude oil (0.41 tonnes) and meal (0.58 tonnes) and that the refined oil (0.40 tonnes) when reacted with methanol will furnish 0.04 tonnes of glycerol and 0.38 tonnes (432 litres) of methyl esters. With an oilseed yield of 3 t/ha (common in Europe, but not elsewhere) this means that 1300 litres of biodiesel can be obtained with the crop from one hectare of land. Obviously, the energy recovered when the esters are used as a biofuel must exceed that required to produce the fuel with calculations starting from the planted seed. Walker has cited energy output/input ratios for rapeseed methyl esters generally between 2.0 and 2.6 after allowing for the value of the meal and the glycerol. One of the most energydemanding steps in the process is the fertiliser requirement. Walker (2004) has also discussed the economics of biodiesel production from rapeseed and produced figures of 30 to 60 pence/litre without allowance for distribution costs, taxation, or profit. The range of cost is related to the size of the operating plant. Several manufactures have designed new or adapted existing plants for efficient biodiesel production. Inputs are methanol, oil (extracted and appropriately pretreated), and catalyst (NaOH, KOH, or NaOMe). The steps involved include interesterification, separation of the glycerol and ester phases, and recovery of glycerol (for further

9.6.4

Specifications for biofuels

Biodiesel must meet specifications (which may differ between countries and regions of the world) that have been accepted by most vehicle manufactures. The EU biodiesel specification (EN 14214) provides minimum and/or maximum values for ester content, density, viscosity, flash point, sulfur content, cetane number, water content, oxidation stability, acid value, iodine value, content of methanol, glycerol, monoacylglycerol, diacylglycerol, linolenate ester, phosphorus, and some other properties. These relate to rapeseed methyl esters and the iodine value with an upper limit of 120 would exclude biodiesel from sunflower oil or soybean oil. ASTM standard D6751 in the U.S. covers flash point, water and sediment, carbon residue, sulfated ash, viscosity, sulfur, cetane, cloud point, acid number, and free and total glycerol. Iodine value is not included. Cetane numbers are an important index of ignition quality. For biodiesel these are affected by chain length, unsaturation, and branching and generally have higher minimum values (47 in ASTM D6751 and 51 in EN 14214) than those specified for petrodiesel. Other important properties are viscosity, pour point, and cloud point, which, if not appropriate, can affect behaviour at low temperatures. Low temperature problems may be overcome by using isopropyl esters in place of methyl esters or by winterisation of methyl esters to remove higher melting components. The latter solution adds cost to the manufacturing process by virtue of the extra step involved and leaves a higher melting fraction for which an alternative use has to be developed. Because biodiesel contains unsaturated centres, it is liable to oxidation during storage and it is important to have adequate storage conditions and possibly to add antioxidant. Reduction of sulfur dioxide emissions from vehicles has been achieved by use of ultra-low sulfur fuels, but this is disadvantageous insofar as lubricity is also reduced. However, lubricity in ultra-low sulfur diesel fuels can be restored by the addition of some 1 to 2% of biodiesel. Knothe and Steidley (2005) have shown that improved lubricity following addition of a little 628

Nonfood Uses of Oils and Fats

biodiesel is a consequence mainly of the free acid and monoacylglycerols present in this material. These have been considered as undesirable contaminants and there is perhaps a case for considering minimum as well as maximum limits for these components. Compared to regular diesel, biodiesel is considered to produce lower emissions of SO2, CO, hydrocarbons, PAH (polycyclic aromatic hydrocarbons), particulates, and smoke. Depending on engine tuning, NOx emissions are usually higher with biodiesel. Of course, carbon dioxide is produced as in the combustion of all organic products, but it is argued that this was trapped from the atmosphere during the growing season and, therefore, does not add to global supplies of this gas. Combustion of petrodiesel on the other hand liberates carbon dioxide trapped in fossil fuels millennia previously and, therefore, adds to the global carbon dioxide supply.

9.6.5

to meet manmade targets to reduce carbon dioxide emissions. The production of biodiesel in India is confined to nonedible oils by government decree (Kale, 2005a,b). Gunstone (2006) has forecast a demand for 40 to 50 million tonnes of biodiesel in 2020.

References Anon., BioX plans palm oil use for green energy, Inform, 16, 748, 2005. Dumelin, E.E., Biodiesel — a blessing in disguise, Eur. J. Lipid Sci. Technol., 107, 63–64, 2005. Fry, J., The world’s oil and fat needs in the 21st century: lessons from the 20th century. Lecture presented in 2001 and cited by Gunstone, F.D., Ed., Vegetable Oils in Food Technology, Blackwell Publishing, Oxford, U.K., 2002, pp. 15–16. Gunstone, F.D., Chang Lecture delivered at the AOCS Exhibition in St. Louis, May 2006. Kale, V., Biodiesel gaining popularity in India, Inform, 16, 466–468, 2005a. Kale, V., Jatropha — India’s crop for biodiesel production, Inform, 16, 532–533, 2005b. Knothe, G. and Dunn, R.O., Biofuels derived from vegetable oils and fats, in Oleochemical Manufacture and Applications, Gunstone, F.D., Hamilton, R.J., Eds., Sheffield Academic Press, Sheffield, U.K., 2001, Chap. 5. Knothe, G., et al., Ed., The Biodiesel Handbook, AOCS Press, Champaign, IL, 2004. Knothe, G. and Steidley, K.R., Lubricity of components of biodiesel and petrodiesel. The origin of biodiesel lubricity, Energ. Fuels, 19, 11921200, 2005. Meyer, S. and Koerbitz, W., Worldwide review of biodiesel production and studies on biodiesel production plants in Europe, Malay. Oil Sci. Technol., 13, 11–16, 2004. Mittelbach, M. and Remschmidt, C., Biodiesel — The Comprehensive Handbook, (published by Mittelbach), Graz, Austria, 2004. Reaney, M.J.T., Vegetable oils as biodiesel, in Bailey’s Industrial Oil and Fat Products, 6th ed., Shahidi, F., Ed., Wiley Interscience, New York, 2005, Chap. 6. Walker, K., Non-food uses, in Rapeseed and Canola Oil, Gunstone, F.D., Ed., Blackwell Publishing, Oxford, U.K., 2004, Chap. 7.

Present and future demand for biodiesel

Targets for the replacement of petrodiesel by biodiesel have been set in the EU, though not all member counties have accepted these. It is considered that in 2005 the level of replacement should be 2.5% of total vehicle consumption and that this should rise at the rate of 0.75% a year to 5.75% of total consumption. Targets set by individual countries for 2005 were 2.0% or above for Austria, Czech Republic, France, Germany, Italy, Latvia, Lithuania, Slovakia, Spain, Sweden, and the U.K., but not all these were met. Some other countries with targets of 1% or below are working actively to increase production and there are regular reports of new plants being planned and coming on stream. These levels of 2.5 and 5.75% will require about 2.5 and 6 tonnes of biodiesel. Currently about 80% of European biodiesel comes from rapeseed oil, but other sources will have to be found and this proportion must fall. Production of rapeseed in EU-25 in 2004/05 was around 5.5 million tonnes, 1 million tonnes higher than in the preceding year. Interest in producing biodiesel is not confined to Europe and many other countries are beginning to produce this material. Where consumption targets have been set (and not met), a growing export–import trade in biodiesel methyl esters will probably develop. For example, Malaysia and Indonesia with their ample supplies of starting material should be a good source of biodiesel and coco-biodiesel is being developed in the Philippines for export to Japan. Malaysia is expected to meet about 10% of the world demand for biodiesel. It is expected that the U.S. will use 0.5 million tonnes of biodiesel in 2006 and that a similar amount will be produced and used in Brazil by 2010. These figures relate to biodiesel and do not include the burning of around 0.5 to 1.0 million tonnes of vegetable oils and animal fats to produce electricity (Anon., 2005). This use is driven in part by low prices for oils and fats (especially palm oil) and in part by pressure

9.7

Surface coatings and inks

It has long been known that when thin layers of unsaturated fatty oils are exposed to air they harden to solid impervious films. Artists used this knowledge to provide a protective transparent film for their paintings. With incorporation of appropriate pigments, it is possible to produce a coloured layer. Surface coatings may be applied to wood, paper, metal and plastic surfaces to protect against moisture, oxygen, sunlight, radiation and pollutants, such as sulfur dioxide. They may also serve to decorate or disguise. About half the annual production of paint is used for internal and external use in buildings 629

9.7

Surface coatings and inks

from a polybasic acid and a polyhydric alcohol with unsaturated fatty acids acylated to additional hydroxyl groups. The polybasic acid is generally phthalic acid (benzene-1,2dicarboxylic acid) used in the form of its anhydride. The polyhydric alcohol may be glycerol or pentaerythritol [C(CH2 OH)4]. Other possible reactants are listed in Table 9.27. The fatty acids are mainly unsaturated and may be supplied as free acids, triacylglycerols, or often as monoacylglycerols derived from appropriate vegetable oils like linseed oil, soybean oil, or dehydrated castor oil. Castor, tung, coconut, safflower, sunflower, and tall oil may also be used. Alkyds usually contain 30 to 70% of fatty acids. Because the drying process involves oxidative polymerization, it is desirable that the vegetable oil serving as a source of the fatty acids contain as little natural antioxidant as possible. The fatty acids may also be modified to improve their properties as in reaction with maleic anhydride to increase functionality or by prior conjugation of the polyunsaturated system with a proprietary catalyst to speed drying. Alkyds are divided into three classes depending on their content of unsaturated oil, which may be >60% (long oil), 40 to 60% (medium oil), and 10-hydroxy-decanoic acid and 2octanol, resulting from caustic fusion with 1 mole of NaOH at 180-200oC; > sebacic acid and 2-octanol, resulting from caustic fusion with 2 moles of NaOH at 250-275oC; >10-undecylenic acid and heptaldehyde, resulting from pyrolysis in superheated steam at 550oC; >12-hydroxy-octadec-9-ynoic acid resulting from bromination and dehydrobromination of ricinoleate; and >9,10-oxy-12-hydroxyoctadecanoic acid, resulting from epoxidation or hot air oxidation.

634

Nonfood Uses of Oils and Fats

production of undecylenate, heptaldehyde is also produced, and is used in flavors and aromas. It is also oxidized to heptanoic acid. Esters of heptanoate remain liquid at low temperatures, retain viscosity, and are not volatile, so they are useful in such demanding applications as jet engine lubrication (Caupin, 1997). Esters of heptanoate are also used as plasticizers. Methyl ricinoleate can be converted to a hydroxyacetylenic fatty acid (Figure 9.9) by bromination, then dehydrobromination by sonic irradiation in ethanolic KOH (Lie Ken Jie et al., 1996). This compound presents three reactive moieties for polymerization reactions and has potential for more advanced applications, for example, in building nanomaterials. Ricinoleate can be epoxidized by hot air oxidation or by oxidation with peroxyacetic acid (Figure 9.9). The product formed can be used as a plasticizer and in paints or other coatings (Naughton and Vignolo, 1992) as a low volatile organic carbon (VOC) component.

9.8.4

Geller, D.P. and Goodrum, J.W. Effects of specific fatty acid methyl esters on diesel fuel lubricity. Fuel, 83, 2351–2356, 2004. Goodrum, J.W. and Geller, D.P. Influence of fatty acid methyl esters from hydroxylated vegetable oils on diesel fuel lubricity. Biores. Technol., 96, 851855, 2005. Guhanathan, S. et al. Studies on castor oil-based polyurethane/ polyacrylonitrile interpenetrating polymer network for toughening of polyunsaturated polyester resin. J. Appl. Polymer Sci., 92, 817–829, 2004. Hofer, R. et al. Oleochemical polyols — a new raw material source for polyurethane coatings and floorings. J. Coatings Tech., 69, 65–72, 1997. Kuhn, H. et al. Mechanism of the odor-absorption effect of zinc ricinoleate. A molecular dynamics computer simulation. J. Surf. Deterg., 3, 335–343, 2000. Lie Ken Jie, M.S.F. et al. Ultrasound-assisted synthesis of santalbic acid and a study of triacyl-glycerol species in Santalum album (Linn) seed oil. Lipids, 31, 1083–1089, 1996. Lin, J.T. et al. Identification and quantification of the molecular species of castor oil by HPLC and ELSD. J. Liq. Chrom. Rel. Tech., 26, 773–780, 2003. Magerl, A. et al. Allergic contact dermatitis from zinc ricinoleate in a deodorant and glyceryl ricinoleate in a lipstick. Cont. Derma., 44, 119–121, 2001. McKeon, T.A. et al. Developing a safe source of castor oil. Inform 13, 381−385, 2002. McKeon, T.A. et al. Hydroxy fatty acids. In Nutraceutical and Specialty Lipids and Their Co-Products, F. Shahidi, Ed., CRC Press, Boca Raton, FL, 2006. Naughton, F.C. and Vignolo, R.L. The chemistry of castor oil and its derivatives and their applications. Int. Castor Oil Assoc., Westfield, NJ, 1992. Qu, J. et al. Synthesis of castor oil water-borne polyurethane-acrylate hybrid emulsions. J. Chem. Ind. Eng., 56, 168–173, 2005. Roethli, J.C. et al. Lesquerella as a Source of Hydroxy Fatty Acids for Industrial Products, USDA-CSRS Office of Agricultural Materials. Growing Industrial Materials Series (unnumbered), Washington, D.C., 1991. Rojas-Barros, P. et al. Inheritance of high oleic/low ricinoleic content in the seed oil of castor mutant OLE-1. Crop Sci., 45, 157–162, 2005. Shikanov, A. et al. Poly(sebacic acid co-ricinoleic) biodegradeable carrier for paclitaxel — effect of additives. J. Cont. Rel., 105, 52–67, 2005. Slivniak, R. and Domb, A.J. Macrolactones and polyesters from ricinoleic acid. Biomacromolecules, 6, 1679–1688, 2005a. Slivniak, R. and Domb, A.J. Lactic acid and ricinoleic acid based copolyesters. Macromolecules, 38, 5545–5553, 2005b. Wurdack, K.J. et al. Molecular phylogenetic analysis of uniovulate Euphorbiaceae (Euphorbiaceae sensu stricto) using plastid rbcL and trnL-F DNA sequences. Am. J. Bot., 92, 1397–1420, 2005. Yeganeh, H. and Mehdizadeh, M.R. Synthesis and properties of isocyanate curable millable polyurethane elastomers based on castor oil as a renewable resource polyol. Eur. Polymer J., 40, 1233–1238, 2003.

Summary

Castor oil is a unique natural product, used in many industries and many types of products. It is generally thought that the uses of castor oil in industry are limited by the availability of the oil. Certainly, the presence of a midchain hydroxyl group and the polyol nature of castor oil provide nearly endless possibilities for chemists. More widespread cultivation of castor is very likely to expand utilization of castor oil by industry.

References Altafim, R.A.C. et al. The effects of fillers on polyurethane resinbased electrical insulators. Mat. Res., 6, 187–191, 2003. Anon., USDA Agricultural Statistics, Government Printing Office, Washington, D.C., 2005. Auld, D.L. et al. Development of castor with reduced toxicity. J. New Seeds, 3, 61–69, 2001. Bodalo-Santoyo, A. et al. Enzymatic biosynthesis of ricinoleic acid estolides. Biochem. Eng. J., 26, 155–158, 2005. Caupin H.J. Products from castor oil: past, present, and future. In Gunstone, F.D., Padley, F.B., Eds., Lipid Technologies and Applications. Marcel Dekker, New York, pp. 787–795. Cermak, S.C. et al. Synthesis and physical properties of estolides from lesquerella and castor fatty acid esters. Ind. Crops Prod., 23, 54–64, 2006. Clay, A.F. et al. Hydroxydecanoic acid for greasy and acne-prone skin. COSSMA, 1(14), 12–14, 2000. Dierig, D. et al. Improvement in hydroxy fatty acid seed oil content and other traits from interspecific hybrids of three Lesquerella species: Lesquerella fendleri, L. pallida, and L. lindheimeri. Euphytica, 139, 199–206, 2004.

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10 LIPID METABOLISM

J.L. Harwood

10.1

Fatty acids

10.1.1

Fatty acid thiolesters

By contrast, mammals, fungi, and plant cytosols contain large (200 to 250 kDa) multifunctional polypeptides. The structures, regulation, and enzymatic mechanisms of the multisubunit ACCases are reviewed by Cronan and Waldrop (2002), while Kim (1997) has described mammalian ACCases and their regulation. In the first part of the ACCase reaction, the biotin moiety of BCCP (biotin carboxyl carrier protein) is carboxylated in an (adenosine 5-triphosphate) ATP-mediated process. Recent evidence has implicated carboxyphosphate as an intermediate. The carboxyl moiety is then transferred to the acceptor acetyl-CoA, probably by the use of carbon dioxide as the electrophile. Study of the halfreactions of acetyl-CoA carboxylase has allowed proposals concerning the mechanism of biotin participation to be made (De Titta et al., 1980). The whole subject of the mechanism of biotin-containing enzymes has been excellently reviewed (Knowles, 1989). Acetyl-CoA carboxylase has been purified to homogeneity from various animal tissues including rat liver (Wakil et al., 1983), chicken liver (Wada and Tanabe, 1983), rat and rabbit mammary gland (Wakil et al., 1983), and goose uropygial gland (Rainwater and Kollattukudy, 1982). It is a multifunctional protein of mass 220 to 260 kDa (kilodaltons).1 By means of combined protein, chemical, and molecular cloning techniques, Takai et al. (1987) elucidated the primary structure around the biotin-binding site of

For most of the reactions in which fatty acids participate in cells, they have to be “activated” (Gurr et al., 2002). This usually involves the generation of a thiolester, such as acylCoA or acyl-ACP. Because of the importance of thiolesters, the synthesis of CoA (coenzyme A) and ACP is critical and, indeed, the CoA biosynthetic pathway has been suggested as a possible target for antibacterials (Leonardi et al., 2005). Activation of fatty acids to coenzyme A esters is discussed by Watkins (1997) and, for a general discussion of the production of acyl-thioesters in different organisms, refer to chapters in Vance and Vance (2002). Because of the poor aqueous solubility of fatty acids, even as their acyl-thioesters, and because the latter may have potentially harmful (detergent-like) effects on cells, there are binding proteins present in cells. These include fatty acid-binding proteins (FABP) as well as acyl-CoA binding proteins (ACBP) (Veerkamp and Maatman, 1995; Glatz and van der Vusse, 1996; Bernlohr et al., 1997).

10.1.2

De novo synthesis

De novo synthesis of fatty acids requires the concerted action of two multiprotein complexes or multifunctional proteins. These are acetyl-CoA (coenzyme A) carboxylase and fatty acid synthase. Acetyl-CoA carboxylase (ACCase) is a type I biotin-containing carboxylase. ACCases can have a multiprotein structure or exist as multifunctional proteins (Table 10.1). ACCase catalyses the first committed step of fatty acid and, hence, acyl lipid synthesis. Bacteria and most plant chloroplasts (Harwood, 1996) contain a multisubunit form that is readily dissociated into its component proteins.

1

637

The dalton (Da) is equal to 1/12 of the mass of an atom of 12C. Many biologists use the dalton as a unit of mass, although it is not a “recognised” unit. It is especially convenient for structures where the word “molecule” is incorrect, but is used more generally. One should write that the molecular mass (not weight) of a protein, for example, is 220,000 Da or 220 kDa.

10.1

Fatty acids

TABLE 10.1 Examples of different acetyl-CoA carboxylases Species

Protein structure

E. coli

Multiprotein complex

Yeast Dicotyledon plants

Multifunctional protein Multiprotein complex Multifunctional protein

Grasses (Poaceae)

Multifunctional proteins

Animals

Multifunctional proteins

Details Four proteins: biotin carboxylase, BCCP and carboxyltransferase (a heterodimer); transcription of all four acc genes is under growth rate control 190–230 kDa; activated but not polymerised by citrate In chloroplasts; similar properties to E. coli enzyme; probably three of the subunits coded by nucleus, one by chloroplast Presumed to be cytosolic; concentrated in epithelial cells in pea; molecular mass 200–240 kDa; functions as dimer Graminicide insensitive Two isoforms, both 200–240 kDa, which function as dimers Chloroplast isoform is graminicide sensitive, but cytosolic form (concentrated in epithelial cells) is insensitive Both are nuclear encoded Cytosolic; about 250 kDa, but functions as polymer of up to 107 kDa; aggregation increased by citrate; also regulated by phosphorylation in response to hormones

Source: From Gurr, M.I., Harwood, J.L. and Frayn, K.N. (2002). Lipid Biochemistry, 5th ed., Blackwell Scientific, Oxford, U.K. With permission.

chicken liver acetyl-CoA carboxylase, and they (in 1988) described the complete amino acid sequence of the chicken liver enzyme by cloning and sequencing the DNA complementary to the appropriate messenger RNA. The chicken liver acetyl-CoA carboxylase was found to be composed of 2324 amino acid residues with a calculated molecular mass of 262,706 Da. The BCCP domain was in the middle region of the polypeptide. The amino terminal portion showed a primary structure homologous to that of carbamyl phosphate synthase, and was thought to be the site of the biotin carboxylase for which carboxyl phosphate is the postulated reaction intermediate (see Knowles, 1989). Although animal acetyl-CoA carboxylases have molecular masses of about 250 kDa, they function as polymers of molar masses 4 × 106 to 8 × 106 g/mol (Wakil et al., 1983). The regulation of mammalian acetyl-CoA carboxylase has been well studied owing to the key role of this enzyme in controlling overall fatty acid (and fat) synthesis. Control occurs in two ways: short-term control, which involves allosteric regulation and covalent enzyme modification, or long-term control, where the amounts of the carboxylase are changed (Gurr et al., 2002). Metabolite control is due to citrate (Moss and Lane, 1971) and long-chain acyl-CoAs. The latter cause depolymerisation of mammalian carboxylases. Palmitoyl-CoA, stearoyl-CoA, and arachidoyl-CoA are the most effective at inhibiting the carboxylase (Nikawa et al., 1979). Other effectors have been reported to regulate mammalian acetyl-CoA carboxylase (cf. Wakil et al., 1983), but their physiological importance is less certain. Mammalian acetyl-CoA carboxylase is also subject to regulation by phosphorylation/dephosphorylation (Kim, 1997). Phosphorylation with one mole of phosphate per mole of rat liver carboxylase subunit causes complete inactivation. The reactions have also been studied using preparations from the rat epididymal fat pad. Long-term regulation of the carboxylase is caused by diet, thyroxine,

and insulin. Total enzyme amounts also change during cell differentiation and development (reviewed by Volpe and Vagelos, 1976). Use of antibodies to rat liver acetylCoA carboxylase revealed unexpectedly that the mitochondrial outer membrane contained a major pool of rather poorly active enzyme. It was suggested that this protein represented a reservoir of acetyl-CoA carboxylase that could be released and activated under lipogenic conditions (Allred and Roman-Lopez, 1988). However, the role of ACCase-generated malonyl-CoA in the control of mitochondria β-oxidation (see below) should be noted. The control of mammalian acetyl-CoA carboxylases has been summarised well by Goodridge (1991). Animal acetyl-CoA carboxylases have been recently reviewed by Rangan and Smith (2002), who give further information about domain organisation. They also draw attention to the two major isoforms of ACCase, α and β, which are found in animals. The α-isoform is found in the cytosol of lipogenic tissues (e.g., adipose) where it is important for de novo synthesis of fatty acids. The βisoform is present in tissues, which generally have a low lipogenic activity and is associated with the outer mitochondrial membrane. The malonyl-CoA produced by ACCase-β functions primarily as a negative regulator of carnitine palmitoyltransferase I and, therefore, regulates the flux of fatty acids into mitochondria for β-oxidation. Until fairly recently the molecular nature of plant acetylCoA carboxylase was unclear. However, it now seems that the enzyme is present as a multifunctional protein of mass 220 to 240 kDa (see Harwood, 1996) in all plant cytosols. Reference to the purification of higher molecular mass forms of the enzyme from parsley and oilseed rape as well as earlier work will be found in Harwood (1988). A similar form has also been purified to near homogeneity from the diatom Cyclotella cryptica (Roessler, 1990). In most plants (the gramineae are an exception), the major ACCase isoform, 638

Lipid Metabolism

however, is a multiprotein complex located in the chloroplast where it is used for de novo fatty acid synthesis. In gramineae (grasses), the chloroplast isoform is a multiprotein complex. In addition to acetyl-CoA carboxylase, cell-free extracts from several monocotyledonous (monocot.) and dicotyledonous (dicot.) plant species have been found to contain three additional biotin-containing enzymes — 3-methylcrotonyl-CoA carboxylase, propionyl-CoA carboxylase and pyruvate carboxylase (Wurtele and Nikolau, 1990) — the presence of which can complicate purification protocols making use of avidin affinity columns. However, propionylCoA carboxylase activity is also present in many purified plant acetyl-CoA carboxylases. Regulation of the plant acetyl-CoA carboxylase is not as well understood as it is for mammals. Tricarboxylic acids do not seem to function at all, and the enzyme in leaves may be activated, at least in part, through changes in the stroma medium as photosynthesis occurs (see Harwood, 1988). Nevertheless, recent measurement of the pool sizes of intermediates during lightactivated fatty acids synthesis in spinach leaves shows clearly that the activity of acetyl-CoA carboxylase is a key regulatory component (Post-Beittenmiller et al., 1991). Recent updates on plant acetyl-CoA carboxylase and its regulation are given by Schmid and Ohlrogge (2002) and by Harwood (2005). These refer to the different forms of ACCase found in graminaceae (grasses) compared to dicotyledons and the importance of ACCase in the regulation of lipid synthesis (see Section 10.8). In addition to the animal and some plant acetyl-CoA carboxylases, that from the yeast Saccharomyces cerevisiae also seems to be a multifunctional protein of about 250 kDa. It appears to be regulated by nutrient supply and coordinately with the fatty acid synthase genes (Trotter, 2001). In contrast to the above, the acetyl-CoA carboxylase from Escherichia coli contains four separate proteins: BCCP, biotin carboxylase and a heterodimer, carboxyl transferase (Cronan and Waldrop, 2002). In this regard, E. coli shares the same characteristics as the transcarboxylase from Propionibacterium shermanii (Samols et al., 1988). Early work on the structure of the component proteins of E. coli acetylTABLE 10.2

CoA carboxylase has been summarised (see Volpe and Vagelos, 1976; Cronan and Waldrop, 2002). The DNA sequence of the gene encoding the biotin carboxylase subunit has been reported and encodes a protein of 449 residues. The sequence is strikingly similar to the amino terminal sequence of two biotin-dependent carboxylase proteins, yeast pyruvate carboxylase and the subunit of rat propionyl-CoA carboxylase (Li and Cronan, 1992). Neither citrate nor phosphorylation plays any role in regulating the bacterial enzyme. Instead, the guanosine nucleotides, guanosine 3′-diphosphate 5-di (and tri-) phosphate, were suggested for use. However, these observations have been questioned (see Cronan and Waldrop, 2002). On the other hand, it is clear that nutrient supply is important in controlling total ACCase activity. Fatty acid synthases can be categorised as Type I, Type II, and Type III. The distribution and some of the properties of the Type I and Type II enzymes are summarised in Table 10.2. The Type I synthases tend to occur in higher organisms, and all of the purified synthases of eukaryotes, with the exception of plants, are of this type. Two bacterial genera, Mycobacterium and Corynebacterium, also contain Type I synthases. These synthases are large molecular mass multifunctional proteins containing covalently bound acyl carrier protein (ACP) (see Wakil et al., 1983). On the other hand, Type II synthases, such as that from E. coli, consist of individual enzymes that can be isolated in an active form. The ACPs of Type II synthases also readily dissociate and can be purified and characterised (see Volpe and Vagelos, 1973). Type III synthases elongate preformed acyl chains and will be dealt with later. The partial reactions of fatty acid synthases are shown in detail in Table 10.3, where assay details for measuring activities in plants are given. A comparison of some features of the Type I and Type II fatty acid synthases is made in Table 10.4. The Type I fatty acid synthases from rat liver, adipose and lactating mammary glands, rabbit mammary glands, and uropygial glands have been purified and found to have many common features (Table 10.4). The two subunits appear to be identical and of molecular mass 200 to 250 kDa

Distribution and major properties of Type I and Type II fatty acid synthases Distribution

Type I

Type II

Animals Birds Yeast Euglena gracilis (cytoplasm) Fungi Mycobacterium smegmatis Corynebacterium diphtheriae Plants Euglena gracilis (chloroplasts) Most bacteria Cyanobacteria

Major characteristics High-molecular-mass multifunctional proteins. Covalently bound ACPa. Release unesterified fatty acids (animals) or acyl-CoA (yeast).

Dissociable enzymes in a complex with ACP. Dissociable ACP. Release acyl thioesters or transfer final products directly to lipid.

ACP, acyl carrier protein. Source: From Harwood, J.L. and Russell, N.J. (1984) Lipids in Plants in Microbes, Allen and Unwin, Hemel Hempstead, U.K. With permission. a

639

10.1

Fatty acids

TABLE 10.3

Partial reactions of fatty acid synthase (FAS) and assay principles for measuring their activity in plants

Enzyme

Method

Activities common to all FASs Acetyl-CoA:ACP transacylase [14C]Acetyl-CoA + ACP [14C]Acetyl-ACP + CoA

Precipitate acetyl-ACP Count

Malonyl-CoA:ACP transacylase [14C]Malonyl-CoA + ACP [14C]Malonyl-ACP + CoA

Precipitate malonyl-ACP Count

β-Ketoacyl-ACP synthasea Acetyl-ACP + Malonyl-ACP

Acetoacetyl-ACP + CO2 + ACP

Measure absorbance at 303 nm (acetoacetate formation)

or nAcyl-ACP + Malonyl-ACP

(n + 2)Ketoacyl-ACP + ACP + CO2

In presence of NADPHb and other FAS enzymes, measure absorbance at 340 nm With [14C]malonyl-CoA, measure counts in acyl chains Use of NaH[14C]CO2 permits a CO2 exchange assay

β-Ketoacyl-ACP reductase β-Ketoacyl-ACP + NAD(P)H

β-Hydroxyacyl-ACP + NAD(P)

Change in absorbance at 340 nm

β-Hydroxyacyl-ACP dehydrase β-Hydroxylacyl-ACP Enoyl-ACP + H2O Enoyl-ACP reductase Enoyl-ACP + NAD(P)H

Back-reaction followed with crotonyl-ACP and decrease in absorbance at 263 nm

Acyl-ACP + NAD(P)

Change in absorbance at 340 nm

Additional activities present in E. coli and higher plants Acetoacetyl-ACP synthase [14C]Acetyl-CoA + Malonyl-ACP

Reaction in presence of cerulenin Precipitate acetoacetyl-ACP and count

[14C]Acetoacetyl-ACP + CoA + CO2

β-Ketoacyl-ACP synthase II Palmitoyl-ACP + [2-14C]Malonyl-ACP ACP + ACP + CO2

[2-14C] β-ketooctadecanoyl-

Reaction products reduced and counts in acyl chain measured

β-Ketoacyl-ACP synthase I in plants. NAD(P)(H), nicotinamide adenine dinucleotide (phosphate), (reduced). Source: From Harwood, J.L. et al. (1990). a

b

TABLE 10.4

Types of fatty acid synthases in different organisms

Source

Subunit types

Subunit (mol. mass)

Native (mol. mass)

Major products

α

220–270 × 103

450–550 × 103

16:0 free acid

βα

200–270 × 103

400–550 × 103

4:0–16:0 free acids

Type I: Multicatalytic polypeptides Mammalian, avian liver Mammalian mammary gland Goose uropygial gland M. smegmatis S. cerevisiae Dinoflagellates

α

2,4,6,8-tetramethyl10:0 16:0-, 24:0-CoA 16:0, 18:0-CoA

290,000 185,000, 180,000 180,000

2 × 106 2.3 × 106 4 × 105

Separate enzymes

–

–

16:0-, 18:0-ACP

Separate enzymes

–

–

Separate enzymes

–

–

12:0-, 14:0-, 16:0-, 18:0-ACP 16:0-, 18:1-ACP

α α, β α

Type II: Freely dissociable enzymes Higher plant chloroplasts E. gracilis chloroplast E. coli

Source: From Gurr, M.I., Harwood, J.L. and Frayn, K.N. (2002). Lipid Biochemistry, 5th ed., Blackwell Scientific, Oxford, U.K. With permission.

640

Lipid Metabolism

N

400

320

140

600

220

230

75

300

KS

MAT

DH

core

ER

KR

ACP

TE

C161 K326A

S581

H878

NBD-1673 S2151A NBD-1886

C

S2302A

FIGURE 10.1 Linear domain map of the animal FAS. The approximate number of residues in each domain is indicated above the map. Below are shown the locations of nucleophilic residues involved in the formation of covalent acyl-O-serine, acyl-S-cysteine, and acyl-S-phosphopantetheine intermediates, the dehydrase active-site histidine residue, and the beginning of the two glycine-rich motifs in the nucleotide-binding domains (NBD). The introduction of mutations at the critical sites indicated compromise activity of the specific domains. Residue numbering is for the rat FAS. Abbreviations: ACP, acyl carrier protein; DH, dehydrase; ER, enoyl reductase; KR, β-ketoacyl-ACP reductase; KS, β-ketoacyl-ACP synthase; MAT, malonyl acetyl transferase; TE, thioesterase. (From Smith, S., Witkowski, A. and Joshi, A.K. (2003) Prog. Lipid Res. 42, 289–317. With permission.)

(cf. Wakil et al., 1983). In contrast, the Type I synthase of yeast is not only larger (2.4 × 106 Da) than those of animals (0.4 ×106 to 5 × 106 Da), but contains two nonidentical subunits of molecular masses 208 and 220 kDa. The native yeast synthase is, thus, an α6 β6 complex (cf. Schweizer et al., 1973). The 208 kDa α subunit of the yeast FAS, encoded by the FAS2 gene, is trifunctional and contains domains for β-ketoacyl-ACP synthase, β-ketoacyl-ACP reductase, and acyl carrier protein. The 220 kDa β subunit, encoded by the FAS1 gene, possesses acetyl-, malonyl-, and palmitoyl transferase, dehydratase, and enoyl-ACP reductase activities (Trotter, 2001). The partial reactions of the yeast Type I FAS were elucidated in a classic series of experiments by Lynen (1967). Since then we have much more information, including the mechanism of reaction termination by which the acyl chain is transferred from ACP on the α-chain of FAS to CoA (Schweizer, 1984). For a general summary, see Gurr et al. (2002). Animal FAS complexes consist of homodimers of native molecular masses of 450 to 550 kDa. The earliest attempts to generate a domain map of FAS utilised limited proteolysis. As the entire sequences of several animal FASs were deduced, a more detailed domain order emerged. The order of domains has now be confirmed by the use of mutants, compromised in specific activities and is shown in Figure 10.1 (Smith et al., 2003). Various models for animal FAS have been proposed with two identical polypeptide chains arranged antiparallel that together form two centres for palmitate synthesis at the subunit interface. By the use of modified FASs in which the activity of one of the functional domains was specifically compromised by mutations, details of the dimeric structure began to be revealed (Smith et al., 2003). In a recent update, Astrurias et al. (2005) carried out cryo-EM analysis of single FAS particles and showed that the images were of two coiled monomers in an overlapping arrangement. Only limited local rearrangements were needed for catalytic interaction among different functional domains. Monomer coiling was suggested to be useful for FAS. The above papers give important information of animal fatty acid synthase (FAS), which is summarised by Rangan and Smith (2002).

The Type I fatty acid synthase of Mycobacterium smegmatis is unusual in several respects. First, the two reductases have different reduced pyridine nucleotide specificities: the β-ketoacyl-ACP reductase requires NADPH and the enoyl-ACP reductase requires NADH, whereas the reductases of other Type I synthases only use NADPH. The products of the M. smegmatis synthetase have a bimodal distribution with peaks at C16 and C24, and the overall rate of fatty acid synthesis is increased by two types of polymethylsaccharides found in the mycobacterial cell wall (Bloch, 1977). Mycobacteria also contain a Type II FAS and details of the action of both types of FAS will be found in Barry et al. (1988). The Type II synthase of E. coli was the first FAS studied. The individual proteins have all been isolated, purified, and characterised (see Volpe and Vagelos, 1973) and the genes coding for these proteins identified (White et al., 2005). The malonyl group of malonyl-CoA (produced by ACCase) is transferred to ACP by malonyl-CoA: ACP transacylase (FabD). All of the subsequent intermediates in E. coli FAS are attached to the terminyl sulfhydryl of ACP, which is one of the most abundant proteins in the bacterium. Acetoacyl-ACP is then formed by acetoacetylACP synthase (FabH), which, it should be noted, uses acetyl-CoA as the other substrate in contrast to the usual priming reaction for FAS (in Type I enzymes) (Figure 10.2). The remaining cycle of reactions continues via reduction (FabG), dehydration (FabA or FabZ), and a second reduction (FabI) to create a saturated fatty acid. Condensation then takes place with malonyl-ACP and is catalysed by FabB (β-ketoacyl-ACP synthase I) or, at later stages, with FabF (β-ketoacyl-ACP synthase II). There are two other isoforms of enoyl reductase formed in bacteria termed FabK and FabL. The FabK protein of Grampositive bacteria has an additional flavin cofactor (White et al., 2005). The E. coli FAS is also interesting in that it can produce monounsaturated (mainly cis-vaccenic) acids as well as saturated products. The ratio of the three principal products — palmitate, palmitoleate, and cis-vaccenate — is controlled by the activity of the enzymes shown in Figure 10.3. Regulation of the pathway is discussed well by Jackowski et al. (1991).

641

10.1

Fatty acids

O RCH2CHCH2C ACP

O CH3C CoA

OH Fab G

Fab A Fab Z

Acc –OOCCH C 2

CoA

O Fab D

Fab H

O RCH2CH2 = CHC ACP

O RCH2CCH2C ACP O Fab B Fab F

Fab I

O RCH2CH2CH2 C ACP

–OOCCH C ACP 2

O

FIGURE 10.2 Pathway of fatty acid synthesis in E.coli. The initial reaction is condensation between acetyl-CoA and malonyl-ACP, catalysed by FabH. After that a cycle of reduction, dehydration, and a second reduction generates a fatty acid. Later condensation utilises the gradually lengthening fatty acid and uses FabB except for the final stages when FabF is used (see text and Heath et al. (2001) for more details.) Abbreviations: Acc, acetyl-CoA carboxylase; Fab A, β-hydroxyacyl-ACP dehydratase isomerase; Fab B, βketoacyl-ACP synthase I; Fab D, malonyl-CoA: ACP transacylase; Fab F, β-ketoacyl-ACP synthase II; Fab G, β-ketoacyl-ACP reductase; Fab H, β-ketoacyl-ACP synthase III; Fab I, enoyl-ACP reductase; Fab Z, β-hydroxyacyl-ACP dehydratase. OH-10:0 β-hydroxydecanoyl-ACP 1 1 ∆2-10:1 trans-2-decenoyl-ACP

∆3-10:1 cis-3-decenoyl-ACP 2

3

∆9-16:1 palmitoleoyl-ACP

16:0 palmitoyl-ACP

4 ∆11-18:1 cis-vaccenoyl-ACP

FIGURE 10.3 Product diversification in fatty acid biosynthesis. Three main fatty acids are produced by the Escherichia coli fatty acid synthase system. The ratio of these fatty acids is controlled by the activity of three enzymes: (1) 3-hydroxydecanoyl-ACP dehydrase (encoded by the fabA gene) is a specific dehydrase that introduces the double bond into the acyl chain; (2) 3-ketoacyl-ACP synthase I (encoded by the fabB gene) catalyses an essential step in the unsaturated fatty acid elongation pathway; (3) both 3-ketoacyl-ACP synthases I and II can elongate saturated fatty acids; and (4) 3-ketoacyl-ACP synthetase II (encoded by the fabF gene) is responsible for the elongation of 9-16:1 to 11-18:1. (From Jackowski et al. (1991).)

The fatty acid synthase of Brevibacterium ammoniagenes has a high molecular mass (like Type I synthases), but produces both saturated and unsaturated acids (like E. coli type II synthetase). In most Gram-positive and some Gram-negative bacteria, branched-chain acids are made by Type II synthases that use short-chain branched acyl-CoA primers instead of acetyl-CoA and so produce iso or anteiso fatty acid products (Kaneda, 1977). The topic of lipid synthesis (and, especially, fatty acid synthesis) in relation to existing and new antibacterials has been reviewed by Heath et al. (2001, 2002) who give a summary of fatty acid synthesis in E. coli with reference to some other bacteria. Plants also contain a Type II FAS. The enzymology of the complex has been reviewed (Stumpf, 1987; Harwood, 1988, 2005). Like E. coli, there are several condensing enzymes. β-Ketoacyl-ACP synthase I (KAS I) is responsible for forming keto acids of up to 16 carbons. KAS II

then allows the condensation of palmitoyl-ACP with malonyl-ACP and, therefore, controls the production of stearate. These enzymes differ in their sensitivity to cerulenin and arsenite (see Harwood, 1988). A third condensing enzyme, KAS III, has recently been discovered and, like its counterpart in E. coli, is responsible for acetoacetyl-ACP formation (Jaworski et al., 1989; Walsh et al., 1990). Although it is believed generally that de novo synthesis of fatty acids in plants is concentrated in plastids (see Harwood, 1988), a report of acyl-ACP in plant mitochondria (Chuman and Brody, 1989), together with fatty acid synthesis mediated by ACP in Neurospora crassa mitochondria (Mikolajczyk and Brody, 1990), raise the possibility for some activity in this organelle. Much attention has been paid to plant ACP. Earlier work on its purification and properties has been well reviewed (Ohlrogge, 1987). ACP is synthesized in the cytosol and posttranslationally imported into plastids, where

642

Lipid Metabolism

proteolytic processing to the mature protein takes place. The attachment of the prosthetic group needs holo-ACP synthetase, which is cytosolic (El Hussein et al., 1988). Further aspects of the biochemistry of plant ACP are covered by Ohlrogge et al. (1991) and by Slabas and Fawcett (1992). All of the proteins catalysing the partial reactions of plant FAS have been purified and, in many cases, genes coding for them have been identified and sequenced. Most of these proteins occur as isoforms (see Harwood, 1988). Updates on the genetics of fatty acid synthesis (Ohlrogge et al., 1991) and its molecular biology (Slabas and Fawcett, 1992) have been published. Fatty acid synthesis in plants has been summarised by Schmid and Ohlrogge (2002) and reviewed in some detail by Harwood (2005). Regulation of fatty acid synthesis in plants has been discussed by Harwood (1996), Ohlrogge and Jaworski (1997), and, more recently, by Harwood (2005). The regulation of fatty acid synthesis in animals is described by Rangan and Smith (2002). Euglena gracilis is an interesting organism because, when it grows heterotrophically (when it is “animallike”), it contains a Type I synthase. When it is grown photoautotrophically, it contains, in addition to its cytoplasmic Type I enzyme, a Type II synthase in its chloroplasts (Ernst-Fonberg and Bloch, 1971). See also Worsham et al. (1988).

Formation of the very long-chain saturated fatty acids involved in the surface coverings of plants is subject to inhibition by thiocarbamate herbicides (Harwood, 1990), probably as their sulfoxide metabolites (Abulnaja and Harwood, 1991; see Section 11.8). Plant fatty acid elongation was updated by Harwood (1996) and summarised in Schmid and Ohlrogge (2002). Elongation products in yeast have been extensively studied and three separate elongase genes ELO1, ELO2, and ELO3 identified in Saccharomyces cerevisiae. All three genes are for condensing enzymes in the elongation systems (Dittrich et al., 1998). More recently, progress has been made in identifying other components. The yeast systems elongate saturated and monounsaturated fatty acids with different chain-length specificities (Leonard et al., 2004). Although there are some animal elongases, which have substrate preferences for saturated or monounsaturated fatty acids, many of them are used to elongate polyunsaturated fatty acids (PUFAs). This is not surprising because most dietary PUFA are 18C molecules, whereas most biologically effective metabolites are 20 or 22C (see Sections 10.1.8 and 11.1). Very long-chain PUFAs are particularly important components of mammalian brain (especially 22:6n-3) and it is not surprising that some of the first studies identified different elongases in this tissue (Goldberg et al., 1973; Bourre et al., 1975). Details of the ELOV (very long-chain elongase) genes from humans, rats, and mice are given in Leonard et al., (2004). Like the plant enzymes, the mammalian elongases are microsomally located (endoplasmic reticulum) and use malonyl-CoA in four steps (like FAS) to produce products 2C longer (see Cook and McMaster, 2002; Leonard et al., 2004). Fatty acids can also be elongated in mammals by a mitochondrial system, which uses acetyl-CoA as the unit for C2 addition and NADH for reduction. In general, the mitochondrial system elongates fatty acids in the range C10 to C14, whereas the microsomal system uses C16 and longer acids. At first it was thought that the mitochondrial elongation system could operate by a reversal of β oxidation, but this is now not considered thermodynamically feasible. Indeed, the (flavin adenine dinucleotide) FAD-dependent acyl-CoA dehydrogenase of oxidation is substituted by a more thermodynamically favorable enzyme, enoyl-CoA reductase, which is rate-limiting for the overall process in mitochondria (Cook and McMaster, 2002). In contrast to animals and plants, bacteria do not usually contain significant amounts of acids longer than C18. A notable exception is the long-chain (up to C56) fatty acids found in mycobacteria. These are formed by an elongation system using acetyl-CoA and NADH, which may be a reversal of β oxidation (Harwood and Russell, 1984). Fatty acid elongation systems in lower eurkaryotes (the nematode Caenorhabditis elegans, fungi, microalgae, moss) are described by Leonard et al. (2004).

10.1.3 Elongation systems As mentioned in Section 10.1.2, the elongation of fatty acids is generally carried out by Type III fatty acid synthases, which use malonyl-CoA as the source of C2 units for addition. In plants, the elongation of stearate to form very longchain saturated fatty acids (which are precursors of the various components of waxes, cutin, and suberin; see Kolattukudy, 1980, 1987) takes place via several chain length-specific systems (Walker and Harwood, 1986). Malonyl-ACP and NADPH are required (see Harwood, 1988, 1996) and a cycle of condensation, reduction, dehydration, and second reduction reactions using acyl-CoAs are established. Proteins catalysing these partial reactions have been demonstrated in leek (Lessire et al., 1989) and Lunaria annua (Fehling et al., 1992). In the latter case, monounsaturated acyl-CoAs are also substrates, and the elongation of such acids is important in brassicas, such as rapeseed, where erucate (∆13-22:1) is a major component of the seed oil (Section 2.2). The genetics of the rape elongation system are well described by Ohlrogge et al., (1991). Elongation in jojoba (which accumulates lipid as wax esters) uses a system with oleoyl-CoA and malonyl-CoA as substrates, and this plant has been studied in some detail (Pollard and Stumpf, 1980).

643

10.1

Fatty acids

10.1.4

Desaturases

cis double bond and results in the synthesis of a series of monoenoic fatty acids (Figure 10.4). Details of the enzymes and the position of the genes involved in their synthesis are summarised in Heath et al. (2002). The ratio of the three main products of E. coli FAS is controlled by the relative activity of three enzymes, as illustrated in Figure 10.3. Aerobic desaturation involves the stereospecific removal of two hydrogen atoms from an acyl chain. Along with reducing equivalents from NAD(P)H or other reductants, the hydrogen atoms are used to reduce molecular oxygen to water. The desaturases are membrane-bound multienzyme complexes, with the exception of the stearoyl-ACP desaturase found in chloroplasts. The basic details of aerobic desaturation appear to be the same in all organisms in that oxygen and a reduced cofactor are necessary, although the nature of the carriers varies in different systems (Figure 10.5) as well as their susceptibility to cyanide

Unsaturated fatty acids can be produced by anaerobic or aerobic pathways — the latter being the most usual mechanism. Aerobic desaturases have been studied in a large number of cases, although successful purifications have only been made a few times. A general summary is found in Gurr et al. (2002). The anaerobic method, which is used by many members of the Eubacteriales, including all the anaerobes as well as some aerobes and facultative aerobes, has already been summarised in Section 10.1.2. It relies on the activity of the β-hydroxy-decanoyl-ACP β, γ-dehydrase whose isomerase activity allows the synthesis of a cis-3-decenoyl-ACP from the usual trans-2-decenoyl-ACP. cis-3-Decenoyl-ACP cannot be reduced by the enoyl-ACP reductase, but can be condensed by the β-keto-acyl-ACP synthase. This reaction, therefore, allows chain lengthening, but preserves the n-7 Acetyl-ACP

β-Ketoacyl-ACP synthetase I (fabB) or II (fabF )

β-Hydroxydecanoyl-ACP β, γ-Dehydrase

trans-2-Decenoyl-ACP

(fabA)

cis-3-Decenoyl-ACP β-Ketoacyl-ACP synthetase I (fabB)

β-Ketoacyl-ACP synthetase I (fabB) or II (fabF)

∆9-16:1 β-Ketoacyl-ACP synthetase II (fabF, Cvc−)

16:0 18:0

∆11-18:1

Saturated fatty acids

Unsaturated fatty acids

FIGURE 10.4 Anaerobic pathway of fatty acid biosynthesis in bacteria showing mutants of Escherichia coli. Here fab refers to deficient mutants described for E.coli. (From Harwood, J.L. and Russell, N.J. (1984) Lipids in Plants in Microbes, Allen and Unwin, Hemel Hempstead, U.K. With permission.) O (R1)CH Electron donor (reduced) Electron donor (oxidised)∗

Oxidised cytochrome Oxidoreductase protein Reduced cytochrome∗∗

Cyanide sensitive protein

CH(R2)C X 2H2O

O2 O (R1)CH2CH2(R2)C X∗∗∗

FIGURE 10.5 A generalized scheme for aerobic fatty acid desaturation: *e.g., NADH, NADPH, reduced ferredoxin; **e.g., cytochrome b5; ***e.g., acyl-ACP (stearoyl-ACP ∆9-desaturase in plants); acyl-CoA (stearoyl-CoA ∆9-desaturase in animals); oleoylphosphatidylcholine (∆12-desaturase in yeast or plants); linoleoyl-monogalactosyldiacylglycerol (∆15-desaturase in plant chloroplasts). (From Gurr, M.I., Harwood, J.L. and Frayn, K.N. (2002). Lipid Biochemistry, 5th ed., Blackwell Scientific, Oxford, U.K. With permission.)

644

Lipid Metabolism

inhibition. There is also some controversy as to whether the two hydrogen atoms are removed sequentially (with the involvement of a hydroxy intermediate) or in a concerted mechanism. Evidence with Corynebacterium diphtheria using tritiated substrates suggested a stepwise mechanism (Schroepfer and Bloch, 1965), whereas experiments with 2H-Iabelled substrates and Chlorella vulgaris indicated concerted hydrogen removal (Morris, 1970). The first double bond inserted into an acyl chain is usually ∆9. The stearoyl-CoA ∆9-desaturase was first purified from rat liver (Strittmatter et al., 1974). The complex consists of three major proteins: NADH-cytochrome b5 reductase, cytochrome b5, and a terminal desaturase (or cyanide-sensitive) protein. Usually the activity of the terminal desaturase limits the overall speed of desaturation. The NADH-cytochrome b5 oxidoreductase is a flavoprotein of mass 43 kDa, cytochrome b5 has a molecular mass of 16.7 kDa, and the terminal desaturase is 53 kDa (see Cook, 1991 ). The approximate location of the three components in the endoplasmic reticulum has been deduced from their amino acid sequence and the use of specific chemical reagents. A useful summary of fatty acid desaturation in animals is given in Cook and McMaster (2002). The ∆9-desaturases from plants and algae (and Euglena gracilis) use stearoyl-ACP (the final product of their FASs) as substrate. The enzyme was first purified substantially from developing safflower seeds by McKeon and Stumpf (1982). The 9-(stearoyl) desaturase has been cloned from castor seeds (Knutzon et al., 1991) and from safflower (Thompson et al., 1991). The cDNA from safflower includes a 33 amino acid transit peptide. Modulation of the steroyl-ACP desaturase level, using antisense technology in transgenic rapeseed, resulted in a marked decrease in oleate with a commensurate increase in stearate (Kridl et al., 1991). For summaries of the stearoyl-ACP desaturases from plants, refer to Harwood (1996). Mutations affecting the levels of oleic acid in crop plants are discussed in Schmid and Ohlrogge (2002). In contrast to animals and plants, bacteria are unique in producing ∆10-monoenoic fatty acids. Bacilli commonly possess ∆5- or ∆10-desaturases, and Bacillus licheniformis contains both enzymes when it grows at low temperatures (Fulco, 1974). Under these conditions, it synthesizes small amounts of ∆5,10-16:2. It will be noted that, although this acid is polyunsaturated, it does not possess the usual methylene-interrupted structure. In fact, a general distinction is often made between bacteria and other organisms in that the former are unable to synthesize methylene-interrupted polyunsaturated fatty acids. This is not always true. For example, the filamentous gliding bacteria (Flexibacter spp.) contain considerable amounts of (n-3)20:5, which in some species are the major fatty acids (Johns and Perry, 1977). Morever, marine bacteria may be able to produce methylene-interrupted PUFAs (see below). Other features of aerobic desaturation in bacteria are discussed by Schweizer (1989). However, it should be

noted that the “anaerobic” pathway utilizing fatty acid synthase is characteristic of many bacteria, particularly those of the orders Pseudomonadales and Eubacteriales. It is now established that several species of Gramnegative bacteria, mainly marine, can produce PUFAs (Tocher et al., 1998). Attention has been particularly focused on Shewanella spp. (Metz et al., 2001) where a polyketide synthase seems to be used (see also Valentine and Valentine, 2004). Most mammalian tissues can modify acyl chain composition by introducing more than one bond. Like ∆9-desaturation, further desaturation requires molecular oxygen and an associated electron transport system. Animal systems cannot generally insert double bonds beyond the ∆9 position. Consequently, double bonds are inserted at the ∆6, ∆5, and ∆4 positions. However, synthesis of linoleate from oleoyl-CoA has been demonstrated in some insects (de Ronobales et al., 1987). Protozoa, such as Tetrahymena (Umeka and Nozawa, 1984) and Acanthamoeba castellanii, also contain desaturases capable of producing linoleate. The latter system has been studied in some detail and shown to take place on phosphatidylcholine and probably is an n-6 (rather than a ∆12-) desaturase (Jones et al., 1993; Rutter et al., 2002). Interestingly, the Acanthamoeba n-6 desaturase is induced by low temperature (Avery et al., 1995) and, independently, by oxygen changes (Thomas et al., 1998). Polyunsaturated fatty acids in all organisms usually contain methylene-interrupted double bonds and conjugated systems are rare. However, so-called conjugases (that produce conjugated PUFAs) have been found in a number of plants and their genes have been cloned (e.g., Cahoon et al., 1999; Iwabuchi et al., 2003). The animal desaturases are also used to further modify polyunsaturated fatty acids from the diet (e.g., linoleate, α-linolenate), and the major pathways are depicted in Figure 10.6. Cook and McMaster (2002) have summarised the metabolism of acids of the n-6 family (linoleate, etc.) and the n-3 family (α-linolenate) and competition between the pathways. In contrast to the stearate desaturases discussed above, the enzymes forming linoleate and α-linolenate in lower and higher plants use complex lipids as substrates. The use of phosphatidylcholine in this way was proposed originally following experiments with the yeast Candida albicans (Pugh and Kates, 1975) and with the green alga Chlorella vulgaris (Nichols et al., 1967). However, some yeasts contain both oleoyl-CoA and oleoyl-phospholipid desaturases (Pugh and Kates, 1975). More recent work covering the ∆9, ∆12, and ∆6-desaturases from various yeasts or fungi is summarised by Tocher et al. (1998). In the fungus, Mortierella alpina, a number of desaturases including a ∆5 enzyme must be present because this organism can produce arachidonate. Other species can also produce 20:5n-3 (EPA) and, therefore, must have a ∆15 (n-3) desaturase as well (Tocher 645

10.1

Fatty acids

Diet

Linolenic acid

Endogenous synthesis Oleic acid

18:1(n - 9) ∆9

20:1(n - 9) ∆11

Linoleic acid

18:2(n - 6) ∆9, 12

∆6 18:2(n - 9) ∆6, 9

20:2(n - 6) ∆11, 14

∆5

20:2(n - 9) ∆8, 11

18:3(n - 3) ∆6 ∆9, 12, 15

18:4(n - 3) ∆6, 9, 12, 15

∆6 18:3(n - 6) ∆6, 9, 12

20:3(n - 6) ∆5 ∆8, 11, 14

20:3(n - 9) ∆5, 8, 11

20:4(n - 3) ∆5 ∆8, 11, 14, 17

20:5(n - 3) ∆5, 8, 11, 14, 17

20:4(n - 6) ∆5, 8, 11, 14 22:5(n - 3) ∆7, 10, 13, 16, 19

22:1(n - 9) ∆13

24:1(n - 9) ∆15

22:6(n - 3) ∆4, 7, 10, 13, 16, 19

Desaturation

β-oxidation 24:5(n - 3) ∆6 24:6(n - 3) ∆9, 12, 15, 18, 21 ∆6, 9, 12, 15, 18, 21

2-Carbon chain elongation

22:4(n - 6) ∆7, 10, 13, 16

22:5(n - 6) ∆4, 7, 10, 13, 16 β-oxidation

24:4(n - 6) ∆6 24:5(n - 6) ∆9, 12, 15, 18 ∆6, 9, 12, 15, 18

34:6(n - 3) ∆16, 19, 22, 25, 28, 31

34:4(n - 6) ∆19, 22, 25, 28

FIGURE 10.6 Major pathways for polyunsaturated fatty acid synthesis in animals. Note the alternating sequence of desaturation in the horizontal direction and chain elongation in the vertical direction in the formation of polyunsaturated fatty acids from dietary essential fatty acids. Type size for individual fatty acids reflects, in a general way, relative accumulation in tissues. (From Gurr, M.I,. Harwood, J.L. and Frayn, K.N. (2002). Lipid Biochemistry, 5th ed., Blackwell Scientific, Oxford, U.K. With permission.)

et al.,1998). Genes isolated from Mortierella, as well as from various algae, have proven useful in the genetic manipulation of higher plants to produce very long-chain PUFAs (see Section 11.8). In plants, desaturation of oleate to linoleate uses phosphatidylcholine (in the endoplasmic reticulum) as the major substrate. The enzyme in safflower has been shown to use cytochrome b5 as an electron source (Kearns et al., 1991; Smith et al., 1992). However, oleate desaturation in chloroplasts uses ferredoxin (Schmidt and Heinz, 1991). Other complex lipid substrates probably are also used (see Harwood. 1988), especially as demonstrated for chloroplast lipids (see Jones and Harwood, 1980). Further desaturation of linoleate to α-linolenate can take place either using monogalactosyldiacylglycerol (Jones and Harwood, 1980) or with phosphatidylcholine. The relative importance of these two substrates depends on the type of tissue: leaves use monogalactosyldiacylglycerol mainly, but seeds (which have poorly developed plastids) use the nonchloroplastic phosphatidylcholine as the main substrate. These aspects are discussed by Harwood (1996)

and by Browse and Somerville (1991). The use of complex lipids for substrates, the cooperation of extraplastidic with plastid pathways and the site of Arabidopsis mutations in the pathways are shown in Figure 10.7. A recent update of plant fatty acid desaturation is by Hildebrand et al. (2005). The fatty acid desaturation systems in cyanobacteria have been studied in detail by Murata and colleagues. This work was particularly in relation to chilling sensitivity. The research has been summarized in Murata and Wada (1995). The possible roles of signaling pathways in desaturase induction are reviewed by Mikami and Murata (2003).

10.1.5 Hydroxylation Hydroxy fatty acids are formed as intermediates in various metabolic sequences (e.g., fatty acid biosynthesis, β-oxidation) as a result of specific hydroxylation reactions, and following other activities, such as those of lipoxygenase (see Section 10.1.7). The hydroxyl group is usually introduced close to one end of the acyl chain 646

Lipid Metabolism

Endoplasmic reticulum PI, PG

CDP·DAG

PE fad 2

PA 18:1 18:1 (16:0)

16:0·CoA 18:1·CoA G3P LPA

PA 18:1 16:0 PG

MGD

act 1 18:1 16:0 18:1 16:0

DAG 18:1 18:1 (16:0)

PC 18:1 18:1 (16:0)

DAG 18:1 16:0 DGD

fad 3

PC 18:2 18:2 (16:0)

PC 18:3 18:3 (16:0)

DAG 18:2 18:2 (16:0) SL

18:1 16:0 18:1 16:0

MGD 18:2 18:2

DGD 18:2 18:2 (16:0)

SL 16:0 18:2

fad A 18:1·ACP

fad B

G3P

18:1 t16:1 18:1 16:1 (16:0) 16:0·ACP fad C 18:0·ACP

fad C

18:2 t16:1 18:2 16:2 (16:0)

18:2 16:0 18:2 16:0

18:3 t16:1 18:3 16:3 (16:0)

18:3 16:0 18:3 16:0

Fatty acid fad D synthase

fad D

fad D 18:3 18:3 18:3 18:3 (16:0)

16:0 18:3

PLASTID

FIGURE 10.7 An abbreviated diagram of the two-pathway scheme of glycerolipid biosynthesis in the 16:3 plant Arabidopsis. Widths of the lines show the relative fluxes through different reactions. (From Browse and Somerville, 1991.)

(e.g., α,β) and less commonly in the middle of the chain. A good example of the latter in plants is the formation of ricinoleic acid (the major acid of castor oil, Section 2.2.2 and Section 9.8) by hydroxylation of oleoyl-phosphatidylcholine substrate using NADH and oxygen as co-factors (Moreau and Stumpf, 1981; Smith et al., 1992). Few of the hydroxylases have been characterised, so it is difficult to compare their properties. However, they appear to be mixed-function oxidases and require molecular oxygen and a reduced pyridine nucleotide. Most of the enzymes appear to require cytochrome P450, although ferredoxin (or a related haem protein) may substitute in some bacterial or plant systems (see Gaillard, 1980). Cellfree hydroxylating systems have been studied extensively in Bacillus megaterium and Pseudomonas oleovorans and some enzymes have been purified (see Schweizer, 1989). α-Oxidation systems producing 2-hydroxy fatty acids have been demonstrated in yeasts, bacteria, plants (see Harwood and Russell, 1984), and animals, while ω-oxidation systems introduce a hydroxyl group to the methyl end of the acyl chain. These oxidations are described more fully in Section 10.1.6. The major hydroxy fatty acids in plants have an ω-OH and an in-chain OH group (e.g., 10,16-dihydroxypalmitic acid). Their synthesis seems to involve ω-hydroxylation with NADPH and O2 as cofactors, followed by in-chain hydroxylation using the same co-substrates. If the

precursor is oleic acid, then the double bond is converted to an epoxide, which is then hydrated to yield 9,10-hydroxy groups. These conversions involve CoA esters. In-chain plant hydroxylase is sensitive to inhibition by O-phenanthroline and by CO in a reaction that is reversed by 420 to 460 nm light (Kolattukudy, 1980, 1987). It should be noted that a few hydroxylations occur without a mixed-function oxidase enzyme. For example, the ergot fungus Claviceps purpurea forms 12-hydroxyoleic (ricinoleic) acid by hydration of linoleic acid (Harwood and Russell, 1984).

10.1.6

Oxidation of fatty acids

Oxidation of fatty acids can occur in a number of ways, depending on the position of oxidation (e.g., α-, β-, or ω-oxidation) and the nature of the substrate (e.g., lipoxygenase attack on polyenoic fatty acids). 10.1.6.1

α-Oxidation

The removal of a single carbon atom from the carbonyl end of a fatty acid is carried out by α-oxidation. This process is particularly active in plants, but is also found in mammals (notably brain tissue) and bacteria. The removal of a single carbon may be important when degradation of a fatty acid by β-oxidation (see below) is blocked by the presence of a methyl branch at position 3, such as in phytanic acid. Defects in α-oxidation of 647

10.1

Fatty acids

CH3 RCH2CH

CH3 CH2COOH

α-oxidation (4 steps)

Phytanic acid

RCH2CH

CH3 CO

SCoA

β-oxidation

Pristanoyl-CoA RCO

FIGURE 10.8

(acyl-CoA synthetase)

Pristanic acid

CH3 RCH2CH

COOH

RCOCHCO (hiolase)

SCoA

SCoA + CH3CH2CO

SCoA

Phytanic acid metabolism.

(2H) RCH2COOH

O2

[RCH(OOH)COOH]

H 2O RCH(OH)COOH

etc. RCOOH NADH

CO2 + H2O RCHO NAD+

FIGURE 10.9

The α-oxidation of fatty acids in plants. Adapted from Galliard (1980).

phytanic acid give rise to Refsum’s disease and there are other disorders giving rise to defects in α-oxidation (Mukherji et al., 2003). Human metabolism of phytanic and pristanic acids is reviewed by Verhoevan and Jakobs (2001) (see Section 11.6) and is shown in simplified form in Figure 10.8. α-Oxidation is also important because α-hydroxy fatty acids are intermediates in the process and these acids are components of certain sphingolipids (Bowen et al., 1974). Thus, brain cerebroside fatty acids are highly enriched in cerebronic acid (α-OH 24:0). Furthermore, tissues that accumulate large amounts of fatty acids almost invariably also contain significant amounts of odd chain-length fatty acids (e.g., see Mcllwain, 1966). In plants the mechanism of α-oxidation in leaves and seeds is identical. The fatty acid substrate is nonesterified (cf. β-oxidation) and is usually C12 to C18. It is attacked by molecular oxygen to generate an unstable 2-hydroperoxy intermediate (Figure 10.9), which decomposes to an aldehyde with release of carbon dioxide. Under certain conditions (e.g.,, in the presence of an enzyme, such as glutathione peroxidase, which will reduce peroxides), then a D-2-hydroxy fatty acid may be produced, which cannot be metabolized easily. Under normal conditions, though, the aldehyde is oxidized in the presence of a source of reducing power (pyridine nucleotide or flavoprotein depending on the tissue) to give a fatty acid one carbon atom shorter than the original fatty acid. A significant breakthrough to our understanding of α-oxidation in plants has come from the discovery of a pathogeninducible oxygenase, which has significant homology to

prostaglandin endoperoxide H synthase (see Section 10.1.8) (Graham and Eastmond, 2002). This has led to the suggestion that a major role for α-oxidation in plants is in defence responses to pathogens (Hamberg et al., 1999). The occurrence of bacterial fatty acid α-oxidation is not firmly established and, if present, represents a pathway of minor importance (Finnerty, 1989). It has been studied in few bacterial species. However, in E.coli, the D-2-hydroxy fatty acid is preferentially decarboxylated, unlike in plants and animals where it is the L form that is metabolised preferentially. Thus, the 2-hydroxy acids tend not to accumulate in bacteria (Lekakis, 1977). 10.1.6.2

b-Oxidation

The β-oxidation of fatty acids was the first metabolic process in which labeled compounds were used for its investigation. Knoop’s classic experiments at the turn of the century were later confirmed and extended by others to reveal the details of the process (for references, see Greville and Tubbs, 1968; Wakil, 1970; Kunau et al., 1995; Schulz, 2002). Key steps in the pathway are the activation of the fatty acid to a coenzyme A thioester, the α,β-dehydrogenation of the acyl-CoA, the hydration of the resultant double bond, oxidation of the β-hydroxyacylCoA, and thiolytic cleavage of the β-ketoacyl-CoA (Figure 10.10). The mechanism of fatty acid uptake by animal cells has not been fully elucidated. Plasma membrane carrier proteins responsible for saturable high-affinity uptake have been identified, but some workers argue that spontaneous and nonspecific diffusion of fatty acids across the 648

Lipid Metabolism

R CH2 CH2 COOH Cell membrane R CH2 CH2 COOH FABP?

CYTOSOL

R CH2 CH2 COOH ATP AS

CoASH

CoASH

AMP + PPi

Outer mitochondrial membrane

CPTI

R CH2 CH2 CO Carnitine

R CH2 CH2 COSCoA Carnitine

Inner mitochondrial membrane

T Carnitine R CH2 CH2 CO Carnitine CoASH

R

CH

AD

CH COSCoA H 2O

CPTII

R CH2 CH2 COSCoA R COSCoA CH3 COSCoA

EH

KT CoASH

R CH OH

CH2 COSCoA

HAD

NAD+

R C CH2 COSCoA O

MATRIX

NADH + H+

FIGURE 10.10 Pathway of mitochondrial fatty acid oxidation. Enzymes of the pathway are AS, acyl-CoA synthetase; CPT I, carnitine palmitoyltransferase I; T, carnitine:acylcarnitine translocase; CPT II, carnitine palmitoyltransferase II; AD, acyl-CoA dehydrogenase; EH, enoyl-CoA hydratase; HAD, L-3-hydroxyacyl-CoA dehydrogenase; KT, 3-ketoacyl-CoA thiolase. Other abbreviation: FABP, fatty acid binding protein. (From Schulz, 1991b.)

plasma membrane is also important (see Schulz, 2002). β-Oxidation is preceded by activation of fatty acids to their coenzyme A (CoA) thioesters. A group of acyl-CoA synthetases have been identified that differ in their chainlength specificities, but which catalyse the same type of reaction: R-COOH + ATP + CoASH

Three enzymes (differing in chain-length specificity) that catalyse α,β-dehydrogenation of acyl-CoAs have also been purified. The overlapping specificities of these enzymes allow efficient oxidation of fatty acids in the C4 to C20 range (Greville and Tubbs, 1968). The acyl-CoA dehydrogenases (EC 1.3.99.3) are flavoproteins containing FAD (flavin adenine dinucleotide) as a prosthetic group. They have molecular masses of 170 to 190 kDa and are composed of four identical subunits, each of which carries a noncovalently bound FAD. Nucleotide sequences for several rat liver and human acyl-CoA dehydrogenases have been determined, with a greater than 90% homology observed for the same enzyme from rat compared to human (see Schulz, 2002). Hydration of the α,β-trans double bond is catalysed by enoyl-CoA hydratase (EC 4.2.1.17; crotonase). This enzyme will also attack cis double bonds and 3-enoyl-CoAs. An L-OH fatty acid is produced (∆4, in the latter case, and ∆3, in β-oxidation) from the trans isomers. For more details, see Schulz (2002). The L-3-hydroxyacyl-CoA obtained in the above step is oxidized by NAD+ in the presence of 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35). The enzyme is specific for the L form and usually has little chain-length specificity (Wakil, 1970). The enzyme has been purified from pig

RCO-SCoA + AMP + PPi

The individual acyl-CoA synthetases are grouped as short-chain, medium-chain, or long-chain synthetases, depending on their substrate specificities. Short-chain synthetases, which act on acetate or propionate, are present in the mitochondria of most animal tissues, but, in addition, are found in the cytosol of lipogenic tissues, such as liver, intestine, adipose tissue, and mammary gland. Medium-chain synthetases are also located in the mitochondrial matrix. By contrast, long-chain acyl-CoA svnthetases in animals are membrane-bound and associated with the endoplasmic reticulum, mitochondrial outer membrane or peroxisomes (see Schulz, 2002). Acyl-CoAs that need to be taken up by mitochondria do so via a carnitine-dependent mechanism. Two carnitine palmitoyltransferases and a carnitine:acylcarnitine translocase are needed, as illustrated in Figure 10.10. 649

10.1

Fatty acids

SCoA 13 12

10 9

O hree cycles of β oxidation SCoA

7

6

4

3

O Enoyl-CoA isomerase O

3

SCoA 2 One cycle of β oxidation

7 6 O

SCoA

5 4

Acyl-CoA dehydrogenase O

3

SCoA

2

5 4

2, 4-Dienoyl-CoA reductase

4

SCoA 3

O Enoyl-CoA isomerase 2

3

SCoA O Four cycles of β oxidation SCoA O

FIGURE 10.11

The β-oxidation of linoleoyl-CoA. (From Schulz, 1991b.)

heart and rat liver. It has a molecular mass of 65 kDa, is composed of two identical subunits, and its conformation at 2.8 Å resolution has been determined (Schulz, 1991). Some enzymes will utilize NADP+ more slowly and these dehydrogenases will also oxidize S-3-hydroxyacyl-Nacylthioethanolamine and S-3-hydroxyacyl-hydrolipoate. Thiolytic cleavage of the 3-ketoacyl-CoA is catalysed by acetyl-CoA acyltransferase (EC 2.3.1.16), which liberates acetyl-CoA and an acyl-CoA two carbons shorter than the original substrate (see Figure 10.10). The above enzyme has a broad chain-length specificity, but a second enzyme, acetyl-CoA acetyltransferase (EC 2.3.1.9), catalyses the same reaction with acetoacetyl-CoA as substrate. Kinetic and exchange studies indicate that these thiolase reactions occur in two stages (see Greville and Tubbs, 1968). Both types of thiolases are homotetramers of mass about 170 kDa. While the 3-ketoacyl-CoA thiolase with its broad chain-length specificity is essential for β-oxidation, the acetoacetyl-CoA thiolase probably functions mainly in ketone body metabolism (Schulz, 1991). All mitochondrial enzymes of β-oxidation are synthesised in the cytosol on free polysomes (see Ozasa et al., 1984).

Oxidation of saturated straight-chain fatty acids proceeds smoothly using the above series of enzymes, but there are problems in the breakdown of other fatty acids. For example, odd-chain acids finally liberate propionyl-CoA, which is converted to succinyl-CoA for further metabolism. Unsaturated fatty acids must also be modified because their double bonds may be in the wrong position and because of incorrect configuration for normal catabolism. Epimerase and isomerase enzymes are present to cope with these problems (see Gurr et al., 2002 and Schulz, 2002; Figure 10.11). Apart from β-oxidation in mitochondria, peroxisomes are also important. The reactions and enzymes involved have been characterised by Hashimoto (1990), with the research being aided by the use of drugs, such as clofibrate or di(2-ethyl-hexyl)phthalate, which cause peroxisome proliferation and also induce the enzymes of peroxisomal β-oxidation (Lazarow and Moser, 1989). Although the intermediates of peroxisomal β-oxidation are the same as in mitochondria, there are differences in the enzymes concerned. The first reaction is catalysed by acyl-CoA oxidase, which generates hydrogen peroxide as a product. 650

Lipid Metabolism

The latter is rapidly destroyed by peroxisomal catalase activity. The hydration and second dehydrogenation steps are catalysed by a trifunctional enzyme. The essential features are illustrated in Figure 10.12. Because the acylCoA oxidase is almost inactive towards octanoyl-CoA (or shorter chains), peroxisomes are incapable of complete oxidation of fatty acids. For the oxidation of unsaturated fatty acids, such as linoleate, a 2,4-dienyl-CoA reductase is present and a ∆3-cis, ∆2-trans-enoyl-CoA isomerase (as the third activity of the trifunctional enzyme) is also needed (see Schulz, 1991; Osmundsen et al., 1991). The main function of peroxisomal β-oxidation in animals seems to be in the chain shortening of very long-chain fatty acids, prostaglandins, dicarboxylic acids, and various xenobiotic compounds (see Schulz, 1991). AMP + PP

In plants, it was observed a number of years ago that peroxisomes, such as the glyoxysomes of germinating oil seeds, contained enzymes necessary for fatty acid β-oxidation (see Galliard, 1980; Kindl, 1987). Early aspects of the work were discussed by Beevers (1980). A thorough review has covered aspects of β-oxidation in plants (Gerhardt, 1992). The author concluded that there was little evidence for any other location for β-oxidation apart from in peroxisomes. Nevertheless, the possibility that mitochondria could be involved for the oxidation of straight chain acids is still not resolved and these organelles seem to be utilized for β-oxidation of short branched-chain 2-oxo acids (Graham and Eastmond, 2002). The latter authors have made a thorough review of peroxisomal β-oxidation in plants and its regulation.

(HOOC) CH3(CH2CH2)nCH2CH2COOH ATP + CoASH

(HOOC) CH3(CH2CH2)nCH2CH2CO H 2O

CoA

S

O2 Acyl-CoA oxidase

Catalase H2O2

(HOOC) CH3(CH2CH2)nCH CHCO H2 O

S

CoA

Bi(tri)functional enzyme

(HOOC) CH3(CH2CH2)nCHCH2CO

S

CoA

OH NAD+

RH2 R

Bi(tri)functional enzyme

NADH

(HOOC) CH3(CH2CH2)nCCH2CO

S

CoA

O CoA

hiolase

(HOOC) CH3(CH2CH2)nCO Chain-shortened acyl-CoA

S

CoA + CH3CO

S

CoA

Acylcarnitine/FFA/acyl-CoA Acetylcarnitine/acetate/acetyl-CoA

FIGURE 10.12 Essential features of peroxisomal β­oxidation. A schematic representation of the main metabolic features of peroxisomal ­ oxidation. The enzymes of peroxisomal β­oxidation are contained within the peroxisomal membrane (rectangular box). Possible end products of β­oxidation are indicated at the bottom. The [HOOC] to the left of the substrate acyl-CoA ester indicates that monoCoA esters of dicarboxylic acids are also substrates for peroxisomal β­oxidation. An incorrect stoichiometry of catalase-dependent decomposition of H2O2 is used for the sake of brevity. The arrows pointing down to Acylcarnitine/FFA/acyl-CoA, or to Acetylcarnitine/ acetate/acetyl-CoA are meant to indicate possible alternative forms of export of chain-shortened fatty acids out of the peroxisome (FFA = free fatty acid). The oxidant (R) of NADH active in vivo remains to be established. With isolated peroxisomes, pyruvate (and lactate dehydrogenase) can function in this capacity. (From Osmundsen et al., 1991.)

651

10.1

Fatty acids

O H3CCH2CSCoA

O

O CH2

CHCSCoA

HOCH2CH2CSCoA

O

O CoASCCH3 + CO2

FIGURE 10.13

HOCH2CH2COH

O

O

CoASCCH2COH

O

HC CH2COH

Propionate metabolism in plants.

Plants operate a different mechanism from animals when removing the propionate liberated from odd chainlength fatty acid oxidation. The plant system involves a hydratase and two hydrogenase enzymes, and the overall scheme is summarized in Figure 10.13. The pathway is superficially similar to modified β-oxidation in bacteria, but the two systems differ in the fate of the individual carbon atoms (Stumpf, 1970). Bacteria take up nonesterified fatty acids from the growth medium by a process that probably involves the formation of acyl-CoA. The fatty acid taken up, therefore, is in a form directly available for β-oxidation. In E. coli there has been some study of the genes coding for fatty acids degradation (fad). These genes, which code for the enzymes of uptake/activation and β-oxidation, are located in three sites on the chromosome and comprise a regulon (Klein et al., 1971). Several types of fad mutants are known and another class of mutants, fadR, are constitutive for β-oxidation and are probably repressor mutants. In addition to being a repressor for fad genes, the fadR gene product may also control isocitrate lyase and malate synthetase (Maloy et al., 1980), which are key enzymes of the glyoxylate shunt. There are some differences in the details of β-oxidation enzymes in E. coli compared to those from animals. For example, only one acyl-CoA synthetase is present and this can activate both medium-chain and long-chain fatty acids (Schulz, 2002). Even though E. coli does not synthesize polyunsaturated fatty acids, it can easily oxidize them by the reductasedependent pathway (above). Details of the enzymes involved in E. coli fatty acid β-oxidation and available mutants that are defective in individual steps are given by Finnerty (1989). The same author also discusses the features of peroxisomal β-oxidation in microbial eukaryotes, such as Candida tropicalis. Other sources of information on E. coli are Nunn (1986) and Black and Dirusso (1994) and for other bacteria (Kunau et al., 1995). The control of mitochondrial β-oxidation flux is reviewed by Eaton (2002). 10.1.6.3

O

long-chain acyl-CoA dehydrogenases; and deficiencies in the electron transferring flavoprotein or its dehydrogenase. Several disorders (e.g., Zellweger syndrome) associated with impairment of peroxisomal β-oxidation have also been described. See Schulz (2002) and Section 11.6 for more details. 10.1.6.4

Ketone bodies

In mammalian liver, the excessive amounts of acetyl-CoA liberated by β-oxidation of fatty acid are converted to various ketone bodies. This conversion takes place at high rates when an elevation in the glucagon/insulin ratio occurs, such as during fasting or in uncontrolled diabetes. Several enzymes are involved, as shown below. These enzymes are also found in extrahepatic tissues, such as heart, kidney and intestine. Acetoacetyl-CoA

2 Acetyl-CoA

Acetoacetyl-CoA + CoA thiolase HMG-CoA

Acetoacetyl-CoA + Acetyl-CoA

Hydroxymethylglutaryl-CoA + CoA synthase HMG-CoA

Hydroxymethylglutaryl-CoA

Acetoacetate + Acetyl-CoA lyase

D-3-Hydroxybutyrate

Acetoacetate + NADH + H+

3-Hydroxybutyrate + NAD+ dehydrogenase

The ketone bodies produced by various tissues can readily diffuse into the blood and be taken up by other extra hepatic tissues and converted back to acetyl-CoA for complete combustion in the tricarboxylic acid cycle. A thorough review of these reactions and of the regulation of ketogenesis (Figure 10.14) has been provided by McGarry and Foster (1980) and see also Gurr et al. (2002) for a summary. Ketone bodies provide important alternative fuels to body tissues when carbohydrate is in short supply or cannot be efficiently utilised. A particular example is the central nervous system, which cannot utilise plasma fatty acids for energy. Thus, in prolonged starvation, ketone bodies become more important than glucose as a fuel source. The possibility of the utilisation of ketone bodies obviates the harmful degradation of muscle protein for gluconeogenesis. In addition, acetoacetate and 3-hydroxybutyrate are thought to be important precursors for lipid synthesis in neonatal brain (Webber and Edmond, 1979).

Inherited diseases of b-oxidation

A number of diseases that compromise the functions of liver, muscle, and other organs have been ascribed to deficiencies of β-oxidation. These include myopathic carnitine deficiency; deficiencies of short-chain, medium-chain, or 652

Lipid Metabolism

VLDL Triacylglycerol

Carnitine

Fatty acyl-CoA

Fatty acid

Glucose Malonyl-CoA Glucagon Fatty acylcarnitine

Pyruvate

Acetyl-CoA

Glucagon Acetyl-CoA

Citrate

Ketone bodies

FIGURE 10.14 Interactions between fatty acid synthesis and oxidation in liver. In the fed state, malonyl-CoA levels are high. This allows rapid fatty acid synthesis and inhibits β-oxidation by lowering carnitine acyltransferase I activity. If triacylglycerol synthesis is impaired, then acyl-CoA will feed back to inhibit acetyl-CoA carboxylase. In the fed state, this does not normally happen, and triacylglycerols are incorporated into very low density lipoprotein (VLDL) for export to extrahepatic tissues. Glucagon excess in fasting leads to a suppression of glycosis, cessation of lipogenesis, and activation of β-oxidation and ketogenesis. (From McGarry, J.D. and Foster, D.W. (1980). Annu. Rev. Biochem. 49, 395–420. With permission.)

Although ketone bodies serve useful functions, excessive accumulation of such compounds in blood can cause clinical problems, such as ketoacidosis. This may be severe in diabetes or alcoholism. A comprehensive review of clinical aspects of ketone bodies is that of Soling and Seufert (1978). 10.1.6.5

rubredoxins), a flavoprotein, and a final component required for hydroxylase activity called the ω-hydroxylase. The latter has a molecular mass of 42,000 Da with 1 atom of iron. It also contains a large amount of phospholipid, and activity is lost if this lipid is removed. Other studies have included electron paramagnetic resonance (EPR) experiments on the spectra shown by the nonhaem ironprotein (for reviews, see Coon et al., 1972; Gunsalus et al., 1975). In plants, the ω-hydroxylase system is responsible for synthesis of ω-hydroxy fatty acyl components of cutin and suberin (see Sections 1.1.2.10 and 1.2.12). The reaction has been studied in preparations from Vicia faba with NADPH and oxygen as the required cofactors (Kolattukudy, 1980). The true substrate for ω-hydroxylation of palmitate is the free acid, and the active subcellular preparation is the microsomal fraction. The reaction showed the properties of a classic mixed-function oxidase, being inhibited by O-phenanthroline, 8-hydroxyquinoline (metal-ion chelators), sodium azide, and thiol-directed reagents. The involvement of cytochrome P450 in the V. faba system is unproven. Although the hydroxylation is inhibited by carbon monoxide, this inhibition was not reversed by light at 420 to 460 nm. Thus, if a cytochrome P450 is involved in the system, it must have unusual properties when compared to other cytochrome-P 450 -containing enzymes (Kolattukudy, 1980). The recent description of three long-chain fatty acid oxidase genes from Candida has led to the identification of a gene family involved in ω-oxidation in yeast with homologues in bacteria and plants (Van Hanen et al., 2000). In Candida the omega carbon is oxidised successively by a cytochrome P450 alkane/fatty acid oxidase, a H2O2-generating alcohol oxidase, an aldehyde dehydrogenase producing

x-Oxidation

Microsomal preparations from many tissues can oxidise fatty acids in the ω-position. An ω-hydroxy fatty acid is formed first, and this can be oxidised by NAD+ and cytoplasmic enzymes to yield a dicarboxylic acid, which can be further attacked by β-oxidation (Greville and Tubbs, 1968). The enzyme(s) responsible for the oxidation of the ω-hydroxy fatty acid have been studied much less than the ω-hydroxylation system. There may be an ω-hydroxy acid dehydrogenase, although liver alcohol dehydrogenase can convert such acids to semialdehyde derivatives. Further oxidation of the latter would be catalysed by an NADlimited aldehyde dehydrogenase (cf. Greville and Tubbs, 1968). ω-Oxidation is important for the further metabolism of fatty acids that are di-substituted in the 2- or 3-positions as well for the catabolism of various xenobiotics that have alkyl chains (see Lenk, 1972). The role of peroxisomes in the ω-oxidation of fatty acids and xenobiotics is discussed by Osmundsen et al. (1991). The ω-hydroxylase system (alkane 1-mono-oxygenase, EC 1.14.15.3) has nonhaem irons as, apparently, the only prosthetic groups directly involved in the hydroxylation reactions. The system from Pseudomonas oleovorans has been isolated by Coon and coworkers (Kusunose et al., 1964). The system was fractionated into three components: a nonhaem iron protein (similar to the 653

10.1

Fatty acids

iron-containing fatty acid dioxygenases, which are ubiquitous in plants and animals (Brash, 1999). LOXs catalyse the regio- and stereo-specific dioxygenations of PUFAs containing a 1Z, 4Z-(cis, cis-1,4) pentadiene system to produce conjugated hydroperoxydiene derivatives:

TABLE 10.5 Major products of ruminant biohydrogenation of C18 unsaturated fatty acids Substrate

Products

Oleic acid Linoleic acid

18:0 9c, 11t-18:2 11t-18:1 18:0 9c, 11t, 15c-18:3 11t, 15c- and 9c, 11t-18:2 11t-18:1 18:0

α-Linolenic acid

RCH = CHCH2CH = CHR´

↓ O2 RCH = CHCH = CHCH(OOH)R´ Thus, typical substrates for lipoxygenases would be the PUFAs linoleic, α- linolenic, arachidonic and eicosapentaenoic acids.

ω-alcohols, ω-aldehydes, and ω-fatty acids. Four genes with high homology to Candida ω-oxidation genes are present in the Arabidopsis genome (Graham and Eastmond, 2002). 10.1.6.6

10.1.7.1

Fatty acid catabolism in ruminants

Because 20C PUFAs are only minor fatty acids in plants, the plant LOXs are classified according to their positional specificity of linoleate oxygenation, either at the 9-(9-LOX) or the 13-carbon (13-LOX) (Feussner and Wasternack, 2002). Lipoxygenase activity is widespread in the plant kingdom, often in very high amounts. The enzymes are particularly important in the food plants, where they destroy the essential polyunsaturated fatty acids to produce derivatives with characteristic tastes and flavours. They are also used for bleaching natural pigments, such as wheat flour carotenoids or alfalfa chlorophyll. General reviews on lipoxygenases are those by Gaffney (1996) and Piazza (1996). Plant lipoxygenases are discussed by Galliard and Chan (1980) and by Siedow (1991), while the lipoxygenase pathway is reviewed by Grechkin (1998) and Feussner and Wasternack (2002). Other useful reviews cover the commercial importance of lipoxygenases (Eskin et al., 1977), their role in olive oil quality (Harwood and Aparicio, 2000) and in plant defence (Blée, 1988). The animal lipoxygenases are dealt with separately in section 10.1.8. Plant tissues containing high levels of lipoxygenase activity are shown in Table 10.6. Leguminous seeds generally contain lipoxygenase, but the absence of measured

A specialized situation for lipid breakdown is the rumen of such animals as sheep and ox. A large number of microorganisms, such as bacteria of the genera Ruminococcus, Bacteroides, and Butyrivibrio, as well as protozoa, play a part in the breakdown of leaf lipids (see Section 2.10). Thus, α- and β-galactosidases are present to cleave the galactose residues of galactosylglycerides. Active lipases hydrolyse the acyl residues, and the liberated fatty acids are often biohydrogenated to give various mixtures of conjugated fatty acids containing trans as well as cis double bonds (Table 10.5) (see Garton, 1977; Harwood and Russell, 1984; and Gurr et al., 2002). Fermentation results in the production of large quantities of acetic, propionic, and butyric acids — the proportions of which vary with the ruminants’ diet. These water-soluble, shortchain fatty acids are absorbed well, and much of their total quantity is metabolised to ketone bodies in the rumen wall. Other features of fatty acid metabolism in ruminants are discussed by Garton (1977).

10.1.7

Lipoxygenase

Lipoxygenases (LOXs) (linoleate: oxygen oxidoreductase (EC 1.13.10.12)) constitutes a large family of nonheme, TABLE 10.6

Lipoxygenases in plants

Plant tissues containing high levels of lipoxygenase activity

Plant

Family

Tissue

Lipoxygenase activity (µl O2 consumed in 10 min/gram fresh weight)

Yellow bean Potato Eggplant Soybean Artichoke Pea Cauliflower Avocado Tomato Lettuce

Leguminosae Solanaceae Solanaceae Leguminosae Compositae Leguminosae Cruciferae Lauraceae Solanaceae Compositae

Seed Tuber Fruit Seed Heart Seed Floret Fruit Fruit Leaf

6480 4560 4320 4150 3360 1769 1440 720 360 120

654

Lipid Metabolism

activity in certain plant tissues does not mean that the enzyme is absent, since there are often inhibitors present. The enzyme has been detected in particulate, cytosolic, and vacuolar fractions (Feussner and Wasternack, 2002). Several types of lipoxygenase have been described. These differ in various properties, and the “classic” soybean lipoxygenase (Theorell et al., 1947) may be atypical in several respects. Two main types are present in soybeans. The Theorell enzyme acts on free acids only and is sometimes called an “acid” lipoxygenase (Verhue and Francke, 1972). The same enzyme is referred to as an “alkaline” lipoxygenase by Grosch et al. (1977) because its optimal activity is found at pH 9. It is better referred to as a Type I enzyme. Similarly, a second lipoxygenase with an optimum pH of 6.5 has been referred to as an “ester” enzyme a “neutral” enzyme, or a “b” enzyme and it is better referred to as the Type II enzyme (Galliard and Chan, 1980). Isoenzymic forms of lipoxygenase have been purified to homogeneity from many different plants (see Eskin et al., 1977). Most of the enzymes have properties similar to the soybean Type II enzyme. Tissues that have been used for study include wheat, alfalfa, potato, barley, pea, and various other legumes. Most lipoxygenases appear to be single polypeptides of molecular masses in the region 70,000 to 100,000 Da. A single atom of nonhaem iron per molecule is found (Chan, 1973) and the pH optima are in the range of 5.5 to 7.0. Because lipoxygenase lacks cofactors other than nonhaem iron, the number of inhibitors is small. The trans-unsaturated fatty acids, acetylenic fatty acids, α-bromo fatty acids, and fatty alcohols will all inhibit, and antioxidants or oxygen scavengers are effective in certain cases (see Hamberg et al., 1974; Eskin et al., 1977). The remarkable increase in sequence information has allowed phylogenetic tree analysis of multigene families. For LOXs, the Type I and Type II enzymes, as well as those classified as the 9- or 13-LOXs, form individual groups in separate branches of the tree (Feussner and Wasternack, 2002). Soybean Type I lipoxygenase is particularly stable. Other lipoxygenases are less stable and activity is lost during purification. Heat treatment is often used in the food industry to cause inactivation so as to prevent offflavours. The purified lipoxygenases are generally unstable at 70°C, but higher temperatures may be necessary for inactivating the enzymes in foodstuffs. A number of assay methods have been used for measuring lipoxygenase. These techniques use radioisotopes or the oxygen electrode and colorimetric methods. These assays and necessary precautions are discussed by Galliard and Chan (1980). Lipoxygenases will also catalyse co-oxidation reactions. This is used both in assay methods for the enzyme and in commercial applications. An example of the latter is the addition of soybean or broad bean flours (both rich in

lipoxygenase activity) to wheat flour in order to bleach pigments for white bread production. Enzymes from different sources differ in their co-oxidation ability, e.g., soybean Type I enzyme has poor activity in this regard while soybean Type II enzyme has high co-oxidation activity. The reaction probably proceeds by a free radical process (Veldink et al., 1977) and requires the presence of a substrate (e.g., linoleic acid) as well as the cosubstrate. The extent of the co-oxidation may depend on the lifetime of the radical intermediates and the relative efficiency of the lipoxygenase-mediated radical reduction (Weber and Grosch, 1976). Whereas in animals, arachidonate is a major substrate for LOX attack, in plants α-linolenate is the major substrate. Breakdown of this acid is known variously as the α-linolenic acid cascade or lipoxygenase pathway (see Figure 10.15). The fatty acid hydroperoxides produced by LOXs can be converted to three main types of products (see Figure 10.15) with important functions (Gurr et al., 2002) (Fig. 10.16): 1. Co-oxidative reactions with peroxygenase give a mixture of epoxy and hydroxyl fatty acids (depending on the nature of the acceptor fatty acid for the monooxygenation) and the products have roles in cutin biogenesis and defence against pest attack (Blée, 1988). 2. Hydroperoxide lyase cleaves the hydroperoxide into an aldehyde and an oxo-unsaturated fatty acid. The products have a role in pest defence and appear to act as pollinator and herbivore attractants, especially for flowers and fruits. They are also important flavour and aroma components in foods, drinks, and perfumes (see e.g., Sanchez and Salas, 2000). 3. Allene oxide synthase gives rise to the precursor of jasmonic acid. The latter and associated compounds are commonly known as jasmonates although some workers prefer the term “jasmonins” to differentiate from jasmonic acid esters. The jasmonins have important effects on plant growth, development, and senescence. Hydroperoxide lyase activity under 2 (above), usually occurs with the 13-hydroperoxide derivative thus giving rise to 6C compounds, which are volatile. Combinations of the volatiles produced are effective attractants for both pollinators and herbivores and the easily detected smells of various fruits, cut grass, cucumber, etc. derive from them. Profiles of the volatiles produced can be easily analysed by headspace gas chromatography and even used for identification purposes (see Harwood and Aparicio, 2000). Physiological roles for lipoxygenase-derived products in plants are shown in Table 10.7 (Gurr et al., 2000). For more detail, refer to Seidow (1991), Blée (1998), Feussner and Wasternack (2002) and Rosahl and Feussner (2005).

655

10.1

Fatty acids

Other 9C compounds Nonadienal +9-oxononanoic acid Lyase 9-lipoxygenase 9-Hydroperoxylinolenic acid O2 COOH

α-Linolenic acid

Epoxy FA Peroxygenase

O2 13-lipoxygenase H O O

Hydroxy FA Hydroxy FA

Peroxygenase

COOH Allene oxide synthase 13-hydroperoxylinolenic acid

Epoxy FA Allene oxide COOH

Lyase O

Allene oxide cyclase

12-Oxo-dodeca3-cis 9-enoic acid hexenal

COOH

Traumatic acid

O 12-oxophytodienoic acid Reduction

Other 6C volatile compounds

β-Oxidation

COOH Jasmonic acid O Tuberonic Cucurbic Other jasmonates acid acid or jasmonins

Conjugates

FIGURE 10.15 The α-linolenic acid cascade and oxylipin formation in plants. (From Gurr, M.I., Harwood, J.L. and Frayn, K.N. (2002). Lipid Biochemistry, 5th ed., Blackwell Scientific, Oxford, U.K. With permission.)

10.1.7.2

Lipid peroxidation

lyase with 9-hydroperoxylinoleic acid. Other aldehydes or conjugated derivatives can also have significant activity (Uchida, 2003).

A constant problem with unsaturated lipids, particularly PUFAs, is the ease of their oxidation. This, in general, not only destroys the beneficial effects of the parent molecule, but often gives rise to products with harmful properties. For further discussion of this subject, see Section 11.1 and Gurr et al. (2002). Specific useful reviews are those of Morrow and Roberts (1997) on isoprostanes and by Itabe (1998) on oxidized phospholipids and atherosclerosis. Some of the products initiated by enzyme activity, such as in the lipoxygenase pathway, may also have important pathophysiological effects. For example, there is much interest in 4-hydroxy-2-nonenal as a product and mediator of oxidation stress. This compound originates from the reaction of hydroperoxide

10.1.8

Production and function of the eicosanoids

Eicosanoids are oxygenated 20-carbon fatty acids. Because the major precursor of these compounds in animals is arachidonic acid, the pathways leading to the eicosanoids are also often known as the “arachidonate cascade.” There are three reactions involved in the initial metabolism of arachidonic acid, which itself must be released from membrane lipids through the activity of phospholipases. The enzymes involved are cyclooxygenase, 5-, 12-, or 15-lipoxygenases and various cytochrome P450

656

Lipid Metabolism

R1

R2

Lipoxygenase

R1

R1

R2

OOH

R2

OOH

Hydroperoxides (fungicidal, induce proteinases)

Lyase pathway

Aldehydes Oxo-acids

Allene oxide synthase pathway

Cyclised products α and γ ketols

(Trigger fungicide production. (Jasmonic acid best known. Acts in signalling transduction Important as flavour and volatile components of foods during wounding, stress and pathogen attack. and beverages) Developmental roles)

Peroxygenase pathway

Epoxides, epoxy alcohols, dihydrodiols, triols (Important, cutin precursors, may have defence roles also)

FIGURE 10.16 Lipoxygenase products and physiological roles in plants. (From Gurr, M.I., Harwood, J.L. and Frayn, K.N. (2002). Lipid Biochemistry, 5th ed., Blackwell Scientific, Oxford, U.K. With permission.)

TABLE 10.7

Some physiological roles for lipoxygenase-derived products in plants

Phenomenon

Notes

Compounds involved

Plant resistance to pathogens

Activation of several defence systems in “hypersensitive response”. Lipoxygenase expression rapidly induced following infection 13-Lipoxygenase induced, translocates to lipid body and dioxygenates storage lipids, which are then hydrolysed and the oxygenated fatty acid catabolised Widespread phenomenon in plants is the drought-induced accumulation of 9hydroperoxy derivatives of membrane lipids; function unknown Lipoxygenase may be involved directly in photosystem inactivation and chlorophyll oxidation; jasmonate as senescencepromoting substance, by inducing production of certain proteins; has complementary effects with ABA (abscisic acid) and may influence ethylene production Differential effects of jasmonates on ethylene formation; several fruit-specific lipoxygenase genes identified Stimulate a number of associated phenomena, such as cell expansion, cytoskeleton structure and carbohydrate accumulation

Jasmonic acid; phytoalexins (e.g., hexenal); eicosanoids from pathogen arachidonate

Mobilisation of storage lipids

Drought stress

Senescence

Fruit ripening

Tuber induction

13-Hydroperoxygenated lipids

9-Hydroperoxygenated lipids

Jasmonins

Jasmonins

Tuberonic acid and other jasmonins

Source: From Gurr, M.I., Harwood, J.L. and Frayn, K.N. (2002). Lipid Biochemistry, 5th Blackwell Scientific, Oxford, U.K. With permission.

657

10.1

Fatty acids

Stimulus

Phospholipid

Precursor fatty acid (e.g., Arachidonate) Cyt P450 epoxygenase

Lipoxygenases Cyclooxygenase Leukotrienes + hydroxyfatty acids lipoxins hepoxilins

Cyclic endoperoxides Prostacyclin synthetase

Prostacyclin (PGl)

Reductase or isomerases Prostaglandins (PGD, PGE, PGF mainly)

Hydroxy fatty acids, fatty acid epoxides hromboxane synthetase

hromboxanes (TXs)

FIGURE 10.17 Overall pathway for conversion of essential fatty acids into eicosanoids. (From Gurr, M.I., Harwood, J.L. and Frayn, K.N. (2002). Lipid Biochemistry, 5th ed., Blackwell Scientific, Oxford, U.K. With permission.)

epoxygenases (Figure 10.17). For prostanoid structures, see Smith and Murphy (2002) and, for a general discussion, Gurr et al.(2002). Eicosanoid synthesis is initiated following the interaction of a stimulus with the cell’s plasma membrane. Interaction of the agonist with its receptor leads to the activation of one or more phospholipase. The mobilisation of arachidonate could be caused, in theory, by a number of lipid degradative enzymes. In practice, various combinations of cytosolic or secretory phospholipase A2 enzymes appear to be used, depending on the circumstance (Smith and Murphy, 2002). Activation of the phospholipase(s) requires or is accompanied by a significant rise in intracellular Ca2+. The release of arachidonate is selective (Dennis, 1987). Once arachidonic acid (or an equivalent PUFA, such as EPA, 20:5n-3) is released, it can be metabolised by prostaglandin endoperoxide (PGH) synthase, also known as cyclooxygenase (COX) or prostaglandin H synthase. This enzyme has two catalytic activities: a cyclooxygenase, which catalyses the formation of PGG2, and a peroxidase, which converts this intermediate to PGH2 (Figure 10.18). Subsequent metabolism of PGH 2 to one of the major prostanoids then takes place via a cell-specific pathway. Prostanoids with a “2” subscript are derived from arachidonic acid, those with a “1” subscript from 8,10,14eicosatrienoic acid and those with a “3” subscript from 5,8,10,14,17-eicosapentaenoic acid. The structure and catalytic properties of prostaglandin endoperoxide synthase (Smith and Marnett, 1991) and regulation of its expression (De Witt, 1991) have been reviewed. For a recent summary, see Kulmacz et al. (2003). Vertebrates, from humans to fish, have two main isoforms of prostaglandin H synthase, termed PGHS-1 and -2(COX-1, COX-2). These two isoforms are structurally very similar, but have very different physiological roles and are regulated very differently (Kulmacz et al., 2003). The nonstearoidal anti-inflammatory drugs, such as

aspirin or ibuprofen, act against the cyclooxygenase binding site. Since PGHS-2 (rather than PGHS-1) is implicated in chronic inflammatory complaints, there has been much interest in developing specific inhibitors against this isoform (Gurr et al., 2002). However, unexpected sidereactions have led to withdrawal of the first (specific) PGHS-2 (COX-2) inhibitors (Drazen, 2005). Once PGH 2 has been formed by PGH synthase, further metabolism is cell-specific. For example, platelets form mainly TxA2, endothelial cells form PGI2 as their major prostanoid, while PGH2 is the major product in renal collecting tubule cells. A number of these enzymes have been purified and characterised (see Smith and Murphy, 2002). PGE2 is the best-studied prostanoid and the molecular biology and physiology of its biosynthetic pathway has been reviewed recently (Murakami and Kudo, 2004). Prostanoids (prostaglandins, thromboxane, prostacyclin) exert a wide variety of actions in the body, which are mediated by specific receptors on plasma membranes. These receptors are classified into five basic types, termed DP, EP, FP, IP, and TP, on the basis of sensitivity to the five primary prostanoids formed from arachidonate. Details of these receptors and their physiological role will be found in Sagimoto et al. (2000). Prostanoids are local hormones that act very near to the site of synthesis (Smith and Murphy, 2002) and which have a very short half-life (a few minutes) due to their rapid catabolism. For PGE2, the initial reaction is oxidation to the relatively inactive 15-keto derivative. Further catabolism involves reduction of the double bond between C-13 and C-14, ω-oxidation and β-oxidation. β-Oxidation of eicosanoids is summarised by Diczfalusy (1994). As an alternative to PGH attack, arachidonic acid can be metabolised through the action of one of three lipoxygenases. These enzymes catalyse reactions analogous to the well-known plant enzymes (Section 10.1.7). The 658

Lipid Metabolism

Stimulus Cell surface Phospholipid Phospholipase COOH

Arachidonic acid Cyclooxygenase COOH

O

PGH synthase

O OOH PGG2 Peroxidase COOH

O O

HOOC

HO OH PGH2

COOH O

O

O

OH PGD2

HO

COOH

PGI2 OH

HO OH PGE2

COOH

HO

COOH

O O

HO OH PGF2α

FIGURE 10.18 1991a.)

OH TxA2

Structures and biosynthetic relationships among prostanoids derived from arachidonic acid. (From Smith et al.

lipoxygenases constitute a family of closely-related, nonhaem, iron-containing dioxygenases. The immediate products of their reaction with various eicosaenoic acids are hydroperoxy fatty acids. In the case of arachidonic acid, the products are peroxyeicosatetraenoic acids (HpETEs). Subsequent metabolism generates hydroxyeicosatetraenoic acids (HETEs) (Figure 10.19). The biochemistry and functional activity of HETEs have been reviewed by Spector et al. (1988). The activity of the 5-lipoxygenase has been studied more thoroughly than that of the other lipoxygenases, mainly because the leukotrienes are the end products of this metabolic pathway. The production of leukotrienes LTA4, LTB4, and LTC4 is shown in Figure 10.20. For summaries of the actions of leukotrienes, see Smith and Murphy (2002) and Gurr et al. (2002). Depending on the

exact chemical structure concerned and the tissue tested, the leukotrienes are potent bronchoconstrictors, arterioconstrictors, vasodilators, and chemotactic agents. 5HETE is a major product of 5-LOX activity in all cells. While 5-HETE has its own acute biological potency (Spector et al., 1988), it can also be dehydrogenated to 5oxo-ETE. The latter compound is a strong chemoattractant and its biochemistry and functions have been reviewed (Powell and Rokach, 2005). 12-LOX is another active enzyme, giving rise initially to 12-HPETE from arachidonate (see Figure 10.19). 12HPETE, together with its metabolite 12-HETE, has a variety of important effects on neurotransmission, white blood cells, airways, and other tissues. 12-HPETE can be converted to hepoxilins via hepoxilin synthase. Alternatively, 12-LOX can act as a lipoxin synthase in converting LTA4 659

10.1

Fatty acids

COOH

Arachidonic acid 5-Lipoxygenase

15-Lipoxygenase

12-Lipoxygenase COOH

COOH

OOH COOH

HOO HOO

5-HPETE

15-HPETE

12-HPETE OH

COOH

COOH

COOH HO 5-HETE

HO 15-HETE 12-HETE

FIGURE 10.19

Lipoxygenase pathways for the synthesis of the major HETE isomers. (From Spector, et al. 1988.) COOH

Arachidonic acid 5-Lipoxygenase OOH COOH 2e– Reduction

O2 4e–

5-HPETE Lipoxygenation

OH

OH

COOH

COOH Dehydration

H 2O OH

5-HETE COOH O

5S, 15S-diHETE

Non-enzymic

H2O 5, 12-diHETE + 5,6-diHETE OH

Leukotriene A4

Hydration (enzymic)

Glutathione S-transferase

H2O

GSH OH COOH S

COOH

CH2 CHCONCH2COOH

NHCO(CH2)2CHCOOH Leukotriene C4 NH2

OH Leukotriene B4

FIGURE 10.20

Formation of leukotrienes from arachidonic acid. (From Gurr and Harwood 1991.)

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into lipoxin A4. Hepoxilins are known to function in relation to the release of intracellular calcium and the opening of potassium channels, while lipoxins have roles as immunologic and hemodynamic regulators. Receptors have been identified for some of these products of 12-LOX metabolism (Yamamoto et al., 1997). 15-LOX produces 15-HPETE from arachidonate and 13-HPODE (13-hydroperoxy, 9Z,10E-octadecadienoic acid) from linoleate. These products can go on to produce 15-HETE and 13-HODE, respectively, which can then initiate various biological effects. Both 15-HETE and 13HODE are bound to cell membrane receptors and these or related metabolites have action on erythropoiesis, the cardiovascular system, skin, respiration, and the reproductive system (Kuhn, 1996). The third metabolic pathway for PUFAs to produce biologically active metabolites is via P450-mediated reactions (see Figure 10.17). Arachidonic and linoleic acids can be oxygenated by P450 in four main ways: epoxidation, hydroxylation of the ω-side chain, hydroxylation of allylic or bis-allylic carbons, and hydroxylation with double bond migration (Oliw, 1994). Epoxyeicosatrienoic acids (EETs) with epoxy groups in the 5/6, 8/9, 11/12, or 14/15 positions (Figure 10.21), which are produced from arachidonate, have important functions in vascular smooth muscle, endothelium, myocardium, and other tissues (Spector et al., 2004). EETs are rapidly metabolised including being β-oxidised. The hydroxy products have been less well studied

than the EETs but varieties of physiological functions have been noted (Oliw, 1994). For a good general summary of the eicosanoids, refer to Smith and Murphy (2002).

10.1.9

Other conversions

Fatty acids can be modified in a number of other ways. For example, cyclopropane fatty acids are formed by the addition of a methylene group from S-adenosylmethionine across the double bond of a monounsaturated fatty acid. The latter is esterified to a phospholipid, so that the actual substrate is a membrane lipid. The aldehyde residues in plasmalogens and the alcohol residues in alkyl ether lipids, such as those in Clostridium butyricum also act as acceptors for the methyl group — in this case, forming the corresponding cyclopropane aldehydes and alcohols (Goldfine and Panos, 1971). The control of cyclopropane fatty acid synthase in bacteria, such as E. coli, has been examined in some detail (see Harwood and Russell, 1984). Cyclopropane fatty acids in plants seem to be made by the same mechanism as for bacteria (Mangold and Spener, 1980). These authors have also reviewed work on the synthesis of cyclopentenyl fatty acids, such as chaulmoogric (13-(2-cyclopentenyl) tridecanoic) acid. Branched, cyclic, and unsaturated hydrocarbons in higher plants are formed from appropriate fatty acids by decarboxylation (see Kolattukudy, 1980, 1987). The process COOH

Arachidonic acid O

O COOH

5,6-EpETrE (5,6-EET)

COOH

O 14,15-EpETrE (14,15-EET)

11,12-EpETrE (11,12-EET)

HO OH COOH

COOH

5,6-DiHETrE

O

8,9-EpETrE (8,9-EET)

HO OH

COOH

COOH

COOH

HO OH 11,12-DiHETrE

8,9-DiHETrE COOH

COOH

HO OH 14,15-DiHETrE COOH

CH2OH OH 19-Hydroxyarachidonate

20-Hydroxyarachidonate

FIGURE 10.21

Structures of products of epoxygenase pathways of the arachidonate cascade. (From Smith et al. 1991a.)

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Fatty acids

De novo synthesis

C16

Free fatty acids C16–C32 Aldehydes C22–C32

C18 + C2 units

C16–C34 fatty acyl chains

Alkan-1-ols C22–C32

CO2 Alkanes C21–C33

Alkan-1-ol esters C36–C58 Secondary alcohols C29–C31

Ketones C29–C31

FIGURE 10.22 Conversion of fatty acids to other wax components. (From von Wettstein-Knowles, P. (1979). In Advances in the Biochemistry and Physiology of Plant Lipids (Eds. L-A Appelqvist and C. Liljenberg) Elsevier, Amsterdam, pp. 126. With permission.)

has also been studied in cyanobacteria, insects, and other species (see Kolattukudy, 1976). However, the exact reaction mechanism is ill defined. Plants use long-chain and very long-chain fatty acids as sources of hydrocarbon and β-diketones. These and other conversions involved in the generation of plant wax constituents are reviewed by von Wettstein-Knowles (1995), while Nelson and Blomquist (1995) discuss the formation of insect waxes. The relationship of fatty acids to the other plant wax components is shown in Figure 10.22. A recent update on the formation of the plant cuticle’s components is that by Kunst et al. (2005).

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667

10.2

Glycerophospholipids

acid, is a key intermediate in glycerolipid metabolism as illustrated in Figure 10.23. In addition to providing the backbone of the glycerophospholipids, phosphatidate (via diacylglycerol) also acts as a precursor of triacylglycerols (Section 10.3) and the glycosylglycerides (Section 10.4). The biosynthesis of phosphatidate in animals begins with the activation of fatty acids to their acyl-CoAs by one of several chain-length-dependent acyl-CoA ligases (synthetases) (Bloch and Vance, 1977; Groot et al., 1976; Brindley, 1991). The most important locations of the longchain acyl-CoA ligases are the endoplasmic reticulum and mitochondria (Brindley, 1991). Some ligases show preferences towards saturated or unsaturated fatty acids. Of course, some of the enzymes play a role more in degradation (by providing acyl-CoAs for β-oxidation) than in biosynthesis. The peroxisomal acyl-CoA synthetases come into this category (Section 10.1.6). A review of the biochemistry of acyl-CoAs is that by Waku (1992). The main acceptor for acyl-CoA in most tissues is snglycerol 3-phosphate, derived from glycolysis. Glycerol 3phosphate acyltransferase is divided equally between mitochondrial and endoplasmic reticulum in mammalian liver, but in other tissues the endoplasmic reticulum is the main site. The latter enzyme is on the cytoplasmic face. The mitochondrial and endoplasmic reticulum enzymes can be distinguished by their relative sensitivities to heat, proteolytic enzymes, and SH-reagents (Brindley, 1991). The substrate selectivities of different enzymes are discussed by Waku (1992). In general, a variety of saturated and unsaturated acyl-CoAs are used. The second acyltransferase (lysophosphatidate acyl transferase) has a strong preference for unsaturated fatty acids in animals. In most subcellular fractions (and most animal tissues), the second acyltransferase has much higher activity than the first acyltransferase, so that lysophosphatidate does not accumulate. The substrate specificities of the two enzymes are such that the glycerolipids of animals show a preferential location of saturated fatty acids at the sn-1 position and unsaturated fatty acids at the sn-2 position. Some properties of the lysophosphatidate acyltransferase are covered in Brindley (1991). See also Coleman et al. (2002) for details of the above two acyltransferases. The acylation of dihydroxyacetone phosphate and the subsequent reduction of acyldihydroxyacetone phosphate to monoacylphosphatidic acid provide an alternative route for phosphatidic acid synthesis (Brindley, 1991). Acyltransferases that utilize dihydroxyacetone phosphate have been detected in several tissues (Bell and Coleman, 1980) and purified from guinea pig liver peroxisomes (Webber and Hajra, 1992). Another possible alternative pathway for phosphatidic acid synthesis (involving diacylglycerol kinase) has been demonstrated in rat liver (see Bell and Coleman, 1980). However, this enzyme is probably not very important quantitatively for phosphatidic acid formation. See also

Wakil, S.J. (1970). Fatty acid metabolism. In Lipid Metabolism, Ed. S.J. Wakil, Academic Press, New York, pp. 1–48. Wakil, S.J. et al. (1983). Fatty acid synthesis and its regulation. Annu. Rev. Biochem. 52, 537–579. Walker, K.A. and Harwood, J.L. (1986). Evidence for separate elongation enzymes for very-long-chain-fatty-acid synthesis in potato (Solanum tuberosum). Biochem. J. 237, 41–46. Walsh, M.C. et al. (1990). The short chain condensing enzyme has a widespread occurrence in the fatty acid synthetases from higher-plants. Phytochemistry. 29, 3797–3799. Wardale, D.A. et al. (1978). Localization of fatty-acid hydroperoxide cleavage activity in membranes of cucumber fruit. Phytochemistry. 17, 205–212. Watkins, P.A. (1997) Fatty acid activation. Prog. Lipid Res. 36, 55–83. Webber, R.J. and Edmond, J. (1979). The in vivo utilization of acetoacetate, D-(-)-3-hydroxybutyrate, and glucose for lipid synthesis in brain in the 18-day-old rat. Evidence for an acetyl-CoA bypass for sterol synthesis. J. Biol. Chem. 254, 3912–3920. Weber, F. and Grosch, W. (1976). Co-oxidation of a carotenoid by the enzyme lipoxygenase: influence on the formation of linoleic acid hydroperoxides. Z. Lebensm, Unters. Forsch. 161, 223–230. White, S.W. et al. (2005). The structural biology of Type II fatty acid biosynthesis. Ann. Rev. Biochem. 74, 791–831. Witkowski, A. et al. (1987). Molecular cloning and sequencing of a cDNA encoding the acyl carrier protein and its flanking domains in the mammalian fatty acid synthetase. Eur. J. Biochem. 165, 601–606. Witkowski, A. et al. (1991). Structural organization of the multifunctional animal fatty acid synthase. Eur. J. Biochem. 198, 571–579. Worsham, L.M.S. et al. (1988). Chemical cross-linking and its effect on fatty acid synthetase activity in intact chloroplasts from Euglena gracilis. Biochim. Biophys. Acta. 963, 423–438. Wurtele, E.S. and Nickolau, B.J. (1990). Plants contain multiple biotin enzymes: discovery of 3-methylcrotonyl-CoA carboxylase, propionyl-CoA carboxylase and pyruvate carboxylase in the plant kingdom. Arch. Biochem. Biophys. 278, 179–186. Yamamoto, S. (1989). Mammalian lipoxygenases: molecular and catalytic properties. Prostaglandins, Leukotrienes Essent. Fatty Acids 35, 219–229. Yamamoto, S. (1991). “Enzymatic” lipid peroxidation: reactions of mammalian lipoxygenases. Free Radical Biol. Med. 10, 149–159. Yamamoto, S. (1992). Mammalian lipoxygenases: molecular structures and functions. Biochim. Biophys. Acta. 1128, 117–131. Yamamoto, S. et al. (1997). Arachidonate 12-lipoxygenases. Prog. Lipid Res, 36, 23–41. Zimmerman, D.L. and Coudron, C.A. (1979). Identification of traumatin, a wound hormone, as 12-oxo-trans-10-dodecanoid acid. Plant Physiol. 63, 536–541.

10.2

Glycerophospholipids

10.2.1

Biosynthesis

The first reactions in glycerophospholipid synthesis can be regarded as the stepwise acylation of glycerol 3-phosphate. The product of these acylations, phosphatidic 668

Lipid Metabolism

Brindley and Sturton (1982) for a review of phosphatidic acid formation. Once phosphatidate has been formed, it can be converted to diacylglycerol through the action of phosphatidate phosphohydrolase. This is a key enzyme in glycerolipid metabolism, controlling, on the one hand, the supply of carbon for the major membrane phospholipids, phosphatidylcholine and phosphatidylethanolamine, and the major storage lipid, triacylglycerol, and, on the other, controlling the relative production of anionic phosphoglycerides by a competing cytidylyltransferase (see Figure 10.23 and below). Phosphatidate phosphohydrolase has been well reviewed (Brindley, 1988; see also Brindley, 1991; Coleman et al., 2000). Phosphatidate phosphohydrolase in animal tissues seems to be subject to control by translocation between the endoplasmic reticulum and cytosolic compartments (Brindley, 1991). In rat liver it may catalyse the rate-limiting step in glycerolipid synthesis because it has the lowest in vitro activity of all the enzymes in the pathway, varies directly with dietary or hormonal regimes

that affect triacylglycerol synthesis and decreases with some drugs that inhibit nonpolar lipid formation (Bell and Coleman, 1980). As shown in Figure 10.23, phosphatidylcholine (the major animal phosphoglyceride) and phosphatidylethanolamine are both synthesized by a (cytidine 5'-diphosphate) CDP-base pathway. This is their major route of formation (Bell and Coleman, 1980; Ansell and Spanner, 1982). Three enzyme steps are required. First, the base is phosphorylated by a kinase enzyme. Choline kinase (EC 1.7.1.32) and ethanolamine kinase (EC 2.7.1.82) are soluble enzymes and have been purified from several tissues. The activities may reside in the same protein (Ulane, et al., 1977), although separate enzymes have been purified from rat liver (Brophy and Vance, 1976). The purified choline kinase is a dimer of mass 42 kDa (Vance, 1991). The second enzyme of the pathway is the cytidylyltransferase (EC 2.7.7.15), and this enzyme is partly soluble and partly associated with the endoplasmic reticulum (see Bell

S-Adenosylmethionine

OH P-Etn P-Choline (PtdCho) (PtdEtn) (DAG) CMP-Phosphorylcholine CMP-Phosphorylethanolamine N-Acyl sphingosine Serine Pi PPi (ceramide) PPi ∗ CTP CTP Phosphorylcholine ATP Sphingomyelin

Ethanolamine

Phosphorylethanolamine

ADP

P (PtdOH)

Choline CTP Acyl-CoA PPi

ADP ATP Ethanolamine ATP

Inositol

Acyl-CoA Glycerol 3-phosphate

P-Ser (PtdSer)

Ptdlns P-CMP

PtdilnsP

CMP PtdilnsP2 P OH

P Pi CDP-DAG OH OH P (PtdGly)

CMP

P OH P (DiPtdGly)

FIGURE 10.23 Phospholipid metabolism in animals. The relative thickness of lines shows the approximate carbon flow down each pathway. The asterisk (*) shows that other phospholipids can act as acceptors for serine in base exchange.

669

10.2

Glycerophospholipids

and Coleman, 1980). The soluble protein in animals can be aggregated under various conditions and its activity raised by association with membranes (see Vance, 1991). The enzyme seems to be the rate-limiting step in the CDPcholine pathway in animals (Vance and Choy, 1979). The enzyme has been purified (Feldman and Weinhold, 1987), and the cDNA coding for its protein identified and expressed (Vance, 1990). The equivalent enzyme, CDPethanalominephosphate cytidylyltransferase (EC 2.7.7.14), has been partly purified from rat liver (see Vance, 1991). CDP-choline cytidylyltransferase is normally thought to be the most important enzyme for regulation of phosphatidylcholine formation (Vance, 2002). Choline phosphotransferase (EC 2.7.8.2) and ethanolamine phosphotransferase (EC 2.7.8.1) are located in the endoplasmic reticulum and catalyse the final step in the CDP-base pathway. Their complete purification has yet to be achieved from animals. The two enzymes seem to have somewhat different properties and appear to be distinct proteins (see Vance, 1991). Phosphatidylcholine can also be synthesized by the stepwise methylation of phosphatidylethanolamine (Figure 10.23). Methyl groups are transferred from S-adenosyl-Lmethionine, and this pathway is a minor one in animals (20% of liver phosphatidylcholine synthesis, but undetectable in other tissues; Bell and Coleman, 1980). In liver, the three transmethylation reactions are catalysed by a single enzyme with a molecular mass of about 18 kDa. The rate of conversion of phosphatidylethanolamine to phosphatidylcholine appears to be regulated by substrate supply (Vance, 1991). A review of phosphatidylethanolamine methylation has been published (Vance and Ridgway, 1988) and Vance (2002) has updated information and speculated on the role of the methylation pathway in forming phosphatidylcholine. Phosphatidylserine accounts for 5 to 15% of the total phospholipids in animal cells. It is made by a base exchange reaction (see Figure 10.23) in which the head group of a preexisting phospholipid is exchanged for serine. The enzyme has been purified, and its base exchange activity is not due to phospholipase D activity (see Vance, 1991). Studies in CHO cells showed that there were two phosphatidylserine synthases, one using phosphatidylcholine and a second, phosphatidylethanolamine (Kuge and Nishijima, 1997). Instead of being dephosphorylated, phosphatidate can be converted to CDP-diacylglycerol. This reaction, catalysed by phosphatidate:CDP-diacylglycerol cytidylyltransferase (EC 2.7.7.41), is important for the synthesis of negatively charged phospholipids, such as phosphatidylglycerol, diphosphatidylglycerol, and the inositol phospholipids (see Figure 10.23). The cytidylyltransferase has been purified from animals (Bell and Coleman, 1980) and cDNAs identified for two isoforms (Vance, 2002). Phosphatidylglycerol can be made by mitochondrial and microsomal fractions from most animal tissues. However, the mitochondrial enzymes are probably

more physiologically relevant (Bell and Coleman, 1980), since most of the phosphatidylglycerol (except in lungs; Section 11.7) is used for diphosphatidylglycerol formation. The phosphatidylglycerol phosphate synthetase has been localised in the inner mitochondrial membrane, but the phosphatase (see Figure 10.23) is soluble. Together with CDP-diacylglycerol, phosphatidylglycerol is a cosubstrate for diphosphatidylglycerol synthesis in animals (see Figure 10.23). This contrasts with the bacterial pathway (see Figure 10.28). Diphosphatidylglycerol (cardiolipin) synthesis in different organisms has been discussed in detail by Schlame et al. (2000) who also discuss the lipid’s function in mitochondria and relevance to a number of human diseases. A third polyglycerophospholipid, bis(monoacylglycerol) phosphate, comprises less than 1% of the phospholipids of most animal tissues except for alveolar (lung) macrophages, where it represents 14 to 18% of the phospholipids. Its pathway of formation was proposed following experiments with macrophage-like cells. Phosphatidylglycerol was hydrolysed by phospholipase A2 and the lyso product acylated using another phospholipid. This yields bis(monoacylglycero)phosphate (BMP). After reorientation of the glycerol backbone, a second acylation (again using a phospholipid as donor) forms sn-1: sn-1'BMP. Spontaneous rearrangement causes the acyl residues to move to the sn-3 positions (Amidon et al., 1995). Phosphatidylinositol is also formed from CDP-diacylglycerol, though in this case only one enzyme step is involved (see Figure 10.23). Further phosphorylation to phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate are catalysed by kinase reactions. The first kinase appears to be present in isoforms and these are integral membrane proteins. One enzyme with a low Km for ATP (20-70 µM) and a molecular mass of 55 kDa has been purified to homogeneity (Downes and MacPhee, 1990). Phosphatidylinositol 4-phosphate kinase can be isolated from cytosolic and membrane sources. It appears to be a peripheral membrane protein (Vance, 1991). The above two kinases are important in the “phosphatidylinositol cycle” (Figure 10.24), which is connected with the important function of phosphatidylinositol 4,5-bisphosphate as a precursor of second messages that activate many regulatory processes in animal cells (see Section 10.2.2 and Section 10.6.3; also McPhee, 2002). Over recent years, a large number of inositol phospholipids have been identified, particularly those with phosphates at the 3-position and which have important signaling functions (Section 10.6) and Rameh and Cantley, 1997). Interconversions of inositol phospholipids through the action of various kinases and phosphatases are shown in Figure 10.25. See Freeman et al. (1998) for a review on phosphoinositide kinases and Vanhaesebroeck et al. (2001) for one on the synthesis and function of the 3-phosphorylated inositol lipids. Phospholipids cannot only be remodeled by base exchange reactions (Taki et al., 1978), but also by reacylation 670

Lipid Metabolism

R1

ATP

R1

ADP

R2

ATP

ADP

R2

R2

I

P

R1

4P

I

P

P

I

4,5P2

CMP I

I

P

I

P2

I

P3

R1 2

R

P

CMP R1

PPi

R1

ATP R2

R CTP

FIGURE 10.24

ADP

2

OH

P

The phosphatidylinositol cycle. I = inositol. (From Vance 1991.)

PI

PI-5-P PI-3,5-P2

PI-3-P PI-4-P

PI-3,5-P2

PI-4,5-P2 PI-3,4-P2

PI-3,4,5-P3

FIGURE 10.25 Interconversion of phosphoinositides. PI=phosphatidylinositol. The connecting recactions are catalysed by 3-, 4-, or 5- kinases and 3-, 4-, and 5- phosphatases.

reactions. The latter are extremely important in determining the final molecular species of lipid that are synthesised as well as allowing the participation of specific fatty acids in particular metabolic processes (e.g., release of arachidonate for prostaglandin formation (see van den Bosch, 1982). The entire subject of reacylation and molecular species turnover has been well reviewed by one of the major research workers in this field, Bill Lands (Hills and Lands, 1970). More recent discoveries are covered by Bell and Coleman (1980) and by Hawthorne and Ansell (1982) (see also MacDonald and Sprecher, 1991). Remodelling can occur on either the sn-1 or sn-2 positions (Vance, 2002). The above discussion has centred on the formation of diacylglycerophospholipids, but many phospholipids in nature have O-alkyl bonds. These include such important molecules as the plasmalogens and platelet-activating factor (PAF). For reviews on the metabolism, regulation and function of ether-linked glycerolipids, refer to Synder et al. (1985, 2002). Enzymes of the acyl dihydroxyacetone phosphate pathway for glycerolipid synthesis are located in peroxisomes as well as the endoplasmic reticulum. Their physiological function in peroxisomes as well as

their role in the production of both acyl and alkyl glycerolipids has been reviewed (Hajra, 1995). The coregulation of sterol and phospholipid metabolism is covered by Ridgeway et al. (1991). The overall pathways for phosphoglyceride synthesis in plants have been reviewed (Mudd, 1980; Moore, 1982; Harwood, 1979, 1989). For a recent update, see Dormann (2005). In general, the formation of these lipids is similar to that in animals, but there are some points of difference (Figure 10.26). First, there are two systems for the initial acylation of glycerol 3-phosphate. In the chloroplast-located pathway, acyl-ACPs are used and the substrate specificity of the enzymes results in the preferential formation of sn-1-oleoyl, sn-2-palmitoyl molecular species. Subsequent utilisation of such phosphatidate for lipid synthesis retains this (sn-1 C18, sn-2 C16) distribution of fatty acids and gives rise to what have been called “prokaryotic” molecular species by analogy with cyanobacterial lipid metabolism (see Roughan and Slack, 1984; Harwood, 1989; Joyard and Douce, 1987). A second glycerol 3-phosphate acylation system is located in the endoplasmic reticulum, uses acyl-CoAs, and has a substrate specificity such that more saturated fatty acyl groups 671

10.2

Glycerophospholipids

S-Adenosylmethionine (1)

P-Etn P-Choline OH (DAG) (PtdEtn) (PtdCho) CMP-Phosphorylethanolamine CMP-Phosphorylcholine Serine Pi PPi PPi CTP CTP (2) Phosphorylcholine ATP

ADP

Choline

Phosphorylethanolamine ADP

P (PtdOH)

ATP Ethanolamine

CTP PPi Acyl-CoA (Acyl-ACP)(3) Glycerol 3-phosphate Acyl-CoA (Acyl-ACP)

Ethanolamine

P-Ser (PtdSer) ATP

Inositol Ptdlns P-CMP

PtdlnsP

CMP PtdlnsP2 P OH

P

Pi OH

CDP-DAG

P OH

OH P

CMP

(PtdGly)

P (DiPtdGly)

FIGURE 10.26 Phospholipid metabolism in plants. The relative thickness of arrows indicates the carbon flux along individual reactions. Notes: (1) Other pathways for incorporating ethanolamine into PtdCho exist (see text); (2) other phospholipids can serve for base exchange; (3) acyl-ACPs are used by chloroplasts, but acyl-CoAs by the endoplasmic reticulum.

are usually located at the sn-1 position, just as in animals (see Harwood, 1989). The endoplasmic reticulum acylations are responsible for extrachloroplastic phospholipid synthesis and triacylglycerol formation (Harwood and Page, 1993), as well as some chloroplast lipid synthesis (see Roughan and Slack, 1984; Browse and Somerville, 1991). The major plant extrachloroplastic lipid, phosphatidylcholine, is mainly made by the CDP-base pathway (Harwood, 1979; Moore, 1982). Separate choline and ethanolamine kinases have been purified from soybean (Harwood, 1979), and the cytidylyltransferase step appears to be rate-limiting (Price-Jones and Harwood, 1986). Separate base transferases for choline and for ethanolaminephosphate appear to be present in soybean (see Harwood, 1979). Although evidence has been produced that plants can convert phosphatidylethanolamine to phosphatidylcholine by stepwise methylation (Mudd, 1980; Moore, 1982), some recent experiments suggest that methylation occurs

on various water-soluble intermediates, such as ethanolaminephosphate, which are then incorporated into phosphatidylcholine via phosphatidyldimethyl- (or methyl-) ethanolamine (see Williams and Harwood, 1994). Phosphatidylserine, phosphatidylglycerol, and phosphatidylinositol seem to be synthesized in plants by pathways similar to those discussed above for animals (Mudd, 1980; Moore, 1982; and see Figure 10.23 and Figure 10.26). Phosphatidylglycerol synthesis has been demonstrated in chloroplasts (see Harwood, 1989) in keeping with the major role of this lipid in thylakoid membranes. The metabolism of inositol lipids and their function in cellular regulation in plants has been reviewed recently (Drobak, 2005). N-Acylphosphatidylethanolamine is a minor phospholipid in plants (Dormann, 2005), but there is some interest in it as a source of signalling metabolites (see Chapman, 2004). 672

Lipid Metabolism

Pathways for the biosynthesis of phospholipids in yeast are generally similar to those demonstrated in other eukaryotes, except that phosphatidylserine is made exclusively from CDP-diacylglycerol (Figure 10.27) and not by base exchange. Phosphatidylserine is also converted to phosphatidylethanolamine by decarboxylation. Work with yeast has been particularly useful as a model system for other eukaryotes because of a large knowledge base in classical genetics and the ability to elucidate and manipulate individual genes coding for enzymes in the pathways. Purification and properties of CDP-diacylglycerol synthetase, phosphatidylserine synthetase, phosphatidylinositol synthetase, and phosphatidylinositol kinase are described by Carman and Henry (1989). Carman and Henry (1999)

reviewed the pathways of synthesis in S.cerevisiae in detail with information on the genes encoding individual enzymes. Particularly informative sections deal with the genetic and biochemical regulation of phospholipid synthesis and its interaction with other metabolic pathways. Phospholipid biosynthesis in microorganisms has been well reviewed by Pieringer (1989). Major pathways for Escherichia coli are shown in Figure 10.28. Several points are of note. First, the acylation of glycerol 3-phosphate uses acyl-ACPs and, thus, phospholipid synthesis is coupled directly to fatty acid formation (Jackowski et al., 1991). Secondly, as in yeasts (see Figure 10.27), phosphatidylserine is made from CDP-diacylglycerol and can be decarboxylated to phosphatidylethanolamine. Thirdly, the

Glycerol 3-phosphate Acyl CoA 1 CoA 1-Acylglycerol 3-phosphate

Glucose 6-phosphate

Acyl CoA

15

CoA Inositol-1-P

Phosphatidic acid

CTP

Pi 14

CoA Acyl CoA

Diacylglycerol

Triacylglycerol Ethanolamine ATP 11

Ethanolamine-P

Inositol CMP 16

3

Phosphatidylinositol Phosphatidylserine

CDP-Ethanolamine

12

Pi

CDP-Diacylglycerol Serine

CMP CTP PPi

ADP

PPi

2

13

CMP

4

Glycerol 3-phosphate

CO2 Phosphatidylethanolamine AdoMet

17 CMP

5 AdoHcy Phosphatidylmonomethylethanolamine AdoMet

Phosphatidylglycerophosphate 18

6 AdoHcy

ATP ADP Choline

8

CTP PPi

Choline-P

9

Phosphatidyldimethylethanolamine AdoMet 7 10 CDP-Choline AdoHcy CMP

Phosphatidylcholine

Phosphatidylglycerol 19 Cardiolipin

FIGURE 10.27 Phospholipid biosynthetic pathways in Saccharomyces cerevisiae. The indicated reactions are catalysed by the following enzymes: (1) glycerol 3-phosphate acyltransferase; (2) CDP-DG synthetase; (3) PS synthetase; (4) PS decarboxylase; (5) PE methyltransferase; (6) and (7) phospholipid methyltransferase; (8) choline kinase; (9) cholinephosphate cytidylyltransferase; (10) cholinephosphotransferase; (11) ethanolamine kinase; (12) ethanolaminephosphate cytidylyltransferase; (13) ethanolaminephosphotransferase; (14) PA phosphatase; (15) I-1-P synthetase; (16) PI synthetase; (17) PGP synthetase; (18) PGP phosphatase; (19) CL synthetase. (From Carman and Henry (1989).)

673

10.2

Glycerophospholipids

O O

OCR

RCO OH Diacylglycerol

OH HO OH Glycerol

Pi

ATP ADP

OH 2RC ACP

HO

H2O

O

ATP O

ADP

O O

O

HO

OCR

OCR CTP RCO RCO P PP CYT PPi P Glycerol 3-phosphate Phosphatidic acid CDP-diacylglycerol

OH

OCR P OH RCO CMP P Phosphatidylglycerophosphate P

NAD+

H2O

L-Serine Pi

OH NADH (NADPH) O O CMP P O + OCR NH 3 Dihydroxyacetone RCO phosphate PCH2CHCO2– Phosphatidylserine

O O

OCR

RCO

PG

OCR

CO2 + PCH2CH2NH3 Phosphatidylethanolamine RCO

O O

OCR

RCO P

P OH O

Oligosaccharide Diacyglycerol

Glycerol O OCR

RCO Diphosphatidylglycerol (cardiolipin)

FIGURE 10.28

OH OH

P Phosphatidylglycerol

O O

O O

Membrane-derived oligosaccharide(glycerol-1-P)n

Phospholipid metabolism in Escherichia coli. (From Pieringer (1989).)

CDP-base pathway for phosphatidylethanolamine is not used. And, finally, diphosphatidylglycerol is made by a reaction involving two phosphatidylglycerol molecules (Figure 10.28). See also Gurr et al. (2002) and Heath et al. (2002). Most research in bacteria has concentrated on E.coli, a Gram-negative bacterium with a rather restricted habitat. It cannot be emphasized too strongly that pathways and enzymes studied in E.coli may not be typical (or even present) in all bacteria (Harwood and Russell, 1984). The overall pathways for phospholipid synthesis in a Gram-positive bacterium, Streptococcus falcium, are shown in Figure 10.29. There has been recent interest in the biosynthesis and occurrence of phosphatidylcholine in bacteria. The occurrence of this lipid in bacteria is thought to be underestimated and, indeed, in Acetobacter aceti represents 73% of the phospholipids. Although it was always thought to be made by methylation of phosphatidylethanolamine, many bacteria use a phosphatidylcholine synthase, which is a member of the CDP-alcohol phosphatidyltransferase group of enzymes. Moreover, the CDP-choline pathway may be present in some bacteria (Sohlenkamp et al., 2003). Individual features of lipid metabolism in bacteria and other prokaryotes or microbes are covered by Pieringer (1989), Jackowski et al. (1991), and Cronan (2003). Smith (1993) has reviewed phospholipid synthesis in protozoa.

Methods for the isolation and study of numerous enzymes of phospholipid metabolism are given in Dennis and Vance (1992). A summary of phospholipid biosynthesis in different organisms is found in Gurr et al. (2002) and in various chapters of Vance and Vance (2002).

10.2.2

Breakdown

The breakdown of glycerophospholipids is catalysed by a series of phospholipases designated A, B, C and D depending on their positions of attack (Figure 10.30). In addition, phosphatidic acid phosphatase is important, but this has already been dealt with (Section 10.2.1). Two phospholipase A specificities are recognized, and these are named A1 and A2, depending on the position of the ester hydrolysed in the diacylphosphoglyceride. Phospholipase B is the name often used for an enzyme that is a monoacylphosphoglyceride acylhydrolase and can be active at either position. However, they are active on diacylphosphoglycerides, whereas lysophospholipases remove the remaining acyl group from a monoacyl phospholipid (see below for further discussion). For reviews of the basic features of phospholipase action, the reader is referred to Hill and Lands (1970), Dawson (1973), van den Bosch (1982), Dennis (1983), Waite (1987), and Wilton and Waite (2002).

674

Lipid Metabolism

O RC ACP

O H 2O OCR

O

OH HO

Pi

O

O

RCO

RCO

P P Glycerol-3-phosphate Phosphatidic acid CTP

OH ADP ATP Diacyglycerol

O

CH2OH O O RCO O

OCR UDP- UDP glucose

Monoglucosyldiacylglycerol

O

OCR

RCO P

O

Kojibiosyldiacylglycerol

OCR

RCO Diphosphatidylglycerol (cardiolipin)

OCR

RCO PP cyt CDP-diacylglycerol

Diacylglycerol

Glycerol

Glycerophosphate Phosphatidyglycerol CMP O O

OCR

RCO

O CH2OH P O

O

H2O

O

P OH

OCR

RCO

P Phosphatidylglycerophosphate

O OH OH

O RCO

RCO

OCR

OCR

Phosphatidylkojibiosyldiacylglycerol

O CH2OH P O

OAA OH

O

P Aminoacylphosphatidylglycerol HO

O RCO O

O

O

O

tRNA

OCR

HO

OOCR OOCR O

CH2OH O

P Phosphatidylglycerol

Pi

AminoacyltRNA O

OCR

O

O

P OH O

O

CH2OH O O RCO O CH2OH O

O

PPi O

UDP- UDP OCR glucose

P

OH P

CH2OH O O n

HO

O

O OCR

RCO O

O

Lipoteichoic acid

FIGURE 10.29 Lipid metabolism in Streptococcus faecium (= faecalis) ATCC9790. For simplicity not all hydroxyl groups on glucose are shown. (From Pieringer (1989).)

B

A2 R2

C O

been published. The interpretation of the kinetics of phospholipase inhibition has been the subject of considerable attention (see Harwood and John, 1990) and suicideinhibitory bifunctionally-linked substrates have been developed as phospholipase A2 inhibitors (Washburn and Dennis, 1991). Phospholipase A enzymes — particularly those from snake venoms or digestive secretions — have been widely studied. Phospholipase A1 is found in microsomal and liposomal fractions (cf. Newkirk and Waite, 1971; Gatt, 1968). It specifically deacylates phosphatidylcholine or phosphatidylethanolamine at the 1-position. Both of these substrates are hydrolysed at the same rate by the adrenal medulla lysosomal enzyme, but that from brain prefers phosphatidylcholine as substrate. Detergents will increase phosphatidylethanolamine hydrolysis by the brain enzyme. A phospholipase A1 (which is relatively specific for phosphatidylglycerol) has been reported from the spores of some bacteria (Raybin et al., 1972), but most bacterial enzymes are unspecific for either the 1- or the 2-positions. Examples of phospholipase A (unspecific) enzymes from bacteria include the so-called detergent-resistant

A1

O

CH2O

C

CH

O O

CH2O

P

R1

O

X

O– C

D

FIGURE 10.30 Position of phosphoglyceride hydrolysis for different phospholipases.

In common with many other lipid-metabolising enzymes, the assay of phospholipases deserves careful consideration. Principally, the potential difficulties are due to the water-insoluble nature of the substrate, the effects of various incubation additions (e.g., solvents, detergents), which can make a big difference to activity, and the problems in kinetic interpretation. Various assay methods have been discussed (Waite, 1987) and an important volume of Methods in Enzymology (Dennis, 1991) has 675

10.2

Glycerophospholipids

enzyme from the outer membrane of E. coli and a cytoplasmic enzyme from the same bacterium (Nakagawa et al., 1991). These enzymes differ considerably in their properties (Harwood and Russell, 1984). A number of Gram-negative bacteria have phospholipase A activity in their outer membranes, but it is often not determined whether they are phospholipase A1 or A2. Several other lipases have been noted to have phospholipase (usually phospholipase A1) activity. These include rat hepatic lipase (Waite et al., 1991) and milk lipoprotein lipase (BengtssonOlivecrona and Olivecrona, 1991). The specificity of phospholipase A2 enzymes varies considerably even among those from snake venoms (see Dawson, 1973). The enzymes remove a fatty acyl group on the glycerol carbon adjacent to the phosphoryl substituent — i.e., position 2 in a natural phosphoglyceride. If the phosphoryl substituent is at the 2-position, then only a fatty acid with the correct steric configuration is hydrolysed. Substrate specificities also vary markedly with the chain length and degree of unsaturation. For the snake venom enzyme hydrolysing phosphatidylcholine, the rate of hydrolysis is in the order: (1-unsat., 2-sat.) > (1-unsat., 2unsat.) > (1-sat., 2-polyunsat.) > (1-sat., 2-mono-unsat.) > (1-sat., 2-sat.). In contrast, the pancreatic enzyme exhibits no preference for chain length or degree of unsaturation. The hydrolysis of certain phospholipids can be increased in different ways for the various phospholipase A2 enzymes, e.g., by detergent or diethyl ether addition. This aspect and comments on the underlying physicochemical mechanisms are discussed by Dawson (1973), Slotboom et al. (1982), and Waite (1987), where further references will be found. Specific details of purification and characterization of individual phospholipases are given in Dennis (1991). Receptors for phospholipase A2 enzymes of various types have been discovered and studied. They are thought to play crucial roles in the physiological actions of phospholipases A2 (Ohera et al., 1995). Based on sequence data, phospholipase A2 enzymes from animals are classified into 10 groups, which can be simplified into three major types, based on their physiological properties and function. These are (1) the secretory, low-molecular weight PLA2; (2) cytosolic Ca2+-dependent PLA2; and (3) the intracellular Ca2+-independent PLA2 (Balsinde et al., 1991). Groups (1) and (3) are also reported from plants (Wang, 2001). Enzymes belonging to (1), secretory phospholipase A2 enzymes from plants and their regulatory and catayltic properties, have been reviewed by Lee et al., (2005). Because of the ready availability of the pancreatic phospholipase A2, this enzyme has been thoroughly examined by enzymologists. The phospholipase is secreted as a zymogen and activated by cleavage of a peptide of three to seven amino acids. The amino acid sequence of the active enzyme has been determined for a number of different preparations (pig, ox, horse, and human). The different preparations show a high degree of homology,

e.g., the human phospholipase differs from the others in only a single amino acid residue out of 123 to 125 in each case (Verheij et al., 1983). Further details are given in Waite (1987). A gene encoding the cobra venom phospholipase A2 has been expressed in E. coli and the recombinant enzyme shown to have the expected activity characteristics (Kelley et al., 1992). Lysophospholipases are probably distributed as widely as the phospholipases A (Waite, 1987). A true definition of lysophospholipases is difficult, as discussed by van den Bosch (1982). Indeed, a number of enzymes in this group also have activity against diacyl phospholipids and, therefore, are more properly referred to as phospholipase-B or -A1 enzymes (van den Bosch et al., 1974). Recent examples are a human phosphatidylserine — specific phospholipase A1 that also has lysophosphatidylserine lysophospholipase activity (Nagai et al., 1999) and two phospholipase B lysophospholipases from S. cerevisiae (Merkel et al., 1999). Also, there are several lysophospholipases that show esterase activity and whose classification is difficult. For more discussion, see Waite (1987) and specific chapters on individual lysophospholipases or the so-called phospholipase B from Penicillium notatum in Dennis (1991). Phospholipase C enzymes are secreted by several bacteria, particularly pathogens, such as Clostridium spp. These proteins are zinc metalloenzymes and some also require Ca2+ for activity. The phospholipase C enzymes vary widely in substrate specificity (van den Bosch, 1982). The enzyme from C. perfringens attacks choline-containing lipids most readily. Such hydrolysis requires a positive zetapotential brought about by long-chain cations or by the addition of Ca2+ or Mg2+. Phosphatidylcholine breakdown in the presence of Ca2+ is greatly increased by the addition of specific concentrations of sodium deoxycholate. The phospholipase C from Bacilus cereus attacks a large number of phospholipids (e.g., PG, DPG), which are poorly hydrolysed by the C. perfringens enzyme. It will also hydrolyse short-chain phosphoglycerides, which are poor substrates for the latter enzyme (Dawson, 1973). The use of phospholipase C enzymes in the preparation of diacylglycerols for molecular species examination is discussed by Christie (1982). Mammalian phospholipase C enzymes are now known to be central in regulatory processes (Waite, 1987). Most, if not all, of these enzymes are specific for phosphatidylinositol or its phosphorylated derivatives. Specific enzymes are discussed in Dennis (1991) and their role in transmembrane signaling in various organisms (Berridge and Irvine, 1989) and in higher plants (Hetherington and Drobak, 1992) has been reviewed (see also Section 10.6). Specific examples of important functional aspects of such phospholipase C activity are given by Turk et al. (1987) and by Cockcroft (1992) in relation to insulin secretion by pancreatic islets and in neutrophils, respectively. Plant phospholipase C enzymes can be divided into three groups: (1) enzymes acting on the phosphoinositides, 676

Lipid Metabolism

(2) nonspecific phospholipase Cs that act on phosphatidylcholine and some other phospholipids, and (3) a phospholipase C that hydrolyses glycosylphosphatidylinositol anchors on proteins. The first type of phospholipase C has many important regulatory functions, including responses to stimuli, in plants (Wang, 2001). There are few examples of phospholipase D (EC 3.1.4.4) in bacteria (Harwood and Russell, 1984; Cockcroft, 1996), but many plant tissues exhibit high activity. Plant phospholipase D is also a transferase enzyme that catalyses transphosphatidylation to an acceptor molecule. If phospholipase D enzymes are not fully inactivated, then this reaction will lead to the artifactual formation of phosphatidylmethanol in plant tissues extracted with methanolic solutions (Harwood, 1980). A variety of water-soluble primary alcohols can be used as acceptors (e.g., propanol, ethanol, ethylene glycol, glycerol). Most enzymes are stimulated in the presence of linear aliphatic ethers (Dawson, 1973; Galliard, 1980). A possible function in plants for phospholipase D in synthesizing phospholipids has now been largely discounted because the phosphatidylglycerol thus made is a racemic mixture, unlike the naturally occurring phospholipid (Yang et al., 1967). However, Batrakov et al. (1975) found evidence for stereospecificity in the transfer. Roughan and Slack (1976) have claimed that phospholipase D is a structural protein that, under certain nonphysiological conditions, possesses enzymic activity. In plants, phospholipase D (PLD) enzymes can be divided into three groups, based on their properties: (1) a conventional PLD that is most active at 20 to 100 mM Ca++, (2) a polyphosphoinositide-dependent PLD that is active at micromolar Ca++, and (3) a phosphatidylinositolspecific PLD that is independent of Ca++. For Arabidopsis, five groups based on gene sequence were identified. PLDs in plants present interesting properties for activation and regulation and appear to play physiological roles in a wide range of stress conditions. These include freezing, drought, wounding, pathogen infection, nutrient deficiency and air pollution (Wang, 2001). There has been some recent interest in the possible role of mammalian phospholipase D in signal transduction, in that a wide variety of agents (including hormones, neurotransmitters and growth factors) have been shown to activate a phospholipase D to hydrolyse phosphatidylcholine (see e.g., Hurst et al., 1990; Xie and Dubyak, 1991). Various GTPases are known to activate the enzyme in mammals and its physiological relevance has been discussed (Cockcroft, 1996). This article also provides information on the enzymology of phospholipase Ds from different organisms and earlier reviews on the subject. Yamane et al. (1989) discuss the commercial use of phospholipase D enzymes for transphosphatidylation purposes. Phosphatidate phosphohydrolase, although a phospholipid degradative enzyme, is mainly involved in the biosynthesis of lipids (Gurr and Harwood, 1991). In mammalian

tissues the enzyme may control the overall rate of lipid (triacylglycerol) deposition. A comprehensive review of the enzyme in different organisms and tissues has been published by Brindley (1988) (see also Dennis, 1991). Many plant tissues possess high activities of nonspecific lipid acyl hydrolases, which can also give rise to problems with the solvent extraction of plants. Acyl hydrolases will hydrolyse fatty acids from a large number of lipids including phosphoglycerides (cf. Galliard, 1980) The enzymes have been purified from several tissues and their substrate specificities and other properties determined. Potato tubers and leaves from Phaseolus spp. are particularly rich sources (cf. Galliard, 1980) and the position of bond cleavage has been determined for the latter enzyme to be on the fatty acid side of the oxygen ester bond (Burns et al., 1980). An important example of a plant acyl hydrolase is patatin, which is a storage protein from potato tubers. Patatin-like proteins are produced in a number of plants in response to stresses, such as virus or fungal infection (Meijer and Munnik, 2003). For an overall review of the functions of phospholipases in such areas as the phosphatidylinositol cycle, the archidonate cascade, in the digestion of dietary fat in lipoprotein metabolism and in snake venoms, see Waite (1987) and also Vance (1991). Phospholipase A2 has also been invoked in the protection of membranes from lipid peroxidation damage (van Kuijk et al., 1987).

References Amidon, B. et al. (1995). Biosynthetic conversion of phosphatidylglycerol to sn-1:sn-1 bis(monoacylglycero) phosphate in a macrophage-like cell line. Biochemistry. 34, 5554–5560. Ansell, G.B. and Spanner, S. (1982). Phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine. In Phospholipids, Eds. J.N. Hawthorne and G.B. Ansell, Elsevier, Amsterdam, pp. 1–49. Balsinde, J. et al. (1999). Regulation and inhibition of phospholipase A2. Annu. Rev. Pharmacol. Toxicol. 39, 175–189. Batrakov, S.G. et al. (1975). Steric analysis of glycerophospholipids by circular dichroism. Stereospecificity of phospholipase D catalyzed transesterification. Biochem. Biophys. Res. Commun., 66, 755–762. Bell, R.M. and Coleman, R.A. (1980). Enzymes of glycerolipid synthesis in eukaryotes. Annu. Rev. Biochem. 49, 459–487. Bengtsson-Olivecrona, G. and Olivecrona, T. (1991). Phospholipase activity of milk lipoprotein lipase. In Methods in Enzymology, vol. 197, Ed. E.A. Dennis, Academic Press, New York, pp. 345–356. Berridge, M.J. and Irvine, R.F. (1984). Inositol triphosphate, a novel second messenger in cellular signal transduction. Nature, 312, 315–321. Berridge, M.J. and Irvine, R.F. (1989). Inositol phosphates and cell signalling. Nature, 341, 197–205. Bloch, K. and Vance, D. (1977). Control mechanisms in the synthesis of saturated fatty acids Annu. Rev. Biochem. 46, 263–298.

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Brindley, D.N. Ed. (1988) Phosphatidate Phosphohydrolase, vols. I and II, CRC Press, Boca Raton, FL. Brindley, D.N. (1991). Metabolism of triacylglycerols. In Biochemistry of Lipids, Lipoprotein and Membranes, Eds. D.E. Vance and J. Vance, Elsevier, Amsterdam, pp. 171–203. Brindley, D.N. and Sturton, R.G. (1982). Phosphatidate metabolism and its relation to triglycerol biosynthesis. In Phospholipids, Eds. J.N. Hawthorne and G.B. Ansell, Elsevier, Amsterdam, pp. 179–213. Brophy, P.J. and Vance, D.E. (1976). Copurification of choline kinase and ethanolamine kinase from rat liver by affinity chromatography. FEBS Lett., 62, 123–125. Browse, J. and Somerville, C.R. (1991). Glycerolipid synthesis: biochemistry and regulation. Annu. Rev. Plant Physiol., 42, 467–506. Burns, D.D. et al. (1980). Properties of acyl hydrolase enzymes from Phaseolus multiflorus leaves. Phytochemisty. 19, 2281–2285. Carman, G.M. and Henry, S.A. (1989). Phospholipid biosynthesis in yeast. Annu. Rev. Biochem., 58, 635–669. Carman, G.M. and Henry, S.A (1999). Phospholipid biosynthesis in the yeast, Saccharomyces cerevisiae and interrelationship with other metabolic processes. Prog. Lipid Res. 38, 361–399. Chapman, K.D. (2004). Occurrence, metabolism and prospective functions of N-acylethanolamines in plants. Prog. Lipid Res. 43, 302–327. Christie, W.W. (1982) Lipid Analysis, 2nd ed., Pergamon, Oxford. Cockcroft, S. (1992). G-protein-regulated phospholipases C, D and A 2 -mediated signalling in neutrophils. Biochim. Biophys. Acta, 1113, 135–160. Cockcroft, S. (1996) Phospholipase D: regulation by GTPases and protein kinase C and physiological relevance. Prog. Lipid Res. 35, 345–370. Cronan, J.E. (2003). Bacterial membrane lipids: where do we stand? Ann. Rev. Microbiol. 57, 203–224. Dawson, R.M.C. (1973). Specificity of enzymes involved in the metabolism of phospholipids. In Form and Function of Phospholipids, Eds. G.B. Ansell, R.M.C. Dawson and J.N. Hawthorne, Elsevier, Amsterdam, pp. 97–116. Dennis, E.A. (1983). Phospholipases. In The Enzymes, vol. XVI, Ed. P. Boyer, Academic Press, New York, pp. 307–353. Dennis, E.A. Ed. (1991) Methods in Enzymology, vol. 197, Academic Press, New York. Dennis, E.A. and Vance, D.E. Eds. (1992) Methods in Enzymology, vol. 209, Academic Press, New York. Dormann, P. (2005). Membrane lipids. In Plant Lipids: biology Utilisation and Manipulation, Ed. D.J. Murphy, Blackwell Publishing, Oxford, U.K., pp. 123–161. Downes, C.P and MacPhee, C.H. (1990). Myo-inositol metabolites as cellular signals. Eur. J. Biochem. 193, 1–18. Drobak, B.K. (2005) Inositol-containing lipids: roles in cellular signalling. In Plant Lipids: Biology, Utilisation and Manipulation, Ed. D.J. Murphy, Blackwell Publishing, Oxford, U.K.. pp. 303–328. Feldman, D.A. and Weinhold, P.A. (1987). CTP: phosphorylcholine cytidylyltransferase from rat liver. Isolation and characterization of the catalytic subunit. J. Biol.Chem. 262, 9075–9081.

Fruman, D.A. et al. (1998). Phosphoinositide kinases. Annu. Rev. Biochem. 67, 481–507. Galliard, T. (1980). Degradation of acyl lipids: hydrolytic and oxidative enzymes. In Biochemistry of Plants, vol. 4, Eds. P.K. Stumpf and E.E. Conn, Academic Press, New York, pp. 85–116. Gatt, S. (1968). Purification and properties of phospholipase A-1 from rat and calf brain. Biochim. Biophys. Acta. 159, 304–316. Groot, P.H.E. et al. (1976). Fatty acid activation: specificity, localization, and function. Adv. Lipid Res. 14, 75–126. Gurr, M.I. and Harwood, J.L. (1991). Lipid Biochemistry, 4th ed., Chapman & Hall, London. Gurr, M.I. et al. (2002). Lipid Biochemistry, 5th ed., Blackwell Scientific, Oxford, U.K. Hajra, A.K. (1995). Glycerolipid biosynthesis in peroxisomes (microbodies). Prog. Lipid Res. 34, 343–364. Harwood, J.L. (1979). The synthesis of acyl lipids in plant tissues. Prog. Lipid Res. 18, 55–86. Harwood, J.L. (1980). Plant acyl lipids: structure, distribution and analysis. In Biochemistry of Plants, vol. 4, Eds. P.K. Stumpf and E.E. Conn, Academic Press, New York, pp. 1–55. Harwood, J.L. (1989). Lipid metabolism in plants. Crit. Rev. Plant Sci. 8, 1–43. Harwood, J.L. and John, R.A. (1990). Evaluation of inhibitors of lipolytic enzymes. Trends Biochem. Sci. 15, 409–410. Harwood, J.L. and Page, R. (1993). Biochemistry of oil synthesis. In Designer Oilseed Crops, Ed. D.J. Murphy, VCH Press, Weinheim, Germany, pp. 165–194. Harwood, J.L., and Russell, N.J. (1984) Lipids in Plants and Microbes, Allen and Unwin, Hemel Hempstead, U.K. Hawthorne, J.N. and Ansell, G.B. Eds. (1982) Phospholipids, Elsevier, Amsterdam. Heath, R.J. et al. (2002). Fatty acid and phospholipid synthesis in prokaryotes. In Biochemistry of Lipids, Lipoproteins and Membranes, Eds. D.E. Vance and J.E. Vance, 4th ed., Elsevier, Amsterdam, pp. 55–92. Heller, M. (1978). Phospholipase D. Adv. Lipid Res., 16, 267–326. Hetherington, A.M. and Drobak, B.K. (1992). Inositol-containing lipids in higher plants Prog. Lipid Res. 31, 53–63. Hill, E.E. and Lands, W.E.M. (1970). Phospholipid metabolism. In Lipid Metabolism, Ed. S.J. Wakil, Academic Press, New York, pp. 185–275. Huang, K.-S., Li, S. and Low, M.G. (1991). Glycosylphosphatidylinositol-specific phospholipase D. In Methods in Enzymology, vol. 197, Ed. E.A. Dennis, Academic Press, New York, pp. 567–575. Hurst, K.M. et al. (1990). The roles of phospholipase D and a GTP-binding protein in guanosine 5'-[gamma-thio]triphosphate-stimulated hydrolysis of phosphatidylcholine in rat liver plasma membranes. Biochem. J. 272, 749–753. Jackowski, S. et al. (1991). Lipid metabolism in prokaryotes. In Biochemistry of Lipids, Lipoproteins and Membranes, Eds. D.E. Vance and J. Vance, Elsevier, Amsterdam, pp. 43–85. Jackson, R.L. and McLean, L.R. (1991). Human postheparin plasma lipoprotein lipase and hepatic triglyceride lipase. In Methods in Enzymology, vol. 197, Ed. E.A. Dennis, Academic Press, New York, pp. 339–345.

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Joyard, J. and Douce, R. (1987). Galactolipid synthesis. In The Biochemistry of Plants, vol. 9, Eds. P.K. Stumpf and E.E. Conn, Academic Press, New York, pp. 215–274. Kelley, M.J. et al. (1992). Renaturation of cobra venom phospholipase A2 expressed from a synthetic gene in Escherichia coli. Biochim. Biophys. Acta. 1118, 107–115. Kent, C. (1995). Eukaryotic phospholipid biosynthesis. Annu. Rev. Biochem. 64, 315–343. Kobayashi, M. and Kanfer, J.N. (1991). Solubilization and purification of rat tissue phospholipase D. In Methods in Enzymology, vol. 197, Ed. E.A. Dennis, Academic Press, New York, pp. 575–583. Kuge, O. and Nishijima, M. (1997). Phosphatidylserine synthases I and II of mammalian cells. Biochim. Biophys. Acta. 1348, 151–156. Lee, H.Y. et al. (2005). Multiple forms of secretory phospholipase A2 in plants. Prog. Lipid Res. 44, 52–67. MacDonald, J.I.S. and Sprecher, H. (1991). Phospholipid fatty acid remodeling in mammalian cells. Biochim. Biophys. Acta. 1084, 105–121. Martin, T.F.J. (1998). Phosphoinositide lipids as signalling molecules: common themes for signal transduction, cytoskeletal regulation and membrane trafficking. Annu. Rev. Cell Dev. Biol.14, 231–264. McPhail, L.C. (2005). Glycerolipids in signal transduction. In Biochemistry of Lipids, Lipoproteins and Membranes, Ed. D.E. Vance and J.E. Vance, 4th ed., Elsevier, Amsterdam, pp. 315–340. Meijer, H.J.G. and Munnik, T. (2003). Phospholipid-based signalling in plants. Annu. Rev. Plant Biol. 54, 265–306. Merkel. O. et al. (1999). Characterisation and function in vivo of two novel phospholipases B lysophospholipases from S a ch a ro myc e s c e revi s i a e. J. B i o l . C h e m . 2 7 4 , 28121–28127. Moore, T.M. (1982). Phospholipid biosynthesis. Annu. Rev. Plant Physiol. 33, 235–259. Mudd, J.B. (1980). Phospholipid biosynthesis. In Biochemistry of Plants, vol. 4, Eds. P.K. Stumpf and E.E. Conn, Academic Press, New York, pp. 249–282. Mudd, S.H. and Datko, A.H. (1986). Phosphoethanolamine bases as intermediates in phosphatidylcholine synthesis by Lemna. Plant Physiol. 82, 126–135. Munnik, T. et al. (1998). Phospholipid signalling in plants. Biochim. Biophys. Acta. 1389, 222–272. Nagai, Y. et al. (1999). An alternative splicing form of phosphatidylserine-specific phospholipase A1 that exhibits lysophosphatidylserine-specific lysophospholipase activity in humans. J. Biol. Chem. 274, 11053–59. Nakagawa, Y. et al. (1991). Detergent-resistant phospholipase A1 from Escherichia coli membranes. In Methods in Enzymology, vol. 197, Ed. E.A. Dennis, Academic Press, New York, pp. 309–315. Newkirk, J.D. and Waite, M. (1971). Identification of a phospholipase A1 in plasma membranes of rat liver. Biochim. Biophys. Acta. 225, 224–233. O’Doherty, P.J.A. (1978). Metabolic studies with natural and synthetic fatty acids and enantiomeric acylglycerols. In Handbook of Lipid Research, vol. 1, Ed. A. Kuksis, Plenum, New York, pp. 289–339.

Ohara, O. et al. (1995). Structure and function of phospholipase A2 receptor. Prog. Lipid Res. 34, 117–138. Pieringer, R.A. (1989). Biosynthesis of non-terpenoid lipids. In Microbial Lipids, vol. 2, Eds. C. Ratledge and S.G. Wilkinson, Academic Press, London, pp. 51–114. Price-Jones, M.J. and Harwood, J.L. (1986). The control of CTP:choline phosphate cytidylyltransferase activity in pea (Pisum sativum L.). Biochem. J. 240, 837–842. Rameh, L.E. and Cantely, L.C. (1999). The role of phosphoinositide 3-kinase lipid products in cell function. J. Biol. Chem. 274, 8347–8350. Raybin, D.M. et al. (1972). A phospholipase in Bacillus megaterium unique to spores and sporangia. Biochemistry. 11, 1754–1760. Ridgeway, N.D. et al. (1999). Integration of phospholipid and sterol metabolism in mammalian cells. Prog. Lipid. Res., 38, 337–360. Roughan, P.G. and Slack, C.R. (1976). Is phospholipase D really an enzyme? A comparison of in situ and in vitro activities. Biochim. Biophys. Acta. 431, 86–95. Roughan, P.G. and Slack, C.R. (1984). Glycerolipid synthesis in leaves. Trends Biochem. Sci. 9, 383–386. Sambanthamurthi, R. et al. (2002). Chemistry and biochemistry of palm oil. Prog. Lipid Res., 39, 507–558. Schlame, M. et al. (2000). The biosynthesis and functional role of cardiolipin. Prog. Lipid Res. 39, 257–288. Slotboom, A.J. et al. (1982). On the mechanism of phospholipases A2. In Phospholipids, Eds. J.N. Hawthorne and G.B. Ansell, Elsevier, Amsterdam, pp. 359–434. Smith, J.D. (1993). Phospholipid synthesis in protozoa. Prog. Lipid Res., 32, 47–60. Snyder, F. (1991). Metabolism, regulation, and function of etherlinked glycerolipids and their bioactive species. In Biochemistry of Lipids, Lipoproteins and Membranes, Eds. D.E. Vance and J. Vance, Elsevier, Amsterdam, pp. 241–267. Snyder, F., et al. (1985). Ether-linked glycerolipids and their bioactive species: enzymes and metabolic regulation. In The Enzymes of Biological Membranes, Ed. A.N. Martinosi, Plenum, New York, pp. 1–58. Snyder, F. et al. (2002). Ether-linked lipids and their bioactive species. In Biochemistry of Lipids, Lipoproteins and Membranes, Eds. D.E. Vance and J.E. Vance, 4th ed., Elsevier, Amsterdam, pp. 233–262. Sohlenkamp, C. et al. (2003). Biosynthesis of phosphatidylcholine in bacteria. Prog. Lipid Res., 42, 115–162. Taki, T. et al. (1978). The “base-exchange” reaction: the serine enzyme. Adv. Exp. Med. Biol. 101, 301–318. Turk, J. et al. (1987). The role of phospholipid-derived mediators including arachidonic acid, its metabolites, and inositoltrisphosphate and of intracellular Ca2+ in glucose-induced insulin secretion by pancreatic islets. Prog. Lipid Res. 26, 125–181. Ulane, R.E. et al. (1977). A rapid accurate assay for choline kinase. Anal. Biochem. 79, 526–534. van den Bosch, H. (1982). Phospholipases. In Phospholipids, Eds. J.N. Hawthorne and G.B. Ansell, Elsevier, Amsterdam, pp. 313–357. van den Bosch, H. et al. (1974). Isolation and properties of a phospholipase A1 activity from beef pancreas. Biochim. Biophys. Acta. 348, 197–209.

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van Kuijk, F.J.G.M. et al. (1987). A new role for phospholipaseA2 — protection of membranes from lipid-peroxidation damage. Trends Biochem. Sci. 12, 31–34. Vance, D.E. (1990). Phosphatidylcholine metabolism: masochistic enzymology, metabolic regulation and lipoprotein assembly. Biochem. Cell Biol. 68, 1151–1165. Vance, D.E. (1991). Phospholipid metabolism and cell signalling in eukaryotes. In Biochemistry of Lipids, Lipoproteins and Membranes, Eds. D.E. Vance and J. Vance, Elsevier, Amsterdam, pp. 205–240. Vance, D.E. (2005). Phospholipid biosynthesis in eukaryotes. In Biochemistry of Lipids, Lipoproteins and Membranes, Eds. D.E. Vance and J.E. Vance, 4th ed., Elsevier, Amsterdam, pp. 205–232. Vance, D.E. and Choy, I.C. (1979). How is phosphatidylcholine biosynthesis regulated? Trends Biochem.Sci. 4,145–148. Vance, D.E. and Ridgway, N.D. (1988). The methylation of phosphatidylethanolamine. Prog. Lipid Res. 27, 61–79. Vance, D.E. and Vance, J.E. Eds. (2002). Biochemistry of Lipids, Lipoproteins and Membranes. Elsevier, Amsterdam. Vance, J.E. (1998). Eukaryotic lipid biosynthetic enzymes: the same but not the same. Trends Biochem. Sci. 23, 423–428. Vanhaesebroeck, B. et al. (2001). Synthesis and function of 3phosphorylated inositol lipids. Annu. Rev. Biochem.70, 535–602. Verheij, H.M. et al. (1983). The complete primary structure of phospholipase A2 from human pancreas. Biochim. Biophys. Acta. 747, 93–99. von Wettstein-Knowles, P. (1979). Genetics and biosynthesis of plant epicuticular waxes. In Advances in the Biochemistry and Physiology of Plant Lipids, Eds. L-A Appelqvist and C. Liljenberg, Elsevier, Amsterdam, pp. 1–26. Waite, M. (1987). The phospholipases. In Handbook of Lipid Research, vol. 5, Ed. D.J. Hanahan, Plenum, New York, pp. 111–133. Waite, M. et al. (1991). Purification and substrate specificity of rat hepatic lipase. In Methods in Enzymology, vol. 197, Ed. E.A. Dennis, Academic Press, New York, pp. 331–339. Waku, (1992). Origins and fates of fatty acyl-CoA esters. Biochim. Biophys. Acta. 1124, 101–111. Wang, X. (2001). Plant phospholipases. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, 211–231. Washburn, W.N. and Dennis, E.A. (1991). Suicide-inhibitory bifunctionally linked substrates (SIBLINKS) as phospholipase A2 inhibitors. Mechanistic implications. J. Biol. Chem. 266, 5042–5048. Webber, K.O. and Hajra, A.K. (1992). Dihydroxyacetone phosphate acyltransferase. In Methods in Enzymology, vol. 209, Eds. E.A. Dennis and D.E. Vance, Academic Press, New York, pp. 92–98. Williams, M. and Harwood, J.L. (1994). Alternative pathways of phosphatidylcholine synthesis in olive (Olea europeea L.) callus cultures. Biochem. J. 304, 463–468. Wilton, D.C. and Waite, M. (2002). Phospholipases. In Biochemistry of Lipids, Lipoproteins and Membranes, Eds. D.E. Vance and J.E. Vance, 4th ed., Elsevier, Amsterdam, pp. 291–314. Xie, M. and Dubyak, G.R. (1991). Guanine-nucleotide- and adenine-nucleotide-dependent regulation of phospholipase D in electropermeabilized HL-60 granulocytes. Biochem. J. 278, 81–89.

Yamane, T. et al. (1989). Conversion of phospholipids by phospholipase D. In Fats for the Future (Ed. R.C. Cambie), Ellis Horwood, Chichester, U.K., pp. 128–138. Yang, S.F. et al. (1967). Transphosphatidylation by phospholipase D. J. Biol. Chem. 242, 477–484.

10.3

Glyceride metabolism

10.3.1

Triacylglycerol synthesis

The lipid stores of animals and almost all plants are triacylglycerols. These compounds, therefore, are of very great importance in physiology as well as to the food industry. One difference between animals and plants is in the relative amounts of de novo synthesized acyl chains in the triacylglycerols. Plants from necessity must make all of the glyceride molecules from simple starting materials (ultimately from photosynthetically fixed CO2). In contrast, animals make use of dietary fatty acids, which to a large degree determine the fatty acid composition of the triacylglycerol stores (e.g., copepods consuming phytoplankton; Bauermeister and Sargent, 1979). In oil seeds the triacylglycerol stores are located in oil bodies. These intracellular structures are surrounded by a half-unit phospholipid membrane containing unique proteins called oleosins. The latter have been isolated and sequenced from a number of plants and probably serve both to stabilize the oil bodies as well as to provide binding sites for lipases to function during germination (for reviews, see Murphy, 1990; Huang, 1992). In animals most triacylglycerol is stored in adipose tissue, though, in times of metabolic stress, significant amounts may build up in liver, heart and skeletal muscle. Also, it should be noted that the variable distribution of adipose tissue stores and the metabolic properties of such tissue in different compartments of the body has important implications for health (Abate and Garg, 1995). The formation of triacylglycerols removes the potentially harmful effects of fatty acids or fatty acyl-CoAs (Brindley, 1991). A major function of animal triacylglycerols is to allow the transport of acyl moieties about the body, in the form of the serum lipoproteins. Two major classes of lipoprotein are relevant. Chylomicrons carry absorbed dietary fat from the intestine to other organs, while very low density lipoproteins carry triacylglycerol from the liver to other tissues (Section 7.5 and Section 11.2). There has been considerable recent interest in the use of oleaginous microorganisms as sources of triacylglycerols, particularly those with unusual fatty acids. In general, yeasts and moulds offer the best possibilities for industrial exploitation, and few bacteria accumulate appreciable amounts of triacylglycerols (actinomycetes are the most noticeable exceptions). The whole topic of the biotechnology of oils and fats in microorganisms is reviewed by Ratledge (1989) and Cohen and Ratledge (2005). See also Davies and Holdsworth (1992). 680

Lipid Metabolism

The major pathways for triacylglycerol synthesis are shown in Figure 10.31. The formation of phosphatidic acid by the glycerol phosphate pathway or the dihydroxyacetone phosphate pathway has already been discussed (Section 10.2.1). In addition, see reviews by Gurr (1980) and O’Doherty (1978). Phosphatidic acid can also be formed from monoacylglycerol or diacylglycerol, but, as will be seen, this is not of relevance for triacylglycerol synthesis. Attempts to estimate the relative contribution of different pathways for phosphatidic acid formation (cf. Hill and Lands, 1970; O’Doherty, 1978) indicate that about 50% of lipid glycerol enters by the dihydroxyacetone pathway (e.g., Manning and Brindley, 1972; Pollock et al., 1975). In some tissues (e.g., Ehrlich ascites tumour cells), the acyldihydroxyacetone pathway is the major route of synthesis (Synder, 1972). The enzymes responsible for the acylation of glycerol phosphate have been purified and studied many times. For reviews of their purification and properties, see Lennarz (1970), Hill and Lands (1970), O’Doherty (1978), Gurr (1980), and Stymne and Stobart (1987). The glycerol 3-phosphate acyltransferase and monoacylglycerol 3-phosphate acyltransferase enzymes in both animals and plants differ in their substrate specificities. Typically, the glycerol 3-phosphate acyltransferase uses more saturated acyl-CoAs than are subsequently attached to the sn-1 position. The major site of both enzymes is the endoplasmic reticulum, though animal mitochondria (particularly those of liver) also contain a second glycerol 3-phosphate acyltransferase (Brindley, 1991). In plant chloroplasts, glycerol 3-phosphate acylating enzymes (which use acyl-ACP substrates and have different substrate specificities to their endoplasmic reticulum counterparts) are present. Although triacylglycerol synthesis can take place in chloroplasts (particularly under adverse environmental conditions; see Sakaki et al., 1985), the chloroplast enzymes are mainly important for the generation of thylakoid acyl lipids (Ohlrogge et al., 1991). For the dihydroxyacetone pathway (see Figure 10.31), acyltransferases have been found in both the endoplasmic reticulum and in peroxisomes (Brindley, 1991). As indicated in Figure 10.31, diacylglycerol can originate from two sources. Either it is formed by phosphatidate phosphohydrolase or it is synthesised from monoacylglycerol. The first demonstration of an enzyme system capable of converting monoacylglycerols to triacylglycerols was the work of Clark and Hübscher (1960) with preparations from rabbit intestine. Indeed, the reactions have been studied most thoroughly in intestine, although activity has been detected in a large number of mammalian tissues including kidney, pancreas, adipose tissue, arterial walls, ascites tumour cells and mammary glands (cf. O’Doherty, 1978; Brindley, 1991). The first enzyme in the pathway is monoacylglycerol acyltransferase (EC 2.3.1.22). The enzyme is particulate and is found in microsomal fractions — probably arising from

the endoplasmic reticulum. Evidence for the monoacylglycerol pathway in plants is poor, and it seems probable that diacylglycerol arises from phosphatidate phosphatase in these phyla (Gurr, 1980). For comments on the extensive literature concerning the substrate specificities and other properties of mammalian monoacylglycerol acyltransferases, refer to O’Doherty (1978) and Dircks and Sul (1999). Phosphatidate phosphohydrolase has already been briefly discussed in relation to glycerophospholipid synthesis (Section 10.2.1). The enzyme was first discovered in plants (Kates, 1955) and, subsequently, identified in animal tissues. It has been purified from a large number of mammalian tissues including liver, kidney, intestinal mucosa, adipose tissue, erythrocyte membranes, and avian salt glands (cf. O’Doherty, 1978). The intracellular distribution of mammalian phosphatidate phosphohydrolase is complicated. It has been found in several particulate fractions — mitochondrial, lysosomal, and microsomal (cf. Sedgwick and Hübscher, 1967). However, quantitatively, the most important of these fractions is the endoplasmic reticulum. In addition, there is significant cytosolic activity in animals. The cytosolic phosphatidate phosphohydrolase is able to translocate to the endoplasmic reticulum and mitochondria. The response of such a Mg2+dependent enzyme to hormones and metabolites and variations in its intracellular distribution between soluble and particulate compartments, are compatible with an important role in controlling triacylglycerol synthesis. For a full discussion of this function, see Brindley (1988, 1991). In plants, phosphatidate phosphohydrolase has a less obvious role in controlling the rate of triacylglycerol synthesis, since the latter occurs usually at a specific developmental period when it is not in competition with other metabolic pathways, such as membrane lipid synthesis (Harwood and Griffiths, 1992). The enzyme has been detected in several subcellular compartments (see Harwood and Price-Jones, 1988), and, moreover, its activity in chloroplast envelopes appears to be important in controlling the flux of lipid carbon between the extrachloroplastic and the chloroplast compartments. This is important in giving rise to different molecular species of lipids in various plants (see Heinz and Roughan, 1987; Ohlrogge et al., 1991). There have been some suggestions that the activity of phosphatidate phosphohydrolase may be important in triacylglycerol accumulation in seeds (Ichihara et al., 1990) even though the enzyme, unlike in animals, is not under acute (hormonal) control. Synthesis of triacylglycerols requires the enzyme diacylglycerol acyltransferase (EC 2.3.1.30). The enzyme is located in the endoplasmic reticulum (Brindley, 1991) and its activity is especially high in lypogenic tissues like adipose and liver. Its properties and those of other acyltransferases involved in the glycerol phosphate pathway for triacylglycerol formation were reviewed by Dircks and Sul (1991). Diacylglycerol acyltransferase is unique to 681

10.3 Glyceride metabolism

C

H

H2C

O

HO

H2 C

OH

H2 C

H

H2C

O

1

CoA

2

O

C

H

R1

C

3

O

NADPH

H 2C O P Glycerol phosphate 1-Acylglycerol-3-snpathway phosphate R2

4

R1CoA

O

O H 2C HO

P

Dihydroxyacetone phosphate

Glycerol-3-snphosphate R1

C

O P

OH

H 2C

O

C

H

H 2C

O

C

R1

P

1-Acyldihydroxyacetone phosphate

Dihydroxyacetone phosphate pathway

CoA O

O R2

C

H2C

O

C

H

H2C

O

O

R1

C

P

1,2-Diacylglycerol-3-snphosphate (phosphatidic acid) 5

P O

O R2

C

H 2C

O

C

H

O

C

O 1

R

R3

R2 7

OH

H 2C

O

CoA

C

O

H2C

O

C

C

H

O

H2C

O

C

R1

R3

1,2-sn-Diacylglycerol Monoacylglycerol pathway

H2C

O R2

C

R2

6

O

C H2C

CoA

OH H OH

2-sn-monoacylglycerol

FIGURE 10.31 Major pathways of triacylglycerol synthesis. Reactions (1), (4), (5), and (7), glycerol phosphate pathway; reactions (2), (3), (4), (5), and (7), dihydroxyacetone phosphate pathway; reactions (6) and (7), monoacylglycerol pathway. Enzymes: (1) glycerol phosphate 1-acyltransferase; (2) dihydroxyacetone phosphate acyltransferase; (3) acyldihydroxyacetone phosphate reductase; (4) 1acylglycerol phosphate 2-acyltransferase; (5) phosphatidate phosphohydrolase; (6) monoacylglycerol acyltransferase; (7) diacylglycerol acyltransferase.

triacylglycerol synthesis and, as such, might be expected to be important for regulation, at least under certain conditions (Mayorek et al., 1989). So far as can be ascertained, it competes with the choline and ethanolamine phosphotransferases (that form zwitteronic phospholipids; Section 10.2.1) for a common pool of diacylglycerol (Brindley, 1991). Diacylglycerol acyltransferase can use a wide range of saturated and unsaturated acyl-CoAs (Dircks and Sul, 1991), but has considerably better activity

with its natural 1,2-diacylglycerol substrate than the 1,3isomer (Hill et al., 1968). The diacylglycerol acyltransferase of plants has been studied in several tissues, though not yet purified. In many plants, it seems less specific for its acyl-CoA substrate than are the other acytransferases (Stymne and Stobart, 1987). It is located in endoplasmic reticulum and has low activity when measured in vitro (e.g., Berneth and Frentzen, 1990). The latter fact, together with the significant buildup of 682

Lipid Metabolism

diacylglycerol during triacylglycerol formation in vivo (Perry and Harwood, 1990) and in vitro (Perry and Harwood, 1991), has led to suggestions that diacylglycerol acyltransferase may exert significant flux control over oil accumulation in some crops (see, e.g., Griffiths and Harwood, 1990). Apart from diacylglycerol acyltransferase being used to form triacylglycerol, evidence for acyl-CoA independent pathways has been obtained in animal tissues and oilseeds. Thus, in mammals, a diacylglycerol transacylase has been reported (Lehner and Kuksi, 1996). In oilseeds, the same enzyme activity is also present, but, in addition, phospholipid: diacylglycerol acyltransferase is found (see Weselake, 2005), although the quantitative importance of the latter in many tissues seems to be slight (see Ramli et al., 2005). Mammalian triacylglycerol synthesis is affected by a large number of factors. These include nutritional, hormonal, and pharmacological effects (cf. O’Doherty, 1978, for review). A discussion of the control of triacylglycerol synthesis in animals is given by Brindley (1991). General accounts of triacylglycerol synthesis in plants are given by Gurr (1980), Stymne and Stobart (1987), Harwood and Page (1993), and Weselake (2005). Specialist accounts of lipid synthesis in the two important oil crops, olive and palm, are given in Salas et al. (2000) and Sambanthamurthi et al. (2000), respectively. Weselake and Taylor (1999) detail the use of microspore-derived cultures to examine triacylglycerol biosynthesis in oilseed rape. Another subject worthy of mention are the efforts by breeders to increase oil yields. These have been quite successful (e.g., maize varieties have been changed from an average 4 to 5% oil content to give IHO lines (20%) or ILO (0.5%) lines). However, the complexity of the phenotype precludes elucidation of the exact genetic basis of these changes (Ohlrogge et al., 1991). On the other hand, a 20% increase in seed fatty acid content (per unit seed weight) has been induced in Arabidopsis by a single gene variation (James and Dooner, 1990).

10.3.2

lipase is the best known and most investigated of all lipolytic enzymes. It acts on mono-, di-, and triacylglycerols, although the reaction rate is slower with partial glycerides (Brockerhoff and Jensen, 1974). It probably lacks stereospecificity. For a thorough review of pancreatic lipase, see Jensen (1971). Metabolism of triacylglycerols in animals requires the interaction of lipoprotein lipase (involved in uptake of acyl chains from plasma) and hormone-sensitive lipase (involved in release of fatty acids from lipid stores). Some aspects of lipoprotein lipase action are discussed in Section 11.3, and the reader is also referred to Brockerhoff and Jensen (1974), Jensen (1971), and Frayn et al. (1995). The enzyme is also known as clearing factor lipase, requires apo-CII for activity, and may be bound via heparin sulfate proteoglycan at the endothelial surface in vivo (Williams et al., 1983). Considerable work has been carried out on intracellular processing of the enzyme in active tissues (Cryer, 1981) and on the action of hormones in controlling the adipose and heart tissue enzymes (Ashby and Robinson, 1980; de Gasquet et al. 1975). There is now considerable sequence information of lipoprotein lipase. The sequence is extraordinarily conserved (87 to 94% with different mammalian enzymes). Comparisons with other lipases have also been made (see Wang et al., 1992). For further information on the structural and functional domains of lipoprotein lipase, the structure and function of apo-CII, and the reaction kinetics of the enzyme, see Wang et al., (1992). Hormone-sensitive lipase tissue activity is stimulated by adrenaline (epinephrine, glucagons, ACTH (corticotrophin), TSH (thyrotropin) and serotonin (Jensen, 1971). These hormones are presumed to exert their effects on adipose tissue by stimulating adenylate cyclase. Certainly, an increased formation of cAMP (cyclic AMP) is brought about by lipolytic hormones and cAMP has been shown to stimulate lipase activity in cell-free preparations (cf. O’Doherty, 1978). Phosphorylation/dephosphorylation has been implicated (Belfarage et al., 1983). The cDNA for rat hormone-sensitive lipase has been reported (Holm et al., 1988) and increased expression of the mRNA for this enzyme found in the adipose tissue of cancer patients (Thompson et al., 1993). A good review is that of Langin et al. (1996) and Bernlohr et al. (2002), also summarises this topic. One typical feature of the mobilisation of fatty acids from adipose tissue, which has not yet been fully explained, is that their release is selective for both chainlength and unsaturation. Part of the reason is due to selectivity properties of hormone-sensitive lipase, but other factors appear important (Raclot, 2003). Other mammalian lipases that have been studied are milk lipase (cf. Jensen, 1971) and monoacylglycerol lipase. The latter enzyme occurs in several tissues including intestine, liver, and adipose tissue. It has high activity with monoacylglycerols when compared to diacylglycerols or triacylglycerols (see O’Doherty, 1978).

Triacylglycerol breakdown

The breakdown of triacylglycerol is catalysed by lipases. A large number of such enzymes have been purified from animals, plants, and microbes (cf. Brockerhoff and Jensen, 1974). It should be noted that the term “lipase” is frequently misused. A true lipase is one that attacks triacylglycerols and acts only at an oil–water interface. This definition, therefore, excludes enzymes acting on watersoluble esters (esterases) or those preferentially hydrolysing other lipids (acyl hydrolases). Since triacylglycerols are important dietary constituents (Section 11.1), there is interest in digestive lipases. Although pharyngeal lipases have been found and studied, their importance during overall digestion remains to be established (cf. O’Doherty, 1978). In contrast, pancreatic 683

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(L) involved in triacylglycerol biosynthesis. Plant Sci. 67, 21–28. Bernlohr, D.A. et al. (2002). Adipose tissue and lipid metabolism. In Biochemistry of Lipids, Lipoproteins and Membranes, Ed. D.E. Vance, 4th ed., Elsevier, Amstedam, p. 257–281. Brindley, D.N. (1978). Some aspects of the physiological and pharmacological control of the synthesis of triacylglycerols and phospholipids. Int. J. Obes. 2, 7–16. Brindley, D.N. Ed. (1988). Phosphatidate Phosphohydrolase, vols. I and II, CRC Press, Boca Raton, FL. Brindley, D.N. (1991). Metabolism of triacylglycerols. In Biochemistry of Lipids, Lipoprotein and Membranes, Eds. D.E. Vance and J. Vance, Elsevier, Amsterdam, pp. 171–203. Brokerhoff, H. and Jensen, R.G. (1974). Lipolytic Enzymes, Academic Press, New York. Clark, B. and Hübscher, G. (1960). Biosynthesis of glycerides in the mucosa of the small intestine. Nature. 183, 35–37. Cohen, Z. and Ratledge, C (2005). Single cell oils. Am. Oil Chem. Soc. Champaign, IL. Coleman, R.A. et al. (2000). Physiological and nutritional regulation of enzymes of triacylglycerol synthesis. Annu. Rev. Nutr. 20, 77–103. Cryer, A. (1981). Tissue lipoprotein lipase activity and its action in lipoprotein metabolism. Int. J. Biochem. 13, 525–541. Davies, R.J. and Holdsworth, J. E. (1992). Synthesis of lipids in yeasts, biochemistry, physiology and production. Adv. Appl. Lipid Res. 1, 119–159. de Gasquet, P. et al. (1975). Effect of glucocorticoids on lipoprotein-lipase activity in rat-heart and adipose-tissue. Horm. Metab. Res. 7, 152–157. Dircks, L. and Sul H.S. (1999). Acyltransferases of de novo glycerolphospholipid biosynthesis. Prog. Lipid Res. 38, 461–479. Douce, R. and Joyard, J. (1980). Plant galactolipids. In Biochemistry of Plants, vol. 4, Eds. P.K. Stumpf and E.E. Conn, Academic Press, New York, pp. 321–362. Eigtved, P. (1992). Enzymes and lipid modification. Appl. Lipid Res. 1, 1–64. Finnerty, W.R. (1989). Microbial lipid metabolism. In Microbial Lipids, vol. 2, Eds. C. Ratledge and S.G. Wilkinson, Academic Press, London, pp. 525–566. Frayn, K.N. et al. (1995). Coordinated regulation of hormonesensitive lipase and lipoprotein lipase in human adepose tissue in vivo. Adv. Enz. Regul. 35, 163–178. Galliard, T. (1980). Degradation of acyl lipids: hydrolytic and oxidative enzymes. In Biochemistry of Plants, vol. 4, Eds. P.K. Stumpf and E.E. Conn, Academic Press, New York, pp. 85–116. Gibbons, G.F. et al. (2000). Mobilisation of triacylglycerol stores. Biochim. Biophys. Acta. 1483, 37–57. Griffiths, G. and Harwood, J.L. (1990). Triacylglycerol synthesis in maturing cotyledons of cocoa. In Plant Lipid Biochemistry, Structure and Utilization, Eds. P.J. Quinn and J.L. Harwood, Portland, London., pp. 216–218. Gurr, M.I. (1980). The biosynthesis of triacylglycerols. In Biochemistry of Plants, Eds. P.K. Stumpf and E.E. Conn, Academic Press, New York, pp. 205–248. Harwood, J.L. and Griffiths, G. (1992). Biochemistry of plant lipids. In Advances in Plant Cell Biochemistry and Biotechnology, Ed. I.M. Morrison, JAI Press, London, pp. 1–52.

Lipase activity has been found in a wide range of plant materials with most work on oil seeds and cereals. One of the best characterized enzymes is the castor bean acid lipase. Ory and coworkers (Ory, 1969) have characterized the enzyme, which is associated with the spherosome membrane. Huang and Moreau (1978) studied lipolytic activity in a range of germinating oil seeds. The enzyme from peanuts seems to be mainly associated with microsomal fractions (Theimer and Rosnitschek, 1978) and is clearly distinguished from the glyoxysomal monoacylglycerol lipase (Huang and Moreau, 1978). Several lipases have been purified from cereal seeds, such as wheat, oat, and rice. Galliard (1980) has reviewed this work (cf. also Jensen, 1971) and has discussed the role of plant growth regulators in controlling activity. For a more recent review, see Huang (1987). Although bacteria do not store energy as triacylglycerol, a number of bacterial lipases have been discovered and studied. An extensive review of microbial lipases (and esterases) has been made by Lawrence (1967). It should be emphasized also that many of the microbial “lipases” (e.g., the widely studied Rhizopus arrhizus enzyme) are, in fact, acyl hydrolases. More recent information is available in Finnerty (1989). The features of lipases useful for industry in lipid modifications have been discussed by Eigtved (1992), and the monograph by Alberghina et al. (1991) contains many specific examples of individual lipases, their characteristics and potential (or actual) industrial use.

References Abate, N. and Garg, A (1995). Heterogeneity in adipose tissue metabolism: causes, implications and management of regional adiposity. Prog. Lipid Res. 34, 53–70. Akesson, B. et al. (1970). Initial incorporation into rat liver glycerolipids of intraportally injected (9,10-3H2) palmitic acid. Biochim. Biophys. Acta. 18, 44–56. Alberghina, L. et al., Eds. (1991). Lipases: Structure, Mechanism and Genetic Engineering, VCH Press, New York. Appelqvist, L.A. (1975). Biochemical and structural aspects of storage and membrane lipids in developing oil seeds. In Recent Advances in Chemistry and Biochemistry of Plant Lipids, Eds. T. Gallaird and E.I. Mercer, Academic Press, London, pp. 247–254. Ashby, P. and Robinson, D.S. (1980). Effects of insulin, glucocorticoids and adrenaline on the activity of rat adipose-tissue lipoprotein lipids. Biochem, J. 188, 185–192. Bauermeister and Sargent, J. (1979). Wax esters: major metabolites in the marine environment. Trends Biochem. Sci. 4, 209–211. Belfrage, P. et al. (1983). Hormonal regulation of adipose tissue lipolysis by reversible phosphorylation of hormone-sensitive lipase. In Cell Function and Differentiation, Part C, Eds. G. Akoyunoglou, et al., Alan R. Liss, New York, pp. 213–223. Berneth, K. and Frentzen, M. (1990). Utilization of erucoyl-CoA by acyltransferases from developing seeds of Brassica-napus

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Harwood, J.L. and Page, R. (1994). Biochemistry of oil synthesis. In Designer Oilseed Crops, Ed. D.J. Murphy, VCH Press, New York, pp. 165–194. Harwood, J.L. and Price-Jones, M.J. (1988). Phosphatidate phosphohydrolase in plants and microorganisms. In Phosphatidate Phosphohydrolase, vol. II, Ed. D.N. Brindley, CRC Press, Boca Raton, FL, pp. 1–37. Heinz, E. (1977). Enzymatic reactions in galactolipid biosynthesis. In Lipids and Lipid Polymers in Higher Plants, Eds. M. Tevini and H.K. Lichtenhaler, Springer-Verlag, Berlin, pp. 102–120. Heinz, E. and Roughan, P.G. (1983). Similarities and differences in lipid-metabolism of chloroplasts isolated from 18:3 and 16:3 plants. Plant Physiol. 72, 273–279. Hill, E.E. and Lands, W.E.M. (1970). Phospholipid metabolism. In Lipid Metabolism, Ed. S.J. Wakil, Academic Press, New York, pp. 185–275. Hill, E.E. et al. (1968). The selective incorporation of 14C-glycerol into different species of phosphatidic acid, phosphatidylethanolamine, and phosphatidylcholine. J. Biol. Chem. 243, 4440–4451. Hitchcock, C. and Nichols, B.W. (1971). Plant Lipid Biochemistry, Academic Press, London. Holm, C. et al. (1988). Hormone-sensitive lipase: sequence, expression, and chromosomal localization to 19 centq13.3. Science, 241, 1503–1506. Huang, A.H.T. (1987). Lipases. In The Biochemistry of Plants, Eds. P.K. Stumpf and E.E. Conn, vol. 9, Academic Press, New York, pp. 91–119. Huang, A.H.C. (1992). Oil bodies and oleosins in seeds. Annu. Rev. Plant Physiol. 43, 177–200. Huang, A.H.C. and Moreau, R.A. (1978). Lipases in storage tissues of peanut and other oil seeds during germination. Planta. 141, 111–116. Ichihara, K. et al. (1990). Intracellular translocation of phosphatidate phosphatase in maturing safflower seeds — a possible mechanism of feed forward control of triacylglycerol synthesis by fatty acids. Biochim. Biophys. Acta. 1043, 227–234. James, D.W. and Dooner, H.K. (1990). Isolation of ems-induced mutants in arabidopsis altered in seed fatty-acid composition. Theor. Appl. Genet. 80, 241–245. Jensen, R.G. (1971). Lipolytic enzymes. Prog. Chem. Fats Other Lipids. 11, 347–394. Kanoh, H. and Ohno, K. (1973). Studies on 1,2-diglycerides formed from endogenous lecithins by back-reaction of ratliver microsomal CDP-choline-1,2-diacylglycerol cholinephosphotransferase. Biochim. Biophys. Acta. 326, 17–25. Kates, M. (1995). Hydrolysis of lecithin by plant plastid enzymes. Can. J. Biochem. Physiol. 33, 575–589. Langin, D. et al. (1996). Adipocyte hormone-sensitive lipase: a major regulator of lipid metabolism. Proc. Nutr. Soc. 55, 93–109. Lawrence, R.C. (1967). Microbial lipases and related esterases. Part II. Estimation of lipase activity, characterization of lipases — recent work concerning their effect on dairy products. Dairy Sci. Abstr. 29, pp. 59–70. Lehner, R. and Kuksis, A (1996). Biosynthesis of triacylglycerols. Prog. Lipid Res. 35, 169–202. Lennarz, W.J. (1970). Bacterial lipids. In Lipid Metabolism, Ed. S.J. Wakil, Academic Press, New York, pp. 155–184.

Manning, R. and Brindley, D.N. (1972). Tritium isotope effects in the measurement of the glycerol phosphate and dihydroxyacetone phosphate pathways of glycerolipid biosynthesis in rat liver. Biochem. J. 130, 1003–1012. Mayorek, N. et al. (1989). Triacylglycerol synthesis in cultured rat hepatocytes. The rate-limiting role of diacylglycerol acyltransferase. Eur. J. Biochem. 182, 395–400. Mitchell, M.P. et al. (1971). Properties of phosphatidate phosphohydrolase. Eur. J. Biochem. 18, 214–220. Moore, T.J. et al. (1973). Enzymes of phospholipid metabolism in endoplasmic-reticulum of castor bean endosperm. Plant Physiol. 52, 50–53. Murphy, D.J. (1990). Storage lipid bodies in plants and other organisms. Prog. Lipid Res. 29, 299–324. O’Doherty, P.J.A. (1978). Metabolic studies with natural and synthetic fatty acids and enantiomeric acylglycerols. In Handbook of Lipid Research, vol. 1, Ed. A. Kuksis, Plenum Press, New York, pp. 289–339. O’Doherty, P.J. and Kuksis, A. (1975). Stimulation of triacylglycerol synthesis by Z protein in rat liver and intestinal mucosa. FEBS Lett. 60, 256–258. O’Doherty, P.J. et al. (1974). Effect of phosphatidylcholine on triacylglycerol synthesis in rat intestinal mucosa. Can J Biochem. 52, 726–733. Ohlrogge, J.B. et al. (1991). The genetics of plant lipids. Biochim. Biophys. Acta. 1082, 1–26. Ory, R.L. (1969). Acid lipase of the castor bean. Lipids. 4, 177–185. Perry, H.J. and Harwood, J.L. (1990). Studies of lipid metabolism in developing oilseed rape. In Plant Lipid Biochemistry, Structure and Utilization, Eds. P.J. Quinn and J.L. Harwood, Portland, London, pp. 204–206. Perry, H.J. and Harwood, J.L. (1991). Lipid metabolism during seed development in oilseed rape (Brassica napus L. cv. Shiralee). Biochem. Soc. Trans. 19, 243S. Pollock, R.J. et al. (1975). The relative utilization of the acyl dihydroxyacetone phosphate and glycerol phosphate pathways for synthesis of glycerolipids in various tumors and normal tissues. Biochim. Biophys. Acta. 380, 421–435. Quinn, P.J. and Harwood, J.L. Eds. (1990) Plant Lipid Biochemistry, Structure and Utilization, Portland Press, London. Raclot, T. (2003). Selective mobilisation of fatty acids from adipose tissue triacylglycerols. Prog. Lipid Res. 42, 257–288. Ramli, U.S. et al. (2005). Metabolic control analysis reveals an important role for diacylglycerol acyltransferase in olive but not in oil palm lipid accumulation. FEBS J. 222, 5764–5770. Ratledge, C. (1989). Biotechnology of oils and fats. In Microbial Lipids, vol. II, Eds. C. Ratledge and S.J. Wilkinson, Academic Press, London, pp. 567–613. Roughan, P.G. (1987). On the control of fatty acid compositions of plant glycerolipids. In The Metabolism, Structure and Function of Plant Lipids, Eds. P.K. Stumpf, J.B. Mudd and W.D. Nes, Plenum, New York, pp. 247–254. Roughan, P.G. and Slack, C.R. (1982). Cellular organization of glycerolipid metabolism. Annu. Rev. Plant Physiol. 33, 97–132. Sakaki, T. et al. (1985). Polar and neutral lipid changes in spinach leaves with ozone fumigation — triacylglycerol synthesis from polar lipids. Plant Cell Physiol. 26, 253–262.

685

10.4

Glycosylglycerides

Salas, J.J. et al. (2000). Biochemistry of lipid metabolism in olive and other oil fruits. Prog. Lipid Res. 39, 151–180. Sedgwick, B. and Hubscher, G. (1967). Partial purification and properties of a soluble phosphatidate phosphohydrolase from rat liver. Biochim. Biophys. Acta. 144, 397–408. Smith, M.E. et al. (1967). The role of phosphatidate phosphohydrolase in glyceride biosynthesis. Eur. J. Biochem. 3, 70–77. Snyder, F.L. (1972). Ether Lipids: Chemistry and Biology, Academic Press, New York. Stymne, S. and Stobart, A.K. (1987). Triacylglycerol biosynthesis In The Biochemistry of Plants, vol. 9, Eds. P.K. Stumpf and E.E. Conn, Academic Press, New York, pp. 175–214. Sul, H.S. and Wang, D. (1998). Nutritional and hormonal regulation of enzymes in fat synthesis. Studies of fatty acid synthase and mitochondrial glycerol 3-phosphate acyltransferase gene transcription. Annu. Rev. Nutr. 18, 331–351. Theimer, R.R. and Rosnitscheck, I. (1978). Development and intracellular-localization of lipase activity in rapeseed (Brassica-napus-L) cotyledons. Planta. 139, 249–256. Thompson, M.P. et al. (1993). Increased expression of the mRNA for hormone-sensitive lipase in adipose tissue of cancer patients. Biochim. Biophys. Acta.1180, 236–242. Wang, C.-S. et al. (1992). Structure and functional properties of lipoprotein lipase. Biochim. Biophys. Acta. 1123, 1–17. Weselake, R.J (2005). Storage lipids. In Plant Lipids: Biology, Utilisation and Manipulation, Ed. D.J. Murphy, Blackwell Publishing, Oxford, U.K., pp. 162–225. Weselake, R.J. and Taylor, D.C. (1999). The study of storage lipid biosynthesis using microspore-derived cultures of oilseed rape. Prog. Lipid Res. 38, 401–460. Williams, M.P. et al. (1983). Heparan sulphate and the binding of lipoprotein lipase to porcine thoracic aorta endothelium. Biochim. Biophys. Acta, 756, 83–91. Wilson, R.F. and Rinne, R.W. (1976). Involvement of phospholipids in triglyceride biosynthesis by developing soybean cotyledons. Plant Physiol. 57, 556–559.

10.4

Glycosylglycerides

10.4.1

Galactosylglycerides

Kennedy pathway

DAG +UDP-SQD

+UDP-gal 2

1 MDGD SQDG

+MGDG 4

+UDP-gal 3

DGDG

FIGURE 10.32 Biosynthesis of plant glycosylglycerides. Abbreviations: DAG, diacylglycerol; MGDG, monogalactosyldiacylglycerol; DGDG, digalactosyldiacylglycerol; SQD, sulfoquinovose; SQDG, sulfoquinovosyldiacylglycerol. Reactions: (1) UDP-SQD: diacylglycerol sulfoquinovosyltransferase; (2) UDPgalactose: diacylglycerol galactosyltransferase; (3) UDP-galactose:MGDG galactosyltransferase; (4) galactolipid: galactolipid galactosyltransferase.

fractions was due to its envelope content. Plastid envelope membranes from nonphotosynthetic tissues, such as the chromoplasts of Narcissus pseudonarcissus (Leidvogel and Kleinig, 1977) and potato tuber amyloplasts (Fishwick and Wright, 1980) are also active. The first galactosyltransferase is specific for the formation of a β-glycosidic bond while the other forms an α-glycosidic bond (Douce and Joyard, 1980). There are two digalactosyldiacylglycerol synthetases in Arabidopsis and details of the formation of galactosyldiacylglycerol and the galactosyltransferases will be found in Dormann and Benning (2002) and in Dormann (2005). As an alternative to the use of UDP-galactose, digalactosyldiacylglycerol may be formed by the action of galactolipid: galactolipid galactosyltransferase (see Figure 10.32), first demonstrated by Von Besouw and Wintermans (1978). Comparative aspects of these alternative pathways have been discussed (Joyard and Douce, 1987) and methods for the analysis of galactolipids and their metabolites reviewed (Douce et al., 1990). Recent evidence, discussed by Dormann (2005), suggests that the galactolipid transferase is not important for digalactosyldiacylglycerol synthesis in vivo, but can be activated under certain conditions, such as during membrane isolation. The formation of polyunsaturated fatty acids (mainly α-linolenate) associated with the galactosylglycerides has been reviewed (Harwood, 1996). Depending on the plant type, there seem to be differences between the “16:3 species” and the “18:3 species,” as discussed by Heinz and Roughan (1982). The distinctive features of the fatty acid distributions of galactolipids in 16:3 and 18:3 plants is illustrated in Section 2.10 and discussed thoroughly by

The two galactosylglycerides, monogalactosyldiacylglycerol (MGDG), and digalactosyldiacylglycerol (DGDG) are the major lipid components of the photosynthetic membranes of oxygen-evolving organisms. Because of that, they are the most prevalent membrane lipids in the world. They are rare or only found in trace amounts in other organisms. Trigalactosyl and tetragalactosyl derivatives are minor components of some chloroplasts, and in marine algae and in some bacteria the sugar residue(s) may be glucose. The pathway for galactosylglyceride synthesis is outlined in Figure 10.32. UDP-galactose is generated in the cytoplasm of plant leaf cells and is used by two galactosyltransferase enzymes, which are located in the outer half of the envelope membrane. Joyard and Douce (1976) showed that the galactosyltransferase activity was located in the plastid envelope and that any activity in microsomal 686

Lipid Metabolism

Browse and Somerville (1991). For 18:3 plants the desaturation of oleate in association with phosphatidylcholine is followed by release of the diacylglycerol for galactolipid formation (Joyard and Douce, 1987). Whether diacylglycerol itself or phosphatidylcholine moves from the endoplasmic reticulum to the chloroplast envelope is not known, although Tanaka and Yamada (1982) demonstrated the latter process mediated via a phospholipid transfer protein. After the formation of monogalactosyldiacylglycerol, further desaturation can then take place (Jones and Harwood, 1980; Harwood, 1996; see also Section 10.1.4). The fatty acid combinations (Heinz, 1977; Rullkötter et al., 1975) and turnover (Heinz et al., 1979) in galactosylglycerides have been studied thoroughly. In 16:3 plants and cyanobacteria the galactosylglycerides

appear to be substrates for successive desaturations of oleate and linoleate to α-linolenate (see Harwood and Jones, 1989). Initial breakdown of galactosylglycerides is catalysed by nonspecific acylhydrolase enzymes (see Galliard, 1980, for review). The enzymes from runner bean leaves (Burns et al., 1979) and potato tubers (Hirayama et al., 1975) have been purified. Two enzymes that differed slightly in substrate specificity were isolated from runner bean leaves (Burns et al., 1979) and the position of hydrolysis identified by mass spectrometry as the bond between the fatty acyl carboxy carbon and the oxygen of glycerol (Burns et al., 1980). Acyl hydrolase enzymes are particularly active; homogenization of potato tubers in water at 4°C for a few seconds being sufficient for most of the endogenous

Epimerase CH2OH O

CH2OH O HO

O

OH

O’UDP

CH2OH O OH

OH

O’UDP OH

OH UDP-Glc

O

OH

CH2OH O HO O’UDP OH

−H2O

O’UDP OH −H2O

CH2 O O

OH

CH2SO3− O

SO2− 3

O

OH

O’UDP

O’UDP OH

OH

+2H CH2SO3− O HO

OH

O’UDP OH

UDP-sulfoquinovose +Diacylglycerol

SULFOLIPID

FIGURE 10.33 Synthesis of sulfoquinovosyldiacylglycerol. (See Pugh et al. (1995), Harwood and Okanenko, (2003) and Dormann (2005) for details.)

687

10.4

Glycosylglycerides

membrane lipids to be destroyed. Some recent information is given in Dormann (2005) and see Section 10.2.2.

10.4.2

Burns, D.D. et al. (1980). Properties of acyl hydrolase enzymes from Phaseolus multiflorus leaves. Phytochemistry, 19, 2281–2285. Dormann, P. (2005). Membrane lipids. In Plant Lipids:Biology, Utilisation and Manipulation, Ed. D.J. Murphy, Blackwell Publishing, Oxford, U.K., pp. 123–161. Dormann, P. and Benning, C. (2002). Galactolipids rule in seed plants. Trends Plant Sci. 7, 112–118. Douce, R. and Joyard, J. (1980). Plant galactolipids. In Biochemistry of Plants, vol. 4, Eds. P.K. Stumpf and E.E. Conn, Academic Press, New York, pp. 321–362. Douce, R. et al. (1990). Glycolipid analyses and synthesis in plastids. In Methods in Plant Biochemistry, vol. 4, Eds. J.L. Harwood and J.R. Boyer, Academic Press, London, pp. 71–103. Fishwick, M.J. and Wright, A.J. (1980). Isolation and characterization of amyloplast envelope membranes from Solanum tuberosum. Phytochemistry, 19, 5559. Galliard, T. (1980). Degradation of acyl lipids: hydrolytic and oxidative enzymes. In Biochemistry of Plants, vol. 4, Eds. P.K. Stumpf and E.E. Conn, Academic Press, New York, pp. 85–116. Harwood, J.L. (1980). Sulpholipids. In The Biochemistry of Plants, vol. 4, Eds. P.K. Stumpf and E.E. Conn, Academic Press, New York, pp. 301–320. Harwood, J.L. (1996). Recent advances in the biosynthesis of plant fatty acids. Biochim. Biophys. Acta. 1301, 7–56. Harwood, J.L. and Jones, A.L. (1989). Lipid metabolism in algae. Adv. Bot. Res. 16, 1–53. Harwood, J.L. and Okanenko, A.A. (2003). Sulphoquinovosyldiacylglycerol (SQDG) – the sulpholipid of higher plants. In Sulphur in Plants, Eds. Y.P. Abrol and A. Ahmad, Kluwer, Dordrecht, pp. 189–219. Heinz, E. (1977). Enzymatic reactions in galactolipid biosynthesis. In Lipids and Lipid Polymers of Higher Plants, Eds. M. Tevini and H.K. Lictenthaler, Springer-Verlag, Berlin, pp. 102–120. Heinz, E. and Roughan, P.G. (1982). De novo synthesis, desaturation and acquisition of monogalactosyldiacylglycerol by chloroplasts of 16:3 and 18:3 – plants. In Biochemistry and Metabolism of Plant Lipids, Eds. J.F.G.M. Wintermans and P.J.C. Kuiper, Elsevier, Amsterdam, pp. 169–182. Heinz, E. et al. (1979). Investigations on the origin of diglyceride diversity in leaf lipids. In Recent Advances in the Biochemistry and Physiology of Plant Lipids, Eds. L.A. Appelqvist and C. Liljenberg, Elsevier, Amsterdam, pp. 99–120. Heinz, E. et al. (1989). Synthesis of different nucleoside 5´diphosphosulphoquinovoses and their use for studies of sulpholipid synthesis in chloroplasts. Eur. J. Biochem. 184, 445–454. Hirayama, O. et al. (1975). Purification and properties of a lipid acyl-hydrolase from potato tubers. Biochim. Biophys. Acta. 384, 127–137. Jones, A.V.M. and Harwood, J.L. (1980). Desaturation of linoleic acid from exogenous lipids by isolated chloroplasts. Biochem. J. 190, 851–854. Joyard, J. and Douce, R. (1976). Preparation and enzymatic activities of spinach chloroplast envelope. Physiol. Veg. 14, 31–48. Joyard, J. and Douce, R. (1987). Galactolipid synthesis. In The Biochemistry of Plants, vol. 9, Eds. P.K. Stumpf and E.E. Conn, Academic Press, New York, pp. 215–274.

Sulfolipid (diacylsulfoquinovosylglycerol)

Early work on possible pathways for the formation of the plant sulfolipid were reviewed by Harwood (1980) and by Mudd and Kleppinger-Sparace (1987). The immediate precursor, UDP-sulfoquinovose, was shown to transfer its sugar to diacylglycerol using SQDE synthase in the envelope membrane of chloroplasts (Heinz et al., 1989). The UDP-sulfoquinovose itself is synthesised from UDP-glucose and sulfite in a series of reactions whose theory is explained by Pugh et al. (1995). Apparently, a single gene codes for the protein needed to carry out all the conversions to UDP-sulfoquinovose (Sanda et al., 2001) (Figure 10.33). The metabolism, genetics and possible physiological functions of sulfoquinovosyldiacylglycerol have been reviewed by Benning (1998) and by Harwood and Okanenko (2003). Plant tissues are capable of, at least, partial catabolism of sulfolipid. Deacylation appears to be the first step and may take place with two enzymes, such as in green algae (e.g., Yagi and Benson, 1962) or by the action of a single acyl hydrolase (cf. Harwood, 1980). Cleavage of sulfoquinovosylglycerol to sulfoquinovose was reported in leaves of Medicago sativa (Lee and Benson, 1972) where sulfolactaldhyde and, later, sulfolactic acid accumulated. In contrast, catabolism by cell-free preparations from Phaseolus multiforus stopped at sulfoquinovose (Burns et al., 1980). Further aspects of sulfolipid catabolism are discussed by Harwood (1980) and in Harwood and Okanenko (2003). Studies on the turnover of molecular species of sulfolipid have been reported in Vicia faba and Hordeum vulgare. In both these plants, the more saturated species were turned over at high rates, whereas the predominant 1-linolenoyl, 2-palmitoyl species had a low rate of turnover. These differences in metabolism may be related to the function of the sulfolipid — the fast turning over species being involved in a metabolic function, while the trienoic species have a structural role (cf. Harwood, 1980).

References Benning, C. (1998). Biosynthesis and function of the sulfolipid sulfoquinovosyldiacylglycerol. Ann. Rev. Plant Physiol. Plant Mol. Biol. 49, 53–75. Benson, A.A. (1963). The plant sulfolipid. Adv. Lipid Res. 1, 382–394. Browse, J. and Somerville, C. (1991). Glycerolipid synthesis: biochemistry and regulation. Annu. Rev. Plant Physiol. 42, 467–506. Burns, D.D. et al. (1979). Purification of acyl hydrolase enzymes from the leaves of Phaseolus multiflorus. Phytochemistry. 18, 1793–1797.

688

Lipid Metabolism

Lee, R.F. and Benson, A.A. (1972). The metabolism of glyceryl 35S-sulfoquinovoside by the coral tree, Erythrina cristagalli, and alfalfa, Medicago sativa. Biochim. Biophys. Acta. 261, 35–37. Leidvogel, B. and Kleining, H. (1977). Lipid metabolism in chromoplast membranes from daffodil — glycosylation and acylation. Planta, 133, 249–253. Mudd, J.B. and Kleppinger-Sparace, K. (1987). Sulfolipids. In The Biochemistry of Plants, vol. 9, Eds. P.K. Stumpf and E.E. Conn Academic Press, New York, pp. 275–289. Pugh, C.E. et al. (1995). A new pathway for the synthesis of the plant sulpholipid, sulphoquinovosyldiacylglycerol. Biochem. J. 309, 513–519. Rullkötter, J. et al. (1975). Combination and positional distribution of fatty acids in plant digalactosyl diglycerides. Z. Pflanzenphysiol. 76, 163–175. Sanda, S. et al. (2001). Recombinant Arabidopsis SQD1 converts UDP-glucose and sulphite to the sulfolipid head group precursor UDP-sulphoquinovose in vivo. J. Biol. Chem. 276, 3941–3946. Tanaka, T. and Yamada, M. (1982). Properties of phospholipid exchange proteins from germinated castor bean endosperm. In Biochemistry and Metabolism of Plant Lipids, Eds. J.F.G.M. Wintermans and P.J.C. Kuiper, Elsevier, Amsterdam, pp. 99–110. Van Besouw, A. and Wintermans, J.F.G.M. (1978). Galactolipid formation in chloroplast envelopes. I. Evidence for two mechanisms in galactosylation. Biochim. Biophys. Acta 529, 44–53.

Yagi, T. and Benson, A.A. (1962). Plant sulfolipid. V. Lysosulfolipid formation. Biochim. Biophys. Acta 57, 601–603.

10.5

Sphingolipids

10.5.1

Biosynthesis

Sphingolipid chemistry and biosynthesis have been reviewed by Carter et al. (1965), Wiegandt (1971), Stoffel (1971), Kanfer and Hakomori (1983), Sweeley (1991), and Merrill and Sandhoff (2002). Biosynthesis of long-chain bases has been studied in a series of experiments by Snell and coworkers (e.g., Brady et al., 1969) and by Stoffel and coworkers (see Stoffel, 1971). A condensing enzyme, requiring pyridoxal phosphate, is able to condense serine with palmitoyl-CoA to produce 3-oxosphinganine (3-keto sphinganine). The reaction, which is probably the ratelimiting step in sphingoid base biosynthesis, proceeds with overall retention of configuration of the C-2 carbon of serine. L-Cycloserine (4-amino-3-isoazolidinone) is an irreversible inhibitor of the palmitoyltransferase and depresses the level of central nervous system sphingolipids if administered to mice (Sweeley, 1991). Other inhibitors of this and succeeding reactions in sphingolipid metabolism are listed in Merrill and Sandhoff (2002). The next reaction is a reduction using NADPH to produce sphinganine (Figure 10.34). The reductase responsible is probably COO−

O

Palmitoyl-CoA + Serine

SCoA

+ H C CH2OH NH3+ Serine palmitoyltransferase

CO2 O

3-ketosphinganine NADPH + H+

CH2OH

NH3+ 3-Ketosphinganine reductase

NADP OH

CH2OH NH +

Sphinganine

3

Fatty acyl-CoA

Ceramide synthase

CoASH OH Dihydroceramide

CH2OH

NH O Cofactors?

“Desaturase” OH

Ceramide

NH O

FIGURE 10.34

CH2OH

Biosynthesis of ceramide.

689

10.5 Sphingolipids

closely associated with the palmitoyltransferase in the endoplasmic reticulum. Both the palmitoyltransferase and the reductase exhibit chain-length specificity, with C14 to C18 CoA esters and C14 to C20 3-dehydrosphinganines being utilized. The amino acid may form a Shiffbase complex with the pyridoxal phosphate coenzyme and Mg 2+ during the condensation reaction. Some researchers have also suggested that palmitaldehyde is an intermediate in the reaction, although this has not been proven. Stoffel et al. (1968) have studied the direct transformation of sphinganine into 4-hydroxysphinganine in the yeast, Hansenula ciferri. The origin of the hydroxyl group is obscure, since Thorpe and Sweeley (1967) concluded that it did not arise from either molecular oxygen or water. For further discussion of the formation of sphingoid bases, see Sweeley (1991) and for their structures and nomenclature, see Merrill and Sandhoff (2002). Ceramides can be rapidly formed from erythro or threo long-chain bases. The specificity of this acylation by acyl-CoAs has been studied in brain and other tissues (see Stoffel, 1971). The significance of the nature of the fatty acid moiety in ceramides is well seen when the glycosylation of ceramides is considered: hydroxyl fatty acid-containing ceramides accept predominantly galactose, while nonhydroxy fatty acid ceramides accept glucose. Reversal of ceramidase to yield ceramide does not appear to be important (Merrill and Sandoff, 2002). Genes for ceramide synthase have been identified in yeast and animals. Microorganisms can produce inhibitors of the enzyme and fumonisins from Fusaria spp. are notable as causes of a number of human pathologies. The last step in ceramide synthesis is the insertion of a 4,5-trans-double bond into the sphingoid base (see Figure 10.34). Desaturase genes have been identified in plants (Sperling et al., 2000; Dunn et al., 2004) and animals. For the 4-hydroxysphinganines, insertion of the 4-hydroxyl group occurs at the level of sphinganine (see Dunn et al., 2004). Cerebrosides are made from ceramide using glycosyltransferase enzymes, which are specific for UDP-galactose or UDP-glucose. The latter also recognises ceramides with nonhydroxy fatty acids, as mentioned above. Thus, galactocerebrosides (and sulfatides) are enriched in α-hydroxy fatty acids (Merrill and Sandhoff, 2002). As expected, the formation of sulfatides involves transfer of sulfate from phosphoadenosine phosphosulfate (PAPS). Galactosylceramide and lactosylceramide were both acceptors for the sulfate from PAPS and the reaction is catalysed by the Golgi enzyme, galactosylceramide sulfotransferase. Sulfate transfer is preceded by receptormediated translocation of PAPS from the cytosol across the Golgi membrane, and this process can be inhibited by 3′-P-AMP, palmitoyl-CoA, or atractyloside (Sweeley, 1991). Sulfatide synthesis is most rapid in the period 20 to 25 days after birth in rat brain (Stoffel, 1971). The

Man-Man-Glc-Cer (mollu)

Man-Glc-Cer Glc-Cer

GlcNAc-Man-Glc-Cer (arthro) Gal-Glc-Cer GalNAc-Gal-Glc-Cer (ganglio) Gal-Gal-Glc-Cer (globo)

Cer GlcNAc-Gal-Glc-Cer (lacto) Gal-Cer

Gal-Gal-Cer (gala)

FIGURE 10.35 Biosynthesis of different root glycosphingolipids from ceramide. (From Sweeley, 1991.)

cDNA encoding the sulfotransferase has been cloned (Honke et al., 1997) and activity of this enzyme appears important in controlling overall sulfatide formation. See also Vos et al. (1994) for metabolic and functional aspects of sulfogalactolipids. Synthesis of the neutral glycosphingolipids begins from glucosylceramide or galactosylceramide. Specific glycosyltransferases are involved, and the activated forms of the sugar substrates (UDP-Glc, UDP-Gal, UDP-GlcNAc, GDP-Man, and GDP-Fuc) are produced in the cytosol from nucleoside triphosphates and hexose 1-phosphates. The formation of different root glycosphingolipids from ceramide is illustrated in Figure 10.35 and occurs in the lumen of the Golgi apparatus. The active sites of the glycosyltransferases are localized on the lumenal surface of the Golgi membrane and may be organized in several multiglycosyltransferases, which could account for the various types of products that are formed (Sweeley, 1991). This contrasts to glycosylceramide, which is made on the cytosolic face of the endoplasmic reticulum and/or early Golgi membranes. Thus, glucosylceramide must undergo trans-bilayer movement before further metabolism (Merrill and Sandhoff, 2002). Gangliosides are formed by stepwise elongation of the carbohydrate chain by the action of various glycosyltransferases. Several of the individual enzymes have been studied (see Stoffel, 1971) and assay methods are detailed by Basu et al. (1987). The individual sugars are transferred from their UDP derivatives, while sialic acid residues are donated by CMP-N-acetylneuraminic acid (CMP-NANA). CMP-NANA itself is produced by a reaction of NANA with CTP. Initial reactions in the formation of gangliosides are shown in Figure 10.36. Ganglioside synthesis involves the use of relatively few enzymes (Table 10.8), which can 690

Lipid Metabolism

NeuAc

NeuAc Gal (GT3)

NeuAc

CMP-NeuAc

Glc

Glc

Gal

Other c-series gangliosides

Cer

Cer Other b-series gangliosides

NeuAc (GD3)

NeuAc CMP-NeuAC

UDP-GalNAc Gal

CMP-NeuAc

Glc

NeuAc Lactosylceramide

Cer

GalNAc

Gal

Glc

Glc

Cer

Other a-series gangliosides

NeuAc (GM2)

(GM3)

UDP-GalNAac

GalNAc

Gal

Cer UDP-Gal

Gal

GalNAc

Gal

Glc

Cer

CMP-NeuAc

Gal

GalNAc

Gal

Glc

Cer

(GM1b)

NeuAc

Other O-series gangliosides

FIGURE 10.36 Key steps in the initial formation of gangliosides. (From Gurr, M.I., Harwood, J.L. and Frayn, K.N. (2002). Lipid Biochemistry, 5th ed., Blackwell Scientific, Oxford, U.K. With permission.)

TABLE 10.8

Glycosyltransferases catalysing the biosynthesis of gangliosides

Abbreviation

Name

Linkage created

Substrates

GalT-1 SAT-1 SAT-2 SAT-3 SAT-4 SAT-5 GalNAcT GalT-2

β-Galactosyltransferase Sialyltransferase Sialyltransferase Sialyltransferase Sialyltransferase Sialyltransferase β-N-Acetylgalactosaminyltransferase β-Galactosyltransferase

Gal(β1­4)Glc Neu5Ac(­2­3)Gal Neu5Ac(­2­8)Neu5Ac Neu5Ac(­2­8)Neu5Ac Neu5Ac(­2­3)Gal Neu5Ac(­2­8)Neu5Ac GalNAc(β1­4)Gal Gal(β1­3)GalNAc

Glc-Cer Lac-Cer GM3 GD3 GA1, GM1a, GD1b GM1, GD1a, GT1b Lac-Cer, GM3, GD3 GA2, GM2, GD2

From Sweeley (1991).

work in various combinations. Thus, the final ganglioside composition can be influenced by the relative activity of these enzymes as well as substrate availability (Muramatsu, 2000). Regulation of ganglioside biosynthesis occurs at transcriptional and post-transcriptional levels (Merrill and Sandhoff, 2002). As with neutral glycosphingolipid synthesis, the glycosyltransferases are membrane-bound and predominantly located in the Golgi apparatus. Sphingomyelin, which is both a phospho- and sphingolipid, is synthesised by transfer of phosphorylcholine from phosphatidylcholine to ceramide, liberating diacylglycerol. De novo sphingomyelin synthesis occurs on

both the plasma membrane and in the Golgi apparatus with the proportions depending on the cell type. An analogous sphingolipid, ceramide phosphorylehanolamine, is made in a similar way but using phosphatidylethanolamine as the donor. Inositolphosphoceramides are made similarly (Merrill and Sandhoff, 2002). A comprehensive review of sphingomyelin metabolism, intracellular transport and various aspects of its biological functions has been published (Koval and Pagano, 1991). The biological function (Stoffel, 1971) and immunochemistry of the sphingolipids — especially the gangliosides — have been reviewed (Hakomori, 1981). Kanfer 691

10.5 Sphingolipids

and Hakomori (1983) have published a comprehensive review on sphingolipid biochemistry and see Bell et al. (1993) for aspects of metabolism. Merrill and Hannun (2000) have edited a useful volume of Methods in Enzymology and Lynch (1993) describes plant sphingolipids.

10.5.2

further by a neuraminidase to give ceramide lactoside (Figure 10.37) (see Brady, 1978). The cleavage of ceramide lactoside needs a β-galactosidase. Two β-galactosidases have been demonstrated in mammalian tissues, both of which have acidic pH optima (pH 4.2 and 4.8). When assayed in vitro, either of these enzymes catabolizes ceramide lactoside, depending on the detergent used in the experiment. The pH4.2 galactosidase also hydrolyses galactose from galactocerebroside. The pH 4.8 enzyme preferentially catalyses the removal of the terminal galactose of GM1 as well as catabolizing ceramide lactoside. There are also two liver β-galactosidases with more neutral pH optima that catabolize ceramide lactoside, but are inactive with galactocerebroside or GM1 (BenYoseph et al., 1977). For degradation of sphingolipids with 4 or less carbohydrate residues, there is often a requirement for sphingolipid activator proteins (SAPs or saposins) in vivo. Some inherited diseases are caused by mutation of the domains of the exoglycosidases that interact with SAPs (Merrill and Sandhoff, 2002). Glucocerebroside is hydrolysed by glucocerebrosidase to give glucose and ceramide. Catabolism of ceramide is catalysed by acid (Gatt, 1963), neutral (Sugita et al., 1975) or alkaline ceramidases in mammalian tissues. A sphingosine

Breakdown

Breakdown of sphingolipids occurs by stepwise hydrolytic cleavage of the various substituents, starting with the terminal hydrophilic portions of the molecules. As an example, catabolism of ganglioside GM1[Gal(β1→3) GalNAc(β1→4)Gal-(3→2αAcNeu)(β1→4)Glc(β1→1′) Cer] begins by the action of a β-galactosidase that cleaves the terminal galactose to give ganglioside GM2 as the other product. Ganglioside GM2 can be cleaved by two pathways. Either a hexosaminidase removes the molecule of GalNAc or a neuroaminidase hydrolyses AcNeu. These reactions have been demonstrated in vivo, but because more GM2[GalNAc(β1→4) Gal-(3→2AcNeu) (β1→4) Glc(β1→1′)Cer] accumulates in Tay-Sachs disease than GA2[GalNAc(β1→4)-Gal(β1→4)Glc(β1→1′)Cer], the hexosaminidase reaction appears the more important. The latter enzyme attacks GM2 to yield GM3[(AcNeu2→3)Gal(β1→4)Glc(β1→1′)Cer], which is then catalysed β

Cer-Glc(4

1)Gal (4

β

1)GalNAc (3

β

1)Gal

3 2 NANA Ganglioside GM1 Galactosidase Cer-Glc-Gal-GalNAc NANA Tay-sachs ganglioside, GM2 Hexosaminidase

Neuraminidase

Cer-Glc-Gal-NANA

Cer-Glc-Gal-GalNAc Hexosaminidase

Neuraminidase Cer-Glc-Gal

β-Galactosidase Cer-Glc β-Glucosidase Cer Ceramidase Sphingosine + Fatty acid

FIGURE 10.37 Breakdown of ganglioside GMI-galactosidase.

692

Lipid Metabolism

Gangliosides Sphingomyelin Sphingomyelin synthase Neutral glycosphingolipids

Glucosyl ceramide β -glucosidase

Galactosyl ceramide

Ceramide

β -galactosidase

Arylsulphatase A

Phosphatidate phosphohydrolase

Ceramide kinase

Ceramide synthase

Sulphatide

Sphingomyelinase

Ceramidase

Sphingosine

Sphingosine phosphatase Ethanolamine phosphate

Ceramide 1- phosphate

Sphingosine-N-methyl transferase

Sphingosine kinase

N,N-dimethyl Sphingosine

Sphingosine 1- phosphate Sphingosine 1- phosphate lyase

Hexadecenal

FIGURE 10.38

Interconversions between and catabolism of simple sphingolipids.

base and a nonesterified fatty acid are liberated. The former is phosphorylated to give a 1-phosphate derivative and then hydrolysed to yield a long-chain aldehyde and phosphoethanolamine (Stoffel, 1971). Sulfatides are catabolised by a sulfatase and the resultant galactocerebroside hydrolysed by a β-galactosidase. In addition, sphingomyelin is hydrolysed by sphingomyelinase to yield ceramide and phosphocholine (see Barenholz and Gatt, 1982) (Figure 10.38). Sphingolipid breakdown is reviewed by Kanfer and Hakomori (1983) and deficiency diseases of sphingolipid catabolism are covered in Section 11.5. Sweeley (1991) and Merrill and Sandhoff (2002) discuss some aspects of sphingolipid breakdown and regulation of their turnover. In the latter connection, glycosphingolipids have important functions during cellular differentiation and oncogenic transformation. Recent interest has also included their function as modulators of transmembrane signalling and as mediators for cellular interactions (Hakomori, 1990). Sphingolipids as cell signalling molecules are discussed in Section 10.6 (see also Aue et al., 2000). Apart from the main compounds (ceramide, sphingoid bases, and sphingosine-1-phosphate), a number of other lysosphingolipids have acute activity. Thus, lysosphingomyelin is a potent mitogen while psychosine (lyso-GlcCer or lyso-GalCer) is highly cytotoxic. Ceramide-1-phosphate is an active Ca++-mobilising agent (see Merrill and Sandhoff, 2002).

For a simple review of sphingolipid metabolism and function see Gurr et al. (2002).

References Aue, N. et al. (2000). Sphingomyelin metabolites in vascular cell signalling and atherogenesis. Prog. Lipid Res. 39, 207–229. Barenholz, Y. and Gatt, S. (1982). Sphingomyelin: metabolism, chemical synthesis, chemical and physical properties. In Phospholipids (Eds. J.N. Hawthorne and G.B. Ansell), Elsevier, Amsterdam, pp. 129–177. Basu, M. et al. (1987). Glycolipids. In Methods in Enzymology, vol. 138 (Ed. V. Ginsburg), Academic Press, Orlando, FL, pp. 575–607. Bell, R.M. et al. Eds. (1993). Sphingolipids, Part B: regulation and function of metabolism. Adv. Lipid Res. 26 (special issue). Ben-Yoseph, Y. et al. (1977). Purification and properties of neutral beta-galactosidases from human liver. Arch. Biochem. Biophys, 184, 373–380. Brady, R.N. et al. (1969). Biosynthesis of sphingolipid bases. 3. Isolation and characterization of ketonic intermediates in the synthesis of sphingosine and dihydrosphingosine by cell-free extracts of Hansenula ciferri. J. Biol. Chem. 244, 491–496. Brady, R.O. (1978). Sphingolipidoses. Annu. Rev. Biochem. 47, 687–713. Carter, H.E. et al. (1965). Glycolipids. Annu. Rev. Biochem. 34, 109–142.

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10.6

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10.6

Dunn, T.M. et al. (2004). A post-genomic approach to understanding sphingolipid metabolism in Arabidopsis thaliana. Ann. Bot.. 93, 483–497. Gatt, S. (1963). Enzymic hydrolysis and synthesis of ceramides. J. Biol. Chem. 238, 3131–3133. Gurr, M.I. and James, A.T. (1980). Lipid Biochemistry, Chapman & Hall, London. Gurr, M.I. et al. (2002). Lipid Biochemistry, 5th ed., Blackwell Scientific, Oxford, U.K. Hakomori, S.-I. (1981). Glycosphingolipids in cellular interaction, differentiation, and oncogenesis. Annu. Rev. Biochem. 50, 733–764. Hakomori, S.-I. (1990). Bifunctional role of glycosphingolipids. Modulators for transmembrane signaling and mediators for cellular interactions. J. Biol. Chem. 265, 18713–18716. Honke, K. et al. (1997). Molecular cloning and expression of cDNA encoding human 3-phosphoadenylsulfate: galactosylceramide 3-sulfotransferase. J. Biol. Chem. 272, 4864–4868. Kanfer, J.N. and Hakomori, S-I. (1983). Sphingolipid Biochemistry, Plenum, New York. Koval, M. and Pagano, R.E. (1991). Intracellular transport and metabolism of sphingomyelin. Biochim. Biophys. Acta. 1082, 113–125. Lynch, D.V. (1993). Sphingolipids. In Lipid Metabolism in Plants, Ed. T.S. Moore, CRC Press, Boca Raton, FL, pp. 285–308. Merrill, A.H. and Hannun, Y.A. Eds. (2000). Sphingolipid metabolism and function Part A, Part B. In Methods of Enzymology, vols. 311 and 312. Merrill, A.H. and Sandhoff, K. (2002). Sphingolipids: metabolism and cell signalling. In Biochemistry of Lipids, Lipoproteins and Membranes, Eds. D.E. Vance and J.E. Vance, 4th ed., Elsevier, Amsterdam, pp. 373–407. Muramatsu, T. (2000). Essential roles of carbohydrate signals in development, immune response and tissue functions, as revealed by gene targeting. J. Biochem (Tokyo). 127, 171–176. Spence, M.W. (1989). Sphingomyelin biosynthesis and catabolism. In Phosphatidylcholine Metabolism, Ed. D.E. Vance, CRC Press, Boca Raton, FL, pp. 185–203. Sperling, P., Blume, A., Zahringer, U. and Heinz, E. (2000). Further characterisation of ∆8-desaturases from higher plants. Biochem. Soc. Trans. 28, 638–641. Stoffel, W. (1971). Sphingolipids. Annu. Rev. Biochem. 40, 57–82. Stoffel, W. et al. (1968). Metabolism of sphingosine bases. IX. Degradation in vitro of dihydrospingosine and dihydrospingosine phosphate to palmitaldehyde and ethanolamine phosphate. Hoppe-Seyler’s Z. Physiol. Chem. 349, 1745–1748. Sugita, M. et al. (1975). Ceramidase and ceramide synthesis in human kidney and cerebellum. Description of a new alkaline ceramidase. Biochim. Biophys. Acta. 398, 125–131. Sweeley, C.C. (1991). Sphingolipids. In Biochemistry of Lipids, Lipoproteins and Membranes, Eds. D.E. Vance and J. Vance, Elsevier, Amsterdam, pp. 327–361. Thompson, G.A. (1973). Phospholipid metabolism in animal tissues. In Form and Function of Phospholipids, Eds. G.B. Ansell, R.M.C. Dawson, and J.N. Hawthorne, Elsevier, Amsterdam, pp. 67–96. Thorpe, S.R. and Sweeley, C.C. (1967). Chemistry and metabolism of sphingolipids. On the biosynthesis of phytosphingosine by yeast. Biochemistry, 6, 887–897. Wiegandt, H. (1971). Glycosphingolipids. Adv. Lipid Res. 9, 249–289.

Lipids as signalling molecules

The first lipids to be recognised as giving rise to lipid second messengers were the inositol-containing phosphoglycerides. Since that time a multitude of lipids with acute biological activity have been recognised and this general area is one of the most active in biochemistry. Signalling molecules include intact phosphoglycerides (e.g., phosphatidic acid (PA), platelet-activating factor (PAF), membrane-soluble hydrolysis products (e.g., diacylglycerol (DAG)), water-soluble products (e.g., inositol1,4,5-tris-phosphate) and other degradative metabolities (e.g., lysoPA, fatty acids). The role of polyunsaturated fatty acids themselves is described in Section 10.1 and Section 11.1. Two general reviews about lipids as signalling molecules or bioactive lipids are Bell et al. (1996) and Nicolaou and Kokotos (2004). Methods for analysis are described in Christie (2003) and in Laychock and Rubin (1999). Simpler summaries of some aspects of lipid signalling are in Gurr et al. (2002) and Vance and Vance (2002).

10.6.1

Platelet activating factor (PAF)

Intact phospholipids have various important roles in signalling. One of the first such lipids to be recognised was platelet activating factor (PAF; 1-O-alkyl-2-acetyl-snglycerol-3-phosphocholine). PAF is produced by many types of cells in response to stimuli. In inflammatory cells, such as monocytes or macrophages, it can be rapidly synthesised by a deacylation-acetylation pathway (Tokumura, 1995). It can also be produced by de novo synthesis, which seems particularly important for maintaining PAF levels in the central nervous and reproductive systems. Because of its very potent biological activity, PAF levels in cells and in the circulation are strictly regulated (Tokomura, 1995). PAF-acetylhydrolase plays a primary role in inactivation. There are a family of PAF-acetylhydrolases that consist of two intracellular isoforms and one secreted (plasma) isoform. Details of these enzymes, physiological function and role in human disease are given by Karasawa et al. (2003). PAF elicits responses in many cells and organs. Originally it was shown to aggregate platelets (hence, the name), but it is now known to affect vasodilation/constriction, bronchial responsiveness and many acute responses connected to inflammation. PAF acts by binding to a unique G-protein-coupled seven transmembrane receptor that links it to various signalling pathways (Ishii and Shimizu, 2000). A series of PAF-like lipids have been identified, many of which have biological activity and some can also be formed under oxidising conditions such as those produced by smoking (see Tokumura, 1995). A general review of the biochemistry of PAF is that by Synder (1995) and Honda et al. (2002) describe PAF

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receptors. Farooqui and Horrocks (2004) discuss general aspects of PAF and provide up-to-date references.

Lysophosphatidic acid (LPA) was first identified as the active ingredient of Darmstoff (smooth muscle-stimulating substance) in 1957. Tokumura (1995) reviewed the activity of LPAs as a vasopressor, a platelet agonist, growth factor, and putative second messenger. LPA can be generated from phosphatidic acid (phospholipase A1 or A2), from lysophospholipids (phospholipase D), or by oxidative modification of low-density lipoprotein. LPA can bind to receptors, a number of which have been reported (Fukushima et al., 2001). It is thought to have important pathophysiological roles in cancer, cell survival apoptosis, and vascular activity (Tigyi and Parrill, 2003). See Pyne (2004) for an update on the metabolism and function of LPA. The biochemistry of the parent compound for LPA, phosphatidic acid, is discussed thoroughly by Bocckino and Exton (1996), who pointed out that many of the purported physiological effects of phosphatidic acid can be explained by traces of LPA. Phosphatidic acid, itself, has many important functions in plants that are well reviewed by Wang (2006) and by Testerink and Munnik (2005).

Reviews of the roles of different intact inositol phospholipids (as well as their biologically active metabolites) in plants have been made recently (Meijer and Munnik, 2003; Drøbak, 2005). Although plant proteins, such as those with PH- or PX-domains, can bind to certain phosphoinositides, there are unique features of plants compared to mammals. Moreover, the pathways for phosphorylation and, hence, the spectrum of plant phosphoinositides (Drøbak, 2005) differ from animal systems (Vanhaesebroeck et al., 2001). Phosphoinositides can also exert their role in the control of cellular processes by being hydrolysed and giving rise to second messengers. The classic reaction is the hydrolysis of PIP2 to give rise to the dual second messengers diacylglycerol and inositol 1,4,5-trisphosphate (Berridge, 1987). For more recent description of these effectors and general descriptions of the importance of phosphoinositides, refer to Payrstre et al. (2001), Toker (2002), and Payrastre (2004). The product of phosphoinositide hydrolysis with phospholipase C (above) is diacylglycerol (DAG), which has a well-known function with protein kinase C (Takai et al., 1979). While hydrolysis of phosphoinositides produces a quick elevation of DAG, it can be produced from phosphatidylcholine in a more sustained manner via phosphatidic acid (Nishizuka, 1995). Other aspects of DAGs are covered in Becker and Hannun (2004).

10.6.3 Inositol lipids

10.6.4 Plasmalogens

Although phosphatidylinositol-4,5-bis-phosphate (PIP2) is an important precursor of the twin second messengers inositol-1,4,5-trisphosphate and diacyglycerol, it has functions as an intact lipid also. These include functions in ion channel function (e.g., Kobrinsky et al., 2000) and in membrane trafficking (see Hilgemann, 2003). There are also roles for phosphoinositides in cytoskeletal function (Yin and Janmey, 2003) and in the activity and binding of phospholipase D. Phosphatidylinositol 4-phosphate may have a function in binding the cytoskeletal protein, talin (Payrastre, 2004). Recent work has also revealed a number of important signalling roles for phosphoinositides that does not involve their hydrolysis. Thus, in addition to PIP2, there are well-evidenced roles for phosphatidylinositol-3phosphate and phosphatidylinositol-3,4,5-trisphosphate as membrane lipids for the recruitment and/or activation of various proteins. In turn, this can influence the action of a large number of proteins and, hence, signalling pathways that underpin mechanisms for signal transduction, cytoskeletal, and membrane trafficking events (Martin, 1998). Additional information on the synthesis and function of 3-phosphorylated inositol phospholipids is given by Vanhaesebroeck et al. (2001) and Katso et al. (2001). Vivanco and Sawyers (2002) discuss phosphatidylinositol 3-kinases and cancer.

Plasmalogens are thought to have a number of important roles in controlling tissue functions. General reviews are those of Farooqui and Horrocks (2001) and Nagan and Zoeller (2001). Functions include those in ion transport, membrane fusion, protection of membranes against oxidative stress, cholesterol efflux, and cellular differentiation. Other ether lipids have been reported to have various physiological functions and to be involved in different human diseases (Farooqui and Horrocks, 2004).

10.6.2 Lysophosphatidic acid (LPA)

10.6.5 Sphingolipids Sphingolipids can have various signalling roles or can act as bioactive lipids. Their relationship with each other is illustrated in Figure 10.39 (see also Gurr et al., 2002). Ceramide was the first such lipid to be described as a second messenger (Hannun et al., 1996). Its biochemistry and signalling actions have been reviewed by Hannun and Obeid (2002) and by de Avalos et al. (2004). Ceramide plays a very important role in apoptosis (Pettus et al., 2002), in oxidative and heat stress, and in various diseases (de Avalos et al., 2004). Ceramides can be hydrolysed to produce sphingosine, which itself has important properties in regulating cellular systems (Merrill et al., 1996). Importantly, sphingosine can be phosphorylated to produce another signalling

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Lipids as signalling molecules

Gangliosides, Complex neutral ceramides Sphingomyelin Sphingomyelin synthase Sphingomyelinase

Glucosyl ceramide

Ceramide glucosyl transferase

β - glucosidase

Ceramide kinase

Ceramide

Phosphatidate phosphohydrolase Ceramide synthase

Ceramidase Sphingosine kinase

Sphingosine

Sphingosine 1- phosphatase

FIGURE 10.39

Sphingosine 1- phosphate

Biosynthetic and catabolic pathways of the core sphingolipid signaling molecules.

molecule, sphingosine 1-phosphate. This compound was reviewed by Pyne and Pyne (2000) and has two major intracellular functions. First, it acts in a “sphingolipid rheostat” where the balance between such lipids determines cellular fate (Pyne, 2004). Second, it is involved in calcium homeostatis. Sphingosine 1-phosphate binds to specific G-protein coupled receptors (Kluk and Hla, 2002). The general aspect of sphingolipids and signalling was reviewed by Smith and Merrill (2002). A large proportion of the sphingolipids that are present in cells and tissues are the complex glycosphingolipids. These lipids accumulate in particular patterns in different cell types and species (Hakomori, 1981). The pattern of glycosphingolipids changes with cell growth, differentiation, viral transformation, ontogenesis, and oncogenesis (Kolter, 2004). Because of their widespread functions, there is increasing interest in them as therapeutic agents or targets (Gagnon and Saragovi, 2002). The role of sphingolipids in storage diseases is covered in Section 11.5.

10.6.6

Ceramide 1- phosphate

found was 2-arachidonylglycerol and most other compounds contain the arachidonyl component (Kokotos, 2004). The endocannabinoids can be chemically synthesised (Razdan and Mahadevan, 2002) and have therapeutic possibilities (Goutopoulos and Makriyannis, 2002). A general review is that of Kokotos (2004). N-acylethanolamines that occur in significant amounts in certain plant tissues and which also bind to endocannabinoid receptors appear to have a role in plant defence (for review, see Chapman, 2004).

References Becker, K.P. and Hannun, Y.A. (2004). Diacylglycerols. In Bioactive Lipids (Eds. A. Nicolaou and G. Kokotos), The Oily Press, Bridgwater, U.K., pp. 37–61. Bell, R.M. et al. Eds. (1996). Lipid Second Messengers, Handbook of Lipid Research, Plenum Press, New York. Berridge, M.J. (1987) Inositol triphosphate and diacylglycerol: two interacting second messengers. Annu. Rev. Biochem. 56, 159–193. Bocckino, S.B. and Exton, J.H. (1996). Phosphatidic acid. In Lipid Second Messengers, Eds. R.M. Bell, J.H. Exton and S.M. Prescott, Plenum Press, New York, pp. 1–58. Chapman, K.D. (2004). Occurrence, metabolism and prospective function of N-acylethanolamines in plants. Prog. Lipid Res. 43, 302–327. Christie, W.W. (2003). Lipid Analysis, 3rd ed., The Oily Press, Bridgwater, U.K. De Avalos, S.V. et al. (2004). Ceramides. In Bioactive Lipids, Eds. A. Nicolaou and. G. Kokotos, The Oily Press, Bridgwater, U.K., pp. 135–168.

Endocannabinoids

The identification, in the 1960s, of brain-specific receptors for one of the major components of Cannabis sativa preparations led to the discovery of endogenous ligands for such receptors. These are known as the endocannabinoids. The first determination and characterisation of a cannabinoid receptor in brain was by Devane et al. (1988). The first endocannabinoid identified and the best studied is anandamide (Devane et al., 1992). The second endocannabinoid 696

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Devane, W.A. et al. (1988) Determination and characterization of a cannabinoid receptor in rat brain. Mol. Pharmacol., 34, 605–613. Devane, W.A. et al. (1992) Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science, 258, 1946–1949. Drøbak, B.K. (2005). Inositol-containing lipids: roles in cellular signalling. In Plant Lipids: Biology, Utilisation and Manipulation, Ed. D.J. Murphy, Blackwell Publishing, Oxford, U.K., pp. 303–328. Farooqui, A.A. and Horrocks, L.A. (2004). Plasmalogens, plateletactivating factor and other ether glycerophospholipids. In Bioactive Lipids, Eds. A. Nicolaou and G. Kokotos, The Oily Press, Bridgwater, U.K., pp. 107–134. Fukushima, N. et al. (2001) Lysophospholipid receptors. Ann. Rev. Pharmacol. Toxicol. 41, 507–534. Gagnon, M. and Saragovi, H.U. (2002). Gangliosides: therapeutic agents or therapeutic targets? Expert Opin. Ther. Patents, 12, 1215–1223. Goutopoulos, A. and Makriyannis, A. (2002) From cannabis to cannabinergics: new therapeutic opportunities. Pharmacol. Ther. 95, 103–117. Gurr, M.I. et al. (2002). Lipid Biochemistry, 5th ed., Blackwell Scientific, Oxford, U.K. Hakomori, S. (1981) Glycosphingolipids in cellular interaction, differentiation and oncogenesis. Annu. Rev. Biochem. 50, 733–764. Hannun, Y.A. et al. (1996). Ceramide: a novel second messenger and lipid mediator. In Lipid Second Messengers, Eds. R.M. Bell, J.H. Exton and S.M. Prescott, Plenum Press, New York, pp. 75–123. Hannun, Y.A. and Obeid, L.M. (2002) The ceramide-centric universe of lipid-mediated cell regulation: stress encounters of the lipid kind. J. Biol. Chem. 277, 25847–25850. Hilgemann, D.W. (2003). Getting ready for the decade of the lipids. Annu. Rev. Physiol. 65, 697–700. Honda, Z. et al. (2002) Platelet-activating factor receptor. J. Biochem. 131, 773–779. Ishii, S. and Shimizu, T. (2000). Platelet-activating factor (PAF) receptor and genetically engineered PAF receptor mutant mice. Prog. Lipid Res. 39, 41–82. Katso, R. et al. (2001) Cellular function of phosphoinositide 3-kinase: implication for development, immunity, homeostasis and cancer. Ann. Rev. Cell Dev. Biol. 17, 615–675. Karasawa, K. et al. (2003). Plasma platelet activating factoracetylhydrolase (PAF-AH). Prog. Lipid Res. 42, 93–114. Kluk, M.J. and Hla, T. (2002) Signalling of sphinosine 1-phosphate via the SIP/EDG-family of G-protein coupled receptors. Biochim. Biophys. Acta. 1582, 72–80. Kobrinsky, E. et al. (2000). Reception-mediated hydrolysis of plasma membrane messenger PIP2 leads to K+ current desensitisation. Nat. Cell Biol. 2, 507–514. Kokotos, G. (2004). Endocannabinoids. In Bioactive Lipids, Eds. A. Nicolaou and G. Kokotos, The Oily Press, Bridgwater, U.K., pp. 245–264. Kolter, T. (2004). Glycosphingolipids. In Bioactive Lipids (Eds. A. Nicolaou and G. Kokotos), The Oily Press, Bridgwater, U.K., pp. 169–196. Laychock, S. and Rubin, R.P. Eds. (1999). Lipid Second Messengers. CRC Press, Boca Raton, FL.

Maijer, H.J.G. and Munnik, T. (2003). Phospholipid-based signalling in plants. Annu. Rev. Plant Biol. 54, 265–306. Martin, T.F.J. (1998). Phosphoinositide lipids as signalling molecules: common themes for signal transduction, cytoskeletal regulation and membrane trafficking. Annu. Rev. Cell Dev. Biol. 14, 231–264. Merrill, A.H. et al. (1996). Bioactive properties of sphingosine and structurally related compounds. In Lipid Second Messengers, Eds. R.M. Bell, J.H. Exton and S.M. Presscott, Plenum Press, New York, pp. 205–237. Nagan, N. and Zoeller, R.A. (2001) Plasmalogens: biosynthesis and functions. Prog. Lipid Res. 40, 199–229. Nicolaou, A. and Kokotos, G. Eds. (2004). Bioactive Lipids, The Oily Press, Bridgwater, U.K. Nishizuka, Y. (1995) Protein kinase C and lipid signalling for sustained cellular responses. FASEB J. 9, 484–496. Payrastre, B. et al. (2001) Phosphoinositides: key players in cell signalling, in time and space. Cell Signal. 13, 377–387. Payrastre, B. (2004). Phosphoinositides. In Bioactive Lipids, Eds. A. Nicolaou and G. Kokotos, The Oily Press, Bridgwater, U.K., pp. 63–84. Pettus, B.J. et al. (2002) Ceramide in apoptosis: an overview and current perspectives. Biochim. Biophys. Acta. 1585, 114–125. Pyne, S. and Pyne, N.J. (2002) Sphingosine 1-phosphate signalling and termination at lipid phosphate receptors. Biochim. Biophys. Acta. 1582, 121–131. Pyne, S. (2004). Lysolipids: sphingosine 1-phosphate and lysophosphatidic acid. In Bioactive Lipids, Eds. A. Nicolaou and G. Kokotos, The Oily Press, Bridgwater, U.K., pp. 85–106. Razdan, R.K. and Mahadevan, A. (2002). Recent advances in the synthesis of the endocannabinoid-related ligands. Chem. Phys. Lipids. 121, 21–33. Rubin, R.P. and Laychock, S. Eds. (1999). Lipid Second Messengers. CRC Press, Boca Raton, FL. Smith, W.L. and Merrill, A.H. (2002) Sphingolipid metabolism and signalling, mini review series. J. Biol. Chem. 277, 25841–25842. Snyder, F. (1995) Platelet-activating factor: the biosynthetic and catabolic enzymes. Biochem. J. 305, 689–705. Takai, Y. et al. (1979) Unsaturated diacylglycerol as a possible messenger for the activation of calcium-activated, phospholipid-dependent protein kinase system. Biochem. Biophys. Res. Commun. 91, 1218–1224. Testerink, C. and Munnik, T. (2005). Phosphatidic acid: a multifunctional stress signalling lipid in plants. Trends Plant Sci. 10, 368–375. Tigyi, G. and Parrill, A.B. (2003). Molecular mechanisms of lysophosphatidic acid action. Prog. Lipid Res. 42, 93–114. Toker, A. (2002) Phosphoinositides and signal transduction. Cell Mol. Life Sci. 59. 761–779. Tokumura, A. (1995). A family of phospholipid autacoids: occurrence, metabolism and bioactions. Prog. Lipid Res. 34, 151–184. Vance, D.E. and Vance, J.E. Eds. (2002). Biochemistry of Lipids, Lipoproteins and Membranes, 4th ed., Elsevier, Amsterdam. Vanhaesebroceck, B. et al. (2001). Synthesis and function of 3-phosphorylated inositol lipids. Annu. Rev. Biochem. 70, 535–602. Vivanco, I. and Sawyers, C.L. (2002) The phosphatidylinositol 3-kinase-Akt pathway in human cancer. Nature Rev. 2, 489–501.

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protein (Gu et al., 2000). Selective uptake from HDL needs SR-B1 binding. The receptor also promotes cholesterol efflux from the plasma membrane by an unknown mechanism. SR-B1 has been localised to cholesterol-rich microdomains called calveoli (Fielding and Fielding, 2002). LCAT may also participate in the movement of cholesterol out of cells by esterifying excess cholesterol in the intravascular circulation (cf. Marcel, 1982). Schneider (2002) has written a useful review on lipoprotein receptors and their importance in plasma lipid metabolism. The purification and properties of LCAT, together with a discussion of its mechanism of reaction, are given by Marcel (1982). A number of disease states involve LCAT activity. Two LCAT deficiencies have been found. In one, no cholesteryl esters are formed in plasma and cholesterol accumulates as droplets in peripheral tissues. In a second disease, LCAT can transesterify cholesterol from VLDL and LDL, but not from exogenous HDL (Fielding and Fielding, 2002). A discussion of cholesteryl ester metabolism in relation to other liver diseases and dyslipoproteinaemia has been reported (Marcel, 1982). Similarly, the metabolism of cholesteryl esters in relation to arteries and arterial disease has been fully discussed (Kritchevsky and Kothari, 1978). Mammalian steroid sulfates have been reviewed by Farooqui (1981). Sterol esters and acylated sterol glycosides have been detected in a number of plant tissues and, in some cases, can be quite significant components (Mudd, 1980). For sterol esters any one of palmitate, oleate, linoleate, or α-linoleate could be the principal fatty acyl component depending on the tissue. In acylated sterol glycosides, palmitate or linoleate are the most abundant fatty acids. Synthesis of sterol esters by preparations from spinach leaves have been studied by Mudd’s group (Mudd, 1980). Enzyme activity may have been associated with mitochondria and diacylglycerol was found to be the best acyl donor, although other lipids including phosphatidylcholine could also serve. The acyl donor for sterol ester synthesis in Phycomyces blakesleanus was also found to be phosphatidylcholine, as in animal tissues (Bartlett et al., 1974). For a summary of plant sterol ester biochemistry, refer to Goad et al. (1987). The acylation of sterol glucosides in plant tissues has been studied by several workers. The research has been reviewed by Eichenberger (1977) and by Mudd (1980). The acylating enzymes are usually particulate and have been partly purified following solubilization. The best purification has been with an enzyme from Gossipium spp. (Forsee et al., 1976). Soluble preparations have been studied from carrot roots (cf. Eichenberger, 1977) and bean leaves (Heinz et al., 1975). The acyl donor for acylated sterol glucoside synthesis seems to vary with the preparation being studied. The particulate enzymes tend to use various phosphoglycerides, whereas the soluble enzymes utilise diacyldigalactosylglycerol much better. The bean leaf

Wang, X. et al. (2006). Signalling functions of phosphatidic acid. Prog. Lipid Res. 45, 250–278. Yin, H.L. and Janmey, P.A. (2003). Phosphoinositide regulation of the actin cytoskeleton. Annu. Rev. Physiol. 65, 761–789.

10.7

Sterol esters

The esterification of cholesterol in animals has attracted considerable research because of the possible involvement of cholesterol and its ester in various disease states (see Glomset and Norum, 1973; Section 11.1, Section 11.3 and Section 11.6). Cholesteryl esters are formed by the action of lecithin cholesterol acyltransferase (LCAT, EC 2.3.1.43), which is particularly active in plasma; see Sabine (1977) for a review of cholesterol metabolism. The reaction involves transfer of a fatty acid from position-2 of the lecithin (phosphatidylcholine) to the 3-hydroxyl group of cholesterol with the formation of monoacylphosphatidylcholine. LCAT is a glycoprotein of mass 60 kDa (Fielding, 1990). LCAT consumes unesterified cholesterol and the cholesteryl ester is retained in the high-density lipoprotein (HDL) core while lysophosphatidylcholine is transferred to albumin. LCAT plays a critical role in the genesis of HDL. In addition, it may be able to directly reactivate lipid-poor HDL (Kendrick et al., 2001). The enzyme is a 416-amino acid serine hydrolase with rather limited sequence homology to other plasma lipases. Aspects of its structure and regulation are discussed by Fielding and Fielding (2002). ApoA1 is needed for both its acyltransferase and phospholipase activities. When LCAT interacts with low-density lipoprotein (LDL), it can catalyse phosphatidylcholine acyl exchange. LCAT is reviewed by Jonas (2000). Cholesterol ester transfer protein (CETP) catalyses the movement of cholesteryl esters, triacylglycerols, and nonpolar lipids (such as retinyl esters) between plasma lipoproteins. CETP expression in hepatocytes is PPARdependent (Luo et al., 2001). Physiologically, the main effect of CETP may be to promote the transfer of LCATderived cholesteryl esters out of HDL (where they were formed) into VLDL and LDL, in exchange for triacylglycerol (see Fielding and Fielding, 2002); CETP is a glycoprotein of mass 53 kDa (Drayna et al., 1987). Its physiological function and mechanism of activity are discussed by Fielding and Fielding (1991). Activity of CETP in plasma is regulated by an inhibitor protein, which acts by displacing it from its lipoprotein binding sites (Morton and Zilverschmidt, 1981). It is structurally related to phospholipid transfer protein (PLTP), which transfers phospholipids between serum lipoprotein classes. Cholesteryl esters, in contrast to free cholesterol, are taken up by cells mostly via specific receptor pathways (Brown et al., 1981), are hydrolysed by lysosomal enzymes and eventually re-esterified and stored within cells. Scavenger receptor B1 (SR-B1) is the important trans-membrane 698

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enzyme would also use diacylgalactosylglycerol efficiently. The overall pathway seems to involve glucosylation of the sterol before acylation (see Mudd, 1980). Work on sterol ester metabolism in insects has been reviewed by Thompson et al. (1973).

Marcel, Y.L. (1982). Lecithin: cholesterol acyltransferase and intravascular cholesterol transport. Adv. Lipid Res. 19, 85–136. Morton, R.E. and Zilverschmidt, D.B. (1981). A plasma inhibitor of triglyceride and cholesteryl ester transfer activities. J. Biol. Chem. 256, 11992–11995. Mudd, J.B. (1980). Sterol interconversions. In Biochemistry of Plants, vol. 4, Eds. P.K. Stumpf and E.E. Conn, Academic Press, New York, pp. 509–534. Sabine, J. R. (1977). Cholesterol, Marcel Dekker, New York. Schneider, W.J. (2002) Lipoprotein receptors. In Biochemistry of Lipids, Lipoproteins and Membranes, Eds. D.E. Vance and J.E. Vance, 4th ed., Elsevier, Amsterdam, pp. 553–572. Thompson, M.J. et al. (1973). Metabolism of steroids in insects. Adv. Lipid Res. 11, 219–265.

References Bartlett, K. et al. (1974). Biosynthesis of sterol esters in Phycomyces blakesleanus. Phytochemistry, 13, 1107–1113. Brown, M.S. et al. (1981). Regulation of plasma cholesterol by lipoprotein receptors. Science. 212, 628–635. Drayna, D. et al. (1987). Cloning and sequencing of human cholesteryl ester transfer protein cDNA. Nature. 327, 632–634. Eichenberger, W. (1977). Steryl glycosides and acylated steryl glycosides. In Lipids and Lipid Polymers from Higher Plants, Eds. M. Tevini and H.K. Lichtenthaler, SpringerVerlag, Berlin, pp. 167–179. Farooqui, A.A. (1981). Metabolism of sulfolipids in mammalian tissues. Adv. Lipid Res. 18, 159–202. Fielding, C.J. (1990). Lecithin: cholesterol acyltransferase. In Advances in Cholesterol Research, Eds. M. Esfahani and J.B. Swaney, Telford, Caldwell, NJ, pp. 271–314. Fielding, P.E. and Fielding, C.J. (1991). Dynamics of lipoprotein transport in the circulatory system. In Biochemistry of Lipids, Eds. D.E. Vance and J. Vance, Elsevier, Amsterdam, pp. 427–459. Fielding, P.E. and Fielding, C.E. (2002). Dynamics of lipoprotein transport in the human circulatory system. In Biochemistry of Lipids, Lipoproteins and Membranes, Eds. D.E. Vance and J.E. Vance, 4th ed., Elsevier, Amsterdam, pp. 527–552. Forsee, W.T. et al. (1976). Acylation of steryl glucosides by phospholipids. Solubilization and properties of the acyl transferase. Arch. Biochem. Biophys., 172, 410–418. Goad, L.J. et al. (1987). The steryl esters of higher plants. In The Metabolism, Structure and Function of Plant Lipids, Eds. P.K. Stumpf, J.B. Mudd and W.D. Nes, Plenum, New York, pp. 95–102. Glomset, J.A. and Norum, K.R. (1973). The metabolic role of lecithin: cholesterol acyltransferase: perspectives from pathology. Adv. Lipid Res. 11, 1–65. Gu, X. et al. (2000) Scavenger receptor, class B type 1-mediated 3H-cholesterol efflux to high and low density lipoproteins is dependent on lipoprotein binding to the receptor. J. Biol. Chem. 275, 29993–30001. Heinz, E. et al. (1975). Enzymatic acylation of steryl glycoside. Z. Pflanzenphysiol., 75, 78–87. Jonas, A. (2000) Lecithin cholesterol acyltransferase. Biochim. Biophys. Acta. 1529, 245–256. Kendrick, J.S. et al. (2001) Superior role of apolipoprotein B-48 over apolipoprotein B100 in chylomicron assembly and fat absorption: an investigation of apobec-1 knockout and wild type mice. Biochem. J. 356, 821–827. Kritchevsky, D. and Kothari, H.V. (1978). Arterial enzymes of cholesteryl ester metabolism. Adv. Lipid Res. 16, 221–266. Luo, Y. et al. (2001) The orphan nuclear receptor LRH-1 potentiates the sterol-mediated induction of the human CETP gene by liver X receptor. J. Biol. Chem. 276, 24767–24773.

10.8

Control mechanisms

It is clearly important for organisms to be able to control the activity of individual enzymes and, hence, the rate of flux down a pathway. This may be as an adaptation or response to an environmental stress, during development or because of disturbances in the normal status quo. One important point to note is that there is (contrary to what is said in many textbooks) no such thing as a rate-controlling enzyme in a pathway. All enzymes can contribute to control, but their contribution can vary with circumstances. An excellent review of the control of metabolism is that by Fell (1997). General comments on the regulation of lipid metabolism will be found in Gurr et al. (2002). Individual aspects of control mechanisms are detailed in the earlier sections of this chapter, but a few additional remarks will be made here. For a recent summary of the regulation of different aspects of lipid metabolism, see Vance and Vance (2002); for plant metabolism, refer to Browse and Somerville (1991), Quinn and Harwood (1990), and Murphy (2005); and for microbial aspects, see Ratledge and Wilkinson (1988, 1989). The overall control of lipogenesis has been reviewed by Saggerson (1980) and the mechanisms by which carbohydrates regulate expression of lipogenic genes, by Girard et al. (2002). So far as saturated fatty acid synthesis is concerned, the activity of both acetyl-CoA carboxylase and fatty acid synthase can be altered in various ways (Gurr et al., 2002). Short-term or acute control involves metabolic or allosteric regulation and the covalent modification of enzymes. Long-term control involves alterations in the amounts of enzyme protein (Wakil et al., 1983). Because acetyl-CoA carboxylase catalyses the first committed step in lipid synthesis and because its substrate lies at a crossroads between carbohydrate and lipid metabolism, its regulation is clearly important. Mammalian acetyl-CoA carboxylase is regulated both acutely (by phosphorylation/dephosphorylation, by acyl-CoAs, and by tricarboxylic acids) and chronically due to changes in enzyme amounts (see Gurr et al., 2002). Citrate not only 699

10.8 Control mechanisms

causes polymerisation of acetyl-CoA carboxylase, but also can overcome inhibition caused by the enzyme product malonyl-CoA or the overall products of fatty acid synthesis, acyl CoAs (Allred and Reilly, 1997). See also Kim (1997) for a general discussion. The overall regulation of fatty acid synthesis in plants has been reviewed (Ohlrogge and Jaworski, 1997). The role of acetyl-CoA carboxylase in leaves was specifically addressed by flux control experiments and it was shown that this enzyme could exert up to 60% of the total control of flux towards lipid synthesis in the light (Page et al., 1994). The mechanism of regulation may involve changes in the enzyme’s redox state (Harwood, 1996; Rawsthorne, 2002). Cronan and Waldrop (2002) have given a very good recent survey of multisubunit acetyl-CoA carboxylases with particular emphasis on that from E. coli. They discuss the physiology, catalytic mechanism and function of the enzyme. Heath et al. (2003) also describe the regulation of E. coli acetyl-CoA carboxylase. Mammalian fatty acid synthase is also subject to adaptive changes in enzyme content. Any short-term metabolic control is ill defined (Wakil et al., 1983) and generally thought to be unimportant (Semenkovich, 1997). However, diet, triiodothyronine, hydrocortisone and insulin effects have been noted on the amount of synthase protein. Hydrocortisone and triiodothyronine have no effect alone, but potentiate the insulin induction of synthase (Wakil et al., 1983). The increases in synthase activity on refeeding or insulin administration are due to an increase in transcription of mRNA, which is elevated 70-fold (Morris et al., 1982). The mechanisms underlying fatty acid synthase regulation are reviewed by Semenkovich (1997). Rangan and Smith (2002) also discuss the regulation of fatty acid synthesis. A thorough discussion of the regulation of fatty acid synthase in plants has been made by Ohlrogge and Jaworski (1997) and recently updated (Harwood, 2005). Rawsthorne (2002) also covered some general aspects of fatty acid synthesis in relation to seed oil production. Heath et al. (2002) have described the overall regulation of fatty acid formation in E. coli from a quantitative and qualitative viewpoint. This article mentions the acetyl-CoA carboxylase accBC operon as well as the fab (fatty acid synthase) cluster. The role of the FadR protein in transcriptional control through the regulation of acyl-CoA concentrations is important in altering the balance between fatty acid synthesis and oxidation. It is well known that changes in growth temperature lead frequently to a modification in the pattern of fatty acids made and in those accumulated in the membranes of poikilotherms. Typical changes include an increase in unsaturated or of shorter chain-length fatty acids at lower growth temperatures. The adaptation has been studied in a large number of organisms. In anaerobic bacteria, such as E. coli, it is not possible for desaturases to be induced,

so that alterations in saturated/unsaturated fatty acid synthesis have to be controlled via fatty acid synthase. At lower temperatures the amount of cis-vaccenic acid is rapidly increased due to increased activity of β-ketoacyl-ACP synthase II. Overexpression of β-ketoacyl-ACP I can also increase the amount of vaccinate, but in a temperatureindependent manner. At low temperatures, vaccenate is also transferred to the sn-1 position of glycerolipids (where it competes with palmitate), whereas it is usually concentrated at the sn-2 position. However, the mechanism for the apparent change in acyltransferase selectivity is unknown (Heath et al., 2002). Growth temperature has also been reported to alter the fatty acid products of other fatty acid synthases. For example, the Saccharomyces cerevisiae synthase produces more palmitate rather than stearate at lower growth temperatures (Okuyama et al., 1979). There has been a considerable advance in our understanding of the control of aerobic fatty acid desaturases in recent years. In the simple protozoa, Tetrahymena and Acanthamoeba, low temperatures induce an increase in desaturase activity in order that the organisms can maintain membrane fluidity (Gurr et al., 2002). In the case of Acanthamoeba, the desaturase concerned with temperature adaptation is a ∆12 (ω6) oleate desaturase which produces linoleate (Avery et al., 1995). It is also induced independently by oxygen (Rutter et al., 2002). Another class of organism where there has been considerable study of temperature adaptation is the cyanobacteria. Induction of a number of individual desaturases and their mechanism of control have been well reviewed (Murata and Wada, 1995; Mikami and Murata, 2003). General aspects of temperature adaptation in different organisms will be found in Cossins (1994). For animals, considerable attention has been paid to the effects of diet on desaturase induction. In particular, there has been much study of stearoyl-∆9-desaturase, which can show extreme responses (100-fold changes in activity) to dietary manipulation. A thorough review of the earlier work in this area was made by Ntambi (1995), who discussed tissue-specific expression and the ability of carbohydrate or unsaturated fatty acids to regulate the stearoyl-CoA desaturase genes. The molecular mechanisms by which dietary polyunsaturated fatty acids could regulate genes (including those of glucose or fatty acid metabolism) is discussed by Jump et al. (1996) and developed further by Clarke (2000). Recently Ntambi and Miyazaki (2004) have revisited the topic of the mammalian stearoyl-CoA desaturases including aspects of the specific functions of isoforms and contribution of the enzyme activity to the regulation of metabolism. In addition to fatty acid synthesis, the assembly of complex acyl lipids is also under careful metabolic control. For general remarks, see Gurr et al. (2002) and Vance and Vance (2002). 700

Lipid Metabolism

The regulation of the enzymes of triacylglycerol synthesis has been well reviewed by Coleman and Lee (2004) with reference to earlier work and to the use of sterol regulatory element-binding protein (SREBP), the liver X receptors and PPARs (peroxisome proliferator-activated receptors) in the regulation. SREBP is, of course, also involved in the relative rates of fatty acid and cholesterol biosynthesis (Gibbons, 2003). Interactions between phospholipids and sterol metabolism in mammalian cells are reviewed by Ridgway et al. (1999), while a comprehensive account of the regulation of phosphoglyceride synthesis and degradation in different organisms will be found in Hawthorne and Ansell (1982); see also Kent (1995) and Vance (1998); and see also various chapters in Vance and Vance (2002). Flux control analysis has been applied to the study of lipid accumulation in oil crops. Experiments with olive and palm have shown that fatty acid synthesis exerts more control than lipid assembly (Ramli et al., 2002), but that, within the Kennedy pathway, diacylglycerol acyltransferase may be important in some crops (Ramli et al., 2005). The regulation of storage oil accumulation has been discussed by Voelker and Kinney (2001), Rawsthorne (2002) and, recently, by Weselake (2005). The regulation of plant lipid metabolism was reviewed by Harwood (1989) and, with respect to environmental stress, by Harwood (1994, 1998). For a review of phospholipid metabolism in yeast and its interrelationship with other metabolic processes, see Carman and Henry (1999), and for metabolic regulation of phospholipids in E. coli consult Shibuya (1992) and Cronan (2003).

Cronan, J.E. (2003) Bacterial membrane lipids: where do we stand? Annu. Rev. Microbiol. 57, 203–224. Cronan, J.E. and Waldrop, G.L. (2002) Multi-subunit acetylCoA carboxylases. Prog. Lipid Res. 41, 407–435. Fell, D.A. (1997) Understanding the Control of Metabolism. Portland Press, London. Gibbons, G.F. (2003) Regulation of fatty acid and cholesterol synthesis: co-operation or competition? Prog. Lipid Res. 42, 479–497. Girard, J. et al. (1997) Mechanisms by which carbohydrates regulate expression of genes for glycolytic and lipogenic enzymes. Annu. Rev. Nutr. 17, 325–352. Gurr, M.I. et al. (2002) Lipid Biochemistry, 5th ed., Blackwell Scientific, Oxford, U.K. Hardie, D.G. (1992) Regulation of fatty acid and cholesterol metabolism by the AMP-activated protein kinase. Biochim. Biophys. Acta. 1123, 231–238. Harwood, J.L. (1989) Lipid metabolism in plants. Crit. Rev. Plant Science. 8, 1–44. Harwood, J.L. (1994) Environmental factors affecting lipid metabolism. Prog. Lipid Res., 33, 193–202. Harwood, J.L. (1996) Recent advances in the biosynthesis of plant fatty acids. Biochim. Biophys. Acta. 1210, 369–372. Harwood, J.L. (1998) Involvement of chloroplast lipids in the reaction of plants submitted to stress. In Lipids in Photosynthesis: Structure, Function and Genetics, Eds. P-A Siegenthaler and N. Murata, Kluwer, Dordrecht, pp. 287–302. Harwood, J.L. (2005) Fatty acid biosynthesis. In Plant Lipids: Biology, Utilisation and Manipulation, Ed. D.J. Murphy, Blackwell Publishing, Oxford, U.K., pp. 27–66. Hawthorne, J.N. (1982). Inositol phospholipids. In Phospholipids, Eds. J.N. Hawthorne and G.B. Ansell, Elsevier Biomedical Press, Amsterdam, pp. 263–278. Hawthorne, J.N. and Ansell, G.B. Eds. (1982). Phospholipids, Elsevier, Amsterdam. Heath, R.J. et al. (2002) Fatty acid and phospholipid metabolism in prokaryotes. In Biochemistry of Lipids, Lipoproteins and Membranes, Eds. D.E. Vance and J.E. Vance, 4th ed., Elsevier, Amsterdam, pp. 55–92. Jump, D.B. et al. (1996) Dietary polyunsaturated fatty acid regulation of gene transcription. Prog. Lipid Res. 35, 227–241. Kent, C. (1995) Eukaryotic phospholipid biosynthesis. Annu. Review Biochem. 64, 315343. Kim, K-H. (1997) Regulation of mammalian acetyl-CoA carboxylase. Annu. Rev. Nutr. 17, 77–99. Lane, M.D. et al. (1979). Hormonal regulation of acetyl-CoA carboxylase activity in the liver cell. CRC Crit. Rev. Biochem. 2, 121–141. Mikami, K. and Murata, N. (2003) Membrane fluidity and the perception of environmental signals in cyanobacteria and plants. Prog. Lipid Res. 42, 527–543. Morris, S.M. et al. (1982). Molecular cloning of gene sequences for avian fatty acid synthase and evidence for nutritional regulation of fatty acid synthase mRNA concentration. J. Biol. Chem. 257, 3225–3229. Murata, N. and Wada, H. (1995) Acyl-lipid desaturases and their importance in the tolerance and acclimatization to cold of cyanobacteria. Biochem. J. 308, 1–8. Murphy, D.J. Ed. (2005) Plant Lipids: Biology, Utilisation and Manipulation. Blackwell Publishing, Oxford, U.K.

References Allred, J.B. and Reilly, K.E. (1997). Short-term regulation of acetyl-CoA carboxylase in tissues of higher animals. Prog. Lipid Res. 35, 371–385. Avery, S.V. et al. (1995). Temperature dependent changes in plasma membrane lipid order and phagocytotic activity of the amoeba Acanthamoeba castellanii are closely related. Biochem. J. 312, 811–816. Browse, J. and Somerville, C.R. (1991). Glycerolipid synthesisbiochemistry and regulation. Annu. Rev. Plant Physiol. 42, 467–506. Carman, G.M. and Henry, S.A. (1999). Phospholipid synthesis in the yeast Saccharomyces cerevisiae and interrelationships with other metabolic processes. Prog. Lipid Res. 38, 361–399. Clarke, S.D. (2000). Polyunsaturated fatty acid regulation of gene transcription: a mechanism to improve energy balance and insulin resistance. Brit. J. Nutr. 83 (Supplement 1) S59–S66. Coleman, R.A. and Lee, D.P. (2004) Enzymes of triacylglycerol synthesis and their regulation. Prog. Lipid Res. 43, 134–176. Cossins, A.J. Ed. (1994). The Temperature Adaptation of Biological Membranes, Portland Press, London.

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Ntambi, J.M. (1995) Regulation of stearoyl-CoA desaturase. Prog. Lipid Res. 34, 139–150. Ntambi, J.M. and Miyazaki, M. (2004) Regulation of stearoylCoA desaturases and their role in metabolism. Prog. Lipid Res. 43, 91–104. Numa, S. (1991). Two long-chain acyl-coenzyme A synthetases: their different roles in fatty acid metabolism and its regulation. Trends Biochem. Sci. 6, 113–115. Ohlrogge, J.B. and Jaworski, J.G. (1997) Regulation of fatty acid synthesis. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48, 109–136. Okuyama, H. et al. (1979). Regulation by temperature of the chain length of fatty acids in yeast. J. Biol. Chem. 254, 12281–12284. Quinn, P.J. and Harwood, J.L. Eds. (1990) Plant Lipid Biochemistry, Structure and Utilization, Portland, London. Ramli, U.S. et al. (2002) Control analysis of lipid biosynthesis in tissue cultures from oil crops shows that flux control is shared between fatty acid synthesis and lipid assembly. Biochem. J. 364, 393–401. Ramli, U.S. et al. (2005) Metabolic control analysis reveals an important role for diacylglycerol acyltransferase in olive but not in oil palm lipid accumulation. FEBS J. 272, 5764–5770. Rangan, V.S. and Smith, S. (2002) Fatty acid synthesis in eukaryotes. In Biochemistry of Lipids, Lipoproteins and Membranes, Eds. D.E. Vance and J.E. Vance, 4th ed., Elsevier, Amsterdam, pp. 151–179. Ratledge, C. and Wilkinson, S.G. Eds. (1988) Microbial Lipids, vol. 1, Academic Press, London. Ratledge, C. and Wilkinson, S.G. Eds. (1989) Microbial Lipids, vol. 2, Academic Press, London. Rawsthorne, S. (2002) Carbon flux and fatty acid synthesis in plants. Prog. Lipid Res. 41, 182–196.

Ridgway, N.D. et al. (1999) Integration of phospholipid and sterol metabolism in mammalian cells. Prog. Lipid Res. 38, 337–360. Rutter, A.J. et al. (2002) Oxygen induction of a novel fatty acid n-6 desaturase in the soil protozoan, Acanthamoeba castellanii. Biochem. J. 368, 57–67. Saggerson, E.D. (1980). Regulation of lipid metabolism in adipose and liver cells. Biochemistry of Cellular Regulation, vol. 2, CRC Press, Boca Raton, FL, pp. 207–256. Semenkovich, C.F. (1997) Regulation of fatty acid synthase. Prog. Lipid Res. 36, 43–53. Shibuya, I. (1992) Metabolic regulation and biological functions of phospholipids in E. coli. Prog. Lipid Res. 31, 245–300. Stubbs, C.D. and Smith, A.D. (1984). The modification of mammalian membrane polyunsaturated fatty acid composition in relation to membrane fluidity and function. Biochim. Biophys. Acta. 779, 89–137. Vance, D.E. and Vance, J.E. Eds. (2002) Biochemistry of Lipids, Lipoproteins and Membranes. 4th ed., Elsevier, Amsterdam. Vance, J.E. (1998) Eukaryotic lipid biosynthetic enzymes: the same but not the same. Trends Biochem. Sci. 23, 423–428. Voelker, T. and Kinney, A.J. (2001) Variations in the biosynthesis of seed storage lipids. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, 335–361. Volpe, J.J. and Vagelos, P.R. (1976). Mechanisms and regulation of biosynthesis of saturated fatty acids. Physiol. Rev. 56, 339–417. Wakil, S.J. et al. (1983). Fatty acid synthesis and its regulation. Annu. Rev. Biochem. 52, 537–539. Yeh, L.A. et al. (1981). Coenzyme A activation of acetyl-CoA carboxylase. J. Biol. Chem. 256, 2289–2296.

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11 MEDICAL AND AGRICULTURAL ASPECTS OF LIPIDS

J.L. Harwood, M. Evans, D.P. Ramji, D.J. Murphy and P.F. Dodds

11.1

Human dietary requirements

11.1.1

Introduction

for intakes of >3000 kcal/day, it should represent at least 30% of the calories. This gives minimum intakes of 56 to 140 g/day. Of course, there are circumstances when dietary fat should be limited (see below and Section 11.2 and Section 11.3). In a survey in the U.S., it was found that intake of fat increased with higher incomes. This correlation may reflect a greater consumption of food or, alternatively, the selection of more expensive foods, such as meat or dairy products instead of cereals (Rizek et al., 1974). Pearson and Dutson (1990) give information on meat lipids, including several chapters dealing with dietary lipids and health, while Gregory et al. (1990) provide data on fat intakes and blood lipid levels for U.K. subjects. Major food types that contribute to fat consumption in the U.K. are indicated in Table 11.1. For the U.S., the intake from fats given by Rizek et al. (1974) was 35% for salad and cooking oil, 23% for shortening, 15% for margarine, 8% for butter, and the remaining 19% from animal sources (meat, lard, etc.). It will be clearly seen from Table 11.1 that dairy products, meats, spreads, and cooking oils are the major contributors. Although not reflected in the table, there has been a significant shift in the sources of fats consumed in recent years. Thus, vegetable fats (margarine, cooking oils) are increasingly important when compared to animal fats (dairy products, lard). For example, the contribution of vegetable fats to the total in the U.S. increased from 17 to 38% in the period 1900 to 1950. This shift has raised the contribution of n-6 polyunsaturated fatty acids from 3 to 6% of the total dietary calories. In addition, the reduction in animal fats has lowered cholesterol consumption (cf. Section 11.3). Gold et al. (1992) have discussed the possible connections between cholesterol and coronary heart disease and Gurr (1992a) has

There are two aspects to lipid requirements in the human diet — qualitative and quantitative. First, certain lipids are needed for good health — essential fatty acids and fat-soluble vitamins are good examples. Secondly, it is usually considered that, in the normal diet, some 25 to 30% of the total calories are conveniently supplied as fat (Jones, 1974). Such lipids (in reasonable amounts) also usually make food more palatable. Some comprehensive sources of information on the role of fats in nutrition are Gurr (1992b, 1999), Vergroesen and Crawford (1989), and Akoh and Lai (2005). The human diet has always contained fat, but the amounts and types vary. Typical intakes in Europe and North America are between 80 and 150 g/day, which represents 30 to 40% of dietary calories. The nutrient fat per capita has been maintained, with a slight overall rise since 1900. However, the same foods are not always responsible for the fat consumed (Rizek et al., 1974). Salad and cooking oils have always been major contributors, followed by dairy products and shortening in the period 1910 to 1930, but by margarine, shortening, and meat in the period 1930 to 1960. Since that time, the increase in dietary fat has been due almost entirely to a rise in meat consumption. Moreover, current consumption of fat in different parts of the world varies markedly. In Asian countries, there is minimal dietary fat, whereas Inuits (North American Eskimos) consume 300 g/day, an amount that would nauseate a European. Because a diet lacking in fat tends to be bulky, some rough rules can apply. If total intake is > phospholipid = glycolipid > sterols. In processed food, there is often a small amount of monoacylglycerol and antioxidants, such as α-tocopherol, may be added. Fatsoluble vitamins may be present naturally or added. The process of digestion is beyond the scope of this review, but a clear, simple description of it will be found in Gurr and Harwood (1991). The use of medium-chain triacylglycerols in patients with digestive problems is discussed in Babayan (1974), Vergroesen (1975) and Thomas and Holub (1994). Triacylglycerols are usually 97 to 100% digested and the products of this process are absorbed into intestinal cells where resynthesis takes place. Unsaturated triacylglycerols are hydrolysed faster than saturated ones. Cholesterol is absorbed as the free sterol and then re-esterified, as necessary, for transport in the lymph. Bile may act as a cofactor for cholesterol esterase and improves cholesterol absorption, but plant sterols inhibit this absorption. Indeed, plant sterols (sitosterol, stigmasterol) and ergosterol are themselves rather poorly absorbed (Boyd, 1975). Thus, commercial margarines containing plant sterols and/or their derivatives are increasingly important as a means of lowering blood cholesterol (see Section 11.2 and Section 11.3). Part of their action is to lower cholesterol absorption (see Moreau et al. (2002) for review).

Although dietary fats increase cholesterol absorption by raising bile flow, saturated lipids are more effective in net terms because unsaturated fats also increase cholesterol excretion. The rate of cholesterol absorption is influenced by the age of the subject, their previous dietary history, genetic factors, the amount of cholesterol, the type of fat consumed simultaneously and the frequency of cholesterol intake. Similarly, overall fat absorption is affected by the type and amount of fat consumed, the age of the individual, the presence of emulsifying agents, whether the food has been heated above 250°C (when fat becomes less digestible), and the presence of calcium (Jones, 1974; Vergroesen, 1975). Malabsorption of fats can be measured with 131Ilabelled fats. Major problems of maldigestion or malabsorption, which can give rise to poor assimilation of dietary lipids, are listed in Table 11.2. Of the complaints listed, steatorrhea is the most common, but does not have pathological significance until the stool fat is greater than 10%. However, it causes distress and inconvenience and, when severe, wasting (loss of salts and liquid, and insufficient uptake of nutrients) and deficiencies (e.g., of fatsoluble vitamins, with vitamin D the first to show). For a fuller discussion of these medical aspects, see Petersdorf et al. (1983).

11.1.2

Food processing

The fatty acid of composition of foods can be affected by agricultural practice (see Section 11.8), but, usually, the biggest effects are those produced by industrial processing. Catalytic hydrogenation (see Section 4.2) is carried out to improve oxidative stability and physical properties. If oxidative stability is increased, then there is less chance of oxidation creating poor flavour and colour and giving rise to toxic compounds. Physical properties are mainly changed by “hardening” so that there are better textural

704

Medical and Agricultural Aspects of Lipids

TABLE 11.2 Maldigestion

Disorders leading to poor assimilation of dietary fats Pancreatic insufficiency

Hepatic insufficiency

Malabsorption

Gastric disturbances Ileum abnormality Intestinal defects

Steatorrhea

Pancreatitis Pancreatic tumour Malnutrition (e.g., Kwashiorkor) Cystic fibrosis side-effect Pancreatic lipase mutation Liver disease Bilary obstruction Abnormal acid secretion Poor reabsorption of bile Bacterial invasion (Tropical spruce, Whipple’s disease) Sensitisation (e.g., gluten sensitivity in coeliac disease) Impared chylomicron formation (e.g., Anderson’s disease) Bacterial invasion

11.1.3

properties (Gurr et al., 2002). The analysis of lipid oxidation is covered in Kamal-Eldin and Pokorny (2005). Chemically, there are three main results of hydrogenation: (1) the total number of double bonds are reduced, (2) some of the cis double bonds are isomerised to trans, and (3) the double bonds may be shifted from their original positions. Of these effects, it is the increase in trans-unsaturated fatty acids that has attracted the most attention. Although trans fatty acids are found naturally and are consumed at the rate of 5 to 7 g/day in the U.K. (British Nutrition Foundation, 1987), there is considerable evidence for adverse effects on health (Gurr, 1996). This has led to the increasing use of interesterification for triacylglycerol modification (Gunstone, 1998) or to the use of different dietary lipid sources (see Section 11.8). It has also led to a requirement for labelling food packaging with the trans fatty acid content and to attempts to minimise their intake (Hunter, 2004). Other processes that can cause lipid changes in food include heating and irradiation. Heating, where there is little contact with air (e.g., deep fat fryer), gives rise to a gradual accumulation of polymeric products (see, e.g., Varela and Ruiz-Rozo, 2000). Provided this oil is not reused excessively, these polymers do not cause problems (Gurr et al., 2002). Heating in the presence of oxygen and, particularly, if there are trace metal catalysts (e.g., iron, copper) can cause lipid peroxidation and production of reactive oxygen species. A major harmful side effect is the loss of antioxidant nutrients, such as vitamin E or carotenes (Gurr, 1988). Little peroxidised lipid is thought to be absorbed intact, but there is some evidence for liver toxicity and gut damage (Gurr, 1999). Any absorbed oxidised cholesterol may have health implications (Gurr et al., 2002). Lipid oxidation may be reduced by the presence of lipid-soluble antioxidants, either natural (e.g., carotenoids, vitamin E) or synthetic (butylated hydroxytoluene, BHT or butylated hydroxyanisole, BHA). Irradiation is used to kill pathogens in some types of food and may generate lipid radicals. Vitamins E and K are particularly susceptible to radiation damage, but not, apparently, carotenoids (Gurr et al., 2002).

Specific dietary lipids that may be harmful

The possible deleterious effects of high quantities of medium- or long-chain saturated fatty acids or cholesterol in the diet is touched on in several sections of Chapter 11; however, there are also specific lipids that are not known to have any nutritional benefit and that may be present in the diet. Trans unsaturated fatty acids were mentioned above (Section 11.1.2) and epidemiological studies of human population and controlled dietary experiments with human subjects have been reported. Some, but not all, of these studies show a correlation of the intake of certain types of trans fatty acids and increased risk of coronary heart disease (see Recommendations of the European Atherosclerosis Society, 1992 and Willett et al., 1993). In general, though, the evidence for harmful effects of trans unsaturated fatty acids (at least, at normal dietary concentrations) is not very persuasive (Gurr, 1999; Gurr et al., 2002). Despite this, in 2006 the U.S. Food and Drug Administration (FDA) made it mandatory to declare the amount of trans fat present in foods. These concerns have led to research on solutions or alternatives to trans fatty acids in foods (see Kodali and List, 2005). Cottonseed oil is the only important oil in the human diet that contains cyclopropene fatty acids. Because such acids (as sterculic acid) fed at 5% of dietary energy to rats caused death and at the 2% level caused disturbances of reproduction, there has been concern about the effect in humans. However, their concentration is low (0.6 to 1.2%) in cottonseed oil and reduced to 0.1 to 0.5% by processing. There has been no evidence that consumption of cottonseed oil in manufactured products has any harmful nutritional effects (Gurr et al., 2002). Very long-chain monounsaturated fatty acids (such as erucic acid, 22:1n-9), when fed to rats at 5% or more of their total energy requirements, caused a buildup of triacylglycerols in heart muscle. Other pathological changes were also noticeable (Gurr et al., 2002). Despite lack of evidence for harmful effects in man, breeding programmes were intitiated to replace older varieties of oilseed rape (up to 45% erucate in its oil) with zero erucate or canola

705

11.1

Human dietary requirements

11.1.4

varieties (see Section 11.8.2.3). The use of such varieties in most industrial countries is now mandatory; however, high erucate varieties are still used extensively in China. Furthermore, although fish oils are consumed for their desirable enrichment with the n-3 PUFAs (polyunsaturated fatty acids), eicosapentaenoic acid, and decosahexaenoic acid, they often contain high concentrations of 20:1n-9 and 22:1n-9. Moreover, certain fat spreads that incorporate hardened fish oils may also have significant concentrations of such acids. The long-term consequences (beneficial or otherwise) of consuming marine long-chain monoenes have been poorly researched (Gurr et al., 2002). Conjugated linolenic acids (CLAs) are a group of geometric and positional isomers of linolenic acid and can be formed by biohydrogenation and oxidation processes in nature. They are significant components of dairy products where they are produced in the rumen by microbiological action. The main form of CLA is usually cis-9, trans-1118:2, with trans-10, cis-12-18:2 the next most abundant. Adverse effects of CLAs have been demonstrated in animal studies, but it is not clear whether similar actions are applicable to humans. On the other hand, a surprising number of health benefits have been attributed to CLAs. This topic is well reviewed by Wahle et al. (2004). We should not close this section without referring to the huge amount of evidence in the literature that claims (or not) to correlate dietary saturated fatty acids or cholesterol with an increase in plasma cholesterol (particularly LDLcholesterol; see Section 11.2) and, then, a rise in cardiovascular disease. This work began with the classic study of Keys et al. (1957). Part of the difficulty in interpreting the various studies is that, often, an individual paper concludes that there are rather poor (and/or statistically not significant) correlations between the various components. This has led to the use of meta-analyses with all the inherent problems of selectivity and trying to compare studies where the measurements are not strictly comparable. Gurr (1992) discussed this subject very comprehensively and for an update of his views, see Gurr (1999). He “expresses scepticism for a major role for dietary lipids in the development of ischaemic heart diesease.” Such a view is, of course, contrary to dietary advice as practiced currently by physicians in North America and most of Europe, even though Gurr’s view is based on a careful scientific analysis of data produced over the past 25 years. Nevertheless, a recent prospective meta-analysis of data from 90,056 participants in 14 randomised trials of statins (which reduce LDL cholesterol) showed a lowering (20%) of major coronary events and stroke (Cholesterol Treatment Trialists (2005)). The reader is also referred to Tholstrup et al. (1994) for information on saturated fatty acids, to Riemersma (1994) for a review of antioxidants in coronary heart disease prevention and to Wald (1994) for lipoprotein: CHD relationships.

Specific fat requirements

There is no doubt that PUFAs are necessary for good health, and both α-linolenic (18:3n-3) and linolenic (18:2n-6) are regarded generally as essential. However, in a thought-provoking article, Cunnane (2003) has argued that PUFAs should be regarded as conditionally indispensable or dispensable, depending on the development stage. Thus, during pregnancy, lactation, infancy, and childhood, he regards linoleate, α-linolenate, arachidonate, and docosahexaenoiate (22:6n-3) as conditionally indispensable. During adulthood, α-linolenate is described as conditionally indispensable, but eicosapentaenoic (20:5n-3) and 22:6n-3 could be described thus, depending on the geographical area and lifestyle of individuals (Cunnane, 2003). Current recommendations for PUFAs in the diet are an adequate intake of linolenic acid at 2% energy, a healthy intake of α-linolenic acid at 0.7% energy and, for cardiovascular health, a minimum intake of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) combined of 500 mg/day. It was also recognised that there may be a healthy upper limit for linolenic acid (ISSFAL, 2004). Full references and justification will be found in this publication. The role of PUFAs in human nutrition and metabolism has been reviewed (Neuringer et al., 1988; Galli and Simpoulos, 1989; Heird and Lapillonne, 2005). Polyunsaturated fatty acids may also be involved in a number of other disease states. These include acrodermatis enteropathica, biliary tract disease, kinky hair disease, hepatoma, kwashiorkor, mycobacterial infections and multiple sclerosis (Soderhjelm et al.,1971). Essential fatty acid deficiency leads to problems in practically every tissue of the body (Holman, 1977). Classic symptoms include dermatitis, growth retardation, and infertility (Table 11.3). There are also biochemical changes, such as in mitochondrial efficiency, in various tissues (Holman, 1977; Gurr et al., 2002). Essential fatty acid deficiency is easily recognised because tissue fatty acids of the n-6 group are partly replaced by n-9 unsaturated fatty acids. In particular, arachidonic acid is reduced and eicosatrienoic acid (20:3n-9, the “Mead” acid) increased (Gurr et al., 2002) (Figure 11.1). Many of the effects of essential fatty acids are due to their conversion to eicosanoids. There are three types of enzyme-catalysed conversions — cyclooxygenase, lipooxygenase and oxidations involving cytochrome P450. The reactions give rise to prostaglandins, thromboxanes, prostacyclin, lipoxins, leukotrienes and other important biologically active molecules (Gurr et al., 2002). The n-3 and n-6 PUFAs compete with each other at a number of levels (Table 11.4). First, the main dietary PUFAs, linoleate and α-linolenate, are converted to the 20C eicosanoid precursors (arachidonate, eicosapentaenoate) using the same enzymes (see Figure 11.1). Secondly, the eicosanoids produced from arachidonate are proinflammatory, whereas those from eicosapentaenoate are mildly or 706

Medical and Agricultural Aspects of Lipids

TABLE 11.3

Major effects of n-6 essential fatty acid deficiency in rats

Skin

Dermatosis, water permeability increased Sebum secretion decreased Epithelial hyperplasia

Body weight

Decreased

Circulation

Heart enlargement Capillary resistance decreased

Kidney

Enlargement, intertubular haemorrhage

Lung

Cholesterol accumulation

Endocrine glands

Adrenals: weight decreased in females, increased in males Thyroid: weight increased

Reproduction

Females: irregular oestrus; impaired lactation, reproduction Males: degeneration of the seminiferous tubules

Metabolism

Changes in tissue fatty acid composition Reduced cholesterol concentration in plasma Increased cholesterol concentration in liver, adrenals and skin Mitochondrial swelling and uncoupled oxidative phosphorylation Increased triacylglycerol output by liver

18:1n-9 Diet or endogenous (oleic acid) synthesis

∆6D

Diet only

18:2n-6 (linoleic acid)

∆6D

Diet only

18:3n-3 (α-linolenic acid)

∆6D

E

18:2n-9

E

18:3n-6 (γ-linolenic acid)

E

18:4n-3

20:2n-9

20:3n-6

20:4n-3

∆5D

∆5D

∆5D

20:3n-9 (‘Mead acid’)

20:4n-6 (arachidonic acid)

20:5n-3 (eicosapentaenoic acid)

FIGURE 11.1 Competition between different fatty acids for production of 20 carbon PUFAs. Abbreviations: ∆5D, delta5-desaturase; E, elongase; ∆6D, delta6-desaturase.

noninflammatory. Thirdly, the n-3 and n-6 PUFAs have effects on the expression of many different proteins (Jump, 2002; Sampath and Ntambi, 2005) and their actions are often different (Harwood and Caterson, 2006). It must also be noted that it has been recently discovered that the n-3 PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), can be converted to new types of biologically active and mainly anti-inflammatory metabolites. Thus, EPA can yield resolvins of the E series, while DHA can produce resolvins of the D series and neuroprotectins (Serhan et al., 2004). While small amounts of essential fatty acids are needed to prevent deficiency syndromes, in practice, TABLE 11.4

EFA-deficiency is seldom seen unless very unusual dietary conditions prevail. However, there is special interest now in elucidating the health benefits of substantial intakes of n-3 and n-6 PUFAs such as recommended in a healthy diet (see above). In particular, n-3 PUFAs are needed for the development and proper function of brain and retina (Lauritzen et al., 2001) and there is increasing evidence that they are of benefit in reducing senile dementia (Morris, et al., 2003; Lim et al., 2005) and other cognitive problems (Harwood and Caterson, 2006). There is also a good deal of interest in the role of PUFAs in cancer (Guthrie and Carroll, 1999; Diggle, 2002) and in cardiovascular disease (NHFA, 1999;

How n-3 and n-6 PUFAs can compete with each other

Effect on enzymes during their metabolism Derived eicosanoids have opposing effects

The n-3 and n-6 PUFAs affect gene expression

∆5 and ∆6-desaturases and the 18C PUFA elongase show substrate competion. In general, metabolites from arachidonic acid (AA) are proinflammatory (e.g., PGE2, LTB4), whereas those from eicosapentadecanoic acid (EPA, 20:5 n-3) are non- or antiinflammatory (e.g., PGE3, LTB5) The effects of the two sets of PUFAs are often opposite. For example, COX-2 expression and activity are reduced by EPA, whereas AA has no effect or increases activity in a variety of tissues

707

11.1

Human dietary requirements

Wijendran and Hayes, 2004). Many of these important diseases have chronic inflammation as a major causative factor (Harwood and Caterson, 2006) and the relative roles of n-3 and n-6 PUFAs in this regard is fundamental (Calder, 1997; Calder et al., 1998). Some further sources of information on dietary PUFAs are British Nutrition Foundation (1992), Chow (1992), Forsyth (1998), Garrow et al. (2000), Innes (1991), Lermer and Mattes (1999) and Lands (2005).

11.1.5

important of which are vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). Again, as with vitamin A, the diet is often able to supply provitamins (e.g., ergosterol), which are converted to the active compounds. All of the provitamins require ultraviolet light for their conversion. Like vitamin A, vitamin D is toxic in high doses. Even amounts only five times normal intake can be toxic and cause more calcium to be absorbed than can be excreted, resulting in excessive deposition in and damage to the kidneys (Gurr et al., 2002). Vitamin E activity is possessed by eight tocopherols and tocotrienols (Gurr et al., 2002). The most potent and abundant form is α-tocopherol. Vitamin E is a natural antioxidant, although it may have other roles, such as structural functions in membranes (Wang and Quinn, 1999). The vitamin is needed for mitochondrial electron-transport function and it prevents oxidation of various compounds, including polyunsaturated fatty acids and vitamin A. The dependence of vitamin E requirement on the amount of dietary polyunsaturated fatty acids has been fully discussed by Jager (1975). Traditionally, vitamin E has been known as the “fertility” vitamin. However, deficiency leads to serious changes in skeletal muscle, the blood system and other tissues before reproduction is impaired. The effects on different animals are described by Jager (1975). Vitamin E is discussed by Scott (1978) and Packer and Fuchs (1993). Vitamin K is the generic name given to a group of compounds, having in common a naphthoquinone ring system (menadione) with different side chains (Gurr et al., 2002). Vitamin K1 is made by plants and found in their green tissues, while K2 is synthesised by microorganisms. The best-studied role for vitamin K is in relation to blood clotting where four of the procoagulant proteins of the clotting cascade depend on vitamin K. It also functions as an enzyme cofactor and plays a role in bone metabolism. It is rare to see vitamin K deficiency in adults except where fat absorption is impaired (see Section 11.1.1 and Sickinger, 1975). Vitamin K is discussed by Suttie (1978). Unlike some other fat-soluble vitamins, there is little evidence of harmful effects from high doses of vitamin K. For a general account of vitamin requirements and overdose symptoms, refer to Wilson (1994) and references therein.

Vitamins

Although somewhat outside the scope of this book, it would be wrong to describe the role of lipids in diets without mentioning fat-soluble vitamins. Vitamin A is alltrans-retinol, which is only found in animal fats. However, plant materials often contain abundant quantities of β-carotene (provitamin A). This can be easily converted to alltrans-retinol in the body. Ritinyl esters, mainly retinol palmitate, are stored principally in the liver from where the latter is released by hydrolysis and transported to target tissues bound to retinal-binding protein. Vitamin A has a number of important functions of which its role in vision is the best understood at the molecular level. An early sign of vitamin A deficiency is “night blindness.” When severe deficiency occurs, it can lead to blindness in young children. This tragic disease, xerophthalmia, is one of the four most common preventable diseases in the world (Gurr et al., 2002; see also Section 11.8.2.5 for efforts to prevent this with “golden” rice). The 9-cis-retinoic acid analog is involved importantly in differentiation. It can bind to two high-affinity receptor proteins, called RAR and RXR. Each of these may be present in one of three isoforms. Retinoic acid isomers are known to have extensive effects on gene expression and to interact (via RXR) with the vitamin D receptor or the PPAR (peroxisomal proliferator activated receptor) system. By these means, they have important effects on cellular differentiation (Gurr et al., 2002). Vitamin A is important in preventing degenerative changes in epithelial surfaces, such as keratinization of skin. It is needed for normal bone development and a deficiency in young animals can lead to secondary nervous problems due to compression of the brain and spinal cord (de Luca, 1978). Vitamin A is involved in the immune response, mainly through the T-helper cell. About 750 µg of vitamin A is needed for the average person daily but, like other fat-soluble vitamins, excessive intakes lead to accumulation, particularly in the liver. Chronic overconsumption may cause not only liver necrosis, but also permanent damage to bones, joints, muscles, and vision. Vitamin D is needed for calcium homeostatis and has various other functions for tissue development (Gurr et al., 2002). It is needed to prevent rickets and its deficiency is involved in other pathological states (de Luca, 1978). A number of different structures have activity, the two most

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Calder, P.C. (1997). n-3 Polyunsaturated fatty acids and immune cell function. Adv. Enzyme Regul. 37, 197–237. Calder, P.C. et al. (1998). Symposium of the Nutrition Society comprising twelve reviews on ‘Lipids and the Immune System.’ Proc. Nutr. Soc. 57, 487–585. Cholesterol Treatment Trialists (2005). Efficiency and safety of cholesterol-lowering treatment: prospective meta-analysis of data from participants in 14 randomised trials of statins. Lancet, 336, 1267–1278. Chow, C.K. Ed. (1992). Fatty Acids in Foods and Their Health Implications. Marcel Dekker, New York. Cunnane, S.C. (2003). Problems with essential fatty acids: time for a new paradigm? Prog. Lipid Res. 42, 544–568. De Luca, H.F. (1978a). Vitamin D. In Handbook of Lipid Research, vol. 1, The Fat-Soluble Vitamins, Ed. H.F. De Luca, Plenum, New York, pp. 69–132. De Luca, L.M. (1978b). Historical developments in vitamin A research. In Handbook of Lipid Research, vol. 2, The Fat Soluble Vitamins, Ed. H.F. De Luca, Plenum, New York, pp. 1–67. Diggle, C.P. (2002). In vitro studies on the relationship between polyunsaturated fatty acids and cancer: tumour or tissue specific effects? Prog. Lipid Res. 41, 240–253. Forsyth, J.S. (1998). Lipids and infant formulas. Nutr. Res. Rev. 11, 255–278. Galli, C. and Simpoulos, A.P. Eds. (1989). Dietary ω3 and ω6 fatty acids. In Biological Effects and Nutritional Essentiality. Plenum Press, New York. Garrow, J.S. et al. Eds. (2000). Human Nutrition and Dietetics, Churchill Livingston, Edinburgh, U.K. Gold, P. et al. (1992). Cholesterol and Coronary Heart Disease – The Great Debate, Panthenon, Carnforth and Park Ridge. Gregory, J. et al. (1990). The Dietary and Nutritional Survey of British Adults, HMSO, London. Gunstone, F.D. (1998). Movements towards tailor-made fats. Prog. Lipid Res. 37, 277–305. Gurr, M.I. (1988). Lipids: products of industrial hydrogenation, oxidation and heating. In Nutritional and Toxicological Aspects of Food Processing, Eds. R. Walker and E. Quattrucci, Taylor & Francis, London, pp. 133–155. Gurr, M.I. (1992a). Dietary lipids and coronary heart disease: old evidence, new perspective. Prog. Lipid Res. 31, 195–243. Gurr, M.I. (1992b). The Role of Fat in Food and Nutrition, Elsevier, London. Gurr, M.I. (1996). Dietary fatty acids with trans unsaturation. Nutr. Res. Rev. 9, 259–279. Gurr, M.I. (1999). Lipids in Health and Nutrition: A Reappraisal. The Oily Press, Bridgwater, U.K. Gurr, M.I. and Harwood, J.L. (1991). Lipid Biochemistry, 4th ed., Chapman & Hall, London. Gurr, M.I., Harwood, J.L. and Frayn, K.N. (2002). Lipid Biochemistry. 5th ed., Blackwell, Oxford, U.K. Guthrie, N. and Carroll, K.K. (1999). Specific versus non-specific effects of dietary fat on carcinogenesis. Prog. Lipid Res. 38, 261–271. Harwood, J.L. and Caterson, B. (2006). Dietary omega-3 polyunsaturated fatty acids and inflammation. Lipid Technol. 18, 7–10.

Heird, W.C. and Lapillonne, A. (2005). The role of essential fatty acids in development. Annu. Rev. Nutr. 25, 549–571. Holman, R.T. (1977). The deficiency of essential fatty acids. In Polyunsaturated Fatty Acids, Eds. W.H. Kunau and R.T. Holman, American Oil Chemists’ Society, Champaign, IL, pp. 163–182. Hunter, J.E. (2004). Alternatives to trans fatty acids in foods. INFORM, 15, 510–512. Hwang, D. (2000). Fatty acids and immune responses — a new perspective in searching for clues to mechanism. Annu. Rev. Nutr. 20, 431–456. Innes, S.M. (1991). Essential fatty acids in growth and development. Prog. Lipid Res. 30, 39–103. ISSFAL (2004). Recommendations for intake of polyunsaturated fatty acids in healthy adults. ISSFAL Newsl. 11, 12–25. Jager, F.C. (1975). Linoleic acid intake and vitamin E requirement. In The Role of Fats in Human Nutrition, Ed. A.J. Vergroesen, Academic Press, London, pp. 381–432. Jones, R.J. (1974). Role of dietary fat in health. J. Am. Oil Chem. Soc. 51, 251–254. Jump, D.B. (2002). Dietary polyunsaturated fatty acids and regulation of gene transcription. Curr. Opin. Lipidol. 13, 155–164. Kamal-Eldin, A. and Pokorny, J. (Eds.) (2005). Analysis of Lipid Oxidation. AOCS, Champaign, IL. Keys, A., Anderson, J.T. and Grande, F. (1957). Prediction of serum cholesterol responses in man to changes in fats in the diet. Lancet (ii), 959–966. Kodali, D.R. and List, G.R. Eds. (2005) Trans Fats Alternatives. AOCS, Champaign, IL. Lands, W.E.M. (2005). Fish, Omega-3 and Human Health, 2nd ed., AOCS, Champaign, IL. Lauritzen, L., Hansen, H.S., Jorgensen, M.H. and Michaelsen, K.F. (2001). The essentiality of long chain n-3 fatty acids in relation to development and function of the brain and retina. Prog. Lipid Res. 40, 1–94. Lermer, C.M. and Mattes, R.D. (1999). Perception of dietary fat: ingestive and metabolic implications. Prog. Lipid Res. 38, 117–128. Lim, G.P. et al. (2005). A diet enriched with the omega-3 fatty acid docosahexaenoic acid reduces amyloid burden in an aged Alzheimer mouse model. J. Neurosci. 25, 3032–3040. Morris, M.C. et al. (2003). Consumption of fish and n-3 fatty acids and risk of incident Alzheimer disease. Arch. Neurol. 60, 940–946. Neuringer, M. et al. (1988). The essentiality of n-3 fatty acids for the development and function of the retina and brain. Annu. Rev. Nutr. 8, 517–541. NHFA (National Heart Foundation of Australia) (1999). A review of the relationship between dietary fat and cardiovascular disease. Aust. J. Nut. Diet. 56, 55–522. Packer, L. and Fuchs, J. (1993). Vitamin E in Health and Disease, Marcel Dekker, New York. Pearson, A.M. and Dutson, T.R. (1990). Meat and health. Advances in Meat Research, vol. 6, Elsevier, New York. Petersdorf, R.G. et al. (Eds.) (1983). Principles of Internal Medicine, 10th ed., McGraw-Hill, New York. Recommendations of the European Atherosclerosis Society (1992) Prevention of coronary heart disease: scientific background and new clinical guidelines. Nutri. Metab. Cardiovasc. Dis. 2, 113–156.

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Lipids and cardiovascular disease

Organisation (WHO), an estimated 17 million people die from CVD each year, with heart attacks and stroke responsible for the majority of deaths. It has been predicted that the total number of deaths from CVD may rise to 20.5 million by 2020 and 24.2 million by 2030 as developing countries acquire westernised habits. CVD is clearly a major economic burden due to expenses incurred for hospital care and medication for patients and days lost from work because of illness, death and looking after relations with the disease. According to the American Heart Association and the National Heart, Lung and Blood Institute, the economic cost of CVD in 2005 alone is estimated to be 393.5 billion dollars.

Riemersma, R.A. (1994). Epidemiology and the role of antioxidants in preventing coronary heart disease: a brief overview. Proc. Nutr. Soc. 53, 59–65. Rizek, R.L., Friend, B. and Page, L. (1974). Fat in todays food supply — level of use and sources. J. Am. Oil Chem. Soc. 51, 244–250. Sampath, H. and Ntambi, J.M. (2005). Polyunsaturated fatty acid regulation of genes of lipid metabolism. Annu. Rev. Nutr., 25, 317–340. Scott, M.L. (1978) Vitamin E2. In Handbook of Lipid Research, The Fat-Soluble Vitamins, Ed. H.F. De Luca, Plenum, New York, pp. 133–210. Serhan, C.N. et al. (2004). Resolvins, decosatrienes and neuroprotectins, novel omega-3-derived mediators and their aspirin-triggered epimers. Lipids, 39, 1125–1132. Sickinger, K. (1975). Clinical aspects and therapy of fat malassimilation with particular reference to the use of medium-chain triglycerides. In The Role of Fats in Human Nutrition, Ed. A.J. Vergroesen, Academic Press, London, pp. 116–209. Söderhjelm, L. et al. (1971). The role of polyunsaturated acids in human nutrition and metabolism. Prog. Chem. Fats Lipids, 9, 555–585. Suttie, J.W. (1978). Current advances in vitamin K research. In The Fat-Soluble Vitamins, Ed. J.W. Suttie, William Heinemann, Ltd., London, pp. 211–220. Varela, G. and Ruiz-Rosa, B. (2000). Some nutritional aspects of olive oil. In Handbook of Olive Oil, Eds. J.L. Harwood and R. Aparcio, Aspen Publishers, Gaithersburg, MD, pp. 565–582. Vergroesen, A.J. (Ed.) (1975). The Role of Fats in Human Nutrition, Academic Press, London. Vergroesen, A.J. and Crawford, M. (Eds.) (1989). The Role of Fats in Human Nutrition, Academic Press, London. Wahle, K.W.J. et al. (2004). Conjugated linolenic acids: are they beneficial or detrimental to health? Prog. Lipid Res. 43, 553–587. Wald, N.J. et al. (1994). Apolipoproteins and ischemic-heartdisease — implications for screening. Lancet, 343, 75–79. Wang, X. and Quinn, P.J. (1999). Vitamin E and its function in membranes. Prog. Lipid Res. 38, 225–248. Wijenderan, V. and Hayes, K.C. (2004). Dietary n-6 and n-3 fatty acid balance and cardiovascular health. Annu. Rev. Nutri. 24, 597–615. Willett, W.C. et al. (1993). Intake of trans fatty acids and risk of coronary heart disease among women. Lancet, 341, 581–585. Wilson, J.D. (1994). Vitamin deficiency and excess. In Principles of Internal Medicine, Eds. K.J. Isselbacher, E. Braunwald, J.D. Wilson, J.B. Martin, A.S. Fanci and D.L. Kasper, 13th ed., McGraw-Hill, New York, pp. 472–480.

11.2

Lipids and cardiovascular disease

11.2.1

Cardiovascular disease

11.2.2

Atherosclerosis is the underlying cause of cardiovascular disease

Atherosclerosis, which comes from the Greek words “athero” (meaning gruel or paste) and “sclerosis” (hardness), is the principal cause of CVD. A normal artery consists of three layers, the intima lining the lumen, the middle layer called the media and an outermost layer termed the adventitia. The intima consists of a single layer of endothelial cells that regulate vascular tone. The media consists predominantly of smooth muscle cells, whereas the adventia contains smooth muscle cells, fibroblasts, and a looser connective tissue. Atherosclerosis, which develops during the life span of an individual, causes a buildup of plaques, consisting of cholesterol, other lipids, and debris from cellular death, in the inner lining of the arteries. Although continued growth of such plaques may impede blood flow, the major problem arises when it becomes fragile and ruptures. This leads to a clot that can block blood flow or can break off and get trapped in another part of the body. Heart attack (also called myocardial infarction, coronary occlusion, or coronary thrombosis) occurs when the clot blocks a coronary artery and, thus, deprives the heart of oxygen and nutrients. On the other hand, blockage of a blood vessel to the brain leads to stroke. Atherosclerosis is initiated by damage to the endothelial cells by a number of risk factors (see Section 11.2.3 below), which then triggers a series of changes in the arterial wall (see Figure 11.2 for a summary; and Ross, 1999; Lusis, 2000; Glass and Witztum, 2001; and Lusis et al., 2004 for a detailed description of these changes). First of all, the permeability of the vascular wall is increased because of the synthesis of cell surface adhesion molecules, such as intercellular adhesion molecule-1, E-selectin, P-selectin and vascular cell-adhesion molecule-1. In addition, the damaged endothelial cells secrete chemo-attracting cytokines (chemokines), such as monocyte-chemoattractant protein-1, which in turn attracts monocytes and T-lymphocytes from circulation and stimulates their migration into the intima of the arterial wall. These monocytes then differentiate into macrophages, a process that is associated with the

Cardiovascular disease (CVD) is a major cause of morbidity and mortality in the western world, with the number of individuals with the disease in developing countries increasing all the time. The clinical manifestations of CVD include heart attacks, stroke, and gangrene of the extremities. According to the statistics from the World Health 710

Medical and Agricultural Aspects of Lipids

Normal artery

Fatty streak

Fibrous plaque

Accumulation of LDL and its oxidation to oxLDL Initial damage to ECs by oxLDL and other atherogenic factors Expression of adhesion proteins and cytokines by ECs Infiltration of monocytes and T-lymphocytes Macrophage differentiation and expression of SRs Uptake of oxLDL by macrophages to form foam cells A chronic inflammatory response due to cytokines produced by macrophages and T-lymphocytes Migration of smooth muscle cells from the media to the intima Proliferation of smooth muscle cells Formation of smooth muscle cell-derived foam cells Secretion of extracellular matrix proteins Apoptosis and necrosis of macrophage and smooth muscle cell-derived foam cells Accumulation of extracellular cholesterol Secretion of matrix metalloproteinases by macrophages leading to instability of the fibrous plaque

Plaque rupture

FIGURE 11.2 A schematic representation of the major steps during the pathogenesis of atherosclerosis (see text for more details). Abbreviations: ECs, endothelial cells; LDL, low density lipoproteins; oxLDL, oxidized low density lipoproteins; SRs, scavenger receptors.

expression of so-called scavenger receptors (SRs), such as SR-A and CD36. Although the normal function of these SRs is believed to be in the uptake of pathogens and apoptotic cells, they also take up modified lipoproteins (see Section 11.2.7 below). This latter property causes macrophages to transform into lipid-loaded foam cells. Such accumulation of foam cells in the vascular wall is a critical early step in the pathogenesis of atherosclerosis and is responsible for the formation of a so-called fatty streak. The macrophages also secrete cytokines, which lead to a local inflammatory response that recruits further monocytes and T-lymphocytes and, thereby, amplify foam cell formation. The importance of macrophages in atherogenesis can be gauged by the finding that diet-induced atherosclerosis in a murine model of the disease is significantly reduced in mice bred to have severely reduced monocyte levels (Smith et al., 1995; see Osterud and Bjorklid, 2003 for an in-depth review on the role of monocytes/macrophages in atherogenesis). This fatty streak then progresses to form a fibrous plaque via a number of cellular and biochemical changes. First, an intermediate fibro-fatty lesion is formed, which consists of foam cells, smooth muscle cells (SMC), T-lymphocytes and a relatively poorly developed extracellular matrix. The transition from such fibro-fatty lesions to more complex lesions is associated with the migration of SMC from the media to the intima, where they proliferate and also take up lipoproteins, thereby contributing to further foam cell formation. In addition, such SMC synthesise extracellular matrix proteins leading to the development of a fibrous cap. More advanced lesions contain a dense fibrous cap, which protrudes into the lumen of the artery and covers a core of macrophages,

SMC, T-lymphocytes, extracellular matrix and debris from dying foam cells. Instability of such advanced lesions may lead to plaque rupture and thrombus formation, which may ultimately result in the occlusion of the artery. An inflammatory response is a major contributor to plaque instability and the development of acute CVD has been found to be associated with elevated circulating levels of markers of inflammation, such as C-reactive protein.

11.2.3

Risk factors for CVD

More than 300 risk factors for CVD have been identified from laboratory- and clinical-based research. The major risk factors that have a high prevalence in many different populations are age, gender, hypertension, smoking, physical inactivity, obesity, diabetes, socioeconomic status, Chlamydia pneumoniae infection, hyperhomocysteinemia and high levels of circulating lipids (see Stoker and Keaney, 2004 for a more detailed discussion of the various risk factors). A number of such risk factors often co-exist in atherosclerotic patients where they may act in a synergistic manner. For example, hypercholesterolemia, obesity, hypertension and physical inactivity are often associated in a number of male patients. The risk for the development of CVD increases with age, with the average risk in males aged 65 being about sevenfold greater than those who are 35. In addition, males have a much higher risk for CVD compared to age-matched women (Barrett-Conner and Bush, 1991). It has been suggested that estrogen provides protection in premenopausal women because such gender-specific effects are not seen in postmenopausal women. However, this speculation has not been substantiated further as estrogen treatment does not decrease 711

11.2

Lipids and cardiovascular disease

the incidence of the disease in postmenopausal women. Other factors are thus likely to contribute for such gender differences (see Mendelsohn and Karas, 2005); for example, women generally have higher levels of the protective highdensity lipoprotein (HDL) (see Section 11.2.4) than agematched males. Hypertension, obesity and diabetes often coexist; for example, hypertension and diabetes are relatively common in obese individuals. According to figures from the WHO, 6.3% of individuals aged 20 or above in developed countries and 4.1% in developing countries suffer from diabetes. This figure is expected to increase in the future because sedentary lifestyle, intake of “convenience food” containing high levels of fat and salt, and obesity are increasing at an alarming rate. More than 60% of adults in the U.S. are overweight or obese and even in China, a population that has been previously classed as slim and physically active, there are 70 million overweight individuals. A risk between smoking and CVD was first suggested in 1940 and this has been substantiated by numerous studies. Smoking promotes CVD via several mechanisms, including damage to the endothelial lining of the arterial wall, increase in the circulating levels of the proatherogenic lowdensity lipoproteins (LDL), decrease in the concentration of HDL (see Section 11.2.4) and stimulation of blood clotting. Cigarette smoking also raises the concentration of plasma carbon monoxide, which has a number of additional detrimental effects, such as promotion of both endothelial hypoxia and thrombus formation. The nicotine in cigarettes also accelerates heart rate and raises blood pressure. From the different risk factors identified, hyperlipidemia, particularly high levels of serum cholesterol, has been the subject of intense research for a number of decades and caused the most debate. A number of epidemiological studies have shown a direct correlation between high plasma cholesterol levels and CVD (Martin et al., 1986; Anderson et al., 1987; Gurr, 1992). Additional support for a proatherogenic role of plasma cholesterol is provided by studies on individuals with the autosomal dominant disorder familial hypercholesterolemia, which is characterised by a two- to five-fold increase in plasma LDL cholesterol (see Section 11.2.6). About 80% of patients that are heterozygous for this disorder experience CVD by the age of 60 and this is reduced to 15 years in TABLE 11.5

homozygous individuals. Furthermore, randomised clinical trials of lipid lowering therapy have shown a greater than 30% reduction in CVD (Maron et al. 2000; Grundy et al. 2004). From the different classes of plasma lipids, high levels of fasting cholesterol and LDL and low levels of HDL have been identified as the most proatherogenic. High concentration of fasting serum triacylglycerols also represents an independent risk factor. The different classes of serum lipids are considered below in more detail.

11.2.4

Serum lipids: classification and metabolic roles

All lipids are carried in the plasma as complexes with proteins. Although long-chain fatty acids circulate bound to plasma albumin, the other lipids are carried by a number of lipoprotein particles, which are responsible for the transport of endogenously produced and dietary lipids. Such lipoprotein particles are spherical in shape, with diameter between 5 µm and 1200 µm, and comprised of a hydrophobic core, containing triacyglycerols and cholesterol esters, surrounded by a hydrophilic shell of phospholipids, unesterified (free) cholesterol and apolipoproteins. There are five major classes of lipoproteins: chylomicrons, very low-density (also called pre-β) lipoproteins (VLDL), intermediate density lipoproteins (IDL), low-density (β-) lipoproteins, and high-density (α-) lipoproteins. The classification is based on the hydrated density of the lipoproteins. The different lipoproteins differ in size, electrophoretic mobility, and composition of lipids and apolipoproteins (see Table 11.5). Although each lipoprotein is synthesised with a characteristic set of apolipoproteins, considerable exchange of these apolipoproteins with other lipoprotein particles occur during their metabolism (see Table 11.5). Chylomicrons are synthesised by the mucosal cells of the small intestine and act as a vehicle for the transport of dietary triacylglycerols and cholesterol. VLDL, which is synthesised and secreted by the liver, is also rich in triacylglycerols, but these are derived from endogenous sources. IDL is formed as triacylglycerols are removed from VLDL (see Section 11.2.5). LDL is the main carrier of cholesterol in the plasma and is derived primarily from the catabolism of VLDL. HDL, on the other hand, is formed mainly in the liver as a lipid-poor particle that becomes modified

The composition of plasma lipoproteins Composition (% total)

Lipoprotein

Density (g/ml)

Chylomicrons VLDL IDL LDL HDL

< 0.95 0.95–1.006 1.006–1.019 1.019–1.036 1.063–1.210

a

Diameter (µm)

TAG

Cholesterol

Phospholipid

75–1200 30–80 25–35 18–25 5–12

90 60 27 10 5

5 12 34 50 20

3 18 27 15 25

Protein 2 10 12 25 50

Apolipoproteinsa B48 (A, C, E) B100 (A, C, E) B100, E B100 AI, AII (C, E)

The main apolipoproteins in each of the lipoproteins are shown first, with those that are exchanged with other lipoprotein particles indicated in parenthesis (see text for more details). TAG, triacylglycerol.

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during the catabolism of VLDL. The major function of HDL is to aid the transport of cholesterol from peripheral tissues to the liver where it can be excreted as bile acids (a process called reverse cholesterol transport). In addition to the lipoprotein classes detailed above, Lipoprotein (a) [Lp (a)] is also found in the plasma where its concentration may range from 0.2 mg/dl to 120 mg/dl. LP (a), which is synthesised in the liver, has generated substantial interest because of its presence in atherosclerotic lesions and its identification as a major risk factor for CVD (see Berglund and Ramakrishnan, 2004; Boffa et al., 2004). LP (a) contains an LDL particle with apoB100 to which a genetically polymorphic form of apo (a) is attached by a disulfide bond. The precise mechanism(s) for a proatherogenic role of LP (a) remain to be determined. However, LP (a) is known to be taken up by macrophages and, thus, contributes to foam cell formation. Apo (a) also shows striking homology with the fibrinolytic proenzyme plasminogen. Although LP (a) does not possess the protease activity associated with plasminogen, it may still interfere with its action and, thereby, impair fibrinolysis (clot resolution).

11.2.5

The apolipoproteins play a crucial role in the regulation of lipoprotein metabolism and the stabilisation of the lipoprotein particles. Additionally, they modulate the activity of several key enzymes in lipoprotein metabolism and some of them also bind to specific cell surface receptors and, thereby, regulate the metabolic fates of lipoproteins (Table 11.6). There are at least four receptors for lipoproteins and remnant particles: the VLDL receptor, the remnant receptor(s), the LDL receptor (LDL-R) (also called the apo B/E receptor), and the LDL receptorrelated protein (LRP). Chylomicrons synthesised by the intestine are secreted into the lymphatic system and reach the plasma through the thoracic duct. Chylomicrons contain apoB48, which is synthesised in the intestine, along with apolipoproteins-A, -C, and -E. ApoB48 is found exclusively in chylomicrons and is derived from the apoB100 gene by RNA editing in the intestinal epithelium. ApoB48 lacks the LDL-R-binding domain present in apoB100. The triacylglycerol core of chylomicrons is hydrolysed by LPL present on the surface of capillary endothelial cells. This is accompanied by transfer of phospholipids, via phospholipid transfer protein (PLTP), and apolipoproteins-A and -C to HDL. The loss of apoCII, an activator of LPL, prevents further hydrolyses of the smaller, chylomicron remnant particles. These chylomicron remnants, which contain primarily cholesterol, apoE and apoB48, are then taken up by the liver principally via a chylomicron remnant receptor. The VLDL synthesised and secreted by the liver contains apoB100 and acquires cholesteryl esters and apolipoproteins-A, -C, and -E from circulating HDLs. Similar to chylomicrons, the triacylglycerol component of VLDL is subjected to LPL-mediated hydrolysis. The action of LPL, along with the transfer of phospholipids and apolipoproteins-A and -C to circulating HDLs, converts VLDLs to IDLs (also called VLDL remnants). Approximately half of these VLDL remnants are removed from the circulation by high affinity binding to the LDLR of liver cells due to the presence of both apoB100 and apoE. The remaining remnants transform into cholesterol-rich LDL particles by losing more triacylglycerols, via hydrolysis by HL, and shedding all of their lipoproteins except for apoB100.

Metabolic fates of circulating lipoproteins

At least four enzymes play crucial roles in the metabolism of lipoproteins: lipoprotein lipase (LPL), hepatic lipase (HL), lecithin:cholesterol acyltransferase (LCAT), and cholesteryl ester transfer protein (CETP). LPL and HL are involved in the hydrolysis of triacylglycerol-rich lipoproteins. LPL, which requires apoCII as a specific co-activator, interacts with heparin sulfate proteoglycans (HSPG) on the surface of vascular endothelial cells, whereas HL is associated with the plasma membrane in the liver. LPL hydrolyses triacylglycerols in chylomicrons and VLDL to produce nonesterified fatty acids and 2-monoacylglycerol, which are either re-esterified for storage in the adipose tissue or used as a source of energy in the muscle. On the other hand, HL acts on particles that have already been partially digested by LPL and facilitates the conversion of IDL to HDL. LCAT, which is activated by apoAI, esterifies cholesterol acquired by HDL, whereas CETP catalyses the transfer of cholesterol esters from HDL to triacylglycerol-rich lipoproteins. TABLE 11.6

Properties of major apoliproteins

Apoliprotein

Major lipoprotein

Effect on enzyme activity

AI AII B48 B100 CI CII CIII E

HDL HDL Chylomicrons VLDL, IDL and LDL Chylomicrons, VLDL and HDL Chylomicrons, VLDL and HDL Chylomicrons, VLDL and HDL Remnants

Activates LCAT Activates HL LCAT cofactor – – LCAT? Activates LPL Inhibits LPL –

Putative receptor SR-BI ? LRP LDL-R – – – LDL-R

Note: HL, hepatic lipase; LCAT, lechithin:cholesterol acyltransferase; LDL-R, LDL receptor; LPL, lipoprotein lipase; LRP, LDL receptor-related protein; SR-BI, scavenger receptor-BI.

713

11.2

Lipids and cardiovascular disease

LDL particles are taken up by the liver or peripheral tissues via LDL-R-mediated endocytosis. The endocytosed membrane vesicles fuse with lysosomes where the apolipoproteins are digested and the cholesterol esters are hydrolysed to yield free cholesterol. The excess intracellular cholesterol is then re-esterified for storage by the action of the enzyme acyl-CoA-cholesterol acyltransferase (ACAT), whose activity is enhanced by free cholesterol. The cellular uptake of LDL via LDL-R is under negative feedback inhibition by the concentration of intracellular cholesterol. This regulation occurs through a family of membrane-bound transcription factors called sterol regulatory element-binding proteins (SREBPs). A low level of intracellular cholesterol leads to the activation of SREBPs, which then stimulate the transcription of the LDL-R gene along with a number of other genes implicated in cholesterol biosynthesis (see Brown and Goldstein, 1999; Eberle et al., 2004). SREBPs are produced as an integral membrane-bound precursor form in the endoplasmic reticulum and are activated by a SREBP cleavage-activating protein (SCAP), which contains a sterol-sensing domain. When the intracellular concentration of cholesterol is low, SCAP and the SREBP precursors move from the endoplasmic reticulum to serine proteases located in the Golgi apparatus. The proteases cleave the SREBP precursors and produce the active transcription factor, which can translocate to the nucleus and bind to its recognition sequences in the regulatory regions of target genes. As discussed in detail in Section 11.2.7 below, lipoproteins, such as LDL, are subject to several types of modifications, particularly oxidation. Such modified lipoproteins are readily taken up by scavenger receptors (SRs). Macrophages express at least six different forms of SRs for modified LDL. Unlike the LDL-R, such SRs are not subject to feedback inhibition by intracellular cholesterol concentration and can, therefore, take up cholesterol in an uncontrolled manner, thereby contributing to foam cell formation (see Greaves and Gordon, 2005, for details on SRs).

SR-BI

HDL2

LCAT

HL LCAT

Bile

HDL plays a crucial role in reverse cholesterol transport (Figure 11.3). Nascent HDL is formed in the liver and the intestine as a disk-like particle containing apoAI and some phospholipids. This HDL particle acts as a potent acceptor of cholesterol derived from peripheral cells. The ATPbinding cassette transporter (ABC)-A1, along with a number of other such transporters, play a key role in the efflux of cholesterol from cells using ATP as a source of energy. The importance of ABCA1 is shown by patients with Tangier disease, which lack this transporter and suffer premature CVD because of a massive accumulation of cholesteryl esters in a number of tissues. The cholesterol taken up by HDL becomes esterified to cholesteryl esters through the action of LCAT. The cholesteryl esters then move deeper into the HDL particle, which now assumes a small, spherical shape and is called HDL3. HDL transfers part of its cholesteryl esters to triacylglycerol-rich lipoproteins and acquires phospholipids and apolipoproteins from them to form larger HDL2 particles. The return of HDL cholesterol to the liver occurs via three pathways (see Figure 11.3). First, the entire HDL particle is taken up by the liver through the LDL-R. Secondly, the cholesterol ester in HDL is transferred to other lipoproteins, via the action of CETP, and these lipoproteins are then taken up by the liver through the LDL-R. The importance of this pathway is supported by several lines of evidence. For example, premature CVD is common in individuals with CETP-deficiency despite the presence of high HDL levels (Bruce et al., 1998). In addition, overexpression of CETP in mice, which normally lack this enzyme, is anti-atherogenic (Bruce et al., 1998). Thirdly, selective delivery of cholesteryl esters in HDL to the liver can occur via scavenger receptor class BI (SR-BI) (Acton et al., 1996). SR-BI binds HDL avidly and mediates the selective delivery of cholesteryl esters to the cell membrane in the liver without internalisation and degradation of the HDL particle. This direct uptake of cholesteryl ester from HDL is facilitated by the binding of HDL to cell surface HSPG because of the presence of apoE in the lipoprotein

HDL3 CETP

Preβ-HDL

ABCA1

Cholesteryl esters

VLDL/IDL HL LDL-R

LDL

Liver

LDL-R Peripheral cell

FIGURE 11.3 A schematic representation of reverse cholesterol transport (see text for more details). Abbreviations: ABCA1, ATPbinding cassette transporter 1; CETP, cholesteryl ester transfer protein; HDL, high density lipoproteins; HL, hepatic lipase; IDL, intermediate density lipoproteins; LCAT, lecithin-cholesterol acyltransferase; LDL-R, low density lipoprotein receptor; SR-B1, scavenger receptor B1; VLDL, very low density lipoproteins.

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(Arai et al., 1999). The importance of SR-BI can be gauged by the finding that its inactivation in mice is associated with a dramatic increase in serum cholesterol levels and the size of HDL particles without any significant effect on the HDL protein concentration (Rigotti et al., 1997). In addition, liver-specific expression of SR-BI in mice results in reduced HDL levels and an increase in reverse cholesterol transport (Wang et al., 1998). Cholesterol delivered to the liver by HDL is excreted as a free sterol or as bile acids. The HDL particle shrinks as a result of the transfer of cholesteryl ester to the liver and some of the particles become nascent HDL for another round of transport of cholesterol.

11.2.6

There are a number of rare inborn errors of lipid metabolism. Those that comprise the familial hyperlipoproteinemias are probably the most widely studied (Table 11.7; Levy and Fredrickson, 1968; Fredrickson and Breslow, 1973; Genest, 2003). These are divided into five main types according to the major changes in plasma lipoprotein profiles. Type I hyperlipoproteinemia, also known as familial hyperchylomicronemia, familial exogenous hypertriglyceridemia, or familial fat-induced lipemia, is a rare recessive condition due to a deficiency of the enzyme LPL or, more rarely, by the absence of its activator apoCII. Chylomicron and VLDL metabolism, therefore, is defective and their accumulation results in very high plasma concentration of triacylglycerols. Clinical symptoms include eruptive xanthomas, hepatosplenomegaly, lipemia retinalis, abdominal pain and pancreatitis. Indeed, pancreatitis rather than atherosclerosis is the major reason for early deaths in these patients. Type II hyperlipoproteinemia includes several genetic conditions, including familial hypercholesterolemia, familial combined hyperlipidemia, familial defective apolipoprotein B and polygenic hypercholesterolemia. These disorders are associated with a very high concentration of plasma LDL and, in certain cases, VLDL. Some classify type II hyperlipoproteinemias into type IIA and type IIB depending on whether hypertriglyceridemia is absent or present, respectively (see Table 11.7). The other major symptoms include xanthomas, particularly on the hand and knee tendons. Familial hypercholesterolemia is caused by the absence of fully functional LDL-R, resulting in delayed clearance of LDL. Homozygous individuals have extremely high levels of plasma LDL-cholesterol irrespective of diet or life style and exhibit severe CVD, usually resulting in death at an early age. Heterozygous individuals are a more diverse group with a 50% probability of death from CVD before the age of 50. A form of familial hypercholesterolemia with

Factors affecting the composition and metabolism of circulating lipids

When factors that affect the composition of circulating lipids in humans are considered, the nature of the “normal” subject must be defined as genetic prepositions, age, sex, diet, exercise, and overt or hidden disease may all contribute to discernible differences. Where there is no evidence of malnutrition, the dietary components that most affect the lipid profiles are fats and carbohydrates. In relation to fats, the nature of the fatty acid components has attracted the most attention. When conventional diets are changed by an increased intake of polyunsaturated fats, significant reductions in total plasma cholesterol concen-trations are seen, with a major reduction in the LDL fraction. The beneficial effects of polyunsaturated fatty acids are seen at virtually all stages of the disease, including the control of overall lipid metabolism and transport, regulation of nuclear receptors (see Section 11.2.8), modulation of adhesion protein and cytokine expression by endothelial cells, and control of platelet function (De Caterina et al., 2004; Mori and Beilin, 2004; Vanden Heuvel, 2004; Mori, 2004; Jump, 2004). TABLE 11.7

Hyperlipoproteinemias

Typea

Other designations

Elevated lipoprotein class

I

Familial Hyperchylomicronemia Familial Exogenous Hypertriglyceridemia Familial Fat-Induced Lipemia Familial Hypercholesterolemia Familial Defective Apolipoprotein B Polygenic Hypercholesterolemia Familial Combined Hyperlipidemia Broad Beta Disease Familial Dysbetalipoproteinemia Endogenous Hypertriglyceridemia Hyperprebetalipoproteinemia Mixed Hyperglyceridemia Mixed Hyperlipidemia Hyperprebetalipoproteinemia with Chylomicronemia

II-a

II-b III IV V

a b

[Cholesterol]b

[Triacylglycerols]b

Chylomicrons

↑

↑

LDL

↑

−

LDL, VLDL β-VLDL

↑ ↑

↑ ↑

VLDL

−

↑

VLDL Chylomicrons

↑

↑

The designation is as proposed by Fredrickson and adopted by the WHO. ↑, increase; ↓, decrease; −, no change.

715

11.2

Lipids and cardiovascular disease

a similar clinical phenotype, called familial defective apolipoprotein B, arises because of mutations in apoB100 in regions that represent binding sites for the LDL-R. Familial combined hyperlipidemia is characterised by excess circulating levels of LDL, VLDL, or both. Excessive hepatic production of apoB100, a major protein constituent of VLDL and LDL, is common. It is transmitted in a dominant manner, but does not often manifest until after adolescence. Defects in a number of genes implicated in lipoprotein metabolism and transport is the most frequent cause for this disorder. Finally, polygenic hypercholesterolemia is a heterogenous group of disorders that accounts for the largest number of patients in type II hyperlipoproteinemias. Most patients have elevated levels of LDL due to its impaired clearance. Type III hyperlipoproteinemia, also known as broad beta disease or familial dysbetalipoproteinemia, is associated with elevated levels of triacyglycerols and cholesterol because of abnormalities in VLDL (i.e., presence of an abnormal form of β-migrating VLDL), xanthomas, and premature CVD (Mahley et al., 1999). The major defect is the presence of an abnormal apoE, which does not bind efficiently to hepatic receptors that require this apolipoprotein for interaction with lipoproteins. The patients have the so-called apoE-2/2 phenotype or genotype. There are three common alleles for apoE, apoE-2, -E3, and E-4, with the apoE-2 allele associated with a marked decrease in binding to the LDL-R and premature CVD (Greenow et al., 2005). Type IV hyperlipoproteinemia, also called endogenous hypertriglyceridemia or hyperprebetalipoproteinemia, is a common disorder characterised by elevated levels of circulating VLDL and increased predisposition to CVD. This disorder is frequently associated with insulin resistance and obesity and is particularly common in American middle-aged men. Type V hyperlipoproteinemia, also called mixed hypertriglyceridemia, mixed hyperlipidemia, or hyperprebetalipoproteinemia with chylomicronemia, is a relatively uncommon disorder associated with defective clearance of exogenous and endogenous triacylglycerols. Symptoms include eruptive xanthomas, lipemia retinalis, hepatosplenomegaly, and abdominal pain. Similar to type I hyperlipoproteinemia, acute pancreatitis rather than CVD is the major reason for early deaths. In addition to these relatively common forms of hyperlipoproteinemias, a number of other rare genetic disorders exist that are characterised by marked hypercholesterolemia and/or abnormal lipid and lipoprotein profile (Rader et al., 2003). For example, autosomal recessive hypercholesterolemia (ARH) is caused by mutations of the ARH gene, which codes for a novel adapter protein involved in the internalisation of the LDL-R:LDL complex. Homozygous ApoA1 deficiency results in the virtual absence of HDL and early CVD. Individuals with LCAT deficiency also exhibit extremely low levels of HDL. Sitosterolemia, which is also associated with premature CVD

and caused by mutations of ABCG5 and ABCG8, is characterised by an accumulation of both animal and plant sterols in the body. This is thought to be due to abnormal absorption of plant sterols, cholesterol hyperabsorption and reduced secretion of sterols into bile. Another recessive form of hypercholesterolemia is associated with deficiency of cholesterol 7α-hydroxylase, a key enzyme in the synthesis of bile acids. Finally, Tangier disease, first identified in the island of Tangier in the Chesapeake Bay in the U.S., is an autosomal recessive disorder because of mutations in the ABCA1 gene that results in hypertriglyceridemia and extremely low levels of HDL and apoA1. The most common form of dyslipidemias, however, has multifactorial origins, such as defects in several genes implicated in cardiovascular disease, environmental and life-style influences and other pathological conditions. Hyperlipidemia is known to arise because of medication (e.g., estrogen, oral contraceptives, steroids), excessive alcohol consumption, chronic and uncontrolled diabetes, nephrosis and endocrine disorders (e.g., hyperthyroidism). Infection and inflammatory responses associated with certain pathological conditions can also cause hyperlipidemias, changes in lipoprotein profile and premature CVD. The elevated levels of circulating cytokines are mainly responsible for such changes (see Mead and Ramji, 2002; Mead et al., 2002; Daugherty et al., 2005; Greenow et al., 2005; Harvey and Ramji, 2005). For example, pro-inflammatory cytokines reduce the expression of LPL in the adipose tissue, thereby causing an accumulation of chylomicrons and VLDL because of their defective clearance. In addition, pro-inflammatory cytokines inhibit the expression of apoE and ABCA1 by macrophages, thereby suppressing cholesterol efflux and accelerating foam cell formation. The most frequent cause of hyperlipidemia is the Metabolic Syndrome (Section 11.3.7) seen in obese individuals. This is clearly a major problem at the moment as the number of obese individuals, including young children, is increasing throughout the world. Lack of physical activity, a diet rich in saturated fats and refined sugars, high intake of calories compared to expenditure, and a sedentary life style all contribute to the proatherogenic lipid and lipoprotein profile in these individuals. In addition, they frequently have elevated blood pressure, peripheral insulin resistance, reduced HDL-cholesterol levels, increased plasma triacylglycerols, and onset of type II diabetes. The combination of these factors often acts in a synergistic manner to promote premature CVD.

11.2.7

Circulating lipids and the pathogenesis of atherosclerosis

The transformation of macrophages into foam cells is clearly a key step in the pathogenesis of atherosclerosis and a major target for therapeutic intervention of the disease (Li and Glass, 2002). Native LDL is not taken up by macrophages rapidly enough to form foam cells and numerous studies have shown that modification of the lipid 716

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and apoB100 moiety drives the formation of fatty streaks (Navab et al., 1996). Oxidation of LDL occurs in the arterial wall and becomes prevalent when levels of circulating LDL are raised. LDL diffuses through the endothelial cell junctions to the subendothelial matrix and its retention in the vessel wall may involve interaction with matrix proteoglycans (Stoker and Keaney, 2004). Although the precise mechanisms responsible for the oxidation of LDL remain to be fully elucidated, lipooxygenases, myelo-peroxidases, NADPH oxidases, and inducible nitric oxide synthase are major contributing enzymes (Stoker and Keaney, 2004). For example, there is diminished atherosclerosis in mice lacking 12/15 lipoxygenase. The inducible nitric oxide synthase also contributes to LDL oxidation in vivo and inhibitors of this enzyme have been shown to decrease atherosclerosis in rabbits. The precise action of oxidised LDL depends on the extent of its modification, which ranges from minimal to extensive. Minimal modification allows LDL to be recognised by the LDL receptor as normal, whereas extensive modification results in the particle being bound by SRs expressed on the surface of macrophages and SMCs, primarily SR-A and CD36. The importance of scavenger receptors in atherogenesis can be gauged by the observation that inactivation of SR-A or CD36 leads to reduced atherosclerosis in murine models of the disease (Greaves and Gordon, 2005). OxLDL taken up by macrophage SRs is delivered to lysosomes where its cholesteryl ester content is hydrolysed to free cholesterol and fatty acids. This free cholesterol has a number of potential metabolic fates, including esterification and storage as lipid droplets in foam cells (see Li and Glass, 2002). The cholesteryl ester stores of macrophages have been shown to undergo a continuous cycle of hydrolysis and reesterification. The hydrolytic step of this cycle is carried out by a neutral cytoplasmic cholesteryl ester hydrolase and the reesterification step is mediated by ACAT-1. As intracellular cholesterol levels increase, the proteolytic activation of SREBPs required for cholesterol biosynthesis and LDL-R expression is inhibited. Although this prevents the further accumulation of cholesterol via these pathways, cholesterol is still taken up via the SRs and, therefore, cholesterol homeostasis cannot be maintained. The macrophage can dispose of excess cholesterol by either enzymatic modification to more soluble forms or efflux to acceptors, such as HDL. The enzyme 27-hydroxylase is expressed in macrophages at relatively high levels and may play a role in cholesterol excretion by converting it to the more soluble 27-oxygenated steroid, which can be readily accepted by albumin (Babiker et al., 1997). The efflux of cholesterol involves the ABC family of membrane transporters, particularly ABCA1, and apoA1 and apoE in HDL as major acceptors of the steroid. LDL also undergoes other types of modifications, such as nonenzymatic glycation, enzymatic degradation, and aggregation. All such modifications generate a wide range

of epitopes, resulting in not only a cellular immune response, but also a humoral response (Horkko et al., 2000). For example, oxLDL is known to stimulate endothelial cells to secrete a range of pro-atherogenic cytokines. In addition, modified LDL is able to activate nuclear factor-κB (NF-κB), a master transcription factor implicated in the induced expression of a battery of genes associated with an inflammatory response. Modified LDL has also been shown to act as a chemoattractant for circulating monocytes, modulate vascular tone and cause aggregation of platelets (Stoker and Keaney, 2004).

11.2.8

Therapeutic approaches in cardiovascular disease based on the control of lipid metabolism

Various health organisations are strongly recommending dietary and other lifestyle changes to help to slow down the development of CVD and decrease the pro-atherogenic parameters. These include cessation of smoking; reduced intake of diets rich in fats, particularly saturated fats, and salt; substituting saturated fatty acids in the diet with polyunsaturated fatty acids; eating the recommended daily five servings of fruit and vegetables; at least 30 minutes of moderate exercise on 5 or more days of the week by adults; moderate alcohol consumption (one or two drinks per day); and combating detrimental psychosocial factors, such as stress, depression and anxiety. A number of other dietary supplements, such as antioxidants, which inhibit oxidation of LDL at least in vitro, have also been recommended for the prevention of atherosclerosis. However, the results from clinical studies have not always identified a positive antiatherogenic effect. Dietary and life-style changes are clearly not sufficient when a “clinical horizon” has been reached. Intake of drug(s) that limit the levels of the pro-atherogenic agent(s) in patients is necessary to prevent premature death. Because atherosclerosis is associated with dramatic changes in lipid metabolism and transport, it is not surprising that several drugs, that are either in current use or being developed, target key proteins or enzymes implicated in the maintenance of lipid homeostasis (Choy, 2004; Wierzbicki, 2004). As detailed above, numerous studies in the past 4 decades have supported a strong link between high circulating levels of LDL-cholesterol and atherosclerosis. About 75% of the total cholesterol pool in the body is derived from de novo synthesis with the remainder obtained from dietary intake. Inhibition of cholesterol synthesis, therefore, represents the main approach for reducing levels of circulating LDL-cholesterol. The conversion of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) to mevalonate, which is catalysed by the enzyme HMG-CoA reductase, is the rate-limiting step in the biosynthesis of cholesterol. The activity of this enzyme is inhibited by the statin class of drugs, such as lovastatin, 717

11.2

Lipids and cardiovascular disease

rosuvastatin, atorvastatin, pravastatin and simvastatin (Maron et al., 2000; Grundy et al., 2004). By decreasing cholesterol synthesis, the statins also increase the expression of the LDL-R by relieving the feedback inhibition, thereby further decreasing circulating LDL-cholesterol levels. Statins also cause a moderate decrease in serum triacylglycerol levels, a slight increase in HDL-cholesterol and inhibit the inflammatory response (Linsel-Nitschke and Tall, 2005; Elrod and Lefer, 2005). Statins are generally well tolerated except when taken at high doses where they may affect liver function. Although a reduction of LDL-cholesterol of 50 to 60% can be achieved by statins, doubling of doses produces only a marginal added decrease in LDL-cholesterol levels, but at the expense of increased side-effects. The inhibition of intestinal uptake of dietary cholesterol, which accounts for approximately 25% of this lipid in the body, is necessary to achieve further reduction of circulating LDL-cholesterol. Consumption of products rich in plant sterols and stanols, such as the Pro-ActivTM range of products from FloraR and similar products from BenecolR, which act as inhibitors of intestinal cholesterol uptake, can lower serum LDL cholesterol levels by 10% to 14% (Plat and Mensink, 2005; Cater et al., 2005). Bile acid sequestrants (e.g., colesevelam), which bind to bile acids in the intestine, also cause a 10 to 15% decrease in plasma LDL-cholesterol levels. Such agents interrupt the enterohepatic circulation of bile acids through which most of them are recycled back to the liver and reabsorbed and, thereby, promote their excretion in the faeces (Norata and Catapano, 2004). The use of such sequestrants is, however, limited because of their severe side effects. Direct inhibition of intestinal cholesterol absorption, therefore, offers the most promise. Ezetimibe is a new drug that acts in this manner and can often be taken with statins to achieve maximal reduction of LDLcholesterol (Gagne et al., 2002; Clader, 2005). Increasing circulating levels of HDL represents another, important avenue to limit atherosclerosis and its complications. Low circulating levels of HDL are common in obese individuals and this is likely to be a major problem in the future because of a dramatic worldwide increase in obesity. Nicotonic acid has been used to raise HDL levels indirectly by inhibiting hepatic VLDL synthesis and peripheral lipolysis and stimulating ABCA1 expression (Carlson, 2005). More recently, major advances in devising potential approaches for increasing circulating HDL levels and reducing other pro-atherogenic changes have been made from studies on nuclear receptors, which represent “hot” therapeutic targets for CVD at the moment. Such nuclear receptors are transcription factors that regulate the expression of a battery of genes implicated in the control of triacylglycerol and cholesterol homeostasis and, additionally, have potent antiinflammatory properties. A number of such receptors were originally identified from studies on the action of antidiabetic and lipid-lowering drugs. The major nuclear receptors that are currently

being studied intensely for therapeutic intervention of CVD are peroxisome proliferators-activated receptors (PPARs), liver-X-receptors (LXRs), and the farnesoid X receptor (FXR) (see Li and Glass, 2004; Marx et al., 2004; Ory, 2004; Barish and Evans, 2004; Berger et al., 2005; Claudel et al., 2005; Lehrke et al., 2005). These receptors form obligate heterodimers with retinoid X receptor, another nuclear receptor, and interact with recognition sequences in the regulatory regions of target genes. The PPAR family contains three members; PPAR-α, -γ, and -δ (also called PPAR-β). Fibrates, such as fenofibrate and gemfibrozil, are PPAR-α agonists, which lower concentration of circulating triacylglycerols by stimulating the expression of numerous genes implicated in the cellular uptake and catabolism of lipids. Many natural compounds have also been shown to act as PPAR-α agonists, including polyunsaturated fatty acids, such as eicosapentanoic acid, dodecahexanoic acid and linoleic acid. The action of PPAR-α on lipid homeostasis is mediated, at least in part, through the activation of genes implicated in the uptake, metabolism and β-oxidation of fatty acids. The channeling of fatty acids to the β-oxidative pathway reduces the availability of substrate for the synthesis of triacylglycerols and, thereby, ultimately leads to a decrease in the synthesis and secretion of VLDL by the liver. In addition, PPAR-α agonists stimulate the hydrolysis of circulating lipoproteins by increasing the synthesis of LPL and decreasing the levels of apoCIII, an inhibitor of LPL. Furthermore, PPAR-α agonists elevate HDL levels because of their ability to increase the synthesis of apoA1, thereby enhancing the formation of new HDL particles and inhibiting the expression of SR-BI, thus decreasing the clearance of HDL. Moreover, PPAR-α agonists have been shown to stimulate cholesterol efflux from foam cells. The PPAR-γ gene gives rise to two isoforms, PPARγ1 and γ2, by alternative use of promoters. PPAR-γ2 is expressed specifically in the adipose tissue, whereas PPAR-γ1 is the predominant isoform in other tissues, such as the liver and muscle. PPAR-γ2 is essential for the differentiation of adipocytes and the maintenance of normal glucose metabolism and promotes lipid accumulation by these cells. A number of naturally occurring fatty acid metabolites can activate PPAR-γ, including 15-deoxy∆12,14-prostaglandin J2 and oxidised linoleic acid (9- and 13-hydroxyoctadecadienoic acids). Pharmacological activators of PPAR-γ, such as glitazones (e.g., rosiglitazone, pioglitazone), have been used widely to improve insulin sensitivity in type II diabetes. Several actions of PPAR-γ are likely to contribute to improved insulin sensitivity, including induced expression of insulin-dependent glucose transporter GLUT4, stimulation of insulin signalling, inhibition of lipolysis and increased uptake of fatty acids and synthesis of triacylglycerols. In addition, PPAR-γ agonists regulate the secretion of several proteins by the adipose tissue, such as adiponectin, which then affects insulin signalling 718

Medical and Agricultural Aspects of Lipids

in the liver and muscles. PPARγ activators have been shown to inhibit atherosclerosis in murine models of the disease. Activators of both PPAR-α and PPAR-γ are also known to inhibit the inflammatory response indirectly by antagonising the actions of key transcription factors, such as NF-κB. As both PPAR-α and -γ have beneficial effects on lipid metabolism, inflammation, and insulin sensitivity, combined use of agonists for both these nuclear receptors promises to be a more effective approach at limiting atherosclerosis than agonists for individual receptors. Studies on the third PPAR member, PPAR-δ (previously called PPAR-β), have generally lagged behind the other two family members, but recent studies have suggested that it also represents another promising therapeutic target for limiting atherosclerosis. PPAR-δ is expressed in a ubiquitous manner and regulates energy homeostasis and fatty acid catabolism. PPAR-δ-deficient mice are susceptible to obesity, whereas transgenic mice that overexpress an activated form of this transcription factor are resistant to genetically or diet-induced hyperlipidemia and obesity. There are two LXRs, LXR-α and LXR-β, with the latter being expressed ubiquitously and the former present at high levels in the liver, intestine, adipose tissue, and macrophages. Both LXRs are activated by oxidised derivatives of cholesterol (e.g., 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, 24(S), 25-epoxycholesterol, 27hydroxycholesterol) and, therefore, act as intracellular sensors of cholesterol. Target genes for the action of LXRs include those implicated in the efflux of cellular cholesterol (e.g., apoE, ABCA1, ABCG1, ABCG4), modification of HDL (e.g., CETP, PLTP), secretion of cholesterol into bile (cholesterol 7α-hydroxylase, ABCG5, ABCG8), and fatty acid metabolism (e.g., sterol response element binding protein SREBP1c, fatty acid synthase, sterol-CoA desaturase-1). The expression of the LXR-α gene is also subject to autoregulation. Overall, LXRs restore cellular cholesterol balance and prevent lipotoxicity by activating cholesterol catabolic and efflux pathways along with those involved in de novo lipogenesis. In addition, LXR activators inhibit the expression of genes implicated in inflammation by antagonising the actions of key transcription factors, such as NF-κB and activator protein-1 (AP-1), largely via a mechanism that does not require sequencespecific DNA binding by the LXRs. A number of in vitro and in vivo studies have revealed a potent anti-atherogenic action of LXRs. However, a major current limitation of employing LXRs as therapeutic targets for atherosclerosis is that they increase the synthesis of fatty acids and cause the accumulation of triacylglycerols. Development of synthetic agonists that increase HDL levels without causing hypertriglyceridemia will clearly be necessary. This is a possibility since ongoing research suggests differences in the action of LXR-α and LXR-β, with the former having a dominant effect on hepatic lipogenesis.

Thus, selective LXR-β agonists could represent potential therapeutic agents for limiting atherosclerosis. FXR was initially identified as a nuclear receptor that is activated by bile acids and products of cholesterol metabolism. FXR regulates the expression of a number of genes implicated in the synthesis, transport and detoxification of bile acids. In addition, recent studies have suggested a role for FXR in the control of lipid metabolism and implicate its activation as a new means for limiting atherosclerosis. For example, polyunsaturated fatty acids, such as arachidonic, linolenic and docosahexaenoic acid, have all been shown to act as FXR ligands in vitro. Additionally, FXR-deficient mice have increased circulating levels of total triacylglycerols and cholesterol and decreased expression of SR-BI in the liver. Other targets for preventing atherosclerosis by modulating circulating lipid levels include inhibitors of CETP and ACAT and infusion of HDL particles or apolipoproteins that act as acceptors of cholesterol (Choy, 2004). CETP deficiency in humans is characterised by increased HDL levels and slightly reduced LDL levels. Moderate consumption of alcohol can increase circulating HDL levels and reduce atherosclerosis, at least in part, by inhibiting CETP. Inhibitors of CETP (e.g., CP-529/414 (torcetrapib), JTT-705) have been developed and are undergoing advanced clinical trials. The esterification of cholesterol by ACAT is critical for macrophage foam cell formation and lipoprotein synthesis in the liver and the intestine. Inhibitors of ACAT, such as avasimibe and CS-505, offer a further avenue for limiting atherosclerosis. Interest in apoA1 as a therapeutic target has emerged from the finding that its deficiency in humans is associated with premature atherosclerosis and extremely low levels of circulating HDL. Although a search of synthetic molecules that specifically induce apoA1 expression has not been fruitful, some success has been achieved, at least in murine models of atherosclerosis, by infusion of apoAI or synthetic peptides that mimic the actions of this lipoprotein. Overall, it appears that patients with CVD will be treated in the future with combination of drugs that lower cholesterol synthesis and induce the expression of the LDL-R (statins), inhibit the absorption of dietary cholesterol (e.g., ezetimibe) and enhance cholesterol efflux from foam cells and raise circulating HDL levels (e.g., agonists of nuclear receptors).

References Acton, S. et al. (1996). Identification of scavenger receptor SRBI as a high-density lipoprotein receptor. Science, 271, 518–520. Anderson, K.M. et al. (1987). Cholesterol and mortality: 30 years of follow-up from the Framingham study. JAMA, 257, 2176–2180. Arai, T. et al. (1999). Decreased selective uptake of highdensity lipoprotein cholesteryl esters in apolipoprotein

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E knock-out mice. Proc. Natl. Acad. Sci. U.S.A. 96, 12050–12055. Babiker, A. et al. (1997). Elimination of cholesterol in macrophages and endothelial cells by the sterol 27-hydroxylase mechanism. Comparison with high density lipoproteinmediated reverse cholesterol transport. J. Biol. Chem. 272, 26253–26261. Barish, G.D. and Evans, R.M. (2004). PPARs and LXRs: atherosclerosis goes nuclear. Trends Endocrinol. Metab. 15, 158–165. Barrett-Connor, E. and Bush, T.L. (1991). Estrogen and coronary heart disease in women. JAMA 265, 1861–1867. Berger, J.P. et al. (2005). PPARs: therapeutic targets for metabolic disease. Trends Pharmacol. Sci. 26, 244–251. Berglund, L. and Ramakrishnan, R. (2004). Lipoprotein (a): an elusive cardiovascular risk factor. Arterioscler. Thromb. Vasc. Biol. 24, 2219–2226. Boffa, M.B. et al. (2004). Lipoprotein (a) as a risk factor for atherosclerosis and thrombosis: mechanistic insights from animal models. Clin. Biochem. 37, 333–343. Brown, M.S. and Goldstein, J.L. (1999). A proteolytic pathway that controls the cholesterol content of membranes, cells, and blood. Proc. Natl. Acad. Sci. U.S.A. 96, 11041–11048. Bruce, C. et al. (1998). Plasma lipid transfer proteins, high-density lipoproteins, and reverse cholesterol transport. Annu. Rev. Nutr. 18, 297–330. Carlson, L.A. (2005). Nicotinic acid: the broad-spectrum lipid drug. A 50th anniversary review. J. Intern. Med. 258, 94–114. Cater, N.B. et al. (2005). Responsiveness of plasma lipids and lipoproteins to plant stanol esters. Am. J. Cardiol. 96, 23D–28D. Choy, P.C. (2004). Lipids and atherosclerosis. Biochem. Cell Biol. 82, 212–224. Clader, J.W. (2005). Ezetimibe and other azetidinine cholesterol absorption inhibitors. Curr. Top. Med. Chem. 5, 243–256. Claudel, T. et al. (2005). The farnesoid X receptor: a molecular link between bile acid and lipid and glucose metabolism. Arterioscler. Thromb. Vasc. Biol. 25, 2020–2030. Daugherty, A. et al. (2005). Thematic review series: the Immune system and atherogenesis. Cytokine regulation of macrophage functions in atherogenesis. J. Lipid Res. 46, 1812–1822. De Caterina, R. et al. (2004). Effects of omega-3 fatty acids on cytokines and adhesion molecules. Curr. Atheroscler. Rep. 6, 485–491. Eberle, D. et al. (2004). SREBP transcription factors: master regulators of lipid homeostasis. Biochimie. 86, 839–848. Elrod, J.W. and Lefer, D.J. (2005). The effects of statins on endothelium, inflammation and cardioprotection. Timely Top. Med. Cardiovasc. Dis. 9, E20. Fredrickson, D.S. and Breslow, J.L. (1973). Primary hyperlipoprotenemia in infants. Annu. Rev. Med. 24, 315–324. Gagne, C., Gaudet, D. and Bruckert, E. (2002). Efficacy and safety of ezetimibe added to ongoing statin therapy for treatment of patients with primary hypercholesterolemia. Am. J. Cardiol. 90, 1084–1091. Genest, J. (2003). Lipoprotein disorders and cardiovascular risk. J. Inherit. Metab. Dis. 26, 267–287. Glass, C.K. and Witztum, J.L. (2001). Atherosclerosis. The road ahead. Cell. 104, 503–516. Greaves, D.R. and Gordon, S. (2005). Recent insights into the biology of macrophage scavenger receptors. J. Lipid Res. 46, 11–20.

Greenow, K. et al. (2005). The key role of apolipoprotein E in atherosclerosis. J. Mol. Med. 83, 329–342. Grundy, S.M. et al. (2004). Implications of recent clinical trials for the National Cholesterol Education Program Adult Treatment Panel III Guidelines. J. Am. Coll. Cardiol. 44, 720–732. Gurr, M.I. (1992). Dietary lipids and coronary heart disease: old evidence, new perspective. Prog. Lipid Res. 31, 195–243. Harvey, E.J. and Ramji, D.P. (2005). Interferon-γ and atherosclerosis: pro- or anti-atherogenic? Cardiovasc. Res. 67, 11–20. Horkko, S. et al. (2000). Immunological responses to oxidized LDL. Free Radic. Biol. Med. 28, 1771–1779. Jump, D.B. (2004). Fatty acid regulation of gene transcription. Crit. Rev. Clin. Lab. Sci. 41, 41–78. Lehrke, M. et al. (2005). Gaining weight: the Keystone symposium on PPAR and LXR. Genes Dev. 19, 1737–1742. Levy, R.I. and Fredrickson, D.S. (1968). Diagnosis and management of hyperproteinemia. Am. J. Cardiol. 22, 576–583. Li, A.C. and Glass, C.K. (2002). The macrophage foam cell as a target for therapeutic intervention. Nat. Med. 8, 1235–1242. Li, A.C. and Glass, C.K. (2004). PPAR- and LXR-dependent pathways controlling lipid metabolism and the development of atherosclerosis. J. Lipid. Res. 45, 2161–2173. Linsel-Nitschke, P. and Tall, A.R. (2005). HDL as a target in the treatment of atherosclerotic cardiovascular disease. Nat. Rev. Drug Discov. 4, 193–206. Lusis, A.J. (2000). Atherosclerosis. Nature. 407, 233–241. Lusis, A.J. et al. (2004). Genetics of atherosclerosis. Annu. Rev. Genomics Hum. Genet. 5, 189–218. Mahley, R.W. et al. (1999). Pathogenesis of type III hyperlipoproteinemia (dysbetalipoproteinemia). Questions, quandaries, and paradoxes. J. Lipid. Res. 40, 1933–1949. Maron, D.J. et al. (2000). Current perspectives on statins. Circulation. 101, 207–213. Martin, M.J. et al. (1986). Serum cholesterol, blood pressure and mortality: implications from cohort of 361,662 men. Lancet, 2, 933–936. Marx, N. et al. (2004). Peroxisome proliferators-activated receptors and atherogenesis: regulators of gene expression in vascular cells. Circ. Res. 94, 1168–1178. Mead, J.R. and Ramji, D.P. (2002). The pivotal role of lipoprotein lipase in atherosclerosis. Cardiovasc. Res. 55, 261–269. Mead, J.R. et al. (2002). Lipoprotein lipase: structure, function, regulation, and role in disease. J. Mol. Med. 80, 753–769. Mendelsohn, M.E. and Karas, R.H. (2005). Molecular and cellular basis of cardiovascular gender differences. Science, 308, 1583–1587. Mori, T.A. (2004). Effect of fish and fish oil-derived omega-3 fatty acids on lipid oxidation. Redox Rep. 9, 193–197. Mori, T.A. and Beilin, L.J. (2004). Omega-3 fatty acids and inflammation. Curr. Atheroscler. Rep. 6, 461–467. Navab, M. et al. (1996). The yin and yang of oxidation in the development of the fatty streak. A review based on the 1994 George Lyman Duff Memorial Lecture. Arterioscler. Thromb. Vasc. Biol. 16, 831–842. Norata, G.D. and Catapano, A.L. (2004). Lipid lowering activity of drugs affecting cholesterol absorption. Nutr. Metab. Cardiovasc. Dis. 14, 42–51. Ory, D.S. (2004). Nuclear receptor signalling in the control of cholesterol homeostasis: have the orphans found a home? Circ. Res. 95, 660–670.

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11.3.2

Osterud, B. and Bjorklid, E. (2003). Role of monocytes in atherogenesis. Physiol. Rev. 83, 1069–1112. Plat, J. and Mensink, R.P. (2005). Plant stanol and sterol esters in the control of blood cholesterol levels: mechanism and safety aspects. Am. J. Cardiol. 96, 16D–22D. Rader, D.J. et al. (2003). Monogenic hypercholesterolemia: new insights in pathogenesis and treatment. J. Clin. Invest. 111, 1795–1803. Rigotti, A. et al. (1997). A targeted mutation in the murine gene encoding the high density lipoprotein (HDL) receptor scavenger receptor class B type I reveals its key role in HDL metabolism. Proc. Natl. Acad. Sci. U.S.A. 94, 12610–12615. Ross, R. (1999). Atherosclerosis — an inflammatory disease. N. Engl. J. Med. 340, 115–126. Smith, J. D. et al. (1995). Decreased atherosclerosis in mice deficient in both macrophage colony stimulating factor (op) and apolipoprotein E. Proc. Natl. Acad. Sci. U.S.A. 92, 8264–8268. Stocker, R. and Keaney, J.F., Jr., (2004). Role of oxidative modifications in atherosclerosis. Physiol. Rev. 84, 1381–1478. Vanden Heuvel, J.P. (2004). Diet, fatty acids, and regulation of genes important for heart disease. Curr. Atheroscler. Rep. 6, 432–440. Wang, N. et al. (1998). Liver-specific overexpression of scavenger receptor BI decreases levels of very low-density lipoprotein apoB, low density lipoprotein apoB and high density lipoprotein in transgenic mice. J. Biol. Chem. 273, 32920–32926. Wierzbicki, A.S. (2004). Lipid-altering agents: the future. Int. J. Clin. Prac. 58, 1063–1072.

11.3

Clinical aspects of lipids with emphasis on cardiovascular disease and dyslipaemia

11.3.1

Objectives of cardiovascular disease prevention in clinical practice

Dyslipidaemia and cardiovascular disease

As concentrations of total and LDL-cholesterol increase, so does the risk of cardiovascular disease (Stamler et al., 2000). The relationship between cholesterol and cardiovascular risk is continuous, while reduction of total and low density lipoprotein-cholesterol (LDL-C) is associated with numerous sequelae (which attenuate atherogenesis) including improved endothelial function, reduced oxidative stress and reduced inflammation (Ross, 1993; Vogel, 1997). In the context of lipids, cardiovascular risk is principally determined by the concentrations of total, LDL- and high density lipoprotein-cholesterol (HDL-C) (inversely) and, to a lesser extent, plasma triacylglycerol concentrations. Indeed, data from various studies including Framingham and MRFIT (Kannel and Larson, 1993; Stamler et al., 1993) have demonstrated that low levels of HDL-C independently predict increased cardiovascular risk, irrespective of total or LDL-C levels (Stamler et al., 1993). The ratio of total or LDL-C to HDL-C has been consistently demonstrated as the strongest determinant of cardiovascular risk in prospective studies. This was further illustrated by the recent INTERHEART study, in which the ratio of apo Bto apo AI-containing lipoproteins was demonstrated to be the single most important determinant of risk for first myocardial infarction from over 10,000 subjects from varying geographical locations and ethnic origins (Yusuf et al., 2004). Meta-analysis of prospective studies has consistently demonstrated that a 1% decrease in HDL-C is associated with a 2 to 3% increase in cardiovascular risk, while the relationship between HDL-C and cardiovascular risk may be stronger in women (Expert Panel, 2001). The reduction of total LDL and LDL-cholesterol is, however, currently the primary goal of lipid-lowering therapy with respect to cardiovascular risk reduction. The reduction of cholesterol whether by diet, drugs, or other means is associated with a reduced risk of CVD (Brady and Betteridge, 2003) and since lipoproteins are only one element of cardiovascular risk, which is determined overall by the presence of other risk factors, the absolute benefit of cholesterol reduction, is a function of baseline cardiovascular risk. The MRC/BHF Family Heart Protection Study (2002) demonstrated that the benefits of cholesterol lowering therapy extend into all forms of atherosclerotic vascular disease including peripheral vascular disease (PVD) and cerebrovascular disease. In a systematic review and meta-analysis evaluating the effects of cholesterol reduction on coronary heart disease (CHD) and stroke, 58 clinical trials of cholesterol reduction by any means were included (Law et al., 2003). Reduction in CHD death and nonfatal myocardial infarction for a 1.0 mmol/l reduction in cholesterol was 11% in the first year, 24% in the second, 33% in the third to fifth, and 36% in the sixth and subsequent years. After standardisation for reduction in LDL-C and duration of treatment, the reduction in risk of fatal and nonfatal events was similar for different

The specific objective of cardiovascular disease (CVD) prevention for all high-risk people in clinical practice is to reduce the risk of the disease and its associated complications. These include the need for percutaneous or surgical revascularisation procedures in any arterial territory, along with improvements in both quality of life and life expectancy. Cardiovascular disease risk reduction in practice involves a multifactorial approach involving appropriate lifestyle changes, such as weight loss, dietary modification, increasing exercise and smoking cessation. Pharmacological interventions aimed at reducing cardiovascular risk also revolve around a multifactorial approach including antiplatelet therapy, anticoagulation and a target-driven approach to blood pressure reduction. Although optimal cardiovascular disease risk reduction requires a multifactorial approach, prospective epidemiological data have consistently demonstrated the preeminence of abnormalities in lipid metabolism in the aetiology of atherosclerotic vascular disease, in particular, coronary heart disease. Hence, the management of dyslipidaemia in clinical practice represents a central pillar in the approach to cardiovascular disease risk reduction. 721

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Clinical aspects of lipids with emphasis on cardiovascular disease and dyslipaemia

methods of reducing cholesterol and for people with and without CHD. The reduction in cholesterol and the duration of treatment were the primary determinants of risk reduction, with a reduction in LDL-C of 1.6 mmol/l or more after 2 years resulting in a 51% reduction in risk. The most compelling evidence for cholesterol lowering comes from clinical trials using statins. The early major statin trials in people with established CVD using simvastatin and pravastatin and in people at risk of developing CVD using pravastatin and lovastatin, demonstrated reduction in coronary morbidity and mortality, and in all-cause mortality where statistical power was sufficient. A meta-analysis of these studies demonstrated reductions in coronary events of 31%, coronary mortality of 29%, and all-cause mortality of 21% (Baienet et al., 2005). More recent trials have extended the evidence base for this class of drug into many populations including women, the elderly, acute coronary disease patients, people with diabetes and renal transplantation, and people previously thought to be at low risk due to low baseline cholesterol levels. In the recent cholesterol trialists collaboration (Baienet et al., 2005), a meta-analysis of data from 90,056 individuals in 14 randomised trials of statins, has shown that statin therapy can safely reduce the 5-year incidence of major coronary events, coronary revascularisation and stroke by about one-fifth per mmol/l reduction in cholesterol. During a mean of 5 years, there were 8186 deaths, 14,348 individuals had major vascular events, and 5103 developed cancer. Mean LDL cholesterol differences at 1 year ranged from 0.35 mmol/l to 1.77 mmol/l (mean 1.09) in these trials. There was a 12% proportional reduction in all-cause mortality per mmol/l reduction in LDL cholesterol. This reflected a 19% reduction in coronary mortality, and nonsignificant reductions in noncoronary vascular mortality and nonvascular mortality. There were corresponding reductions in myocardial infarction or coronary death, in the need for coronary revascularisation, in fatal or nonfatal stroke, and, combining these, of 21% in any such major vascular event. These benefits were significant within the first year, but were greater in subsequent years. Taking all years together, the overall reduction of about one-fifth per mmol/l, LDLC reduction translated into 48 fewer participants having major vascular events per 1000 among those with preexisting CHD at baseline, compared with 25 per 1000 among participants with no such history. A similar proportional reduction in risk is seen for all people with atherosclerotic disease regardless of the vascular territory. Women demonstrated a similar proportionate reduction in risk to men, with no age at which these benefits have not been demonstrated, with older people who are at higher absolute risk having similar risk reduction as younger people. The absolute benefit of statin therapy is related chiefly to an individual’s absolute cardiovascular risk and to the absolute reduction in cholesterol achieved. These observations reinforce the need to consider prolonged statin treatment with

substantial LDL-C reductions in all patients at high risk of any type of major vascular event. Furthermore, in a recent study of the safety and efficacy of intensive cholesterol reduction in 1825 patients with acute coronary syndrome, continuing outcome benefits were demonstrated in association with reductions in LDL-C as low as