Arginine Amino Acid [1 ed.] 9781611222548, 9781617619816

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MICROBIOLOGY RESEARCH ADVANCES

Copyright © 2011. Nova Science Publishers, Incorporated. All rights reserved.

ARGININE AMINO ACID

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MICROBIOLOGY RESEARCH ADVANCES

ARGININE AMINO ACID

NATHAN L. JACOBS

Copyright © 2011. Nova Science Publishers, Incorporated. All rights reserved.

EDITOR

Nova Science Publishers, Inc. New York

Copyright © 2011 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers‟ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works.

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Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Additional color graphics may be available in the e-book version of this book. Library of Congress Cataloging-in-Publication Data Arginine amino acid / [edited by] Nathan L. Jacobs. p. ; cm. Includes bibliographical references and index. ISBN:  (eEook)

1. Arginine. I. Jacobs, Nathan L. [DNLM: 1. Arginine. QU 60] QP562.A7A74 2010 612.3'98--dc22 2010034003

Published by Nova Science Publishers, Inc. † New York

Contents

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Preface

vii

Chapter 1

Analytical Methods of the Determination of Arginine Amino Acid R.M. Callejon, C. Ubeda, A.M. Troncoso and M.L. Morales,

Chapter 2

Alternative Metabolic Pathways of Arginine and their Pathophysiological Roles András Hrabák and Zoltán Kukor

Chapter 3

Free Amino Acid Analysis in Natural Matrices Graciliana Lopes, Patrícia Valentão and Paula B. Andrade

Chapter 4

Discovery of Argininosuccinate Synthetase and Argininosuccinate Lyase Olivier Levillain

Chapter 5

Expression and Localization of Argininosuccinate Synthetase and Argininosuccinate Lyase in the Female and Male Rat Kidneys Olivier Levillain and Heinrich Wiesinger

Chapter 6

Chemical Structure and Toxicity in Arginine-Based Surfactants. Aurora Pinazo, Lourdes Pérez, María Rosa Infante, María Pilar Vinardell, Montse Mitjans, María Carmen Morán and Verónica Martínez

Chapter 7

Arginine: Physico-Chemical Properties, Interactions with Ion-Exchange Membranes, Recovery and Concentration by Electrodialysis T. Eliseeva, E. Krisilova, G. Oros and V. Selemenev

Chapter 8

Chapter 9

1

33 67

91

111 125

143

Central Functions of L-Arginine and its Metabolites for Stress Behavior Shozo Tomonaga, D. Michael Denbow and Mitsuhiro Furuse

163

Arginine Requirement and Metabolism in Marine Fish Larvae-Review of Recent Findings Margarida Saavedra

191

vi Chapter 10

Arginine-Rich Cell-Penetrating Peptides in Cellular Internalization Betty Revon Liu, Huey-Jenn Chiang and Han-Jung Lee

Chapter 11

Effects of Deep Sea Water on Changes in Free Amino Acids and Tolerance to Fusarium Root Rot in Mycorrhizal Asparagus Plants Abu Shamim Mohammad Nahiyan, Mika Yokoyama and Yoichi Matsubara

219

Influence of Arginine-Containing Peptides on the Haemostasis System Maria Golubeva and Marina Grigorjeva

229

Newly Identified Transcriptional Regulation by Mcm1p at ARG1 Promoter Sungpil Yoon

237

Chapter 12

Chapter 13

Index

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Contents 207

253

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Preface Arginine is a conditionally nonessential amino acid, meaning most of the time it can be manufactured by the human body, and does not need to be obtained directly through the diet. Arginine plays an important role in cell division, the healing of wounds, removing ammonia from the body, immune function, and the release of hormones. This book presents topical research in the study of arginine amino acids, including the techniques and methodologies used for the measurement of arginine in different matrixes; alternative metabolic pathways of arginine and their pathophysiological roles; the biosynthesis of arginine in the mammalian liver and kidney; the relationship between the structure and toxicity evaluated by in vitro methods of a series of arginine-based surfactants; the physico-chemical properties of arginine; and the central functions of L-arginine and its metabolites for stress behavior. Chapter 1 - L-Arginine is one of the most versatile amino acids from the metabolic and physiological point of view. Known funtions for arginine include: substrate for protein and biologically active peptide synthesis, intermediate in ammonia detoxification, hormone liberation, and poliamine and creatine biosynthesis. All these funtions have been recently increased with the discovery of its role as the substrate for the synthesis of nitric oxide, a multifuntional effector involved in vasodilation, neurotransmission, and inmune responses. From the nutritional point of view, L-arginine must be considered as a “semiessential” or “conditionally essential” amino acid in mammals, depending on the developmental stage and health status of the individual. Therefore, the endogenous synthesis and dietary supply both contribute to satisfy arginine needs. In man, biosynthesis seems to be high enough to fulfil normal physiological arginine demand consumption of the amino acid. Such a situation suggests a potential therapeutic use for dietary arginine administration that is just starting to be analyzed. Hence, there is a need for the accurate and reliable quantification of arginine in food and various biological fluids. This current chapter gives a comprehensive overview of the techniques and methodologies currently available for the measurement of arginine in different matrixes. Arginine, as well as the rest of the amino acids, usually needs to be derivatized to make it more detectable. Several derivatizing reagents have been employed for the determination of arginine and each has its advantages and disadvantages. Chapter 2 - Arginine is not only a protein constituent but it is metabolized through various alternative pathways in mammalian cells. The author review is focused on the two most important alternative pathways: the nitric oxide (NO) synthesis and the arginase reaction. In cells where both pathways are active, their regulation is generally reciprocal due

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viii

Nathan L. Jacobs

to the common substrate. This reciprocal regulation was described at the level of both enzyme activities and expressions. Cross inhibition between arginase and NO synthase metabolites can be observed and different cytokines act differently on the expression of both enzymes as well. Arginase and NO synthase isoenzymes are involved in various important physiological processes such as urea cycle, vasodilation, immune defense against certain invaders and neurotransmission. However, the overexpression of these enzymes and overproduction of NO may contribute to the onset of various pathophysiological processes. NO overproduction may be responsible for neurotoxicity, septic shock, type 1 diabetes mellitus or various inflammatory diseases in numerous organs. In addition, inflammatory responses involving the inducible NO synthase may also contribute to the etiology of metabolic syndrome, while impaired NO production by the endothelial NO synthase may be in the background of cardiovascular disorders or preeclampsia. On the other part, the involvement of high arginase expression was observed in cardiovascular disorders and several pulmonary diseases, as pulmonary hypertension, silicosis and asthma. In conclusion, alternative arginine metabolic pathways and their regulation are important factors both in the maintenance of healthy state and in the pathogenesis of various diseases. Chapter 3 - Amino acids constitute a class of biologically active compounds found either in the free form or as linear chains in peptides and proteins. In addition to their primary function as protein components, they have several biological roles, being important as neurotransmitters, hormones, precursors of complex nitrogen containing molecules and as metabolic intermediates. Amino acids are molecules containing an amine group, a carboxylic acid group and a side chain that varies between different amino acids. There are 20 amino acids commonly found in proteins, which are classified depending on the polarity of the side chain. According to this criterion they can be non-polar and neutral, polar and neutral, acidic or basic. In plants they are also involved in secondary metabolism, namely in the biosynthesis of phenolic compounds, glucosinolates, cyanogenic heterosides and alkaloids. The amino acids composition is a reliable indicator of the nutritional value of matrices used for human consumption, but also a useful tool in natural products authenticity. In this work the authors provide an overview on the application of HPLC-UV-vis and GC-FID analysis on the determination of the amino acids profile of several natural matrices: wild edible mushroom species, Brassica oleracea var. costata, Catharanthus roseus, Cydonia oblonga and red wine inoculated with different Dekkera bruxellensis strains. The influence of different factors, such as the collection date, geographical origin and vegetal tissue, in the amino acids composition of the samples are also discussed. Chapter 4 - This review summarizes the impressive work performed by several teams of research to discover the biosynthesis of arginine in mammalian liver and kidney and the different steps which led to identify the metabolites involved in this reaction. Successive purification steps allowed the isolation and characterization of two enzymatic proteins, namely argininosuccinate synthetase and argininosuccinate lyase. New approaches including molecular biology gave insights into the molecular characteristics of ASS and ASL genes, mRNAs, and proteins. Furthermore, new techniques such as Northern blot, Western blot, and immunocytology became excellent tools to analyze the expression on ASS and ASL genes in the different organs of several mammalian species. ASS and ASL are homotetramers with subunits of 46 and 51 kDa, respectively. ASS and ASL are generally co-localized in the same cell type and are widely distributed in various organs.

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Preface

ix

Chapter 5 - The enzymes argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) convert L-citrulline into L-arginine. In mammalian kidneys, L-arginine is essentially produced in the proximal convoluted tubules (PCT). Almost all the studies performed on this renal metabolic pathway were restricted to male animals. The authors experiments were first conducted to determine whether female rat kidneys express ASS and ASL genes and the results were compared to those of the males. The expression of ASS and ASL was analyzed by Western blot analyses in the different zones dissected from rat kidneys. ASS protein was localized by immunofluorescence. Finally, the authors tested whether sex influences the renal level of ASS and ASL proteins, as determined by Western blot analyses. The author results reveal that high levels of ASS and ASL proteins were detected in female as well as in male rat kidneys. The relative abundance of ASS and ASL proteins was higher in the cortex (superficial > deep) than in the outer stripe of the outer medulla. Immunolocalization studies clearly showed that ASS expression was restricted to the proximal tubules. PCT exhibited the highest level of ASS compared with the proximal straight tubules (PST). The levels of ASS and ASL proteins were similar in female and male rat kidneys. In conclusion, female rat kidneys express the two enzymes involved in the production of the conditionally essential amino acid L-arginine. No sexual dimorphism in ASS and ASL expression was found in the rat kidney. Chapter 6 - Surfactants are one of the most representative chemical products which are consumed in large quantities every day on a worldwide scale. The use of surfactants in everyday life is almost unavoidable. The development of less irritant, less toxic, consumerfriendly surfactants or surfactant systems is, therefore, of general interest. During the last 20 years, the author group has been developing new biocompatible surfactants derived from amino acids. Among them, arginine derivative surfactants constitute a novel class that can be regarded as an alternative to conventional cationic surfactants due to their multifuncional properties and the renewable source of raw materials used during the synthesis process. These characteristics make them candidates of choice as additives in pharmaceutical, food and cosmetic formulations. Evaluation of the irritant potential in vivo, of new products or ingredients, for pharmaceutical use, is required by law in most EU countries, prior to human exposure. However, due to increasing concern over animal use and in lights of its potential ban in the near future, alongside with the obvious ethical implications of using directly human subjects, in vitro alternative methods should now be encouraged. This review reports on the relationship between the structure and toxicity evaluated by in vitro methods of a series of arginine-based surfactants including surfactants with one single chain, gemini surfactants, and surfactants with glycerolipid-like structure. Chapter 7 - L-Arginine (2-amino-5-guanidinpentanoic acid) is a product of great importance in medicine, food and pharmaceutical industry. L-Arginine is a basic, genetically coded α-amino acid, one of the twenty most common natural amino acids. It is essential for human ("semi-essential") [1-3]. Arginine plays a significant role in cell division, healing of wounds, removing ammonia from a body, immune function, and release of hormones [2,4-5]. Chapter 8 - L-Arginine is an essential amino acid for birds, carnivores and young mammals and a conditionally essential amino acid for adults. L-Arginine can be catabolized by four sets of enzymes in mammalian cells, resulting in the production of urea, L-ornithine, L-proline, L-glutamate, polyamines, nitric oxide, creatine, agmatine, etc.. Unlike mammals, birds lack carbamyl phosphate synthetase, one of the urea cycle enzymes necessary for the synthesis of L-citrulline from L-ornithine in the liver and kidney. Therefore, it is impossible

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Nathan L. Jacobs

to synthesize L-arginine in birds, and L-arginine is classified as an essential amino acid for birds. In this chapter, the authors introduce recent studies about central functions of Larginine and its metabolites for stress behavior. In particular, the functions in avian species are focused upon. In neonatal chicks, centrally injected L-arginine induces sedative and hypnotic effects under social separation stress. Among L-arginine metabolites, L-ornithine, Lproline and L-glutamate would especially contribute to these effects. Chapter 9 - Amino acids (AA) are the most important energetic substrate during fish larval development. Estimation of AA requirements is, therefore, crucial to formulate suitable diets for aquaculture species in order to obtain better larval survival, growth and general fish performance. AA larval requirements can be estimated using the AA profile of fish carcass as it is a good indicator of fish larval requirements. The AA profile can later be corrected with the AA bioavailability and a fair estimation of larval AA requirements is achieved. Once the AA requirements are determined they need to be compared to the AA profile of the fish larval diets. Most fish larval stages are still dependent on live feed, such as rotifers and Artemia, to survive and grow. Therefore, correction of the AA profiles of the live feed must be done enriching or supplementing the live feed diet. The effect of feed supplementation with AA can be tested at long term through zootechnique trials or at short term using the tube-feeding technique. This review intends to evaluate the current knowledge of arginine requirements and the effect of arginine supplementation on the metabolism of this AA in several marine fish larvae such as white seabream, Diplodus sargus, and Senegalese sole, Solea senegalensis. Arginine was chosen because it is an indispensable AA involved in metabolic pathways such as protein synthesis, urea production, metabolism of glutamic acid and proline, and synthesis of creatine and polyamines. It is also considered to be in deficiency in the diet of some fish. Chapter 10 - Polypeptides composed of arginine-rich domains play a key role in gene regulation, such as nuclear localization signal which can penetrate nuclear membrane. Recent studies indicated that arginine-rich cell-penetrating peptides (CPPs) possess the ability to penetrate plasma membrane without receptors. Length of peptides with arginine residues determines the efficiency of cell internalization. Also, D- or L-form of arginine residues have different effects on internalization efficiency. In the author investigations, arginine-rich CPPs could serve as efficient shuttles to deliver proteins, DNAs, RNAs or nanoparticles (such as quantum dots) into animal or plant cells in a covalent (CPT), noncovalent (NPT), or covalent and noncovalent (CNPT) protein transduction manner. The penetrating mechanism of CPPs was still controversial, but more evidences indicated that these peptides enter cells through multiple internalization pathways. No cytotoxicity and high transduction efficiency highlight great advantages of these CPPs in cargoes or drug delivery. Therefore, studies of argininerich CPPs could launch a new page in pharmaceutics, therapeutics or cell biology. Chapter 11 - Asparagus (Asparagus officinalis L., cv. Welcome) plants inoculated with arbuscular mycorrhizal fungi (AMF) (Glomus intraradices and Gigaspora margarita). Dry weight of shoots and roots were increased in deep-sea-water added asparagus plants with or without presence of AMF, though higher dry weight was observed in deep-sea-water added AMF-inoculated plots. Thus, plant growth promotion via symbiosis appeared in mycorrhizal asparagus plants. As for disease tolerance, incidence and severity of root rot was lower in AMF plants than in non-AMF plants. On the other hand, several free amino acids , such as asparagine, glutamine, aspartic acid, alanine, GABA, tyrosine, ornithine and lysine were increased in shoots and roots of most of the mycorrhizal asparagus plants. From these findings, it is suggested that plant growth promotion through AMF and tolerance to Fusarium

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Preface

xi

root rot occurred in mycorrhizal asparagus plants. In addition, several free amino acid contents are stimulated by the symbiosis, supposing that the changes might be associated with the disease tolerance. Chapter 12 - It is known that many regulatory peptides take part in haemostatic reactions of the organism, influence on all phases of blood coagulation and fibrinolysis. At the same time formed in process degradation of regulatory peptides fragments have independent biological activity. For example, many short peptides have fibrinolytic, anticoagulant and antitrombotic effects in blood. By stage-by-stage proteolysis it is possible to define structural conditionality of most regulatory peptides effects and to define the minimum fragment, responsible for its biological activity. Any modification of peptide molecule can cause changes of its biological efficiency. Therefore the study of peptides degradation ways, and also structure and effects of formed fragments, has the great interest and significance. In the present study summarizes the results of own researches about the influence on various links of the haemostasis system of same regulatory peptides fragments and different olygopeptides, containing the arginine amino acid in different positions of peptides chain. Chapter 13 - Recent studies with chromation immunoprecipitation assay and mutational analysis in binding sites of the regulators demonstrated unexpected biological mechanisms in the transcriptional regulation of ARG1. Two roles of Mcm1p were identified at ARG1: a Gcn4p-mediated positive transcriptional role and a negative role involving Arg80p, Arg81p, and Arg82p. Mcm1p contributed to active transcription at the ARG1 promoter by increasing the binding of the activator Gcn4p and by recruiting the co-activator complex SWI/SNF at ARG1 under Gcn4p-induced conditions. Mutational analysis of the ARG1 promoter also revealed a positive role for the Mcm1p binding site in ARG1 transcription and growth to overcome arginine starvation in the absence of Gcn4p. A transcriptional negative role for Mcm1p was apparent in the recruitment of the whole repressor ArgR/Mcm1p complex, which contributed to dampening the activating function of Gcn4p at ARG1 in arginine-replete cells. Concerning the mechanism of ARG1 transcription, the most interesting finding was that ARG1 transcription could be controlled by different mechanisms with the Mcm1p binding sites through either the presence or absence of complete amino acids under condition of arginine starvation.

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In: Arginine Amino Acid Editor: Nathan L. Jacobs

ISBN 978-1-61761-981-6 © 2011 Nova Science Publishers, Inc.

Chapter 1

Analytical Methods of the Determination of Arginine Amino Acid R.M. Callejon, C. Ubeda, A.M. Troncoso and M.L. Morales Área de Nutrición y Bromatología, Facultad de Farmacia, Universidad de Sevilla, Sevilla, Spain

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Abstract L-Arginine is one of the most versatile amino acids from the metabolic and physiological point of view. Known funtions for arginine include: substrate for protein and biologically active peptide synthesis, intermediate in ammonia detoxification, hormone liberation, and poliamine and creatine biosynthesis. All these funtions have been recently increased with the discovery of its role as the substrate for the synthesis of nitric oxide, a multifuntional effector involved in vasodilation, neurotransmission, and inmune responses. From the nutritional point of view, L-arginine must be considered as a “semiessential” or “conditionally essential” amino acid in mammals, depending on the developmental stage and health status of the individual. Therefore, the endogenous synthesis and dietary supply both contribute to satisfy arginine needs. In man, biosynthesis seems to be high enough to fulfil normal physiological arginine demand consumption of the amino acid. Such a situation suggests a potential therapeutic use for dietary arginine administration that is just starting to be analyzed. Hence, there is a need for the accurate and reliable quantification of arginine in food and various biological fluids. This current chapter gives a comprehensive overview of the techniques and methodologies currently available for the measurement of arginine in different matrixes. Arginine, as well as the rest of the amino acids, usually needs to be derivatized to make it more detectable. Several derivatizing reagents have been employed for the determination of arginine and each has its advantages and disadvantages.

2

R.M. Callejon, C. Ubeda, A.M. Troncoso and M.L. Morales

Introduction

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Arginine (abreviated as Arg) is an α-amino acid whose L-form is one of the 20 most common natural amino acids. L-arginine has been characterized as a semi-essential amino acid, in that it is non-essential in the healthy adult organism of most mammals, but has to be supplemented in the growing organism, after trauma or during various disease states [1-3]. Normal L-arginine plasma levels are in the range of 100–200 µM [4-6]. It is important to note that even in adult mammalian organisms not all of the enzymes required for de novo synthesis of L-arginine are expressed in every tissue. Arginine was first isolated from a lupin seedling extract in 1886 by the Swiss chemist Ernst Schultze [7] and since then, the biochemistry and physiology of this amino acid has been a field of active research. However, the discovery about 100 years later that L-arginine is the only physiologically significant substrate for the synthesis of nitric oxide (NO) [8-10], has markedly stimulated the interest in the complex metabolism of this amino acid. This is due to NO being identified as an important intra- and transcellular signalling molecule involved in the regulation of many physiological and pathophysiological processes in the mammalian organism [11-15]. As shown in Figure 1, the amino acid side chain of arginine consists of a 3-carbon aliphatic straight chain, the distal end of which is capped by a complex guanidinium group. This group is positively charged in neutral, acidic and even most basic environments, and thus imparts basic chemical properties to arginine. The specific characteristics of this amino acid are shown in Table 1.

Figure 1. Structure of Arginine.

Sources Dietary Sources Since arginie is a conditionally nonessential amino acid, meaning most of the time it can be manufactured by the human body, it does not have to be obtained directly through the diet. However, the biosynthetic pathway does not produce sufficient arginine, and hence, the requirements of this amino acid must be provided by diet. Individuals who have poor

Analytical Methods of the Determination of Arginine Amino Acid

3

nutrition or certain physical conditions may be advised to increase their intake of foods containing arginine [17,18]. Table 1. Characteristics of arginine [16] Chemical name Empirical formulae Molecular weight Appearance Physical properties Optical rotation Melting point Hygroscopicity Solubility Characteristics Residue on Ignition Vibrational spectroscopy

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Dissociation constant

Stability

2-amino-5-guanidinovaleric acid (S)-2-amino-5-[(aminoiminomethyl)amino]pentanoic acid C6H14N4O2 174.202 White or almost white powder, obtained as practically odorless crystals The specific rotation of a 80mg/ml of Arg dissolved in 6N HCl is between +26.5º and +26.9º 235ºC (decomposition) Arg is not a hygroscopic substance Arg dried at 105ºC for 3 hours does not lose more than 0.5% of its weight Freely soluble in water (1g dissolves in 5 ml of water), sparingly or very sparingly soluble in alcohol and practically insoluble in ether ≤ 0.05% 400-4000 cm-1 pKa1 (-COOH) = 2.17 pKa2 (α-NH3) = 9.04 pKa3 (R-group) = 12.48 The isoelectric point of arginine is found to be pH = 10.8 When recrystallized in water, the dehydrate form is obtained, but usually the water of crystallization is eliminated by drying at 105 ºC. When hydrolyzed with hot alkali, citrullina and ornithina are formed.

Arginine is found in a wide variety of foods including: animal sources, such as dairy products (e.g., cottage cheese, ricotta, milk, yogurt, whey protein drinks), beef, pork (e.g., bacon, ham), poultry (e.g., chiken and turkey light meat), wild game (e.g., pheasant, quail), or seafood (e.g., halibut, lobster, salmon, shrimp, snails, tuna); vegetable sources such as wheat germ and flour, buckwheat, granola, oatmeal, peanuts, nuts, seeds, chick peas, cooked soybeans, onion, garlic, asparagus, cabbages, radishes, cucumbers, lettuce, bananas, peaches,etc. [19-21]. In addition, arginine is also present in fermented beverages such as wine [22,23], beers [24-26] and vinegars [27-29]. In fact, arginine is the most abundant amino acid in wine after proline and it is used by yeasts and bacteria as a nitrogen source. As a consequence of consumption and metabolism of this amino acid by these bacteria, some biogenic amines and precursors of ethyl carbamate can be formed, which are considered perjudicial for human health [30]. Hence, arginine is closely related to the levels of ethyl carbamate in wine [31]. Ethyl carbamate, also known as urethane, occurs naturally in wines and some other fermented food and beverages, and is classified as a posible human carcinogen [32]. On the other hand, biogenic amines are also undesirable in all foods and beverages because if they are absorbed

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R.M. Callejon, C. Ubeda, A.M. Troncoso and M.L. Morales

in high concentration, they can produce a headache, respiratory distress, heart palpitations, hyper-or hypotension and allergic effects.

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Figure 2. Figure2. Metabolic pathways of L-arginine ARG: arginase; ASL: argininosuccinat lyase; ASS1: argininosuccinat synthetase 1; OTC: ornithine carbamoyltransferase; CPS1: carbamoyl-phosphate synthetase 1, mitochondrial; ADC: arginine decarboxylase; GATM: glycine aminotransferase; RARS: arginyl-tRNA-synthetase; NOS: nitric oxide synthase; ODC1: ornithine decarboxylase; OAT: ornithine aminotransferase; PYCR1: pyrroline-5carboxylate reductase 1; P5CDh = ALDH4A1, aldehyde dehydrogenase family 4, member A1; P5CS: pyrroline-5-carboxylase synthetase; PRODH: praline dehydrogenase (oxidase) 1; SRM: spermidine synthase; SMS: spermine synthase.

Biosynthesis L-arginine is synthesized by three sequential steps differentially compartmented in the mammalian organism: biosynthesis of i) L-ornithine, ii) L-citruline, and iii) L-arginine (Figure 2). i)

L-ornithine is synthesized from L-glutamine and L-proline of food or blood derivates. This biosynthesis occurs almost exclusively in the small intestine [33] by the action of some enzymes such as L-1-pyrroline-5-carboxylate synthetase (P5CS) or proline dehydrogenase (PROHD) [34,35].

Analytical Methods of the Determination of Arginine Amino Acid

5

ii) The biosynthesis of L-citruline from L-ornithine depends on the presence of ornithine carbamoyltransferase (OTC) and carbamoylphosphatase (CPS1). The expression of both enzymes is restricted to the mitochondrial matrix of hepatocytes and epithelial cells of the small and to a minor extent large intestine [36]. iii) The biosynthesis of L-arginine from L-citruline is performed by the cytosolic enzymes arginimosuccinase synthetase 1 (ASS1) and argininosuccinate lyase (ASL). This is energetically costly, as the synthesis of each molecule of argininosuccinate requires hydrolysis of adenosine triphosphate (ATP) to adenosine monophosfate (AMP). Furthermore, the reaction catalysed by ASS requires L-aspartate as cosubstrate and is the rate-limiting step [37,38]. On a whole-body basis, synthesis of arginine takes place mainly via the intestinal-renal axis, where epithelial cells of the small intestine, which produce citruline, collaborate with the proximal tubule cells of the kidney, which extract citruline from the circulation and convert it to arginine, which is returned to the circulation. Synthesis of arginine from citrulline also occurs at a low level in many other cells, whose capacity for arginine synthesis can be markedly increased under circunstances that also induce nitric oxide synthase. Thus, citrulline, a coproduct of the nitric oxide synthase (NOS)catalyzed reaction can be recycled to arginine in a pathway known as the citrulline-NO or arginine-citrulline pathway. This is demonstrated by the fact that in many cells typed, citrulline can substitute for arginine to some degree in supporting NO synthesis [35, 39].

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Functions and Importance of Arginine Arginine is involved in multiple areas of human physiology and metabolism. Arginine plays an important role in cell division, the healing of wounds because it accelerates the collagen synthesis, removing ammonia from the body, the release of hormones such as growth hormone and immune function, [40,41]. L-arginine is used by the immune system to help regulate the activity of the thymus gland, which is responsible for manufacturing T lymphocytes. Therefore, this amino acid acts as an immune system enhancer since it stimulates the thymus gland increasing white blood cell production. Furthermore, L-arginine is involved in the synthesis of creatine, polyamines and DNA and it can decrease the cholesterol level [40]. Because arginine is produced naturally by the body, most people do not need to take extra supplements. However, during times of unusual stress or injury, the body may not be able to produce the necessary amount of arginine. Deficiency produces symptoms of muscle weakness, similar to muscular dystrophy. Arginine-deficiency impairs insulin production, glucose production, and liver lipid metabolism. Conditional deficiencies of arginine or ornithine are associated with the presence of excessive ammonia in the blood, excessive lysine, rapid growth, pregnancy, trauma, or protein deficiency and malnutrition [40]. Arginine deficiency is also associated with rash, hair loss and hair breakage, poor wound healing, constipation, fatty liver, hepatic cirrhosis, and hepatic coma. According to results of many research studies, the oral supplementation of L-arginine provides a range of benefits:

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R.M. Callejon, C. Ubeda, A.M. Troncoso and M.L. Morales

Arginine is the immediate precursor of NO, which is an endogenous messenger molecule involved in a variety of endothelium-dependent physiological effects in the cardiovascular system [40, 42, 43]. For being a precursor of NO, which relaxes blood vessels, arginine is used in many conditions where vasodilation is required such as in angina pectoris, congestive heart failure, hypertension, coronary heart disease, preeclampsia, intermittent claudication, and erectile dysfunction [40]. Arginine supplements appear to reduce mildly elevated blood pressure by enhancing the synthesis of nitric oxide in the cells that line the blood vessels. This helps dilate vessel walls and improve blood flow around the heart. In addition, arginine supplementation may reduce the risk of atherosclerosis since it prevents the production of plaques in blood vessel walls [44]. Every day there is more evidence according to the clinical experience, that we can use this amino acid, arginine, as a preventive agent for heart attacks. In addition, several clinical studies have shown that L-arginine, as food supplement has therapeutic value in a painful bladed disease known as interstitial cystitis because it avoids these strong contractions of the bladed which cause pain [45]. Nowadays, we know a lot of disease produced by strong muscle contractions where L-arginine plays an important role. For example, this amino acid has shown to have a therapeutic effectiveness as a nutrient in the treatment of hemorrhoids, which can be caused by anal sphincter spasms [46]. On the other hand, inadequate concentration of nitric oxide leads to vasoconstriction of the intestinal vessels, which might lead to ischemia and a predisposition to neuroendocrine carcinoma (NEC). Furthermore, nitric oxide acts as a neurotransmitter for enteric nonadrenergic non-cholinergic neurons that regulate peristalsis. Hence, lack or inadequacy of nitric oxide can alter intestinal motility. According to several studies, L-arginine has shown to play a prominent role in agerelated degenerative diseases such as Alzheimer´s disease [47]. In addition, L-arginine administration may improve disordered nitric oxid metabolism associated with allergic airway inflammation, and alleviates some features of asthma [48, 49]. Arginine also plays an important role in both innate and acquired immunity. Available evidence shows that arginine is required for defense against viruses, bacteria, fungi, malignant cells, intracellular protozoa, and parasites in mammals, birds, terrestrial animals, lower vertebrates and intervertebrates [40]. In fact, several studies have shown that L-arginine may be of benefit in individuals with human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS), and in the treatment of diseases caused by virus, such as herpes simplex virus or influenza A virus [49, 50]. As mentioned above, arginine may help stimulate the activity and increase the size of the thymus gland, which begins to decrease in size after puberty. Arginine is known to stimulate growth hormone release and has been said to increase muscle mass and fat loss [40]. Human growth hormone is secreted by a gland in the brain and has a direct effect on metabolism by increasing the levels of fat and glucose burnt for energy. For this reason, L-arginine is frequently used in growth hormone stimulation tests. Other studies have reported the success obtained by using arginine supplements for the treatment of male infertility. Seminal fluid is particularly abundant in polyamines (putrescine, spermidine and spermine), polycationic products of arginine degradation, that are essential for cell growth and differentiation [40]. Dietary arginine supplementation to boards increased sperm counts and sperm motility [21]. Thus, enhanging arginine provision may improve fertility in males.

Analytical Methods of the Determination of Arginine Amino Acid

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Dietary arginine supplementation inhibits the progression of colon cancer possibly by increasing NO secretion and consequently enhancing NK cell activity [51]. However, certain cancers may be auxotrophic for arginine and arginine deprivation is a novel approach to target tumors with lack argininosuccinate synthase expression such as melanoma, hepatocellular carcinoma, some renal cell cancers…etc [52]. Thus, whether arginine suppresses or enhances tumor growth depends on the relative activities of NOS and arginase pathways, whose expression may vary with the stage of carcinogenesis. This may explain apparently conflicting findings in the literature that arginine both stimulated and inhibited the growth of tumors [53]. Growing evidence indicates that arginine supplementation may be a novel therapy for obesity and the metabolic syndrome. First, dietary supplementation with arginine decreased plasma levels of glucose, homocysteine, fatty acids and triglycerides, and improved insulin sensitivity in chemically induced diabetic rats [54], genetically obese Zucker diabetic fatty rats [55] and diet-induced obese rats [56]. Similar results have been reported for obese humans with type-II diabetes receiving oral [57] or intravenous [58]. Hence, arginine may provide novel and effective therapies for obesity, diabetes, and the metabolic syndrome [40]. Apart from the benefits of arginine, there are several works about the toxicity of this amino acid. The first one reported on the stimulation of lymphocyte natural cytotoxicity by Larginine [35] and another work reported the use of high doses of dietary arginine during repletion impair weight gain and increased infectious mortality in protein-malnourished mice [59].

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Methods of Analysis Arginine is one of the most versatile amino acids from the metabolic and physiological point of view, as mentioned above. Hence, there is a need for the accurate and reliable quantification of this amino acid in food and various biological fluids. The quantitative determination of arginine in various biological fluids is of crucial importance to scientific progress in the field of cellular NO production and to investigate the clinical implications of disorders related to NO [42]. On the other hand, the determination of amino acids, such as arginine, is also of great importance to the food industry because of nutritional labelling requirements [60]. Microbial catabolism of amino acids produces flavour compounds of importance for foods, such as cheese, wine, honey and fermented sausages. The arginine supply in an infant´s first month of life must be sufficient in quantity and quality to fulfil the needs of this critical period. Accurate, fast and precise analytical methods are therefore needed for the detection and analysis of amino acids and oligopeptides compatible with the practical requirements of routine analysis and high sample throughput. In recent years, the evolution of instrumental analysis has allowed the detection and quantification of more and more free amino acids with increasing sensitivity and accuracy. The first qualitative test for arginine, was the diacetyl reaction of Harden and Norris [61], this test was applied later to its colorimetric estimation by Lang [62]. In 1911, Van Slyke [63] developed a method that estimated the content of arginine based on the decomposition of the guanidine group with the formation of ammonia. This method was slightly improved by

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R.M. Callejon, C. Ubeda, A.M. Troncoso and M.L. Morales

Plimmer [64] and Koekler [65]. As the determination of arginine by Van Slyke's method demands a considerable amount of material, colour tests for small quantities of arginine would be very useful. Arginine has a guanidin group in its chemical structure. In 1925, Sakaguchi [66] observed the extremely sensitive color reaction given by certain guanidine derivatives with α-naphthol and sodium hypochlorite and this reaction may be applied as a qualitative test for arginine. Sakaguchi's reaction for arginine is not only specific and very sensitive. As shown by Weber [67], the Sakaguchi reaction can be used for the quantitative determination of 0.05-0.005 mg arginine. The applicability of Sakagushi reaction and its various modifications to the detection and determination of arginine in solution has been documented. The different modifications of Sakaguchi reaction carried out, even by the author himself, have been aimed to improve the color stability and sensitivity. Weber, in 1930 [67], observed that when sodium hypobromite is employed the color development is practically instantaneous and the addition of urea stabilizes the color sufficiently to permit colorimetric determination. The molecule used as chromogen in this reaction has been the second point more modified [68-71]. The majority of published methods for the direct colorimetric determination of arginine residues in proteins have largely been based on Sakaguchi reaction [31]. Nowaday, this reaction continues to be used for the L-arginine analysis in food and biological materials [31, 72, 73]. Recently, Francis et al. [74], found that the reaction between arginine and hypobromite is chemiluminescent which has been used to develop an analytical procedure (rapid, simple and selective) for arginine in presence of other amino acids. Modern methods for separation and quantification of free amino acids either before or after protein hydrolysis include liquid chromatography (LC), gas chromatography (GC) and capillary electrophoresis (CE). Due to the zwitterionic character of these compounds it is necessary the use of adequate derivatization methods for LC, GC and CE assays [60]. Most of the LC methods proposed for the determination of plasmatic concentration of arginine and methyl derivates used pre-column derivatization and fluorescence detectors. However, these methods are often time-consuming due to tedious derivatization procedures and long running time [75, 76]. Although different methods are available, the LC with fluorescence detector remains the method of choice for research. Recently, the quantification of arginine in various biological fluids by chromatographicmass spectrometric means has reached a high degree of maturity, increasing the specificity and sensitivity of the methods and reducing the sample pre-treatment step [76].

Liquid Chromatography Ion Exchange Separation Traditionally the analysis of amino acids was carried out using ion exchange chromatography, where the amino acids were separated and then reacted with ninhydrin in a post-column derivatization system. Finally, they were detected by absorbance in the UVvisible region at one or two wavelengths [30]. The stationary phases of ion exchange columns, also known as ion exchange resins, are sulfonated polymers with a particle diameter of 5 to 10 µm. The separation mechanism is

Analytical Methods of the Determination of Arginine Amino Acid

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based on ionic interaction with a strongly acidic medium, which is the basis of the exchanger. First the acidic amino acids are eluted, followed by the hydroxylic, then neutral and finally the basic ones [77]. Nowadays, the columns are very efficient, so the analysis time is shorter, but they are quite expensive. Although this method has shown good reliability and an excellent resolving power, the analysis times are too long, the sensitivity is limited, the peaks that appear may be too wide, and post-derivatization systems are difficult to manage and maintain. This method gives good results but requires lengthy sample preparation [78]. It has also presented other problems such as matrix interferences and high detection limits [79]. Despite its drawbacks, ion-exchange chromatography with post-column derivatization gives more repeatable results than precolumn derivatization followed by reverse-phase liquid chromatography, because the chromatography and derivatization are two separate events that can be individually optimized [80].

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Reverse-Phase Liquid Chromatography In most studies dealing with the analysis of amino acids by liquid chromatography (LC), this has been carried out using reverse-phase columns that have a silica stationary phase with C-8 or C-18 groups. These columns, packed with very small silica particles (3-10 m) and small column diameter (2-5 mm), are subjected to high pressure and have a very controlled flow rate of mobile phases [30]. The mobile phases used most frequently in reverse phase are mixtures of water and an organic solvent (methanol, acetonitrile, or oxolane). When the sample has substances containing acidic or basic groups such as amino acids, the pH of the mobile phase is controlled through a buffer [77]. Reverse-phase liquid chromatography uses the solubility properties of the sample to partition it between a hydrophilic and a lipophilic solvent. The partition of the sample components between the two phases will depend on their respective solubility characteristics. Less hydrophobic components will associate primarily with the hydrophilic phase, while more hydrophobic components will be found in the lipophilic phase. The whole process depends on the extractive power of the hydrophilic phase. In the reverse phase, silica particles coated with chemically-bonded hydrocarbon chains represent the lipophilic phase, while the aqueous mixture of an organic solvent that surrounds the particles represents the hydrophilic phase. Since all the amino acids show a wide range of polarities, to resolve them in a single chromatogram it is necessary to vary the composition of the mobile phase to increase its elution power. In this way, using a gradient of polarity, it is possible to achieve the separation of all the compounds in a reasonable time, which would be impossible to achieve with an isocratic elution [77]. Reverse-phase LC uses pre-column derivatization methods. This technique is simpler, faster, has greater sensitivity, and uses somewhat less expensive LC systems that operate at higher pressures compared to dedicated, ion exchange-based amino acid analyzers [80]. This technique has been widely employed for the determination of arginine in biological samples, such as human plasma [16], serum, urine, and cerebrospinal fluid (CSF) [81]; and

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R.M. Callejon, C. Ubeda, A.M. Troncoso and M.L. Morales

foods and beverages such as strawberry fruits [82], honey [83], cheese [84], wines [22] [85,86] [87, 88], beers [24, 26] and vinegars [27, 29, 89]. Although many published LC methods coupled to conventional detectors as ultraviolet or fluorescence have been adequately validated [81], is growing more and more the use of LC methods involving mass spectrometry (MS) detection for the determination of arginine in biological fluids and foods [60,75,90]. Atmospheric pressure ionization is the currently most used ionization technique for quantitative LC-MS methods. Two general substypes of this technology exist: electrospray (ESI) and atmospheric pressure chemical ionization (APCI). Both are considered as “soft” ionization procedures and both produce mainly protonated or deprotonated ions without fragmentation [42]. In both ESI and APCI, the ionization rate of analytes depends strongly on the physicochemical environment in the ion source. ESI is reported to be more susceptible to subtle changes in the characteristics of the LC-effluent than APCI. However, in both methods, samples with a complex matrix like biological fluids can cause MS signal suppressions or enhancements, which are termed “matrix effects”. It is widely believed that these effects are due to ionization competition between different species eluting from HPLC column. On the other hand, a sophisticated detection technique that provides high sensitivity is LC coupled to two consecutive MS detectors (LC-MS-MS). Vishwanathan et al. [91] reported the first LC-MS-MS method for the determination of arginine. After protein precipitation with acetonitrile and solvent evaporation, the underivatized amino acids were separated on a straight phase silica column and the chromatographic run time was about 15 min. The retention of arginine in its underivatized state on a reverse phase 18 column was achieved by Huang et al. [92] from urine as well as from plasma samples [93]. Both methods are quite similar with regard LC separation and MS detection. As it was demonstrated by Vishwanathan et al. [91] and Huang et al. [92, 93], the very polar endogenous amino acids were not easily to retain and separate on reversed phase LC columns. As a consequence of these findings, Martens-Lobenhoffer and Bode- Böger [94] developed an assay applicable to human plasma and urine, which derivatized the analytes with ortho-phtalaldehyde (OPA) and 2-mercaptoethanol as co-reagent prior to LC separation, following similar approaches developed for fluorescence detection [42]. Besides, LC-OPA methods with fluorescence detection, the OPA/2-mercaptoethanol derivatives of arginine were analyzed by ESI-MS [94]. Because of the superior selectivity of the mass spectrometric detection, the laborious sample cleanup necessary for the relatively unselective fluorescence detection could be avoided, and sample preparation was reduced to protein precipitation for plasma and dilution for urine samples. An LC-MS-MS method by reverse phase ion-pairing chromatography was described by Piraud et al [95] for the separation and quantitative determination of 76 underivatized amino acids and related compounds including arginine. The ion-pairing agent was tridecafluoroheptanoic acid, and the separation took place on a reversed phase C18 column with an acetonitrile gradient. Recently, Matns-Lobenhoffer et al. [96] reported an LC-MS-MS method for the separation of Arginine by hydrophilic interaction liquid chromatography (HILIC). In this method, the amino acids were separated in their underivatized state on a straight phase silica column in only 8 min. HILIC has great advantages in LC-MS analysis of polar substances over reserved phase chromatographic approaches. The most important one is that the mobile phase greatly favors the ionization process in the ESI ion source [97].

Analytical Methods of the Determination of Arginine Amino Acid

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Gas Chromatography Gas chromatography (GC) is another alternative for the analysis of amino acids. This technique is fast and has a high power of resolution and high sensitivity, but its performance requires considerable experience. Since free amino acids are not sufficiently volatile, they have to be converted into volatile derivatives to be determined. The production of several different esters of amino acids has been used for the profiling and quantification of amino acids in food products by GC [80]. Hence, forty seven biological amino acids were derivatized by a single-step reaction using N-methyl-N-(tert-butyldimethyl-silyl) trifluoroacetamide, and successfully separated on a HP-1 capillary column [98]. A procedure for the quantitative determination of arginine in biological fluids like human plasma and cell culture supernatant utilizing such a derivatization for subsequent GC-MS analysis has been reported by Tsikas et al. [81]. The sample cleanup step in this method was ultrafiltration of plasma samples to remove porteins and subsequent evaporation of the biological liquid. Derivatization of the acid- and amino-functions of the amino acids took place in a first step by esterification with acidic methanol and in a second step by the conversion of the amino moieties into pentafluoropropionic acid amides with pentafluoropropionic anhydride (PFPA). After such a derivatization, arginine was suitable for GC separation, resulting in sharp and symetric peak shapes on a medium polar Optima-17 capillary column (Macherey Nagel, Germany). Another procedure for the determination of arginine in human plasma and cell culture supernatant has been published by Albsmeier et al. [99]. Sample cleanup consisted of protein precipitation with acetone for plasma and solid phase extraction with carboxyl acid ion exchange columns for cell culture supernatant. A two-step derivatization with acidic methanol and PFPA was performed prior to GC separation, similar to the procedure described by Tsikas et al [81]. After separation on an Optima-17 capillary column (Macherey Nagel, Germany), the analytes were detected by single stage negative-ion chemical ionization (NICI)-MS. No interferences from endogenous substances were observed in the chromatograms. The main disadvantage of GC seems to be the procedure of derivatization, due to the complexity of reactions and types of reagents used. The speed at which derivatization takes place differs from one amino acid to another, and strict reproduction of reaction conditions is essential for all samples. Moreover, most of the volatile derivatives of amino acids may be lost during the concentration of the sample. Furthermore, this technique requires a careful sample extraction and preconcentration, and these requirements must be taken in account when assessing the reliability of final results [79]. Despite these drawbacks, GC is considered selective, sensitive, precise, accurate, inexpensive, and versatile, relative to ion-exchange methodology, and its ability to interface with MS allows identifications based on more than just retention times [80]. GC coupled with mass spectrometry (GC-MS) can be a useful alternative to other methods of amino acid analysis, especially when the sample quantities are limited and high sensitivity is required [100]. This technique has been applied in atmospheric aerosols [101].

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R.M. Callejon, C. Ubeda, A.M. Troncoso and M.L. Morales

Capillary Electrophoresis

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Capillary electrophoresis (CE) has been used to separate and detect amino acids, peptides and proteins. This technique allows the analysis of extremely small sample volumes, and therefore, it requires detection of high sensitivity. Methods of detection used include laserinduced fluorescence, which has limits of detection of about 10-20 moles for the separation by CE of derivatized amino acids. One drawback of this technique is the length of time taken in sample preparation, since it requires a process of derivatization and pre-concentration prior to analysis [102]. The derivatized analyte may have additional properties that enhance separation. Pre-column, post-column and on-column derivatizations have been used for CE separations, although on- and post-electrophoretic derivatizations are mostly employed. In addition, CE can be applied with a laser-induced fluorescence detector for post-column reactions [103] and with electrochemical detection [104]. The analysis of amino acids by CE gives extremely low detection limits but, in practice, these low limits of detection are not required in routine analysis of amino acids in food and drink products. However, levels of amino acids, specifically arginine, in biological fluids are much lower and in future methods of food processing, new compounds may occur at levels sufficiently low to recommend the application of this technique. Relative to LC methods for separation of amino acids, CE is cheaper, involves shorter analysis times without the need for gradient elution, uses less solvent (mL/day), produces less solvent waste, and its mass detection capability is 1000-times lower due to smaller injection volumes. It also offers different selectivity and higher efficiency compared to LC [105]. There are many applications of this technique for the determination of arginine in human plasma [76] and in foods [80,106].

Nuclear Magnetic Resonance Spectroscopy Nuclear magnetic resonance (NMR) spectroscopy offers some outstanding advantages in the field of chemical analysis of food products because it is non-destructive, selective and capable of simultaneous detection of a great number of low molecular mass components, such as arginine, in complex mixtures [102]. The popular and widely-used LC and CE techniques are more sensitive than highresolution NMR spectrometry, but they require time-consuming sample preparation before measurements can be made. Separation, derivatization, and preconcentration in the case of compounds in low concentration, are usually common steps in these procedures. However, sample preparation for NMR spectroscopy is simpler and less time-consuming. Another great advantage of high-resolution NMR is the possibility of detecting the magnetic resonance of different nuclei present in a molecule in different electronic and spatial environments. This technique has proven to be useful for assessing wine quality; for example, it has been used in the verification of the origin and age of wine, and the effects of adulteration. In recent years the use of high-resolution NMR techniques in the study of wine has attracted the interest of several groups, and, as result, 1-dimensional and 2-dimensional NMR experiments have been conducted to characterize and classify a wide variety of wines [107,108]. In addition, proton nuclear magnetic resonance (1HNMR) spectroscopy with pattern recognition

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13

methods has been used to investigate the metabolic differences in grape pulp, skin, seed and wines from different regions [108].

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Methodological Considerations Regarding the Quantification of L-Arginine in Biological Matrices The quantification of arginine in biological fluids is associated with numerous analytical difficulties. In plasma, most physiological amino acids are present in concentrations of the same order of magnitude as arginine, i.e. they are of the order of 50 µM. Hence, this condition requires assays of high specificity and sensitivity. Arginine is lacking of chromophores, so that no specific identification of this substance by UV absorbance detection is possible. Furthermore, as amino acids are polar, thermally labile and non-volatile compounds, their analysis without derivatization is difficult by means of reversed phase LC and impossible by GC. Likewise, effective separation and detection in CE methods requires derivatization of the analytes [42]. Nevertheless, because all physiological amino acids result in similar derivatives, the problem of unspecific detection persists if the analytes are monitored by detectors of limited selectivity such as UV absorbance and fluorescence detectors. On the other hand, use of mass spectrometers adds additional dimensions of selectivity because analytes are identified due their characteristics molecular mass-to-charge (m/z) ratio, and, if applicable, by the fragmentation pattern, in addition to their characteristic retention times in LC or GC. Therefore, the selectivity of mass spectroscopy-based approaches is considerably higher in comparison to conventional detectors used in LC and GC, and quantification errors due to interferences are minimized [42]. Another potential problem in the accurace quantification of arginine and its derivates is the availability of a suitable internal standard. Most of the conventional LC methods use homoarginine as an internal standard, which is, however, also an endogenous substance [42]. This may lead to systematic errors in the quantification, since the total concentration of the internal standard, i.e. endogenous plus added, is unknown and variable within individual samples. Calibration of assay for endogenous substances such as arginine is also associated with analytical problems and pitfalls. No biological matrix without an endogenous content of the analytes is available. When calibration in a matrix such as plasma is done by adding up the analyte in increasing concentrations, the resulting calibration curve will not go through the origin, but it will intercept the y-axis at a value corresponding to the endogenous concentration of the analyte in the matrix. Therefore, the true analyte concentration in the matrix is within the range of the lowest level of the calibration curve, where the analytical imprecision is in general most prominent. On the other hand, when the calibration curve is prepared in aqueous phase free of analyte, a possible matrix effect of the biological fluid will not be taken into account at all [109]. Matrix effects are generally not reproducible nor repeatable between various samples or even between different injections of the same sample and, thus, can severely compromise quantitative analysis [110]. Therefore, the careful evaluation of matrix effects has to be an integral part of the validation of quantitative methods in LC-MS.

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R.M. Callejon, C. Ubeda, A.M. Troncoso and M.L. Morales

Matrix effects can be minimized by improving the sample preparation to achieve as clean as possible extracts, by optimizing the chromatographic procedure to separate the analytes from the matrix effects, by changing the ionization conditions, or by a combination of the above. Table 2. Advantages and disadvantages of pre-column and post-column derivatization [30] Derivatization

Post-column

Advantages

It is possible to use different detection systems and elute the compounds detecting them by nondestructive methods prior to derivatization. The reaction is reproducible without the need to form a single derivative.

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The only limitation to the conditions of the reaction is that it must be completed in a reasonable time and quantitative

Pre-column

The reaction can be performed in a solvent not compatible with the mobile phase used in chromatographic separation. The secondary product formed in the column or before chromatographic separation can be separated.

Disadvantages Presence of interferences due to excess of reagent or degradation products. Loss of resolution caused by the widening of the chromatographic bands in the reactor where the reaction is performed. It is not allowed to use very long retention times. This method can be expensive due to the changes that have to be performed to carry out the reaction. Presence of interfering peaks in the chromatograms due to the reagent, reaction or degradation products or impurities of the reagents. Hence, it is convenient to remove the excess of reagent, solvents or other components of the reaction mixture prior to injection into the chromatograph. A substantial part of all derivatives will be identical. Minor differences in side chain of amino acids will have less effect on the chromatographic behaviour of the derivatives, making the separation more difficult

Derivatization Reagents for LC Analysis Amino acids, specifically arginine, can be detected directly by ultraviolet (UV) or visible light detection since they absorb at a wavelength between 190-210 nm. However, the majority of solvents and other components of the samples also absorb in this region of the spectrum; hence, the amino acids have to be derivatized prior to analysis. Such derivatization can be undertaken either before (pre-column), or after (post-column) chromatographic separation of amino acids, and more rarely, on the column (on-column). Each way of obtaining the derivative has its own advantages and disadvantages (Table 2).

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In post-column derivatization the separation of amino acids is carried out with a cation exchange resin and a gradient of acidic buffers. After separation, amino acids are converted into coloured ninhydrin derivatives for colorimetric detection, or into ortho-phtaldehyde (OPA) for fluorescence detection. Although pre-column derivatization presents more advantages, traditional post-column methods have not been totally discarded. Various different derivatizing agents have been employed for the determination of amino acids. Those most widely used for the determination of arginine are listed below and their corresponding advantages and disadvantages are shown in Table 3.

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Ninhydrin Nindydrin-based monitoring systems remain among the most widely-used methods for the quantitative determination of amino acids after they have been separated in their native form by ion exchange chromatography. Hence, derivatization with ninhydrin is exclusively of the post-column type [111]. The sensitivity of the reaction with ninhydrin is below 1 pmol but it is rarely reproducible below 100 pmol. This limitation makes it unsuitable for many of the more demanding applications. Ninhydrin can easily deteriorate on exposure to light, atmospheric oxygen and changes in pH and temperature. Therefore, these factors must be considered when this agent is used. Ninhydrin decarboxylates and deaminates the primary amino acids, forming the purple complex known as Ruhemann´s Purple, which absorbs maximally at 570 nm. A yellowcoloured product, which can be monitored at 440 nm, is formed upon reacting ninhydrin with the secondary amino acids, proline and hydroxyproline. When ninhydrin becomes oxidized, its colour does not develop well at 570 nm, but absorption at 440 nm remains fairly constant. A good indicator of reagent degradation is when the height of the proline peak at 440 nm approaches the height of the glutamic acid peak at 570 nm for equal amounts of each. Some authors have applied this technique for the characterization of wines based on the content of amino acids and biogenic amines [112]. It has also employed to examine the rateof-living theory according to age-related changes in amino acids [113] or, for example, to determine amino acid composition of feeds [114].

Dansyl Chloride Dansyl chloride (5-N,N-dimethylamino-naphthalene-1-sulfonyl chloride) (Dns-Cl) is a well-known fluorogenic pre-column derivatization reagent for the determination of primary and secondary amines. Dansylation has been used mainly as a method for determining free amino acids, as well as protein hydrolysate amino acids and terminal amino acids from proteins and peptides. Dansyl amino acids are detected by fluorescence (ex 360 nm; em 470 nm) and UV (max 250 nm). Detection levels are in the pmol or fmol range, depending on the sensitivity of the detector [111].

Table 3. Agents employed in derivatization of arginine Reagent

Derivatization

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Type

Ninhydrin

Postcolumn

Dns-Cl

Pre- and postcolumn

Conditions

High temperature (110ºC)

25-40ºC in the dark

Time (min)

Separation

Run Time (min)

Detection

Advantages

Able to detect pmol.

10

Ion exchange

35-60

Reverse phase: - Phenomenex® Luna C18 - Spherisorb ODS2

30-80

Fluorescence UV

Detection levels are in the pmol or fmol range. Derivatives are stable to hydrolysis. Good reproducibility

50-90

Visible

Derivatives are stable during 4 weeks at room temperature. Able to detect pmol.

10-30

Fluorescence

Detection levels are in the pmol range.

DABS-Cl

Precolumn

70ºC

10-20

Reverse phase: - Spherisorb ODS2 - Lichrospher 100 RP-18

DNFB

Precolumn

50-60 ºC

30 min in the dark

Reverse phase: - Catridge C18

60-120

UV

Disadvantages Rarely reproducible below 100 pmol. Problems of interferences with matrix. Sensitive to light, O2, temperature changes and pH. Reagent excess interferes with amino acid chromatogram peaks. Derivatives are photosensitive. Slow reaction. Not specific and reacts with other compounds. Reagent excess interferes with amino acid chromatogram peaks. Presence of an excess of salt and detergents interferes with the reaction. It produces multiple derivatives. Derivatives are photosensitive. Slow reaction. Destructive method for peptides.

Ref.

[111] [112]

[89]

[88]

[77]

PITCa

OPA

Pre- and postcolumn

Room Temperature

Room Temperature

Precolumn

Room Temperature

DEEMM

Precolumn

25-50ºC in a methanolic medium

AQC

Precolumn

Room Temperature

FMOC-Cl

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Precolumn

10-20

Reverse phase: - Spherisorb ODS2 - Pico Tag Column

1-2

Ion exchange and Reverse phase: - Nova Pack C18 - Hypersil ODS

6-10

30-50

10

Reverse phase: - Nucleodour C18 - Hypersil ODS

Reverse phase: - Nova Pack C18

Reverse phase: - Nova Pack C18 - Phenomenex® Luna C18

10-70

6-40

30-40

UV

Fast reaction. Derivatives are more stable than others. Reagent excess does not interfere.

Fluorescence UV

Reagent excess does not interfere. Fast reaction. Derivatives are highly fluorescent. 10 times more sensitive than reaction with Ninhydrin.

Fluorescence

32-35

UV-Visible

70-80

Fluorescence UV

Fast reaction. Derivatives are stable and highly fluorescent. Very sensitive.

Direct derivatization without prior preparation. Reagent excess does not interfere. Sensitivity of pmol. Reagent excess does not interfere. Derivatives are stable and highly fluorescent. Very sensitive (50-300 fmol). Fast reaction. Direct derivatization without prior preparation. Salts and detergents present in samples do not interfere.

Problems of interferences with the matrix in analysis of grape musts. Not suitable for automation. Less sensitivity than other methods. The procedure consists of several manual steps. Unstable derivatives. Needs a complete automation of the reaction.

Produces multiple derivatives. Highly fluorescent and interferes with amino acid chromatogram peaks. Derivatization process is slow since the reagent has to be eliminated.

[28]

[22] [27] [78] [79]

[87]

Slow derivatization process Not sensitive below pmol level.

[12] [126]

If ammonia is not completely derivatized, the excess may distort the analysis of Arg

[127] [132]

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Dansyl amino acids are formed under optimal conditions within 35-60 min in the dark. On the other hand, any excess of Dns-Cl reacts with dansyl amino acids producing dansyl amide. Dansyl amide formation is an unavoidable limitation of the method and the quantity formed depends on the amino acid concentration and the excess of dansyl chloride. Hence, it is convenient to minimize the emergence of such secondary products, as far as possible. Dansyl derivatives are relatively stable against hydrolysis, which does not occur with the reagent. However, the exposure of the derivatives to light should be avoided because they are photosensitive. Dansyl amino acids are stable for at least 7 days if they are kept at 4ºC. This technique is generally used in pre-column derivatization. Many authors have applied it for the determination of amino acids, including arginine, in food such as chesse, clams, salami, beers [115], grape musts, wines and vinegars [89]. This Dns-Cl method gives a good reproducibility for most arginine due to the low relative fluorescence response of the didansylated adduct and the formation of two dansyl derivatives [111].

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Dabsyl Chloride Another derivatizing agent used for the determination of amino acids is dabsyl chloride (4-dimethylaminoazobenzene-4-sulfonyl chloride) (DABS-Cl). Dabsyl derivatives present maximum absorption at 420 nm (visible region), are highly stable and are readily separated by LC and detected at the pmol level. These derivatives remain quite stable for four weeks at room temperature [30]. A limitation of the DABS-Cl method is that the presence of excess amounts of salt, urea, SDS, phosphate, or ammonium bicarbonate will alter the pH of the buffer and interfere with the dabsylation reaction. For the quantitative analysis of unknown samples, the DABS amino acid standards have to be obtained from an amino acid standard mixture that has been hydrolyzed and dabsylated in parallel with the unknown samples under identical conditions [111]. Krause et al. [45] proposed the use of dabsyl chloride as an alternative method to conventional analysis of amino acids, including arginine, and biogenic amines in food (cheese, meat, sausages, fish) and biological samples (plasma, tissue), and achieved the separation of more than forty compounds simultaneously.

1-Fluoro-2, 4-Dinitrobenzene 1-Fluoro-2,4-dinitrobenzene (FDNB) is a pre-column derivatizing agent used for the characterization of terminal amino acids of peptide chains. This agent reacts with primary and secondary amino acids to form compounds that are fluorescent at 365 nm. The reaction is carried out at 50 ºC for 30 minutes. Dinitrophenyl amino acids are known to be photosensitive but, when shielded from light, are stable for 48 hours. FDNB can detect small amounts of amino acids, in the low pmol range, and is suitable for determining amino acids that are poorly resolved in other systems [111].

Analytical Methods of the Determination of Arginine Amino Acid

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Phenylisothiocyanate The derivatizing agent isothiocyanatobenzene, also known as phenylisothiocyanate (PITC), has been used for more than 30 years in the Edman degradation method for sequencing peptides and proteins. PITC reacts with free amino acids to yield phenylthiocarbamyl (PTC) amino acids. This agent has been applied to determine free amino acids, including arginine, in serum and organs (liver, brain and heart) (Shervington and AlTayyem 2001) and in food samples such as orange juices [116], wines [117] and vinegars [28]. Under mild conditions, the reaction is essentially complete in less than 10-20 min and, because the reagent is volatile, a large excess can be used. This excess can be readily removed under reduced pressure. The dried samples may then be stored at -20ºC, at which temperature they should be stable for 4 weeks. Since the derivatives do not fluoresce, the technique is limited to UV detection, which normally takes place at 254 nm (max= 269 nm). Although this technique is not as sensitive as some of the fluorescent reagents, the highly UV-absorbing PTC derivatives can be detected at the low pmol level, which represents the practical level of sensitivity for real samples. The limit of sensitivity is 50 pmol at a signal-to-noise ratio of 2.5, which is ca. 50 times less sensitive than detection of OPA or FMOC adducts.

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Ortho-Phtaldehyde Orto-phtaldehyde (OPA) was introduced in 1971 and is probably the most commonly used derivatization agent in reverse-phase LC for the determination of free amino acids. It has been applied in wines [22, 86, 117], vinegars [27], foods [118], honeys [119], beers [120] and biological fluids [94] OPA can also be used in CE and ion-exchange chromatography. Another application of this reagent is the determination of enantiomeric amino acids in foods and beverages [118]. OPA is a reagent that does not have a natural fluorescence, but this develops when OPA reacts with primary amino functions, such as arginine. This reagent may be applied as either a pre-column or a post-column derivatizing agent. The reaction between OPA and amino acids takes place in an aqueous medium at a strongly alkaline pH in the presence of a thiol such as 2-mercapto-ethanol, 3-mercapto-1propanol or ethanothiol to form highly fluorescent derivatives). The isoindol derivative formed is unstable and must be stabilized by acidification. Reaction is fully completed in 1 or 2 min at room temperature [77]. There is no need to remove excess OPA prior to sample injection since OPA itself will not interfere with separation or detection. However, since OPA-amino acid derivatives are unstable, complete automation of the pre-column reaction, with accurate control of reaction time, is essential for reproducible results. The limit of detection of OPA is around the fmol range; hence, this technique is 10 times more sensitive than the ninhydrin reaction. The resulting derivatives can be also detected by UV monitoring at 330 nm, but for higher sensitivity, fluorescence detection is often chosen and emission is measured at wavelengths above 430 nm.

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R.M. Callejon, C. Ubeda, A.M. Troncoso and M.L. Morales

9H-Fluoren-9-Ylmethyl Chloroformate 9H-Fluoren-9-ylmethyl chloroformate (FMOC-Cl) has recently been shown to be a suitable pre-column derivatizing reagent for the determination of primary and secondary amino acids, for which similar responses are given. This reagent results in highly fluorescent and stable derivatives [121]. In contrast to the other pre-column derivatization reagents that yield fluorescent derivatives (i.e. Dns-Cl and OPA), the reagent FMOC-Cl is fluorescent itself. Both FMOC and its hydrolysis products have absorption and fluorescence spectra that are similar to those of FMOC-amino acids, hence, they can interfere in the quantification of amino acid derivatives. However, this property need not be a limiting factor, since the reagent excess and fluorescent side-products can be eliminated without loss of the amino acid derivatives by liquid-liquid extraction. Excess FMOC remaining after derivatization reacts with water to form 9-fluorenemethanol (FMOC-OH). An alternative method of preventing interference of FMOC-OH is the reaction between FMOC and a very hydrophobic amine to form a derivative that elutes after the peaks of interest [111]. This derivatizing agent has been used to determine biogenic amines and their amino acid precursors in wines [121, 117] and foods [122, 123], and has also been applied to determine amino acids in botanical preparations [124].

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Diethyl 2(Ethoxymethylidene)Propanedioate Diethyl 2(ethoxymethylidene) propanedioate, also known as diethyl ethoxymethylene malonate (DEEMM) is another pre-column derivatizing agent which gives amino acid derivatives detectable in the ultraviolet region. The derivatives are easily obtained and are very stable. This agent has been employed to determine arginine and other amino acids in wines and in different types of Spanish honey [125]. In this last-cited research work, the information obtained was examined in order to establish whether free amino acid composition of a honey can explain its botanical origin. The derivatization reaction is carried out in a methanolic medium for 30-50 min. Then, the sample is heated for 2 hours at 70 °C for the complete degradation of excess reagent and its side-products. Most of the derivatives are perfectly stable, at least during the first week. Hence, for quantification purposes, the analysis should be performed in the 24 hours following the derivatization reaction. This is one of the disadvantages of the method. Moreover, limits of detection are under 0.4 mg/L for amino acids and 0.07 mg/L for biogenic amines; hence, this reagent should be used when a sensitivity of below pmol is not required. Another disadvantage is the time required for the derivatizion, since other agents give derivatization in shorter periods of time [126]. However, this reagent presents some advantages such as direct derivatization without previous preparation; simultaneous quantification of 24 amino acids, biogenic amines and the ammonium ion; the UV detector, which is the type available in most laboratories, can be employed, and there is no interference from the reagent excess.

Analytical Methods of the Determination of Arginine Amino Acid

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6-Aminoquinolyl-N-Hydroxysuccinimidyl Carbamate 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) is a derivatizing agent specifically designed for the analysis of amino acids with the object of simplifying the derivatization reaction, increasing the yields of the reaction, and increasing the sensitivity and selectivity of the derivatives formed when fluorescence detection is used [127-129]. This compound reacts quickly with primary and secondary amino acids forming stable and highly fluorescent products at 395 nm. The derivatives are stable at room temperature for at least one week, and they are easily separated by reverse-phase LC using a C18 column. The excess reagent is hydrolyzed during the reaction to form 6-aminoquinoline (AMQ), whose spectral characteristics are different from any of the amino acids derivatized. It allows a wavelength to be selected that maximizes the emission response of derivatives and minimizes the response of the AMQ. During the hydrolysis of the reagent Nhydroxysuccinimide and carbon dioxide are also formed, but they do not interfere in the chromatographic analysis. Destruction of excess reagent is completed in less than 1 min. The protocol for derivatization is simple and direct. The reagent is added to the sample pre-buffered and then is heated to carry out the reaction. Derivatives are injected without additional preparation of the sample, since salts present in the sample do not interfere in the reaction or in the reproducibility of results [129]. AMQ absorbs about 200 times more than any of the derivatized amino acids using a UV detector at 250 nm. This can cause difficulties in the quantification of aspartic acid, which is the first amino acid that elutes. This does not happen when the detection is performed by fluorescence, because the signal from the AMQ is much smaller than that obtained in UV detection. Another characteristic of AMQ is that its retention time can vary depending on the pH of the mobile phase. This reagent has been used as an alternative to the most common derivatization agent, OPA, to determine biogenic amines and amino acids in wines [130-133] and to study the evolution of amino acids and peptides during alcoholic fermentation and autolysis of wines [134]. In addition, AQC has been applied to determine amino acids in vinegars in submerged and surface acetifications [29]. Recently, this compound has shown to be suitable for the determination of arginine and other amino acids in beers [24], cheese [135], infant food [136] and biological materials [137].

Conclusion Arginine is a component of dietary protein and body fluids. Based on nitrogen balance and functional needs beyond tissue protein synthesis, arginine is classified as a nutritionally “semiessential” amino acid in certain conditions, including inflammation, dysfunction of small bowel or kidney, or in neonates and premature infants. In animals and humans, this amino acid fulfills versatile physiological functions and it has shown a beneficial effect in treating many developmental and health problems. Hence, there is a widespread interest in the determination of arginine. Different methodologies for the analysis of this amino acid have been outlined, and specific applications for the analysis of arginine are discussed. Modern methods for separation and quantification of arginine include LC, GC and CE. However,

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R.M. Callejon, C. Ubeda, A.M. Troncoso and M.L. Morales

these methods are often time-consuming due to tedious derivatization procedures and long running time. Recently, LC coupled to MS has shown to reach more sensitivity and selectivity. This technique has been used with and without a previous derivatization. The reagents most widely used for derivatization are: Dns-Cl, DABS-Cl, DNFB, PITC, FMOC-Cl, OPA, EMMDE and AQC. However, none of these reagents is considered “the universal agent” since none of them meets all the requirements [30].     

It must react rapidly under mild conditions to provide quantitative yields of derivative. The derivatives should remain stable for several days, preferably at room temperature, to permit automated analysis of multiple samples. There should be no interference from the reagent, breakdown products, or side reactions. Response should be linear over the concentration ranges typical of most applications. The derivatives should have reasonably similar molar response factors.

Each of the methods has its own advantages and disadvantages. Therefore, the predominant criteria that will influence selection of the most suitable type of derivatization and analytical procedure are the degree of resolution, sensitivity and speed required in the analysis, in each case.

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[72] Li, H; Liang, X; Feng, L; Wang, H. Determination of L-arginine content in fermented liquor by thymol-spectrophotometric method. Zhongguo Shipin Xuebao, 2007, 7(4), 126-131. [73] Liang, X; Zhao, G; Zhao, R. Spectrophotometric determination of L-arginine in fermentation broth. Shipin Yu Fajiao Gongye, 2007, 33(4), 122-125. [74] Francis, PS; Barnett, NW; Foitzik, RC; Gange, ME; Lewis, SW. Chemiluminescence from the Sakaguchi reaction. Analytical Biochemistry, 2004, 329(2), 340-341. [75] Wang, HY; Hu, P; Jiang, J. Rapid Determination of Underivatized Arginine, Ornithine, Citrulline and Symmetric/Asymmetric Dimethylarginine in Human Plasma by LC-MS. Chromatographia, 2010, 71(9/10), 933-939. [76] Desiderio, C; Rossetti, DV; Messana, I; Giardina, B; Castagnola, M. Analysis of arginine and methylated metabolites in human plasma by field amplified sample injection capillary electrophoresis tandem mass spectrometry. Electrophoresis, 2010, 31(11), 1894-1902. [77] Cáceres, I; Barahona, F; Polo, C. El análisis íntegro de los vinos. Cromatografía de líquidos de alta eficacia. Alimentos, Equipo y Tecnología, 1986, 5, 141-152. [78] Pripis-Nicolau, L; De Revel, G; Marchand, S; Beloqui, AA; Bertrand, A. Automated HPLC method for the measurement of free amino acids including cysteine in musts and wines; first applications. Journal of the Science of Food and Agriculture, 2001, 81(8), 731-738. [79] Herbert, P; Barros, P; Ratola, N; Alves, A. HPLC determination of amino acids in musts and port wine using OPA/FMOC derivatives. Journal of Food Science, 2000, 65(7), 1130-1133. [80] Peace, RW; Gilani, GS. Chromatographic determination of amino acids in foods. Journal of AOAC International, 2005, 88(3), 877-887. [81] Tsikas, D. A critical review and discussion of analytical methods in the Larginine/nitric oxide area of basic and clinical research. Analytical Biochemistry, 2008, 379(2), 139-163. [82] Zhang, J; Yang, H; Wang, X; Hu, Y; Fang, C. Determination of the major amino acid components by high performance liquid chromatography coupled with fluorescence detection during the development of strawberry fruits. Anhui Nongye Daxue Xuebao, 2009, 36(3), 377-381. [83] Pereira, V; Pontes, M; Câmara, JS; Marques, JC. Simultaneous analysis of free amino acids and biogenic amines in honey and wine samples using in loop orthophthaldehyde derivatization procedure. Journal of Chromatography A, 2008, 1189, 435-443. [84] Korös, A; Varga, ZS; Molnár-Perl, I. Simultaneous analysis of amino acids and amines as their o-phthalaldehyde-ethanethiol-9-fluorenylmethyl chloroformate derivatives in cheese by high performance liquid chromatography. Journal of Chromatography A, 2008, 1203, 146-152. [85] Martínez-Rodríguez, AJ; Carrascosa, AV; Martín-Álvarez, PJ; Moreno-Arribas, V; Polo, MC. Influence of the yeast strain on the changes of the amino acids, peptides and proteins during sparkling wine production by the traditional method. Journal of Industrial Microbiology & Biotechnology, 2002, 29(6), 314-322. [86] Villamiel, M; Polo, MC; Moreno-Arribas, MV. Nitrogen compounds and polysaccharides changes during the biological ageing of sherry wines. Food Science and Technology, 2008, 41, 1842-1846.

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[87] Lozanov, V; Petrov, S; Mitev, V. Simultaneous analysis of amino acid and biogenic polyamines by high-performance liquid chromatography after pre-column derivatization with N-(9-fluorenylmethoxycarbonyloxy)succinimide. Journal of Chromatography, A, 2004, 1025(2), 201-208. [88] Krause, I; Bockhardt, A; Neckermann, H; Henle, T; Klostermeyer, H. Simultaneous determination of amino acids and biogenic amines by reversed-phase high-performance liquid chromatography of the dabsyl derivatives. Journal of Chromatography, A, 1995, 715(1), 67-79. [89] Valero, E; Berlanga, TM; Roldán, PM; Jiménez, C; García, I; Mauricio, JC. Free amino acids and volatile compounds in vinegars obtained from different types of substrate. Journal of the Science and Food Agriculture, 2005, 85, 603-608. [90] Erxleben, BT; Mreyen, M. Analysis of amino acids in foods using LC-MS. LC-GC Europe, 2009, 15-16. [91] Vishwanathan, K, Tackett, R.L; Stewart, J.T; Bartlett, MGJ. Determination of arginine and methylated arginines in human plasma by liquid chromatography-tandem mass spectrometry. Journal of Chromatography, B: Biomedical Sciences and Applications, 2000, 748(1), 157-166. [92] Huang, L.F; Guo, FQ; Liang, YZ; Hu, QN; Cheng, BM. Rapid simultaneous determination of arginine and methylated arginines in human urine by highperformance liquid chromatography-mass spectrometry. Analytica Chimica Acta, 2003, 487(2), 145-153. [93] Huang, L.F.; Guo, F.Q.; Liang, Y.Z.;Li, B. Y.; Cheng, B.M. Simultaneous determination of L-arginine and its mono- and dimethylated metabolites in human plasma by high-performance liquid chromatography-mass spectrometry. Analytica Chimica Acta, 2004, 380(4), 643-649. [94] Martens-Lobenhoffer, J; Bode- Böger, SMJ. Simultaneous detection of arginine, asymmetric dimethylarginine, symmetric dimethylarginine and citrulline in human plasma and urine applying liquid chromatography-mass spectrometry with very straightforward sample preparation. Journal of Chromatography, B: Analytical Technologies in the Biomedical and Life Sciences, 2003, 798(2), 231-239. [95] Piraud, M; Vianey-Saban, C; Bourdin, C; Acquaviva-Bourdain, C; Boyer, S; Elfakir, C; Bouchu, D. A new reversed-phase liquid chromatographic/tandem mass spectrometric method for analysis of underivatised amino acids: Evaluation for the diagnosis and the management of inherited disorders of amino acid metabolism. Rapid Communications in Mass Spectrometry, 2005, 19(22), 3287-3297. [96] Martens-Lobenhoffer, J; Bode-Böeger, SM. Fast and efficient determination of arginine, symmetric dimethylarginine, and asymmetric dimethylarginine in biological fluids by hydrophilic-interaction liquid chromatography-electrospray tandem mass spectrometry. Clinical Chemistry, 2006, 52(3), 488-493. [97] Guo, Y; Gaiki, S. Retention behavior of small polar compounds on polar stationary phases in hydrophilic interaction chromatography. Journal of Chromatography A, 2005, 1074, (1-2), 71-80. [98] Woo, KL; Lee, DS. Capillary gas chromatographic determination of proteins and biological amino acids as N(O)-tert.-butyldimethylsilyl derivatives. Journal of Chromatography, B, 1995, 665, 15-25

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[99] Albsmeier, J; Schwedhelm, E; Schulze, F; Kastner, M; Böger, RHJ. Determination of NG, NG-dimethyl-L-arginine, an endogenous NO synthase inhibitor, by gas chromatography-mass spectrometry. Journal of Chromatography, B: Analytical Technologies in the Biomedical and Life Sciences, 2004, 809(1), 59-65. [100] Duncan, MW; Poljak, A. Amino acids analysis of peptides and proteins on the femtomole scale by gas chromatography/mass spectrometry. Analytical Chemistry, 1998, 70, 890-896. [101] Mandalakis, M; Apostolaki, M; Stephanou, EG. Trace analysis of free and combined amino acids in atmospheric aerosols by gas chromatography-mass spectrometry. Journal of Chromatography, A, 2010, 1217(1), 143-150. [102] Kosir, IJ; Kidric, J. Use of modern nuclear magnetic resonance spectroscopy in wine analysis: determination of minor compounds. Analytica Chimica Acta, 2002, 458(1), 77-84. [103] Kilgore, JA; Smith, JT. Abstracts of Papers, 225th ACS National Meeting, New Orleans, LA, United States, March 23-27, 2003 CHED-174. [104] Ding, X; Gao, L; Han, R; Ye, J. Determination of amino acids in vinegar and soy sauce by capillary electrophoresis with electrochemical detection. Huadong Shifan Daxue Xuebao, Ziran Kexueban, 2002, (1), 56-60. [105] Poinsot, V; Bayle, C; Couderc, F. Recent advances in amino acid analysis by capillary electrophoresis. Electrophoresis, 2003, 24(22-23), 4047-4062. [106] Nouadje, G; Simeon, N; Dedieu, F; Nertz, M; Puig, Ph; Couderc, F. Determination of twenty eight biogenic amines and amino acids during wine aging by micellar electrokinetic chromatography and laser-induced fluorescence detection, Journal of Chromatography A, 1997, 765(2), 337-343. [107] Kosir, IJ; Kidric, J. Identification of Amino Acids in Wines by One- and TwoDimensional Nuclear Magnetic Resonance Spectroscopy. Journal of Agricultural and Food Chemistry, 2001, 49(1), 50-56. [108] Son, H; Hwang, G; Kim, KM; Ahn, H; Park, W; Van Den Berg, F; Hong, Y; Lee, C. Metabolomic Studies on Geographical Grapes and Their Wines Using 1H NMR. Journal of Agricultural and Food Chemistry, 2009, 57(4), 1481-1490. [109] Martens-Lobenhoffer, J; Bode-Böger, SM. European Journal of Clinical Pharmacology. 62 (2006) 61.Measurement of asymmetric dimethylarginine (ADMA) in human plasma: from liquid chromatography estimation to liquid chromatography-mass spectrometry quantification. European Journal of Clinical Pharmacology, 2006, 62, 61–68. [110] Souverain, S; Rudaz, S; Veuthey, JL. Matrix Effect in LC-ESI-MS and LC-APCI-LCMS with off-line and on-line extraction procedures, Journal of Chromatography A, 2004, 1058, 61-66. [111] White, JA; Hart, RJ. HPLC of amino acids. In: Fennema, OR; Karel, M; Sanderson, GW; Tannebaum, SR; Pieter, W; Whitaker, JR. (Eds.), Analysis by HPLC, Madrid; 1992; 75-11. [112] Heberger, K; Csomos, E; Simon-Sarkadi, L. Principal Component and Linear Discriminant Analyses of Free Amino Acids and Biogenic Amines in Hungarian Wines Journal of Agricultural and Food Chemistry, 2003, 51(27), 8055-8060. [113] Osanai, M; Yonezawa, Y. Age-related changes in amino acid pool sizes in the adult silkmoth, Bombyx mori, reared at low and high temperature; a biochemical

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examination of the rate-of-living theory and urea accumulation when reared at high temperature. Experimental gerontology, 1984, 19(1), 37-51. [114] Bonomi, A; Anghinetti, A; Lucchelli, L; Darecchio, D; Bonomi, A. Amino acid determination in feeds by high-performance liquid chromatography (HPLC). Rivista della Societa Italiana di Scienza dell'Alimentazione, 1989, 18(3), 155-62. [115] Mazzucco, E; Gosetti, F; Bobba, M; Marengo, E; Robotti, E; Gennaro, MC. HighPerformance Liquid Chromatography-Ultraviolet Detection Method for the Simultaneous Determination of Typical Biogenic Amines and Precursor Amino Acids. Applications in Food Chemistry. Journal of Agricultural and Food Chemistry, 2010, 58(1), 127-134. [116] Naulet, N; Tesson, P; Couler, N; Martin, GJ. Optimization of analytical methods for origin assessment of orange juices. II. Determination of the amino acid composition. Analusis, 1997, 25(2), 42-47. [117] Lehtonen, P. Determination of amines and amino acids in wine: a review. American Journal of Enology and Viticulture, 1996, 47(2), 127-133. [118] Brückner, H; Langer, M; Luepke, M; Westhauser, T; Godel, H. Liquid chromatographic determination of amino acid enantiomers by derivatization with o-phthaldialdehyde and chiral thiols. Applications with reference to food science. Journal of Chromatography, A, 1995, 697, 229-46. [119] González-Paramás, AM; García-Villanova, RJ; Gómez Bárez, JA; Sánchez Sánchez, J; Ardanuy Albajar, R. Botanical origin of monovarietal dark honeys (from heather, holm oak, pyrenean oak and sweet chestnut) based on their chromatic characters and amino acid profiles. European Food Research and Technology, 2007, 226(1-2), 87-92. [120] Zheng, X; Wang, Y; Huang, Y; Li, H; Luo, H; Wu, Y; Zhang, W. HPLC determination of amino acids in beer with internal standard methods. Niangjiu, 2005, 32(1), 89-91. [121] Bauza, T; Blaise, A; Daumas, F; Cabanis, JC. Determination of biogenic amines and their precursor amino acids in wines of the Vallee du Rhone by high-performance liquid chromatography with precolumn derivatization and fluorimetric detection. Journal of Chromatography, A, 1995, 707(2), 373-9. [122] Kirschbaum, J; Luckas, B; Beinert, WD. HPLC analysis of biogenic amines and amino acids in food after automatic pre-column derivatization with 9-fluorenylmethyl chloroformate. American Laboratory, 1994, 26(15), 28C-288, 28F, 28H [123] Buetikofer, U; Bosset, JO; Fuchs, D. Quantitative determination of amino acids in protein hydrolysates. Evaluation of an interlaboratory test with classical ion-exchange amino acid analyzers and HPLC systems with OPA/FMOC precolumn derivatization. Mitteilungen aus dem Gebiete der Lebensmitteluntersuchung und Hygiene, 1992, 83(5), 457-466. [124] Carratu, B; Boniglia, C; Giammarioli, S; Mosca, M; Sanzini, E. Free amino acids in botanicals and botanical preparations. Journal of Food Science, 2008, 73(5), 323-328. [125] Hermosín, I; Chicón, RM; Cabezudo, MD. Free amino acid composition and botanical origin of honey. Food Chemistry, 2003, 83(2), 263-268. [126] Alaiz, M; Navarro, JL; Girón, J; Vioque, EJ. Amino acid analysis by high-performance liquid chromatography after derivatization with diethyl ethoxymethylenemalonate. Journal of Chromatography, A, 1992, 591, 181-186. [127] Cohen, SA; Michaud, DP. Synthesis of a fluorescent derivatizing reagent, 6aminoquinolyl-N-hydroxysuccinimidyl carbamate, and its application for the analysis

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of hydrolysate amino acids via high-performance liquid chromatography. Analytical Biochemistry, 1993, 211(2), 279-87. [128] Cohen, SA; De Antonis, KM. Applications of amino acid derivatization with 6aminoquinolyl-N-hydroxysuccinimidyl carbamate. Analysis of feed grains, intravenous solutions and glycoproteins. Journal of Chromatography, A, 1994, 661(1-2), 25-34. [129] Wandelen, C; Cohen, SA. Using quaternary high-performance liquid chromatography eluent systems for separating 6-aminoquinolyl-N-hydroxysuccinimidyl carbamatederivatized amino acid mixtures. Journal of Chromatography, A, 1997, 763, 11-22. [130] Hernández-Orte, P; Ibarz, MJ; Cacho, J; Ferreira, V. Addition of amino acids to grape juice of the Merlot variety: Effect on amino acid uptake and aroma generation during alcoholic fermentation. Food Chemistry, 2006, 98(2), 300-310. [131] Hernández-Orte, P; Lapeña, AC; Peña-Gallego, A; Astrain, J; Baron, C; Pardo, I; Polo, L; Ferrer, S; Cacho, J; Ferreira, V. Biogenic amine determination in wine fermented in oak barrels: Factors affecting formation. Food Research International, 2008, 41(7), 697-706. [132] Hernandez-Orte, P; Ibarz, MJ; Cacho, J; Ferreira, V. Amino acid determination in grape juices and wines by HPLC using a modification of the 6-aminoquinolyl-Nhydroxysuccinimidyl carbamate (AQC) method. Chromatographia, 2003, 58(1/2), 2935. [133] Busto, O; Guash, J; Borrull, F. Determination of biogenic amines in wine after precolumn derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate Journal of Chromatography, A, 1996, 737, 205-213. [134] Alexandre, H; Heintz, D; Chassagne, D; Guilloux-Benatier, M; Charpentier, C; Feuillat, M. Protease A activity and nitrogen fractions released during alcoholic fermentation and autolysis in enological conditions. Journal of Industrial Microbiology & Biotechnology, 2001, 26, 235-240. [135] Giraudo, M; Sanchez, H; Muset, G; Pavesi, R; Castaneda, R; Fernandez, M; Noseda, D; Markowski, I; Guirin, G. Quantitative determination of free amino acids in Argentinian Reggianito cheese by 6-AQC derivatization and RP-HPLC. Alimentaria, 2002, 337, 121-126. [136] Bosch, L; Alegria, A; Farre, R. Application of the 6-aminoquinolyl-Nhydroxysuccinimidyl carbamate (AQC) reagent to the RP-HPLC determination of amino acids in infant foods. Journal of Chromatography, B: Analytical Technologies in the Biomedical and Life Sciences, 2006, 831(1-2), 176-183. [137] Chang, B; Liu, H; Yan, H; Yu, F; Liu, X.An ideal precolumn derivatization-highperformance liquid chromatography for the determination of amino acids. Fenxi Huaxue, 1995, 23(1), 100-3.

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In: Arginine Amino Acid Editor: Nathan L. Jacobs

ISBN 978-1-61761-981-6 © 2011 Nova Science Publishers, Inc.

Chapter 2

Alternative Metabolic Pathways of Arginine and their Pathophysiological Roles András Hrabák and Zoltán Kukor* Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, H-1094, Tűzoltó u. 37-47, POB 260, Hungary

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Arginine is not only a protein constituent but it is metabolized through various alternative pathways in mammalian cells. Our review is focused on the two most important alternative pathways: the nitric oxide (NO) synthesis and the arginase reaction. In cells where both pathways are active, their regulation is generally reciprocal due to the common substrate. This reciprocal regulation was described at the level of both enzyme activities and expressions. Cross inhibition between arginase and NO synthase metabolites can be observed and different cytokines act differently on the expression of both enzymes as well. Arginase and NO synthase isoenzymes are involved in various important physiological processes such as urea cycle, vasodilation, immune defense against certain invaders and neurotransmission. However, the overexpression of these enzymes and overproduction of NO may contribute to the onset of various pathophysiological processes. NO overproduction may be responsible for neurotoxicity, septic shock, type 1 diabetes mellitus or various inflammatory diseases in numerous organs. In addition, inflammatory responses involving the inducible NO synthase may also contribute to the etiology of metabolic syndrome, while impaired NO production by the endothelial NO synthase may be in the background of cardiovascular disorders or preeclampsia. On the other part, the involvement of high arginase expression was observed in cardiovascular disorders and several pulmonary diseases, as pulmonary hypertension, silicosis and asthma. In conclusion, alternative arginine metabolic *

Postal Address: H-1444 POB 260, Hungary

34

András Hrabák and Zoltán Kukor pathways and their regulation are important factors both in the maintenance of healthy state and in the pathogenesis of various diseases.

Key words: alternative pathways, arginase, arginine, asthma, cardiovascular disorders, inhibitors, metabolic syndrome, nitric oxide synthase, preeclampsia, pulmonal hypertension, reciprocal regulation, silicosis.

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Abbreviations ADMA – asymmetric N,N‟-dimethyl-L-arginine CAT – cationic amino acid transporter cAMP – 3‟-5‟-cyclic adenosine monophosphate C/EBP – CCAAT enhancer binding protein FAD – flavin adenine dinucleotide FMN – flavin mononucleotide GM-CSF – granulocyte-monocyte colony stimulating factor GLUT – glucose transporter HbA1 – hemoglobin A1 IP-10 – interferon inducible protein-10 IL - interleukin IKKβ - inhibitor of nuclear factor κB kinase subunit β IRS-1 – insulin receptor substrate-1 JNK – Jun-N-terminal kinase LDL – low density lipoprotein LPS – lipopolysaccharide LXR – liver X-receptor MCP – monocyte chemotactic protein NADPH – nicotinamide adenine dinucleotide phosphate NF-κB – nuclear factor κB PGE2 – prostaglandin E2 PMN – polymorphonuclear cells PPAR – peroxisome proliferator activated receptor STAT - signal transducers and activator of transcription TNF-α - tumor necrosis factor-α TGF-β - transforming growth factor-β VEGF – vascular endothelial growth factor

Introduction - Arginine, a Functional Amino Acid with Several Metabolic Roles Amino acids are known mainly as building blocks of proteins. However, certain amino acids, characterized as functional amino acids [Wu, 2009] are involved in various metabolic processes as well. Arginine is one of those: in addition to its structural role in proteins, it

Alternative Metabolic Pathways of Arginine and their Pathophysiological Roles

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serves as a precursor of urea, nitric oxide, agmatine, polyamines and other amino acids. From nutritional aspect, arginine is a semi-essential amino acid for mammals as it can be synthesized from available precursors; however, the amount of de novo produced arginine is not sufficient for each function the in vivo demand is partly provided from exogenous sources. A simplified summary of arginine metabolism is shown in Figure 1. A more detailed representation of these metabolic pathways can be found in the paper of Wu et al [2009]. Minor routes were observed in some species or in certain mammalian tissues, while the arginase and NO synthase reactions are regarded as major routes. Our study is focused on the two most important alternative pathways of arginine metabolism, catalyzed by the arginase and nitric oxide synthase enzymes.

Figure 1. Summary of the most important alternative arginine pathways Cit – L-citrulline, Orn – L-ornithine, Asp – L-aspartic acid, Put – putrescine, Gly – glycine, ASS – argininosuccinate synthase, ASL – argininosuccinate lyase, ASE – arginase, OTC – ornithine transcarbamoylase, ADC – arginine decarboxylase, ODC – ornithine decarboxylase, NOS – nitric oxide synthase, SAM – S-adenosyl-methionine.

Nitric Oxide Synthase Nitric oxide synthase (NOS) was discovered at the end of the 1980‟s, when several lines of evidence suggested that the endothelium-derived relaxing factor (EDRF) is actually identical with nitric oxide (NO) [Furchgott & Zawadzki, 1980, Furchgott, 1999]. The de novo

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András Hrabák and Zoltán Kukor

nitrite/nitrate formation in mammalian organisms was also described [Stuehr and Marletta, 1985]. Three isoforms of NOS have been identified: a neural or brain enzyme (nNOS, bNOS, NOS I), a macrophage-derived inducible enzyme (macNOS, iNOS, NOS II) and an endothelial form (eNOS, NOS III). Later, these isoforms were found in many other tissues, frequently more than one isoform in the same cell type. NOS I and NOS II are soluble, cytosolic proteins, while NOS III is anchored to membranes by myristoyl and palmitoyl fatty acid side chains. From another aspect, NOS I and NOS III are Ca2+-dependent, constitutively expressed enzymes, while NOS II is an inducible, Ca2+-independent isoform. This Ca2+insensibility may be due to the tight binding of calmodulin to NOS II even at very low Ca 2+ concentrations. Although NOS I and NOS III are considered as „constitutive” isoforms in vitro, numerous data suggest that their expression in cells is controlled in different ways. These regulatory mechanisms were summarized in a review previously [Förstermann et al., 1998]. NOS I expression is mainly regulated by physical stimuli, neurotransmitters and steroid hormones in function of the actual developmental state. NOS III expression is upregulated by shear stress [Xiao et al., 1997], but reduced by hypoxia [Liao et al., 1995a]. In endothelial cell lines, TNF- reduced the NOS expression [Yoshizumi, 1993]. The up-regulation of NOS III by estrogens in rats [Goetz, 1994] may be physiologically important in the protection of the vascular system during pregnancy. On the contrary, oxidized LDL and other atherogenic lipoproteins usually decreased NOS III expression [Liao et al., 1995b], but contradictory observations were also published [Hirata et al., 1996]. In most cases, these effects are not mediated transcriptionally, like in the case of NOS II, rather by the stabilization/ destabilization of mRNA of the „constitutive” NOS. The activation of protein kinase C was also shown to enhance NOS III expression [Ohara et al. 1995]. The phosphorylation status of the enzyme plays an important role in the regulation of eNOS activity. Several eNOS phosphorylation sites have been described: Tyr81, Ser114, Thr495, Ser615, Ser633 and Ser1177 (phosphorylation sites are numbered according to the human eNOS sequence). Activity of eNOS is increased principally by phosphorylation at Ser1177 and dephosphorylation at Thr495 [Mount et al. 2007]. The regulatory mechanisms that govern the synthesis of NOS proteins are summarized in Table 1. The three NOS isoenzymes have very similar biochemical properties: low KM value (1-20 M), identical reaction mechanism [Stuehr 1997, Griffith et al., 1995, Ghosh & Salerno, 2003] and cofactors (NADPH, FAD, FMN, tetrahydrobiopterin, calmodulin), the presence of an oxygenase domain containing a porphyrine iron, requirement of dimeric structure for full activity [Abu-Soud et al., 1994, 1995,], identical or similar inhibitors. Schematic structures of the domains, gene and mRNA of the NOS II isoenzyme are shown in Figure 2, while the schematic dimeric enzyme can be seen in Figure 3. As depicted in Figure 2, the polypeptide chain consists of an oxygenase and a reductase domain, the first being responsible for the NOS reaction, while the latter is the binding site for the oxidoreductase coenzymes. The oxygenase reaction is quite unique: in the first reaction, a NADPH-dependent hydroxylation step occurs at the guanidino nitrogen of arginine, forming a NG-hydroxy-arginine, while in the second reaction only a half NADPH molecule is involved, leading to the production of NO and citrulline. (Figure 4.). In comparison to arginase, the efficiency (characterized by Vmax) of NOS is rather low (100-1000 pmoles of citrulline per mg per min), although that of arginase and NOS II expressed as Vmax/KM are not very far from each other.

Alternative Metabolic Pathways of Arginine and their Pathophysiological Roles

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Table 1. Regulators of the expression of NOS isoforms Level of regulation Transcriptional

NOS isoform NOS II

regulation increase

NOS II

regulator LPS, IL-1, IL-2, IL-6, IFN-γ TNF-α, GM-CSF, uv. light, O3, cAMP glucocorticoids, indomethacin, chloroquine IFN-γ, TNF-α

Transcriptional

NOS II

Post-transcriptional and translational Post-transcriptional and translational Post-transcriptional and translational Post-transcriptional and translational Post-translational

NOS II

TGF-β

mRNA destabilized

NOS III

estrogens

mRNA stabilized

NOS III

TNF-α, oxidized LDL

mRNA destabilized

NOS I, NOS III

calmodulin binding, phosphorylation tetrahydrobiopterin, oxygen, L-arginine

increase

Post-translational

NOS I, NOS II, NOS III

inhibition

mRNA stabilized

Dimerization, activation

Figure 2. Schematic structure of human NOS II protein (up), NOS II gene (middle) and its mRNA (down). Gene is located on chromosome 17, size of mRNA is 4.4 kb molar mass of NOS II: 131 kD. Binding site abbreviations: BH4 = tetrahydrobiopterin, CaM = calmodulin, RE = responsive elements, TATA = TATA box.

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András Hrabák and Zoltán Kukor

Figure 3. The scheme of NOS dimers.

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Assembly of dimers requires L-Arg and tetrahydrobiopterin (BH4), the active dimer is formed only if both of them are present. The direction of electron transport is indicated by arrows.

Figure 4. The schematic mechanism of NOS reaction. Protoporphyrin-IX rings including Fe-atom are symbolysed by squares with the corresponding Fe-forms.

Alternative Metabolic Pathways of Arginine and their Pathophysiological Roles

39

In the absence of the L-arginine substrate or tetrahydrobiopterin, the NOS reaction is uncoupled from NO generation and the enzyme produces hydrogen peroxide and, more importantly, superoxide, a well-known free radical [Vasquez-Vivar et al., 1998]. Superoxide is generated by the oxygenase domain via dissociation of the ferrous-dioxygen complex. The addition of L-arginine and tetrahydrobiopterin abolishes superoxide generation by eNOS. The generation of superoxide may lead to the formation of peroxynitrite (OONO-), which is responsible for numerous effects of NO, e.g. eliciting the nitrosylation of tyrosine in protein targets. In addition, peroxynitrite itself can oxidize tetrahydrobiopterin that results in further uncoupling of NOS III, but ascorbate may prevent this effect [Kuzkaya et al., 2003, Tóth et al. 2002]. The role of chronic hypoxia in the stimulation of NOS III may partly be mediated by the heat shock protein hsp90, which forms a complex with NOS III, thus preventing the uncoupling of NOS. It has been shown that inhibition of hsp90 caused NOS III uncoupling and prevented the NO-dependent proliferation of endothelial cells [Shi et al., 2002, Ou et al., 2003].

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Arginase Arginase is a key enzyme of the urea cycle in mammalian liver. It hydrolyses arginine into ornithine (Orn; the first compound of the cycle) and urea. The full cycle is functional only in the liver in order to convert the excess nitrogen to urea. However, certain enzymes of the cycle are present in each cell type. In kidneys, another arginase isoenzyme is found. While the liver-type arginase I is cytosolic, the kidney-type arginase II isoform is localized in mitochondria. The two isoenzymes are very similar in their kinetic properties, inhibitors, molar masses and subunit structures (homotrimeric); differences were observed with respect to their electrophoretic mobility and immune specificity [Jenkinson et al., 1996]. The presence of arginase was proven in macrophages and its possible function in the immune response was extensively investigated, but finally it turned out that the arginase-like processes were mainly due to nitric oxide synthase. After twenty years of ignorance, the involvement of arginase in numerous disorders has brought it recently back into the focus of biochemical research (see later). Arginase I can be easily isolated from liver of mammals, using various precipitation methods (acetone and ammonium sulfate) in combination with heat treatment and cationic exchange chromatography. The heat stability of arginase in the presence of Mn2+ ions is particularly important during its isolation from other heat-sensitive proteins. A 30-min heat treatment at 60oC causes activation of arginase [Schimke, 1964]. The isolation and purification of arginase II from kidneys is more difficult. First, due to its mitochondrial localization the mitochondrial fraction should be isolated by differential centrifugation and lysed by sonication in the presence of Mn2+-ions. This is followed by ultracentrifugation and the supernatant should be purified further by a series of precipitations, short heat treatment (60oC, 1 min), and ion-exchange chromatography [Kaysen and Strecher, 1973]. The importance of manganese ions in the reaction mechanism has been explained by Xray studies. Since arginase I is a stable, crystallizable enzyme that can be isolated in high amounts from the liver or kidney, its structure and reaction mechanism were discovered by Xray diffraction studies. The trimeric rat liver enzyme contains a spin-coupled Mn2+-Mn2+

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cluster that is essential for catalytic activity at the bottom of a 15 Å deep active site cleft. The hydrolysis of arginine is catalyzed by a metal-activated water molecule which forms a symmetrical bridge between both Mn2+-ions [Kanyo et al., 1996]. Both isoenzymes consist of 40-45 kDa subunits which form homotrimers in the active enzyme. Arginases have a low substrate affinity (2-10 mM), but high efficiency (Vmax in the range of 100-1000 nmoles of urea per mg protein per min). Most of their inhibitors are also efficient in the millimolar range only. The first exception was the NG-hydroxy-L-arginine, the unstable intermediate of the NOS reaction [Daghigh et al., 1994].

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Alternative Pathways of Arginine Metabolism In most tissues, only one of these two alternative pathways was observed at measurable rates. However, in some cell types, both arginase and NOS II may be expressed and their activities and expressions are both regulated. Macrophages represent a model cell type for studying these competitive alternative arginine pathways which were characterized earlier mainly in rodents. The role of the two alternative pathways in the general arginine metabolism was reviewed in details [Wu & Morris 1998, Boucher et al., 1999, Morris, 2004]. As mentioned above the two enzymes have contradictory biochemical features. NOS has high substrate affinity but low efficiency, while arginase has low substrate affinity and high efficiency. Therefore, NOS can efficiently catalyze the reaction even at micromolar arginine concentrations. However, at higher arginine concentrations, the very efficient arginase consumes the substrate molecules therewith preventing their conversion by NOS. These properties can explain why arginase added to the culture medium of macrophages can deplete arginine substrate and prevent NO formation, in spite of the high substrate affinity of NOS. Of course, this paradoxical regulation can only be demonstrated on cell types which can express both enzymes (e.g. macrophages). The reciprocal relationship of the alternative pathways was already demonstrated in our early experiments. Elicited macrophages isolated from mice and rats showed different NOSarginase patterns: in mice, high arginase activity was associated with moderate NOS activities, while in rats a high NOS activity was measured along with very low, almost undetectable arginase levels [Hrabák et al., 1992, 2006]. The inhibitory effect of nitrite (the stable end-product of the NOS reaction) on arginase activity was also observed [Hrabák et al., 1996a] suggesting a cross-inhibition between the two metabolic pathways. In addition to the alternative regulations at the enzyme activity level, reciprocal regulatory effects on enzyme expressions were also observed. Different effects of certain cytokines (IL-4, IL-10), prostaglandin E2 and LPS versus interferon-γ (IFN-) on arginase expression were observed in bone marrow derived macrophages [Corraliza et al., 1995; Modolell et al., 1995]. Based on these results, these authors suggested that NO synthase and arginase appeared to define two alternate functional states of macrophages, induced by Th-1 and Th-2 cytokines, respectively. Nevertheless, upregulation of the expression of both enzymes by LPS suggests that the two enzymes cannot be associated unequivocally with proor anti-inflammatory responses, respectively. Further experiments on bone marrow-derived macrophages revealed that the effect of interleukin-10, a Th-2 type cytokine on LPS-induced arginase expression was mediated by protein kinase A through increased cAMP levels.

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Interestingly, suppressors of NOS expression (e.g. protein kinase C inhibitors) did not affect the LPS-induced arginase [Corraliza et al. 1997]. These experiments were extended further, investigating the interaction of Th-1 and Th-2 clones of CD4+ T-cells and macrophages, where the latter were used as antigen presenting cells. Th-1 cells induced NOS expression exclusively while Th-2 cells induced the arginase. The effect of cytokine mixtures in the supernatants of Th-2 cells was synergistic as compared to individual cytokines particularly between IL-4/IL-13 and IL-10, and direct cell interactions were not required for massive arginase induction [Munder et al., 1998]. Studies on the arginase isoenzyme expression pattern of bone marrow-derived macrophages or dendritic cells showed the specific induction of the arginase I isoform by Th-2 cytokines, while the expression of arginase II was not changed, if detectable at all [Munder et al., 1999]. Although the direct reciprocal relationship between arginase and NOS II were proven mainly in macrophages and related cells, where both enzymes are expressed, additional data suggest that this relationship can also be observed in other tissues. Low arginase and Mn2+ ion (arginase cofactor) levels associated with high NO in the blood plasma from patients with various neurological disorders (in schizophrenia and bipolar disease [Yanik et al., 2003, 2004] and in Alzheimer disease [Vural et al., 2009]) were observed. In a mouse model of Duchenne muscular dystrophy, the proportion of pro-inflammatory M1 macrophages increased, which could be prevented by the genetic ablation of the NOS II gene. In addition, the presence of arginase-producing M2 macrophages reduced the lysis of muscle cells via competing for arginine. IL-4 and IL-10 cytokines were involved in the activation of M2 macrophages associated with the deactivation of M1 phenotype probably due to a reduced expression of iNOS, IL-6, MCP-1 and IP-10. These results suggest that distinct macrophage subpopulations can promote muscle injury or repair in muscular dystrophy, and the balance between these macrophage populations may influence the course of muscular dystrophy [Villalta et al., 2009]. The arginase-inducing effect of the IL-4 cytokine required a composite response element containing STAT6 and C/EBP binding sites. STAT6 and C/EBPβ can bind to the STAT6 and C/EBP sites noncooperatively as it was demonstrated in the RAW 264.7 macrophage cell line [Gray et al., 2005]. The M1-M2 paradigm has been explained in details by Mills et al [2000]. These authors revealed earlier that the arginase/NOS balance influenced the response of various mouse strains against tumors [Mills et al., 1992]. After further investigations, different strains were defined as Th-1 strains (e.g. C57BL/6) or Th-2 strains (e.g. BALB/c) and the strains may exhibit different activities of the two alternative arginine metabolic pathways. Thus, M-1/M-2 do not simply describe activated or not unactivated macrophages but cells operating distinct metabolic programs. Macrophages of Th-1 strain can be stimulated mainly by inflammatory cytokines as interferon-γ, TNF-α, IL-1β, IL-6, leading to the induction of NOS II that inhibits cell division and causes an inflammatory response. On the contrary, Th-2 strains are stimulated by different cytokines such as IL-4, IL-10, TGFβ-1, inducing arginase and downregulating NOSII. Taken together, these results indicate that M-1 or M-2-dominant macrophage responses can influence whether Th-1/Th-2 or other types of inflammatory responses occur [Mills et al, 2000]. The role of Th-1 cytokines in the induction of NOS II was also observed in retinal pigment epithelial cells [Liversidge et al., 1994]. The Th-1/Th-2 cytokine balance is also critical in infections: Schistosoma infections in mice provoke a Th-2 dominant immune defense (mediated by IL-4, IL-10, IL-13), while Mycobacterium avium

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infections develop a Th-1 dominant NOS II-response [Hesse et al., 2001]. The reciprocal regulations of NOS II and arginase are summarized in Table 2. Table 2. Reciprocal regulations of NOS II and arginase Level of regulation Expression

Enzyme activity

NOS II

Arginase

Inducers LPS; IL-1; IL-2, IL-6, IFNγ; TNFα; GM-CSF

Blockers IL-4; IL-10; IL-13; TGFβ; PGE2 glucocorticoids

Inducers LPS; IL-4; IL10; IL-13; TGFβ; PGE2

Blockers IL-1; IL-2, IL-6, IFNγ; TNFα

activators Ca2+, BH4

inhibitors Putrescine, NO

activators Mn2+

Inhibitors Val, Orn, NOHA, NO2-

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Inhibition of NOS and Arginase Isoforms Since the increased activity and/or expression of both NOS and arginase may be involved in the etiopathogenesis of various disorders, their inhibitors might be of potential therapeutic value. The endogenous NOS inhibitors (NG-methylarginines) may be responsible for the interesting „arginine paradox”, i.e. increasing arginine concentration in plasma can enhance NO production, in spite of the high plasma arginine concentration compared to the low KM of NOS [Tsikas et al., 2000]. For this reason, here we summarize the most important inhibitors of NOS and arginase isoenzymes.

NOS Inhibitors The most known and abundant group of NOS inhibitors are substrate analogs, that usually cause competitive inhibition. These compounds are NG-substituted L-arginines (methyl-, nitro-, amino derivatives) and their esters; although their effects on various isoforms are not identical, no isoform-specificity was observed. Although only L-isomers are active inhibitors, their guanidino forms without the arginine-specific carbon chain are also inhibitory, but to a much less extent. This effect can be explained by the mechanism of substrate binding and oxidation. The guanidinium group is bound to a tryptophan and a glutamate side chain of the NOS active site by H-bonds. The substrate and its inhibitory analogs are oriented in the active site to place –NH2 and =NH2+ groups in the distal guanidium nitrogen pocket. The electric charges are not very important in the binding. However, replacement of a CH2-group with an O-atom in the isosteric L-canavanine leads to a change in the orientation, preventing the formation of the H-bond between the glutamate side chain and the N5-atom [Babu et al., 1999].

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Another group of NOS inhibitors were developed from L-thiocitrulline. These sulfurbased inhibitors usually do not contain the arginine carbon chain and their effect is based on the binding of the guanidino group to the Trp-Glu side chains and the binding of their S-atom to the Fe-atom coordinated by the porphyrine backbone [Southan et al., 1995, 1996]. Several compounds not related to arginine or guanidine are also strong inhibitors of NOS isoforms. 2-amino-thiazol and 2-aminopyrimidine are partly specific for NOS II, while indazol and 7-nitroindazol act mainly on NOS I. They are considered as heme ligands. 2iminopyrrolidine and 2-imino-piperidine are „non-amino acid” inhibitors, mainly on NOS II, but without a strong specificity. In addition to NOS inhibitors, the expression of inducible NOS may also be influenced pharmacologically. The most important inhibitor of NOS II expression is the glucocorticoid family [Matsumura et al., 2001], but we also observed the inhibitory effect of chloroquine [Hrabák et al., 1998] and – in higher doses – indomethacin [Hrabák et al., 2001]. More recently, the up-regulating effect of various statins on the expression of NOS III and endothelial NO production was described, probably by a multitarget mechanism including the decrease of superoxide production, potentiation of tetrahydrobiopterin synthesis, phosphorylation of NOS III by Akt and promoting the hsp90 binding to NOS III [Laufs, 2003].

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Arginase Inhibitors Classical arginase inhibitors are the basic and neutral L-amino acids (L-ornithine, Llysine, L-valine etc.). However, the affinity of these compounds to arginase is low (KM is in mM range) and their binding properties suggest that the guanidino side chain is not very important in the binding [Hrabák et al., 1996b]. The role of α–amino and α–carboxyl groups and side chain length were more significant. These results suggested a tighter fit between substrate/inhibitor and the active site of arginase compared to NOS [Hrabák et al., 1994, 1996b]. The first high-affinity arginase inhibitor was the NG-hydroxy-L-arginine (NOHA), the intermediate of the NOS reaction [Daghigh et al., 1994]. The physiological role of this effect can be explained well; this inhibition might play a role in the cross-inhibition of the alternative routes, similarly to the lower affinity inhibition of arginase by nitrite, the stable NO end-product [Hrabák et al., 1996a]. The hydroxylic group of NOHA may be bound to the metal cluster, forming a bridge between the Mn2+ ions and thus preventing the binding of water to arginase. Based on this mechanism, numerous other hydroxyamino acids were synthesized with strong inhibitory effect (0.5 μM-0.5 mM), e.g. Nω-hydroxy-indospicine, Nωhydroxy-L-norvaline and other related derivatives [Custot et al., 1996, 1997, Hey et al., 1997, Tenu et al., 1999], for examples, see Figure 5. Boronic acid-based transition-state analogues, like 2(S)-amino-6-boronohexanoic acid (ABH) and S-(2-boronoethyl)-L-cysteine (BEC) are also efficient competitive arginase inhibitors, particularly at pH 7.5 with Ki values of 0.25 and 0.31 μM, respectively [Colleluori & Ash 2001], indicating that the design of arginine analogues with uncharged, tetrahedral functional groups will lead to the development of more potent inhibitors of arginases at physiological pH. It should also be noted that α-difluoromethylornithine (DFMO), a strong inhibitor of ornithine decarboxylase, has an inhibitory effect on arginase as well.

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Figure 5. Hydroxy-amino acids, high affinity inhibitors of arginases. A = NG-hydroxy-L-arginine, B = NG-hydroxy-L-indospicin, C = N-hydroxy-L-lysine, D = N-hydroxy-L-ornithine, E = NG-hydroxy-nor-L-arginine.

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NOS Isoforms in Diseases As summarized above, NO plays important roles in vasodilation, neurotransmission, immune defense and in other functions. For this reason, its production is tightly regulated. Disturbed regulation of NO synthesis in various tissues may lead to severe diseases. The most frequently seen disturbance is the uncontrolled NO production by induced NOS II leading to cytotoxic effects due to the free radical nature of NO. The most thoroughly studied disorder elicited by this mechanism is the septic shock, that is due to the systemic induction of NOS II in various blood cells, resulting in a generalized NO overproduction in the whole body, with a consequent dramatic decrease of blood pressure that leads to cardiovascular shock and death [Vallance & Moncada, 1993, Thiemermann 1994, Kilbourn et al., 1997, Rubanyi, 1998, Vincent et al., 2000] (see Figure 6A). Overproduction of NO by the inducible NOS II is also involved in the etiology of type 1 diabetes mellitus. The NOS II isoform may be induced either in macrophages infiltrating the pancreatic islets or in islet cells themselves, leading to a considerable islet cell death with the inability of insulin production (see Figure 6B). The induction of NOS II may be provoked by an insulitis, which is mediated by inflammatory cytokines, mainly by interleukin-1β, with the synergistic effects of interferon-γ and TNF-α [Cetkovic-Cvrlje & Eizirik, 1994, Eizirik et al., 1996, Mandrup-Poulsen, 1996]. This process may be considered as an inflammatory autoimmune response. The involvement of NOS II induction in the etiology of diabetes is well documented both in rodent models and in humans [Flodström et al., 1995, 1996]. However the latter is less pronounced, due to the lower sensitivity of human islet cells for the NO free radical [Eizirik et al., 1994]. The role of NOS induction in these diseases and inflammatory processes has been reviewed by several authors in the past years; therefore we focus here on other disorders involving the NOS II isoform.

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Figure 6. Schematic background of septic shock (A) and diabetes mellitus type I (B).

The overproduction of NO by NOS I was observed in neurocytotoxicity, possibly leading to stroke and other neurological diseases. NO overproduction causes DNA damages which activate the poly(ADP)ribose synthesis [Moncada & Bolanos, 2006]. This process requires NAD, transforming it to nicotinamide mononucleotide. Its restoration to NAD utilizes phosphoribosyl-pyrophosphate and ATP, leading to energy depletion and cell death (Figure 7). In addition, NO overproduction is a possible etiological factor in neurodegenerative diseases such as Parkinson‟s, Alzheimer‟s and Huntington‟s diseases [Chabrier et al., 1999]. The cause of NOS III-related diseases is mainly the impairment of enzyme activity or expression. This impairment is detectable in most endothelial disorders, cardiovascular diseases and deficiencies of blood coagulation. Here we concentrate on another important field, the role of NOS III in pre-eclampsia.

The Involvement of NOS Isoforms in the Obesity and Metabolic Syndrome Obesity is a chronic imbalance between energy intake and expenditure both in humans and animals. This disorder represents an important risk factor for insulin resistance (i.e. type 2 diabetes mellitus), dyslipidemia, cardiovascular disorders (atherosclerosis, stroke, hypertension), summarized as „metabolic syndrome”. In the last decade, a large body of new information became available concerning the role of L-arginine and NO in the metabolic syndrome. Dietary arginine supplementation resulted in decrease of plasma glucose and lipid levels and in improvement of insulin sensitivity in rats with chemically induced or genetically

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determined (Zucker) diabetes [Kohli et al., 2004, Wu et al., 2007]. In contrast to drugs utilized in diabetic therapy, arginine supplementation also reduced adiposity and improved insulin sensitivity [Fu et al., 2005].

Figure 7. Scheme of NO-mediated neurocitotoxicity. NA = nicotinamide, NMN = nicotinamide mononucleotide, PRPP = phosphoribosyl-pyrophosphate.

The putative underlying mechanisms may involve multiple cyclic guanosine-3',5'monophosphate-dependent pathways. NO stimulates the phosphorylation of AMP-activated protein kinase that in turn inhibits acetyl-CoA carboxylase, decreases the expression of genes related to lipogenesis and gluconeogenesis, stimulates lipolysis by the phosphorylation of hormone-sensitive lipase. In addition, NO activates expression of PPAR-γ coactivator-1α (PGC-1α) enhancing mitochondrial biogenesis and increases blood flow to insulin-sensitive tissues promoting substrate uptake and product removal [Jobgen et al., 2006]. The role of tetrahydrobiopterin in NO production and its relation with arginine availability is a plausible explanation for the arginine paradox. While diets with a high saturated fat content induce high plasma fatty acid levels, endothelial NO production is often impaired due to a reduction in NOS phosphorylation. Increasing the arginine availability and/or arginase inhibition was proposed as a potential therapy to treat hypertension. Since inadequate de novo arginine

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production may reduce NO production, citrulline (Cit) supplementation may also be a good therapeutic approach in conditions of arginine deficiency [Luiking et al., 2010]. While the higher activity of NOS III seems to be beneficial against metabolic syndrome, that of NOS II is not favorable. It has been demonstrated that insulin resistance is associated with a chronic low-grade inflammation, and several mediators involved in the development of insulin resistance are released from immune cells and adipocytes. Several pro-inflammatory cytokines as TNF-α, IL-1, IL-6 and various adipocytokines are among these mediators. Furthermore, several transcription factors and kinases (JNK, IKKβ) also participate in this process. Hepatocyte-specific overexpression of NF-κB is associated with insulin resistance and can mimic all features of fatty liver disease. Since NF-κB is also involved in the induction of NOS II, its higher expression is an indicator of the inflammatory state [Tilg & Moschen, 2008]. Macrophage-secreted inflammatory factors blocked insulin action in adipocytes via downregulation of GLUT4 and IRS-1, leading to a decrease in Akt phosphorylation and impaired insulin-stimulated GLUT4 translocation to the plasma membrane. These changes are similar to those observed in adipose tissue from insulin-resistant humans and animal models [Lumeng et al., 2007]. The role of NOS II in inflammatory state leading to insulin resistance was further supported by using obese mice lacking iNOS gene. Rosiglitazone improved whole-body insulin sensitivity and insulin signaling to Akt/PKB in skeletal muscle of obese iNOS(-/-) and obese iNOS(+/+) mice. However, rosiglitazone further improved glucose tolerance and liver insulin signaling only in obese mice lacking iNOS. This genotypespecific effect of rosiglitazone was associated with the ability of the drug to raise plasma adiponectin levels. Rosiglitazone also increased AMP-kinase activation in muscle and liver only in obese iNOS(-/-) mice. PPAR-γ transcription increased in adipose tissue of iNOS (-/-) mice, while treatment of 3T3-L1 adipocytes with a NO donor reduced PPAR-γ activity. These results suggest that the iNOS/NO pathway is a critical modulator of PPAR-γ activation and circulating adiponectin levels [Dallaire et al., 2008]. A partly contradictory result was published when mice lacking all NOS isoforms were studied. The triply n/i/eNOS(-/-) mice, but not singly eNOS(-/-) mice, exhibited markedly reduced survival, due to spontaneous myocardial infarction and severe coronary arteriosclerotic lesions. In addition, the same mice manifested phenotypes resembling metabolic syndrome in humans, including visceral obesity, hypertension, hypertriglyceridemia, and impaired glucose tolerance [Nataka et al., 2008]. Liver X receptors (LXRs) and their ligands are negative regulators of macrophage inflammatory gene expression. LXR ligands inhibited the expression of inflammatory mediators such as NOS II, COX-2 and IL-6 in response to bacterial infection or LPS stimulation. In vivo, LXR agonists reduce inflammation in a model of contact dermatitis and inhibit inflammatory gene expression in the aortas of atherosclerotic mice. These findings identify LXRs as lipid-dependent regulators of inflammatory gene expression that may link lipid metabolism to immune responses in macrophages [Joseph et al., 2003].

Nitric Oxide and Preeclampsia Preeclampsia is a gestational disorder with hypertension and proteinuria after 20 weeks‟ gestation (blood pressure> 140/90 Hgmm and proteinuria > 0.3 g in 24 hours [Stella & Sibai 2006] frequently with edema and intrauterine degradation. Preeclampsia affects 3-10 % of

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pregnancies and it is the leading cause of obstetric morbidity and mortality. The only successful “treatment” for preeclampsia is delivery. Despite intensive research, the cause of disorder is still unknown. The placenta has a crucial role in the development of preeclampsia. Preeclampsia evolves in molar pregnancies in the absence of a fetus [Billieux et al. 2004]. Although the initiating events of preeclampsia are unclear, some observations indicate the potential key role of impaired production of NO in pregnancy. The endothelial eNOS is the primary isoenzyme expressed in human placenta [Kukor & Tóth 1994, Kukor et al. 1996, Tóth et al. 1997]. NO production is decreased in preeclamptic pregnancy, thus the dysfunction of eNOS may have a pivotal role in the development of preeclampsia. The reason for eNOS dysfunction may be of genetic, environmental or behavioural origin and often associates with other diseases (diabetes, metabolic syndrome). Diminished levels of NO could be related to deficiencies of calcium, arginine and the enzyme cofactor BH4, to reduced BH4 affinity, decreased eNOS expression, or to elevate free fatty acid (FFA), soluble VEGF receptor, glucose, reactive oxygen species (ROS) and ADMA levels. The low level of NO could be associated with the presence of certain eNOS polymorphisms (Figure 8.).

Figure 8. Preeclampsia and dysfunction of NOS III. The eNOS produces NO from arginine in physiological circumstances (above, empty part). The enzyme is phosphorylated by VEGF on Ser1177 (S1177) and dephosphorylated on Thr495. The arginine, Ca2+, BH4 levels are adequate to NO production. The NO production is insufficient in preeclampsia (down, filled part). The elevated FFA levels and E298D mutation cause decreased eNOS expression. The increased ADMA and decreased BH4, arginine and Ca2+ levels may cause reduced NO production. Elevated ADMA level and deficiency of BH4 and arginine lead to O2-. production. The phosphorylation on Ser1177 is inhibited by FFA and sVEGFR. (explications in the text) BH4 = tetrahydrobiopterin, FFA = free fatty acids, sVEGFR = soluble vascular endothelial growth factor receptor; other abbreviations see in the List of Abbreviations.

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The eNOS is Ca2+-dependent NOS isoform. Deficiency of dietary calcium is associated with low levels of plasma Ca2+, being a crucial determinant of eNOS activity. The concentration of arginine in umbilical blood and in villous tissue was lower in preeclampsia than in normotensive pregnancy, and it was not attributable to defective Larginine transport. Arginase expression increased in the maternal vasculature of women with preeclampsia. Moreover, arginase decreases the eNOS substrate arginine level in endothelial cells. Therefore, arginase not only reduces NO formation that competes with NOS for Larginine but also increases superoxide production by NOS. L-arginine supplementation to combat oxidative stress could lead to a further increase in peroxynitrite generation [Noris et al. 2004, Sankaralingam et al., 2010]. BH4 plays a physiological role in the regulation of eNOS activity and the enzyme affinity to BH4 could be decreased in preeclamptic placentas [Kukor et al. 2000]. In contrast no decreased affinity to BH4 was detected in control samples. BH4 deficiency not only decreases NO production by eNOS but can affect superoxide (O2-.) production as well [Gao et al., 2007]. In primordial and mature placentas the BH4 levels are only sufficient to reach half maximal enzyme activities (Sankaralingam et al., 2010]. Changes in BH4 levels or BH4 affinity may affect the enzyme activity of eNOS substantially. However, BH4 levels are not altered in preeclamptic placentas, therefore decreased BH4 affinity may cause the relative lack of BH4. This may in turn lead to decreased NO synthesis and increased O2- production. Decreased BH4 levels can elevate reactive oxidative free radical concentrations as observed in preeclamptic pregnants [Rodrigo et al., 2005]. Preeclampsia could be associated with diabetes. High glucose levels have been shown to reduce BH4 by enhancing the proteasome-dependent degradation of GTP cyclohydrolase I (GTPCH), a rate-limiting enzyme in the synthesis of BH4, parallel with increased formation of O2-. [Xu et al. 2007]. Apoptosis of trophoblast is accelerated in preeclamptic placenta. Arginine and BH4 deficiencies cause decreased NO and increased O2-. production by eNOS. However, peroxynitrite is formed by the very fast, diffusion controlled reaction of nitric oxide and superoxide radicals. ONOO- is a strong oxidizing and nitrating agent that is known to inhibit the mitochondrial electron transport chain, resulting in an increased mitochondrial superoxide production, exacerbation of cellular oxidative stress and further mitochondrial damage and ONOO- production. Impairment of the electron transport chain may lead to a loss of membrane potential, opening of the permeability transition pore and release of proapoptotic factors. Plasma FFA levels are commonly elevated during late pregnancy. Elevated FFA levels cause insulin resistance in all pregnant women. Serum samples of pregnant women with preeclampsia, metabolic syndrome or diabetes contain higher levels of FFA than those of healthy control pregnants. High FFA levels induce oxidative stress which results in conversion of NO into ONOO-. Furthermore, the FFA block phosphorylation of eNOS Ser1177 by inhibition of Akt, which causes decreased eNOS activity. Finally, elevated FFA levels result in insulin resistance (inhibition of phosphorylation on eNOS Ser1177). Ceramide synthesis is specifically potentiated by free palmitic acid. Ceramide causes apoptosis and could decrease transcription and activation of eNOS, [Lain & Catalano 2007, Xiao-Xun et al. 2009]. The vascular endothelial growth factor receptor-1 (VEGFR-1) is essential for the normal development and function of the placenta. VEGF increases eNOS activity through inducing

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phosphorylation of Ser1177 and dephosphorylation of Thr495. The soluble VEGFR (sVEGFR) level increases in preeclamptic pregnancy. The sVEGFR binds VEGF in plasma, effective VEGF levels will decrease [Mutter & Karumanchi 2008, Kimura & Esumi 2003]. Several studies have reported elevated levels of the endogenous NOS inhibitor ADMA in preeclampsia. The IC50 depends on the arginine concentration. Arginine levels may decrease in preeclampsia, enhancing the effect of ADMA. In addition to blocking NO production, ADMA may uncouple NOS and lead to the generation of superoxide. The ADMA concentration in plasma is regulated by the dimethylarginine dimethylaminohydrolase (DDAH). Some single nucleotide polymorphisms in the DDAH1 gene were also associated with preeclampsia [Böger et al. 2010]. Some studies suggested that ADMA is elevated in early stages of pregnancy in women who later develop preeclampsia, making it a potential biomarker to identify pregnant women who are at risk of developing preeclampsia. Although the identification of NOS polymorphisms has been pursued for years now, no mutations have been found so far which are unequivocally associated with preeclampsia. However, statistically significant associations have been identified with the mutations E298D, -786(T→C) in eNOS gene and G300A and G274T in iNOS gene. Studies of recombinant eNOS Asp298 and Glu298 showed no discernible difference either in the Km, or the Vmax and in Ki values for ADMA. The E298D mutant eNOS could be degraded faster than the wild type eNOS. The T(-786)→C promoter polymorphism may associate with reduced eNOS expression [Doshi et al. 2010]

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Arginase in Diseases Although arginase is an enzyme in the hepatic urea cycle which is crucial in the removal of excess nitrogen, arginase deficiency, - in contrast to that of other enzymes of the cycle - is not fatal. Following the discovery of macrophage arginase [Currie, 1978], its role in the immune response and wound healing was extensively studied [Schneider and Dy, 1985, Albina et al., 1990], but without significant results. Moreover, some results based on arginine consumption could be due to the presence of NOS. Nevertheless, after two decades of low interest, in the last years, new data have been accumulated concerning the key roles of arginase isoforms in the pulmonary and cardiovascular disorders, infectious diseases, immune cell function and cancer. These roles are usually due to the competition of the two alternative metabolic routes (i.e. NOS and arginase) for the common substrate. The expression of arginase isoforms in endothelial and vascular smooth muscle cells, the post-translational modulation of arginase activity and application of arginase inhibitors in vivo are reviewed by Morris (2009).

Arginase in Cardiovascular Diseases The role of arginase in vascular disorders was studied both in smooth muscle and endothelial cells. Arginase has been reported to exert pleiotropic effects on multiple targets. First, arginase can compete for its common substrate with eNOS, decreasing NO production which may lead to abnormal vasodilation and increased blood pressure. In addition, the

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deviating metabolic pathway may cause cell proliferation, because ornithine is the precursor of polyamines involved in proliferative processes. Endothelial cells transfected with arginase I and II cDNA produced high amounts of putrescine and spermidine, This effect could be blocked by an inhibitor of ornithine decarboxylase. Exogenously added putrescine enhanced the proliferation of endothelial cells even if they were not transfected by arginase [Li et al., 2002]. Similar results were obtained using rat aortic smooth muscle cells transfected with cDNA of arginase I: cell proliferation rates were increased and this effect could be inhibited with NG-hydroxy-L-arginine and S-(2-boronoethyl)-l-cysteine, high affinity inhibitors of arginase [Wei et al., 2001]. In addition, arginase-transfected endothelial cells produced proline, an amino acid involved in collagen synthesis, which may lead to collagen deposition in the vasculature [Li et al., 2001]. Summarizing these results, the deteriorating effect of arginase in the vascular system may be due to its ability to suppress NO production (leading to hypertension), to increase ornithine production which can be transformed into polyamines by ornithine decarboxylase and into proline (a component of collagen) by the ornithine aminotransferase. Polyamines can stimulate smooth muscle cell proliferation which – together with the increased collagen production in endothelial cells – leads to intimal thickening (Figure 9).

Figure 9. The role of arginase in the pulmonal/vascular disorders. The increased arginase activity (and superoxide production derived from inflammation, not shown) results in a decreased NO availability and smooth muscle cell proliferation, leading to hypertension and intimal thickening. OAT = ornithine aminotransferase, ODC = ornithine decarboxylase, SMC = smooth muscle cell.

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The protective effect of arginase inhibition in myocardial infarction was also observed. The mechanism of this phenomenon is based again on the shift of arginine utilization towards NOS, suggesting a possible therapeutic application [Jung et al., 2010]. In rats, nor-NOHA, an efficient arginase inhibitor reduced the infarct size which was completely abolished by NOS inhibitors. Nor-NOHA treatment also increased plasma nitrite levels and the citrulline/ornithine ratio. The uncoupling of NOS III in endothelial cells of old rats was associated with the upregulation of arginase. Increased superoxide production of old rats was inhibited by using NOS and arginase inhibitors, which were not efficient in young animals. In addition, the ratio of eNOS dimer to monomer in old rats was significantly decreased as compared to young rats. Furthermore, S-nitrosylation of arginase I was higher in old rats, supporting that NOS II nitrosylates and activates arginase I [Santhanam et al., 2007]. Arginase inhibition in old rats preserved the dimer/monomer ratio of NOS III reducing O2production as well. These data indicate that iNOS-dependent S-nitrosylation of arginase I and the increase in arginase activity lead to eNOS uncoupling, contributing to nitroso-redox imbalance, endothelial dysfunction, and vascular stiffness typical features of vascular aging [Kim et al., 2009]. Hypertension has been observed in arginase II knock-out mice, in spite of their impaired vasoconstrictory response, representing a hypertensive phenotype [Huynh et al., 2009], and suggesting that loss of arginase activity may generally result in hypertension.

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Arginase in Pulmonary Hypertension The first observations concerning the etiological role of arginase in pulmonary hypertension were described after a liver transplantation (Langle et al., 1995). Some years later, arginine therapy was tried successfully in patients with sickle cell disease which is associated with pulmonary hypertension (Morris et al., 2003). Further investigations revealed the competition between arginase and NOS, leading to decreased bioavailability of arginine substrate for NO synthesis. Pulmonary artery endothelial cells derived from the lung of patients had higher arginase II expression and produced lower NO than control cells in vitro. Thus, substrate availability affects NOS activity and vasodilation, implicating arginase II and alterations in arginine metabolic pathways in the pathophysiology of this disease (Xu et al., 2004). The CAT-2 arginine transporter was induced and the metabolism of arginine on both routes (NOS and arginase) were increased by LPS and TNF-α in the same cell line due to increased availability of Arg [Nelin et al., 2001]. The role of impaired NO synthesis was further supported by the observations that endogenous NOS inhibitors (e.g. dimethylarginine) associated with elevated arginase activity also provoked pulmonary hypertension in rats [Sasaki et al, 2007]. In hypoxia, arginase II (but not arginase I) is overexpressed in the smooth muscle of pulmonary artery causing a decreased bioavailability of arginine for NO synthesis and an increased proliferation of these cells as well. The role of arginase was also proven by using its inhibitor, S-(2-boronoethyl)-l-cysteine, which prevented the endothelial dysfunction in hypoxia-induced cells. In addition, the utilization of arginase II-targeted siRNA also prevented cellular proliferation [Chen et al., 2009]. As arginase II is co-localized with Hsp47, a collagen-specific chaperone, this also supports the role of arginase in collagen synthesis leading to lung fibrosis [Endo et al. 2003]. Chloroquine, an anti-malarial drug was found to be a competitive inhibitor of arginase in vitro in sickle erythrocytes suggesting a

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possible therapeutical utilization in pulmonary hypertension [Iyamu et al., 2007]. The results concerning the impairment of arginine metabolism associated with pulmonary hypertension were summarized and reviewed recently [Morris et al., 2008] and the major metabolic alterations are summarized in Figure 10.

Figure 10. Arginine metabolic alterations in pulmonal hypertension caused by sickle cell disease. L-arginine availability decreases because of renal dysfunction. NOS and arginase are induced by cytokines or released from damaged liver or red cells. NO is consumed by hemoglobin (hemolysis), and NOS uncoupling (forming O2-, leading to OONO-, which are cytotoxic). Arginase pathway becomes overwhelming, resulting in the formation of proline and collagen (leading to lung fibrosis) and polyamines (leading to cell proliferation and airway remodeling). The final consequences are lung injury, pulmonal hypertension and asthma.

Arginase in Silicosis Silicosis is an occupational disease, caused by the persistent inhalation of silica powder (SiO2), which leads to inflammation, lung damage and fibrosis. It is common among workers in mining, construction, stone cutting and electronics. First results suggesting the role of arginase in silica-induced lung inflammation were published by Schapira et al. who found that the uptake of L-arginine and both NOS II and arginase I expressions were increased in alveolar macrophages and neutrophils in lung inflammation [Schapira et al., 1998]. These results were supported further by the same group, showing the increased expression of CAT-1

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and CAT-2 arginine transporters, NOS II and arginase in lungs, without affecting NOS III [Nelin et al., 2002]. Some authors could not reinforce the role of arginase as a marker of silicosis in studies on alveolar macrophages [Misson et al., 2004]. Further PCR and immunohistochemical studies on rats revealed that arginase I but not arginase II expression has been increased in lungs and alveolar macrophages and NOS II has also been induced and co-expressed in most cells clustered in the inflammatory foci, compared to control animals. The rapid induction of arginase I expression in inflammatory lung cells, similarly to other inflammatory lung diseases, suggests the implication of elevated arginase activity in the development of lung damage following silica exposure [Poljakovic et al., 2007].

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Arginase in Asthma Experimentally provoked asthma in guinea pigs was found to be associated with elevated arginase activity and the arginase inhibitor nor-NOHA normalized the hyperresponsiveness of the challenged airways. A competition between NOS III and arginase for the common substrate has also been demonstrated in this study [Meurs et al., 2002]. These results, i.e. the competition of arginases and NOS for arginine substrate have been validated more recently by using NOS2 knockout mice: ovalbumin-induced lung inflammation and airway hyperreactivity was associated with lowered NO and a markedly increased expression of both arginase isoenzymes in knockout mice compared to control mice [Bratt et al., 2009]. The role of arginase in competition with NOS for arginine was also observed in asthmatic patients when their serum arginine and arginase levels were determined. High arginase activity in patients with asthma may lower circulating arginine levels, thereby limiting arginine bioavailability and causing NO deficiency leading to hyperreactive airways. Therefore, arginine metabolism is impaired in asthmatic patients and the treatments aiming the alterations in arginine metabolism may serve as new potential therapy of asthma [Morris et al., 2004]. Inhibition of phosphodiesterase 4, leading to the increase of cAMP level also results in enhanced arginase expression (mainly for arginase I compared to arginase II) in IL4 and TGF-β induced RAW 264.7 macrophage cells. The synergetic effect of IL-4 and a phosphodiesterase inhibitor was also observed in human alveolar macrophages as well. This suggests that phosphodiesterase inhibitors should be used carefully in the treatment of inflammatory diseases [Erdely et al., 2006]. A microarray study revealed 6.5 % of all genes showed altered in asthma and key factors of arginine metabolism, like CAT-2 arginine transporter and the two arginase isoenzymes were among the prominent asthma signature genes. Since arginase can regulate the generation of NO, polyamines, and collagen, pharmacological intervention in arginine metabolism may be therapeutically relevant in allergic disorders [Zimmermann et al., 2003]. Increased arginase activities in the airways reduced bioavailability of L-arginine for the constitutive and inducible nitric oxide synthases, causing a deficiency of the bronchodilating and anti-inflammatory NO, as well as increased formation of peroxynitrite, which may be involved in allergen-induced airways obstruction and inflammation. Increased arginase activity may also be involved in airway remodeling by promoting cell proliferation and collagen deposition. Therefore, arginase inhibitors may have therapeutic potential in the treatment of acute and chronic asthma as reviewed recently by Maarsingh et al. (2009a). The same authors proposed that increased peroxynitrite formation may be due to the decreased bioavailability of arginine caused by arginase. It might lead to

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superoxide formation by NOS III (see above) and NO and superoxides are the sources of peroxynitrite (Maarsingh et al, 2009b). The increased expression of arginase as a hypoxiasensitive protein was also demonstrated in allergic airway inflammations [Fajardo et al., 2004]. Nevertheless, other authors observed that inhibition of arginase caused inflammation in mouse airways via increasing the availability of arginine for NOS, and increased NO synthesis led to the nitration of tyrosine and nitrosylation of cysteine side chains in proteins [Ckless et al., 2008]. The role of alterations in arginine metabolism in asthma and airway inflammations was reviewed and the key role of CAT-2 arginine transporter and the two arginase isoenzymes was demonstrated earlier [King et al., 2004, Zimmermann & Rothenberg 2006].

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Arginase in other Disorders Recent findings suggest that arginases have significant roles in various diseases. Arginase II reduces the translation of inducible NOS in macrophages and blocks the defense against Helicobacter pylori [Lewis et al., 2010]. The unique role of increased arginase activity and protein was described in rheumatoid arthritis, but not in other immune-related diseases [Huang et al., 2001]. The role of NO in the uveoretinitis has been known for years, but recently the role of arginase in retinal neovascularization has also been demonstrated recently. Competing for the common L-arginine substrate, the decreased bioavailability of arginine leads to the production of reactive oxygen species by NOS III causing vascular inflammation. In addition, angiogenic cytokines and increased polyamine and proline synthesis (as further products of ornithine from the arginase reaction) may lead to cell proliferation and fibrosis in blood vessels. Inhibition of arginase may form the basis of a new therapeutic approach to treat retinopathy, a potential cause of blindness [Caldwell et al., 2010]. The same researchers demonstrated that the increase in arginase activity was associated with increases in both mRNA and protein levels of arginase I but not arginase II, localized mainly to glia and microglia. Their studies suggest that retinal inflammation in endotoxin-induced uveitis is mediated by NADPH oxidase-dependent increases in arginase activity [Zhang et al, 2009]. Arginase is induced in murine myeloid cells mainly by Th2 cytokines and inflammatory agents and participates in a variety of inflammatory diseases by down-regulating NO synthesis, inducing fibrosis and tissue regeneration. Arginase I is constitutively expressed in PMN cells and liberated during inflammation. Arginase-mediated L-arginine depletion suppresses T cell immune responses as a basis of inflammation-related immunosuppression. Therefore, the pharmacological modulation of L-arginine metabolism may be utilized in the treatment of cancer, autoimmunity and other immunological disorders. [Munder, 2009]. Contrasted to NOS, the role of arginase in diabetes mellitus is not well documented. However, since the role of NO in the etiology of type 1 diabetes is known, and the impairment of endothelial NOS may lead to endothelial dysfunction, the influence of the competitive pathway seems very likely. In blood samples of diabetic children, high arginase activity was associated with hyperglycemia and increased HbA1 in contrast to lower serum Mg2+ levels. This reciprocal relationship between arginase and Mg2+-levels has been suggested to be a consequence of reduced insulin action and increased protein catabolism [Bjelakovic et al., 2009].

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As mentioned above, the role of arginase in wound healing has already been described in animal models [Albina et al., 1990], but recent observations in human wounds supported again the involvement of arginase (and NOS) isoenzymes in these processes [Debats et al. 2009]. In a pig model, after a surgical intervention, early supplementation of arginine disturbed the reciprocal regulation of NOS II and arginase, favoring excess NO production, with consequent reduction in angiogenesis and granulation tissue formation (Haffernan et al., 2006]. NOS II gene transfer reversed impaired wound healing in NOS II-deficient mice [Yamasaki et al., 1998]. Lower arginase activities associated with higher NO synthesis were observed in fibromyalgic patients, suggesting the inflammatory role of NO in the etiology of this disorder and the possible utilization of NOS inhibitors in its therapy [Cimen et al., 2009].

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Conclusion The reciprocal regulation of the two arginine-utilizing metabolic pathways, catalyzed by arginase and nitric oxide synthase plays an important role in numerous physiological and pathophysiological processes. When the pathological process is based on the overproduction of NO, the use of NOS inhibitors is a potential pharmacologic tool, although none of them could be developed to a therapeutically suitable drug. When low NO production is the cause of the disorder, dietary L-arginine supplementation may be a useful therapy, because the „Larginine paradox” [Bode-Boger et al., 2007] makes possible the increase of NO production possible by raising the substrate concentration. Similarly, when the pathological process is due to high expression or activity of arginase which utilizes the substrate of NOS, oral or intravenous supplementation of L-arginine was applied successfully. Although L-arginine is tolerated in a broad dosage range in general, some patients cannot tolerate it because of side effects. Alternatively, in this case, L-arginine may be replaced by L-citrulline, as its precursor [Wu et al., 2007b]. Therefore, the L-arginine or L-citrulline supplementation is a possible therapeutical tool in the case of disorders affecting the alternative arginine metabolic routes.

Acknowledgment The authors wish to express their thanks to dr. Gergely Keszler (Department of Medical Chemistry, Molecular Biology and Pathobiochemisty, Semmelweis University) for the helpful discussion.

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Hrabák, A; Bajor, T; Temesi, Á. Comparison of substrate and inhibitor specificity of arginase and nitric oxide synthase for arginine analogues and related compounds in murine and rat macrophages. Biochem. Biophys. Res. Commun. 1994, 198, 206-212. Hrabák, A; Bajor, T; Temesi, Á; Mészáros Gy. The inhibitory effect of nitrite, a stable product of nitric oxide(NO) formation on arginase. FEBS Lett. 1996a, 390, 203-206. Hrabák, A; Bajor, T; Temesi Á. Computer-aided comparison of arginase and nitric oxide synthase in macrophages by amino acids not related to arginine. Comp. Biochem. Physiol. 1996b, 113B, 375-381. Hrabák, A; Sefrioui, H; Vercruysse, V; Temesi, Á; Bajor, T; Vray B. Action of chloroquine on nitric oxide production and parasite killing by macrophages. Eur. J. Pharmacol. 1998, 354, 83-90. Hrabák, A; Vercruysse, V; Kahán, LI; Vray, B. Indomethacin prevents the induction of inducible nitric oxide synthase in murine peritoneal macrophages and decreases their nitric oxide production. Life Sci. 2001, 68, PL 1923-1930. Hrabák, A; Bajor, T; Csuka I. The effect of various inflammatory agents on the alternative metabolic pathways of arginine in mouse and rat macrophages. Inflamm. Res. 2006, 55, 23-31. Huang, LW; Chang, KL; Chen, CJ; Liu, HW. Arginase levels are increased in patients with rheumatoid arthritis. Kaohsiung J. Med. Sci. 2001, 17, 358-363. Huynh, NN; Andrews, KL; Head, GA; Khong, SML; Mayorov, DN; Murphy, AJ; Lambert, G; Kiriazis, H; Xu, Q; Du, XJ; Chin-Dusting, JPF. Arginase II knockout mouse displays a hypertensive phenotype despite a decreased vasoconstrictory profile. Hypertension 2009, 54: 294-301. Iyamu, EW; Ekekezie, C; Woods, GM. In vitro evidence of the inhibitory capacity of chloroquine on arginase activity in sickle erythrocytes. Br. J. Haematol. 2007, 139, 337343. Jenkinson, CP; Grody, WW; Cederbaum, SD. Comparative properties of arginases. Comp. Biochem. Physiol. 1996, 114B, 107-132. Jobgen, WS; Fried, SK; Fu, WJ; Meininger, CJ; Wu, G. Regulatory role for the argininenitric oxide pathway in metabolism of energy substrates. J. Nutr. Biochem. 2006, 17, 571-588. Joseph, SB; Castrillo, A; Laffitte, BA; Mangelsdorf, DJ; Tontonoz, P. Reciprocal regulation of inflammation and lipid metabolism by liver X receptors. Nature Med. 2003, 9, 213219. Jung, C; Gonon, T; Sjoquist, PO; Lundberg, JO; Pernow, J. Arginase inhibition mediates cardioprotection during ischaemia-reperfusion. Cardiovascular Res. 2010, 85, 147-154. Kanyo, ZF; Scolnick, LR; Ash, DE; Christianson, DW. Structure of a unique binuclear manganese cluster in arginase. Nature 1996, 383, 554-557. Kaysen, GA; Strecker, HJ. Purification and properties of arginase of rat kidney. Biochem. J. 1973, 133, 779-788. Kilbourn, RG; Szabo, C; Traber DL. Beneficial versus detrimental effects of nitric oxide synthase inhibitors in circulatory shock, lessons learned from experimental and clinical studies. Shock 1997, 7, 235-246. Kim, JH; Bugaj, LJ; Oh, YJ; Bivalacqua, TJ; Ryoo, S; Soucy, KG; Santhanam, L; Webb, A; Camara, A; Sikka, G; Nyhan, D; Shoukas, AA; Ilies, M; Christianson, DW; Champion,

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HC; Berkowitz, DE. Arginase inhibition restores NOS coupling and reverses endothelial dysfunction and vascular stiffness in old rats. J. Applied Physiol. 2009, 107, 1249-1257. Kimura, H; Esumi, H. Reciprocal regulation between nitric oxide and vascular endothelial growth factor in angiogenesis. Acta Biochim. Pol. 2003, 50, 49-59. King, NE; Rothenberg, ME; Zimmermann, N. Arginine in asthma and lung inflammation. J. Nutr. 2004, 134, 2830S-2836S. Kohli, R; Meininger, CJ; Haynes, TE; Yan, W; Self, JT; Wu G. Dietary L-arginine supplementation enhances endothelial nitric oxide synthesis in streptozotocin-induced diabetic rats. J. Nutr. 2004, 134, 600-608. Kukor, Z; Tóth, M. Ca(2+)-dependent and Ca(2+)-independent NO-synthesizing activities of human primordial placenta. Acta Physiol. Hung. 1994, 82, 313-319. Kukor, Z; Mészáros, G; Hertelendy, F; Tóth M. Calcium-dependent nitric oxide synthesis is potently stimulated by tetrahydrobiopterin in human primordial placenta. Placenta 1996, 17, 69-73. Kuzkaya, N; Weissmann, N; Harrison, DG; Dikalov, S. Interactions of peroxynitrite, tetrahydrobiopterin, ascorbic acid, and thiols, implications for uncoupling endothelial nitric-oxide synthase. J. Biol. Chem. 2003, 278, 22546-22554. Lain, KY; Catalano, PM. Metabolic changes in pregnancy. Clin. Obstet. Gynecol. 2007, 50, 938-948. Langle, F; Roth, E; Steininger, R; Winkler, S; Muhlbacher, F. Arginase release following liver reperfusion. Evidence of hemodynamic action of arginase infusions. Transplantation 1995, 59, 1542-1549. Laufs, U. Beyond lipid-lowering, effects of statins on endothelial nitric oxide. Eur. J. Clin. Pharmacol. 2003, 58, 719-731. Lewis, ND; Asim, M; Barry, DP; Singh, K; de Sablet, T ; Boucher, JL ; Gobert, AP; Chaturvedi, R; Wilson, KT. Arginase II restricts host defense to Helicobacter pylori by attenuating inducible nitric oxide synthase translation in macrophages. J. Immunol. 2010, 184, 2572-2582. Li, H; Meininger, CJ; Hawker, JR Jr; Haynes, TE; Kepka-Lenhart, D; Mistry, SK; Morris, SM Jr.; Wu, G. Regulatory role of arginase I and II in nitric oxide, polyamine, and proline syntheses in endothelial cells. Amer. J. Physiol. Endocrinol & Metab. 2001, 280, E75-82. Li, H; Meininger, CJ; Kelly, KA; Hawker, JR Jr; Morris, SM Jr; Wu, G. Activities of arginase I and II are limiting for endothelial cell proliferation. Amer. J. Physiol. Regul. Integr. & Comp. Physiol. 2002, 282, R64-69. Liao, JK; Zulueta, JJ; Yu, FS; Peng, HB; Cote, GG; Hassoun, PM. Regulation of bovine endothelial constitutive nitric oxide synthesis by oxygen. J. Clin. Invest. 1995a, 96, 26612666. Liao, JK; Shin, WS; Lee, WY; Clark, SL. Oxidized low-density lipoprotein decreases the expression of endothelial nitric oxide synthase. J. Biol. Chem. 1995b, 270, 319-324. Liversidge, J; Grabowski, P; Ralston, S; Benjamin, N; Forrester, JV. Rat retinal pigment epithelial cells express an inducible form of nitric oxide synthase and produce nitric oxide in response to inflammatory cytokines and activated T cells. Immunology 1994, 83, 404-409. Luiking, YC; Engelen, MPKJ; Deutz, NEP. Regulation of nitric oxide production in health and disease. Curr. Opinion Clin. Nutr. Metab. Care 2010, 13, 97-104.

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Mount, PF; Kemp, BE; Power DA. Regulation of endothelial and myocardial NO synthesis by multi-site eNOS phosphorylation J. Mol. Cell. Cardiol. 2007, 42, 271–279. Munder, M; Eichmann, K; Modolell, M. Alternative metabolic states in murine macrophages reflected by the nitric oxide synthase/arginase balance, competitive regulation by CD4+ T cells correlates with Th1/Th2 phenotype. J. Immunol. 1998, 160, 5347-5354. Munder, M; Eichmann, K; Moran, JM; Centeno, F; Soler, G; Modolell, M. Th1/Th2regulated expression of arginase isoforms in murine macrophages and dendritic cells. J. Immunol. 1999, 163, 3771-3777. Munder, M. Arginase, an emerging key player in the mammalian immune system. Br. J. Pharmacol. 2009, 158, 638-651. Mutter, WP; Karumanchi, SA. Molecular mechanisms of preeclampsia. Microvasc Res. 2008, 75, 1-8. Nakata, S; Tsutsui, M; Shimokawa, H; Suda, O; Morishita, T; Shibata, K; Yatera, Y; Sabanai, K; Tanimoto, A; Nagasaki, M; Tasaki, H; Sasaguri, Y; Nakashima, Y; Otsuji, Y; Yanagihara, N. Spontaneous myocardial infarction in mice lacking all nitric oxide synthase isoforms. Circulation 2008, 117, 2211-2223. Nelin, LD; Nash, HE; Chicoine, LG. Cytokine treatment increases arginine metabolism and uptake in bovine pulmonary arterial endothelial cells. Amer. J. Physiol. Lung Cell. Mol. Physiol. 2001, 281, L1232-1239. Nelin, LD; Krenz, GS; Chicoine, LG; Dawson, CA; Schapira, RM. L-Arginine uptake and metabolism following in vivo silica exposure in rat lungs. Amer. J. Resp. Cell Mol. Biol. 2002, 26, 348-355. Noris, M; Todeschini, M; Cassis, P; Pasta, F; Cappellini, A; Bonazzola, S; Macconi, D; Maucci, R; Porrati, F; Benigni, A; Picciolo, C; Remuzzi G. L-arginine depletion in preeclampsia orients nitric oxide synthase toward oxidant species. Hypertension 2004, 43; 614-622. Ohara, Y; Sayegh, HS; Yamin, JJ; Harrison, DG. Regulation of endohelial constitutive nitric oxide synthase by protein kinase C. Hypertension 1995, 25, 414-420. Ou, J; Ou, Z; Ackerman, AW; Oldham, KT; Pritchard, KA Jr. Inhibition of heat shock protein 90 (hsp90) in proliferating endothelial cells uncouples endothelial nitric oxide synthase activity. Free Rad. Biol. Med. 2003, 34, 269-276. Poljakovic, M; Porter, DW; Millecchia, L; Kepka-Lenhart, D; Beighley C; Wolfarth, MG; Castranova, V; Morris, SMJr; Cell- and isoform-specific increases in arginase expression in acute silica-induced pulmonary inflammation. J. Toxicol. Environm. Health Part A. 2007, 70, 118-127. Rodrigo, R; Parra, M; Bosco, C; Fernández, V; Barja, P; Guajardo, J; Messina, R. Pathophysiological basis for the prophylaxis of preeclampsia through early supplementation with antioxidant vitamins. Pharmacol Ther. 2005, 107, 177-197. Rubanyi, GM. Nitric oxide and circulatory shock. Adv. Exp. Med. Biol. 1998, 454, 165-172. Sankaralingam, S; Xu, H; Davidge, ST. , Arginase contributes to endothelial cell oxidative stress in response to plasma from women with preeclampsia. Cardiovasc Res. 2010, 85, 194-203. Santhanam, L; Lim, HK; Miriel, V; Brown, T; Patel, M; Balanson, S; Ryoo, S; Anderson, M; Irani, K; Khanday, F; Di Costanzo, L; Nyhan, D; Hare, JM; Christianson, DW; Rivers, R; Shoukas, A; Berkowitz, DE. Inducible NO synthase dependent S-nitrosylation and

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activation of arginase1 contribute to age-related endothelial dysfunction. Circ. Res. 2007, 101, 692-702. Sasaki, A; Doi, S; Mizutani, S; Azuma, H. Roles of accumulated endogenous nitric oxide synthase inhibitors, enhanced arginase activity, and attenuated nitric oxide synthase activity in endothelial cells for pulmonary hypertension in rats. Amer. J. Physiol. Lung Cell. Mol. Physiol. 2007, 292, L1480-L1487. Schapira, RM; Wiessner, JH; Morrisey, JF; Almagro, UA; Nelin, LD. L-arginine uptake and metabolism by lung macrophages and neutrophils following intratracheal instillation of silica in vivo. Amer. J. Resp. Cell Mol. Biol. 1998, 19, 308-315. Schneider, E; Dy, M. The role of arginase in the immune system. Immunol. Today 1985, 6, 136-140. Schimke, RT. The importance of both synthesis and degradation in the control of arginase levels in rat liver. J. Biol. Chem. 1964, 239, 3808-3817. Shi, Y; Baker, JE; Zhang, C; Tweddell, JS; Su, J; Pritchard, KA Jr. Chronic hypoxia increases endothelial nitric oxide synthase generation of nitric oxide by increasing heat shock protein 90 association and serine phosphorylation. Circ. Res. 2002, 91, 300-306. Southan, GJ; Szabó, C; Thiemermann, C. Isothioureas, potent inhibitors of nitric oxide synthase with variable isoform selectivity. Br. J. Pharmacol. 1995, 114, 510-516. Southan, GJ; Zingarelli, B; O‟Connor, M; Salzman, AL; Szabó, C. Sponteneous rearrangement of aminoalkylisothioureas into mercaptoalkylguanidines, a novel class of nitric oxide synthase inhibitors with selectivity towards the inducible isoform. Br. J. Pharmacol. 1996, 117, 619-632 Stella, CL; Sibai, BM. Preeclampsia, Diagnosis and management of the atypical presentation. J. Matern Fetal Neonat. Med. 2006, 19, 381-386. Stuehr, DJ; Marletta, MA. Mammalian nitrate biosynthesis, mouse macrophages produce nitrite and nitrate in response to Escherichia coli lipopolysaccharide. Proc. Natl. Acad. Sci. USA 1985, 82, 7738-7742. Stuehr, DJ. Structure-function aspects in the nitric oxide synthases. Annu. Rev. Pharmacol. Toxicol. 1997, 37, 339-359. Tenu, JP; Lepoivre, M; Moali, C; Brollo, M; Mansuy, D; Boucher JL. Effects of the new arginase inhibitor N(omega)-hydroxy-nor-L-arginine on NO synthase activity in murine macrophages. Nitric Oxide 1999, 3, 427-438. Thiemermann, C; The role of the L-arginine, nitric oxide pathway in circulatory shock. Adv. Pharmacol. 1994, 28, 45-79. Tilg, H; Moschen, AR. Inflammatory mechanisms in the regulation of insulin resistance. Mol. Med. 2008, 14, 222-231. Tóth, M; Kukor, Z; Sahin-Tóth, M. Activation and dimerization of type III nitric oxide synthase by submicromolar concentrations of tetrahydrobiopterin in microsomal preparations from human primordial placenta. Placenta 1997, 18, 189-196. Tóth, M; Kukor, Z; Valent, S. Chemical stabilization of tetrahydrobiopterin by L-ascorbic acid, contribution to placental endothelial nitric oxide synthase activity. Mol. Hum. Reprod. 2002, 8, 271-280. Tsikas, D; Böger, RH; Sandmann, J; Bode-Böger, SM; Frölich, JC. Endogeneous nitric oxide synthase inhibitors are responsible for the L-arginine paradox. FEBS Lett. 2000, 478, 1-3. Vallance, P; Moncada, S. Role of endogenous nitric oxide in septic shock. New Horizons 1993, 1, 77-86.

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Vasquez-Vivar, J; Kalyanaraman, B; Martasek, P; Hogg, N; Masters, BS; Karoui, H; Tordo, P; Pritchard, KA Jr. Superoxide generation by endothelial nitric oxide synthase, the influence of cofactors. Proc. Natl. Acad. Sci. USA 1998, 95, 9220-9225. Villalta, SA; Nguyen, HX; Deng, B; Gotoh, T; Tidball, JG. Shifts in macrophage phenotypes and macrophage competition for arginine metabolism affect the severity of muscle pathology in muscular dystrophy. Human Mol. Genet. 2009, 18, 482-496. Vincent, JL; Zhang, H; Szabo, C; Preiser, JC. Effects of nitric oxide in septic shock. Amer. J. Resp. Crit. Care Med. 2000, 161, 1781-1785. Vural, H; Sirin, B; Yilmaz, N; Eren, I; Delibas, N. The role of arginine-nitric oxide pathway in patients with Alzheimer disease. Biol. Trace Element Res. 2009, 129, 58-64. Wei, L H; Wu, G; Morris, SMJr; Ignarro, LJ. Elevated arginase I expression in rat aortic smooth muscle cells increases cell proliferation. Proc. Natl. Acad. Sci. USA 2001, 98, 9260-9264. Wu, G, Morris, SMJr. Arginine metabolism, nitric oxide and beyond. Biochem J. 1998, 336, 1-17. Wu, G. Amino acids: metabolism, functions, and nutrition. Amino Acids 2009, 37, 1-17 Wu, G; Collins, JK; Perkins-Veazie, P; Siddiq, M; Dolan, KD; Kelly, KA; Heaps, CL; Meininger, CJ. Dietary supplementation with watermelon pomace juice enhances arginine availability and ameliorates the metabolic syndrome in Zucker diabetic fatty rats. J. Nutr. 2007a, 137, 2680-2685. Wu, G; Bazer, FW; Cudd, TA; Jobgen, WS; Kim, SW; Lassala, A; Li, P; Matis, JH; Meininger, CJ; Spencer, TE. Pharmacokinetics and safety of arginine supplementation in animals. J. Nutr. 2007b, 137, 1673S-1680S. Wu, G; Bazer, FW; Davis, TA; Kim, SW; Li, P; Marc Rhoads, J; Carey, SM; Smith, SB; Spencer, TE; Yin, Y. Arginine metabolism and nutrition in growth, health and disease. Amino Acids 2009, 37, 153-168. Xiao, Z; Zhang, Z; Diamond, SL. Shear stress induction of the endothelial nitric loxide synthase is calcium-dependent, but not calcium-activated. J. Cell. Physiol. 1997, 171, 205-211. Xiao-Yun, X; Zhuo-Xiong, C; Min-Xiang, L; Xingxuan, H; Schuchman, EH; Feng, L; HanSong, X; An-Hua, L. Ceramide mediates inhibition of the AKT/eNOS signaling pathway by palmitate in human vascular endothelial cells. Med Sci Monit. 2009, 15, BR 254-261. Xu, W; Kaneko, FT; Zheng, S; Comhair, SAA; Janocha, AJ; Goggans, T; Thunnissen, FBJM; Farver, C; Hazen, SL; Jennings, C; Dweik, RA; Arroliga, AC; Erzurum, SC. Increased arginase II and decreased NO synthesis in endothelial cells of patients with pulmonary arterial hypertension. FASEB J. 2004, 18, 1746-1748. Xu J, Wu Y, Song P, Zhang M, Wang S, Zou MH. : Proteasome-dependent degradation of guanosine 5'-triphosphate cyclohydrolase I causes tetrahydrobiopterin deficiency in diabetes mellitus. Circulation 2007, 116, 944-953. Yamasaki K. Edington HD. McClosky C. Tzeng E. Lizonova A. Kovesdi I. Steed DL. Billiar TR. Reversal of impaired wound repair in iNOS-deficient mice by topical adenoviralmediated iNOS gene transfer. J. Clin. Invest. 1998, 101, 967-971. Yanik, M., Vural, H., Kocyigit, A., Tutkun, H., Zoroglu, SS., Herken, H., Savas, H., Asuman. KA., Akyol, O. Is the arginine-nitric oxide pathway involved in the pathogenesis of schizophrenia? Neuropsychobiology 2003, 47, 61-65.

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Yanik, M., Vural, H., Tutkun, H., Zoroglu, SS., Savas, HA., Herken, H., Kocyigit, A., Keles, H., Akyol, O. The role of the arginine-nitric oxide pathway in the pathogenesis of bipolar affective disorder. Eur. Arch. Psych. Clin. Neurosci. 2004, 254, 43-47. Yoshizumi M, Perrella MA, Burnett JC, Lee ME. Tumor necrosis factor downregulates an endothelial nitric oxide synthase messenger RNA by shortening its half-life. Circ. Res. 1993, 73, 205-209. Zhang, W., Baban, B., Rojas, M., Tofigh, S., Virmani, SK., Patel, C., Behzadian, MA., Romero, MJ., Caldwell, RW., Caldwell, RB. Arginase activity mediates retinal inflammation in endotoxin-induced uveitis. Am. J. Pathol. 2009, 175, 891-902. Zimmermann N. King NE. Laporte J. Yang M. Mishra A. Pope SM. Muntel EE. Witte DP. Pegg AA. Foster PS. Hamid Q. Rothenberg ME. Dissection of experimental asthma with DNA microarray analysis identifies arginase in asthma pathogenesis. J. Clin. Invest. 2003, 111, 1863-1874. Zimmermann N. Rothenberg ME. The arginine-arginase balance in asthma and lung inflammation. Eur. J. Pharmacol. 2006, 533, 253-262.

In: Arginine Amino Acid Editor: Nathan L. Jacobs

ISBN 978-1-61761-981-6 © 2011 Nova Science Publishers, Inc.

Chapter 3

Free Amino Acid Analysis in Natural Matrices Graciliana Lopes, Patrícia Valentão and Paula B. Andrade* REQUIMTE/Department of Pharmacognosy, Faculty of Pharmacy, Porto University, R. Aníbal Cunha 164, 4050-047 Porto, Portugal

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Abstract Amino acids constitute a class of biologically active compounds found either in the free form or as linear chains in peptides and proteins. In addition to their primary function as protein components, they have several biological roles, being important as neurotransmitters, hormones, precursors of complex nitrogen containing molecules and as metabolic intermediates. Amino acids are molecules containing an amine group, a carboxylic acid group and a side chain that varies between different amino acids. There are 20 amino acids commonly found in proteins, which are classified depending on the polarity of the side chain. According to this criterion they can be non-polar and neutral, polar and neutral, acidic or basic. In plants they are also involved in secondary metabolism, namely in the biosynthesis of phenolic compounds, glucosinolates, cyanogenic heterosides and alkaloids. The amino acids composition is a reliable indicator of the nutritional value of matrices used for human consumption, but also a useful tool in natural products authenticity. In this work we provide an overview on the application of HPLC-UV-vis and GC-FID analysis on the determination of the amino acids profile of several natural matrices: wild edible mushroom species, Brassica oleracea var. costata, Catharanthus roseus, Cydonia oblonga and red wine inoculated with different Dekkera bruxellensis strains. The influence of different factors, such as the collection date, geographical origin and vegetal tissue, in the amino acids composition of the samples are also discussed.

Keywords: Free amino acids, red wine, Cydonia oblonga, Brassica oleraceae var. costata, edible mushrooms, Catharanthus roseus.

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Introduction Amino acids constitute a fundamental alphabet with several billion years old. They supply the required building blocks for protein biosynthesis, directly contributing to the flavour of foods and beverages (Berg et al., 2001). Amino acids consist of a central carbon atom (α carbon) linked to an amino group, a carboxylic acid group, a hydrogen atom and a “R” group also known as the side chain (Figure 1). The four different substituents of the asymmetric carbon atom make α-amino acids chiral molecules (Berg et al., 2001).

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Figure 1. Amino acids basic structure.

In the presence of a chiral carbon atom optic isomers can occur. Amino acids have optical activity, as they have the ability to rotate the plane of polarized light to the right or to the left. The optical isomers are mirror images of each other, which result from the tetrahedral geometry around the chiral carbon centre. Optical isomers are designated with the letters “D” and “L” to indicate the rotation of the polarized light to the right or left, respectively. Only L isomers are constituents of proteins (Berg et al. 2001). The ionization state of amino acids is altered by a change in pH. In acid solution, only the amino group is protonated. On the other hand, in basic solution, the carboxylic group is deprotonated and the amino group is not dissociated. At neutral pH, dipolar ions (also called zwitterions) are predominant, where the amino group is protonated and the carboxyl group is deprotonated (Figure 2) (Berg et al., 2001).

Figure 2. Ionization state of amino acids as a function of pH.

All proteins in all species are constructed from the same set of 20 amino acids (Figure 3 and 4). The twenty different side chains vary in size, shape, charge, hydrogen bonding capacity, hydrophobic character and chemical reactivity. There are a number of ways of classifying amino acids. Since their side chains are the deciding factors for intra and intermolecular interactions in proteins, and hence, for protein properties, they can be classified as (Belitz et al., 1999):

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Amino acids with nonpolar and uncharged side chain (glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan and methionine) Amino acids with charged side chains (aspartic acid, glutamic acid, histidine, lysine and arginine) Amino acids with polar and uncharged side chain (serine, threonine, cysteine, tyrosine, asparagine and glutamine)

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Another classification system is based on amino acids nutritional/physiological roles. The essential amino acids are arginine (only required for the young), histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine (Figure 3). These amino acids have to be obtained from the diet. The amino acids produced by human organism are alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine and tyrosine (Figure 4). Tyrosine is produced from phenylalanine so, if the diet is deficient in this last, tyrosine will be required as well (Belitz et al., 1999).

Figure 3. Essential amino acids. (Arg) arginine; (His) histidine; (Ile) isoleucine; (Leu) leucine; (Lys) lysine; (Met) methionine; (Phe) phenylalanine; (Thr) threonine; (Trp) tryptophan and (Val) valine.

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Graciliana Lopes, Patrícia Valentão and Paula B. Andrade

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Essential Amino Acids Essential amino acids (Figure 3) are those needed for protein synthesis, which cannot be synthesized by humans. Phenylalanine is an essential amino acid with an important metabolic role in the human organism. It may help reducing chronic pain by stimulating pain nerve pathways in the brain. It can be used for vitiligo treatment when combined with UV radiation. It also acts as analgesic and enhances the production of dopamine and nor-epinephrine, giving relief to patients suffering of depression (Meletis et al., 2005; Belitz et al., 1999). Valine is needed for muscle metabolism, tissue repair and the maintenance of a proper nitrogen body balance. It is found in high concentrations in muscle tissue, being used as an energy source for muscles and then preserving the use of glucose (Belitz et al., 1999). Tryptophan is a precursor of serotonin, (a neurotransmitter which regulates appetite, sleep patterns and mood), being applied in insomnia, anxiety and depression conditions. It is also converted in vitamin B3 in the liver (Meletis et al., 2005; Belitz et al., 1999). Threonine is required for the production of collagen and elastin in the skin. It helps the stabilization of blood sugar, being converted into glucose in the liver by the process of gluconeogenesis. It is also a precursor of phosphatidyl serine (Meletis et al., 2005; Belitz et al., 1999). Isoleucine is important for the production of energy, helping the muscle recovery (together with valine and leucine) after physical exercise. It is also needed for the formation of haemoglobin (Belitz et al., 1999). Methionine has an important function in the breakdown of fats, avoiding their accumulation in arteries. Besides its great antioxidant potential, it also helps to maintain a healthy skin tone, strong hair and nails. The capacity of increasing bile flow leads to a more efficient elimination of toxins from the liver. It is also important for cystitis and allergy situations (Meletis et al., 2005; Belitz et al., 1999). Histidine is a precursor of histamine, which is released by immune system cells during an allergic reaction. It is needed for growth and tissue repair, acting as nerve cells protector by the maintenance of myelin sheaths. Histidine is required for blood cells formation and is also helpful in arthritis (Meletis et al., 2005; Belitz et al., 1999). Arginine is a precursor of nitric oxide (important for blood vessels dilatation), being important in people with angina and congestive heart failure. It also acts by stimulating immune function and promoting the secretion of several hormones, such as glucagon, insulin and growth hormone (Meletis et al., 2005). Arginine is a semi-essential amino acid for humans and is present in all proteins at an average level of 3-6%, being biochemically important as intermediary in urea synthesis. Arginine is of great importance in babies, as they cannot produce it in their first few months. In adults, this semi-essential amino acid becomes more important when body is submitted to great physical stress. In this situation, additional arginine is required (Belitz et al., 1999). Leucine helps regulating blood sugar levels, growth and repair of muscle tissues, growth hormone production and energy regulation. Its anabolic effect can prevent the breakdown of muscle proteins after trauma or severe stress (Belitz et al., 1999). Lysine is essential for the production of carnitine, which promotes the conversion of fatty acids into energy (preventing obesity), thus helping to maintain normal blood cholesterol

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levels. Lysine promotes calcium absorption and is important for the formation of collagen (Belitz et al., 1999).

Nonessential Amino Acids

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Nonessential amino acids are also needed for protein synthesis, but they can be synthesized by the human organism (Figure 4).

Figure 4. Nonessential amino acids. (Ala) alanine; (Asn) asparagine; (Asp) aspartic acid; (Cys) cysteine; (Gln) glutamine; (Glu) glutamic acid; (Gly) glycine; (Pro) proline; (Ser) serine and (Tyr) tyrosine.

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The simplest amino acid is glycine (important as a neurotransmitter), with a hydrogen atom as the side chain, which makes it achiral. It is useful in aiding the absorption of calcium in the body. Because it supplies the body additional creatine, it is important for retarding muscles degeneration (Meletis et al., 2005). Alanine is present in prostate fluid, playing a role in supporting prostate health. It is an important source of energy for muscle tissue, brain and central nervous system, being a strengthener of the immune system by producing antibodies; it also helps in the metabolism of sugars and amino acids (Meletis et al., 2005, Belitz et al., 1999). Proline is needed for the production of collagen and cartilage. It keeps muscles and joints flexible and helps to reduce the formation of wrinkles due to UV exposition. This amino acid is essential to the maintenance of healthy skin and connective tissues, especially at the site of traumatic tissue injury (Meletis et al., 2005; Belitz et al., 1999). Aspartic acid is important in the metabolism during construction of other amino acids and biochemicals in the citric acid cycle. It also acts in removing ammonia and toxins from blood stream, and in the synthesis of immunoglobulin and antibodies (Belitz et al., 1999). Glutamine is converted into glutamic acid in the brain, which is essential for cerebral functions and increases the amount of gamma amino butyric acid (GABA), required for brain function and mental activity. Glutamine is used in the muscles for the synthesis of proteins and treatment of muscles after illness or post-operative care. Glutamic acid is important as an excitatory neurotransmitter, contributing to potassium transport across the blood brain barrier (Meletis et al., 2005). Cysteine is essential for collagen production, skin elasticity and texture. It is a detoxificant that protects brain and liver from alcohol and drugs damage, and acts in the metabolism of a number of essential biochemicals, such as coenzyme A, heparin, biotin, lipoic acid and glutathione (Meletis et al., 2005; Belitz et al., 1999). Asparagine is involved in the biosynthesis of glycoproteins. The nervous system needs this amino acid to maintain the equilibrium. It increases the resistance to fatigue and enhances the smooth functioning of the liver (Belitz et al., 1999). Serine plays a major role in the biosynthesis of purines, pyrimidines and porphyrins. It is also the precursor of several amino acids, including glycine and cysteine. Serine occurs in the active sites of enzymes as trypsin and chymotrypsin. These enzymes catalyze the hydrolysis of peptide bonds in polypeptides and proteins, a major function in the digestive process. Serine is also important in psychiatric disorders, mood and memory (Meletis et al., 2005; Belitz et al., 1999). Tyrosine is a precursor of the neurotransmitters epinephrine and dopamine. It is also useful as antidepressant, in the suppression of appetite and reduction of body fat and production of skin and hair pigment. Tyrosine improves the function of thyroid, pituitary and adrenal glands (Meletis et al., 2005). In opposition to humans, who do not have all the enzymes required for the biosynthesis of all of the amino acids, plants must be able to synthesize all of them. Amino acids are critical to life and have many functions in plant metabolism. They are involved in secondary metabolism, namely in the biosynthesis of phenolic compounds, glucosinolates, cyanogenic heterosides and alkaloids. These biomolecules have attracted a great deal of attention, mainly concentrated on their role in preventing diseases (Galili et al., 2008; Hounsome et al., 2008). Amino acids participate in many metabolic networks that control both plant growth and adaptation to the environment (Stitt et al., 2010). Secondary plant metabolites have important

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roles in cellular function, in signalling, and in adaptation to abiotic and biotic stress, playing an important role in the interaction between plants and environment and also in human health. They serve as transport molecules among tissues, including transport of nitrogen from vegetative to reproductive ones (Galili et al., 2008). In young plants, amino acids biosynthesis is ruled by a complex metabolic network that links nitrogen assimilation with carbon metabolism. The carbon/nitrogen network is strongly regulated by the metabolism of glutamine, glutamic acid, aspartic acid and asparagine, which are then converted into all other amino acids by various biochemical processes (Galili et al., 2008). The importance of amino acids in plants does not only stem from being central regulators of plant growth and responses to environmental signals, but for being also effectors of the nutritional quality of human foods and animal feeds. Although some amino acids are present in low concentrations, all vegetables, fruits and grains contain all the essential amino acids. Thus, a combined diet with different vegetable sources can provide all the essential amino acids (Galili et al., 2008; Hounsome et al., 2008). Besides the importance for human metabolism, free amino acids contribute to the taste of vegetables. Glycine and alanine are sweet, valine and leucine are bitter, and aspartic acid and glutamate have sour and “savory” (umami in Japanese) tastes (Hounsome et al., 2008). The dietary patterns that prevail in the Mediterranean area have many common characteristics. Mediterranean diets are characterized by abundant intake of plant foods, such as legumes, vegetables and fruits, and a moderate consumption of wine. Around the Mediterranean region, some very famous fortified wines are produced, especially in the north of Portugal, having socio economical importance (Matalas et al., 2000). Considering the potential beneficial effects of many natural matrices, in this chapter the amino acids composition of red wine, Brassica oleracea var. costata, Cydonia oblonga, wild edible mushroom species and Catharanthus roseus will be considered. Quantification methods for free amino acids determination will also be discussed.

1. Amino Acids Analysis For the analysis of the amino acids profile in natural matrices, different procedures have been proposed. In general, they are based in chromatographic techniques. Such techniques frequently need a previous step of derivatization, in order to enhance the sensitivity of the determination by high performance liquid chromatography (HPLC), or to increase the volatility of the analytes in gas chromatography (GC) (Belitz et al., 1999). Although direct analysis is not always simple, because these compounds lack natural strong chromophore or fluorophore groups, it is also possible to perform a direct determination of underivatized amino acids. In recent years, electrochemical detection schemes, coupled to ion-exchange chromatographic techniques, have gained prominence as sensitive analytical procedures for the direct detection of amino acids without the use of any derivatization procedure (Casella et al, 2003).

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1.1. Derivatization

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Amino acids analysis is based in the reaction of the α-amino group with several derivatization reagents, which improve the amino acids ultraviolet response at a higher wavelength or confer visible or fluorescent characteristics to them. A linear relationship is generally assumed between the absorbance/emission intensity and the concentration of αamino group. The derivatization reaction can be done after separating the amino acids (postcolumn derivatization) or before separation (pre-column derivatization). Post-Column Derivatization Post-column derivatization involves the separation of the free amino acids themselves through a liquid chromatographic column. The derivatizing reagent is introduced into the effluent from the column. Post-column derivatization has been used after ion paired reversed phase HPLC amino acids separation. This type of derivatization requires additional equipment, such as additional pumps to introduce the reagent and mixing and, sometimes, heating devices. The reagents that have been usually employed for post-column derivatization are ninhydrin, fluorescamine and o-phtaldehyde (OPA) (Aristoy et al., 2004). OPA is a fluorophore that reacts with primary amino acids to form an isoindole derivative, which is amenable to reversed phase chromatography and sensitive to small changes in the mobile phase conditions. It can be detected either spectrophotometrically (UV at 338 nm) or with fluorescence for a higher sensitivity. When OPA was introduced to amino acids analysis it was used exclusively in the post-column mode. Nowadays, most applications are performed with the pre-column technique. The major disadvantage of this reagent is the lack of reaction with amino acids with a secondary amino group (proline) or cysteine, and the low stability of their derivatives (Soufleros et al, 2003; Furst et al., 1990). In post-column derivatization it is usual to promote oxidation of secondary amines into primary amines, using hypochlorite or chloramine T, prior to OPA derivatization (Aristoy et al., 2004). Ninhydrin reacts with all 20 amino acids to give coloured derivatives. Derivatives with a primary amino group give a blue reaction product with a maximum of absorbance at 570 nm, while secondary amines give a brownish derivative that absorbs around 440 nm. Ninhydrin has also been used as coloured derivatizing reagent alone or in conjunction with Cd for the analysis of amino acids (Aristoy et al., 2004). Fluorescamine reaction with amines is almost instantaneous at room temperature in aqueous media. The products are highly fluorescent, whereas the reagent and its degradation products are non fluorescent. It potentially improves the sensitivity achieved with ninhydrin. It forms a fluorescent derivative with primary amino acids, but not with those with a secondary amino group. The fluorescence is recorded at a wavelength emission of 475 nm after excitation at 390 nm. The derivatization reaction takes place under alkaline conditions, while the separation on the ion-exchange column takes place under acidic conditions. This makes necessary the addition of a second post-column pump to introduce an alkaline buffer before fluorescamine (Aristoy et al., 2004). Pre-Column Derivatization The molecule formed with pre-column derivatization allows sensitivity and selectivity in the detection. Additionally, the derivatizing agent confers hydrophobicity to the amino acid

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molecule, making it suitable for separation by partition chromatography on a reversed phase column. Phenylisothiocyanate (PITC) converts the amino acids in their phenylthiocarbamyl (PTC) derivatives, detectable by UV (254 nm). The PTC amino acids are moderately stable at room temperature for one day and more stable in freezer, especially if dryed. Sample preparation requires basic medium with triethylamine. As the reagent is highly fluorescent, its excess may interfere with the chromatographic determination. To avoid this disadvantage, it must be extracted or converted into a non interfering adduct prior to injection (Aristoy et al., 2004; Furst et al., 1990). OPA, also used in post-column derivatization, allows a fast derivatization which is performed at room temperature in alkaline buffer (ph 9.5). As OPA derivatives are not stable, the time between sample derivatization and column injection must be short, which can be overcome by automation. In order to solve the lack of reaction with amino acids with a secondary amino group, it is common to use a double derivatization with 9-fluorennylmethyl chloroformate (OPA/FMOC) (Aristoy et al., 2004; Furst et al., 1990). Iodoacetic acid (IDA) is a monohaloacetate used for the carboxymethylation of amino acids before derivatization with OPA. These two derivatizations can be followed by a third one with FMOC, allowing the determination of the secondary amino acids in only one step. IDA is usually applied for the determination of amino acids in physiological fluids (PripisNicolau et al, 2001). 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) reacts with primary and secondary amines of amino acids, peptides and proteins, resulting in very stable derivatives with fluorescent properties (λex=250 nm, λem=395 nm), easily separated by HPLC. The excess of the reagent is consumed during reaction to form aminoquinoline (AMQ), which does not interfere in the chromatogram. Derivatization is fast, but heating is necessary if a tyrosine monoderivative is required. The derivatives are very stable at room temperature. The main drawback of this method is the poor fluorescence of tryptophan and the incomplete resolution of amino acids in physiological fluids (Aristoy et al., 2004). 1, 2-Naphthoquinone-4-sulfonate (NQS) has several important characteristics. It is soluble in water at any pH and is able to react with amino acids with primary and secondary amino groups. Both types of derivatives can be detected fluorimetrically at the same wavelength (305 nm), avoiding the need for multiple wavelength detection. NQS has also been applied to amino acids determination as post-column derivatization reagent, in ion-pair liquid chromatography (Saurina et al., 1996; Saurina et al., 1994; Kivi, 2000). Ethylchloroformate (ECF) is a suitable derivative agent for a broad array of low molecular weight metabolites. It is a simple derivatization reagent, as it reacts rapidly in water-ethanol-pyridine and shows excellent resolution. There is no requirement for a dry residue, multiple reaction steps and sample heating. The greatest disadvantage is that it is not selective for amino acids, reacting with amines and organic acids under the same reaction conditions. Other chloroformate reagents, methyl chloroformate (MCF) and menthyl chloroformate (MenCF) have been used for the derivatization of seleno and sulphur amino acids (Huseck et al., 2005). Dabsyl chloride (4-dimethylaminoazobenzene-4‟-sulfonyl chloride) is one of the most widely used agents for the derivatization of amino acids, as it allows a simple procedure, good stability, good reproducibility and good limits of detection. Additionally, a complete

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HPLC separation of all the amino acids is obtained, with a specific detection at a wavelength in the visible region (usually 420 nm) (Kivi, 2000). Dabsyl derivatization reagent can be prepared by dissolving dabsyl chloride in acetone or acetonitrile and mixing this solution with a buffer (pH 8.5-9.5). The derivatization reaction is carried out at elevated temperatures (70 ºC) and consists of an incubation of the sample with dabsyl reagent during 15-30 minutes. During the derivatization process, dabsyl chloride reacts with primary and secondary amino groups of amino acids, leading to the formation of monodabsyl derivatives and bis-dabsyl derivatives with lysine, tyrosine and histidine (Kivi, 2000).

1.2. Separation

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The separation of individual amino acids in a mixture requires very efficient separation techniques, such as chromatography (liquid or gas chromatography) or capillary electrophoresis (Aristoy et al., 2004). High Performance Liquid Chromatography (HPLC) The HPLC methods for amino acids analysis fall predominately into two types: ion exchange and reversed phase. Ion exchange chromatography (IEC) is predominately used in post-column derivatization, while reversed phase chromatography (RPC) is used with precolumn derivatization (Massom, 2002). The IEC used for amino acid analysis is strong cation exchange (stationary phase with a negative charge), which attracts positively charged cations and repels negatively charged anions. In acidic conditions, the net charge of the amino acids will be positive, being retained by the stationary phase. By increasing the pH of the mobile phase (from 2.7 to over 11), carboxyl and amine groups will lose protons, becoming negative and less positive, respectively, which results in their elution. The stationary phase used in amino acids analysis is usually sulfonated styrene instead of silica. Citrate is commonly used as a buffering agent (0.2 M). Since citrate loses its buffering action above pH 7.5, some mobile phase systems use the borate ion as the final mobile phase buffering agent, which is excellent at very high pH but has no buffering action at neutral or acidic pH. To reduce the elution time of basic amino acids, a salt gradient occurs simultaneously. Sodium and lithium are used as the cation, with chlorine being the salt‟s anion. Research has shown that sodium works better for most matrices, while lithium is more suitable for physiological fluids. A gradient elution allows small variations in pH and salts concentration, which improves the amino acids separation (Massom, 2002). Reversed phase HPLC based methods have the advantage of being accessible to most analytical laboratories, since they do not require expensive dedicated instruments. The most used column packaging consists of alkyl-bonded silica particles, mainly octadecylsilane. The presence of residual uncapped silanol groups on the silica surface can cause unwanted tailing of peaks. The addition of triethylamine, a strong cation, to the mobile phase, can overcome this problem. The most commonly employed solvent systems involve acetonitrile, methanol and/or tetrahydrophuran (Aristoy et al., 2004; Furst et al., 1990). Buffers are typically acetate or phosphate at approximately 100 µM. For the analysis of a small number of free amino acids, isocratic elution is often possible. For the determination of an overall amino acid profile from a hydrolysed sample, complicated ternary gradients are often necessary. In

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general, ultraviolet and fluorometric detectors are the more widely used detectors in liquid chromatography of amino acids (Kivi, 2000).

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Mass Spectrometry (MS) Mass spectrometry is based in the conversion of components of a sample into gaseous ions that can be resolved in the basis of their mass-to-charge ratios that are characteristic of each ion, allowing its identification. MS has been used in detection after HPLC, offering the additional advantage of analyzing the amino acids without derivatization, that means a minor sample manipulation and thus, reducing problems related to matrix interferences or poor resolution betweens peaks. One of the main requirements for samples to be analyzed by MS is that amino acids must be ionized, which can be done by atmospheric pressure microwaveinduced plasma ionization (AP-MIPI), atmospheric pressure chemical-ionization (APCI) or electrospray ionization (ESI) (Aristoy et al., 2004). ESI is a soft ionization technique used to transfer ions from solution into the gas phase at atmospheric pressure. Since ESI is a flow device, it is ideal for interfacing HPLC with MS. ESI-MS has got many advantages. It increases sensitivity and provides better on-column retention for highly hydrophilic, ionic and polar compounds. Significant time savings, due to feasibility of using higher flow rates, are an advantage obtained from lower back pressures encountered when using low aqueous/ high organic solvent mobile phases (Nguyen et al., 2008). Gas Chromatography (GC) Separation of amino acids has always been a challenge because of the heterogeneous nature of this series of compounds, due to the wide range of functional groups they possess. Although the analysis of amino acids is generally performed by HPLC, GC has some advantages due to its capillary flexibility, high resolution and speed of analysis (Silva et al., 2003). The analysis of amino acids requires a derivatization step to produce volatile adducts. The most commonly used procedure consists of a fast reaction in aqueous solution in which amino acids react with a solution of ethylchloroformate, pyridine and ethanol. The most common GC detectors are the flame ionization detector and the thermal conductivity detector (Huseck, 1991). Capillary Electrophoresis The most fundamental mechanism of separation in electrophoresis utilizes the migration of molecules in an applied electric field, according to their differences in charge, size, and hydrophobicity. Capillary zone electrophoresis (CZE) is an efficient technique for separation of charged solutes. In this technique the entire separation matrix is filled with an excess of buffered electrolyte. An infinitely thin zone of sample is introduced at one end of the system, and a potential is applied. Despite the high efficiency, speed and low sample amount requirements, the amino acids structure turns its separation difficult. Under the conditions of the electro-osmotic flow in capillary zone electrophoresis, the species with different charge can be simultaneously analyzed. The primary limitation of CZE is its inability to separate neutral compounds from each other. It is possible to introduce surfactant-formed micelles in the running buffer to provide a two phase chromatographic system to separate neutral compounds. This technique

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is used to improve separation of amino acids and to enhance ultraviolet detection or to allow fluorescence detection (Aristoy et al., 2004).

2. Free Amino Acids in Natural Matrices

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2.1. Wild Edible Mushroom Species Edible mushrooms are the fleshy and edible fruiting bodies of several species of fungi. They belong to the macro fungi and can appear either above ground or below it (Mattila et al., 2000). Mushrooms not only provide a nutritious, protein rich food, but some species also produce medicinal effective products. Edibility may be defined by criteria that include absence of poisonous effects on humans and desirable taste and aroma. Due to their texture and high content of flavour components, but also because they are rich in proteins and amino acids and poor in calories, cultivated mushrooms have now become popular all over the world (Chang et al., 1989). The nutritional value of mushrooms is closely associated with their amino acids profile, which contribute to their delicious taste and make them attractive for consumption. Trás-os-Montes region, in the north of Portugal, is recognized as one of the richest ones in wild edible mushrooms. The study of 11 wild mushrooms species (Tricholomopsis rutilans, Suillus belini, Hygrophorus agathosmus, Russula cyanoxantha, Tricholoma equestre, Fistulina hepatica, Amanita rubescens, Suillus luteus, Suillus granulatus, Boletus edulis and Cantharellus cibarius) belonging to this region was conduced in what concerns to their free amino acids composition, in order to achieve their qualitative and quantitative profile. A derivatization procedure with dabsyl chloride was performed, followed by HPLCUV-vis analysis, which allowed the detection of 20 free amino acids in almost all studied species (Figure 5). Total amino acids content varied from 1531 to 22673 mg/Kg dry matter (Ribeiro et al., 2008). B. edulis is considered one of the safest wild mushrooms. This species, followed by T. equestre (one of the tastier edible species, very appreciated in Portugal) proved to be the ones with the highest amino acid amounts (22673 and 20304 mg/Kg dry matter, respectively). On the other hand, F. hepatica revealed to have the minor amino acid contents. Although the amino acid contents in mushrooms were considerably divergent, alanine was the main compound in almost all studied species, representing 18-45% of the total analyzed compounds. T. rutilans, B. edulis and C. cibarius presented glutamine as the major compound, with 31, 26 and 25%, respectively, while, in F. hepatica, valine was the most representative (18%). Alanine, the predominant free amino acid in H. agathosmus, A. rubescens, R. cyanoxantha, T. equestre, S. bellini, S. luteus and S. granulatus, is responsible for the sweet taste of these species. On the other hand, S. bellini, S. granulatus and F. hepatica were also rich in valine, which can confer these species a bitter taste, turning them less acceptable by consumers. Considering Suillus genus, some differences were observed between the several species. Glutamic acid was not detected in S. luteus, nor was aspartic acid in S. granulatus. Cysteine was not detected in either S. bellini or S. granulatus. Although alanine was the main compound in the three species, it was more representative in S. luteus (45%), which also

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presented the higher aspartic acid relative amount (18%). Thus, the amino acid distribution pattern found does not seem to reflect any phylogenetic relationship among the three Suillus species, as they presented different amino acids profile.

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Ribeiro et al., 2008. Figure 5. HPLC-UV amino acids profile of R. cyanoxantha mushroom. Detection at 436 nm: (1) aspartic acid; (2) glutamic acid; (3) asparagine; (4) glutamine; (5) serine; (6) threonine; (7) glycine; (8) alanine; (9) valine; (10) proline; (11) arginine; (12) isoleucine; (13) leucine; (14) tryptophan; (15) phenylalanine; (16) cysteine; (17) ornithine; (18) lysine; (19) histidine; (20) tyrosine.

The amino acids composition found in literature for edible mushrooms is very distinct (Mattila et al., 2002), but it seems that glutamic and aspartic acid appear more often. It also seems that the geographical origin influences the amino acid composition (Mdachi et al., 2004). Despite alanine, glutamic acid was among the major compounds (1-12%). Within the studied species, S. luteus and C. cibarius were those containing the highest amounts of aspartic and glutamic acid. These amino acids are the ones that contribute the most to the typical mushroom taste. Despite the different geographical origin and stage of development of the analyzed species, the differences in the qualitative and quantitative amino acid profiles can be explained by the diversity of extraction, derivatization or quantification methods used in the different studies (Chang et al., 1989; Mattila et al., 2002). Although the amino acids contents in mushrooms are considerably divergent, the studied species proved to be a good source of amino acids when compared with common vegetables. Conceivably, these results indicate the importance of combining different species of mushrooms in a diet, in order to obtain a good amount and variety of all the essential amino acids.

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2.2. Brassica oleraceae var. costata DC (Tronchuda Cabbage)

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Cruciferous vegetables, such as cabbage, are among the most important dietary vegetables consumed in Europe owing to their availability in local markets, cheapness and consumer preference (Kusznierewicz et al., 2008). Brassica oleraceae var. costata is a species native of the Mediterranean region and southern Europe, extending northward to southern England. It is one of the major vegetable crops grown, constituting part of a well balanced diet in Mediterranean countries, especially in Portugal. Due to its weak susceptibility to pests and diseases and its good adaptation to a wide range of climates, tronchuda cabbage has the capacity to grow with little or no agrochemical input (Vaughan and Geissler, 1997). The free amino acids profile of tronchuda cabbage internal and external leaves was determined. The internal and external leaves are considerably different with regard to organoleptic characteristics, which may influence the preferences of consumers. Internal leaves are pale yellow and are tender and sweeter than the external ones, which present a dark green colour. The variations along three different harvesting months (from November 2005 to January 2006) were studied in order to achieve if there is a period in which the production of amino acids is clearly higher, constituting a nutritional advantage. Pre-column derivatization with dabsyl chloride followed by HPLC-UV-vis was applied to the analysis of free amino acids in aqueous hydrolysed extracts of tronchuda cabbage internal and external leaves, which allowed the detection of 20 compounds (Oliveira et al., 2008). Internal Leaves Tronchuda cabbage internal leaves presented a high content of free amino acids, varying from 4.64 g/Kg to 10.16 g/Kg (fresh weight). Taking into account the different collection times studied, significant increases were registered in internal leaves amino acids amounts from November to January. Arginine was the amino acid present in higher amounts (Figure 6A), achieving its maximum concentration in November (49% relative amount). Threonine and glutamine are the second major constituents in internal leaves (Figure 6A). Threonine amounts varied from 9 to 12% relative amount, achieving its higher concentration in December (17% relative amount), while glutamine, a nonessential amino acid, decreased along winter, exhibiting the maximum concentration in November (10% relative amount) (Oliveira et al., 2008). Cysteine was also representative and its concentration remained almost constant along the different collection times. The other amino acids detected in internal leaves were present in very small proportions (Figure 6A).

External Leaves Tronchuda cabbage external leaves presented a free amino acids amounts ranging from 8.31 g/Kg in November to 8.16 g/Kg (fresh weight) in January. Although arginine was present in external leaves in considerably high amounts (from 20 to 32% of total compounds), the most representative amino acid in this vegetal material was proline (Figure 6B). This nonessential amino acid suffers minor concentration differences

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during the collection months, achieving its maximum value in December (38%). In plants, proline seems to have a protective effect by forming complexes with heavy metals, thus, reducing the free metal ion activity (Sharma et al., 1998). Due to its capacity of protection against UV radiation, this amino acid might have an important role in external leaves, as they are more exposed than internal ones. The lowest heavy metal content that proline detoxification provides can be beneficial for human‟s health. Threonine, glutamine, glutamic acid and lysine are the third major amino acids of external leaves, varying from 4% (lysine) to 8% (glutamic acid). In opposition to internal leaves, where lysine is present in trace concentrations, in external leaves it can be found in higher amounts (Figure 6). Due to its capacity to fixate calcium, this amino acid may have an important role avoiding the deterioration of external leaves, as the cell walls are more exposed to climatic variations. All other amino acids were detected in external leaves, although, they were present in very small proportions (Figure 6B) (Oliveira et al., 2008).

Figure 6. Amino acids relative content of B. oleraceae var. costata (A) internal leaves and (B) external leaves. (asp) Aspartic acid; (glu) glutamic acid; (asn) asparagine; (gln) glutamine; (ser) serine; (thr) threonine; (gly) glycine; (ala) alanine; (val) valine; (pro) proline; (arg) arginine; (ile) isoleucine; (leu) leucine; (trp) tryptophan; (phe) phenylalanine; (cys) cysteine; (orn) ornitine; (lys) lysine; (his) histidine and (tyr) tyrosine.

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The results obtained also revealed that free amino acids profile presented some significant differences according to the position of the leaf (internal or external) in what concerns to valine, leucine, cysteine, lysine, histidine and tyrosine contents (Figure 6). The two kinds of tronchuda leaves supply different proportions of free amino acids, although it is important to consume both kinds of leaves to obtain the appropriate amounts of amino acids, especially the essential ones, which are indispensable in a healthy diet. Carlotti and co-workers (1997) studied the synergistic effect of some amino acids (tryptophan, cysteine, alanine and glycine) on the antioxidant effect of vitamin E and C against lipoperoxidation of linoleic acid. Among them, only tryptophan and cysteine exerted a noteworthy synergistic effect with vitamin E and vitamin C. All the referred amino acids were found in the tronchuda cabbage analysed samples and, since this matrix contain large amounts of free amino acids, its antioxidant capacity can be enhanced. In fact, the previously analysed aqueous lyophilized extracts of B. oleraceae var. costata leaves revealed a great antioxidant potential (Ferreres et al., 2006). Once internal leaves are richer than external ones in what concerns to tryptophan and cystein contents, these free amino acids might enhance the antioxidant capacity of this matrix.

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2.3. Red Wine Dão is a unique region located in the north central Portugal, with important viticulture traditions with secular reputation, from which the excellent edapho-climatic conditions are turned to advantage for vineyard culture. The Dão region is characterized by a temperate climate, varying from cold and rainy in winter to frequently hot in summer. The vineyards from Dão are established in granite land, between 400 and 500 m altitude. These vineyards give origin to grape varieties destined to the elaboration of wines with “Denomination d‟Origine Controllée” (DOC). Touriga Nacional is the most important grape cultivar because of its capacity to produce high quality Dão red wines. This grape variety is responsible for the organoleptic characteristics and the prestige that Dão wines have gained in the course of time (Silva et al., 2007; Valentão et al., 2007). Wine is the product of complex interactions between fungi, yeasts and bacteria that start in the vineyard and continue throughout the fermentation process until packaging (Fleet, 2003). The amino acids profile of wines plays an important role in their organoleptic characteristics and economical value. Once they constitute an important fraction of nitrogen in must and wine, they are used as nutrients for the growth of microorganisms like yeast and bacteria, during alcoholic and secondary fermentation (Alcaide-Hidalgo et al., 2007; Chatonnet et al., 1995). Free amino acids present in wines can arise from different sources. Besides those present in grapes, they can result from the enzymatic degradation of grape proteins, the excretion by yeasts at the end of fermentation and from proteolysis during the autolysis of death yeasts. Furthermore, the amino acids content of grapes can vary according to different factors, including the type of fermentation, grape variety, vintage, region of cultivation, fertilization and climatic conditions and with different viticultural and enological practices adopted during wine making (Soufleros et al., 2003).

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Dekkera bruxellensis Dekkera bruxellensis is a yeast often found in wine, a poor environment for most microorganisms due to minute amount of sugars and high ethanol levels. It constitutes an economical problem associated with wine industry, as it has the capacity to adulterate wine. The presence of D. bruxellensis in wine during barrel ageing is often associated with detrimental organoleptic effects. This yeast has the capacity to grow and survive in wine with high ethanol levels and low sugar supply. The growth of this type of yeast is uncommon during fermentation if elementary hygiene precautions are taken (Fleet, 2003; AlcaideHidalgo et al., 2006). Amino Acids Profile of Dão Red Wine and D. bruxellensis Effects The determination of free amino acids in Dão red wine obtained from Touriga Nacional grapes was studied in order to evaluate the effect of the inoculation with the spoilage D. bruxellensis. Samples were analyzed by reversed phase HPLC-UV-Vis after pre-column derivatization with dabsyl chloride. A wine sample was taken from a stainless steel tank, sterilized by filtration and distributed by several flasks. Nine of them were inoculated with different D. bruxellensis strains, one was inoculated with Saccharomyces cerevisiae and used as a control, and witness flasks, with and without refrigeration, were also analysed (Silva et al., 2007). All the samples presented twenty free amino acids, ranging from 1.4 to 620.1 mg/L, with proline being the major one. Proline has been found to be the major free amino acid in other Portuguese elementary red wines, which confirms the inadequacy of proline test for general wine authenticity (Vasconcelos and Chaves das Neves, 1989). Refrigeration didn‟t influence the total free amino acids amount, although a higher content of asparagine was observed in the sample stored at low temperature. The inoculation with D. bruxellensis seemed to increase the contents of glutamic acid, asparagine, glutamine and isoleucine. Some differences in the free amino acids profile resultant from the inoculation with the different D. bruxellensis strains were observed. With the exception of one strain, the inoculation of D. bruxellensis in Dão red wine lead to an increase in total free amino acids content (Silva et al., 2007). Tryptophan content was reduced from 0.6% (in the witness sample) to 0.2% by one strain, while another one caused its increase to 1.5%. Arginine was also influenced by some strains: two strains lead to its reduction, when compared with the witness sample, while two others resulted in a higher relative amount. In fact, arginine content in inoculated samples varied from half to a double when compared with the witness sample, which shows the different influence of D. bruxellensis strains in this amino acid. According to the results, the inoculation with this yeast seemed to mainly affect the contents of aspartic and glutamic acids, asparagine, glutamine and isoleucine (Silva et al., 2007). The inoculation with S. cerevisiae significantly increased the amounts of arginine and tryptophan. Asparagine, which was one of the main free amino acids affected by D. bruxellensis, was not affected by S. cerevisiae. The amino acids analysis of wines could be effective in wine quality control determinations, namely in the evaluation of the spoilage wine yeast D. bruxellensis influence upon the contents of free amino acids in Dão red wine (Silva et al., 2007).

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2.4. Quince (Cydonia oblonga Miller) Fruit and Jam

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Quince fruit is a pome with a yellow pulp, generally acid and astringent, and numerous seeds. The fruit has variable dimensions and asymmetric shapes, exhibiting a characteristic fragrance. The peel is covered by an abundant hair, which disappears with fruit ripening. Due to its astringent properties, it can be consumed when processed as jam or jelly, which are much appreciated in Portugal and economically important. According to the Portuguese Legislation (Decreto-Lei no. 97/84 de 28 de Março), quince jam, known as “marmelada”, is the food product of the homogeneous and consistent mixture of boiling quince mesocarp with sugars. When quince production is scarce, industry manufacturers are tempted to adulterate quince jam by adding apples or pears, because of their low cost and similar texture and rheological properties. Once the stronger odour of quince masks the sweet flavour of both fruits, sensory evaluation cannot be used to detect their presence. All fruits show typical free amino acids pattern and the analysis of quince free amino acids profile can be useful for the determination of quince products authenticity (Silva et al., 2000). A GC/FID methodology with pre-column derivatization was developed for free amino acids determination in quince fruit (pulp and peel) and jam (Silva et al., 2003). The precolumn derivatization procedure was performed with ECF. Quince Fruit All quince fruit samples presented an identical qualitative profile, composed by twenty one amino acids (Figure 7). The two major free amino acids in this fruit were asparagine and aspartic acid. Samples of quince fruit from seven different geographic origins from Portugal were analyzed. Generally, the amount of free amino acids was higher in peels (1010 µg/Kg, mean value) than in the corresponding pulps (807 µg/Kg, mean value). Glycine was the most representative free amino acid in almost all peels (217 µg/Kg, mean value) (Figure 7) (Silva et al., 2004).

Figure 7. Free amino acids composition of quince pulps, peels and jams (relative content) (Mean values). (Ala) alanine; (Gly) glycine; (Val) valine; (Leu) leucine; (Ile) isoleucine; (Pro) proline; (Thr) threonine; (Ser) serine; (Glu) glutamic acid; (Asn) asparagine; (Asp) aspartic acid; (Met) methionine; (Hypro) hydroxiproline; (Phe) phenylalanine; (Cys) cysteine; (Gln) glutamine; (Orn) ornitine; (Lys) lysine; (His) histidine; (Tyr) tyrosine and (Trp) tryptophan.

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The three major free amino acids present in quince pulps were aspartic acid (24%), hydroxyproline (13%) and asparagine (17%), while in quince peels were glycine (22%), aspartic acid (20%) and asparagine (12%). The mean value of the sum of the twenty one free amino acids was ca. 0.8 and 1.0 mg/Kg for pulp and peel, respectively. Hydroxyproline content was significantly higher in pulp, while glutamic acid contents were significantly lower in this part of the fruit. Although some free amino acids varyed significantly between the harvesting years, the free amino acid profile was similar in pulp and peel (Silva et al., 2004). Besides cultural practices and degree of maturation, it seems that the geographical origin influences the free amino acids content of quince fruit, as fruits collected in different places, presented a somewhat distinct amino acids profile. Quince Jam Quince jams were rich in aspartic acid (32%) and asparagine (30%). Medium values of glycine, hydroxyproline, threonine, alanine, glutamic acid and cysteine were found. All other free amino acids were found in very small proportions. For all homemade quince jams, the two most abundant free amino acids were aspartic acid and asparagine and the third was cysteine, hydroxyproline, or glycine. In industrially manufactured quince jam samples the three major free amino acids were generally aspartic acid, asparagine and, glycine or hydroxyproline. According to this, it was possible to conceive that samples in which glycine was the major amino acid were prepared with unpeeled quinces. As some authors reported, the main free amino acid of pear was proline, followed by considerable percentages of aspartic acid, asparagine, glutamic acid, serine and alanine (Belitz et al., 1999). On the other hand, apple mainly contained asparagine, aspartic acid, glutamic acid and serine (Gomis et al., 1992). According to this information, a high content of proline in quince jam might be related to pear addition. The proportions of glutamic acid, glutamine and lysine were significantly higher in industrial quince jam, while aspartic acid was significantly higher in homemade quince jam. Some free amino acids content (valine, threonine and glutamic acid) varyed significantly according to the year of commercialization. Glutamine content was also significantly distinct according to the quince jam brand. Quince fruit and quince jam levels of asparagine and aspartic acid exhibited an inverse correlation. Quince fruit composition was higher in asparagine than in aspartic acid, but quince jam had higher aspartic acid than asparagine content. This can probably be explained by the fact that asparagine can be converted into aspartic acid or due to hydrolysis of proteins, peptides and other compounds with amino acids in their constitution, which can occur during thermal processing in acid medium, as happens in the jam preparation.

2.5. Catharanthus roseus Catharanthus roseus is an evergreen perennial herb, widely cultivated for its medicinal properties. It is a popular ornamental plant found in gardens and homes across the warmer parts of the world. As it is also known as the source of alkaloids now used in the treatment of cancer, its discovery led to one of the most important medical breakthroughs of the last

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century. Some of its alkaloids are approved as antineoplasic agents to treat leukemia, Hodgkin's disease, malignant lymphomas, neuroblastoma, rhabdomyosarcoma, Wilms' tumor, and other cancers (Nayak et al., 2006). The plant has a flower adapted to pollination by a long-tongued insect, such as a moth or butterfly. It is also able to self-pollinate. Self-compatibility and a relatively high tolerance to disturbance have enabled the plant to spread from cultivation and to become naturalised in many parts of the world. Its seeds have been seen to be distributed by ants (Misra et al., 1978). The consumption of this species is mainly done by means of decocts and infusions for various applications, such as diabetes mellitus, fever, bleeding arresting and also to suppress the sensation of hunger and fatigue (Ross et al., 2003). The qualitative and quantitative amino acids profile of C. roseus was achieved by HPLCUV, after pre-column derivatization with dabsyl chloride (Pereira et al., 2009). Samples of several vegetable tissues (seeds, petals, stems and leaves) were analyzed. With the exceptions of glycine, alanine, valine, proline and cysteine, the remaining compounds were found in all materials (Figure 8). The total amino acids content followed the order leaves > seeds > petals > stems, varying between 31557 and 159697 mg/Kg. Seeds were the sample with higher number of compounds, with valine being found in trace amounts. On the other hand, petals revealed to be the sample with lower diversity of compounds, being glycine, alanine, valine and cysteine vestigial constituents (Pereira et al., 2009).

Figure 8. Amino acids relative content of C. roseus plant parts aqueous extract. (Asp) aspartic acid; (Glu) glutamic acid; (Asn) asparagine; (Gln) glutamine; (Ser) serine; (Thr) threonine; (Gly) glycine; (Ala) alanine; (Val) valine; (Pro) proline; (Arg) arginine; (Leu) leucine; (Trp) tryptophan; (Phe) phenylalanine; (Cys) cysteine; (Orn) ornitine; (Lys) lysine.

Arginine was the major amino acid, varying from 24 % in seeds to 60% in leaves. The second major amino acid was asparagine in seeds (11%), glutamic acid in stems (10%), glutamine in petals (10%) and leucine in leaves (5%). Valine was found in vestigial amounts in all vegetal tissues, excepting in stems. The same was observed for cysteine, which was able to be quantified only in seeds and leaves. Proline was present in trace amounts in stems and leaves. The high arginine content in leaves can be an explanation for the use of this plant material in decocts for diabetes mellitus, as arginine acts by stimulating immune function and promoting the secretion of several hormones, such as glucagon and insulin (Meletis et al., 2005).

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Threonine was also present in high amounts in leaves, and, like arginine, it helps the stabilization of blood sugar, being this activity a possible explanation for the use of C. roseus leaves in decocts by diabetics. The representative amounts of glutamic acid and glutamine may be a reason for also using C. roseus decocts in fatigue situations (Meletis et al., 2005; Belitz et al., 1999).

Conclusion

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Vegetables are an important part of the human diet and a major source of biologically active substances, such as vitamins, dietary fibre, antioxidants, cholesterol-lowering compounds and amino acids. Metabolites like amino acids are essential for plant growth, development, stress adaptation, and defence. When using amino acids as therapeutics, a dietary concern should be kept in mind. Amino acids have potential for addressing a range of conditions either for prevention or treatment. Besides the importance for human metabolism, free amino acids contribute to the taste of vegetables. Nutrient composition of vegetables is very complex and difficult to assess. The application of chromatography allows qualitative and quantitative determinations of a large number of plant metabolites. Amino acid analysis is a suitable tool for precise determination of protein quantities, but also provides detailed information regarding the relative amino acid composition and free amino acids. Amino acids are often derivatized for sensitive detection and separated by HPLC. Whereas post-column derivatization was typical earlier, pre-column derivatization has gained importance and can be achieved by a broader range of derivatization reagents.

Acknowledgments Reviewed by Prof. Dr. Branca M. Silva (Faculdade de Ciências da Saúde, Universidade Fernando Pessoa, Rua Carlos da Maia 296, 4200-150 Porto, Portugal). G. Lopes (SFRH/BD/61565/2009) acknowledges Fundação para a Ciência e a Tecnologia for the grant.

References Alcaide-Hidalgo, J. M., Moreno-Arribas, M. V., Martín-Álvarez, P. J. and Polo, M. C. (2007). Influence of malolactic fermentation, postfermentative treatments and ageing with lees on nitrogen compounds of red wines. Food Chem. 103, 572-581. Aristoy, M. C. and Toldrá, F. (2004). Amino acids. In: Handbook of food analysis: Physical characterization and nutrient analysis. Marcel Dekker: USA, pp 83-103. Belitz, H. D. and Grosch, W. (1999). Amino acids, peptides and proteins. In: Food Chemistry. Springer-Verlag: Berlin, pp 8-34.

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Berg, J. M., Tymoczko, J. L. and Stryer, L. (2001). Protein structure and function, In: Biochemistry 5th ed.; USA, pp 41-76. Carlotti, M. E., Gallarate, M., Gasco, M. R., Morel, S., Serafino, A. and Ugazio, E. (1997). Synergistic action of vitamin C and amino acids on vitamin E in inhibition of the lipoperoxidation of linoleic acid in disperse systems. Int. J. Pharmaceut. 155, 251-261. Casella, I. G. and Contursi, M. (2003). Isocratic ion exchange determination of underivatized amino acids by electrochemical detection. Anal. Chim. Acta 478, 179-189. Chatonnet, P., Dubourdieu, D. and Boidron, J. N. (1995). The influence of Brettanomyces/Dekkera sp. Yeasts and lactic acid bacteria on the ethylphenol content of red wines. Am. J. Enol. Vitic. 46, 463-468. Chang, S. T. and Miles, P. G. (1989). Mushrooms: cultivation, nutritional value, medicinal effect, and environmental impact. CRC Press. pp. 4-13. Ferreres, F., Sousa, C., Vrchovská, V., Valentão, P., Pereira, J. A., Seabra, R. M. and Andrade, P. B. (2006). Chemical composition and antioxidant activity of tronchuda cabbage internal leaves. Eur. Food Res. Technol. 222, 88-98. Fleet, G. H. (2003). Yeast interactions and wine flavor. Int. J. Food Microbiol. 86, 11-22. Furst, P., Pollack, L., Graser, T. A., Godel, H. and Stehle, P. (1990). Appraisal of four precolumn derivatization methods for the high-performance liquid chromatographic determination of free amino acids in biological materials. J. Chromatogr. A 499, 557569. Galili, S., Amir, R. and Galili, G. (2008). Genetic engineering of amino acid metabolism in plants. Advances in Plant Biochemistry and Molecular Biology 1, 49-80. Gomis, D. B., Lobo, A. M. P. and Alonso, J. M. (1992). Determination of amino acids in ripening apples by high performance liquid chromatography. Z. Lebensm.-Unters. Forsch 194, 134-138. Hounsome, H., Hounsome, B., Tomos, D. and Edwards-Jones, G. (2008). Plant metabolites and nutritional quality of vegetables. J. Food Sci. 73, R48-R65. Huseck, P. (1991) Rapid derivatization and gas chromatographic determination of amino acids. J. Chromatogr. A 552, 289-299. Huseck, P. (2005). Quantification of amino acids as chloroformates – A return to gas chromatography, In: Quantification of amino acids and amines by chromatography. Elsevier, Netherlands, pp. 2-39. Kivi, J. T., (2000). Amino acids, In: Food analysis by HPLC (2000). Marcel Dekker, USA, pp 74-84. Kusznierewicz, B., Bartoszek, A., Wolska, L., Drzewiecki, J., Gorinstein, S. and Namiesnik, J. (2008). Partial characterization of white cabbages (Brassica oleraceae var. capitata f. alba) from different regions by glucosinolates, bioactive compounds, total antioxidant activities and proteins. LWT 41, 1-9. Massom, L. R. (2002). Amino acid analysis, In: Methods of analysis for functional foods and nutraceuticals. CRC Press, USA, pp 261-271. Matalas, A. L., Zampelas, A., Stavrinos, V. and Wolinsky, I. (2000). The Mediterranean diet: Constituents and health promotion. CRC Press, USA, pp. 181-190. Mattila, P., Suonpää, K. and Piironen, V. (2000). Functional properties of edible mushrooms. Nutrition 16, 694–6.

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Mattila, P., Salo-Vaananen, P., Konko, H. A. and Jalava, T. (2002). Basic composition and amino acid contents of mushrooms cultivated in Finland. J. Agric. Food Chem. 50, 64196422. Mdachi, S. J. M., Nkunya, M. H. H., Nyigo, V. A. and Urasa, I. T. (2004). Amino acid composition of some Tanzanian wild mushrooms. Food Chem. 86, 179-182. Meletis, C. D. and Barker, J. E. (2005). Therapeutic uses of aminoacids. Alternative & Complementary Therapies. 11, 24-28. Misra, R., Singh, J. S. and Gopal, B. (1978). Glimpses of ecology: Professor R. Misra commemoration volume. International Scientific Publications: Universidade de Cornell, USA, pp 472. Nayak, B. S. and Pereira, L. M. P. (2006). Catharanhtus roseus flower extract has woundhealing activity in Sprague Dawley rats. BMC Complement. Altern. Med. 6:41 (Doi:10.1186/1472-6882-6-41). Nguyen, H. P. and Schug, K. A. (2008). The advantage of ESI-MS detection in conjunction with HILIC mode separations: Fundamentals and applications. J. Sep. Sci. 31, 14651480. Oliveira, A. P., Pereira, D. M., Andrade, P. B., Valentão, P., Sousa, C., Pereira, J. A., Bento, A., Rodrigues, M. A., Seabra, R. M. and Silva, B. M. (2008). Free amino acids of tronchuda cabbage (Brassica oleraceae var. costata DC): Influence of leaf position (internal and external) and collection time. J. Agric. Food. Chem. 56, 5216-5221. Pereira, D. M., Ferreres, F., Oliveira, J., Valentão, P., Andrade, P. B. and Sottomayor, M. (2009). Targeted metabolite analysis of Catharanthus roseus and its biological potential. Food Chem. Toxicol. 47, 1349-1354. Pripis-Nicolau, L., Revel, G., Marchand, S., Beloqui, A. A. and Bertrand, A. (2001). Automated HPLC method for the measurement of free amino acids including cysteine in musts and wines; first applications. J. Sci. Food. Agric. 81, 731-738. Ribeiro, B., Andrade, P. B., Silva, B. M., Baptista, P., Seabra, R. M. and Valentão, P. (2008). Comparative study on free amino acid composition of wild edible mushroom species. J. Agric. Food Chem. 56, 10973-10979. Ross, I. A. (2003). Catharanthus roseus. Medicinal Plants of the world, vol. I. Humana Press, Totowa, NJ, pp. 175-196. Saurina, J. and Hernández-Cassou, S. (1996). Chromatographic determination of amino acids by pre-column derivatization using 1,2-naphthoquinone-4-sulfonate as reagent. J. Chromatogr. A 740, 21-30. Saurina, J. and Hérnandez-Cassou, S. (1994). Determination of amino acids by ion-pair liquid chromatography with post-column derivatization using 1,2-naphthoquinone-4-sulfonate. J. Chromatogr. A 676, 311-319. Sharma, S. S., Schat, H. and Vooijs, R. (1998). In vitro alleviation of heavy metal-induced enzyme inhibition by proline. Phytochemistry 49, 1531-1553. Silva, B. M., Casal, S., Andrade, P. B., Seabra, R. M., Oliveira, M. B. and Ferreira, M. A. (2003). Development and evaluation of a GC/FID method for the analysis of free amino acids in quince fruit and jam. Anal. Sci. 19, 1285-1290. Silva, B. M., Casal, S., Andrade, P. B., Seabra, R. M., Oliveira, M. B. P. P. and Ferreira, M. A. (2004). Free amino acid composition of quince (Cydonia oblonga Miller) fruit (pulp and peel) and jam. J. Agric. Food Chem. 52, 1201-1206.

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Silva, B. M., Silva, L. R., Valentão, P., Seabra, R. M., Andrade, P. B., Trujillo, M. E. and Velázquez, E. (2007). HPLC determination of free amino acids profile of Dão red wine: effect of Dekkera bruxellensis contamination. J. Liq. Chromatogr. Rel. Technol. 30, 1371-1383. Silva, B. M., Andrade, P. B., Valentão, P., Mendes, G. C., Seabra, R. M. and Ferreira, M. A. (2000). Phenolic profile in the evaluation of commercial quince jellies authenticity. Food Chem. 71, 281-285. Soufleros, E. H., Bouloumpasi, E., Tsarchopoulos, C. and Biliaderis, C. G. (2003). Primary amino acid profiles of Greek white wines and their use in classification according to variety, origin and vintage. Food Chem. 80, 261-273. Stitt, M., Sulpice, R. and Keurentjes, J. (2010). Metabolic networks: How to identify key components in the regulation of metabolism and growth. Plant Physiol. 152, 428-444. Valentão, P., Andrade, P. B., Lopes, G., Cardoso, L., Silva, L. R., Martins, D., Trujillo, M. E., Velázquez, E. and Seabra, R. M. (2007). Influence of Dekkera bruxellensis on the contents of anthocyanins, organic acids and volatile phenols of Dão red wine. Food Chem. 100, 64-70. Vasconcelos, A. M. P. and Chaves das Neves, H. J. (1989). Characterization of elementary wines of Vitis vinifera varieties by pattern recognition of free amino acids profile. J. Agric. Food Chem. 37, 931-937. Vaughan, J. G. and Geissler, C. A. (1997). The new Oxford book of food plants. New York: Oxford University press, 166-169.

In: Arginine Amino Acid Editor: Nathan L. Jacobs

ISBN 978-1-61761-981-6 © 2011 Nova Science Publishers, Inc.

Chapter 4

Discovery of Argininosuccinate Synthetase and Argininosuccinate Lyase Olivier Levillain* Université Claude Bernard Lyon I, Physiologie Intégrative, Cellulaire et Moléculaire UMR 5123 CNRS, Bâtiment. R. Dubois, 43, Bvd du 11 Novembre 1918 F-69622 Villeurbanne Cedex, France

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Abstract This review summarizes the impressive work performed by several teams of research to discover the biosynthesis of arginine in mammalian liver and kidney and the different steps which led to identify the metabolites involved in this reaction. Successive purification steps allowed the isolation and characterization of two enzymatic proteins, namely argininosuccinate synthetase and argininosuccinate lyase. New approaches including molecular biology gave insights into the molecular characteristics of ASS and ASL genes, mRNAs, and proteins. Furthermore, new techniques such as Northern blot, Western blot, and immunocytology became excellent tools to analyze the expression on ASS and ASL genes in the different organs of several mammalian species. ASS and ASL are homotetramers with subunits of 46 and 51 kDa, respectively. ASS and ASL are generally co-localized in the same cell type and are widely distributed in various organs.

*

Email: [email protected] Fax 33-04-72-43-11-72

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1. Introduction to Arginine The amino acid arginine (Arg or R), also named 2-amino-5-guanidino pentanoïc acid, was discovered in 1886 by Schulze and Steiger [70]. Arginine is characterized by a basic isoelectric point of 10.76, a molecular weight of 174, and contains a guanidino group. In 1895, Hedin reported that arginine is a component of animal protein [26]. This amino acid is involved in several metabolic pathways and plays key roles in cell physiology. Arginine is not only supplied by food but also synthesized by a myriad of organisms including plants and animals. The discovery of the different steps involved in arginine synthesis required more than one decade and are summarized below. Interestingly, the progresses in the understanding of this metabolic pathways occurred simultaneously and/or alternatively in the liver and the kidney.

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2. History of Arginine Synthesis The biochemical synthesis of arginine was reported for the first time in 1932 [35]. Krebs and Henseleit proposed that one step of hepatic ornithine cycle involves the conversion of citrulline into arginine. In 1940, Klose and Almquist reported that citrulline is as effective as arginine in sustaining chicken growth [32]. These authors concluded that citrulline was presumably first converted to arginine. In 1941, Borsook and Bubnoff, who analyzed the formation of glycocyamine in animal tissues, observed that rat kidney slices were able to synthesize glycocyamine from arginine and glycine [9]. More interestingly, when the amide donor was replaced by citrulline, rat kidney slices produced quite similar amounts of glycocyamine as they did with arginine. These results led the authors to hypothesize that citrulline might be converted into arginine in rat kidney slices [9]. On the basis of these two reports, Borsook and Bubnoff tested the formation of arginine from citrulline and glutamic acid or aspartic acid in rat and guinea pig kidneys [8]. Renal arginine synthesis occurred in the presence of these three substrates, but glutamine was as effective as glutamic acid because the kidney expresses an active glutaminase [34]. In contrast, replacement of aspartic acid by asparagine led to poor arginine production because a very low asparaginase activity was found in the rat kidney. Further experiments were performed to identify the substrates involved in arginine synthesis. Arginine synthesis in the rat kidney was tested with other amino acids as nitrogen donors. Proline, ornithine and, in a lesser extent, hydroxyproline permitted significant production of arginine [8]. Weil-Malherbe and Krebs showed that guinea-pig, rabbit, and rat kidneys metabolize proline and hydroxyproline to glutamic acid [76]. Lysine was reported to contribute to arginine synthesis [8]. The role of lysine in arginine synthesis was clarified in 1948. Indeed, Dubnoff and Borsook reported the formation of arginine from -aminoadipic acid, a by-product of lysine, and citrulline in rat kidney slices [21]. Thus, -aminoadipic acid is used for transamination and probably as a source of nitrogen for citrulline. Further work identified activators and inhibitors of arginine synthesis. The renal synthesis of arginine was inhibited by cyanide [8]. The inhibitory effect of cyanide is indirect since it prevents the oxidation of cytochrome by blocking the flow of electrons. This indicates that an oxidative step took place in the reaction mechanism. The authors could not explain why

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cyanide inhibited arginine formation probably because the biochemical equation and the enzymes involved in the conversion of citrulline into arginine were not yet established [8]. Cyanide inhibition can be prevented by adding sufficient concentration of -keto-acid derivative of methionine, oxalacetic acid, and pyruvic acid [8]. The effect of metallic ions was also investigated. Fe3+, Zn2+, and Fe2+ inhibited the reaction to the extent of 20%, while Mn2+ and Ca2+ each inhibited to the extent of 56% [61]. Mn2+ and Ca2+ might compete with Mg2+ for the enzyme. The formation of arginine from citrulline was also inhibited by pyruvate whether the nitrogen donor is aspartic acid, glutamic acid, proline, ornithine, or lysine. Indeed, in the kidney, pyruvate is preferentially converted in alanine at the expense of glutamic acid [8]. Moreover, the renal capacity to synthesize arginine is reduced by 60-90% when the kidney is homogenized to produce cell-free suspension [8, 13]. Thus, renal arginine synthesis requires cell integrity. In 1946, Cohen and Hayano analyzed and compared in detail the molecular mechanisms required for the conversion of citrulline into arginine in slices and homogenates prepared from liver and kidney of adult rats [13, 14]. Their findings showed that homogenates of both organs required ATP (0.8 mM), magnesium ions (3 mM), and oxygen to sustain arginine synthesis. Under anaerobic conditions, neither slices nor homogenates of either liver or kidney synthesized arginine. In addition, they observed that liver homogenate contained a very « active system » for the synthesis of arginine, 80% greater than in kidney slices whereas liver slices showed a very low activity [13]. However, we point out that the liver is not a net arginine producing organ because the arginine produced in the ornithine cycle is immediately hydrolysed by a very active cytosolic arginase. In contrast, the kidney does produce net arginine [20]. In liver homogenates, glutamic acid was 4 times as effective as aspartic acid in supporting arginine synthesis. Cohen and Hayano demonstrated that arginine synthesis in liver homogenates from glutamic acid and citrulline originates from nitrogen transfer from an amino acid rather than from NH3 [13, 14]. In 1949, Ratner and Pappas suggested that the mechanism of nitrogen transfer involves a preliminary condensation of the amino group of aspartic acid with the ureido carbon of citrulline to form C-NH-C linkage [60]. No amino acid other than aspartic acid has been found to react with citrulline. The transamination of glutamic acid with oxalacetic acid to form aspartate accounts for the fact that glutamate can replace aspartic acid. Therefore, glutamic acid functions in arginine synthesis indirectly rather than by an interaction with citrulline. They also found the fundamental role of Mg++ in the condensation step which occurs with the first enzyme and a number of phosphate-transferring enzymes [60]. Finally, the oxygen dependence in respiring preparations must be associated with the generation of ATP by oxidation of respiratory substrates. At first, the reaction mechanism was formulated as follows : Step 1 citrulline + aspartic acid + ATP

---------->

intermediate + ADP + Pi

---------->

arginine

Step 2 intermediate

+ malic acid

The intermediate compound corresponds to a new amino acid, extremely water-soluble, which was named argininosuccinic acid [63].

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Although the molecular partners of the biochemical reaction converting citrulline into arginine have been clearly listed, no efforts were undertaken to isolate and characterize the enzymes before 1947. To the best of our knowledge, this was first attempted by Ratner [53]. She isolated a partially purified enzyme system from beef liver which catalyzed the anaerobic formation of arginine and malic acid from citrulline and aspartic acid in the presence of Mg++ and catalytic amounts of ATP [53]. Ratner also understood that aerobic conditions are required for the generation of ATP. Investigations were undertaken to elucidate fundamental aspects of the enzymatic mechanism involved in arginine synthesis. Two unknown enzymes were separated by repeated ammonium sulfate fractionation of acetone powder extracts of ox liver [61]. One of them led to the disappearance of citrulline and the simultaneous appearance of inorganic phosphate. The second enzyme led to the formation or arginine and malic acid, most probably by a hydrolytic step [60, 61]. It was also of interest to know whether the enzymatic mechanism occurring in liver applies also to tissues which lack the other enzymes of the ornithine cycle. To document this point, Ratner and Petrack investigated arginine formation in respiring pig kidney homogenates [62]. The specific activities of acetone-dried tissue extracts are about the same in liver and kidney of ox and pig. As reported for the liver, dialyzed pig kidney extracts require citrulline, aspartic acid, Mg++, and ATP. The enzyme-substrate affinity (Km) for the pig kidney and liver were in the same range (1 - 1.5 mM for the substrates and Mg++) suggesting that the two enzyme systems have similar enzymatic properties [62]. The characteristics of the condensing and splitting enzymes observed in the liver were also found in the kidney. In addition, in the forward reaction, the intermediate compound, argininosuccinic acid, is cleaved into arginine and fumaric acid by the latter enzyme and seems to be identical with the reaction reported by Davison and Elliott [17]. These authors observed that unknown enzymes from aqueous extracts of dry pea meal metabolized almost exclusively arginine and fumarate into a new ninhydrin-reacting amino compound. Their results suggested that the amidine group of arginine was involved in the reaction with fumarate. This back reaction was found in a wide variety of cells including rat kidney, sheep heart, sheep pancreas, yeast, and Proteus vulgaris [17]. The liver and the kidneys possess similar enzyme systems to produce arginine. However, the source of the substrates remained an open debate. The liver produces endogenous aspartate via aspartate aminotransferase (EC 2.6.1.1) and citrulline in the ornithine cycle (Figure 1). The second step of this cycle is catalysed by ornithine transcarbamylase (OTC; EC: 2.1.3.3), a mitochondrial enzyme that transfers a carbamyl phosphate group onto ornithine to generate citrulline [15]. In contrast, the kidney is unable to metabolize ornithine into citrulline [15]. Thus, it was suggested that citrulline is supplied to the kidneys by the blood stream. This was supported by the discovery of citrulline in plasma of fasted humans (≈ 30 µM), humans with renal disease (≈ 100 µM), and fasted dogs (60-90 µM) [4]. Although the source of citrulline remained unknown in 1941, Borsook and Dubnoff yet established that the kidney is the major organ involved in arginine synthesis [8].

Discovery of Argininosuccinate Synthetase and Argininosuccinate Lyase

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Figure 1. Ornithine cycle.

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The full ornithine cycle also called urea cycle is restricted to hepatocytes. Enzyme abbreviations : N-AGS : N-acetyl glutamate synthetase ; CPS I : carbamyl phosphate synthetase I ; OTC : ornithine transcarbamylase ; ASS : argininosuccinate synthetase ; ASL : argininosuccinate lyase.

3. The Biochemical Reactions of Arginine Synthesis In the fifties, further advances revealed the details of the reaction mechanisms of arginine synthesis. The nature of the biochemical reactions involved is identical in liver and kidney. This reaction consists of two distinct steps. In the first step, aspartic acid and citrulline, in the presence of Mg2+ and ATP, condense to form argininosuccinic acid with the simultaneous formation of inorganic phosphate. In the second step, argininosuccinic acid is hydrolyzed to form arginine and malic acid. Unfortunately, the presence of fumarase in the crude enzyme led to a predominance of malic acid. Removal of fumarase by fractionation revealed the presence of fumarate [57]. A new reaction mechanism was formulated: Step 1 citrulline + aspartic acid + ATP

---------->

argininosuccinate + ADP + Pi

Step 2 argininosuccinate

---------->

arginine

+ fumaric acid

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Olivier Levillain

A highly purified argininosuccinate splitting enzyme was prepared from ox liver and characterized. The activity of the splitting enzyme was optimum at pH 7.5. A Km value of 1.5 mM for argininosuccinic acid was determined. The reaction was found to be reversible, but only at unphysiologically high arginine and fumarate concentrations [57]. Similar splitting enzyme is widely distributed in nature. It has been found in liver, kidney, heart, yeast, pea seeds, and Chlorella [17]. Step 2 argininosuccinate

arginine + fumaric acid

The fate of the ATP used for the condensation of citrulline and aspartic acid was still to be resolved. The possibility was considered that ATP cleavage might liberate pyrophosphate (P-P). To verify this hypothesis, the condensation step was carried out with hog kidney enzyme in the presence of fluoride ion to inhibit pyrophosphatase activity. Under this condition, the reaction led to an accumulation of P-P in amounts equivalent to the utilisation of citrulline [52]. In an additional step 3, P-P is hydrolyzed by the endogenous pyrophosphatase. Step 1 citrulline + aspartic acid + ATP ---- Condensation -----> argininosuccinate + AMP + PPi Step 3

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PPi

---- Pyrophosphatase --->

2 Pi

The velocity of the condensation step is accelerated by the removal of pyrophosphate [52]. Interestingly, the condensation step is also reversible. In the presence of large amounts of the condensing enzyme, AMP, P-P, and Mg2+, argininosuccinate is cleaved as measured by the production of citrulline. ATP is also produced. AMP is specific of the reverse reaction. The velocity of the backward reaction displayed an optimum at pH 6 while that of the forward reaction was at pH 8.7 [52]. Finally, given that pyrophosphatase activity is very high in mammalian liver and kidney, the enzymatic hydrolysis of pyrophosphate pulls the condensation step in the direction of synthesis. The nature of the phosphorylation step is of considerable interest. It was uncertain whether ATP reacts with citrulline or aspartic acid. This was resolved by the use of [O18ureido] citrulline which showed that O18 was recovered exclusively in the AMP moiety of ATP [69]. Although, ATP interacts with the ureido group of citrulline, the presence of O18 in AMP indicated that AMP activates citrulline. This result suggested that adenylocitrulline might be an intermediate in a two-step mechanism [69]. The cleavage of ATP in its  position was quite conclusively demonstrated [65]. Pulse labeling with [14C]-citrulline showed more directly that citrulline activation by ATP precedes the condensation with aspartic acid [65]. Experiments support that an adenylate intermediate was formed. The step 1a controlled by the condensing enzyme consists of two reactions. In the first reaction (step 1a), an anhydride is formed between the phosphate group of AMP and the carbamyl group of citrulline.

Discovery of Argininosuccinate Synthetase and Argininosuccinate Lyase

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Step 1a citrulline + MgATP2-

----->

citrulline-AMP2-

+ MgPPi2- + 2H+

Step 1b citrulline-AMP2-

----->

argininosuccinate

+ AMP2-

+ aspartate

In the second reaction (step 1b), the activated citrulline is transferred to the amino group of aspartate to form an amidine [65]. The production of an intermediary acyladenylate tightly bound to the enzyme has also been reported for several other synthetases (for references see [65]). Finally, at present, in books and publications, authors often summarize the equation of arginine synthesis as follows: Step 1 citrulline + aspartate + Mg-ATP ----- ASS -----> argininosuccinate + AMP + MgPPi + 2H+ Step 2 argininosuccinate

----- ASL ------>

arginine

+ fumarate

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The “condensing enzyme” which metabolizes citrulline and aspartate into argininosuccinate has been named argininosuccinate synthetase or [citrulline:aspartate ligase (AMP-forming)] (ASS; EC:6.3.4.5). The “splitting enzyme” which hydrolyzes argininosuccinate into arginine and fumarate has been named argininosuccinase or argininosuccinate lyase (ASL; EC:4.3.2.1).

4. Catalytic, Physical, and Immunological Properties of ASS Catalytic Properties Highly purified ASS from steer liver and hog kidney was used to determine the affinity constant (Km) for the substrates. Comparative data are summarized in Table 1. Km for citrulline was 46 µM and 44 µM, respectively. Km for aspartate was 35 µM and 38 µM, respectively [65]. These Km values are much lower than those previously reported for the crude enzyme [60, 62]. A Km of 0.32 mM was obtained for ATP. Two analogues of aspartate, -methyl-DL-aspartate and D-aspartate, revealed low affinity for ASS. Their inhibition constants (Ki) were 1.8 mM and 20 mM, respectively [65]. The rat ASS was isolated and crystallized for further characterization [67]. The active rat ASS has an optimum pH of 8.4-8.8 [66, 67]. The affinity for the substrates were determined for the crystalline rat liver ASS. The Km values for citrulline (42 - 44 µM), aspartate (19 - 20 µM), and ATP (0.10 - 0.15 mM) are similar to those reported in steer liver and hog kidney [65-68]. The bovine liver ASS was also purified and crystallized [64]. Kinetic studies gave Km values of 0.2 mM for citrulline, 0.17 mM for aspartate, and 0.27 mM for ATP. These values are somewhat higher than those previously reported [64, 65]. ASS was inhibited by ADP and AMP. Ki

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Olivier Levillain

values were 0.3 to 1.0 mM for ADP and 0.3 to 2.0 mM for AMP [64]. Kinetic studies were also performed with the human liver ASS. O‟Brien published Km values of 16 µM for citrulline, 17 µM for aspartate, and 41 µM for ATP [48]. At saturating concentration of the other substrates, Saheki et al. reported Km values of 24 µM for citrulline, 19 µM for aspartate, and 77 µM for ATP [68]. Kinetic parameters were determined in human lymphoblast cell lines MGL8B2 and MGL8B1 [31]. The affinity constants were calculated for citrulline (Km 61 µM and 89 µM for the MGL8B2 and MGL8B1 cell lines, respectively) and for aspartate (Km 78 µM for both cell lines). In contrast with ASS of other tissues, the affinity constant for ATP depended on its concentration. The Km value for ATP was 0.47 mM at high ATP concentration and 0.094 mM at low ATP concentration [31]. Table 1. Catalytic properties of ASS in different animal species

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Source

Organ

Citrulline

Aspartate

ATP

µM

µM

mM

References

Steer

Liver

46

35

0.32

[65]

Hog

Liver

44

38

0.32

[65]

Rat

Liver

42-44

19-20

0.1-0.15

[67, 68]

Bovine

Liver

200

170

0.27

[64]

Human

Liver

16

17

0.041

[48]

Human

Liver

24

19

0.077

[68]

Human

Lymphoblast line MGL8B2

61

78

0.094-0.47

[31]

MGL8B1

89

78

0.094-0.47

Physical Properties The first value obtained for the molecular weight of the native rat liver ASS was reported to be 192,000 [67]. The molecular weight of the subunit was determined by SDS-PAGE with 7.5% acrylamide. A single band was found with a size of 48 ± 1 kDa [67]. However, sometimes, four or five minor bands were observed except if 5 mM argininosuccinate was added [67]. Nevertheless, the homogeneity of ASS was proven by the sedimentation coefficient value (S20, w) of 8.0 [67]. A similar sedimentation velocity (S20, w) of 7.9 was

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Discovery of Argininosuccinate Synthetase and Argininosuccinate Lyase

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reported for the bovine liver ASS [64]. Later, Saheki et al. re-analyzed the molecular weight of the rat liver ASS by using different techniques [66]. Sizes of 158 ± 3, 192, and 207 kDa were deduced using Sephadex G-200 column, SDS-PAGE, and sedimentation equilibrium centrifugation, respectively [66]. The molecular weight of the active bovine liver ASS was determined by gel filtration. A size of 185 ± 18.5 kDa was estimated [64]. When electrophoresis was carried out with ASS treated with SDS but without mercaptoethanol, a major band of 94,000 was seen. The addition of mercaptoethanol in SDS-PAGE, revealed a peptide with a molecular weight of 46,500 ± 4,650 [64]. The molecular weight of the native ASS purified from human liver was 183,000 and that of the subunit 42,800 [48]. A sedimentation coefficient of 8.2 was found. Later, another group purified the human liver ASS for comparison with the rat liver ASS [68]. The molecular weight of the two native ASS was estimated to be 185,000 by gel filtration. Purified human and rat liver ASS were analyzed by SDS-PAGE and showed a single band indicating a subunit molecular weight of 45,000 [68]. The amino acid composition of the human and rat liver ASS exhibited a high degree of similarity. However, the human liver ASS is more basic than the rat liver ASS. Both subunit of human and rat liver ASS contain 4 half-cystine residues [66, 68]. Surprisingly, Ratner found only 3 half-cystine residues per subunit of bovine ASS [56]. Human lymphoblasts and lymphoblast cell lines MGL8B2 and MGL8B1 express ASS [31]. However, the latter cell line overproduce ASS compared with the former [31]. The molecular weight of the native protein was determined by gel filtration on Sephadex G-200 and that of the ASS subunit by SDSPAGE. In both cell lines, the size of the native ASS was 180 kDa and that of ASS subunit was 45 kDa [31]. Comparative data are summarized in Table 2. Although, the molecular weights reported by the different teams diverge somewhat, all studies support that the enzymatically active ASS is a homotetramer of 175-185 kDa. More recently, a protein of 45 kDa corresponding to ASS was purified from the pig kidney [75]. The National Center for Biotechnology Information (NCBI) database provides the ASS protein sequence of human (accession number : NP_000041 and NP_446464), rat (NP_037289), and mouse (NP_031520). All ASS are composed of 412 amino acid residues with a theoretical size of 46.3 kDa. Western blots analyses performed in several rat tissues confirmed the expected size of 46 kDa for ASS [37, 80]. The attempts to identify the isoelectric point (pI) of ASS gave a value of 7.9 for the rat liver ASS [66] and of 7.60 for the bovine liver ASS [56]. A two-dimensional gel electrophoresis of the purified ASS displayed at least two protein spots at a pI range of 6.3 – 6.8 [75]. Recently, protein isolated from male rat kidney cortex and medulla cytosols were examined by two-dimensional electrophoresis (2-DE) [79]. A large and well resolved protein spot (pI ≈ 6.89) was analyzed by MALDI-MS and ESI-MS/MS for identification. This spot corresponds to ASS.

Immunological Properties Antibodies raised against the human liver ASS cross-react with the beef and rat liver ASS with similar affinity [48]. A few years later, the immunological properties of the rat and human liver ASS were re-examined by Ouchterlony double-diffusion techniques. Immunological analyses showed a high degree of similarity between the enzymes [68].

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Olivier Levillain Table 2. Physical properties of ASS in different animal species

Source

Organ

Molecular

Sedimentation

Molecular

Weight

coefficient

Weight

Native

S20, w

Subunit

References

Rat

Liver

192,000

8.0

48,000 ± 1,000

[67]

Bovine

Liver

185,000 ± 18,500

7.9

46,500 ± 4,650

[64]

Human

Liver

183,000

8.2

42,800

[48]

Human

Liver

185,000

*

45,000

[68]

Human

Lymplocytes

180,000

*

45,000

[31]

Rat

Liver

158,000 ± 3,000

*

192,000

*

207,000

*

[66] 48,000

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5. Molecular Aspects of ASS The human ASS gene was assigned to chromosome 9 using adenylate kinase I (AK-1) as a marker of this chromosome [11]. ASS gene was localized on the distal portion of the long arm of chromosome 9, in the region 9q34 -> 9qter, and AK3 and cis-aconitase (ACONs) on the short arm, in the region 9pter -> 9pl3 [12]. DNA sequences closely homologous to ASS are present at ten or more distinct locations in the human genome, including sites on chromosome 6, 9, and X [5]. Several ASS-like sequences were found in genomic human DNA [72]. ASS pseudogenes in human were confirmed by another group [29]. Finally, it was determined that a single gene is active and none of the pseudogenes is expressed (for reference see [7]). In 1984, it was shown that the entire human ASS gene spans 63 kb and is composed at least of 13 exons [22]. A more recent study indicates that the murine ASS gene contains 16 exons [73]. Data collected from NCBI site indicate that the mouse ASS gene maps to chromosome 2 whereas that of the rat maps to chromosome 3, in the region 3p12. The human ASS gene contains 16 exons. Additional details are summarized in a recent review [16]. The human ASS gene has been sequenced in patients suffering of ASS deficiency to identify mutations. This work will be helpful for molecular diagnostics in families with classical and mild citrullinemia [24]. Experiments were undertaken to clone ASS and characterize its mRNA. Canavanineresistant human cell lines (Canr) isolated from lymphoblasts have very high levels of ASS

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101

activity. These Canr cells were used to clone ASS and a cDNA probe was synthesized to analyse RNA by Northern blot [72]. A major mRNA species of 1.67 kb was observed in wild type and Canr cell lines. The relative intensity of this mRNA species correlated well with the enzyme activity. A minor mRNA of 2.68 kb was detected in Canr cells [72]. These two mRNA species might represent heterogenous products of a single gene. The sequence of these mRNA revealed that, in human liver and fibroblasts, the major ASS mRNA lacks exon 2 [22]. In baboon liver, two ASS mRNA species were detected as previously reported for Canr cells. The major mRNA species contains exon 2 sequence [22]. These experiments show that alternative splicing of ASS mRNA is species-specific in primates and varies among different human cell types. Northern blot analyses for ASS performed with different rat tissues showed a major ASS mRNA species of about 1.5 kb long. Nevertheless, a second mRNA species with a higher size was detected in liver, kidney, and testis when the blots were exposed for a longer time [80]. To characterize and explore the expression ASS mRNA, the next step consists to clone ASS gene. The human ASS was cloned and sequenced [7]. An open reading frame of 1,236 nucleotides encodes a protein with a molecular weight of 46,434 [7]. Later, the rat kidney ASS was cloned and characterized [74]. A clone composed of 1,495 bp was isolated. A protein of 412 amino acids is encoded and shares 97% identity with the human enzyme. At the nucleotide level, there is 89% identity over the translated region [74]. ASS is highly conserved in human, bovine, rat, and mouse [28]. The structure of the human ASS provides new insights into the function of the mutant ASS identified in patients with type I citrullinemia [30]. The promoter of the human and mouse ASS gene has been characterized partially. The reader may refer to a recent review [28].

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6. Catalytic, Physical, and Immunological Properties of ASL To the best of our knowledge, crystalline ASL was purified from steer liver for the first time in 1965 [25]. This new purification procedure greatly facilitated further analysis of the properties of ASL [25]. In addition, given that ASL has been found in various extrahepatic tissues in a given species, it could not be assumed that all ASLs are identical. Indeed, it has been reported that the properties of the enzyme vary among organs (for references see [43]). For this reason, comparative studies were undertaken.

Catalytic and Immunological Properties ASL was purified from bovine kidney and liver to compare their properties. ASL of both organs exhibit similar specific activities (kidney: 1,300 and liver : 920 µmoles substrate split per mg per hr) [10, 25]. The Michaelis constant, determined from Lineweaver-Burk plots at 26°C, showed Km values for argininosuccinate of 146 µM and 148 µM in the kidney and the liver, respectively [10]. Both enzymes were cold inactivated at the same rate and reactivated similarly at 33°C with different pH. Phosphate protects both kidney and liver ASL against cold inactivation [10, 25]. The antigenic properties of both ASL were examined by Ouchterlony double-diffusion techniques. The kidney and liver enzymes share similar

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Olivier Levillain

immunological properties. The comparison of amino acid analyses revealed only few, if any, differences. Both enzymes contain 16 half-cystine residues [10, 38]. ASL of kidney and liver contain 41-43% hydrophobic amino acids.

Physical Properties

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Various experiments were performed to determine the molecular weight of ASL and its quaternary structure. After ultracentrifugation, the sedimentation coefficient value (S20, w) of the bovine kidney ASL was 9.27 and that of the bovine liver ASL was 9.3 [10, 25]. The authors assumed that both ASL have the same molecular weight of 202,000. Inactivation of ASL by lowering the temperature is accompanied by dissociation into subunits (for references see [71]). When ASL was inactivated by cold, the enzyme was dissociated into presumably identical subunits of 100,000 [71]. Lystry and Ratner analyzed in details the quaternary structure of ASL purified from steer liver [38]. An attempt was made to determine the size of the polypeptide chains of ASL. The enzyme was dissociated with SDS and mercaptoethanol and analyzed by the method of SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The results indicated that the polypeptide chains are of identical size (54 kDa) [38]. The molecular weight of ASL was determined with high speed equilibrium method. ASL was dissolved in either 6M guanidine-HCl or 8 M urea. A size of 49.61 ± 1.10 kDa was calculated [38]. Data are summarized in Table 3. Taken together, the data strongly suggested that ASL is a tetramer composed of four subunit polypeptide chains of identical size. Electron microscopic studies were undertaken to characterize the geometry of ASL and to observe the number of subunits. ASL molecules appeared as triangles and squares with an indication of four subunits. Subunits are arranged very compactly and a tetrahedral model was proposed [38].

Comparative Analyses The comparison of the catalytic, physico-chemical, and immunological properties of the hepatic and renal bovine ASL indicated that both enzymes are identical (Table 3) [10, 54]. These findings raise the question as to whether ASL from other tissues of the same species are identical to the renal and hepatic enzymes. Given that ASL occurs in the brain of different species [58], Murakami-Murofushi and Ratner compared the catalytic, physical, and chemical properties of the bovine brain enzyme with those of liver and kidney [43]. The ratio of activities of ASL in homogenates of liver, kidney, and brain is 270:43:3. Gel electrophoresis in 7.5% polyacrylamide showed that liver, kidney, and brain ASL migrated as a single band. A molecular weight of 200,000 ± 10,000 was determined by gel filtration for the brain and liver ASL [43]. This value is quite similar to the value of 202,000 ± 5,000 obtained by sedimentation equilibrium [25]. After dissociation of ASL with SDS, a subunit molecular weight of 50,000 was found as previously reported for the liver and the kidney [43]. The triptic cleavage of the brain and liver ASL revealed 48-49 ninhydrin-positive peptides in agreement with prediction [43]. In addition, peptide maps of cyanogen bromide cleavage products of the brain and the liver showed similar patterns. Cold inactivation of the brain enzyme was identical to that of the liver [43]. The brain, kidney, and liver ASL are composed

Discovery of Argininosuccinate Synthetase and Argininosuccinate Lyase

103

of 16 half-cystine residues [43]. The three enzymes are closely similar in antigenic properties [43]. Table 3. Physical properties of ASL in different tissues and animal species

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Source

Organ

Molecular

Sedimentation

Molecular

Weight

coefficient

Weight

Native

S20, w

Subunit

References

Bovine

Kidney

202,000

9.27

*

[25]

Bovine

Liver

202,000

9.3

*

[10]

(unknown)

Liver

202,000

9.5

*

[71]

Steer

Liver

202,000

*

54,000

[38]

*

49,610 ± 1,100

[38]

Human

Liver

200,000

*

50,000

[51]

Bovine

Brain

200,000

*

50,000

[43]

Bovine

Liver

200,000

*

50,000

[43]

Human

Liver

187,000

*

49,000

[49]

Experiments were also performed to document the human ASL (Table 3). In the liver, the molecular weight of the native ASL was estimated to be 187,400 [49] and 200,000 [51]. When these two ASL preparations were analyzed in SDS-PAGE, the subunit had a molecular weight of 49,000 [49] and 50,000 [51]. Both ASL preparations are composed of four subunits. Additional studies were performed to identify the kinetic properties of human liver ASL. O‟Brien and Barr reported a pH optimum of 7.5, a Vm of 10.3 µmol.min -1.mg-1 and a Km value of 200 µM argininosuccinate [49]. Palekar and Mantagos gave a Km value of 100 µM for argininosuccinate [51]. The predicted polypeptide of ASL is composed of 461 amino acids in rats (NCBI protein : NP_067588.2) and 464 amino acids in humans (P04424.4) and mice (NP_598529.1). The size of the rat, human, and mouse ASL given on NCBI site are 51.26, 51.53, and 51.61 kDa, respectively. Western blots analyses performed in several rat tissues confirmed the expected size of 51 kDa for ASL [37]. All data support that, whatever the mammalian species and the organs studied, the native ASL is a homotetrameric protein of about 200 kDa.

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Olivier Levillain

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7. Molecular Aspects of ASL The human ASL gene has been mapped to the pter region ->q22 of chromosome 7 [47]. O‟Brien et al. confirmed this result and found a pseudogene which might be localized on chromosome 22 [50]. Data found on the NCBI site indicate that the rat ASL gene is localized on chromosome 12 in the region 12q13 whereas the mouse ASL gene is mapped on chromosome 5 in the region 5 74.0 cM. The organization of ASL gene was also examined. The rat ASL gene is about 14 kb and consists of 16 exons [39]. Surprisingly, the exon-intron organization of ASL gene is strikingly similar to that of the chicken -crystallin genes [39, 78]. The -crystallin gene encodes the major protein of the eye lens. This finding suggests that these genes evolved from a common ancestral gene. To our knowledge, the rat liver ASL was first cloned in 1986 [1, 36, 50]. The cDNA probe for ASL was used to analyze the expression of ASL in rat tissues by Northern blot [1]. In the liver, two mRNA species were detected. A major RNA species of 2.1 kb and a minor one of about 1.7 kb. The 2.1 kb mRNA was abundant in the kidney and in minute amounts in the spleen [1]. Neither the small intestine nor the heart contained ASL mRNA. The tissue distribution of ASL and the levels of ASL mRNA in these organs are consistent with the results of ASL activity [1, 59]. A size of 2.0 kb for the major ASL mRNA was reported for several rat hepatoma cell lines [36]. These authors detected a minor unknown band of 4.5 kb. The same year, O‟Brien et al. cloned and sequenced a cDNA for the human ASL [50]. A clone of 1,565 bp was entirely sequenced. An open reading frame of 1,503 bp encoding a protein of 463 amino acids with a predicted molecular weight of 51,663 was identified [50]. The cloned rat liver ASL revealed an open reading frame of 1,383 bp encoding for a polypeptide of 461 amino acid residues with a size of 51,549 Da [2]. The rat liver, kidney, lung, and spleen expressed a major ASL mRNA of 2.0 kb long. However, in the testis, the major ASL mRNA species was 2.5 kb and the minor one 2.0 kb long [80]. The promoter of the rat ASL gene has been characterized partially [40].

8. Tissue Expression of ASS and ASL From 1932 until 1960, almost all the studies designed to analyze ASS and ASL were performed in liver and kidney. However, ASS and ASL expression is not restricted to these two organs. ASS was reported in the brain of ox, monkey (Rhesus, Macaca mulatta), and man [58]. ASL activity was detected in whole brain, cerebellum, white and gray matter of monkey, steer, and man [58]. ASL was identified in whole homogenates of rat, guinea pig, cat, and man brain [58]. The localization of ASS and ASL in the nervous system has been summarized in a review [77]. ASL activity was demonstrated in many normal mammalian organs including liver, brain, kidney, red blood cells, fibroblasts, spleen, muscle, skin, thyroid, thymus, and lung [54, 55]. In rats, ASL activity was very high in the liver, high in the kidney and low in the lung, the intestine, the brain, the spleen, and the heart [59]. In a detailed study, the expression of ASS and ASL was analyzed in several rat tissues at the transcriptional and translational levels [80]. The highest levels of ASS mRNA and protein were observed in liver, kidney, and testis. Lung and spleen were also positive. A quite similar distribution pattern was reported for ASL mRNA and protein [80]. Both ASS and ASL were

Discovery of Argininosuccinate Synthetase and Argininosuccinate Lyase

105

expressed in liver and kidney of Swiss-Webster albino mice [42], ferrets [19], and Swiss mice [46]. ASS is expressed in the liver and kidney of pigs [75]. In children and adult humans, ASL has been reported in red blood cells [31]. ASL is also expressed in the liver, brain, and kidney of young children [31]. Interestingly, in neonatal argininosuccinic aciduria, the liver ASL is absent but the brain and kidney ASL activities were in the control range [31]. The enzymes ASS and ASL are generally co-localized in the same cell type and are widely distributed in various organs including the eye [33], nerve structures [44, 45, 77], intestine [6, 18, 27], kidneys [3, 19, 20, 23, 37, 41, 46, 80], and other tissues [23] of various animal species. ASS and ASL are ubiquitous enzymes. In this review, the vascular localization of both ASS and ASL is not addressed. The literature on these two enzymes is extremely abundant and the author is aware that it is not possible in this review to discuss all publications. Indeed, a search of PubMed for « argininosuccinate synthetase », « argininosuccinate lyase », and « argininosuccinate synthetase and argininosuccinate lyase » gave 805, 569, and 280 references, respectively.

Acknowledgments The author is indebted to Pr John T (Sean) Brosnan (Department of Biochemistry, Memorial University of Newfoundland, St. John's, NL, Canada) for reviewing this manuscript.

References Copyright © 2011. Nova Science Publishers, Incorporated. All rights reserved.

[1]

[2]

[3]

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Amaya, Y., Kawamoto, S., Oda, T., Kuzumi, T., Saheki, T., Kimura, S. & Mori, M. (1986). Molecular cloning of cDNA for argininosuccinate lyase of rat liver. Biochemistry International, 13, 433-438. Amaya, Y., Matsubasa, T., Takiguchi, M., Kobayashi, K., Saheki, T., Kawamoto, S. & Mori, M. (1988). Amino acid sequence of rat argininosuccinate lyase deduced from cDNA. Journal of Biochemistry (Tokyo), 103, 177-181. Aperia, A., Broberger, O., Larsson, A. & Snellman, K. (1979). Studies of renal urea cycle enzymes. I. Renal concentrating ability and urea cycle enzymes in the rat during protein deprivation. Scandinavian Journal of Clinical and Laboratory Investigation, 39, 329-336. Archibald, R. M. (1944). Determination of citrulline and allantoin and demonstration of citrulline in blood plasma. The Journal of Biological Chemistry, 156, 121-142. Beaudet, A. L., Su, T. S., O'Brien, W. E., D'Eustachio, P., Barker, P. E. & Ruddle, F. H. (1982). Dispersion of argininosuccinate-synthetase-like human genes to multiple autosomes and the X chromosome. Cell, 30, 287-293. Blachier, F., M'Rabet-Touil, H., Posho, L., Darcy-Vrillon, B. & Duée, P.-H. (1993). Intestinal arginine metabolism during development. Evidence for de novo synthesis of L-arginine in newborn pig enterocytes. European Journal of Biochemistry, 216, 109117.

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[25] Havir, E. A., Tamir, H., Ratner, S. & Warner, R. C. (1965). Biosynthesis of Urea. XI. Preparation and Properties of Crystalline Argininosuccinase. The Journal of Biological Chemistry, 240, 3079-3088. [26] Hedin, S. G. (1895). Eine Methode, das Lysin zu isoliren, nebst einigen Bemerkungen über das Lysatinin. Zeitschrift für Physiologische Chemie, 21, 297-305. [27] Hurwitz, R. & Kretchmer, N. (1986). Development of arginine-synthesizing enzymes in mouse intestine. American Journal of Physiology: Gastrointestinal Liver Physiology, 251, G103-G110. [28] Husson, A., Brasse-Lagnel, C., Fairand, A., Renouf, S. & Lavoinne, A. (2003). Argininosuccinate synthetase from the urea cycle to the citrulline–NO cycle. European Journal of Biochemistry, 270, 1887-1899. [29] Jinno, Y., Nomiyama, H., Wakasugi, S., Shimada, K., Matsuda, I. & Saheki, T. (1984). Isolation and characterization of phage clones carrying the human argininosuccinate synthetase-like genes. Journal of Inherited Metabolic Disease, 7, 133-134. [30] Karlberg, T., Collins, R., van den Berg, S., Flores, A., Hammarstrom, M., Hogbom, M., Holmberg Schiavone, L. & Uppenberg, J. (2008). Structure of human argininosuccinate synthetase. Acta Crystallographica. Section D, Biological Crystallography, 64, 279286. [31] Kimball, M. E. & Jacoby, L. B. (1980). Purification and properties of argininosuccinate synthetase from normal and canavanine-resistant human lymphoblasts. Biochemistry, 19, 705-709. [32] Klose, A. A. & Almquist, H. J. (1940). The ability of citrulline to replace arginine in the diet of the chicken. The Journal of Biological Chemistry, 135, 153-155. [33] Koshiyama, Y., Mori, M., Miyanaka, K., Kobayashi, T., Negi, A. & Gotoh, T. (2000). Expression and localization of enzymes of arginine metabolism in the rat eye. Current Eye Research, 20, 313-321. [34] Krebs, A. (1935). Metabolism of amino acids. IV. The synthesis of glutamine from glutamic acid and ammonia, and the enzymatic hydrolysis of glutamine in animal tissues. The Biochemical Journal, 29, 1951-1969. [35] Krebs, H. A. & Henseleit, K. (1932). Untersuchungen über die Harnstoffbildung im Tierköper. Zeitschrift für Physiologische Chemie, 210, 33-66. [36] Lambert, M. A., Simard, L. R., Ray, P. N. & McInnes, R. R. (1986). Molecular cloning of cDNA for rat argininosuccinate lyase and its expression in rat hepatoma cell lines. Molecular and Cellular Biology, 6, 1722-1728. [37] Levillain, O. & Wiesinger, H. (2010). Expression and localization of argininosuccinate synthetase and argininosuccinate lyase in the female and male rat kidneys. In: Arginine Amino Acid (Ed. Nathan L. Jacobs), pp. 111-124 ST: NOVAPublishers. [38] Lusty, C. J. & Ratner, S. (1972). Biosynthesis of urea. XIV. The quaternary structure of argininosuccinase. The Journal of Biological Chemistry, 247, 7010-7022. [39] Matsubasa, T., Takiguchi, M., Amaya, Y., Matsuda, I. & Mori, M. (1989). Structure of the rat argininosuccinate lyase gene: close similarity to chicken delta-crystallin genes. Proceedings of the National Academy of Sciences of the United States of America, 86, 592-596. [40] Matsubasa, T., Takiguchi, M., Matsuda, I. & Mori, M. (1994). Rat argininosuccinate lyase promoter: the dyad-symmetric CCAAT box sequence CCAATTGG in the promoter is recognized by NF-Y. Journal of Biochemistry, 116, 1044-1055.

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[41] Mistry, S. K., Greenfeld, Z., Morris Jr, S. M. & Baylis, C. (2002). The "intestinal-renal" arginine biosynthetic axis in the aging rat. Mechanisms of Ageing and Development, 123, 1159-1165. [42] Morris Jr, S. M., Sweeney Jr, W. E., Kepla Jr, D. M., O'brien, W. E. & Avner, E. D. (1991). Localization of arginine biosynthetic enzymes in renal proximal tubules and abundance of mRNA during development. Pediatric Research, 25, 151-154. [43] Murakami-Murofushi, K. & Ratner, S. (1979). Argininosuccinase from bovine brain: isolation and comparison of catalytic, physical, and chemical properties with the enzymes from liver and kidney. Analytical Biochemistry, 95, 139-155. [44] Nakamura, H., Saheki, T., Ichiki, H., Nakata, K. & Nakagawa, S. (1991). Immunocytochemical localization of argininosuccinate synthetase in the rat brain. The Journal of Comparative Neurology, 312, 652-679. [45] Nakamura, H., Saheki, T. & Nakagawa, S. (1990). Differential cellular localization of enzymes of L-arginine metabolism in the rat brain. Brain Research, 530, 108-112. [46] Natesan, S. & Reddy, S. R. (2001). Compensatory changes in enzymes of arginine metabolism during renal hypertrophy in mice. Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology, 130, 585-595. [47] Naylor, S. L., Klebe, R. J. & Shows, T. B. (1978). Argininosuccinic aciduria: assignment of the argininosuccinate lyase gene to the pter to q22 region of human chromosome 7 by bioautography. Proceedings of the National Academy of Sciences of the United States of America, 75, 6159-6162. [48] O'Brien, W. E. (1979). Isolation and characterization of argininosuccinate synthetase from human liver. Biochemistry, 18, 5353-5356. [49] O'Brien, W. E. & Barr, R. H. (1981). Argininosuccinate lyase: purification and characterization from human liver. Biochemistry, 20, 2056-2060. [50] O'Brien, W. E., McInnes, R., Kalumuck, K. & Adcock, M. (1986). Cloning and sequence analysis of cDNA for human argininosuccinate lyase. Proceedings of the National Academy of Sciences of the United States of America, 83, 7211-7215. [51] Palekar, A. G. & Mantagos, S. (1981). Human liver arginiosuccinase purification and partial characterization. The Journal of Biological Chemistry, 256, 9192-9194. [52] Petrack, B. & Ratner, S. (1958). Biosynthesis of urea. VII. Reversible formation of argininosuccinic acid. The Journal of Biological Chemistry, 233, 1494-1500. [53] Ratner, S. (1947). The enzymatic mechanism of arginine formation from citrulline. The Journal of Biological Chemistry, 170, 761-762. [54] Ratner, S. (1973). Enzymes of arginine and urea synthesis. Advances in Enzymology and Related Areas of Molecular Biology, 39, 1-90. [55] Ratner, S. (1976). Enzymes of arginine and urea synthesis. In: Grisolia, S., Baguena, R. & Mayor, F. (Eds.), The Urea Cycle (pp. 181-219). New York, London, Sydney, Toronto, ST: J Wiley & Sons. [56] Ratner, S. (1982). Argininosuccinate synthetase of bovine liver: chemical and physical properties. Proceedings of the National Academy of Sciences of the United States of America, 79, 5197-5199. [57] Ratner, S., Anslow Jr, W. P. & Petrack, B. (1953). Biosynthesis of urea. VI. Enzymatic cleavage of argininosuccinic acid to arginine and fumaric acid. The Journal of Biological Chemistry, 204, 115-125.

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[58] Ratner, S., Morell, H. & Carvalho, E. (1960). Enzymes of arginine metabolism in brain. Archives of Biochemistry and Biophysics, 91, 280-289. [59] Ratner, S. & Murakami-Murofushi, K. (1980). A new radiochemical assay for argininosuccinase with purified [14C] argininosuccinate. Analytical Biochemistry, 106, 134-147. [60] Ratner, S. & Pappas, A. (1949). Biosynthesis of urea I. Enzymatic mechanism of arginine synthesis from citrulline. The Journal of Biological Chemistry, 179, 11831198. [61] Ratner, S. & Petrack, B. (1951). Biosynthesis of urea. III. Further studies on arginine synthesis from citrulline. The Journal of Biological Chemistry, 191, 693-705. [62] Ratner, S. & Petrack, B. (1953). The mechanism of arginine synthesis from citrulline in kidney. The Journal of Biological Chemistry, 200, 175-185. [63] Ratner, S., Petrack, B. & Rochovansky, O. (1953). Biosynthesis of urea. V. Isolation and properties of argininosuccinic acid. The Journal of Biological Chemistry, 204, 95113. [64] Rochovansky, O., Kodowaki, H. & Ratner, S. (1977). Biosynthesis of urea. Molecular and regulatory properties of crystalline argininosuccinate synthetase. The Journal of Biological Chemistry, 252, 5287-5294. [65] Rochovansky, O. & Ratner, S. (1967). Biosynthesis of urea. XII. Further studies on argininosuccinate synthetase: substrate affinity and mechanism of action. The Journal of Biological Chemistry, 242, 3839-3849. [66] Saheki, T., Kusumi, T., Takada, S. & Katsunuma, T. (1977). Studies of rat liver argininosucciante synthetase. I. Physicochemical, catalytic, and immunochemical properties. Journal of Biochemistry, 81, 687-696. [67] Saheki, T., Kusumi, T., Takada, S., Katsunuma, T. & Katunuma, N. (1975). Crystallization and some properties of argininosuccinate synthase from rat liver. FEBS letters, 58, 314-317. [68] Saheki, T., Sase, M., Nakano, K., Azuma, F. & Katsunuma, T. (1983). Some properties of argininosuccinate synthetase purified from human liver and a comparison with the rat liver enzyme. Journal of Biochemistry (Tokyo), 93, 1531-1537. [69] Schuegraf, A., Ratner, S. & Warner, R. C. (1960). Free energy changes of the argininosuccinate synthetase reaction and of the hydrolysis of the inner pyrophosphate bond of adenosine triphosphate. The Journal of Biological Chemistry, 235, 3597-3602. [70] Schulze, E. & Steiger, E. (1886). Ueber das Arginin. Zeitschrift für Physiologische Chemie, 11, 43-65. [71] Schulze, I. T., Lusty, C. J. & Ratner, S. (1970). Biosynthesis of urea. XIII. Dissociation-association kinetics and equilibria of argininosuccinase. The Journal of Biological Chemistry, 245, 4534-4543. [72] Su, T. S., Bock, H. G., O'Brien, W. E. & Beaudet, A. L. (1981). Cloning of cDNA for argininosuccinate synthetase mRNA and study of enzyme overproduction in a human cell line. The Journal of Biological Chemistry, 256, 11826-11831. [73] Surh, L. C., Beaudet, A. L. & O'Brien, W. E. (1991). Molecular characterization of the murine argininosuccinate synthetase locus. Gene, 99, 181-189. [74] Surh, L. C., Morris, S. M., O'Brien, W. E. & Beaudet, A. L. (1988). Nucleotide sequence of the cDNA encoding the rat argininosuccinate synthetase. Nucleic Acids Research, 16, 9352.

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[75] Wakui, H., Komatsuda, A., Itoh, H., Kobayashi, R., Nakamoto, Y. & Miura, A. B. (1992). Renal argininosuccinate synthetase: purification, immunohistochemical localization, and elastin-binding property. Renal Physiology and Biochemistry, 15, 1-9. [76] Weil-Malherbe, H. & Krebs, H. A. (1935). Metabolism of amino acids. V. The conversion of proline into glutamic acid in kidney. The Biochemical Journal, 29, 20772081. [77] Weisinger, H. (2001). Arginine metabolism and the synthesis of nitric oxide in the nervous system. Progress in Neurobiology, 64, 365-391. [78] Wistow, G. & Piatigorsky, J. (1987). Recruitment of enzymes as lens structural proteins. Science (New York, N.Y.), 236, 1554-1556. [79] Witzmann, F. A., Fultz, C. D., Grant, R. A., Wright, L. S., Kornguth, S. E. & Siegel, F. L. (1998). Differential expression of cytosolic proteins in the rat kidney cortex and medulla: preliminary proteomics. Electrophoresis, 19, 2491-2497. [80] Yu, Y., Terada, K., Nagasaki, A., Takiguchi, M. & Mori, M. (1995). Preparation of recombinant argininosuccinate synthetase and argininosuccinate lyase: expression of the enzymes in rat tissues. Journal of Biochemistry, 117, 952-957.

In: Arginine Amino Acid Editor: Nathan L. Jacobs

ISBN: 978-1-61761-981-6 ©2011 Nova Science Publishers, Inc.

Chapter 5

Expression and Localization of Argininosuccinate Synthetase and Argininosuccinate Lyase in the Female and Male Rat Kidneys Olivier Levillain1* and Heinrich Wiesinger2 1

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Université Lyon I, Lyon, France, UMR 5123 CNRS, Laboratoire de Physiologie Intégrative, Cellulaire et Moléculaire, Villeurbanne, France 2 Physiologisch-Chemisches Institut der Universität, Tuebingen, Germany

Abstract The enzymes argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) convert L-citrulline into L-arginine. In mammalian kidneys, L-arginine is essentially produced in the proximal convoluted tubules (PCT). Almost all the studies performed on this renal metabolic pathway were restricted to male animals. Our experiments were first conducted to determine whether female rat kidneys express ASS and ASL genes and the results were compared to those of the males. The expression of ASS and ASL was assayed by Western blot analyses in the different zones dissected from rat kidneys. ASS protein was localized by immunofluorescence. Finally, we tested whether sex influences the renal level of ASS and ASL proteins, as determined by Western blot analyses. Our results reveal that high levels of ASS and ASL proteins were detected in female as well as in male rat kidneys. The relative abundance of ASS and ASL proteins was higher in the cortex (superficial > deep) than in the outer stripe of the outer medulla. Immunolocalization studies clearly showed that ASS expression was restricted to the *

Correspondance to : Dr O. Levillain, Université Claude Bernard Lyon I, Physiologie Intégrative, Cellulaire et Moléculaire, UMR 5123 CNRS, Bâtiment R. Dubois, 43, Boulevard du 11 Novembre 1918, 69622 Villeurbanne cedex, France. [email protected]

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Olivier Levillain and Heinrich Wiesinger proximal tubules. PCT exhibited the highest level of ASS compared with the proximal straight tubules (PST). The levels of ASS and ASL proteins were similar in female and male rat kidneys. In conclusion, female rat kidneys express the two enzymes involved in the production of the conditionally essential amino acid L-arginine. No sexual dimorphism in ASS and ASL expression was found in the rat kidney.

Key words: rat, sex, renal zones, proximal tubules, arginine, Western blot, immunofluorescence

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Introduction The mammalian kidney is the sole organ that produces large amounts of arginine. The biochemical mechanisms involved in arginine synthesis have been detailed in earlier reports [8, 26, 28, 29]. Arginine biosynthesis requires two enzymes, the substrate citrulline, a nitrogen source supplied by aspartate, glutamine, or glutamate, oxygen for energy production in the form of ATP, Mg++ as a cofactor, and cell integrity [4, 8, 27]. The first enzyme argininosuccinate synthetase (ASS; EC 6.3.4.5) condensates aspartate with citrulline to produce argininosuccinate while the second enzyme argininosuccinate lyase (ASL; EC 4.3.2.1) cleaves this compound into arginine and fumarate. The newly formed arginine serves not only locally for renal metabolic needs but, also for those of the other organs. In the latter case, arginine is carried out of the proximal tubular cells and released in the bloodstream. The renal arterio-venous differences for citrulline and arginine clearly prove an uptake of the former, a production and a release of the latter [9]. During the last three decades, the expression of ASS and ASL genes was studied at the transcriptional and protein levels in rodent kidneys. The relative abundance of ASS and ASL mRNAs and proteins was analyzed in male Wistar [37] and Wistar HLA [14] rat tissues. These two genes are predominantly expressed in the liver and the kidneys compared with the other organs. The time course expression of ASS and ASL mRNAs during development was determined in kidneys of fetuses, newborn, and adult Wistar rats. The results revealed that the levels of ASS and ASL mRNAs increased throughout the perinatal life [12]. Our team demonstrated the functionality of this metabolic pathway in several mammalian kidneys. Microdissected viable nephron segments were incubated with aspartate and radiolabeled citrulline and the production of radiolabeled arginine was quantified using a specific assay [19, 20]. This elegant approach allowed us to identify precisely the nephron segments involved in arginine synthesis in male Sprague Dawley rats, mice, lagomorphs, and carnivores [13, 19-21, 23]. In all species studied, arginine synthesis occurs almost exclusively in the proximal tubule. The initial portion of the proximal convoluted tubule (PCT) produces the highest amount of arginine whereas the medullary pars recta of the proximal tubule (OSPST) produces the lowest rate [20]. All experiments focused on the renal expression of ASS and ASL as well as the renal metabolism of arginine were almost exclusively performed with kidneys of male animals. To our knowledge, the renal metabolism of arginine has never been analyzed in females. The present work was designed to document the expression of ASS and ASL in the female rat kidney and to compare the renal expression of these two enzymes in rats of both sexes. To achieve these goals, the distribution and the abundance of ASS and ASL proteins were

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analyzed in the main dissected renal zones of female and male rats. The nephron segments that express ASS were precisely identified by immunofluorescence in rat kidney of both sexes. Finally, the levels of ASS and ASL proteins were quantitated in whole kidneys of male and female rats. Our results show that ASS and ASL genes are expressed in the female rat kidneys. Whatever the sex, these genes are predominantly expressed in the superficial and deep cortex, more precisely in PCT. The levels of ASS and ASL proteins did not differ between female and male rat kidneys indicating the absence of sexual dimorphism in rats.

Material and Methods Animals

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Six week-old female [168 ± 6 g body weight (BW)] and seven week-old male (236 ± 4 g BW) Sprague Dawley rats from Iffa Credo (L'Arbresle sur Orge, France) were used to dissect the renal zones and for histological studies. Six week-old female and male Sprague Dawley rats (163 ± 5 g BW and 167 ± 3 g BW, n = 5 rats respectively) were used to analyze the influence of sex on the renal expression of ASS and ASL proteins. Rats had free access to tap water and standard laboratory food (Souffirat, 20% protein, Genthon S.A., France). Animals were housed in a controlled environment maintained at 20 ± 1°C with a 12-h light, 12-h dark cycle, lights on at 0700 h. Rats were anaesthetized by injecting intraperitoneally 0.1 ml/100 g BW sodium pentobarbital (Nembutal 6%, Clin Midy, Paris, France). Animal care complied with French regulations for the protection of animals used for experimental and other scientific purposes and with European Community regulations. The author (O.L.) is authorised to use animals for these experiments (N° 69-33).

Kidney Preparation and Dissection of the Renal Zones The right and left kidneys were rapidly removed and decapsulated. The blood contained in each kidney was removed with blotting paper (blood-free). Some kidneys were cut along the cortico-papillary axis to dissect six zones [superficial cortex (Cs), deep cortex (Cd), outer stripe of the outer medulla (OS), inner stripe of the outer medulla (IS), inner medulla (IM), and papilla (Pap)] at 4°C under a stereo-microscope. The dissected tissue and whole kidneys were immediately placed in a sterilized Eppendorf tube, frozen, and conserved in liquid nitrogen.

Protein Extraction and Western Blot Analyses Whole kidneys and tissue of each renal zone were mixed at 4°C with a Turrax homogenizer in the proportion of 100 mg tissue/ml of lysing buffer [17] containing 1 mM protease inhibitor cocktail, 1 mM phenylmethylsulphonyl fluoride (PMSF), and 1 mM benzamidine, then submitted to a centrifugation at 10,000 g for 30 min at 4°C. Protein

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concentrations were determined on the supernatant using the Bradford protein assay and bovine serum albumin as standard [6]. Proteins were resuspended in Laemmli buffer (TrisHCl 62.5 mM pH 6.8, 1% sodium dodecyl sulfate (SDS), 10% glycerol, 0.1M dithioerythritol, and bromophenol blue), heated at 95°C for 5 min, and immediately dipped in a bath maintained at 4°C. Fifty-microgram samples of soluble proteins were subjected to a 10% PAGE containing 0.1% SDS using 6 Watt per gel. Ten microliters of a protein ladder (Precision plus protein standards, Bio-Rad, Marnes la Coquette, France) were deposed on each gel to further verify the size of the protein of interest. Proteins were transfered to a polyvinylidene difluoride membrane (0.45 µm, Immobilon-P, Millipore, St Quentin en Yvelines, France) at 150 mA for 90 min. Proteins were visualized on the membrane with Ponceau S solution. Immunoblots were washed twice for 15 min in TBST (20 mM Tris pH 7.6, 137 mM NaCl, 0.1% Tween 20), and immersed twice in a blocking solution consisting of 5% fat-free milk powder in TBST for 30 min. Immunoblots were incubated with polyclonal rabbit antibodies raised against the amino acid sequence 196-222 of the murine hepatic ASS (dilution 1:1,000) [32], the partial sequence of the mouse liver ASL (dilution 1:1,000) [3], and the mouse aldose reductase (AR, dilution 1:3,000) [18] diluted in 5% fat-free milk in TBST. Aldose reductase was used as a marker of the inner medullary and papillary collecting ducts [31]. Immunoblots were washed three times for 20 min in TBST and incubated for 60 min with a peroxidase-conjugated anti-rabbit IgG (dilution 1:10,000) secondary antibody in 5% fat-free milk in TBST. Immunoblots were washed again three-times for 20 min in TBST. Antibody binding was revealed using chemiluminescence (ECL) Western blotting kit. ECL detection was performed using X-MAT films. Low exposure films were scanned and intensity of the bands was estimated using the ImagerMaster Total Lab program (Pharmacia, Orsay, France).

Indirect Immunofluorescence Kidneys were cut along the cortico-papillary axis, dipped in Bouin fixative and embedded in paraffin. Sections of 4-7 μm were collected on glass slides (ChemMate, Dako, Trappes, France). Nonspecific sites were coated with PBS-Triton-BSA for 120 min at room temperature and incubated overnight at 4°C with the rabbit anti-ASS primary antibody (dilution 1:100) in PBS-Triton-BSA. The slides were rinsed three times in PBS for 5 min and incubated for 120 min at room temperature with the secondary Alexa fluor 488 or Alexa fluor 546 conjugated goat anti-rabbit IgG (Interchim, Montluçon, France). Slides were washed three times for 5-10 min in PBS-Triton and incubated for 5 min at room temperature with 4‟, 6-diamidine-2-phenylindole dihydrochloride (DAPI, dilution 1:50 in water). Slides were washed three times in water and mounted with Fluoprep (Biomérieux, Lyon, France). Tissue sections were examined with a fluorescence microscope.

Calculation and Statistical analyses In each renal zone, the relative abundance of ASS and ASL proteins corresponds to the amount of ASS or ASL protein divided by the whole amount of ASS or ASL protein. The results are expressed as a percentage. Statistical differences in protein levels were assessed

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using the U-Mann-Whitney test. Differences were considered when P < 0.05 and were calculated using StatView II SE+Gr 1.04 VF program.

Chemicals Salts, sodium dodecyl sulfate, glycerol, Tween 20, dithioerythritol, bovine serum albumin, benzamidine, phenylmethylsulphonylfluoride, ponceau S solution, peroxidaseconjugated anti-rabbit IgG secondary antibody, X-MAT films were purchased from Sigma (Saint Quentin Fallavier, France). ImagerMaster Total Lab program V 2.01 and ECL Western blotting kit were purchased from Amersham, Buckinghamshire, England. Cocktail of protease inhibitors and DAPI were purchased from Roche, Mannhein, Germany.

Results

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Regional Distribution of ASS Protein in the Rat Kidney The expression of ASS gene was analyzed at the protein level on Western blots prepared with soluble proteins extracted from all renal zones of female and male rats. This approach permits us to quantify the relative abundance of ASS and ASL proteins in each zone whereas immunohistological approaches do not. The specific ASS antibody detected a single band with an apparent molecular mass of about 46 kDa that corresponds to the predicted size of the ASS polypeptide subunit (Swiss-Prot: rat P09034) [34, 35, 37]. ASS protein was detected in female and male rat kidneys (Fig. 1, left). ASS protein exhibited a heterogeneous and characteristic distribution along the cortico-papillary axis. In details, 86-95 % of ASS protein were found in the cortical tissue and about 2/3 of ASS protein were restricted to Cs (Fig. 1, right). The amount of ASS protein found in OS represented only 4-14% of the whole renal content (Fig. 1, right). The level of ASS protein sharply declined from Cs to OS, and was undetectable in the deeper medulla including IS, IM, and Pap. These three zones were characterized by aldose reductase, a protein of 35 kDa whose expression is restricted to the medullary and papillary collecting ducts [31]. The renal cortex is largely composed of proximal tubules. Given that the production of arginine is restricted to the proximal tubules and the intensity of its synthesis decreases from PCT to OSPST [19, 20], we undertook an experiment to establish a correlation between the levels of ASS protein and the amount of arginine produced along the proximal tubule. To achieve this goal, we refined the tubular localization of ASS protein in the rat kidney by indirect immunofluorescence. The quality and the specificity of our ASS antibody allowed us to localize ASS by this technique (Fig. 2). Most prominent ASS immunostaining was observed in tubules with thick epithelium and large tubular diameter, identified as proximal tubules. The PCTs which are mainly gathered together in the labyrinths (Fig. 2), were highly immunostained whereas cortical proximal straight tubules (CPST) localized in the medullary rays were lightly immunoreactive (Fig. 2 left top). In PCTs, immunostaining was observed in whole cells, but appeared to be more intense at the apical pole (Fig. 2 left bottom).

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Figure 1. Regional expression of argininosuccinate lyase (ASL), argininosuccinate synthetase (ASS) and aldose reductase (AR) proteins in renal zones of female (A) and male (B) rats. Fifty-µg soluble proteins were analyzed by 10% SDS-PAGE. Each lane corresponds to 1 rat except for Pap where n = 2. Left: representative Western blot probed with antibodies raised against ASL, ASS, and AR, revealed by ECL, exposed to X-ray film, and quantitated by densitometry. Right: Relative levels of ASL (red bars) and ASS (orange bars) proteins. AR is a marker of the medullary collecting ducts. Abbreviations : superficial cortex (Cs), deep cortex (Cd), outer stripe of the outer medulla (OS), inner stripe of the outer medulla (IS), inner medulla (IM), and papilla (Pap). Blot analyses were repeated at least 3-times to quantify the proteins, each time yielding identical results. Values are means ± SE, n = 3 measurements.

Figure 2. A (continued)

Expression and Localization of Argininosuccinate Synthetase….

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Figure 2. B (continued)

Figure 2. C (continued)

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.

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Figure 2. D Figure 2. Indirect immunofluorescent detection of ASS in female (A-C) and male (D) rat kidneys. ASS immunoreactivity is visualized by red or green dyes, and nuclei are colored in blue by 4‟,6-diamidine-2phenylindole dihydrochloride (DAPI). A: general view of the female rat cortex. Strong immunoreactivity was detected in proximal convoluted tubules (PCT), whereas glomeruli (G) were unlabeled (magnification x 50). B: a weak immunoreactivity was detected in the medullary rays (MR) where are localized the cortical proximal straight tubules (CPST). Intense immunoreactivity was observed in labyrinths (L) which gather together PCTs (magnification x 50). C: at a higher magnification, immunoreactivity was detected in the whole PCT cells, but was more pronounced at the apical pole of the cells (magnification x 200). D: in male rats, intense immunoreactivity was detected in PCTs (magnification x 50).

Regional Expression of ASL Protein in the Rat Kidney To determine whether kidneys of female rats express the ASL gene, proteins extracted from the different renal zones were analyzed on Western blots. The results were compared with those of the males. The specific antibody raised against ASL revealed a single band of 51 kDa that corresponds to the predicted size of the ASL polypeptide subunit (Swiss-Prot : rat P20673) [1-3, 37]. The results clearly indicate that ASL protein was highly expressed in the female rat kidney (Fig. 1, left). The distribution pattern of ASL protein along the corticopapillary axis strongly resembled that of ASS protein. The cortex contained about 77-78% of the whole renal ASL. The level of ASL protein was the highest in Cs of rats (45-54%) as compared to the other renal zones whereas it decreased sharply from Cs to OS to reach the lowest levels in IS, IM, and Pap (Fig. 1, right). It is noteworthy that, in contrast to ASS, ASL protein was detected in almost all renal zones including the Pap. Unfortunately, for an unknown reason, our ASL antibody could not be used for indirect immunological studies.

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Differential Expression of ASS and ASL in the Rat Kidney

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To know whether sex influences the expression of ASS and ASL genes in the rat kidney, we analyzed the level of their proteins and used -actin and G3PDH as internal controls of protein loading and transfer quality (Fig. 3 left). The results of immunoblots show that in rat kidneys, the levels of -actin (44 kDa) and G3PDH (35 kDa) proteins did not statistically differ between the groups of female and male rats (Fig. 3 right). Once this was established, we proceeded to compare the levels of ASS and ASL proteins in these groups. Neither ASS nor ASL protein levels differed between female and male rat kidneys (Fig. 3 right, U-MannWhitney, ASS, P < 0.966 and ASL P < 0.298).

Figure 3. Influence of sex on the differential expression of ASL and ASS proteins in the rat kidneys. Western blot analyses of ASL, ASS, -actin and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) levels in the kidneys of female (F) and male (M) rats. A : Each lane corresponds to 1 rat and 50 g sample of soluble proteins subjected to 10% SDS-PAGE. Representative immunoblots probed with rabbit anti-ASL and ASS antibodies and mouse anti--actin and G3PDH antibodies were revealed by ECL. B : After exposure to X-ray film, the bands corresponding to ASL, ASS, -actin and G3PDH proteins were quantitated by densitometry. -actin and G3PDH were used as controls for protein loading and transfer. Values are means ± SE ; n = 5 rats per group. U-Mann-Whitney, ASL: P < 0.298 and ASS: P < 0.966.

Conclusion The renal anabolism of arginine has been extensively studied in male mammals. For this reason, we undertook experiments to document the renal expression of ASS and ASL genes in females of a common rodent used in laboratories, namely the rat. For the first time, we present data analyzing the expression of ASS and ASL genes at the protein level in rat kidneys of both sexes. An overview of the results proves that kidneys of female rats expressed both ASS and ASL genes. The typical distribution pattern of ASS and ASL proteins depicted within the female rat kidney resembles that of the male. Our results are in a good agreement with the distribution of ASS and ASL activities assayed in renal zones dissected from male Sprague Dawley rats

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[10]. Taken together, the data support that arginine synthesis essentially occurs in the renal cortex (Cs > Cd) compared to OS and the other renal zones. Although we could not measure the plasma concentrations of citrulline and arginine in the renal artery and vein to calculate the renal production of arginine, we expect that the kidney of female rat produce quite similar amounts of arginine as compared to the male kidney. Indeed, we found that the levels of ASS and ASL proteins were similar in whole kidneys of male and female rats. It is well established that the kidney is an heterogeneous organ composed of several nephron segments and cell types and a complex vascular structure [16]. To determine which tubules and cell types express ASS and ASL genes, both proteins were finely localized by using immunofluorescence. As expected, we observed that ASS is heterogeneously distributed along the nephron with the highest expression (i.e. the intensity of fluorescent dyes) occuring in PCTs, followed by CPSTs and in a lesser extent by OSPSTs of female and male rats. Unfortunately, the experiments performed with our ASL antibody were unsuccessful. Nevertheless, our results are is a good agreement with the immunohistochemical localization of ASS and ASL reported in kidneys of male Wistar rats [22]. In addition, the distribution of ASS along the proximal tubule perfectly correlates with the levels of arginine produced by PCTs, CPSTs, and OSPSTs of male rats [19], mice and rabbits [20], cats [21], and gerbil Meriones shawii [13]. Furthermore, all data well corroborate with the distribution of ASS mRNA analyzed by serial analysis of gene expression (SAGE) in microdissected human nephron segments [7]. Indeed, ASS mRNA tags display higher levels in the PCTs than in PSTs and very low levels in the other nephron segments. It is striking that a non negligible level of ASL protein was detected in IS, IM, and Pap of male and female rats while ASS protein remained undetectable. Since arginine synthesis requires both ASS and ASL activities, the expression of only ASL in the medulla and the papilla raises the question of its local physiological role. We cannot envisage a production of arginine in these tubule segments unless argininosuccinate is present in the plasma and can be carried to these cells. This possibility remains to be documented. Our study also analyzed the differential expression of ASS and ASL in whole kidneys of female and male rats. This point is particularly relevant because numerous genes are known to be differentially expressed in male and female rat and mouse kidneys [30]. To the best of our knowledge, there was no information on whether the expression of ASS and ASL genes, as well as the renal production of arginine, are influenced by sex. Given that the relative abundance of ASS and ASL proteins is quite similar in rats of both sexes, we conclude that, in this species and this strain, the expression of ASS and ASL genes is sex-independent and probably not regulated by sex-hormones. One would expect that female rat kidneys produce similar amounts of arginine as compared to those of the males. Unfortunately, this statement could not be verified because the use of radiolabelled molecules was not further allowed in our laboratory. It is generally assumed that the renal production of arginine is controlled by the concentration of citrulline [5, 9, 19]. However, it should be point out that the renal synthesis of arginine constitutes an excellent example of functional adaptation between renal physiology, biochemistry, and hemodymanics. Indeed, we identify at least four factors which interact to facilitate arginine synthesis. The first factor affects the site of citrulline reabsorption. Under healthy physiological conditions, the solutes (e.g. amino acids) filtrated by glomeruli are reabsorbed along the PCT [33]. Citrulline reabsorption and transport have been finely analyzed within the proximal tubules. The initial portion of PCT is the principal

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site of citrulline reabsorption [15]. Almost all citrulline is reabsorbed within the three first millimeter of the PCT. The second factor, which is documented in the present paper, concerns the spatial distribution of ASS and ASL gene expression. The highest levels of ASS and ASL proteins were found in the PCT and the abundance of these enzymes sharply decreases along the proximal tubule towards the OSPST. The distribution of ASS and ASL is in a good agreement with the rate of arginine synthesis along dissected PCTs [13, 20]. The third factor is associated to the renal vasculature. Indeed, each PCT is surrounded by an efferent arteriole [11]. Consequently the molecules reabsorbed and/or synthesized by PCTs are transported at the basolateral membrane and released into the bloodstream towards the renal vein. Thus, most of the newly synthesized arginine escapes the kidney and becomes available for the other organs. The last factor might contribute to enhance the intracellular concentration of citrulline. The proteolysis of post-transcriptionally methylated proteins liberates NG,NGdimethylarginine (asymmetrical dimethylarginine : ADMA) in body fluids and the enzyme NG,NG-dimethylarginine dimethylaminohydrolase (DDAH, EC 3.5.3.18) metabolizes ADMA into citrulline and dimethylamine in the rat kidney [25]. DDAH is localized in vascular structures and several nephron segments including PCTs and PSTs of rat kidneys [36]. A net uptake of ADMA in the rat kidney has been reported [24]. At present, the contribution of DDHA in renal production of citrulline remains to be documented. In summary, the kidneys of female and male Sprague Dawley rats express ASS and ASL genes and share the same distribution pattern of ASS and ASL along the cortico-papillary axis and within the nephron. Based on the similar levels of renal ASS and ASL proteins, we conclude that there is no sexual dimorphism in ASS and ASL expression and that these genes are not regulated by sex hormones.

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Acknowledgments The authors are indebted to Pr John T (Sean) Brosnan (Department of Biochemistry, Memorial University of Newfoundland, St. John's, NL, Canada) for reviewing this manuscript and to Dr Anne-Marie Lefrançois-Martinez (CNRS, UMR 6547, Le Theix, France) who kindly provided us a rabbit anti-mouse aldose reductase primary antibody. The results were presented at the colloque « Cellules rénales et interactions dans les épithéliums: Du gène à la thérapeutique, la physiologie dans tous ses états » Paris, 15-16 June 2005 and the abstract was published in Nephron Physiology 2006, 104 (1), p 44.

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Olivier Levillain and Heinrich Wiesinger Bolla, T., Kalbacher, H., Vogel, D. & Wiesinger, H. (1999). Argininosuccinate lyase: Generation of antisera against peptide sequences of the rat brain enzyme and immunochemical studies on glial cells. Biological Chemistry, 380, S95. Borsook, H. & Dubnoff, J. W. (1941). The conversion of citrulline to arginine in kidney. The Journal of Biological Chemistry, 140, 717-738. Bouby, N., Hassler, C., Parvy, P. & Bankir, L. (1993). Renal synthesis of arginine in chronic renal failure: in vivo and in vitro studies in rats with 5/6 nephrectomy. Kidney International, 44, 676-683. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248-254. Chabardes-Garonne, D., Mejean, A., Aude, J. C., Cheval, L., Di Stefano, A. , Gaillard, M. C., Imbert-Teboul, M., Wittner, M., Balian, C., Anthouard, V., Robert, C., Segurens, B., Wincker, P., Weissenbach, J., Doucet, A. & Elalouf, J. M. (2003). A panoramic view of gene expression in the human kidney. Proceedings of the National Academy of Sciences of the United States of America, 100, 13710-13715. Cohen, P. P. & Hayano, M. (1946). The conversion of citrulline to arginine (transimination) by tissue slices and homogenates. The Journal of Biological Chemistry, 166, 239-250. Dhanakoti, S. N., Brosnan, J. T., Herzberg, G. R. & Brosnan, M. E. (1990). Renal arginine synthesis: studies in vitro and in vivo. American Journal of Physiology Endocrinology and Metabolism, 259, E437-E442. Dhanakoti, S. N., Brosnan, M. E., Herzberg, G. R. & Brosnan, J. T. (1992). Cellular and subcellular localization of enzymes of arginine metabolism in rat kidney. The Biochemical Journal, 282, 369-375. Dworkin, L. D. & Brenner, B. M. (2000). The renal circulations. In: Brenner, B. M. (Eds.), The Kidney (6th, pp. 247-285). Philadelphia, London, Toronto, Montreal, Sydney, Tokyo, ST: W. B. Saunders Company. Goutal, I., Fairand, A. & Husson, A. (1999). Expression of the genes of argininesynthesizing enzymes in the rat during development. Biology of the Neonate, 76, 253260. Hus-Citharel, A., Levillain, O. & Morel, F. (1995). Site of arginine synthesis and urea production along the nephron of a rodent species, Meriones shawii. Pflügers Archiv European Journal of Physiology, 429, 485-493. Kato, H., Oyamada, I., Mizutani-Funahashi, M. & Nakagawa, H. (1976). New radioisotopic assays of argininosuccinate synthetase and argininosuccinase. Journal of Biochemistry (Tokyo), 79, 945-953. Kettner, A. & Silbernagl, S. (1985). Renal handling of citrulline. In: Dzurik, R., Lichardus, B. & Guder, W. G. (Eds.), Kidney metabolism and function (pp. 51-60). Dordrecht, Boston, Lancaster, ST: Martinus Nijhoff Publishers. Kriz, W. & Bankir, L. (1988). A standard nomenclature for structures of the kidney. The Renal Commission of the International Union of Physiological Sciences (IUPS). American Journal of Physiology - Renal Fluid Electrolyte Physiology, 254, F1-F8. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685.

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[18] Lambert-Langlais, S., Pointud, J. C., Lefrancois-Martinez, A. M., Volat, F., Manin, M., Coudore, F., Val, P., Sahut-Barnola, I., Ragazzon, B., Louiset, E., Delarue, C., Lefebvre, H., Urade, Y. & Martinez, A. (2009). Aldo keto reductase 1B7 and prostaglandin F2alpha are regulators of adrenal endocrine functions. PLoS One, 4, e7309. [19] Levillain, O., Hus-Citharel, A., Morel, F. & Bankir, L. (1990). Localization of arginine synthesis along rat nephron. American Journal of Physiology - Renal Fluid Electrolyte Physiology, 259, F916-F923. [20] Levillain, O., Hus-citharel, A., Morel, F. & Bankir, L. (1993). Arginine synthesis in mouse and rabbit nephron: localization and functional significance. American Journal of Physiology - Renal Fluid Electrolyte Physiology, 264, F1038-F1045. [21] Levillain, O., Parvy, P. & Hus-citharel, A. (1996). Arginine metabolism in cat kidney. Journal of Physiology London, 491, 471-477. [22] Miyanaka, K., Gotoh, T., Nagasaki, A., Takeya, M., Ozaki, M., Iwase, K., Takiguchi, M., Iyama, K.-I., Tomita, K. & Mori, M. (1998). Immunohistochemical localization of arginase II and other enzymes of arginine metabolism in rat kidney and liver. Histochemical Journal, 30, 741-751. [23] Morel, F., Hus-citharel, A. & Levillain, O. (1996). Biochemical heterogeneity of arginine metabolism along kidney proximal tubules. Kidney International, 49, 16081610. [24] Nijveldt, R. J., Teerlink, T., van Guldener, C., Prins, H. A., van Lambalgen, A. A., Stehouwer, C. D., Rauwerda, J. A. & van Leeuwen, P. A. (2003). Handling of asymmetrical dimethylarginine and symmetrical dimethylarginine by the rat kidney under basal conditions and during endotoxaemia. Nephrology Dialysis Transplantation, 18, 2542-2550. [25] Ogawa, T., Kimoto, M. & Sasaoka, K. (1989). Purification and properties of a new enzyme, NG, NG-dimethylarginine dimethylaminohydrolase, from rat kidney. The Journal of Biological Chemistry, 264, 10205-10209. [26] Ratner, S. (1973). Enzymes of arginine and urea synthesis. Advances in Enzymology, 39, 1-90. [27] Ratner, S. & Pappas, A. (1949). Biosynthesis of urea I. Enzymatic mechanism of arginine synthesis from citrulline. The Journal of Biological Chemistry, 179, 11831198. [28] Ratner, S. & Petrack, B. (1953). The mechanism of arginine synthesis from citrulline in kidney. The Journal of Biological Chemistry, 200, 175-185. [29] Rochovansky, O. & Ratner, S. (1967). Biosynthesis of urea. The Journal of Biological Chemistry, 242, 3839-3849. [30] Sabolic, I., Asif, A. R., Budach, W. E., Wanke, C., Bahn, A. & Burckhardt, G. (2007). Gender differences in kidney function. Pflügers Archiv - European Journal of Physiology, 455, 397-429. [31] Sands, J. M., Terada, Y., Bernard, L. M. & Knepper, M. A. (1989). Aldose reductase activities in microdissected rat renal tubule segments. American Journal of Physiology Renal Physiology, 256, F563-F569. [32] Schmidlin, A., Kalbacher, H. & Wiesinger, H. (1997). Presence of argininosuccinate synthetase in glial cells as revealed by peptide-specific antisera. Biological Chemistry, 378, 47-50.

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[33] Silbernagl, S. (1992). Amino acids and oligopeptides. In: Seldin, D. W. & Giebish, G. (Eds.), The Kidney: Physiology and Pathophysiology (Second Edition, pp. 2889-2920). New York, ST: Raven Press, Ltd. [34] Su, T. S., Bock, H. G., O'Brien, W. E. & Beaudet, A. L. (1981). Cloning of cDNA for argininosuccinate synthetase mRNA and study of enzyme overproduction in a human cell line. The Journal of Biological Chemistry, 256, 11826-11831. [35] Surh, L. C., Beaudet, A. L. & O'Brien, W. E. (1991). Molecular characterization of the murine argininosuccinate synthetase locus. Gene, 99, 181-189. [36] Tojo, A., Welch, W. J., Bremer, V., Kimoto, M., Kimura, K., Omata, M., Ogawa, T., Vallance, P. & Wilcox, C. S. (1997). Colocalization of dimethylating enzymes and NOS functional effects of methylarginines in rat kidney. Kidney International, 52, 1593-1601. [37] Yu, Y., Terada, K., Nagasaki, A., Takiguchi, M. & Mori, M. (1995). Preparation of recombinant argininosuccinate synthetase and argininosuccinate lyase: expression of the enzymes in rat tissues. Journal of Biochemistry, 117, 952-957.

In: Arginine Amino Acid Editor: Nathan L. Jacobs

ISBN 978-1-61761-981-6 © 2011 Nova Science Publishers, Inc.

Chapter 6

Chemical Structure and Toxicity in Arginine-Based Surfactants Aurora Pinazo1*, Lourdes Pérez1, María Rosa Infante1, María Pilar Vinardell2, Montse Mitjans2, María Carmen Morán2, and Verónica Martínez2 1

Institut de Química Avançada de Catalunya, CSIC, Barcelona, Spain 2 Facultat de Farmàcia, UB, Barcelona, Spain

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Abstract Surfactants are one of the most representative chemical products which are consumed in large quantities every day on a worldwide scale. The use of surfactants in everyday life is almost unavoidable. The development of less irritant, less toxic, consumer-friendly surfactants or surfactant systems is, therefore, of general interest. During the last 20 years, our group has been developing new biocompatible surfactants derived from amino acids. Among them, arginine derivative surfactants constitute a novel class that can be regarded as an alternative to conventional cationic surfactants due to their multifuncional properties and the renewable source of raw materials used during the synthesis process. These characteristics make them candidates of choice as additives in pharmaceutical, food and cosmetic formulations. Evaluation of the irritant potential in vivo, of new products or ingredients, for pharmaceutical use, is required by law in most EU countries, prior to human exposure. However, due to increasing concern over animal use and in lights of its potential ban in the near future, alongside with the obvious ethical implications of using directly human subjects, in vitro alternative methods should now be encouraged. This review reports on the relationship between the structure and toxicity evaluated by in vitro methods of a series of arginine-based surfactants including surfactants with one single chain, gemini surfactants, and surfactants with glycerolipidlike structure. *

E-mail: [email protected]

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1. Introduction Surfactants are one of the most representative chemical products which are consumed in large quantities every day on a worldwide scale. The economic importance of cationic surfactants was realized early in 1935, when their bacteriostatic properties were discovered, leading to many commercial products as sanitizing and antiseptic agents, germicides, fungicides, and as components in pharmaceutical and cosmetic formulations. Is in these latter applications where adverse effects may occur causing eye and skin irritation. Therefore it is paramount to develop new biocompatible surfactants with low toxicity profiles or no toxicity at all. Amino acids are not only essential components of a living body but also interesting raw materials for biocompatible surfactants. The value of amino acids as raw material for the preparation of surfactants was recognized as soon as they were discovered early in the last century (W. Heutrich 1936) and they can be produced by biotechnological and chemical methods (Furutani et al. 1997; Gallot and Hassan 1995; Godfredsen and Bjoerkling 1990; Nagao and Kito 1989; Nnanna and Xia 2001; Vonderhagen et al. 1999; Valivety et al. 1997). Lipoamino acids or lipopeptides compounds constitute an interesting alternative to conventional synthetic surfactants in which the three fundamental requirements for industrial developments are present: (1) multifunctional properties, (2) low toxicity and (3) renewable sources as raw materials. They can be defined as amphiphilic molecules that contain one amino acid residue as the hydrophilic part and at least one long chain as the hydrophobic part. Given the chemical duality of an amino acid molecule, many amino acid derivatives with different structures can be obtained by the reaction of amino acids with long chain compounds such as fatty acids, fatty esters, fatty amines and fatty alcohols, being able to modulate their surface properties (Infante et al. 1985, 1997; Xia et al. 1995). The amino acid or peptide moiety determines the main differences of adsorption, aggregation and biological activity between the amino acid based surfactants. Hence, cationic, anionic, non-ionic and amphoteric surfactants can be obtained depending on the free functional groups. Further modification of these allows a fine-tuning of their properties to meet almost every particular application. The use of surfactants in everyday life is almost unavoidable. The development of less irritant, less toxic, consumer-friendly surfactants or surfactant systems is, therefore, of general interest. During the last 20 years, our group has been developing new biocompatible surfactants derived from amino acids. Among them, arginine derivative surfactants constitute a novel class that can be regarded as an alternative to conventional cationic surfactants due to their multifunctional properties and the renewable source of raw materials used during the synthesis process. These characteristics make them candidates of choice as additives in pharmaceutical, food and cosmetic formulations. Due to their potential application it is necessary to evaluate their adverse local irritation potential. (Martínez et al.,2006). Evaluation of the irritant potential in vivo, of new products or ingredients, for pharmaceutical use, is required by law in most EU countries, prior to human exposure. However, due to increasing concern over animal use and in lights of its potential ban in the near future, alongside with the obvious ethical implications of using directly human subjects, in vitro alternative methods should now be encouraged. This review reports on the relationship between structure and

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toxicity of a series of arginine-based surfactants including with one single chain (a), gemini surfactants (b), and surfactants with glycerolipid-like structure (c) (Scheme 1).

Scheme 1. Structures of arginine-based surfactants. (a) Single chain, (b) Gemini, (c) Glycerolipid like structures.

2. Experimental Methods

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2.1. Adsorption and Aggregation Properties The determination of the value of the critical micellar concentration (cmc) can be made by measuring as a function of concentration, surface tension, conductivity, light scattering, pH among others. The cmc is the concentration at which a break in the curve is observed. The direct determination of the amount of surfactant adsorbed per unit area of liquid is not generally undertaken because of the difficulty of isolating the interfacial region from the bulk phase. Instead, the amount of material adsorbed per unit area of interface (Γmax) is calculated indirectly from surface tension measurements using the Gibbs adsorption equation (Rosen 2004).

2.2. Red Blood Cell Assay Red blood cells (RBC) have a long scientific history of being used in the study of the lysis of plasma membranes. The RBC test was developed to assess initial cellular reactions to the irritation caused by certain chemicals (Vinardell and Mitjans, 2008). Certain classes of chemical irritants damage cell plasma membranes and denature several types of proteins. Nonirritant surfactants will not cause these reactions. It is hypothesized that such reactions can be correlated with the initial events in eye tissue irritation, leading to inflammatory responses of the tissue and changes in protein conformation. Such events occur, for instance, in the opacification of the cornea after contact with chemicals. The method is based on measuring the hemolysis induced in erythrocytes, and the hemoglobin denaturation (ID). The L/D ratio can be calculated from the hemolysis concentration and the ID. This value enables compounds to be classified as a function of their potential ocular irritation. The mechanism of

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the RBC test is clear and the method is simple. It does not require any special techniques or equipment. This method also has the merit of being rapid and inexpensive. It correlates well with the in vivo method (Okamoto et al., 1999) and is particularly valid for the study of surfactants (Mitjans et al., 2003) and cleaning products containing surfactants. The hemolytic activity or inhibitory concentration, HC50, is the concentration of surfactant that causes 50% of hemolysis of the red blood cells. Erythrocyte suspensions were added to tubs containing different surfactant concentrations. The tubs were incubated for 10 min at 25ºC, and then were centrifuged and the percentage of hemolysis was determined by comparison the absorbance (540 nm) of the supernatant with that of control samples totally hemolized with distilled water. The potential ocular irritation of the surfactants was studied with a method based on the use of red blood cells to quantify adverse effects of surfactants and detergents products on the cytoplasmatic membrane (hemolysis) in combination with the damage to liberated cellular proteins (denaturation). The irritation index was determined according to the lysis/denaturation ratio (L/D) obtained dividing the HC50 (μg/ml) by the denaturation index. The denaturation index (DI) of each surfactant was determined by comparing the hemoglobin denaturation induced by the sodium dodecyl sulfate (SDS). The resulting L/D ratio is used instead of the ocular irritancy score in the acute phase of in vivo evaluation. The surfactant can be classified according to this L/D ratio as non-irritant (>100), slight irritant (>10), moderate irritant (>1), irritant (>0.1), and very irritant ( 1000 μg/mL, whatever the chemical structure and the hydrophobicity of the molecule. Thus, we conclude that all these arginine surfactants have a low or very low hemolytic activity. The results of L/D ratio obtained in this study are in agreement with previous in vivo data published (Vinardell et al. 1990, Martinez et al., 2006), in which these surfactants were classified as non or slight eye irritants. This fact also points out the inability of the Draize test to detect differences among surfactants that have a similar irritation potential although differences exist. Thus, in vitro testing seems to be more sensitive to detect subtle differences in irritancy than in vivo visual scores. Table 2. Cytotoxic properties of commercial surfactants studied with red blood cells and cutaneous fibroblasts and keratynocytes. Compound

HC5 0

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SDS

(μg/mL) 43.6

HTAB

11.6

TegoBet

34.4

ID (%) 100 46. 5 14. 4

L/D

0.4 (irritant) 0.2 (irritant) 2.4 (moderate)

IC50 (μg/mL) 3T6 71.1

IC50 (μg/mL) NCTC 53.5

0.5

2.1

165.7

203.2

*data collected from Martínez et al., 2006 Because no major differences in the cmc values and minimum surface tension decreases, different toxicity values should be attributed to other factors. Surfactant interaction into membrane leads to changes in the membrane molecular organization and increase of membrane permeability that concludes with cell lyses (Shalel et al. 2002 a, b). However, hemolysis depends upon the adsorption of the surfactant to the components of the membrane surface which is influenced by electrostatic attraction between surfactant molecules and membrane components, among other factors. One of the most important parameters that determine surfactant–cell interaction is the partition of surfactant between the cell membrane and the suspending solution (Shalel et al., 2003). The observed differences in the values of HC50 in the series a, and series b and c, can be attributed for instance to the number of positive charges in the molecule (one in series a, and two in series b and c) and thus modifying the equilibrium between the bulk surfactant and the membrane-bound surfactant.

Chemical Structure and Toxicity in Arginine-Based Surfactants.

133

Table 3. Cytotoxic properties of single chain arginine surfactants studied in red blood cells. Serie

Compound

Nα-acyl a

CAM LAM MAM

N-alkyl b O-alkyl c

HC50 (μg/mL)

ID (%)

L/D

38.5 58.8 52.4

9.4 22.5 6.2

4.1 (moderate) 2.6 (moderate) 8.4 (moderate)

ACA ALA AMA

>1000 >1000 >1000

n.d. n.d. n.d.

∞ (no irritant) ∞ (no irritant) ∞ (no irritant)

AOE ACE ALE

>1000 >1000 >1000

n.d. n.d. n.d.

∞ (no irritant) ∞ (no irritant) ∞ (no irritant)

*data collected from Moran et al, 2001 and Martínez et al., 2006; n.d. not determined, due to the very low or inexistent hemolytic action at the concentrations assayed, as reflected by the HC50.

Gemini surfactants contain two hydrophobic groups and two polar groups per molecule, connected by a spacer chain close to the hydrophilic groups (Figure 2). An obvious strategy to increase the efficiency of cationic surfactants and reduce their environmental impact and potential toxicity is to build up gemini surfactants from environmentally friendly single-chain arginine-based surfactants (Perez et al.1996). These molecules can be considered dimers of the N-acyl arginine single chain surfactants (Figure 1 a). There has been considerable interest in these compounds, both academic and industrial, since it was pointed out (Rosen, 1993) that the interfacial properties of these surfactants in aqueous media can be orders of magnitude greater than those of comparable conventional surfactants. O H3C n

O

NH

( )

NH NH

( )S

(

NH

O

O

HN

NH

NH2

NH2

NH2

Cl

NH2 Cl

CS(LA)2 , n = 10, S = 0, 1, 2, 4, 7, 8 CS(CA)2 ,n = 8, S = 0, 1, 2, 4, 7, 8 CS(OA)2 ,n = 6, S = 1

Figure 2. Chemical structure of gemini arginine based surfactants

(

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4. Arginine-Based Gemini Surfactants

n CH3

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Aurora Pinazo, Lourdes Pérez, Maria Rosa Infante et al.

4.1. Adsorption and Aggregation Properties Adsorption and aggregation properties of arginine based gemini surfactants were investigated and compared with those of corresponding single-chain counterpart surfactant (Perez 1998, Pinazo 1999, Perez 2007). Values of cmc, γcmc, Γmax and the minimum area per molecule are collected on Table 4. For comparison, values for LAM, CAM (Figure 1 a) and OAM (N-capryl arginine methyl ester), the single chain analogs are also included. Table 4. Adsorption and aggregation properties of gemini arginine surfactants Series

CS (LA)2

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*CS (CA)2

CS (OA)2

Compound

cmcx10-6 mols l-1

γ mNm-1

Γmax x 1014 mol m-2

Aminx102 nm2/molecule

C2 (LA)2 C3 (LA)2 C4 (LA)2 C6 (LA)2 C9 (LA)2 C10 (LA)2 LAM

9.5 4.4 2.8 1.3 2.8 1.9 6000

30 35 30 30 34 34 33

1.8 1.9 1.3 1.5 2.2 2.2 2.5

91 86 130 113 77 74 67

C2 (CA)2 C3 (CA)2 C4 (CA)2 C6 (CA)2 C9 (CA)2 C10 (CA)2 CAM

58 43 48 21 16 7.3 2700

33 32 33 33 35 36 29

2.6 2.5 2.1 2.1 2.8 2.9 3.6

64 66 77 79 60 58 47

C3 (OA)2 OAM

700 16000

35

1.6

105

*Surface properties in 10-2 M NaCl.

Comparing with LAM, the compounds CS (LA)2 in water show very small cmc values. Micellization takes place at concentrations in the range of micro molar, about three orders of magnitude lower than in the case of LAM. This ability to aggregate is a common feature of the gemini type surfactants. The low cmc values of gemini surfatants are due to the greater total number of carbon atoms in the alkyl chains of the molecules and not to the hydrophiliclipophilic balance. As expected, the spacer chain length exerts an influence on the cmc; cmc values decreases as the length spacer chain increases. Compared with CAM, the compounds CS (CA)2 also show very small cmc values, two orders of magnitude less. Compared with those of the CS (LA)2 homologues, the cmc of the CS (CA)2 increase somewhat less than one order of magnitude as the length of the alkyl chain decreases from 12 to 10 carbon atoms. A similar situation was observed in the case of C3 (OA)2, the cmc values for the gemini is on the range of milli molar while for OAM is in the order of molar.

Chemical Structure and Toxicity in Arginine-Based Surfactants.

135

4.2. Toxicity 4.2.1 Hemolysis and Potential Ocular Irritation Hemolytic activity of arginine based gemini surfactants is shown on Table 5, together with cytotoxic activity in 3T6 fibroblasts for some of them. Table 5. Cytotoxic properties of arginine based gemini surfactants studied in red blood cells and cutaneous fibroblasts and keratynocytes.

Compound C3(OA)2 C2(CA)2 C3(CA)2 C4(CA)2 C6(CA)2 C9(CA)2 C3(LA)2

HC50 (μg/mL) >1000 102.6 48.1 50.6 9.0 8.7 12.5

L/D non irritant slight irritant moderate moderate moderate moderate moderate

IC50 (μg/mL) 3T6 341.8

IC50 (μg/mL) NCTC 155.2

41.6

22.8

21.9

10.1

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*data collected from Pérez et al., (2002b) and Martinez et al., (2006)

The hemolysis test showed that the highest HC50 values were obtained for compounds with the highest hydrophobic character, namely, those with the longest alkyl and spacer chain lengths (Perez et al. 2002 b). Comparing the obtained results, dimerization of single chain arginine surfactants (CAM and LAM, with HC50 of 38.5 and 58.8, respectively Table 3) yields gemini surfactants with higher hemolytic activity except for the shorter spacer chain (C2(CA)2) surfactant studied. Concerning the possible mechanism that induced hemolysis, when surfactants are added to the erythrocyte suspension in aqueous medium, they could first distribute between erythrocyte and the solution by adsorption until equilibrium is reached. The surfactanterythrocyte membrane interaction at sublytic concentration could be governed by the affinity of each surfactant for the aqueous or the membrane, this factor is closely related to the hydrophobicity of surfactants and consequently to the critical micellar concentration (Lichtenberg et al. 1983). Hemolysis probably begins when erythrocyte membranes are saturated with the surfactant molecules. Comparing cmc values (Table 4) and hemolytic concentration (HC50) (Table 5) of arginine based gemini surfactants from series Cn(CA)2, it can be observed that there exists a good correlation between the cmc and HC50 (r : 0.946). There are considerable differences between the hemolytic power of gemini surfactants from arginine and those from the conventional monoquats with a quaternary ammonium group in the polar head. Bisquats, which have similar surface activity, are able to lysis red blood cells at low concentrations (0.05-0.1 mg/L), whereas the arginine based surfactants showed hemolytic effects in a concentration range between 8.7–110 mg/L (Devinsky et al. 1987).

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Aurora Pinazo, Lourdes Pérez, Maria Rosa Infante et al.

4.2.2 Cytotoxicity The effect of arginine gemini surfactants on cell membrane integrity was assessed using the NRU method as indirect measure of cell viability, being the commercial surfactants used as a reference compounds (Table 2). The cells were exposed to a wide concentration range of the C3(OA)2, C3(CA)2 and C3(LA)2 for 24 h. We established a clear dose–response relationship from results relative to NRU assay which allowed us to calculate the concentration of surfactant that causes 50% inhibition of growth (IC50) for the fibroblast cell line 3T6 and keratinocyte NCTC, as reported in Table 5. All arginine derivatives surfactants tested showed higher IC50 values for both cell lines than the cationic surfactant HTAB. The C3(OA)2, emerged as the least cytotoxic surfactant when compared with the slight irritant TegoBet. Moreover, the cytotoxicity shown by these gemini surfactants over cutaneous cells (3T6 and NCTC) corroborate the observed trend derived from the hemolysis assay.

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5. Glycerolipid-Like Arginine Based Surfactants Considerable research has been focused on new surfactants and emulsifiers in recent years. The main driving force behind the development of novel surfactants is in one side, the search for environmentally friendly compounds and in the other side the search for new surfactants to combine surface activity and another property in one molecule, i.e., polymerizability (Guyot et al. 1998), susceptibility to cleavage by some specific mechanism (Holmberg 1998) or antimicrobial properties (Seguer 1994, Perez 1996). Food emulsifiers are polar lipids needed to increase colloidal stability and provide interfacial interactions between food components such as lipids, proteins and cabohydrates. Such interactions are important factors in obtaining emulsion stability, foam formation of whipped products, and increased shelf life in many foods (Krog 1997). Lecithin and lysolecithin are well known food and cosmetic additives. They are natural environmentally friendly products, which exhibit a number of desirable properties such as low toxicity, high biodegradability and compatibility with a number of pharmaceutical products. However, they have low water solubility. Surfactants that exhibit the properties of lecithins, along with significant water solubility be valuable compounds. Arginine amino acid glyceride conjugates constitute a novel class of lipoamino acids, which can be considered analogues of mono and diacylglycerides and phospholipids. They consist of one or two aliphatic chains and the arginine amino acid (X0R and XXR) or Nαacetyl-arginine (XXRAc), as polar head, linked together through ester bonds in the glycerol backbone (Perez, L., et al.2002a, Perez, L., et al. 2004a) (Figure 3). These compounds combine in one molecule the physicochemical properties of the glycerol derivatives and those of the polar arginine-based surfactants.

5.1. Adsorption and Aggregation Properties The micelle formation of mono- and diacylglycerol surfactants from arginine was evaluated by conductivity, surface tension, fluorescence and proton activity measurements (Perez et al. 2004 a, 2004b, 2004c, Pinazo et al. 2004).

Chemical Structure and Toxicity in Arginine-Based Surfactants.

137

Micellization of monoglycerides of arginine (X0R) take place at 6 mM for 100R, 1.3 mM for 120R and 0.2 mM for 140R. As expected, the cmc decreases when the alkyl chain length increases as a consequence of the higher hydrophobic content of the molecule. The cmc of lysophosphatidylcholine with same alkyl chain (Yamanaka et al. 1997) are two times lower than that monoglycerides of arginine. The lysophosphatidylcholine compounds are zwiterionic surfactants and generally appear to have slightly lower cmc‟s than those of ionic surfactants with the same number of carbon atoms in the hydrophobic group.

O

OH H3C

( )

O

n

NH2 R'

O

O

X0R

NH NH2 NH2 Cl

n = 8, 10 0R n = 10, 12 0 R n = 12, 14 0 R

O H3C

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H3C

( )

( )

n

O

n

O

O O

XXR, XXRAc

NH2 R' n = 6, 88R n = 8, 1010 R n = 10, 1212 R n = 12, 1414 R

O

NH NH2 NH2 Cl

n= 10, 1212RAc n= 12,1414RAc

Figure 3. Structures of glycerolipid arginine based surfactants. R‟ in the structure can be H or –COCH3. R refers to arginine.

The XXR surfactants differ from short chain lecithines in terms of aggregation behavior. Short chain phospholipids aggregate in water forming micelles at concentrations of about 16.5 mM for di C6-lecithin and 0.24 mM for C8-lecithin (Tausk 1974). Comparing these cmc values with those of the same fatty chain length of XXR homologues, the cmc (Table 6) are one order of magnitude higher. This increment can be attributed to the high solubility of the XXR due to the cationic character of the polar group. The solubility in water of XXR compounds depends on the fatty chain length. Compounds 88R and 1010R at 0.1% w/v form isotropic single phase whereas compounds 1212R and 1414R at 0.1% w/v form liquid crystalline dispersion.

138

Aurora Pinazo, Lourdes Pérez, Maria Rosa Infante et al. Table 6. Aggregation properties of glicerol arginine-based surfactants

Compound 10 0 R 12 0 R 14 0 R 88R 10 10 R 12 12 R 14 14 R 1212RAc 1414RAc

Conductivity (mM) 6 1.3 0.2 5 1.1 0.3 0.25 0.12 0.09

Fluorescence mM

Surface Tension mM

pH mM

7 0.3

0.07 0.006 0.008

0.9

0.002

From the conductivity/concentration curves, the cmc of the diacyl arginine acylglycerol surfactants of 12 carbon atoms is 0.3 mM for 1212R and 0.12 mM for the 1212RAc homolog. The cmc values on Table 6 show that the presence of a second alkyl chain increases the hydrophobic content of the molecule and, consequently, the cmc of these compounds is lower than those corresponding to the monoacyl arginine acylglycerols with the same alkyl chain.

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5.2. Toxicity 5.2.1. Hemolysis The hemolytic activity test (Table 7) performed on human red blood cell suspensions, shows values of similar order for the compounds XXR and XXRAc. The hemolytic action of this class of surfactants showed a clear tendency to decrease with increase in hydrophobic chain length. The most hemolytic were the surfactants with shorter alkyl chains (8 and 10 carbon atoms). When comparing with other commercially available surfactants, as HTAB which produce lysis at concentrations as low as 0.5 mg/L, the HC50 for XXR and XXRAc is ranged between 40 to 150 times higher. Thus, these compounds hold less hemolytic activity which in turn is reflected in the L/D ratio and in their potential ocular irritation, with values similar to that obtained for a soft amphoteric surfactant Tego®-bet (Table 2). Table 7. Cytotoxic properties of XXR and XXRAc compounds* studied in red blood cells and 3T6 cutaneous fibroblasts Compound 88R 1010R 1212R 1414R 1212RAc 1414RAc

HC50 (μg/mL) 24.0 20.5 60.1 64.3 75.1 44.7

ID(%)

L/D

19.1 11.9 11.7 12.8 10.7 4.5

1.3 (moderate) 1.7 (moderate) 5.6 (moderate) 5.0 (moderate) 6.4 (moderate) 9.9 (moderate)

*data collected from Benavides et al, 2004. n.d. not determined

IC50 (μg/mL) 60.5 74.8 345.2 63.7 294.6 n.d.

Chemical Structure and Toxicity in Arginine-Based Surfactants.

139

The obtained potential irritation values for these arginine-derivative surfactants, however, are higher than those shown by lecithin mimics derived from lysine with non-ionic character (Macián et al. 1996). These results suggest that the irritant character is not related to the molecular structure but to the cationic charges in the compounds. 5.2.2. Cytotoxicity The viability and metabolic activity of 3T6 fibroblasts cell, previously incubated with surfactant dilutions, are shown Table 7 expressed as IC50. The cytotoxicity of the surfactants tested show that the cationic HTAB is far more toxic (Table 2) than 88R, 1010R, SDS, TegoBet, 1212RAc and with 1212R being least toxic. The results related to the hemolytic action observed in the present study since the surfactants with larger hydrophobic moiety are less hemolytic than those with smaller carbon chains. However, in the case of 1414R, the expected cytotoxic behavior does not correlate with the observed IC50 value. Thus, we conclude that in this series of surfactants the inclusion of alkyl chain higher than 12 carbon atoms increases dramatically the cytotoxicity (Benavides et al. 2004).

Conclusion Arginine based surfactants constitute a class of surfactants with excellent surface properties and low potential toxicity. These characteristics make them an alternative to conventional surfactants for food, cosmetic and pharmaceutical industries.

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References Benavides T., Martinez V., Mitjans M., Infante M.R., Moran C., Clapés P., Clothier R., Vinardell M.P. (2004). Assessment of the potential irritation and photoirritation of novel amino acid-based surfactants by in vitro methods as alternative to the animal tests. Toxicology, 201, 87-93. Borenfreud, E. and Puerner, J. A. (1985). Toxicity determined in vitro by morphological alterations and neutral red absorption, Toxicol. Lett. 24, 119-124. Clapés P.; Morán M.R.; Inante M.R. (1996). Enzymatic Synthesis of arginine –based cationic surfactants. Biotech. Bioeng. 63, 333-343. Devinsky F., Lacko I., Bittererova F., Mlynarcik D. (1987). Quaternary ammpnium salts XVIII. Preparation and relationship between structure, IR spectral characteristics and antimicrobial activity of some new bis-quaternary isosters of 1,5-pentanediammonium dibromides. Chem. Pap. 41, 803-814. Draize J.H. (1959). Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetic, Assoc. Food and Drug Officials of USA, Austin, TX. Furutani, T; Oshima, H; Kato J (1997). Enzyme Microbiol. Technol. 20, 214. Gallot B. and Hassan H.H., (1989). Lyotropic Lipo-Amino-Acids - Synthesis and Structural Study. Molecular Crystals and Liquid Crystals 170,195-214. Godfredsen, SE; Bjoerkling F (1990). World Patent No. 90/14429. Guyot A. in: K.Holmberg (Ed.), Polymerizable Surfactants in Novel Surfactants, Surfactant Science Series, vol. 74, Marcel Dekker, Inc., New York, 1998, 279.

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Heutrich, W; Keppler,H; Hintzmann, K. German Patent 635522,1936. Holmberg K. in: K.Holmberg (Ed.), Cleavable Surfactants in Novel Surfactants, Surfactant Science Series, vol. 74, Marcel Dekker, Inc., New York, 1998, 333. Infante M.R.; Pinazo, A.; Seguer J. (1997). Non-conv entional Surfactants from Amino Acids and Glycolipids: Structure, Preparation and Properties. Colloids and Surfaces A, 123-124, 49-70. Infante, M.R., Pérez L., Pinazo A., Clapes P., Morán M.C., Angelet M., García M.T., Vinardell M.P. (2004). Amino acid-based surfactantes. C.R.Chimie, 7, 583-592. Infante, MR; Molinero, J; Julia, R; Garcia-Dominguez, JJ (1985). A comparative study on surface active and antimicrobial properties of some N-alfa-lauroyl-L-alpha, omega dibasic amino acids derivatives. Fette Seifen Anstrichmittel, 87, 309-313. Infante, MR; Pinazo, A; Seguer J (1997). Non-conventional Surfactants from Amino Acids and Glycolipids: Structure, Preparation and Properties. Colloids and Sufaces A, 123-124, 49-70. Kirk, O., F.D. Pedersen, and C. Fuglsang, (1998). Preparation adn Properties of a New Type of Carbohydrate-Based Cationic Surfactant. Journal of Surfactants and Detergents, 1, 3740. Krog N. in: F.D. Gunstone, F.B. Padley (Eds.), Food Emulsifiers in Lipid Technologies and Applications, Marcel Dekker Inc., New York, 1997, 521 Lichtenberg, D.,. Robson R.J., Dennis E.A. (1983). Solubilization of Phospholipids by Detergents - Structural and Kinetic Aspects. Biochimica Et Biophysica Acta, 737, 285304. Macián, M., Vives, M.A., Seguer J.,Infante, M. R.,Vinardell P. (1996).Haemolytic Action of Non-ionic Surfactants Derived from Lysine in Rat Erythrocytes. Pharmaceutical Sciences, 2, 1246-1248. Martinez V.; Corsini E.; Mitjans M.; Pinazo, A.; Vinardell, M.P. (2006) Evaluation of eye and skin irritation of arginine-derivative surfactants using different in vitro endpoints as alternatives to the in vivo assays, Toxicol. Lett. 164, 259-267. Mitjans, M., Martinez V., Clapés P., Pérez L., Infante M.R., Vinardell M.P. (2003), Low potential ocular irritation of arginine-based gemini surfactants and their mixtures with nonionic and zwitterionic surfactants. Pharmaceutical Research. 20, 1697-1701. Moran, C., Clapés P., Comelles F., García T., Pérez L., Vinardell M.P. Mitjans M., Infante M.R. (2001). Chemical structure/property relationship in single-chain arginine surfactants. Langmuir, 17, 5071-5075. Moran, M.C., Pinazo A., Pérez L., Clapés P., Angelet M., García T., Vinardell M.P., Infante M.P. (2004). "Green" amino acid-based surfactants. Green Chemistry, 6, 233-240. Nagao, A. and Kito M. (1989). Synthesis of O-Acyl-L-Homoserine by Lipase. Journal of the American Oil Chemists Society, 66, 710-713. Nnanna IA; Xia J., (2001). Protein based surfactants: synthesis, physicochemical properties and applications, Marcel Dekker, New York. Okamoto Y, Ohkoshi K, Itagaki H, Tsuda T, Kakishima H, Ogawa T, et al. (1999). Interlaboratory validation of the in vitro eye irritation tests for cosmetic ingredients. (3) Evaluation of the haemolysis test. Toxicol In Vitro, 13:115–124. Pape W.J.W., Pfannenbecker U., Hoppe U. (1987). Validation of the red blood cell test system as in vivo assay for the rapid screening of irritation potential of surfactants. Mol. Toxicol., 1, 525-536.

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Perez, L., Torres J.L., Manresa A., Solans C., Infante M.R. (1996). Synthesis, aggregation, and biological properties of a new class of gemini cationic amphiphilic compounds from arginine, bis(Args). Langmuir, 12, 5296-5301. Perez L., Aurora .Pinazo, Milton.J.Rosen, Mª Rosa Infante (1998). Surface activity properties at equilibrium of Novel gemini cationic amphiphilic compounds from arginine, bis(Args)..Langmuir, 14, 2307- 2315. Perez L., Pinazo A., Vinardell P., Clapés P., Angelet M., Infante M.R., (2002,a), Synthesis and biological properties of dicationic arginine-diglycerides, New Journal of Chemistry, 2002a, 26, 1221-1227. Perez, L., García M.T., Ribosa I., Vinardell M.P. Manresa A., M.R. Infante (2002, b). Biological properties of arginine-based gemini cationic surfactants. Environmental Toxicology and Chemistry, 21, 1279-1285. Perez, L., Infante M.R., Pons R., Morán C., Vinardell M.P., Mitjans M., Pinazo A., (2004 a). A synthetic alternative to natural lecithins with antimicrobial properties. Colloids Surf. B,. 35, 235-242. Perez L., Infante M. R., Angelet M.,Clapes P., Pinazo A. (2004 b). Glycerolipid argininebased surfactants: synthesis and surface active properties, in Trends in Colloid and Interface Science XVI, M.G. Miguel and H.D. Burrows, Editors., Springer-Verlag Berlin: Berlin. 210-216. Perez L., Pinazo A., García M.T., Morán M.C., Infante M.R. (2004 c). Monoglyceride surfactants from arginine: synthesis and biological properties. New Journal of Chemistry, 28, 1326-1334. Perez, L., Pinazo A., Infante M.R., Pons R. (2007). Investigation of the micellization process of single and gemini surfactants from arginine by SAXS, NMR self-diffusion, and light scattering. Journal of Physical Chemistry B, 111, 11379-11387. Piera E, Comelles F, Erra P, Infante MR. (1998). New alquil amide type cationic surfactants from arginine: Journal of the Chemical Society-Perkin Transactions II, 2, 335-342. Pinazo, A., Wen XY., Pérez L., Infante M.R., Franses E.I. (1999). Aggregation behavior in water of monomeric and gemini cationic surfactants derived from arginine. Langmuir, 15, 3134-3142. Pinazo, A., Perez L., Infante M.R., Pons R. (2004), Unconventional vesicle-to-ribbon transition behaviour of diacyl glycerol amino acid based surfactants in extremely diluted systems induced by pH-concentration effects. Phys. Chem. Chem. Phys., 6, 1475-1481 Riddell R.J., Panacer D.S., Wilde, S.M., Clothier, R.H., Balls, M. (1998). The importance of exposure period and cell type in in vitro cytotoxicity test. ATLA Altern. Lab. Anim. 14, 86-92. Rosen, M. J. (1993) Geminis: a new generation of surfactants. Chemtech 23, 30-33. Rosen, M.M. Surfactants and interfacial phenomena. Third edition. Wiley Science, 2004, 6264. Seguer J., Infante M.R., Allouch M., Mansuy L., Selve C., Vinardell P. (1994) Synthesis and evaluation of non-ionic amphiphilic compounds from amino acids: molecular mimics of lecithins. New J. Chem., 18, 765-774. Shalel S, Streichman S, Marmur A. (2003). The use of hemolysis kinetics to evaluate erythrocyte-bound surfactant, Colloids and Surfaces B: Biointerfaces, 27, 215-222. Shalel, S., Streichman S., Marmur A.,(2002, a). Monitoring surfactant-induced hemolysis by surface tension measurement. Journal of Colloid and Interface Science, 255, 265-269.

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Shalel, S., Streichman S., Marmur A., (2002, b). The mechanism of hemolysis by surfactants: Effect of solution composition. Journal of Colloid and Interface Science, 252, 66-76. Strickland, J.A. et al. 2003. Desing of a validation study to evaluate in vitro cytotoxicity assays for predicting rodent and human acute systemic toxicity. Toxicologist, 72, 157. Tausk, R.J.M., Karmiggelt J., Oudshoorn C., Overbeek J.T.G. (1974) Physical chemical studies of short chain lecithin homologues. I. Influence of the chain length of the fatty acid ester and of electrolytes on the critical micelle concentration. Biophysical Chemistry, 1, 175-183. Valivety, R., Jauregi P., Gill I., Vulfson E. (1997). Chemo-Enzymatic Synthesis of Amino Acid-Based Surfactants. JAOCS, 74, 879-886. Van de Sandt, J., Roguet, R., Cohen, C., Esdaile, D., Panec, M., Corsini, E., Parker, C., Fusenig, N., Liebsch, M., Benford, D., de Brugerolle de Fraissinette, A., Forstach, M., 1999. The use of human keratinocytes and human skin models for predicting skin irritation. ATLA Altern Lab. Anim. 27, 723–743. Vinardell, M.P., Benavides T., Mitjans M., Infante M.R., Clapés P., Clothier R. (2008). Comparative evaluation of cytotoxicity and phototoxicity of mono and diacylglycerol amino acid-based surfactants. Food and Chemical Toxicology, 46,. 3837-3841. Vinardell, M.P., Molinero J., Parra J.L. Infante M.R. (1990). Comparative Ocular Test of Lipopeptidic Surfactants. International Journal of Cosmetic Science, 12, 13-20. Vives M.A., Infante M.R., García E., Selve C., Maugras M., Vinardell M.P. (1999) Erythrocyte hemolysis and shape changes induced by new lysine-derivative surfactants. Chemico-Biol Interact. 118, 1-18. Vonderhagen, A; Raths H-C; Eilers E (1999). German Offen. DE 19749555 A1, Henkel K.G.a.A., Germany, 12 May 1999, p.4. Wang, X-L.; Ramusovic, S.; Nguyen, T.; Lu, Z-R. (2007). Novel polymerizable surfactants with pH-sensitive amphiphilicity and cell membrane disruption for efficient siRNA delivery. Biocomjugate Chem. 18, 2169-2177, 2007. Wihelm, K.P., Böttjer, B., Siegers, C.P. (2001). Quantitative assessment of primary skin irritants in vitro in a cytotoxic model: comparison with in vivo human irritation tests. Br. J. Dermatol. 145, 709–715. Xia, J; Xia, Y; Nnanna, IA (1995). Structure function relationship of acyl amino acid surfactants: surface activity and antimicrobial properties. J Agric Food Chem. 43, 867871. Yamanaka, T., Ogihara N., Ohhori T., Hayashi H., Muramatsu T. (1997). Surface chemical properties of homologs and analogs of lysophosphatidylcholine and lysophosphatidylethanolamine in water. Chemistry and physics of lipids, 90, 97-107.

In: Arginine Amino Acid Editor: Nathan L. Jacobs

ISBN 978-1-61761-981-6 © 2011 Nova Science Publishers, Inc.

Chapter 7

Arginine: Physico-Chemical Properties, Interactions with Ion-Exchange Membranes, Recovery and Concentration by Electrodialysis T. Eliseeva*, E. Krisilova, G. Oros and V. Selemenev Voronezh State University, Universitetskaya pl., 1, Voronezh 394006, Russia

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Key words: arginine, cation-exchange membranes, bipolar membranes, sorption, hydration, IR-spectrum, electrodialysis.

1. Introduction L-Arginine (2-amino-5-guanidinpentanoic acid) is a product of great importance in medicine, food and pharmaceutical industry. L-Arginine is a basic, genetically coded α-amino acid, one of the twenty most common natural amino acids. It is essential for human ("semiessential") [1-3]. Arginine plays a significant role in cell division, healing of wounds, removing ammonia from a body, immune function, and release of hormones [2, 4-5]. This amino acid is used to treat cardiovascular disorders as a precursor for the synthesis of nitric oxide (NO) [6] such as heart failure, intermittent claudication, impotence, sexual disfunction, and interstitial cystitis [7]. The benefits and functions attributed to oral supplementation of L-arginine include: 

Reduces healing time of injuries (particularly, bones)[4-5]

* Tel.: +74732208932; Fax: +74732208755; E-mail: [email protected], [email protected]

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Quickens repair time of damaged tissue [4-5] Helps decrease blood pressure [8-9].

Arginine is the most basic amino acid (pI=10.76). Side chain of arginine consists of a 3carbon aliphatic straight chain, the distal end of which is capped by a complex guanidinium group. Guanidinium group (pKa 12.48) is positively charged in neutral, acidic and even most basic environments, and thus imparts basic chemical properties to arginine. Because of the conjugation between the double bond and the nitrogen lone pairs, the positive charge is delocalized, enabling the formation of multiple H-bonds. Charge and double-bond in guanidinium group of L-Arginine are delocalized, as shown in Table 1 [1]. Some physicochemical characteristics of arginine [10] are also presented in Table 1. Table 1. Physico-chemical characteristics of L-arginine Characteristics

Value

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Structure

М, g/mol pI pK1(-COOH) pK2(-NH2) pK3(guanidinium group) []20D ( 3.5% aq. solution) []20D (8 g. in 100 g. 6M HCl) Solubility, g in 100 g. Н2О at 298 К

174.21 10.76 2.18 9.09 13.20 +12.5 +22.0 15.0

As other amino acids, arginine is a typical ampholyte and exists in a solution in different ionic forms: cations, bipolar ions and anions. At the pH value, equal to isoelectric point, amino acids exist preferably in the form of bipolar ions in individual solution and even in solid state. In acidic solution bipolar ions recharge into cations:

Arginine: Physico-Chemical Properties, Interactions, Recovery… +

H3N-RCH-COO- + H3O+  bipolar ion

+

H3N-RCH-COOH + H2O cation

145 (1)

At high pH value they can exist in negatively charged form: +

H3N-RCH-COO- + OH-  NH2-RCH-COO- + H2O bipolar ion anion

(2)

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Distribution diagram for arginine amino acid ionic forms is shown in Figure 1.

Figure 1. Amount fraction of various arginine ions as pH function.

This diagram allows to determine what forms of amino acid exist at a given pH value and to predict their sorption and transport through the ion-exchange membranes. The aim of this article is to consider the processes of sorption, hydration and mass transfer in the system arginine-ion-exchange membrane-water as well as to show the possibilities of electrodialysis in separation of basic amino acid and concentration of its solutions.

2. Experimental The objects of this study are an amino acid arginine (2-amino-5-guanidinpentanoic acid) and heterogeneous sulfopolysterene cation-exchange membrane MK-40 (produced by “Shchekinoazot”, Russia), perfluorinated sulfocation-exchange membrane MF-4SK (produced by “Plastpolymer”, Russia) - analogue of Nafion 117 and bipolar membranes MB3 (“Shchekinoazot”, Russia). The solutions of amino acid (produced by Ajinomoto) are analyzed by the method of photometry based on cooper complexes formation [11].

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Amino acid sorption by membranes is studied in the static conditions. Water sorption isotherms are obtained by isopiestic method. IR-spectra of membrane samples are obtained using Bruker IR-spectrometer. The electrodialysis experiments are carried out in a laboratory multi-compartment cell with alternating bipolar and cation-exchange membranes. The scheme of the multicompartment cell fragment is shown in Figure 2. The experiments are carried out in a constant current regime. Every compartment has individual input and output for a solution. The height of the compartments is 20 cm. The membrane effective area is 20 cm2. Concentrate compartments has no flow through. In order to prevent amino acids transformations in the electrode compartments they are fed by sodium sulphate.

Figure 2. The fragment of the cell with bipolar and cation-exchange membranes for amino acid solution concentration.

3. Results and Discussion 3.1. Arginine Sorption by Cation-Exchange Membranes Studies of amino acids‟ sorption are very important for the description of mass transfer processes in membranes. They are essential for the development of techniques for the separation and demineralization of amino acids by membrane methods and allow us to improve theoretical concepts of the nature and mechanism of amino acids transport in synthetic and biological membranes. Knowledge of sorption peculiarities is necessary for a mathematical description of transport processes in ion-exchange membranes [12-13]. Sorption of amino acids by granulated ion exchange resins has been studied fairly thoroughly [14-15]. At the same time, data on the sorption of amino acids by ion-exchange membranes are limited [16-17].

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In the present work we determine the influence of membrane structure on the sorption of arginine by cation-exchange membranes. We have obtained the isotherms of equilibrium sorption of this amino acid by the cation-exchange membranes MK-40 and MF-4SK. The distribution coefficients of arginine have been calculated. Both membranes have fixed groups of the same nature (sulfo-groups), the difference between them is in the structure of polymeric matrix. The isotherms of arginine amino acid sorption by MK-40 and MF-4SK cation-exchange membranes are shown in Figure 3.

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Figure 3. The isotherms of arginine sorption by MK-40 and MF-4SK cation-exchange membranes.

The maximum capacity per gram of absolutely dry membrane is 3.40 mmol/g for MK-40 and 3.03 mmol/g for MF-4SK. For both membranes maximum capacity values are close to each other, but form of isotherms is different, so one can propose different sorption mechanism. For heterogeneous sulfopolysterene cation-exchange membrane MK-40 curve reaches saturation as the equilibrium concentration increases. For homogeneous perfluorinated sulfocation-exchange membrane MF-4SK (analogue of Nafion 117) isotherm is S-shaped. At low concentrations of equilibrium solution the curve is convex, that confirms the preferable interactions of bipolar ions and cations of amino acid with functional groups of the membrane. After reaching the plateau ( capacity values 0.3-0.5 mmol/g) at concentrations of equilibrium solution being higher than 0.05 mol/dm3 curve goes up and the quantity of sorbed amino acid becomes greater than the total exchange capacity (0.9 mmol/g ). The concave shape of the curve shows the preferable interactions of sorbed ions among themselves. The analysis of the isotherm makes it possible to draw two important conclusions. First – the quantity of sorbed amino acid that corresponds to the isotherm plateau is lower than the total exchange capacity of membrane (sorption of H+ ions). Second – complex form of the sorption curve can not be described by the Langmuir isotherm and it permits to suppose super-equilibrium uptake of arginine. The first peculiarity can be explained by the influence of steric hindrance factor – ion exchange and protolysis reaction are hindered for large organic ions. From the other hand,

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complex structure of sorbate ions gives the possibility of additional (not ion exchange) interactions sorbate-sorbate and sorbate-matrix. Note, that the maximal amount of sorbed amino acid is higher than exchange capacity for Н+-ions (2.5 mmol/g and 0.9 mmol/g for MK-40 and MF-4SK, respectively), so arginine shows super-equivalent sorption by membranes studied. Excess of sorbed arginine amount related to exchange capacity is presented in Figure 4. From the analysis of Figure 4 one can conclude, that interactions other than ion exchange are more significant in sorption of arginine by the membrane MF-4SK. For the sorption of organic ions such as amino acids, the size of penetrating particles, interactions between the solute, membrane polymeric matrix, and solvent as well as the formation of sorbate–sorbate associates are of great importance along with ion exchange and protolysis. Arginine molecules can form chains with H-bonds between guanidine group nitrogen atoms and carboxyl oxygen atoms [18], so they participate in sorbate–sorbate interactions, that can be a possible reason for exceeding the exchange capacity.

Figure 4. Relation of sorbed arginine amount to exchange capacity of membranes.

The distribution coefficient analysis leads to similar conclusions. The distribution coefficients in the system amino acid solution – cation-exchange membrane are shown in Figure 5. We observe that distribution coefficients decrease as the equilibrium concentration increases because of the saturation of membrane MK-40 active centers. The distribution coefficient increases to some extent for MF-4SK at high concentrations because of sorbate– sorbate and sorbate–matrix hydrophobic interactions. Sorption of amino acids can be treated as the transfer of molecules from one phase to another [19-20]. The dependences of interphase surface tension on the degree of filling of the membrane phase with amino acid ions are shown in Figure 6. The curves for two types of membranes have a different shape. For MF-4SK, surface tension increases sharply even at a small degree of membrane filling. Maximum corresponds to formation of sorbed amino acid monolayer. At x > 0.5, the σxi value remains almost constant, although the concentration of the amino acid in the membrane phase continues to increase. This leads us to suggest the

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formation of compact associates with a minimum contact area with water because of hydrophobic binding [20]. For MK-40, an increase in surface tension is observed only at x > 0.6, likely because of the formation of sorbed amino acid chains.

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Figure 5. Logarithm of distribution coefficient in the system amino acid solution – cation-exchange membrane as a function of equilibrium solution concentration.

Figure 6. The dependences of interphase surface tension on the degree of filling of the membrane phase with arginine ions.

The comparison of two studied membranes shows that the contribution of not-ionexchange interactions is higher for the perfluorinated cation-exchange membrane because of its lower exchange capacity, lower humidity and higher flexibility of matrix polymer chains. On the basis of the obtained difference in the mechanism of sorption it is possible to suppose that the transport of arginine through the membrane MF-4SK under the influence of electric field is hindered in comparison with the membrane MK-40. However, water transport through the membranes that is determined by the hydration of the membranes has also the essential

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influence on the electrodialysis process efficiency. Therefore the next chapter deals with the study of hydration of the membranes MK-40 and MF-4SK in H+ and arginine forms.

3.2. Hydration of Membranes, Saturated with Arginine

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The content and state of water in ion-exchange membranes substantially influence their physicochemical properties and transport characteristics [21-23]. The nature of the polymeric backbone and the type of functional groups and counterions in turn determine the hydration of membranes [23]. In this work the hydration of heterogeneous and homogeneous cationexchange membranes in the arginine and H+- forms is estimated. The studied membranes have identical functional groups, however, distinction in the nature and structure of polymer matrix has an influence on the state and quantity of water in membrane phase and, as consequence, on electrodialysis concentration parameters. The hydration of cation -exchange membranes after sorption of basic amino acid is estimated by the isopiestic method. The isotherms of water sorption by MK-40 and MF-4SK cationexchange membranes in arginine and H+ -forms are shown in Figure 7. The dependences obtained are S-shaped. The first steep region corresponds to the formation of a monolayer of sorbed water and characterizes the hydration of fixed ion exchanger groups on the basis of which the membrane is formed and counterions. The second flatter region corresponds to less strongly bound water. The third region with a steep rise of the curve is related to the absorption of “free” water. The absorption of the solvent at p/p0 > 0.650 is largely caused by osmotic forces. The presence of bipolar amino acid ions limits the sorption of water and changes the amount (Q) and state of water in the membrane phase. It follows from our data that saturation of ion-exchange membranes with arginine causes decrease of water uptake, thus the share of the bonded water increases.

Figure 7. Water sorption isotherms of cation-exchange membranes MF-4SK and МК-40 in H+ and arginine forms.

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At comparison of homogeneous and heterogeneous membranes the largest difference is observed in the region of low water activities, corresponding to hydration of fixed groups and counterions, the heterogeneous membrane keeps in the both forms more strongly connected water. Heterogeneous membrane МК-40 has higher value of total exchange capacity of (that means - more sulfo-groups) – 2.5 mmol/g of absolutely dry membrane, than MF-4SK membrane, which capacity is equal to 0.9 mol/g. Note that arginine sorption influences on hydration of perfluorinated membrane more strongly, because fluorocarbon chains are more flexible than cross-linked polystyrene matrix. From the obtained water sorption isotherms Gibbs free energy change is calculated for membrane hydration by the method described in the monograph [24]. Hydration free energy for the cation-exchange membranes MF-4SK and МК-40 in H+ and arginine forms is shown in Figure 8 as a function of water amount per membrane functional group (n).

Figure 8. Change of Gibbs free energy at water molecules sorption by cation-exchange membranes MF-4SK and МК-40 in H+ and arginine forms.

The values obtained are in correspondence with data of the literature [25] for the membrane Nafion 117 (analogue of the membrane MF-4SK) in forms of mineral ions. The comparison of swelling potential for two investigated membranes shows that heterogeneous membrane МК-40 interacts with water more strongly, than homogeneous membrane MF4SK, both in hydrogen, and in arginine form. Hence, the hydrophobic matrix of perfluorinated membrane influences on MF-4SK hydration essentially. Hydration of homogeneous perfluorinated МF-4SК and heterogeneous МК-40 sulfocation-exchange membranes in H+ and arginine forms is studied by IR-spectroscopy method. The energy of hydrogen bonds between water molecules in ion-exchange membranes is calculated on the basis of bands frequency shift in the IR-spectrum. The existence of structure and energy heterogeneity of water associates in studied membranes is shown. IR-spectra of membrane МF-4SК in arginine and Н+-form are shown in the Figure 9.

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(A)

(B) Figure 9. IR-spectra of membrane МF-4SК in arginine and H+-forms (1 – H+-form, 2 – Arg-form). a) 5001800 cm-1, b) 2500-3800 cm-1.

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The vibrational spectrum of membrane МF-4SК contains set of bands of the absorption characterising various groups of atoms. Minima 625, 717, 970, 982, 1200, 1375, 1414, 1456 and 2924 cm-1 are related to fluorocarbon chains of backbone. Peaks at 1057, 1144 (symmetric stretching) and 1312, 1445 (asymmetric stretching) refer to membrane sulfogroups. Presence of C-S bond of sulfo-group, connected with a backbone, gives minima at 567 and 625 cm-1 [26]. Saturation of membrane in arginine solution changes the spectrum of membrane in H+form (Figure 9). IR-spectrum of the membrane in arginine form contains bands at 1634 cm-1 и 1533 cm-1 – deformation vibrations of NH3+-group of amino acid (I and II amino acids bands) [26]. Stretching vibrations of -NH3+ и -C=NH of guanidinium-group of arginine appear as the minima of transmission at 3366, 3294 и 3227 см-1. Vibrational spectra make it possible not only to reveal the bands of absorption corresponding to matrix, functional groups and counter-ions of membrane but also to establish the existence and force of hydrogen bonding. Deformation vibrations of water molecules appear at 1600-1750 cm-1 and stretching vibrations of –О–Н bonds – at 3000-4000 cm-1. Appearance of distortions in IR-spectrum (shift of frequency and broadening of spectral band, intensity change) accompanying hydrogen bonds formation can be a criterion of their formation and enables to estimate the energy of bonding. For the assessment of hydration properties of membrane gel phase on can use the bands of stretching vibrations of OH-groups at the region 3700-3000 cm-1 which are the most sensitive to hydrogen bonds modification. In the spectrum of H+-form of perfluorinated sulfo-cation-exchange membrane one can observe the shoulder at 3765 cm-1 with low intensity which can be referred to non-associated water molecules and the broad band at 3526-3250 cm-1 characterising the absorption of water associates with various energy of hydrogen bonds. According to G. Zundel [27-28] a continuous absorption in this region appears in the case of existence of two water molecules per one proton, i.e. in the case of Н5О2+ formation. It can be confirmed by the appearance of the band at 1740 cm-1. Spectrum of MF-4SK membrane in arginine form differs from the spectrum of H+-form. The shoulder shifts to the region of lower wave numbers (3666 cm-1), and instead of one broad band one can find several bands: shoulders at 3416 cm-1 and at 3070 cm-1(vibrations of associates water-water) and minima 3368, 3298 и 3227 cm-1 (stretching vibrations of NH3+ и -C=NH). According to the literature data [29-30] IR-spectra of perfluorinated ion-exchange membrane contain minima corresponding to associates water-water being close to C-F groups of polymer matrix or hydrocarbon fragment of amino acid (3514 cm-1 in Н+-form and 3535, 3618 cm-1 in amino acid form). Absorption at 3408, 3416 cm-1 and also at 3040-3070 cm-1 characterises the associates including water molecules, sulfo-groups and ether groups of matrix C-O-C [Ostrowska J.]. In the spectrum of H+-form one can observe the vibrations of associates Н2О…SO3- (3288 cm-1), in arginine form this peak overlaps with the band of amino-group stretching vibrations: 3294 cm-1. Figure 10 shows IR-spectra of MK-40 membrane in arginine and Н+-forms.

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(A)

(B) Figure 10. IR-spectra of membrane МK-40 in arginine and H+-forms (1 – H+-form, 2 – Arg-form). a) 5001800 cm-1, b) 2500-4000 cm-1.

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The dominant minima of transmission in the spectra of MK-40 membrane are minima at 2922, 2851 cm-1, corresponding to asymmetric and symmetric vibrations of -СН and -СН2 groups. Minima at 1005, 1032, 1123, 1155 cm-1 deal with stretching vibrations of –SO3 groups, minima at 1472, 1462 см-1 correspond to deformation vibrations of СН и СН2 groups. Peaks at 773 and 831 cm-1 confirm the appearance of not flat deformation vibrations of -CH in substituted benzene ring [26]. The vibrations of C-S bond of sulfo-group connected with benzene ring are confirmed by the minima at 579 и 731 cm-1. The deformation vibrations of C-H of benzene ring one can observe at 667 cm-1. IR-spectrum of the membrane in arginine form contains bands at 1628 cm-1 и 1514 cm-1 – deformation vibrations of NH3+ in arginine ( I and II amino acids bands) [26]. Also it is possible to reveal the appearance of the minimum at 1323 см-1 – symmetric vibrations of –СОО- groups. In IR-spectrum of MK-40 for the region 3000-4000 cm-1 shoulder is found at 3686 cm-1 (it shifts to 3560 cm-1 for amino acid form indicating the formation of more strong associates) and the wide band including several peaks is situated in the region 3470-3250 cm-1. For the membrane in arginine form one can observe also bands corresponding to the stretching vibrations of guanidinium fragment and amino group: 3331, 3292, 3169 cm-1. Table 2. Parameters of hydrogen bonds for the membranes MK-40 and MF-4SK in H+ and arginine forms МК-40 Membrane form

_



Ен, kJ/mol

_

ОН

cm-1

,

Δ ОН

MF-4SK Membrane form

_



cm-1

,

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-1

Н+

Аrg

3560 3472 3412 3294 3256 3057 3030 3649 3618 3562 3398 3285 3059 3038

cm 140 228 288 406 444 643 670 51 82 138 302 415 641 662

Ен, kJ/mol

_

ОН

,

Δ ОН

, -1

9.9 16.1 20.4 28.7 31.4 45.5 47.4 3.6 5.8 9.8 21.4 29.4 45.3 46.8

Н+

Аrg

3526 3408 3333 3288 3090

cm 174 292 367 412 610

12.3 20.7 26.0 29.1 43.1

3560 3416 3070

140 284 630

9.9 20.1 44.6

The energy of hydrogen bonds (Ен) for H+- and arginine forms of the membranes have been calculated on the basis of the frequency shift value related to the frequency of notassociated OH-group band (3700 cm-1) [31]. These data are presented in the Table 2. The analysis of hydrogen bonds parameters (Ен) leads to the conclusion that water in the phase of cation-exchange membranes saturated in arginine solution forms associates of various

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structure. In the phase of heterogeneous cation-exchange membrane MK-40 along with the associates Н2О…SO3-, Н2О…СОО-, Н2О…Н2О, Н2О…NH3+ there exist water molecules with distorted, weakened and partially destroyed hydrogen bonds (Ен is lower than the following value for liquid water). According to Ен values for MF-4SK membrane (H+-form) it is possible to conclude about the presence of water molecules with destroyed hydrogen bonds, of associates waterwater in various environment (Ен=20.7-26.0 kJ/mol) and of water molecules participating in hydrogen bonding with sulfo-group (Ен > 30 кДж/моль). For arginine form it is evident that energy of hydrogen bonding for water molecules in various states differs substantially. Water molecules with the most strong hydrogen bonding find themselves near the carboxyl-groups of arginine, intermediate values of Ен correspond to water molecules participating in the formation of water-water bonds, i.e. clusters. Molecules of water with destroyed hydrogen bonds are located close to hydrocarbon fragments of amino acid and fluorocarbon chains of membrane matrix. Presence of water with the destroyed hydrogen bonds can lead to an increase in its transport through a membrane during the electrodialysis and, consequently, to a dilution of concentrate. Thus, analysis of vibrational spectra of heterogeneous (MK-40) and homogeneous (MF4SK) membranes confirms earlier drawn conclusions about the different states of water in the phase of membranes with matrix of different chemical nature and structure. In the phase of heterogeneous membrane there exist intergel regions where water structure is the same as in pure solvent. In the phase of perfluorinated membrane variety of water states is small. This manifests itself in a decrease of absorption bands number, water molecules are preferably united into clusters. Uptake of arginine and passage from polysterene to fluorocarbon matrix induce a decrease of total water content in the membrane phase and an increase of fraction of water molecules which are characterized by high energy of hydrogen bonds. To summarize, sorption of arginine substantially affects the content and state of water in the both membranes. The results obtained can be used to optimize membrane separation, isolation, and concentration of arginine.

3.3. Recovery and Concentration of Arginine in an Electromembrane System Amphoteric nature of amino acids one can consider as a basis for their recovery and concentration in the system with bipolar and cation-exchange membrane using reaction (1) and in the system with bipolar and anion-exchange membrane using reaction (2). However, for basic amino acids extraction it is preferable to use the system containing cation-exchange membranes. Bipolar ions do not migrate in the electric field. However, during the interaction with hydrogen and hydroxyl ions they form cations and anions, accordingly, which are able to transfer under the influence of electric current. Some aspects of amino acids separation by electrodialysis with bipolar membranes have been discussed in literature [32-34]. The task of this chapter is the consideration of basic amino acid concentration peculiarities in the electromembrane system with bipolar and cation-exchange membranes.

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The recovery and concentration of arginine is studied in the electromembrane system with bipolar and various cation-exchange membranes – heterogeneous MK-40 and homogeneous MF-4SK. Bipolar membranes generate hydronium and hydroxyl ions in the course of water splitting from the side of dilute compartments.: 2 H2O  H3O+ + OH- (3)

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This process leads to a decrease of pH value in these compartments and to formation of arginine cations that are capable to migrate through a cation-exchange membrane. The distribution diagram (Figure 1) indicates that acidic and even neutral media permit to increase the amount fraction of arginine cations. Cations flux provides the possibility to concentrate amino acids in the adjacent compartments which have no flow through. For the estimation of process efficiency concentration factor Fc=C/C0 is used (C – concentration in concentrate solution, C0 – concentration in feed solution). The dependence of Fc on the current density (i) is shown in the Figure 11.

Figure 11. The dependence of arginine concentration factor on the current density in the system with bipolar membranes MB-3 and heterogeneous membranes MK-40 (1) or with bipolar membranes MB-3 and homogeneous membranes MF-4SK (2) (C0 = 0.01M).

As it follows from the Figure 11 concentration factor increases with current and reaches Fc=27-35 at values of current density i≈ 8-10 mA/cm2. The further growth of current density leads to increase of electrical resistance in the system and warming up of the solution because of Joule heat release. The comparison of two studied membranes shows that in intensive current regime usage of heterogeneous membrane MK-40 allows to reach deeper concentration of amino acid solution, than usage of homogeneous membrane MF-4SK.

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The dependence of concentration factor Fc on feed arginine solution concentration is presented in the next figure. Process was carried out at the current density corresponding to the maximum obtained value of Fc for the definite concentration.

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Figure 12. The dependence of concentration factor on feed solution concentration: 1 – MK-40, 2 – MF-4SK.

Utilization of heterogeneous membrane MK-40 is preferable in comparison with homogeneous MF-4SK. However, at low values of initial concentrations (lgC0= -4) concentration factor (Fc) decreases and one can not observe any difference between membranes. According to [35], electrodialysis concentration of electrolytes is limited basically by electroosmotic transport of water with migrating ions. So we have carried out the measurements of electroosmotic transport of water through the membranes MK-40 and MF4SK. The measurements of electroosmotic water transport have been carried out in the fourthcompartment cell, the electrode compartments were separated from the adjacent compartments by bipolar membranes. The volume of liquid transferred through the studied membrane has been registered by the measurement of the level rise in the capillary connected hermetically with the concentrate compartment. Concentrate compartment had no flow through. The results of measurements permit to calculate electroosmotic permeability of studied membranes, D, cm3/C, according to the following equation:

D

V , StI

(4)

where V – volume of liquid, passed through the membrane, cm3; S – membrane surface, cm2, t – time, s; I - current, A. Figure13 shows the dependence of electroosmotic permeability of the membranes MK-40 and MF-4SK on the current density at initial concentration of arginine solution 0.01M.

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The electroosmotic permeability of the both membranes practically does not depend upon current. The electroosmotic permeability of the membrane MK-40 is higher than of the membrane MF-4SK. The difference in water transport for the studied membranes is explained by the difference of their structure and physico-chemical properties.

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Figure 13. The dependence of electroosmotic permeability of membranes MK-40 and MF-4SK on current density.

The membrane MK-40 contains various pores (1.5-100 nm) in the structure as well as channels with radius up to 1000 nm [36-37]. Size of homogeneous membranes MF-4SK transport channels is 3.5-4.0 nm [12]. Therefore, water is mainly transferred through the membrane MF-4SK in primary hydration shells of migrating ions, for MK-40 membrane along with such mechanism water can transfer by the mechanism of convection. However, electrodialysis concentration efficiency in the case of relatively large organic ion such as arginine, can be limited not only by electroosmotic water transport, but also by the steric factor. Migration of amino acids cations (the size without hydration shell ca. 0.6 nm [18]) through the membrane MK-40 does not have steric hindrance. At the same time, homogeneous membranes MF-4SK have more dense structure that can lead to difficulties of amino acid cations transport and, hence, lower concentration efficiency. That is why the arginine flux through the membrane MK-40 is greater than through the membrane MF-4SK (Figure 14). For the both membranes one can observe some decrease of arginine flux after reaching the limiting current density – barrier effect, then in the intensive current regime amino acid transport increases again – effect of facilitated migration. These effects have been described by us earlier for neutral amino acids [34]. The use of the membrane MK-40 which is characterized by greater total exchange capacity, by broader transport channels and lower influence of additional interactions with arginine makes it possible to provide more effective concentration of this basic amino acid in comparison with the membrane MF-4SK. The value of concentration factor increases also with a decrease of membrane hydration leading to lower water flux through the membrane with arginine cations.

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Figure 14. The dependence of arginine flux on the current density during electrodialysis of 0.01M solution.

Another task of this study is consideration of basic amino acid concentration in the course of its salts conversion into free amino acid with simultaneous concentration. Such task appears at the last stage of amino acid chemical synthesis. Chemical synthesis allows to produce only racemate. So it is necessary to recover L-form. Separation of racemate is carried out taking into account different solubility of D- and L-forms salts which are formed in the reaction of racemate with tartaric acids. At the final stage of the process it is necessary to convert salt into basic amino acid. This task has been solved by the ion-exchange method that requires the solutions of acids and bases for regeneration. The suggested procedure using electrodialysis with bipolar membranes is non reagent, advisable from ecological point of view and it can be used for the recovery of basic amino acid obtained in the course of chemical synthesis [38-39] Bipolar membranes generate hydrogen ions recharging basic amino acid into doubly charged and single-charged cations which transfer through the cation-exchange membrane being free of tartaric acid. Tartaric acid can be returned from even-numbered compartments to the stage of racemate separation. The solution of pure basic amino acid has been collected from all non-even numbered compartments which had no flow through in order to concentrate the product. The influence of current density on the concentration factor is shown in the Table 3 for electrodialysis of arginine tartrates solutions with alternating MK-40/MB-3 membranes. Table 3. The dependence of concentration factor on the current density during the electrodialysis of arginine tartrates solutions i, mA/cm2 2 4 6 8 10

Concentration Factor 22 32 38 42 44

Arginine: Physico-Chemical Properties, Interactions, Recovery…

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Electrodialysis with bipolar membranes permits to increase concentration of basic amino acid 38-44 times at room temperature without any danger of destruction. For arginine we can reach the concentration 1.25 mol/l.

Conclusion

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The processes of sorption, hydration and mass transfer in the system arginine-ionexchange membrane-water are considered. Sorption of arginine by homogeneous and heterogeneous cation-exchange membranes MK-40 and MF-4SK is studied in static conditions. The conclusion about important role of hydrophobic interactions in amino acid uptake is drawn on the basis of sorption isotherms and distribution coefficients analysis. The dependences of interphase surface tension on the degree of filling of the membrane phase with amino acid are obtained. The formation of arginine associates in the phase of membrane is revealed. The hydration properties of the membranes are studied by the isopiestic method and by the method of IR-spectroscopy. Uptake of arginine and passage from polysterene to fluorocarbon matrix induce a decrease of total water content in the membrane phase and an increase of fraction of water molecules which are characterized by high energy of hydrogen bonds. The possibilities of electrodialysis in separation of basic amino acid and concentration of its solutions are shown. The use of the membrane MK-40 which is characterized by greater total exchange capacity, by broader transport channels and lower influence of additional interactions with arginine makes it possible to provide more effective concentration of this basic amino acid in comparison with the membrane MF-4SK. The value of concentration factor increases with a decrease of membrane phase hydration that leads to lower flux of water through the membrane with arginine cations being concentrated.

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References

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Murray, R. K.; Granner, D. K.; Mayes, P. A.; Rodwell V.W. Harper’s Biochemistry. Part 1 . Appleton&Lange : Norwalk/San Mateo, 1988. 381 p. Tapiero, H.; et al. Biomedicine and Pharmacotherapy. 2002, 56, 439–445.

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Wu, G.; et al. J. Nutr. Biochem. 2004, 15, 332–451. Stechmiller, J.K.; et al. Nutrition in Clinical Practice. 2005, 20, 52–61. Witte, M.B.; Barbul, A. Wound Repair and Regeneration. 2003, 11, 419–423. Andrew, P.J.; Myer, B. Cardiovascular Research. 1999, 43, 521–531. http://www.smart-publications.com/sexual_health/l-arginine.php.

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[8] Gokce, N. J. Nutr. 2004, 134, 2807S-2811S. [9] Rajapakse, N.W.; et al. Hypertension. 2008, 52, 1084–1090. [10] Dawson, R.; Elliott, D.; Elliott, W.; Jones, K. Data for Biochemical Research. Claredon Press: Oxford, 1986. 543 p. [11] Roshal E.R. et al. Russian Chem.-pharm. J. 1988, 6, 30. [12] Timashev, S. F. Physicochemistry of Membrane Processes. Khimiya: Moscow, 1988. 240 p. [in Russian]. [13] Zabolotskii, V. I.; Nikonenko, V. V. Ion Transfer in Membranes.Nauka: Moscow, 1996 [in Russian]. [14] G. S. Libinson, Sorbtion of Organic Compounds by Ionits. Meditsina: Moscow, 1979. [in Russian]. [15] Selemenev, V.F. et. al. Interionic and intermolecular Interactions in Ion-Exchange and Sorption Systems involving physiologically active Sustances / in “Ion Exchange” / ed. by D. Muraviov, V. Gorshkov, A. Warshawsky. Marcel Dekker: NY, 2000. 615-689. [16] Kikuchi, K. et al. J. Chem. Eng. Jap. 1994, 21, 391-398. [17] Gotoh, T.; Kikuchi, K. Bioseparation. 2000, 9, 37-41. [18] Gurskaya, G. V. Structures of Aminoacids.Nauka: Moscow, 1966. 157 p. [in Russian]. [19] Hermann, R. B. J. Phys. Chem., 1972, 76 (19), 2754–2759. [20] Krisilova, E.V.; et al. Rus. J. Phys. Chem. A. 2009, 83 (10), 1763–1767. [21] Mauritz, K.A.; Moore, R. B. Chem. Rev. 2004, 104 (10), 4535-4585. [22] Wu, D.; Paddison, S. J.; Elliott, J. A. Energy Environ. Sci. 2008, 1 (2), 284–293. [23] Rowland, S.P., Ed. Water in Polymers. ACS Symposium Series 127: Washington, 1980. 532 p. [24] Helfferich, F. Ionenaustauscher. Weinheim: Verlag Chemie GMBH, 1959. 490 pp. [25] Pushpa, К.К.; Nandan, D.; Iyer, R.M. J. Chem. Soc. Faraday Trans I. 1988, 84 (6), 2047-2056. [26] Bellamy, L. J. Infrared Spectra of Complex Molecules. John Wiley & Sons, Inc.: N.Y., 1958, 280 pp. [27] Zundel, G. J. Membr. Sci. 1982, 11, (3), 249-274; [28] Zundel, G. Angewandte Chemie Int. Edition in English. 1968, 8 (7), 499 – 509. [29] Falk M. Can. J. Chem. 1980, 58, 1495-1501. [30] Ostrowska J.; Narеbska, A. Colloid. Polym. Sci. 1984, 262, 305-310. [31] Johansen, A. V. Infrared Spectroscopy and Spectral Determination of Hydrogen Bond Energy. In: Hydrogen Bond [Russian translation], Moscow, 1981, 112-155. [32] Grib, H.; et al. J. Chem. Technol. Biotechnol. 1998, 73, 64-70. [33] Fischer, A.; Martin, Ch.; Muller, J. Pat. # 19952961, Germany (1999). [34] Shaposhnik V.A.; Eliseeva, T.V. J. Membr. Sci. 1999, 161, 223-227. [35] Zabolotsky, V.I.; Shudrenko, A.A.; Gnusin, N.P. Rus. J. Electrochem. 1988, 24, 744– 750. [36] Berezina, N.P.; Kononenko, N.A.; Dyomina, O.A.; Gnusin, N.P. Adv. Coll. Int. Sci.. 2008, 139 (1-2), 3-28. [37] Karpenko-Jereb, L.V.; Berezina, N.P. Desalination, 2009, 245 (1-3), 587-596. [38] T.V. Eliseeva, V.A. Shaposhnik, E.V. Krisilova, A.E. Bukhovets. Desalination. 2009, 241, 86-90. [39] T.V. Eliseeva, E.V. Krisilova, V.A. Shaposhnik, A.E. Bukhovets. Desalination and Water Treatment. 2010, 14, 196-200.

In: Arginine Amino Acid Editor: Nathan L. Jacobs

ISBN 978-1-61761-981-6 © 2011 Nova Science Publishers, Inc.

Chapter 8

Central Functions of L-Arginine and its Metabolites for Stress Behavior 1

3*

Shozo Tomonaga , D. Michael Denbow2 and Mitsuhiro Furuse 1

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Laboratory of Advanced Animal and Marine Bioresources, Faculty of Agriculture, Kyushu University, Fukuoka 812-8511, Japan 2 Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0306, USA 3 Labotratory of Regulation in Metabolism and Behavior, Faculty of Agriculture, Kyushu University, Fukuoka 812-8511, Japan

Abstract L-Arginine is an essential amino acid for birds, carnivores and young mammals and a conditionally essential amino acid for adults. L-Arginine can be catabolized by four sets of enzymes in mammalian cells, resulting in the production of urea, L-ornithine, Lproline, L-glutamate, polyamines, nitric oxide, creatine, agmatine, etc.. Unlike mammals, birds lack carbamyl phosphate synthetase, one of the urea cycle enzymes necessary for the synthesis of L-citrulline from L-ornithine in the liver and kidney. Therefore, it is impossible to synthesize L-arginine in birds, and L-arginine is classified as an essential amino acid for birds. In this chapter, we introduce recent studies about central functions of L-arginine and its metabolites for stress behavior. In particular, the functions in avian species are focused upon. In neonatal chicks, centrally injected L-arginine induces sedative and hypnotic effects under social separation stress. Among L-arginine

*

Correspondence to: Mitsuhiro Furuse Laboratory of Regulation in Metabolism and Behavior Faculty of Agriculture, Kyushu University Fukuoka 812-8511, Japan Tel/Fax: (81) (92) 642-2953 E-mail: [email protected]

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Shozo Tomonaga, D. Michael Denbow and Mitsuhiro Furuse metabolites, L-ornithine, L-proline and L-glutamate would especially contribute to these effects.

1. Introduction Research on the biochemistry and physiology of L-arginine has remained an active area for scientists due to its diverse physiological functions in mammals. L-Arginine is classified as an essential amino acid for birds, carnivores and young mammals, and a conditionally essential amino acid for adults.

L-Citrulline

L-Glutamate

Creatine

COOH COOH

COOH

H2N C H

L-Proline

H2N C H

CH 2

CH 2

CH 2

H3C

CH 2

CH 2 N H

COOH

COOH

Nitric oxide . NO

CH 2 NH

N C=NH NH 2

C=O

Ornithine aminotransferase

NH 2

NO synthase Arginase

L-Arginine COOH

COOH

H2N C H

H2N C H CH 2 CH 2 CH 2

Agmatine H H2N C H

Urea

CH 2

CH 2

H 2 N C=O

CH 2

CH 2

NH 2

CH 2

CH 2

NH

NH

NH 2

Putrescine Spermidine Spermine Figure 1. L-Arginine metabolism in mammals.

=

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L-Ornithine

Arginine decarboxylase

CNH 2

C=NH 2

NH 2

NH 2

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Central Functions of L-Arginine and its Metabolites for Stress Behavior

165

As shown in Figure 1, L-arginine can be catabolized by four sets of enzymes in mammalian cells resulting ultimately in the production of urea, L-ornithine, L-proline, Lglutamate, polyamines, nitric oxide (NO), creatine, agmatine, etc. (Morris, 2004). In particular, one of the L-arginine metabolites, NO, is produced by NO synthase (NOS) (Palmer et al., 1987). NO, originally called endothelium derived relaxing factor, is a mediator of immune responses, a neurotransmitter, a cytotoxic free radical, and a widespread signaling molecule in the body. In addition, it was shown that NO modulates various behaviors. For instance, NO donors produce defensive reactions characterized by wild running and jumping (De Oliveira et al., 2001). In contrast, NO increased slow wave sleep and reduced waking during the dark phase in adult rats (Monti and Jantos, 2004). Accordingly, it is believed that NO has biphasic effects (Colasanti and Suzuki, 2000, Da Silva et al., 2000). Birds lack carbamyl phosphate synthetase, one of the urea cycle enzymes necessary for the synthesis of L-citrulline from L-ornithine in the liver and kidney (Tamir and Ratner, 1963). Therefore, birds can not synthesize L-arginine, and L-arginine is classified as an essential amino acid for birds. However, the role of L-arginine in the central nervous system (CNS) under stressful conditions has not been investigated in birds which, unlike mammals, lack a urea cycle. Therefore, in an effort to investigate a novel function of L-arginine on stress responses, the effects of L-arginine and its metabolites including L-ornithine, L-proline, Lglutamate, polyamines, NO, creatine, and agmatine, on the stress response in neonatal chicks was studied. In these studies, the social separation stress model was used. This stress model is frequently used for the study of anxiety. Chicks are comfortable when living in a group, but exhibit anxiety when isolated. Social separation stress increases spontaneous activity and vocalization of chicks (Feltenstein et al., 2003). This social separation stress paradigm has been used for developing anti-anxiety agents using vocalization and spontaneous activity as parameters. Additionally, this model has a high utility since chicks are inexpensive to purchase and maintain, and they require small quantities of drugs in the screening process (Watson et al., 1999).

2. Central Functions of L-Arginine, Nitric Oxide, Agmatine, L-Ornithine and L-Citrulline for Stress Behavior in Chicks Under social separation stress, the intracerebroventricular (i.c.v.) injection of L-arginine clearly attenuated spontaneous activity and the number of vocalizations compared with the control in as little as 5 minutes post-injection (Figure 2, Table 1; Suenaga et al., 2008a). In addition, L-arginine increased the time spent in sleeping posture. These results suggest that Larginine has sedative and hypnotic effects. In preliminary experiments, behavioral tests conducted immediately after the i.c.v. injection of L-arginine found no significant effects of L-arginine. These results suggested that a metabolite of L-arginine, rather than L-arginine itself, might participate in the central function.

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Reproduced from Suenaga, R., Tomonaga, S., Yamane, H., Kurauchi, I., Tsuneyoshi, Y., Sato, H., Denbow, D.M. and Furuse, M. (2008a) Intracerebroventricular injection of L-arginine induces sedative and hypnotic effects under an acute stress in neonatal chicks. Amino Acids, 35:139-146, with permission from Springer-Verlag. Figure 2. Effect of i.c.v. injection of of L-arginine on spontaneous activity (upper panel) and vocalizations (lower panel) during a 10 min social separation stress in 6-day-old layer chicks. Values are means with S.E.M. Different letters indicate significant differences at P