Handbook of Chemistry, Biochemistry and Biology : New Frontiers [1 ed.] 9781612094250, 9781607418610

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Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved. Handbook of Chemistry, Biochemistry and Biology : New Frontiers, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved. Handbook of Chemistry, Biochemistry and Biology : New Frontiers, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

HANDBOOK OF CHEMISTRY, BIOCHEMISTRY AND BIOLOGY: NEW FRONTIERS

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, 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 herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services.

Handbook of Chemistry, Biochemistry and Biology : New Frontiers, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved. Handbook of Chemistry, Biochemistry and Biology : New Frontiers, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

HANDBOOK OF CHEMISTRY, BIOCHEMISTRY AND BIOLOGY: NEW FRONTIERS

LUDMILA N. SHISHKINA GENNADY E. ZAIKOV Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

AND

ALEXANDER N. GOLOSCHAPOV EDITORS

Nova Science Publishers, Inc. New York

Handbook of Chemistry, Biochemistry and Biology : New Frontiers, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

Copyright © 2010 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.

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

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. LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA available upon request

ISBN 978-1-61209-425-0 (E-Book)

Published by Nova Science Publishers, Inc.  New York

Handbook of Chemistry, Biochemistry and Biology : New Frontiers, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

CONTENTS Preface Chapter 1

Chapter 2

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Chapter 3

Chapter 4

Chapter 5

Chapter 6

xi Hydrogels in Endovascular Embolization Part I. Spherical Particles of Poly(2-Hydroxyethyl Methacrylate) and their Medico-Biological Properties Daniel Horak, K.Z. Gumargalieva, G.E. Zaikov and M.I. Artsis Hydrogels in Endovascular Embolization Part II. Clinical Use of Spherical Particles Daniel Horak, K.Z. Gumargalieva, G.E. Zaikov and L.A. Zimina

1

13

Hydrogels in Endovascular Embolization Part III. Radiopaque Spherical Particles, their Preparation and Properties Daniel Horak, K.Z. Gumargalieva, G.E. Zaikov and L.L. Madyuskina

23

Hydrogels in Endovascular Embolization Part IV. Morphological Foundation of Hydrogel Use for Vascular Occlusion Daniel Horak, K.Z. Gumargalieva, G.E. Zaikov and N.N. Madyuskin

33

Hydrogels in Endovascular Embolization Part V. Antitumor Agent Methotrexate-Containing P(HEMA) Daniel Horak, K.Z. Gumargalieva, G.E. Zaikov, M.I. Artsis and L.L. Madyuskina Hydrogels in Endovascular Embolization Part VI. Poly(2-Hydroxyethyl Methacrylate) with Intensified Haemostatic Activity as a New Embolic Material Daniel Horak, K.Z. Gumargalieva, G.E. Zaikov, L.A. Zimina and N.N. Madyuskin

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vi Chapter 7

Chapter 8

Chapter 9

Chapter 10

Chapter 11

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Chapter 12

Chapter 13

Chapter 14

Chapter 15

Contents On the Possibility of Using an Embolizing Preparation Derived from Poly(2-Hydroxyethyl Methacrylate) (Poly-HEMA) for Chemoemobolization E.V. Koverzanova, S.V. Usachev, K.Z. Gumargalieva and L.V. Kokov

59

A New Mechanism of E. Coli Resistance to Alkylation Damage Induced by NO-Donating Agent—A “Quasi -Adaptive Response” Svetlana V. Vasilieva, Elena Ju. Moschkovskaya and Michael R.Volkert

67

A Kinetic Approach to Explain the Effects of α-Tocopherol at the Physiological and Ultra-Low Concentrations on the Activity of Protein Kinase C In Vitro E.L. Maltseva, K.G. Gurevich and N.P. Palmina Pharmacological Premises of the Creation of New Antitumor Preparations of the Class of Nitrosoalkylurea J. A. Djamanbaev, Ch. Kamchybekova, J. A. Abdurashitova and G. E. Zaikov Influence on the Oxidation Processes Regulation is the Reason for Biological Activity of the Ecdysteroid-Containing Compounds L.N. Shishkina, O.G. Shevchenko and N.G. Zagorskaya Influence of the Composition and Physicochemical Parameters of Natural Lipids on Properties of Liposomes Formed from Them M.A. Klimovich, L.N. Shishkina, D.V. Paramonov and V.I. Trofimov State of Lipid Component of Soybean Flour Enzymatic Hydrolyzates during Storage L.N. Shishkina, E.V. Miloradova, E.A. Badichko and S.E. Traubenberg XRD Characterization of Superfine Fe Powder and EPR Study of Its Interaction with Lipid Membranes Liudmila D. Fatkullina, Alexey V. Krivandin, Elena B. Burlakova and Alexander N. Goloschapov The Quenching of Intrinsic Fluorescence of Sarcoplasmic Reticulum for the Lipid-Protein Interrelationship Determination O.M. Alekseeva, Yu.A. Kim, V.A. Rykov and N.L.Vekshin

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Contents Chapter 16

New Equipment to Fight Industrial Emissions R.R. Usmanova and G.E. Zaikov

Chapter 17

Synthesis of Peroxy Oligomers Based on Epoxy Compounds Using Tert-Butyl Peroxymethanol Michael Bratychak, Olena Shyshchak, Mikhailo Bratychak and Olena Astakhova

Chapter 18

Chapter 19

Chapter 20

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Chapter 21

167

179

Kinetics of the Fermentative Process in Stationary State for Sunflower−Seed Oil Hydrolysis by Lipase in the Presence of BioSAS A. A Turovsky, R. O. Khvorostetsky, L. I. Bazylyak and G. E. Zaikov

191

Thermal Degradation and Combustion Behavior of the Polyethylene/Clay Nanocomposites Prepared by Intercalative Polymerization L.A. Novokshonova, S. M. Lomakin, P.N. Brevnov, A.N. Shchegolikhin and R. Kozlowski Molecular Design and Reactivity of the 1-Hydroxycyclohexyl Hydroperoxide - Alk4NBr Complexes N.A. Turovskij, E.V. Raksha, E.N. Pasternak, I.A. Opeida and G.E. Zaikov Char Formation Flame Retardant of PVC Plasticates N.A. Khalturinskiy, D.D. Novikov, L.A. Zhorina, L.V. Kompaniets, T.A. Rudakova and S.L. Bobot’ko

Chapter 23

Mobile Structural Defects as Catalyst of Solid State Polymerization A.M. Kaplan and N.I. Chekunaev

Chapter 25

157

Kinetics of the Fermentative Reaction of H2O2 Decomposition under the Action of Catalase in the Presence of bioSAS for the Stationary State A. A. Turovsky, R. O. Khvorostetsky, L. I. Bazylyak and G. E.Zaikov

Chapter 22

Chapter 24

vii

Flame Retardation of Unsaturated Polyester Resins and Glass-Reinforced Polyester Resin Laminates with Use of Halogen Free Modification Ewa Kicko-Walczak and Marzena Półka Absorption of High-Boiling Hydrocarbons from Associated Petroleum Gas at Tubular Devices with Converging-Diverging Construction T.G. Umergalin, F.B. Shevlyakov, V.P. Zakharov, D.H. Kaem and G.E. Zaikov

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viii Chapter 26

Modification of Microparticles via Previous Adsorption of Cobalt (III) Acethylacetonate on their Surface A. R Kytsya, L. I Bazylyak, V. V. Kochubey, Yu. G. Medvedevskikh and G. E.Zaikov

Chapter 27

Antioxidant Properties of Lemon Essential Oils T.A. Misharina, M.B. Terenina, N.I. Krikunova and I.B. Medvedeva

Chapter 28

The Structure and Mobility of Vulcanized Nets of Polysulphide Oligomers V.S. Minkin, Yu.N. Khakimullin, T.R. Deberdeev and G.E. Zaikov

Chapter 29

EPR Detection of γ-Irradiated Green Tea R. Mladenova, N.D. Yordanov, M. V. Motyakin and A.M. Wasserman

Chapter 30

Luminescence Diagnostics of Malignant Tumours in Near Infrared Using Yb-Complexes of Porphyrins N.V. Roshchina, V.D.Rumyantseva, I.P. Shilov, A.F. Mironov and V.A. Kotelnikov

Chapter 31

Chapter 32 Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Contents

Chapter 33

Chapter 34

Chapter 35

The Study of the Atomic Structure and Elastic Properties of the Silicon Carbide Nanowires Pavel B. Sorokin, Pavel V. Avramov, Dmitry G. Kvashnin, Alexander G. Kvashnin and Leonid A. Chernozatonskii The Elastic Properties of Branched Silicon Nanowires: The Theoretical Study Pavel B. Sorokin, Alexander G. Kvashnin, Dmitriy G. Kvashnin, Pavel V. Avramov and Leonid A. Chernozatonskii Thermostability and Coke Formation Ability of Diphenyldiacetylene and P-Diethynylbenzene Polymers Vjacheslav M. Misin, Nikolay N. Glagolev and Michael V. Misin Melting and Non-Isothermal Crystallization Behavior of Polyethylene, Poly(Ethylene-Co-Vinyl-Acetate) and their Blends with Natural Rubber Natalya N. Kolesnikova, Anna V. Baranova, Yulia K. Lukanina and Anatoly A. Popov Influence of the Structure of a Composite Material’s Polymeric Template on the Development of Micromycetes Yu.K. Lukanina, N.N. Kolesnikova, A.V. Khvatov, A.N. Likhachev and A.A. Popov

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Contents Chapter 36

Chapter 37

Effect of Solvent Composition on the Decay Kinetics of Carbocations in the Photolysis of 1,2-Dihydroquinolines O. N. Lygo, T. D. Nekipelova, E. N. Khodot, V. A. Kuzmin

365

Spectroscopic Studies on the Interaction between Chlorine Derivative and Human Serum Albumin G.V. Golovina, V.A. Kuzmin and M.A. Grin

373

Chapter 38

The Most Important Commodity Is Water G.G. Zharikova

379

Chapter 39

Concomitant Microflora of Boletus Edulis S. F. Vladimirova

385

Chapter 40

Germination of Basidiospores and Colony Structure of Boletus Edulis G. G. Zharikova, S. F. Vladimirova and M. V. Nefelova

Chapter 41

Chapter 42

Chapter 43

Chapter 44 Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

ix

391

Changes in Microbial Population of Confectionery Products during Storage I.B. Leonova, K.A.Cravshenko and P.A.Solopova

399

Dependence of Thermodynamic Characteristics upon Spatial-Energy Parameter of Free Atoms G.А. Коrablev and G.Е. Zaikov

407

Formation of Carbon Nanostructures and Spatial-Energy Stabilization Criterion G.А. Korablev and G.E. Zaikov

427

Truth of the Truths and Resonance of the Resonances G.А. Коrablev and G.Е. Zaikov

Index

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439 449

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved. Handbook of Chemistry, Biochemistry and Biology : New Frontiers, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

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PREFACE The majority of chapters in this book were written by scientists of N. M. Emanuel Institute of Biochemical Physics (IBChPh) of Russian Academy of Sciences. Prof. N. M. Emanuel was one of the founders of biochemical physics – a part of natural science. This science borders on the line of physics, chemistry and biology with integration of mathematics and with practical applications in medicine and agriculture. The book is devoted to these topics. The time has come to show the scientific community worldwide what Russian scientists have recently done in this area. Six chapters of this volume have information about hydrogels in endovascular embolization. Special attention devoted to synthesis and properties of spherical particles (SP) of hydrogels and their medico-biological properties, clinical use of SP, radiopaque SP and their preparation and properties, morphological foundation of hydrogels use for vascular occlusion, antitumor agents methotrexate-containing poly(HEMA)-hydrogels and poly(HEMA) with intensified haemostatic activity as a new embolic materials. The volume has very important information about pharmacological premises of the creation of new antitumor preparations of the class of nitrosoalkylurea and investigation of new mechanism of E.coli resistance to alkylation damages induced by NOdonation agent – a “Quasi-adaptive response”. It also includes information about biological activity of different enzymes in process of oxidation in vivo and in vitro, investigation of the properties of lipids in plants and in animals. Some chapters deal with pharmacological criterions for new antitumor drugs, using of Tocopherols as bioantioxidants in vitro and in vivo, creation of new equipment for chemical engineering, investigation of enzyme reactions, thermodegradation and combustion of polymers and polymer composites, formation of char during of combustion, molecular design and reactivity of some chemical compounds, problems of pethrochemistry, preparation and modification of microparticles, investigation of antioxidants in food products, chemistry of rubber and formation of carbon nanostructures. Several chapters include very important information about application of electron spin resonance techniques for investigation of chemical and biochemical reactions. Chapter 1 - Investigation of the responses of tissue surrounding spherical hydrogels, after various periods of time from implantation, revealed that a thrombus had been formed on the surface of the embolus and inside the cavities between the hydrogel and the blood vessel immediately after the occlusion. This thrombus is replaced step by step by connective tissue. Chapter 2 - Experience provided by successful clinical applications of spherical emboli prepared from poly(2-hydroxyethyl methacrylate) and intended for the occlusion of blood

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Ludmila N. Shishkina, Gennady E. Zaikov and Alexander N. Goloschapov

vessels allows the authors to infer that this material, which can be obtained by employing a simple procedure, is nontoxic, perfectly biocompatible, and possesses properties that make it particularly suitable for stopping haemorrhage by the method of transcatheter embolization. Chapter 3 - It can be seen that in practice a compromise should be sought between particles with a high iodine content, which can be roentgenologically perfectly monitored, and those with a low iodine content, which swell well. Moreover, it is necessary for medical applications that emboli should also preserve a sufficient mechanical stability. These requirements are best met by particles with a 25–30 wt.% of iodine which can be adequately monitored when being introduced into the blood vessels and which also enable the checking of their performance in viva without the necessity for angiography. Chapter 4 - Clinical observations give evidence to the fact that hydrogel emboli can be considered as a material that is absolutely suitable and can be successfully used for therapeutic occlusion of blood vessels. Chapter 5 – This chapter allows the authors to assume that emboli made from the modified p(HEMA) hydrogel can be loaded with methotrexate, one of the most frequently used cytostatics. Such a simple MTX-fixation to a p(HEMA)-Hex carrier has the advantage that the pharmacological activity of the drug remains unchanged. The use of p(HEMA)-HexMTX hydrogel emboli should enable us to achieve a maximum concentration and a prolonged effect of the cytostatic in the embolized region (tumor) while reducing its concentration in other tissues of the organism at the same time. Thus, there is a prospect of the targeted local palliative cytostatic effect of the drug on the tumor and of prevention of the possible haematogenic formation of metastases, along with a decreased blood supply to this zone or to the whole organ. In other words, the site-specific function of the released antitumor drug could be combined with the endovascular occlusion of blood supply to the tumor tissue. Chapter 6 - Haemostatic properties of ethamsylate-sorbed hydrogel particles tested on normal healthy plasma were not very distinct. Tests carried out with pathologically changed plasma of the patients suffering from focal alteration of the liver showed a pronounced haemostatic effect of ethamsylate or aminocaproic acid-containing hydrogels. A comparison of haemostatic properties of drug-free PHEMA emboli with those of ethamsylate or aminocaproic acid-treated PHEMA indicate their potential in the treatment to prevent bleeding, particularly for those caused by a disturbed haemostasis system. Chapter 7 - The principal opportunity of uptake of weakly crosslinked hydrogel emboli is shown by Doxorubicin at different temperatures. Optimal time of process is 1.5–2.5 hours. It is revealed that Doxorubicin is able to diffuse from a polymeric matrix, having a targeted medical effect on surrounding tissue, reducing side impacts on other organs. Chapter 8 - All living organisms posses mechanisms protecting cells from toxic and genotoxic effects of alkylation damages, induced from endogenous and environmental sources. O6-methylguanine is the major mutagenic base derivative, which strictly modifies base pairing and leads to mutations. To prevent the effects O6- alkylguanine - DNA alkyltransferase (Ada protein) directly dimethylates O6-meG in cellular DNA by transferring the methyl group onto one of their cysteine residues. In E. coli the protective mechanism involves the ada, alkA, alkB and aidB genes expression as well, which is positively controlled by the Ada protein. This DNA repair pathway (the Ada response) is well known as very specific and ubiquitous. Since cysteine methylation at SH groups is the crucial factor for the Ada activation, we assumed that the protein activity can be alternatively regulated by NO – containing agent, via the S - nitrosyl cysteine, functioning in place of S - methyl cysteine in

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Preface

xiii

key position. In the present work a new original mechanism of E. coli resistance to alkylation damages induced by NO-donating agent—a “quasi -adaptive response”—was verified experimentally. Chapter 9 - The effect of a natural antioxidant α-tocopherol (α-TL) (in concentrations from 10-2 to 10-17 M) on the activity of protein kinase C (PKC) isolated from rabbit hearts was studied. Subsequent modeling was performed in terms of kinetic methods. It was shown that α-TL inhibits PKC to a maximum of 80% by a non-competing mechanism. It was found that the dose dependence yields a bimodal curve with the maxima of inhibition at the α-TL concentrations 10-4 and 10-14 M. It was shown that the substrate (histone H1) dependences of the PKC activity in the absence and in the presence of high (10-4 M) and ultra-low (10-14 M) doses of α-TL exhibit maxima at the same concentration of histone H1 (1 μM). The effect can be described by a formal kinetic scheme of inhibition with an excess amount of the substrate. Identification of the parameters of the system was performed with a conjugate gradient technique; the approximation of the experimental results is 98%. A kinetic scheme of allosteric regulation of the PKC activity under the action of α-TL was suggested; the scheme adequately describes the bimodal dose—effect dependence. A good agreement between the experimental and theoretical constants was obtained. Chapter 10 - Perspectives in the field of creation of highly effective anticancerogenic preparations have been evaluated. For their creation is offered a new regio-selective method of glycosylation of alkylurea in conditions of nucleophilic catalysis with some following nitrosing of glycosyl carbamides of the D- and L-rows. This method opens principally new possibilities for modification of compounds by means of glycosylamides bond allowing us to get preparations, possessing small toxicity and high selectivity. Chapter 11 - Influence of serpisten and inokosterone on the phospholipids composition in liver and blood erythrocytes, intensity of lipid peroxidation in tissues (liver, spleen, blood plasma), catalase activity in the liver and general peroxidase activity of white outbreed mice has been studied. A biological activity of ecdysteroid-containing compounds is shown to be associated with an influence on the parameters of the physicochemical regulatory system of lipid peroxidation (LPO). Possessing pronounced membrane-tropic properties due to alterations in the exchange of predominantly choline-containing fractions of phospholipids, ecdysteroid-containing preparations are capable of modifying a cell membrane phase state. A substantial dependence of a biological effect of the compounds on a dose, duration of their application as well as on an intensity of the LPO processes in the tissues and an animal’s sex require a more detailed research on the properties of the given ecdysteroids. Chapter 12 - The influence of composition and physicochemical parameters (the antiperoxide activity, the amount of the TBA-reactive substances, the content of diene conjugates and ketodienes) of lipids isolated from the liver and brain of outbreed mice on the characteristics of liposomes from these lipids has been studied. The data obtained make it possible to conclude that the phosphatidyl choline/phosphatidyl ethanolamine ratio and the diminution of the share of the more easily oxidizable phospholipids have an important role in the formation of liposomes from the natural lipids, and the [sterols] / [phospholipids] ratio in natural lipids has influenced the sizes formed from the liposomes. Chapter 13 - This chapter is a study of the influence of hydrolysis and centrifugation processes of soybean semifat flour on various indices of lipid components and dynamics of

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Ludmila N. Shishkina, Gennady E. Zaikov and Alexander N. Goloschapov

changes in the composition and characteristics in hydrolyzates within three months of storage. It was shown that processes of hydrolysis and centrifugation, as well as storage, cause reliable changes in the physical and chemical characteristics and lipid composition in hydrolyzates. Chapter 14 - The structural properties of the superfine iron powder and its interaction with lipid membranes of mice erythrocytes and lipid membranes of egg lecithin liposomes were studied by X-ray diffraction (XRD) and electron paramagnetic resonance (EPR) methods. The superfine iron powder was prepared by the method of heterophase interaction. It was shown by XRD analysis that this powder consisted predominantly of the crystalline iron in the α-form (α-Fe) with the crystal lattice parameter a = 0.2866 nm and the average crystal size about 30 nm. The microviscosity variation of liposome membranes and erythrocyte membranes under the action of the superfine iron powder in vitro was analyzed by EPR-spectroscopy. The effect of the iron powder on the lipid membrane microviscosity depended on the powder concentration, the time of the powder interaction with membranes and the type of these membranes. It was shown that the superfine iron powder at the ultra low concentrations had a more pronounced effect on the lipid membrane microviscosity than this powder at high concentrations. Chapter 15 - This investigation deals with the structural properties of sarcoplasmic reticulum (SR) membranes. SR is the main Ca2+-pool in the rabbit skeletal muscle. The principal Ca2+-pool functions of the vesicles of fragmented sarcoplasmic reticulum were greatly varied subject to the source of the vesicles’ origin. The heavy vesicles are the fragmented terminal cistern SR, which mainly released Ca2+. The light ones are the fragmented longitudinal tubules SR, which mainly pumped Ca2+. All tested vesicles have some similar and some different structural and functional characteristics that depend on the arrangements of their lipid and protein molecules in the membranes. The lipid-protein relationships were tested with the tryptophan fluorescence quenchers. Chapter 16 - The problem of clearing of gas emissions is actual now. The efficiency of gas purification can be raised at the expense of working out new more perfect designs of dedusters. In this chapter, new designs of wet dedusters of centrifugal and inertial action are considered. Constructive schemes have resulted. The authors give a description of the principle of how the devices work and investigate their performance in industrial conditions for the clearing of gas emissions. Commercial operation has shown that the developed devices provide high degree of clearing of gas. Chapter 17 - The possibility of peroxy oligomers production has been examined. Chemical modification of epoxy resins or telomerization of diepoxy compounds with tertbutyl peroxymethanol have been used for the synthesis. Reaction conditions have been determined. The synthesis procedure has been developed. The structure of synthesized peroxy oligomers has been confirmed by chemical analysis as well as IR- and PMR-spectroscopy. Chapter 18 - The fermentative stationary kinetics of hydrogen peroxide decomposition under the action of catalase in the presence of bioSAS was investigated. The authors obtained the kinetic parameters of this process. It was shown that the bioSAS has an influence on the fermentative process, which can be explained by the change of the fermentative center activity or by the change of substrate concentration. It was determined that the temperature of a process has an insignificant influence on the value of kinetic parameters. Chapter 19 - The catalytic rate constants for the process in the presence of bioSAS by different concentrations was obtained. It was shown that some constants increase at bioSAS concentration increasing up to the beginning of their micelle−formation. The temperature has

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Preface

xv

a slight influence on the value of catalysis constants, which can be explained by practically zero activation energies and depends on activation entropy. Chapter 20 - A comparative study of thermal and thermal-oxidative degradation processes for polyethylene/organically modified montmorillonite (PE-MMT) nanocomposites, prepared by the ethylene intercalative polymerization in situ, with or without subsequent addition of an antioxidant, is reported in this chapter. The results of TGA and time/temperature-dependent FTIR spectroscopy experiments have provided evidence for an accelerated formation and decomposition of hydroperoxides during the thermal oxidative degradation tests of PE-MMT nanocomposites in the range of 170–200oC, as compared to the unfilled PE, thus indicating a catalytic action of MMT. It has been shown that effective formation of intermolecular chemical cross-links in the PE-MMT nanocomposite has ensued above 200oC as the result of recombination reactions involving the radical products of hydroperoxides decomposition. Apparently, this process is induced by the oxygen deficiency in the PE-MMT nanocomposite due to its lowered oxygen permeability. It is shown that the intermolecular cross-linking and dehydrogenation reactions followed by the shear carbonization lead to appreciable increase of thermal-oxidative stability of the PE nanocomposite as compared to that of pristine PE. Notably, the TGA traces for the antioxidant-stabilized PE-MMT nanocomposites recorded in air were quite similar to those obtainable for the non-stabilized PE-MMT nanocomposites in argon. The results of treatment of the experimentally acquired TGA data in frames of an advanced model kinetic analysis are reported and discussed. Significant decrease of the combustibility of the PE nanocomposite was shown by a cone calorimeter method. Chapter 21 - Chemically activated 1-hydroxycyclohexyl hydroperoxide decomposition in the presence of ammonium salts is proposed to proceed through the complexation stage. Complex structure and reactivity have been investigated by molecular modelling methods. Kinetics of the chemically activated hydroperoxide decomposition in the presence of quaternary ammonium salts (Et4NBr, Pr4NBr, Bu4NBr, and Hex4NBr) has been studied. The correlation between reactivity and structural characteristics of ammonium cations was found. Chapter 22 - The effect of flame retardants on the combustibility and mechanical properties of PVC plasticates based on commercial materials is discussed. The smoke formation of the investigated samples was studied under pyrolysis and combustion modes. It was shown that, by addition of flame retardant to the PVC plasticate, its combustibility can be controlled with retention of the basic performance of the material. It should be noted that the studied method of the plasticate modification allows one to form a strong coke skeleton on the surface of the polymer composition and to avoid the flow of molten material upon burning and, as a result, to prevent flame propagation. Chapter 23 - A model of the solid state polymerization based on the catalytic role of carriers of the surplus free volume, namely mobile structural defects, has been developed. This model succeeded for the first time to explain most of non-trivial kinetic peculiarities of solid state polymerization. Chapter 24 - An evaluation was made of the effectiveness of flame retardancy of halogen free flame retardants (FR) as nitrogen compounds that act with phosphorus or boron in relation to unsaturated polyester resins and glass-reinforced polyester resin laminates. The impact of such FR modifiers (applied in desired quantities and various physical forms) on the flammability of polyester compositions and glass-reinforced polyester resin laminates was determined by defining oxygen indices values, with the use of the thermogravimetric methods

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and an analysis of the combustion process with a cone calorimeter. The analysis of the thermal decomposition process provided proof of good flame retardancy effectiveness of the tested products, including in the first place nitrogen–phosphorus units and boron-nitric compounds with the observed phenomenon of synergic action of compounds of both atoms. No adverse impact of modification was ascertained on basic strength properties of products. Chapter 25 - This chapter shows a method of perfecting the after-extract process of highboiling hydrocarbons from associated petroleum gas at the expense of use of the small-sized tubular turbulent device diverging-converging construction at a stage of absorption by crude oil. Chapter 26 - The method of obtaining functionalized and modified microparticles via adsorption of the β−diketonates of transition metals on their surface was proposed for the first time. It was shown that the β– diketonates of transition metals, in particular Cobalt (III) acethylacetonate, can be used as high effective initiators of the polymerization “from the surface” of the substrate. The authors studied the thermal stability of the product immobilized on the surface of Al2O3. Chapter 27 - Antioxidant properties of lemon essential oils (Citrus limon L.) with different composition, of individual limonene and citral, were investigated by capillary gasliquid chromatography. Antioxidant activity was assessed in model systems by oxidation of aliphatic aldehydes 2-hexenal or 2,4-decadienal to the carboxylic acids. It was found that antioxidant activity of essential oils increased as their concentration increased. Individual citral and limonene had minimum antioxidant activity. The activity of their mixture was more. The differences of stability to oxidation of components of lemon essential oil were determined. The synergistic effects in the antioxidant activity and stability of the main oil component were found. Chapter 28 - By the nuclear magnetic resonance method in a wide temperature interval, molecular mobility in the vulcanizates of liquid thiokols, vulcanized by the various agents, is studied. The influence of the nature of the vulcanizing agents and a structure formed vulcanized grids on change of the character of molecular movement of the vulcanizates of polysulphidic oligomers is established. Correlation of the effective (υeff) and the chemical (υchem) density of vulcanized grids thiokol hermetics with parametres of spectra of a nuclear magnetic resonance of the latter is found. Influence of the nature of a vulcanizing agent on features of formation of vulcanized grids of polysulphidic oligomers is discussed. Chapter 29 - One symmetrical single EPR line centered at g=2.0044±0.0003 with peakto-peak width of 9.0±0.1 G was detected in non-irradiated green tea samples. The γ-ray irradiation led to an appearance of two weak satellite lines situated around the central signal. Detection of these two satellite lines is unambiguous evidence for irradiation treatment. Intensity of the central line decreased, and satellite lines fully disappeared during the storage of irradiated tea samples. To distinguish a difference between irradiated and non-irradiated tea samples, the changes in intensity of EPR signal in the samples during heating were monitored. The decrease of signal intensity after heating was evidence of past irradiation. Chapter 30 - The basic idea of this work is to create and to put into practice a new class of diagnostic photosensitizers, namely non-phototoxic photosensitizers, which do not generate singlet oxygen, and exhibit as high luminescent characteristics and tumour-affinity level as modern therapeutic photosensitizers.

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Chapter 31 - The atomic structure and elastic properties of silicon carbide nanowires were calculated by density functional theory. The effect of the surface reconstruction and relaxation was observed. The splitting of the SiC geometry to hexagonal and cubic phases was found. The Young’s modulus of the nanowires was calculated. Chapter 32 - The atomic structure and elastic properties of Y-silicon nanowire junctions were theoretically studied and effective Young modules were calculated using Tersoff interatomic potential. It was shown that boundary effects at junctions of different parts of the wires determine mechanical properties of the nanostructures. As the final result, the bending of the wires under external stress leads to the formation of new bonds between different parts of the junctions. Chapter 33 - It was determined that homo- and copolymers of diphenyldiacetylene and pdiethynylbenzene have a high thermal and thermooxidation stability. The amount of coke residue obtained from commercial epoxy-resins is essentially increased upon their modification with added polydiphenyldiacetylene and copolymer diphenyldiacetylene with pdiethynylbenzene. Additions of polydiethynylbenzene to commercial olygoetheracrylates improved their thermooxidation stability and strength characteristics of solidified composite materials at high temperatures. Chapter 34 - Non-isothermal crystallization and melting behavior of low density polyethylene (PE), poly(ethylene-co-vinyl-acetate) (CEVA) and their composites with natural rubber (NR) were investigated using differential scanning calorimetry (DSC). The results indicated that melting and crystallization parameters of PE and CEVA composites are not affected significantly by natural rubber, as well as its content. PE and CEVA in the blends with NR crystallized separately to form the isolated crystalline phase. The non-isothermal crystallization data were analyzed by using the Avrami equation for low degrees of crystallinity PE and CEVA. Chapter 35 - The possibility of creating a biodegradable composite material based on propylene-ethylene copolymers supplemented with cellulose, as well as with the woodworking industry waste (powder of different species of wood), was investigated. Investigations into the development of fungi on the given substrata have exhibited a difference in accumulating a biomass for diverse strains. The rate of accretion of micromycetes on composite materials is defined not only by a natural component introduced into the polymeric template, but also by the structure of the polymer material. Chapter 36 - The dependence of the quantum yield and the decay rate constant on the solvent composition for the carbocations generated in the photolysis of 1,2-dihydroquinolines was studied by steady-state and pulse photolysis in binary mixtures of alcohols with solvents of different polarity, proton donating and accepting ability, and capability for hydrogen bonding. The results were discussed on the basis of the reaction mechanism involving competing reactions of the carbocation combination with two nucleophilic particles, the ROH molecule and the RO– anion. The kinetic behavior of the carbocations generated from 1,2dihydroquinolines in the photolysis in solvent mixtures is determined by the polarity, proton donating and accepting ability, nucleophilicity, as well as the ability to form hydrogenbonded associates between the solvent molecules and between the solvent and solute molecules. Chapter 37 - The conjugate between chlorine e6 with D-lactose and human serum albumin was investigated by absorption and fluorescence spectroscopy. The strong interaction of the chlorine e6 derivative and the protein was detected. It was found that the presence of

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albumin causes remarkable changes in the absorption and fluorescence spectra of the chlorine. The association constant (2·105 М–1) was calculated from the fluorescence experiment. Chapter 38 - At the present stage of development of mankind, in a megalopolis, a person faces the choice of whether to drink piped or bottled water. Because, when making a choice, we must take into account such aspects as hygienic safety, health value and our own financial capabilities, as far as the water is a special product that has no substantiations in human nutrition. Water is the greatest value for any inhabitant of the planet and is among those few elements that are the essence of development of civilization as a whole. People began to understand the strength of interdependency of the quality of water and standard of living relatively recently. The authors have studied four types of drinking water: tap water, well water, bottled water and mineral water. The results obtained demonstrate quite a clear picture—maximum amount of sterile samples was found from the number of mineral water samples, while none of the well water samples was characterized by the absence of microorganisms. In their research, they used a deep method of sowing in a dense nourishing environment, incubation of sowing, count of all growing visible colonies. The piped water does not meet the requirements of SanPiN (Sanitary regulations and norms). However, its daily consumption is much cheaper for us than those two cherished litres we need for nutrition purposes. Of course, even when buying the bottled water purified from various types of impurities, there is a risk to buy and drink a counterfeit product, as the growth rate of this market is too attractive for the supporters of a highly marketable business. Chapter 39 - The laboratory of provision in Plekhanov Russian Academy of Economics has been studying the life cycle of Boletus edunis for several years. The research is focused on germination of basidiospores of B. edulis, on their sterile isolation and on studies of hymenophore microflora. Basidiospores were isolated from dried hymenophores of B. edulis with two methods. The first yielded dried fungi powder on white sterile paper after exposure of mycothallus cap with hymenophore downside on the paper. Basidiospores dropped from hymenophore tubes and tightly attached to the paper. Spores could be gathered only with scalpel, and it might contaminate basidiospores with extraneous microflora. The second method of asidiospores isolation: hymenophores were separated from trama, cut into 1 cm pieces and dried in the air on sterile paper, with occasional turning upsidedown. Hymenophore mixture was treated and purified according to the method, developed by us previously and including dispensing, washing, filtration, centrifugation, short treatment with antiseptics. To verify the sterility, isolated basidiospores were applied to Petri dishes in appropriate medium. Colonies of mold fungi were transferred to tubes, both macro- and microscopic characteristics were determined, and fungi were identified. Chapter 40 - Germination of Boletus edulis basidiospores and growth of primary colonies under laboratory conditions are studied in this work. Apparently the determined first mechanism of germination is peculiar solely to this species of higher fungi. In liquid medium basidiospores swell and decay forming small spherical structures that further aggregate into globules covered with membrane and initiate hyphae.

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Unlike the described approaches, the authors studied the process in dynamics: from the onset of basidiospores germination to the formation of microcolonies of vegetating mycelium. The formation of growth tubes has not been detected for Boletus edulis for several years of observation. The study of basidiospores germination was performed only with cultures free of exogenous bacterial or mold fungal microflora which was controlled microscopically and with inoculation on appropriate cultures. Mycothalluses of mature cepes gathered in central Russia in 1999, 2001 and 2007 were used in this work. Chapter 41 - The researchers from The Food Microbiology laboratory of the G. V. Plekhanov Russian Academy of Economics have been studying the quality of various groups of confectionery products for more than 20 years, using quantitative and qualitative microbiological criteria. The study objective was to reveal the pattern of changes of microbiological population by studying the time profile of confectionery product contamination against various microbiological criteria during storage. The objects of the study were chocolate products and chocolate sweets. Standard microbiological test methods were used. The study revealed some regularities in quantitative changes of microorganism population in bitter chocolate during storage (measured with standard quantitative microbiological criteria): Mesophilic Aerobic and Facultative Anaerobic Microorganisms, Escherichia coli group bacteria and mold fungi. The examination of the products’ microorganism population and its changes during the product storage calls for an assumption about a complex system of relations within the population. On getting into the human digestive system, the population begins a “new life”. In addition to a complex system of relations among various groups of microorganisms, the microbial population is subject to quantitative changes in its composition. Chapter 42 - The dependence of some thermodynamic characteristics upon initial spatialenergy parameters of free atoms has been analyzed. The corresponding equations have been obtained, the dissociation energies of binary molecules and enthalpy of single-atom gas formation have been calculated based on them. Chapter 43 - Spatial-energy criterion of structure stabilization was obtained. The computation results for a hundred binary systems correspond to the experimental data. The basic regularity of organic cyclic compound formation is given and its application for carbon nanostructures is shown. Chapter 44 - It is ascertained that struggle and opposition between two basic political and economic systems do not make sense since the most progressive system is the combination of the two. One of the possible variants of such formation is proposed. It is shown that periods of resonances of solar activity have multipronged importance for our planet.

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In: Handbook of Chemistry, Biochemistry … Editors: L. N. Shishkina et al., pp. 1-11

ISBN: 978-1-61209-425-0 © 2010 Nova Science Publishers, Inc.

Chapter 1

HYDROGELS IN ENDOVASCULAR EMBOLIZATION PART I. SPHERICAL PARTICLES OF POLY(2HYDROXYETHYL METHACRYLATE) AND THEIR MEDICO-BIOLOGICAL PROPERTIES Daniel Horak1, K.Z. Gumargalieva2*, G.E. Zaikov3**, and M.I. Artsis**

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Institute of Macromolecular Chemistry, Czech Academy of Sciences, Czech Republic *N.N. Semenov Institute of Chemical Physics, Russian Academy of Sciences, Moscow 119991, Russia **N.M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow 119334, Russia

ABSTRACT Investigation of the responses of tissue surrounding spherical hydrogels, after various periods of time from implantation, revealed that a thrombus had been formed on the surface of the embolus and inside the cavities between the hydrogel and the blood vessel immediately after the occlusion. This thrombus is replaced step by step by connective tissue.

Keywords: embolization, hydrogels, endovascular, medical and biological properties, spherical particles.

1 2 Heyrovskogo sq., Prague-6, Czech Republic. 2 4 Kosygin str., Moscow 119991, Russia. 3 4 Kosygin str., Moscow 119334, Russia, [email protected] . Handbook of Chemistry, Biochemistry and Biology : New Frontiers, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

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1.1. INTRODUCTION Endovascular occlusion of blood vessels is an innovative procedure in surgical practice; it is used to stop bleeding, reduce blood supply to various organs and parts of the body, and the like. However, its application on a major scale is impeded by the restricted choice of suitable materials; moreover, there is no special material specifically suited to endovascular occlusion. Up to now, particles of various materials have been employed in vascular occlusion, such as metals, including ferromagnets, natural and synthetic polymers, e.g., collagen sponge, polyacrylates, polyurethane, polytetrafluoroethylene, cyanoacrylate adhesives (which remain permanently in the organism) or even an autologous precipitate of the organism’s own blood which after some time is resorbed or degraded to soluble components. However, these materials display a number of shortcomings, such as difficulty in processing, electrolytical corrosion and inflexibility of metals, the possibility of malignant growth and also hydrophobicity which in the case of synthetic polymers makes them behave in the blood vessel as a completely foreign body, in a similar way to rare metals, for example. It should also be pointed out that sufficient purity can never be guaranteed. In view of the hydrophilicity of living matter. It is evident that hydrophilic synthetic polymers based on 2-hydroxyethyl methacrylate can remove these shortcomings. Due to their hydrophilicity and high swelling ability, these polymers are soft and permeable to water, which facilitates quick removal of all low-molecular weight, water soluble compounds from the polymer [1 - 3], and consequently its purification. Long-term experience gained in the field of reconstruction and plastic surgery using homogeneous and microporous hydrogels, in particular, has revealed their advantages: nontoxicity, nonirritability, good “healing-in”, antigenicity, excellent compatibility with tissue of the living organism, and also, the facts that they do not cause febrile reactions, and are not subject to fast destruction [4]. Moreover, poly(2-hydroxyethyl methacrylate) is widely applied in the production of soft contact lenses [1]. Although it seemed at first that hydrogels used as part of vascular prostheses were thromboresistant [5], it appeared later that in some cases they behaved to some extent as thrombogenic material [6] which could advantageously be used in embolization. The first approach to the synthesis of particles for endovascular occlusion based on poly(2-hydroxyethyl methacrylate) consisted in bulk polymerization in a cylindrical mould; a spaghetti-like product [7] was obtained, which for application in occlusion was cut into short cylinders which were then introduced by operation. They must be spherical in shape to make simple transcatheteral introduction of particles possible for endovascular occlusion. This includes treatment of haemoptysis and angiodysplasia, occlusion of bronchial, renal, rectal, hepatic and other incoming arteries in vascular surgery, as a primary condition in the preoperative preparation of patients before the excision of vascular tumors and in those cases where, due to the localization of the vascular tumor, its removal is impossible. In this Chapter we describe a new approach to the synthesis of spherical macroporous poly(2-hydroxyethyl methacrylate) particles and discuss their physico-chemical and medicobiological properties.

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1.2. MATERIALS AND METHODS 1.2.1. Materials Monomers and Auxiliary Compounds The chemicals were redistilled before use: ethylene dimethacrylate (Ugilor S.A., France) b.p. 50°C/7 Pa; cyclohexanol (Lachema Brno) - b.p. 68°C/l.6 kPa. Kerosine, decalin, tetralin, butyl acetate, methacrylic acid were also distilled. 2,2'-Azo(bis-isobutyronitrile) (Ferak, Berlin) was recrystallized twice from ethanol. 2-Hydroxyethyl methacrylate (Leciva Modrany), medicinal purity, and 1-dodecanol (Fluka A.G. Switzerland) were used without purification. Poly(vinyl pyrrolidone) K-90 (Fluka A.G.), poly(vinyl alcohol) (Polyviol W 25/140, Wacker, FRG) and the other chemicals were reagent grade.

1.2.2. Methods

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1-Dodecyl methacrylate (Fluka A.G.) was polymerized in the form of a solution in decalin, with 0.005 wt.%, 2,2'-azo(bis-isobutyronitrile) as initiator, in inert atmosphere in an ampoule at 70°C for 30 h.

Preparation of Hydrogel Particles Polymer particles were synthesized by the suspension radical copolymerization of 2hydroxyethyl methacrylate with ethylene dimethacrylate, and/or methacrylic acid in a laboratory reactor (250 ml volume) provided with an anchor-type stirrer. The mixture of monomers, together with an inert solvent and the initiator of the radical polymerization, were dispersed by vigorous stirring in the dispersing medium containing a dissolved suspension stabilizer which prevents agglomeration of particles during stirring. The optimal particles used in the medico-biological tests were obtained by the following procedure: dispersed organic phase consisting of 9.8 ml 2-hydroxyethyl methacrylate, 0.2 ml ethylenedimethacrylate, 7.5 ml 1-dodecanol, 7.5 ml cyclohexanol and 0.l g 2,2'-azo(bis-isobutyronitrile); 1% water solution of poly(vinyl pyrrolidone) K-90 served as a dispersing medium. The polymerization proceeded at 70°C for 8 h under continuous stirring (150 rpm). After cooling the polymer was separated into the beaker and decanted I 0 times each by water, and methanol respectively, 3 times each by acetone and ether respectively (each portion contained ca. 300 ml of solvent and was left stationary for at least 4 h). After drying in vacuum the spherical macroporous particles were sieved into separate fractions: 0.4 - 0.6; 0.6 - 0.8; 0.8 - 1.0; 1.0 - 1.2 and 1.2 - l.5 mm. The particle size distribution curve is obtained from weights of the respective fractions. For the investigation described in this paper, fraction 0.6 - 0.8 mm was used. Before use, 1 ml of particles was immersed in 250 ml water which was changed daily until usually after 5 d (according to the UV spectrum), the concentration of the low-molecular weight impurities in the solution was lower than that obtained by washing-out 10-5 g of impurities from 1 g of dry polymer. Sterilization of the samples was carried out in boiling distilled water for 45 min.

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Porosity (% of pore volume related to the volume of polymer particles) was calculated from an increase in the diameter of the particles due to the equilibrium swelling of hydrogel beads in water, at room temperature, measured by a GM-l horizontal microscope equipped with an ocular micrometer. The specific surface area was measured from the dynamic desorption of nitrogen according to BET isotherm; the sorption of water vapour by the hydrogel was measured using a McBain's balance. The diffusion coefficient was evaluated according to the Equation [8]:

M t 6 ⎛ Dt ⎞ = ×⎜ ⎟ M∞ r ⎝ π ⎠

0.5

,

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where Mt, M∞ are the amounts of sorbed water in time t and at the end of the sorption, respectively; D is the diffusion coefficient, and r the particle radius. Changes in the morphological picture of blood were quantified under the microscope in 1 ml samples prepared as a mixture of 0.1 ml water extract from hydrogel and 0.9 ml donor blood in the cell.

Implantation The hydrogel samples were intravascularly and subcutaneously implanted in rabbits. After preparation of the operation zone and local Novocain anaesthesia, a linear incision was made along the projection of the vessel leash. When the major femoral arteries were isolated, the catheter was installed into the vascular lumen. Then the emboli were introduced into the vessel through the catheter. In order to check their location angiography (Urografin 76%, 3 ml) and X-ray images were performed prior to and after the embolization. If the emboli were subcutaneously introduced into the inguinal zone, X-ray images were taken and sutures were made. After a certain time (2 weeks, 5 weeks or 21 months), samples of the surrounding tissue were cut out and pieces of the tissue were embedded in celloidin. Histological preparations were stained with haematoxylin and eosin and also after van Gieson.

1.3. RESULTS AND DISCUSSION 1.3.1. Synthesis of Particles for Embolization Suspension radical polymerization is the optimal method for the preparation of spherical polymer particles [9]. The principle is that the dispersed monomer phase is stirred and polymerized in a dispersing medium immiscible with it. At the same time, the monomer mixture which was stirred into spherical drops must be protected (stabilized), by means of a suspension stabilizer to prevent aglutination or agglomeration. The stabilizer differs in its chemical character, depending upon whether the medium is water or an organic liquid. If the synthesized beads are to be suitable for the purposes of endovascular occlusion, they have to be soft, possess a sufficient swelling ability and at the same time have satisfactory mechanical properties. For this reason, the suspension polymerization of 2-hydroxyethyl

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methacrylate with a small amount of the crosslinking agent (2 wt.%) was systematically investigated, and the effect of inert components on the product’s properties studied. The presence of inert solvents is a condition of the formation of a heterogeneous porous structure; the fibrous tissue grows into the pores, thus making anchoring of the gel in the vascular lumen possible. Since 2-hydroxyethyl methacrylate is a hydrophilic monomer readily soluble in water, the inverse phase polymerization was tested first. The monomer dissolved in water as an inert solvent, was polymerized by using ammonium persulphate (initiator) after dispersing in an organic liquid immiscible with water, i.e. decalin, medical paraffin oil, kerosine, or tetralin. Only with decalin were spherical particles, up to 2 mm in size, obtained. Preparation of a suitable suspension stabilizer for this system , i.e. poly(1-dodecyl methacrylate) having a molecular weight 4×106, is however time-consuming, while other stabilizing systems, e.g. based on cetyl alcohol, Span 85 or butyl rubber, were found to be unsuitable. For this reason, only polymerization in water as the dispersing medium was studied; the effect of composition, both of the dispersed phase and of the dispersing medium, on the properties of the resulting product were examined. A number of stabilization systems were tested, such as xanthan gum with hydroxypropylmethyl cellulose (also in a saturated sodium chloride solution), sodium polymethacrylate, poly(vinyl alcohol), and gelatine. Poly(vinyl pyrrolidone), molecular weight 360 000, was found to be best for this purpose. With water used as the dispersing medium, the danger arises that the monomer might dissolve in the water. This may be prevented by choosing inert solvents, added to the dispersed phase, with which the monomer has a much higher distribution coefficient than with water, and thus does not pass into it. The mixture of 1-dodecanol and cyclohexanol appeared to be especially suitable. With cyclohexanol alone, a nonporous, hard, glassy polymer was obtained. Other inert systems, such as, e.g. butyl acetate or polystyrene, were equally unsuitable. Only particles of poly(2-hydroxyethyl methacrylate) crosslinked with 2% ethylene dimethacrylate and prepared in the presence of a mixture of cyclohexanol and 1-dodecanol in an aqueous solution of poly(vinyl pyrrolidone) were used in the experiments.

1.3.2. Physico-Chemical Properties Particles of poly(2-hydroxyethyl methacrylate) prepared by suspension polymerization are shown in the dry state in Figure la in which their rough, but prevalently regular spherical shape can be seen. The same granules swollen in water (Figure 1b) are larger and smoother. Most of the particles (88%) have a bead diameter, in the dry state, in the range 0.4 - 0.8 mm; this is evident from the particle size distribution curve (Figure 2). In water, the diameter of the bead increases quickly, i.e. within 10 min by 20%, which is the equilibrium value. If the monomeric mixture contains, in addition to 2-hydroxyethyl methacrylate, 4 wt.% methacrylic acid, a substantial increase in the bead size in the swollen state takes place, e.g. in the Na+ cycle it is by 110%. The amount of sorbed water at a constant vapour pressure is virtually unaffected by temperature (Figure 3). The calculated value of the diffusion coefficient is 4×10-6 cm2/s. This order of magnitude corresponds to the diffusion coefficient of poly(2-hydroxyethyl methacrylate) synthesized earlier by the conventional method [2].

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The porosity of spherical hydrogel particles reaches 60% in water, and the specific surface area amounts to 0.9 m2/g in the dry state. The diffusion and sorption characteristics of the spherical particles ensure their fast swelling in the blood stream and the expectant localization in the blood vessel. The swelling of poly(2-hydroxyethyl methacrylate) beads ensures reliable stability of the occlusive effect in the vascular lumen and excludes their possible migration as a result of blood pressure, on the one hand, and the formation of firm thrombotic masses, on the other.

Figure 1. Microphotographs of spherical particles of poly(2-hydroxyethyl methacrylate): (a) in the dry state; (b) swollen in water. (× 8.5).

1.3.3. Haematological Testing Blood analyses, or analyses of changes in the number of blood elements due to lowmolecular weight compounds released from the hydrogel during its washing, can be advantageously employed in the determination of the quality of purification of artificial emboli prior to their use.

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Figure 2. Differential curve of hydrogel particle size distribution.

Figure 3. Kinetics of water vapour sorption by spherical hydrogel at various temperatures (1 - 30°C; 2 36°C; 3 - 44°C) and constant vapour pressure 3.5 kPa; Mt is the amount of sorted water in time t.

Table 1. Effect of the length of time of washing-out the hydrogel on the morphological structure of donor blood

Blood elements Erythrocytes (×102) Leucocytes (×109) Thrombocytes (×109)

Number of blood elemnts in 1 l Contact between donor blood and water extract after Donor blood washing-out the hydrogel for: 1d 5d 3.85 ± 0.55 2.85 ± 0.23 3.51 ± 0.40 5.60 ± 0.50 2.85 ± 0.37 5.10 ± 0.30 5.20 ± 1.30 5.10 ± 0.20 7.50 ± 0.30

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After washing-out the hydrogel for l day, a decrease in the number of erythocytes caused by their haemolysis, and of thrombocytes due to aggregation of the latter is found (Table 1). At the same time, one can perceive cytolysis and the formation of aggregates of leucocytes accompanied by a decrease in their number. Values in Table 1 show that after washing-out the hydrogel for 5 days there is no further decrease in the number of erythrocytes and leucocytes; hence, the low-molecular weight compound content is nontoxic, amounting to 10-5 g per 1 g of polymer at most, according to spectrophotometric measurements. Below this value all blood cells in contact with the extract retain their natural character. In this way the analysis of the blood morphology indicates the nontoxic amount of residual impurities in the polymer. If the hydrogel is first washed so that the content of low-molecular weight compounds in it drops below the toxic limit (10-5 g/g), only thrombocytopenia can be oberved in the contact of blood with the subsequent extract; this is accompanied by the formation of thrombocytic aggregates (8 - 10 aggregates per 1000 erythrocytes). Each aggregate contains 20 - 30 thrombocytes. This is caused by the effect of the residual monomer and of the other lowmolecular weight compounds inducing aggregation of the thrombocytes. Adhesion of the latter is the first step in the process of thrombus formation. When the amount of impurities in the hydrogel exceeds 10-5 g/g, contact between blood and the hydrogel extract leads to morphological changes in erythrocytes (decrease in their size and changes in shape i.e. micro- anisocytosis). One can see fragments of erythrocytes and their fragmentation. The haemoglobin level decreases by 40 - 50%. The haemolysis level increases to 50 - 60 mg %. At the same time, there is a great change in the morphology of white blood cells - leucocyte artefacts (10 per 100 cells), pathological shape of neutrophiles and shrinkage of the nuclei. The number of thrombocytes decreases 3 - 5 times compared with the standard. The physical character of the surface (roughness) and of the interior of hydrogel particles makes sorption of blood proteins and cell elements possible. In this respect the hydrogel can be regarded as a thrombogenic material. At the same time, one can see selective adsorption of fibrinogen. This facilitates the following adhesion and aggregation of thrombocytes onto the hydrogel surface and rigid fixation in the place of occlusion. Both processes which appear during implantation of the hydrogel, i.e. both desorption of impurities and sorption of proteins help to bring about quick blood coagulation and thrombus formation on the particle surface, i.e. support the process of endovascular embolization. Spontaneous aggregation of thrombocytes plays an important role particularly immediately after embolization.

1.3.4. Histomorphological Investigation The last medico-biological test consisted of a histological investigation of the material which had been implanted in the subcutaneous cellular tissue or blood vessel of the test animal for variously long periods of time. Two weeks afterthe implantation, the hydrogel bead, subcutaneously localized in the rabbit, is surrounded by a fibrous capsule without any dystrophic and (or) inflammatory changes (Figure 4). The hydrogel is in an early stage of organization. Individual fibroblasts fill the pores of the hydrogel embolus, mainly in the peripheral domains of the bead.

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Figure 4. Histological section of subcutaneously localized spherical hydrogel 2 weeks after implantation. EM, embolus material occupying the largest part: fibrous capsule is on the left-hand side. Stained with haematoxylin andeosin. (× 106).

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Five weeks after the implantation the number of fibroblasts inside the pores of the peripheral domains of the bead is increased (Figure 5). Thin bunches of collagen fibres (resulting from the biosynthesis of fibroblasts) can be seen in the pores. There are also some blood capillaries.

Figure 5. Histological section of subcutaneously localized spherical hydrogel 5 weeks after implantation. C, capillaries, arrows indicate collagen fibres. Stained after van Gieson. (× 166).

An earlier investigation [10] of the tolerance of heterogeneous poly(2-hydroxyethyl methacrylate) gels in the living tissue also confirmed that macroporous polymers were encapsulated with a capsule of collagen fibrous tissue, from which newly formed capillaries and numerous cells penetrated into the implants. The more porous the material, the broader the zone of cellulization. On the other hand, however, gels intergrown with the newly formed fibrous tissue are inconvenient as implants in plastic surgery, since calcification occurs in this case which depends on the degree of porosity in heterogeneous macroporous polymers. At the same time, for the purpose of endovascular embolization, a highly porous structure formed by

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interconnected channels is an essential condition in the vascular lumen for anchoring the gel by the fibrous tissue. Twenty-one months after implantation of a hydrogel bead into the bronchial artery its wall had atrophied and sclerosed (Figure 6). There were no inflammatory or destructive changes to the wall. The embolus material is seen to be divided by the accumulation of giant cells of foreign bodies (resorption) and by the streaks of fibrous tissue cells growing into the pores (organization). The recanalization of the vascular lumen is absent.

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Figure 6. Histological section of the bronchial artery with spherical hydrogel 21 months after implantation. AW - artery wall; EM - embolus material; GC - giant cell; FT - fibrous tissue. Stained with haematoxylin and eosin (× 42).

Consequently, investigation of the responses of tissue surrounding spherical hydrogels, after various periods of time from implantation, revealed that a thrombus had been formed on the surface of the embolus and inside the cavities between the hydrogel and the blood vessel immediately after the occlusion. This thrombus is replaced step by step by connective tissue. All indistinct resorption due to the reaction of the giant cells and the partial organization of the hydrogel beads prevailed in the case of medium long periods of time (e.g., 7 weeks) after implantation. These phenomena were more intense with decreasing bead size. Chronic inflammation and destructive-dystrophic changes (necrosis) of the tissue were absent. Resorption of hydrogel emboli does not cause recanalization or reopening of the vascular lumen, which is especially important in the clinical application of hydrogels to endovascular occlusion. The polymeric material did not display any mechanical destruction more than 1 year after implantation [11, 12].

REFERENCES [1] [2]

Wichterle, Hydrogels, in Encyclopedia of Polymer Science and Technology, 15, Supplement, Interscience, J. Wiley and Sons, New York, 1971 , 273-291. M.F. Refojo, Permeation of Water Through Some Hydrogels, J. Appl. Polym. Sci., 1965, 9, 3417.

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Hydrogels in Endovascular Embolization

K.H. Lee, J.G. Jee, M.S. Jhon, T. Ree, Solute Transport Through Crosslinked Poly(2hydroxyethyl methacrylate) Membrane, J. Bioengineering, 1978, 2, 269. [4] L. Sprincl, J. Vacik, J. Kopecek, Biological Tolerance of Ionogenic Hydrophilic Gels, J. Biomed. Mater. Res., 1973, 7, 123. [5] D.F. Williams, R. Roaf, Implants in Surgery, W.B. Saunders, London, 1973, p. 279. [6] H. Tanzawa, in Medical Use of Polymers, Ed. S. Manabu, (Translation into Russian), Medicina, Moscow, 1981, p. 108. (Rus) [7] M. Stol., P. Lopour, I. Cifkova, Process of Preparation of Sponge-like Synthetic Hydrogels with Improved Mechanical and Biological Properties, Czech. Pat. Appl., 1983, 3372-3383. [8] Yu.V. Moiseev, G.E. Zaikov, The Chemical Resistance of Polymers, Khimia, Moscow, 1979, p. 104. (Rus) [9] D. Horak, Z. Pelzbauer, M. Bleha, M. Ilavsky, F. Svec., J. Kalal, Effect of Composition of Polymerization Feed on Morphology and Some Physical Properties of Macroporous Suspension Copolymers Glycidyl Methacrylate-ethylene Dimethacrylate, J. Appl. Polym. Sci., 1981, 26, 411. [10] L. Sprincl, J. Kopecek, D. Lim, Effect of the Structure of the Poly(glycol monomethacrylate) Gel on the Calcification of Implants, Calc. Tiss. Res., 1973, 13, 63. [11] S.D. Varfolomeev, L.P. Krylova, G.E. Zaikov “Molecular and nanosystems for energy conversion”, New York, Nova Science Publ., 2008, 224 pp. [12] S.K. Rakovsky, G.E. Zaikov “Kinetics and mechanism of ozone reactions with organic compounds”, Shawbury, Shrewsbury, Shropshire (UK), Rapra Technology, 2009, 288 pp.

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[3]

11

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In: Handbook of Chemistry, Biochemistry … Editors: L. N. Shishkina et al., pp. 13-21

ISBN: 978-1-61209-425-0 © 2010 Nova Science Publishers, Inc.

Chapter 2

HYDROGELS IN ENDOVASCULAR EMBOLIZATION PART II. CLINICAL USE OF SPHERICAL PARTICLES Daniel Horak1, K.Z. Gumargalieva2*, G.E. Zaikov3**, and L.A. Zimina**

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Institute of Macromolecular Chemistry, Czech Academy of Sciences, Prague, Czech Republic *N.N. Semenov Institute of Chemical Physics, Russian Academy of Sciences, Moscow, Russia **N.M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences Moscow , Russia

ABSTRACT Experience provided by successful clinical applications of spherical emboli prepared from poly(2-hydroxyethyl methacrylate) and intended for the occlusion of blood vessels allows us to infer that this material, which can be obtained by employing a simple procedure, is nontoxic, perfectly biocompatible, and possesses properties that make it particularly suitable for stopping haemorrhage by the method of transcatheter embolization.

Keywords: hydrogels, spherical particles, clinical testing, blood vessels, embolization.

1 2 Heyrovskogo sq., Prague-6, Czech Republic. 2 4 Kosygin str., Moscow 119991, Russia. 3 4 Kosygin str., Moscow 119334, Russia, [email protected]. Handbook of Chemistry, Biochemistry and Biology : New Frontiers, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

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2.1. INTRODUCTION In the preceding part of hydrogels series and the paper [1] we reported the synthesis and physicochemical and medico-biological properties of biocompatible spherical hydrogel particles based on poly(2-hydroxyethyl methacrylate). These particles (called artificial emboli) were prepared for the occlusion of blood vessels, especially in pulmonary haemorrhage. Pulmonary haemorrhage (bleeding and haemoptysis) is one of the most frequent causes for seeking medical assistance; at the same time, it is one of the most serious complications, which affects considerably the course of the disease and the prognosis for both the working ability and for life itself. Pulmonary haemorrhage is a phenomenon accompanying many lung and heart diseases. According to various authors, 7–15% patients admitted to hospital for chest diseases suffer from haemoptysis or haemorrhage [2, 3]. Pulmonary haemorrhage is the main cause underlying increased death which in the case of strong haemorrhage reaches an average of 20% of the total number of patients suffering from it. In those cases where the surgical procedure used to stop the bleeding is rather risky for the patient's life due to his/ her grave health state, or to the extensive character of the process, haemorrhage can be stopped by introducing artificial emboli into the blood stream. A successful course of such treatment is considerably dependent on the properties of emboli chosen for occlusion. Up to now, materials used for this purpose were randomly selected and differed diametrically in their chemical structure and properties. Gelatine sponge emboli are those most widely used [4]. Several other materials are available, compressible as well as noncompressible. Muscle emboli, as well as autogenous blood clots have been used as compressible emboli, but have the disadvantage of being rapidly dissolved. Intravascular tissue adhesives constitute another category of embolic material [5]. These plastic materials, polymerizing after injection into the blood stream, are difficult to handle and so far this has limited their widespread use. The shortcomings of haemostatic sponges based on gelatine (e.g. Gelaspon, manufactured in GDR) are that within 2–4 weeks they undergo lysis, and revascularization commonly occurs. It is recommended that, when Spongostan is used as an embolic material preoperatively in order to decrease the bleeding at a subsequent operation, the operation should be performed within the first few days following embolization, before revascularization has taken place. Some other materials, such as china or steel beads, spherical microparticles made from polystyrene, suspension of barium sulphate, polyurethane sponge, or poly(methyl methacrylate) also did not meet the surgeon’s requirements [1]. At present, special embolization in surgical practice is implemented with Gianturco coils in combination with velour felt from teflon [6]. However, their use is also associated with the following disadvantages. Firstly, the manufacture of the coils, which must be prepared for each patient individually immediately before the operation, is not a simple procedure and secondly, the transcatheter introduction is not easy to use. In addition application of the coil may produce aneurysm of the occluded blood vessel [7]. Consequently development of biocompatible emboli suited to the occlusion of blood vessels and studies of the indications and counter-indications for their clinical application are of great practical importance. Devascularization by embolization is indicated in tumors and vascular malformations either as a complement to surgery or as a single occlusive measure. Facilitation of surgery by preoperative embolization, or the occasional transformation of an inoperable lesion into one that is surgically accessible, is of practical interest, e.g. in

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15

therapeutic transcatheter embolization in the region of the external carotid artery [8]. Embolization offers the possibility of occluding arteries far from the periphery, by selecting emboli of a suitable size. The ideal would be to begin with small ernboli to occlude the peripheral branches and then to continue with larger emboli in order to achieve a more proximal occlusion of the main feeding arteries [9]. In this part of the Chapter some aspects of the preparation of spherical emboli from poly(2-hydroxyethyl methacrylate) and the clinical results obtained from their application in termination or reduction of haemorrhage are discussed.

2.2. EXPERIMENTAL SECTION 2.2.1. Materials The procedure used in the preparation of hydrogel particles and their classification according to size, has been described earlier [1]. A narrow particle size distribution according to the diameter of the vessels to be embolized (see Results and Discussion) is always required by the surgeons. Small particle size is employed in cases where at first small vessels are to be embolized and then larger ones, as a large embolus could prevent embolization of small vessels. Polydispersity is therefore undesirable. Particle size, in the dry and swollen state, was determined on the same particles as those used by surgeons in clinical applications.

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2.2.2. Methods The rate (virtually kinetics) of swelling of spherical particles in blood was determined according to the following method. Samples of spherical emboli on a support made from unwoven linen cloth, I mm thick, were placed on a perforated plate (Figure 2.1) (25 perforations in an area 3.5 cm in diam.; perforation diam. 3 mm) which was placed in a vessel thermostatted to 37°C and filled with citrated blood. An increase in the size of each particle was measured from the side in a horizontal GM 3 microscope equipped with an ocular micrometer. To obtain a statistical viable change in particle size at least 100 particles were measured.

Figure 2.1. Apparatus for the determination of rate of swelling of porous materials: 1 - sample; 2 perforated plate; 3 - thermostatted vessel; 4 - float. Handbook of Chemistry, Biochemistry and Biology : New Frontiers, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

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Spherical particles were purified for medical use [1], in the container sterilized in vapour at 120°C, at 11 Pa, for 8 min; the quality of sterilization of each batch was bacteriologically tested. The following method was used for the introduction of sterile spherical particles: firstly, 10–15 ml of 0.25% Novocaine solution was infused into the vascular lumen through a catheter with an i.d. of 1 mm, then 20 ml of contrast medium (76% solution of Verografin) was injected, so that the final correct positioning of the catheter in the branch to be embolized could be confirmed. The blood vessels were then embolized by injecting a suspension of emboli, particle size 0.4 - 0.6 and 0.6 - 0.8 mm, in saline, through a catheter. The number of emboli needed for occlusion of a main branch vessel varied depending on the type of lesion and size of the vessel. The whole amount of the saline was about 200 g. The end-result was always documented angiographically before withdrawal of the catheter from the artery. In the presence of a pathological hyper-vascularization of large arteriovenous anastomoses in the focus, larger emboli (0.8 mm) were injected first, followed by smaller ones (0.4 - 0.6 mm). In such cases the particles were classified in advance using stainless steel templates with apertures 0.4, 0.5, 0.6, 0.7 and 0.8mm [1]. Used particles were already graded by sieving into fractions 0.4 - 0.6 and 0.6 - 0.8 mm. In the case of pathological hyper-vascularization of small blood vessels without anastomoses the occlusion began with small emboli which were replaced step by step with larger ones, until the vessels were closed from the periphery to the centre. The degree and character of occlusion were continuously checked by angiography.

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2.3. RESULTS AND DISCUSSION One of the main requirements of spherical particles used in embolization is a narrow particle size distribution. The presence of very small particles in the overall amount of emboli may lead to a grave complication, i.e. tissue necrosis due to the intrusion of emboli into blood vessels not intended for embolization. The presence of large particles means the danger of the catheter being occluded and this would necessitate its exchange during the course of the operation. This raises the degree of traumatization of the tissues and extends the operation time. Another, very important parameter in the selection of emboli for the occlusion of blood vessels consists in an ability to increase their volume in contact with blood. This is of extreme importance if the emboli are to be quickly and reliably fixed in the blood stream. Figure 2.2 shows the time dependence of the swelling of the emboli in various liquid media. The increase in size of hydrogel particles in blood is significantly larger than in water or saline (by 10%). This facilitates subsequent quick fixation of the embolus in the blood stream where it arrives from the catheter in saline, since - as shown in Figure 5.2.2 - equilibrium swelling is reached within 2 - 3 min after contact with blood. The increase in the particle size in blood compared with water or saline may be explained by the adsorption of blood proteins and aggregation of thrombocytes on the particle surface and by coagulation of blood.

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Hydrogels in Endovascular Embolization. Part II Clinical Use of Spherical Particles

Figure 2.2. Time dependence of the relative particle size

⎛ d⎞ ⎜ ⎟ ⎝ d0 ⎠

17

in various media: 1 - citrated blood; 2

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- saline; 3 - water: d0 is the particle diam. in the dry state; d the particle diam. in the swollen state.

Spherical emboli made from poly(2-hydroxyethyl methacrylate) were used to stop haemorrhage in patients suffering from lung and heart diseases. Figure 2.3 (a, b) shows arteriograms of bronchial blood vessels before and after the embolization of a patient, aged 22, with a diagnosis of chronic pneumonia and pulmonary abscess. During the previous 3 months he was suffering from pulmonary haemorrhage. Conservative treatment was ineffective. Arteriography showed hyperplasia of bronchial arteries with foci of hypervascularization in the abscess zone (Figure 2.3a). The trunk of the artery is widened and twisted and the number of peripheral branches is abnormally large. One can see foci of hypervascularization of lung tissue. Transcatheter endovascular occlusion of bronchial arteries was carried out with hydrogel beads, 0.4 - 0.6 mm in size. The amount of wet beads measured by volume was 1.5 cm3. After the embolization was completed all pathologically degenerated branches responsible for haemorrhage disappeared; only the beginning of the trunk of the bronchial artery is contrasted (Figure 2.3b). Haemorrhage had stopped. No relapses were recorded. A control check-up after 4 months revealed that the amount of released sputum was much smaller. Sputum changed from purulent to mucous, which suggested a positive effect of occlusion on the inflammatory process in the lungs. Emboli made from poly(2-hydroxyethyl methacrylate) were further used in the transcatheter occlusion of patients suffering from Fallot’s tetralogy. In the pathogenesis of Fallot’s tetralogy an important place is held by the hyperplasia of bronchial and other systemic pulmonary arteries. The hyperplasia of collateral arteries brings about grave complications,

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with pulmonary haemorrhage being the most frequent one. A conservative treatment of pulmonary haemorrhage in Fallot’s tetralogy does not give the desired results, and the existing ways of operative treatment involve a considerable risk to the patient’s life. Figure 2.4 (a, b) shows arteriograms of bronchial arteries before and after the embolization of the patient, aged 14, with diagnosis of Fallot’s tetralogy and dextrocardia. When hospitalized, the patient suffered from frequent haemoptysis. The blood volume in haemoptysis was 50 - 70 ml. Medical examination revealed (Figure 2.4a) a considerably widened trunk of the intercostal artery which supplied blood to the left lung and to the third left intercostal space. Bronchial arteries were considerably twisted and formed loops. In the peripheral parts of the artery one can see foci of hypervascularization with bronchopulmonary shunts.

Figure 2.3. An arteriogram of bronchial blood vessels of a patient suffering from chronic pneumonia and pulmonary abscess before faf and after fbi embolization.

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Figure 2.4. An arteriogram of the trunk of the intercostal artery of a patient suffering from Fallet’s tetralogy and dextrocardia before (a) and after (b) embolization.

Transcatheter occlusion of the intercostal artery was achieved using poly(2-hydroxyethyl methacrylate) beads, 0.4–0.6 mm in diam., in an amount corresponding to 2.5 ml. During the occlusion of the bronchial artery with the emboli the bronchopulmonary anastomoses and the branched network of arteries which were the source of the haemorrhage were blocked. After successful embolization the control angiography (Figure 2.4b) showed that only that part of the basic trunk of the artery which does not contain emboli is contrasted; the blocked blood vessels do not contrast. Haemoptysis did not occur any more. The patient was released in a satisfactory state. A later check-up after 24 months confirmed the correctness of the treatment. Hydrogel spherical particles were used in 187 patients in the occlusion of bronchial, renal, mesenteric, hypogastrica interna and other arteries in order to stop organic haemorrhage and to

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Daniel Horak, K.Z. Gumargalieva, G.E. Zaikov et al.

influence the course of the disease. Due to the large inner diameter of the anastomoses, large ernboli, 0.8 mm in size, were applied to 50 of 175 patients suffering from arterovenous anastomoses in the focus of hypervascularization. An evident haemostatic and clinical effect could be seen in an immediate and later (1 - 2 years) observation. In the cases of haemorrhage and haemoptysis the introduction ofemboli resulted in an immediate haemostatic effect in 86.6% of cases. Unsatisfactory results were caused by incomplete occlusion of the main feeding arteries, by some special features of the disease and in 3 cases by the grave state of the patient. Otherwise even after a considerable time interval, the results of embolization ranged from good to excellent in 92% cases. Experience provided by successful clinical applications of spherical emboli prepared from poly(2-hydroxyethyl methacrylate) and intended for the occlusion of blood vessels allows us to infer that this material, which can be obtained by employing a simple procedure, is nontoxic, perfectly biocompatible, and possesses properties which make it particularly suitable for stopping haemorrhage by the method of transcatheter embolization. A forthcoming part will deal with the preparation and properties of X-ray contrast spherical hydrogel particles [10-12].

REFERENCES [1]

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[2]

[3] [4]

[5]

[6] [7] [8] [9]

D. Horak, F. Svec, J. Kalal, K.Z. Gumargalieva, A.A. Adamyan, N.D. Skuba, M.I. Titova., N.V. Trostenyuk, Hydrogels in Endovascular Embolization. I. Spherical Particles of Poly(2-hydroxyethyl methacrylate) and Their Medico-biological Properties, Biomaterials, 1986, 7, 188-192. M.I. Kuzin, Yu. D. Volynskii, Etiology and Pathogenesis of Pulmonary Hemorrhage, In: Endovascular Therapeutics and Surgery of Pulmonary Hemorrhage, VostochnoSibirskaya Pravda, Irkutsk, 1981, 7-11. (Rus) J.D. Wolfe, D.U. Simmons, Hemoptysis Diagnosis and Management, West. J. Med., 1977, 127, 383-390. I. Kh. Rabkin, M.I. Perelman, Yu.V. Biryukov, J.N. Gotman, R.K. Ambrozaitis, Experiment of Therapeutic Embolization of Bronchial Arteries in Pulmonary Hemorrhage and Hemoptysis, Khirurgiya, 1979, 12, 8-13. (Rus) J. Clarisse, G. Gozet, J.P. Cornil, M. Jomin, J.M. Delandsheer, E. Laine, Les Emboles Plastiques Fluides. Etude Experimentale et Rapport de Deux Cas Cliniques de Fistulas Arterioveineuses Trairtes, J. Neuroradiol., 1975, 2, 29-38. I.Kh. Rabkin, V.A. Klimanskii, L.N. Gotman, G.N. Zakharov, Embolization of Splenic Artery with Disease of Blood System, Khirurgiya, 1981, 2, 53-55. (Rus) L. Vlahos, S. Trakadas, P. Kehyas, G. Pontifex, Transcatheter Arterial Embolization With Gianturco Steel Coils: A Complication, Acta Urol. Belg., 1983, 51, 256-260. J. Brismar, S. Cronquist, Therapeutic Embolization in the External Carotid Artery Region, Acta Radial. Diagn., 1978, 19, 715-731. L. Picard, J.M. Andre, J. Roland, M. Sigiel, J. Montaut, J. Lepoire, L’embolisation dans les Malformations Vasculaires Meningo-cranio-cutanees Complexes, J. Neuroradiol., 1975, 2, 233-256.

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[10] B.A. Howell, N.G. Lekishvili, G.E. Zaikov. Compounds and materials with specific properties. New York: Nova Science Publ., 2008, 386 pp. [11] G.V. Kozlov, G.E. Zaikov. Fractal analysis in nanosystems, New York: Nova Science Publ., 2008, 198 pp. [12] R. Thonggoom, Th. Rochanopruk.. Emerging topics in organic chemistry, New York: Nova Science Publ., 2008, 312 pp.

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In: Handbook of Chemistry, Biochemistry … Editors: L. N. Shishkina et al., pp. 23-32

ISBN: 978-1-61209-425-0 © 2010 Nova Science Publishers, Inc.

Chapter 3

HYDROGELS IN ENDOVASCULAR EMBOLIZATION PART III. RADIOPAQUE SPHERICAL PARTICLES, THEIR PREPARATION AND PROPERTIES

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Daniel Horak1, K.Z. Gumargalieva2*, G.E. Zaikov3**, and L.L. Madyuskina** Institute of Macromolecular Chemistry, Czech Academy of Sciences, Prague, Czech Republic *N.N. Semenov Institute of Chemical Physics, Russian Academy of Sciences Moscow , Russia **N.M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences Moscow , Russia

ABSTRACT It can be seen that in practice a compromise should be sought between particles with a high iodine content, which can be roentgenologically perfectly monitored, and those with a low iodine content, which swell well. Moreover, it is necessary for medical applications that emboli should also preserve a sufficient mechanical stability. These requirements are best met by particles with a 25–30 wt.% of iodine which can be adequately monitored when being introduced into the blood vessels and which also enable the checking of their performance in viva without the necessity for angiography.

Keywords: radiopaque, preparation, properties, hydrogels, spherical particles, iodine compounds. 1 2 Heyrovskogo sq., Prague-6, Czech Republic. 2 4 Kosygin str., Moscow 119991, Russia. 3 4 Kosygin str., Moscow 119334, Russia,[email protected].

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3.1 INTRODUCTION In preceding communications in this series, we have reported the preparation of spherical hydrogel particles, their properties [1] and clinical performance in vascular occlusion [2]. They were employed for the embolization of tumor arterial blood supply, either in inoperable tumors or to prevent haemorrhage during surgery. Embolization is used in the treatment of nonmalignant and malignant neoplasmas, especially haemangiomas, arteriovenous anastomoses and vascular malformations, organ haemorrhage and haemoptysis, and the like. Soft and readily swellable spherical hydrogel microparticles have many advantages in clinical applications: they are perfectly biocompatible, the living tissue grows through them without inflammation or causing destructive dystrophic changes. The thrombus is formed immediately after the application of the embolus which is quickly fixed in the vascular lumen where it has a permanent haemostatic effect without recanalization. Last but not least, the biggest advantage of these particles is the simplicity of their introduction into the bloodstream by means of a syringe and catheter. On the contrary, materials used so far in therapeutical practice present many shortcomings, e.g., only a short-term occlusion effect, the necessity of a complicated operation, insufficient biocompatibility, and also lack of radiopacity. However, in order to keep embolization under control, permanent surveillance of the embolization process and implant’s behaviour in the postoperation period is imperative. Up to now, this process has been monitored only by angiography. Therefore, radiopaque emboli, which can be directly monitored by X-ray camera immediately after their introduction, as well as for a long period of time after implantation, represent a qualitatively higher category of material suitable for the embolization of blood vessels. The first X-ray contrast materials available for the occlusion of an arteriovenous fistula were fluid plastic emboli containing radiopaque oil; however, certain toxicity, and difficult handling during influx [3, 4] limit their application. Recently, the gels for therapeutic embolization, containing X-ray contrast media, have been reported in the Patent literature. They are used to prevent haemorrhage or to produce ischaemia of malignant tumors. Celling agents are based on cellulose [5], gelatin [6] , silicone rubber [6], acetylcellulose [7], or a copolymer of vinyl pyrrolidone and methyl methacrylate [7]. The latter two materials clog the catheter in cases where the addition of poly(ethylene glycol) is omitted. Tantalum powder or Fe2O3⋅6BaO is also recommended [7] as a contrast substance. However, by using these substances in vivo, the radiopaque additive is readily washed out from the embolus. This shortcoming can be overcome by particles with the radiopaque compound chemically bonded to the polymer chain. In this study we report the synthesis arid properties of such spherical hydrogel particles.

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3.2. EXPERIMENTAL 3.2.1. Materials Monomers and Auxiliary Compounds The chemicals were redistilled prior to use: ethylene dimethacrylate (Ugilor S.A., France) - b.p. 50°C/7 Pa, cyclohexanol (Lachema Brno) - b.p. 68°C/l .6 kPa, decahydronaphthalene (Lachema Brno), methacrylic acid (Ferak, Berlin), thionylchloride (Cambrian Chemicals, Great Britain), dioxan. 2,2’-Azo(bis-isobutyronitrile) (Ferak, Berlin) was recrystallized twice from ethanol. 2-Hydroxyethyl methacrylate (Leciva Modrrany), medicinal purity, was used without purification. The other chemicals used were reagent grade. The preparation of poly(1dodecyl methacrylate) has been described in a previous communication [1].

3.2.2. Methods

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Preparation of Particles Containing Barium Sulphate by Inverse Phase Suspension Polymerization The 250 ml glass reactor equipped with an anchor-type stirrer was filled with 25 ml of dispersed phase containing 15 ml of a 20 wt.% aqueous solution of barium chloride, 9.3 ml 2hydroxyethyl methacrylate, 0.3 ml ethylene dimethacrylate, 0.4 ml methacrylate acid and 0.l g 2,2’-azo(bis-isobutyronitrile) and 75 ml of dispersing medium consisting of a 5 wt.% solution of poly(1-dodecyl methacrylate) (molecular weight 4×106) in decahydronaphthalene. The polymerization mixture was degassed by bubbling nitrogen through for l0 min. The polymerization proceeded under continuous stirring (400 rpm) at 70°C for 8 h. After cooling the polymer was obtained as an agglomerate of particles difficult to process. It was separated and decanted by hexane and ether. Barium sulphate was generated by 5% sulphuric acid. Preparation of Particles Containing Iopanoic Acid by Suspension Polymerization 25 ml dispersed phase containing l.3 g iopanoic acid [β-(3-amino-2,4,6-triiodophenyl)-αethylpropionic acid - trade name Jopagnost Spofa] dissolved together with a 0.1 g 2,2’-azo(bisisobutyronitrile) in 7.5 ml 1-dodecanol (Fluka A.G.), 7.5 ml cyclohexanol, 9.8 ml 2hydroxyethyl methacrylate and 0.2 ml ethylene dimethacrylate was placed in the reactor followed by 75 ml of a 1% aqueous poly(vinyl pyrrolidone) K-90 (Fluka A.G.) as the dispersing medium. Turbidity due to the Jopagnost precipitation occurred after the addition of the aqueous dispersing phase. After bubbling nitrogen through the polymerization proceeded at 70°C for 8 h under continuous stirring at 240 rpm. The resulting beads were separated, decanted by water, methanol, acetone and ether, and dried. Preparation of Particles by a Modification of the Glycidyl Methacrylate - Ethylene Dimethacrylate Copolymer with 2-Aminoethylamide of 3-Amino-2,4,6-Triiodobenzoic Acid The preparation of the sample spherical macroporous copolymer of glycidyl methacrylate (75%) and ethylene dimethacrylate (25%) has been described elsewhere [8]. 2Aminoethylamide of 3-amino-2,4,6-triiodobenzoic acid was synthesized according to a

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26

Czechoslovak Patent [9] by reacting ethyl 3-aminobenzoatewith ethylenediamine (Fluka A.G.) followed by iodination. The glycidyl methacrylate beads (150 - 180 mm) (0.1 g) were soaked with 6 ml dimethylformamide containing 0.6 g of 2-amino ethylamide of 3-amino-2,4,6triiodobenzoic acid, and the suspension was shaken in a thermostatted bath at 60°C for 260 h. The product was washed with dimethylformamide, acetone, ether, and dried.

The Acylation of 2-Hydroxyethyl Methacrylate-Ethylene Dimethacfylate Particles with 3-Acetylamino-2,4,6-Triiodobenzoyl Chloride This was according to the Scheme:

COCl I COOCH2

CH2

OH

I

+ NHCOCH3 I

COOCH2

CH2O CO I

I

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NHCOCH3 I Table 3.1.Modification of 2-hydroxyethyl methacrylate-ethylene dimethacryiate particles

No.

Molar ratio ROS/-OH

TEA/ROS

1 2 3 4 5 6 7

2 2 1 1 0.5 0.5 0.5

1.1 ⎯ 1.1 ⎯ 1.1 ⎯ 1.1

Concentration, mmol ROS/ ml dioxan 0.3 0.3 0.2 0.2 0.1 0.1 0.15

Iodine content in product, wt.% 43.5 21.7 34.9 14.4 23.4 8.4 35.1

ROS, Radiopaque substance (3-acetylamino-2,4,6-triiodobenzoyl chloride); -OH, hydroxy groups of poly(2-hydroxyethyl methacrylate): size of sample beads 1 - 1.2 mm; TEA, triethylamine. See Experimental section for details.

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The synthesis of sample hydrogel beads by the suspension radical copolymerization of 2hydroxyethyl methacrylate (98%) with ethylene dimethacrylate (2%) has been described earlier [1]. 3-Acetylamino-2,4,6-triiodobenzoyl chloride was prepared [10] by reacting 3acetylamino-2,4,6-triiodobenzoic acid (obtained from 2,4,6-triiodobenzoic acid, Leciva Modrrany) with thionylchloride. In a typical reaction, 0.13 g (0.225 mmol) 3-acetylamino2,4,6-triiodobenzoyl chloride was dissolved in 2 ml of dry dioxan with stirring at 50°C. After cooling to room temperature, 0.035 ml (0.25 mmol) triethylamine and 0.06 g (0.45 mmol -OH) hydrogel particles, 1 - 1.2 mm in size, were added. The mixture was shaken in a thermostatted bath at 60°C for 150 h. The particles were then washed with dioxan and water until the OD230 of washing water falls below 0.03 (1 cm cell). Finally, the particles were washed 10 times each by acetone and ether, respectively (each portion contained ca. 15 ml of solvent) and then vacuum dried at room temperature. Polymer no. 5 (Table 3.1) was obtained according to this procedure. In the following experiments, the concentration and molar ratio of the radiopaque substance to the hydroxy groups were varied, or triethylamine was omitted (cf. Table 3.1).

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Preparation of Particles by a Modification of the Terpolymer of 2-Hydroxyethyl Methacrylate, Ethylene Dimethacrylate and Methacrylic Acid With 3-Acetylamino2,4,6-Triiodobenzoyl Chloride The sample terpolymer beads were prepared as described above for the preparation of particles by inverse phase suspension polymerization, the only difference being that the addition of barium chloride was omitted. For modification of the final beads, the same procedure was adopted as for the modification of 2-hydroxyethyl methacrylate - ethylene dimethacrylate particles with 3-acetylamino-2,4,6-triiodobenzoyl chloride. At least 100 particles, each of which was treated individually, were measured under the microscope to obtain a particle size in the dry and swollen state. Iodine was analysed by Schoniger’s method.

3.3. RESULTS AND DISCUSSION Several procedures for the preparation of radiopaque emboli have been tested. Firstly the suspension polymerization of 2-hydroxyethyl methacrylate in the inverse phase, where the dispersing phase consists of an organic liquid immiscible with water, and the aqueous solution of monomers constitutes the dispersed phase, has been exploited. In addition, barium chloride was dissolved in the aqueous phase and precipitated with sulphuric acid in the resulting product after the polymerization had been completed. However, this procedure failed because of the instability of the suspension due to the barium salt, and the resulting product in the form of an agglomerate became difficult to process. Another original procedure, in which the radiopaque substance is precipitated in the hydrogel network, uses two water-soluble compounds. The hydrogel swollen in an aqueous solution of one of these compounds is immersed into a solution of the other compound [11]. Yet another method describes the preparation of radiopaque hydrogel by dissolution in a polymerization mixture of a radiopaque substance insoluble in water and its precipitation in the gel after swelling with water [12]. However, when using iopanoic acid [P-(3-amino-2,4,6triiodophenyl)-α-ethylpropionic acid] dissolved in the polymerization mixture for the

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preparation of radiopaque beads according to the latter method, the precipitationoccurred immediately after the aqueous dispersing phase had been added, i.e., before the polymerization had started, and the resulting particles lacked radiopacity. In contrast to all the methods reported here, where the radiopaque substance remains unhanded, we preferred the covalent attachment of the X-ray adsorbing group to the polymer, which prevented its release in vivo and facilitated the washing out of all unreacted low-molecular weight compounds after the preparation.

Figure 3.1. X-ray images of emboli with various iodine contents: (a) 8.3% I; (b) 8.9% I; (c) 14.4% I; (d) 27.6% I; (e) 23.4% I; (f) 34.9% I; (g) 35.7% I; (h) 43.5% I.

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Modification of reactive glycidyl methacrylate - ethylene dimethacrylate beads using 2aminoethylamide of 3-amino-2,4,6-triiodobenzoic acid was attempted, since these beads are available on the market. Although the radiopaque substance was used in a two-molar excess compared with the epoxy groups, only 13.4 wt.% of iodine was detected in the final product, and therefore the particles did not provide enough contrast. Moreover, the glycidyl methacrylate polymers did not prove to be perfectly biocompatible [13]. In the preparation of radiopaque particles based on poly(2-hydroxyethyl methacrylate) by modification with 3-acetylamino-2,4,6-triiodobenzoyl chloride, the amount of bonded radiopaque substance may be markedly affected by the molar ratio of the iodine-containing derivative to the hydroxy groups of hydrogel particles. Also it is markedly affected by the concentration of 3-acetylamino-2,4,6-triiodobenzoyl chloride in dioxan and by the addition of hydrogen chloride acceptor (triethylamine). The amount of the bonded radiopaque substance increases with increasing molar ratio of the iodine-containing derivative with respect to hydroxy groups and with its increasing concentration in the solvent, and is raised by the addition of triethylamine (Table 1). Particles containing 43.5 wt.% iodine were the maximum content product achieved. The amount of bonded radiopaque substance also depends on the size of the hydrogel particles at the start. Under otherwise identical conditions, more of the iodine-containing compound is bonded in smaller particles (Table 2) due to some kind of ‘plugging’ effect (or gradual filling of all pores accessible to the radiopaque compound); the surface layer which is penetrated by the iodinated molecule is limited. This layer represents a higher portion of the total bead mass in smaller particles, and therefore a higher average iodine content can be reached with smaller particles. As shown in Figure 3.1, thecontrast of the emboli on the X-ray screen is directly related to the iodine content. The large volume of the molecule of triiodobenzoic acid derivative is one of the reasons why the size of the hydrogel particles increases with the degree of substitution (Table 3). Thus, e.g., at 43.5 wt.% iodine the particle diameter in the dry state is larger after the first and second drying cycles by 9% and 68%, respectively, compared with unmodified particles. It is known [14], that the porous structure of the original poly(2-hydroxyethyl methacrylate) particles collapses after two drying-swelling cycles and the particles become glassy. Modification by 3-acetylamino-2,4,6-triiodobenzoyl chloride partially suppressed this phenomenon, depending on the degree of substitution. At 43.5 wt.% iodine the radiopaque substance attached to the particles prevents the shrinking of the sample swollen beads during drying. Originally in the dry nonmodified beads polymer chains were coiled. Upon swelling these beads show a 20% increase in diameter and are fixed by acylation, because bulky iodinecontaining substituents prevent the original compact (coiled) arrange ment of polymer chains after drying. After transfer to water there are no more changes in the size of the modified particles i.e. the beads do not swell. However, beads with a lower content of the radiopaque substance still partially retain their swelling ability. Thus, e.g. beads containing 10 wt.% iodine increase their diameter in water by 14%. Moreover, Table 3 shows the tendency to shrinking, i.e. to a decrease in the size of the dry particles after two cycles of drying and swelling which is less pronounced at higher iodine contents. Highly iodinated particles are harder, more brittle and less hydrophilic.

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30

Table 2. The size effect of 2-hydroxyethyl methacrylate - ethyiene dimethacrylate particles on their acylation by 3-acetylamino-2,4,6-triiodobenzoyl chloride (given as the iodine content). Fraction with particle sixe, mm 0.6 - 0.8 0.8 - 1.0 1.0 - 1.2 1.2 - 1.5

Iodine content in product, wt.% 30.6 29.3 28.7 27.9

Reaction conditions: molar ratio ROS/-OH = 0.5; TEA/ROS = 1.1; concentration is 0.12 mmol ROS/ml dioxan. For meaning of abbreviations see Table 1.

The low swelling of the particles impedes medical applications. The size of the emboli should facilitate the passage through the catheter, and at the same time they should swell in the bloodstream so as to be well fixed in the blood vessel lumen. It is known [15] that the connective tissue grows much more easily through high swelling porous particles. The swelling of particles could be aided by copolymerization using several percent of methacrylic acid. Modified particles of such terpolymer in the Na+ form containing 9.2 or 23.5 wt.% iodine increase in size in an isotonic solution of sodium chloride by 33% and 16%, respectively. Preliminary tests have shown, however, that already a small amount (4 vol.%) of methacrylic acid in hydrogel particles leads to blood haemolysis.

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Table 3. Effect of iodine content on the size of radiopaque particles based on poly(2hydroxyethyl methacrylate) in the dry, swollen in water, and the dried again states Iodine content, wt.% 8.9 14.4 21.7 23.4 35.1 43.5

Size of particles, mm Dry 0.66 0.75 0.77 0.84 1.03 1.11

Swollen 0.76 0.81 0.82 0.89 1.07 1.11

Dried again 0.62 0.73 0.76 0.83 1.02 1.10

Size of poly(2-hydroxyethyl methacrylate) sample particles before start of reaction: in the dry state 1.02; swollen in water 1.23; and the dried again state 0.66 mm.

Thus, it can be seen that in practice a compromise should be sought between particles with a high iodine content, which can be roentgenologically perfectly monitored, and those with a low iodine content, which swell well. Moreover, it is necessary for medical applications that emboli should also preserve a sufficient mechanical stability. These requirements are best met by particles with a 25–30 wt.% of iodine which can be adequately monitored when being introduced into the blood vessels and which also enable the checking of their performance in viva without the necessity for angiography. Histological tests and clinical applications of X-ray contrast spherical particles will be reported in a future paper in this series [16-18].

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Hydrogels in Endovascular Embolization Part III…

31

REFERENCES [1]

[2]

[3]

[4] [5] [6]

[7]

[8]

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[9] [10] [11]

[12]

[13] [14] [15]

[16]

D. Horak, F. Svec, J. Kalal, K.Z. Gumargalieva, A.A. Adamyan, N.D. Skuba, M.I. Titova, N.V. Trostenyuk, Hydrogels in Endovascular Embolization. I. Spherical Particles of Poly(2-hydroxyethyl methacrylate) and Their Medico-biological Properties, Biomaterials, 1986, 7, 188-192. D. Horak, F. Svec, J. Kalal, A.A. Adamyan, Yu.D. Volynskii, O.S. Voronkova, L.S. Kokov, K.Z. Gumargalieva, Hydrogels in Endovascular Embolization. II. Clinical Use of Spherical Particles, Biomaterials, 1986, 7, 467-470. J. Clarisse, G. Gozet, J.P. Cornil, M. Jorum, J.M. Delandsheer, E. Laine, Les Emboles Plastiques Fluides. Etude Experimentale et Rapport de Deux Cas Cliniques de Fistules Arterioveineuses Traitees, J. Neuroradiol., 1975, 2, 29-38. R.E. Krall, Radiopaque Cyanoacrylate Compositions, Europ. Patent Appl., No. 50457, 1982. P. Trampont, P. Madoule, B. Bonnemain, F. Puisieux, Gel for Therapeutic Embolization, Fr. Demande, No. 2 548 902, 1985. L. Lazar, K. Palossy-Becker, J. Naggy, E. Pasztor, Pharmaceutically Acceptable Silicone Rubber and Therapeutical Set and Their Use for Surgical Embolization, Can. Patent No. 1 182 124, 1985. V. D. Solodovnik, T.I. Solodkaya, M.T. Litvinova, T.A. Meshkova, A.V. Usova, A.B. Davydov, Composition for the Embolization of Blood Vessels/Radiopaques, Brit. Patent No. 2144327, 1985. D. Horak, Z. Pelbauer, M. Biota, M. Ilavsky, F. Svec, J. Kalal, Reactive Polymers. XXXII. Effect of Composition of Polymerization Feed on Morphology and Some Physical Properties of Macroporous Suspension Copolymers Glycidyl Methacrylate Ethylene Dimethacrylate, J. Appl. Polym. Sci., 1981, 26, 411-421. J. Drobnik, J. Kalal, M. Metalova, F. Rypdcek, Macromolecular X-ray Contrast Substance and Method of Its Preparation, Czech. Patent No. 207858, 1984. H. Priewe, R. Rutkowski, K. Pirner, K. Junkmann, K., Derivate der 2,4,6-triiod-3-aminobenzoesaure, Chem. Ber., 1954, 87, 651-658. N.V. Trostenyuk, K.Z. Gumargalieva, Yu.V. Moiseev, P. Lopour, J. Sulc, J. Vacik, J. Kalal, The Method of Preparation of Hydrogels with X-ray Contrast Properties, Czech. Patent Appl. No. 231700, 1984. P. Lopour, J. Sulc, J. Vacik, J. Kalal, T.T. Daurova, O.S. Voronkova, N.D. Skuba, The Method of Preparation of X-ray Contrast Hydrogel, Czech. Patent Appl. No. 231699, 1984. K. Filip, PhD Thesis, Institute of Clinical and Experimental Medicine, Prague, 1986. J. Hasa, PhD Thesis, Institute of Macromolecular Chemistry, Prague, 1966. L. Sprincl, J. Kopecek, D. Lin, Effect of Porosity of Heterogeneous Poly(glycol monomethacrylate) Gels on the Healing-in of Test Implants, J. Biomed. Mater. Res., 1971, 5, 447-458. G.E. Zaikov, V.G. Zaikov “Methods and theories in physical organic chemistry”, New York, Nova Science Publ., 2008, 209 pp.

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[17] Kukovinets, R.A. Zainullin, M.I. Abdullin, R.V. Kunakova, G.E. Zaikov “Chemical and physical methods for protection of biopolymers against pests“, New York, Nova Science Publ., 2008, 238 pp. [18] A.K. Mikitaev, M. Kh. Ligidov, G.E. Zaikov “Modern tendencies in organic and bioorganic chemistry. Today and tomorrow”, New York, Nova Science Publ., 2008, 428 pp.

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Chapter 4

HYDROGELS IN ENDOVASCULAR EMBOLIZATION PART IV. MORPHOLOGICAL FOUNDATION OF HYDROGEL USE FOR VASCULAR OCCLUSION Daniel Horak1, K.Z. Gumargalieva2*, G.E. Zaikov3**, and N.N. Madyuskin**

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Institute of Macromolecular Chemistry, Czech Academy of Sciences, Prague, Czech Republic *N.N. Semenov Institute of Chemical Physics, Russian Academy of Sciences Moscow, Russia **N.M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia

ABSTRACT Clinical observations give evidence to the fact that hydrogel emboli can be considered as a material that is absolutely suitable and can be successfully used for therapeutic occlusion of blood vessels.

Keywords: morphology, vascular, hydrogels, occlusion, animals, collagen, fibrocytes, emboly.

1 2 Heyrovskogo sq., Prague-6, Czech Republic 2 4 Kosygin str., Moscow 119991, Russia 3 4 Kosygin str., Moscow 119334, Russia, [email protected]. Handbook of Chemistry, Biochemistry and Biology : New Frontiers, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

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4.1. INTRODUCTION In clinical practice, one can often face such situations, when conventional methods of hemorrhage control are not feasible or they require complicated surgical procedures threatening the patient’s life [1]. Bronchial hemorrhage is the most vivid example in this respect, as it is conditioned by corrosion of the blood vessel wall. In such cases the safest and, at the same time, the most reliable method of hemorrhage control is endovascular occlusion by means of injection of different artificial emboli into the vessels [2]. Embolization is also needed in vascular surgery as a primary condition in the preoperative preparation of patients before excision of vascular tumors. In those cases when due to localization of a vascular tumor or developmental anomaly of vessels their removal is impossible, endovascular occlusion is the only possible way of treatment. The possibility of successful therapeutic endovascular occlusion depends upon the correct choice of the material used for embolus production. At present, the question of selection of the material for embolization purposes has not been solved definitively [3]. The aim of the present study is to determine the character of hydrogel embolus interaction with blood, vascular walls and with body soft tissues.

4.2. MATERIALS AND METHODS The material used in this study was:

1) femoral arteries of rabbits (10 animals) occluded with hydrogel emboli; 2) soft periembolar tissues, the subcutaneously implanted emboli to rabbits (10 animals); 3) blood vessels and renal tissue of patients with renal tumors after renal artery occlusion Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

with hydrogel emboli (2 patients);

4) bronchial arteries and pulmonary tissue of patients suffering from chronic nonspecific pulmonary diseases (21patlents).

Figure 4.1. The cross section of the femoral artery 0.5 cm proximal to the embolus. Thrombus is in the vascular lumen, 1 month after the implantation. HE staining. Handbook of Chemistry, Biochemistry and Biology : New Frontiers, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

Hydrogels in Endovascular Embolization Part IV

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Materials were taken at different times after embolization, from a few days to 2 years. The main method of investigation was a histological one. In the early period (from 1 to 7 days after the embolus injection) microscopic studies of the histologic specimens revealed the microporous structure of the hydrogel. Its pores were identified by the presence in them of plasma proteins or polymorphonuclear neutrophils. The hydrogel substance itself was not stained by histologic dyes. The thrombotic masses are observed in sites of vascular lumen, that are not filled with emboli. Thrombosis of the vascular lumen also occurs involving a small distance of the vessel distally and proximally to the emboli localization, as shown in Figure 4.1.

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Figure 4.2. The hydrogel is in the vascular lumen, 1 month after the implantation. The vascular wall is not changed. The hydrogel in the early stage of resorption and organization. Stained after van Gieson.

The vascular wall in the site of embolus localization looked thinned and distended in all observations. It is explained by the fact that hydrogel has a property to swell directly in the vascular lumen or in the depth of body tissues. The swelling of hydrogel emboli is accompanied by a significant increase of their volume, which ensures stability of the occlusive effect and excludes their possible migration as a result of blood pressure. Stability of the obturating effect is ensured in the future by the formation of a firm aggregation of emboli and thrombotic masses in the interspaces between them. In neither of the observations made on the vascular wall, coming in contact with hydrogel, destructive-dystrophic changes, or inflammatory changes were noted (Figure 4.2). It is indicated by the absence of injuring and irritating effects of this material upon viable tissues. Similar results were received at the investigation of soft tissues adjoining the subcutaneously implanted hydrogel emboli: neither destruction, nor inflammatory reaction was noticed in them. The implanted material was gradually incapsulated. In studying the experimental material it was determined that in the late follow-up period (490 days) the surface layers of emboli were grown through with a connective tissue; connective cells (fibroblasts) were determined in the hydrogel pores as well as collagen fibres synthesized by them (Figure 4.3).

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Figure 4.3. Fibrocytes and collagen fibres are within the pores of the hydrogel embolus, 490 days after the occlusion. Destructive-dystrophic changes or inflammatory changes are not observed. HE staining.

A small number of foreign body giant cells was noted in the superficial layers of emboli. The presence of these cells indicated an insignificant embolus resorption. The phenomena of embolus organization and resorption were more marked, when the emboli were subcutaneously implanted, than when they were placed directly into the vascular lumen. In the late time implantation (490 days) a partial embolus destruction of purely mechanical character occurs. It explains the presence of chipped off hydrogel particles in lymphatic fissures close to the autocutaneously implanted emboli. Thus, the experimental studies have shown, that hydrogel is a material with such properties , that allow to recommend its use for blood vessel occlusion. Such features are: absence of injuring or irritating effect on tissues, capability to enhance thrombogenesis, while in contact with blood, the capability to provide a stable obturating effect. The results got in the experimental work allowed us to use hydrogel for blood vessel occlusion in clinical conditions. The successful endovascular occlusion with hydrogel emboli was performed on 110 patients in the Vishnevsky Surgery Institute. The data received on the operational material are essentially the same as in the experiment (Figure 4.4).

Figure 4.4. The lumen of a small bronchial artery filled up by a hydrogel embolus. The wall is thinned, 2 days after endovascular occlusion. HE staining. Handbook of Chemistry, Biochemistry and Biology : New Frontiers, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

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Neither inflammatory reaction, nor any other destructive changes were noted in the wall of bronchial arteries in the hydrogel embolus localization, both in the early and in the late periods [4], (Figure 4.5). Necrotic and destructive areas of pulmonary tissue were not observed in the bronchial wall and pulmonary parenchyma. The difference is only in the degree of the resorptive giant-cell reaction, when hydrogel was used for control of pulmonary hemorrhage in patients with chronic non-specific pulmonary diseases. Endovascular occlusion of bronchial arteries was performed in these patients. The giant-cell response in these observations was significant already a month after the embolization of bronchial arteries. Then it was increasing, so that to the 20th month some smallest hydrogel emboli were completely dissolved, and the vascular lumen was filled with the giant cells of foreign bodies or was overgrown with connective tissue. We are inclined to account this peculiarity for the fact, that here the emboli were more accessible for hematogenous inflow of macrophagous elements. Such accessibility is due to abundant vascularization of the bronchial wall and presence of anastomoses at the precapillary level between the bronchial artery system and pulmonary artery system. When studying the surgical patients, to whom endovasoular occlusion of bronchial arteries had been performed, it was possible to follow the character of rearrangement of the vascular net not only in the embolized zone, but distal and proximal to embolus localization. This process involved vacate hyperplasia of the intima, reorientation of smooth muscle cells from the circular to longitudinal direction, that in the long run resulted in the acute constriction of the blood vessel lumen. The reason for such rearrangement was the reduction of blood flow after endovascular occlusion. The hemostasis was obtained after endovascular occlusion as a result of the decrease of blood filling in the bronchial wall. At the same time fading of the inflammatory process with the reduction of the amount of sputum secretion occurred. Frequently the patients’ condition after endovascular occlusion of bronchial arteries improved so much, that this conservative treatment became sufficient, without the need of surgical intervention [5-8].

Figure 4.5. The cross section of a bronchial artery, 635 days after occlusion by the emboli. The wall of the artery is not infiltrated with inflammatory cells. HE staining.

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The reason for fading of the inflammatory process was the decrease of exudation degree, leucocyte release and reduction of secretion from bronchial glands. It should be emphasized that in our observations after bronchial artery embolization we did not see destructive and necrotic changes, either in the bronchial wall, or in pulmonary parenchyma. It is connected with the presence of anastomoses at the pre-capillary level between the system of bronchial arteries and the pulmonary arterial system. The increase of the bronchial wall sclerosis and pneumosclerosis in the zone of pathological focus was observed in the late time after embolization. It indicated the completion of the cycle of pathological process. Thus, clinical observations give evidence to the fact, that hydrogel emboli can be considered as a material that is absolutely suitable and can be successfully used for therapeutic occlusion of blood vessels.

REFERENCES [1] [2] [3] [4] [5] [6]

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[7] [8]

M.I. Kusin, Yu.D. Volynski, Surgery, 1978, 131-136. M.I. Kusin,Yu.D. Volynski, A.A. Vishnevskiy, P.I. Todua, The Endovagcular Therapy and Surgery of Pulmonary Hemoptysis, Irkutsk, 1981. (Rus) I. Remy, C. Voisin, C. Dupruis, Am. Radiology, 1976, 17, 5-16. N.E. Scuba, F.I. Todua, The Endovascular Therapy and Surgery of Pulmonary Hemoptysis, Irkutsk, 1981. (Rus) G.E. Zaikov. Biotechnology: state of the art and prospect for development, New York: Nova Science Publ., 2008, 228 pp. G.E. Zaikov Progress in chemical and biochemical physics. Kinetics and thermodynamics, New York: Nova Science Publ., 2008, 228 pp. P.E. Stott, G.E. Zaikov, V.F. Kablov, Research progress in biotechnology, New York: Nova Science Publ., 2008, 258 pp. Yu.G. Medvedevskikh, A. Valente, B. Howell, G.E. Zaikov. Organic and physical chemistry. Prospects and developments, New York, Nova Science Publ., 2008, 292 pp.

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Chapter 5

HYDROGELS IN ENDOVASCULAR EMBOLIZATION PART V. ANTITUMOR AGENT METHOTREXATECONTAINING P(HEMA)

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Daniel Horak1, K.Z. Gumargalieva2*, G.E. Zaikov3**, M.I. Artsis**, and L.L. Madyuskina** Institute of Macromolecular Chemistry, Czech Academy of Sciences, Prague, Czech Republic *N.N. Semenov Institute of Chemical Physics, Russian Academy of Sciences Moscow, Russia **N.M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences Moscow, Russia

ABSTRACT This paper allows us to assume that emboli made from the modified p(HEMA) hydrogel can be loaded with methotrexate, one of the most frequently used cytostatics. Such a simple MTX-fixation to a p(HEMA)-Hex carrier has the advantage that the pharmacological activity of the drug remains unchanged. The use of p(HEMA)-Hex-MTX hydrogel emboli should enable us to achieve a maximum concentration and a prolonged effect of the cytostatic in the embolized region (tumor) while reducing its concentration in other tissues of the organism at the same time. Thus, there is a prospect of the targeted local palliative cytostatic effect of the drug on the tumor and of prevention of the possible haematogenic formation of metastases, along with a decreased blood supply to this zone or to the whole organ. In other words, the site-specific function of the released antitumor drug could be combined with the endovascular occlusion of blood supply to the tumor tissue. 1 2 Heyrovskogo sq., Prague-6, Czech Republic. 2 4 Kosygin str., Moscow 119991, Russia 3 4 Kosygin str., Moscow 119334, Russia. [email protected].

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Keywords: antitumor agent, hydrogels, p(HEMA), haematological testing, plasma.

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5.1. INTRODUCTION The widespread occurrence of malignant tumors and often low response rate of current methods of treatment call for new therapeutic approaches. In the literature, testing the effectiveness of a targeted introduction of cytostatic drugs into the neoplastic tissue has been reported [1, 2]. For this purpose endolymphatic introduction of cytostatics was performed or they were infused into the artery which supplies blood to the tumor tissue [3]. However, these procedures for targeted introduction of cytostatics into tumor tissues bring only a short-term effect of the drug unless the catheter is left in the vessel for a long time. A pronounced therapeutic effect usually requires that the procedures must be repeated many times. Such treatment with cytostatics is accompanied by the same toxic side effect on haemopoiesis, on the gastrointestinal tract and other organs as conventional treatments. Finally, since most of the drug given to a patient is “wasted”, unnecessarily high amounts of expensive drugs must be administered. An attractive procedure is that in which the cytostatic drug does not pass through blood or lymphatic vessels of the tumor but its effective concentration is maintained in them for a prolonged time using a single dosage form [4]. This may be achieved by a continuous controlled release, at biological pH, of the reversibly immobilized antitumor agent from a matrix. This can strongly increase the efficiency of the drug while limiting the toxic effects [4]. Among immobilization techniques, reversible electrostatical banding of the drug to a hydrophilic polymer matrix is recommended [5]. In addition to the effect of cytostatic drugs, slowing down of the growth of the tumor may be helped by decreasing blood supply by means of embolization of the supply vessels. A combination of these two complementary procedures seems justified. Moreover, in the preoperative embolization of blood vessels supplying the tumor, these joint effects might help to decrease the release of tumor cells into the blood stream during the following operation and reduce their viability. To put this into effect, we load emboli intended for occlusion of the tumor-supplying vessels with a cytostatic drug. The drug situated in the immediate vicinity of the tumor tissues should slowly diffuse into the tumor and probably ensure a prolonged suppression of its growth. Considerable experience exists in the application of emboli made from the poly(2hydroxyethyl methacrylate) (p(HEMA)) hydrogel for the endovascular occlusion of blood vessels in the treatment of various diseases [6, 7]. Emboli made from the hydrogel are optimal from the therapeutic viewpoint since they prevent recanalization [8], important in ensuring long-term ischaemia of the tumor. Additionally, experience has been gathered with p(HEMA)based spherical emboli as drug carriers, especially for haemostatic drugs (thrombin [9], ethamsylate [10], aminocaproic acid [10]). In this Section we examine the possibility of loading emboli made from p(HEMA) hydrogel with one of the widely applied cytostatics - methotrexate, N-(4-(N-methyl-2,4diamino-6-pteridinyl-methylamino)benzoyl) glutamic acid, the folic acid antagonist. This compound has been in clinical use for the treatment of various malignant as well as nonneoplastic diseases for many years. Furthermore, we investigate the character of histological

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changes in the vessel wall and in the surrounding tissues following the endovascular implantation of such biologically active emboli.

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5.2. MATERIALS AND METHODS The synthesis of spherical p(HEMA) particles and the method of their purification have been described [8]. The beads used in this investigation were 0.4–0.6 mm in size in the dry state. A part (I g) of these particles of medical grade purity was preswelled in dioxane, immersed in a solution of 0.358 g of 4-nitrophenyl chloroformate in 10 ml of dioxane, and the mixture was shaken at room temperature for 4.5 h. The unreacted reactants were removed by thorough washing with dioxane and the activated polymer was added to a solution of 1.5 g of 1,6-diaminohexane in 5 ml of dioxane with shaking at room temperature for 2.5 h. After completion of the reaction the p(HEMA)-Hex beads were thoroughly washed with dioxane, with 0.5 M sodium carbonate until the polymer lost its yellow colour, and with water. The polymer particles, which according to elemental analysis contained 0.75 wt.% of nitrogen, were then immersed in 50 ml of 1.8 wt.% aqueous solution of methotrexate (MTX), which was dissolved by adding sodium hydrogen carbonate. The desorption kinetics of MTX was determined spectrophotometrically (at 234 nm) in a 10 ml cell; water was changed after incubation of 5, 15, 25, 40 and 60 min. After this time there is virtually no further release of MTX. The amount of antimetabolite ionically bound on 1 g of the carrier was 0.19 g and was determined from the balance between the initially loaded and released amounts of antimetabolite. Sterilization of the samples was carried out in boiling distilled water for 45 min. Donor blood analysis was based on measurements of changes in the number of leucocytes, erythrocytes and thrombocytes in 1 μl of blood, followed by determination of the haemolysis level increase [8]. A second method of the in vitro determination of haemostatic properties of materials consisted of an evaluation of indicators of donor plasma clotting in contact with the material: activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT). All these indicators were determined using a Hyland coagulometer in the donor plasma, to which 0.1 ml of a suspension of hydrogel particles in water (1 : 3) was added, i.e. in a ratio analogous to that introduced into the vessel through a catheter during the embolization. The method of implantation of emboli into the rabbit’s femoral artery has been described in a preceding paper [8]. A histological investigation of the blood vessels containing the emboli and of the surrounding tissues was carried out 1, 7 and 14 days after the implantation. Celloidine histological samples were stained with haematoxylin, eosin, and according to van Gieson.

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5.3. RESULTS AND DISCUSSION 5.3.1. Synthesis of P(HEMA)-Hex-MTX Particles To avoid the decrease of methotrexate activity by its chemical modification, we used simple sorption as a method of its fixation to the polymer. In this report p(HEMA), the copolymer of 2-hydroxyethyl methacrylate (98 vol.%) and ethylene dimethacrylate (2 vol.%) in the form of regular spherical porous particles was used as a polymeric carrier. A p(HEMA) carrier as such does not possess the required affinity for MTX or for other cytostatic substances [11]. From MTX solution-saturated p(HEMA), the drug can be quickly and completely eluted with water. It is known that the desired sorbing ability can be attained by introduction of aminohexyl groups to p(HEMA) [11]. Such a p(HEMA)-Hex derivative has been prepared from p(HEMA) by esterification of the hydroxyl groups with nitric acid, which is a dangerous and difficult reaction, followed by a reaction with 1,6-diaminohexane [12]. Other methods of the synthesis of amino derivatives of p(HEMA) are less direct. These include the reaction of an alkylating derivative of p(HEMA), e.g., halide, tosylate, or sulphate, with ammonia or an amine, or a reaction of p(HEMA) with alkylating amines, or activation with cyanogen bromide followed by substitution of active groups with airline. These methods employ compounds which are unsuitable for medical applications because of toxicity, instability, difficult manipulation, etc. To avoid shortcomings, it is advantageous to use solid activated esters of chloroformic acid [13, 14]. In this Section hydroxy groups of poly(2-hydroxyethyl methacrylate) (1) were activated [13, 14] in a nonaqueous solvent using 4-nitrophenyl chloroformate (2) according to:

CH3

O

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CH CH2

+ Cl

C O

NO 2

COOCH2CH2OH (1)

_ OH

(2)

(1) CH3 CH CH2 COOCH2CH2

O O

C O

NO2

(3) The resulting unstable product (3) was reacted with an amino group of 1,6-diaminohexane (4) to form the urethane bond according to:

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(3) + H2N(CH2)6NH2 OH

(4)

NO2

(2) CH3 CH CH2 COOCH2CH2

O O

C NH(CH2)6NH2

(5)

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This activation of hydroxy groups of p(HEMA) leads to products which are easy to prepare, stable and non-hazardous. Another advantage is in an easy check of binding, deactivation, and washing due to the yellow colour of 4-nitrophenol. The aminohexyl derivative of poly(2-hydroxyethyl methacrylate) (p(HEMA)-Hex) (5) thus formed is a weakly basic anion exchanger, characterized by the total exchange capacity, capable of adsorbing MTX from aqueous solution. An alternate mechanism of MTX-fixation, e.g. reversible adsorption, cannot be excluded. At pH 6.8 the quantity of sorbed MTX was linearly proportional to its concentration in the aqueous solution up to a concentration given by the solubility limit of MTX in water at pH 6.8. The amount of MTX absorbed from such a solution was 0.21 g/g of dry polymer. The release of MTX in the organism might be based on the anion exchange. The biological environment is rich in various anions which can replace MTX from the p(HEMA)-Hex-MTX ion exchanger.

5.3.2. Haematolagical Testing p(HEMA)-Hex-MTX spherical particles intended for embolization were kept in an excess of saturated MTX solution and then washed to medical grade purity before being used in experiments [8]. Donor blood analyses were employed as the criteria in evaluating leaching of excessive MTX from the emboli. In this test the morphology of the red blood cells and of elements of white blood (thrombocytes, lymphocytes, erythrocytes) was carefully examined. Blood was examined before and after contact with the hydrogel samples tested. The data thus obtained showed that the first test set of unwashed p(HEMA)-Hex-MTX emboli causes rough changes in blood (haemolysis of blood elements, leucocytopenia, anaemia, thrombocytopenia and a morphological damage to blood cells), which indicate tissue incompatibility. Redundant (unbound) MTX must be desorbed to obtain medical grade particles, because it could produce vast necroses of the artery wall after its embolization. In the p(HEMA)-Hex-MTX hydrogel there are apparently two kinds of sorbed MTX molecules: those that are immobilized by ionic bonds and those that are not. Such immobilization may involve a bond between acid carboxylic substituents of the MTX molecule with basic amino groups of the p(HEMA)-Hex polymer.

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Figure 5.1. Desorption of MTX from PHEMA-Hex-MTX carrier as a function of time.

To remove redundant MTX, p(HEMA)-Hex-MTX spherical particles were placed in distilled water at room temperature, and the release rate was checked using the kinetics of desorption (Figure 5.1).The curve is characterized by a large initial slope corresponding to the fast release rate which takes 40 min and marks the elimination of MTX molecules in excess of the total capacity of the p(HEMA)-Hex polymer. This is followed by a stationary state which presumably signals completion of the elimination of MTX molecules exceeding the exchange capacity. The amount of such MTX molecules released over the whole time per gram of sample and determined by UV spectroscopy was quite small: 2×10-2 g/g. Here, it is worth mentioning that if p(HEMA) emboli are treated with a solution of ethamsylate, which is not very firmly bound with the hydrogel, the drug is desorbed from the hydrogel at a concentration higher than that in the case of MTX by one and a half orders of magnitude [10]. Calculations carried out using the kinetic curve data (Figure 5.1) show that after purification by washing with distilled water the p(HEMA)-Hex-MTX polymer contains 0.19 g of ionically bound MTX per 1 g of the polymer. The efficiency of MTX binding with the p(HEMA)-Hex hydrogel from the MTX saturated solution is 24%). Haemolysis tests after contact of the purified p(HEMA)Hex-MTX material with donor blood show minimal damage to the blood cells and an increase in thrombocytopenia at the expense of the formation of thrombocytic aggregates. Such material satisfies toxicological require ments and can be used in the endovascular occlusion. The method described above, i.e. an analysis of changes in the donor blood elements in contact with the material, is to be used as a technological check of the degree of purification of biologically active emboli after MTX loading for their experimental and, in the future, clinical application.

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5.4. HAEMOSTATIC PROPERTIES The effect on blood coagulation by purified p(HEMA)-Hex-MTX emboli was investigated by means of a coagulogram, which is a general test used for determination of the effect of drugs on the blood coagulation system. Table 5.1 shows some indicators of the coagulogram: APTT, TT and PT both in healthy and in pathologically altered donor blood, and in the presence of MTX-containing and MTX-free hydrogel emboli. The MTX-free emboli were studied since the hydrogel has a haemostatic effect due to the nature of the monomer and the structure of p(HEMA) [8]. Pathological plasma was taken from patients suffering from a focal alteration of the liver, where APTT is extended from 69 to 90 s (at the APTT standard 30 - 35 s). As documented by Table 5.1, the introduction of MTX does not affect APTT, if the plasma is healthy. In the case of pathologically altered plasma characterized by hypocoagulation there is a considerable (virtually double) decrease in APTT compared to the standard value. A still more positive effect following the introduction of MTX can be seen in a three-fold decrease in the TT indicators (patient No. 5 and 6). Results obtained from the interaction between the p(HEMA)-Hex-MTX hydrogel and blood show that the former exerts a weaker haemostatic effect compared with the hydrogel treated with ethamsylate or aminocaproic acid [10].

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5.5. HISTOMORPHOLOGICAL INVESTIGATION The most important medico-biological test consisted of a histological investigation of p(HEMA)-Hex-MTX embolic material implanted in femoral arteries of a rabbit for various periods of time. Samples of vessels with surrounding tissues containing hydrogel emboli of the same size, but MTX-free, taken at the same times were used as control. It was shown that in control experiments the reaction of the vessel wall was virtually absent; only extension of the wall occurred if the latter was tightly filled with the emboli. The implantation of p(HEMA)-Hex-MTX emboli caused necrosis of the intima and a complete or partial necrosis of the muscular envelope of the artery in the section where the embolus adhered to the wall of the vessel. As a result, the emboli had adhered directly to the adventitia [Figs. 5.2(a-c)]. In the skeletal muscles surrounding the vessel moderate necrosis was also observed within the times of investigation (Figure 5.3a), this was followed by regeneration (Figure 5.3b). With increasing time, absorption of necrotized muscular fibrils takes place, and their regeneration phenomena increase. Lysis and regeneration of the muscular tissue took place first near the blood vessel with MTX-containing emboli. Starting from the 7th day after the implantation, one part of the emboli appeared microporous without any cells in the pores of the material (Figure 5.2b). On the other hand, in MTX-free emboli fibroblasts which had penetrated into the pores were found in their lumina; the number of the fibroblasts increased with increasing time of observation.

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Table 5.1.Indicators of donor plasma used in the characterization of properties of the PHEMA-Hex-MTX hydrogel Indicator

APTT(s)

Reported data for normal blood

Experimental data No. of donor

30 - 35

Healthy plasma

Pahological plasma

TT(s)

16 - 21

Healthy plasma

Pathological plasma

PT(s)

12

Healthy plasma Pathological plasma

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 5 6 8

Standard

PHEMA

30 31 32 29 82 89 90 69 24 26 22 17 105 110 112 89 15 18 26 30 32

28 28 30 25 71 76 70 58 18 25 18 14 100 106 82 75 15 18 ⎯ 28 27

PHEMAHex-MTX 28 32 31 23 34 58 54 27 17 25 18 13 32 33 29 55 18 17 18 27 30

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APTT is the activated partial thromboplastin time; TT the thrombin time; PT the prothrombin time.

a - 1 day after the implantation. Artery wall in a state of necrobiosis and necrosis with massive infiltration of leucocytes. Between emboli detritus from necrotized intima.

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b - 7 days after the implantation. Intima and muscular envelope of the artery are lacking. The embolus adheres to the adventitia of the vessel. A small focus of the muscular envelope remains in the angle between two emboli. No penetration of cells into pores of the emboli.

c - 14 days after the implantation. Intima and media necrotized. Sclerosis of the tissue in the perivascular space. Figure 5.2. Histological section of the rabbit’s femoral artery with two emboli (one embolus) from the p(HEMA)-Hex-MTX hydrogel. Stained after van Gieson: a, with haematoxylin and eosin, b, c. (× 160).

The results obtained suggest that in the animal MTX does not remain in an immediate contact with the material of the embolus. It is gradually washed out by the blood plasma and diffuses into the vessel wall and the surrounding tissues. The MTX activity is maintained, as indicated by necrotic changes in the vessel wall and surrounding tissues. After some time the necrotic parts are resorbed throughout the wall and in skeletal muscles. At the same time, the acute inflammation in the vessel wall recedes and changes to chronic inflammation in the tissues surrounding the vessel. It is noteworthy that fibroblasts grow into pores of the hydrogel, particularly by the 14th day of implantation. This indicates a sufficiently complete release of the cytostatic from the embolic material by this time and the absence of a local toxic effect,

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which allows cells of the connective tissue to grow unhampered into the pores of the hydrogel [15, 16].

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a - 7 days after the implantation. Necrosis with demarcation of inflammation.

b - 14 days after the implantation. Necrosis and onset of regeneration. Figure 5.3. Skeletal muscular fibres in the immediate vicinity of the p(HEMA)-Hex-MTX emboli. Stained with haematoxylin and eosin, a, after van Gieson, b. (× 160).

Preliminary data reported in this Section allow us to assume that emboli made from the modified p(HEMA) hydrogel can be loaded with methotrexate, one of the most frequently used cytostatics. Such a simple MTX-fixation to a p(HEMA)-Hex carrier has the advantage that the pharmacological activity of the drug remains unchanged. The use of p(HEMA)-HexMTX hydrogel emboli should enable us to achieve a maximum concentration and a prolonged effect of the cytostatic in the embolized region (tumor) while reducing its concentration in other tissues of the organism at the same time. Thus, there is a prospect of the targeted local palliative cytostatic effect of the drug on the tumor and of prevention of the possible haematogenic formation of metastases, along with a decreased blood supply to this zone or to

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the whole organ. In other words, site-specific function of released antitumor drug could be combined with the endovascular occlusion of blood supply to the tumor tissue.

REFERENCES [1] [2] [3]

[4]

[5]

[6]

[7]

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[8]

[9]

[10]

[11]

[12] [13]

Intra-Arterial and Intfacavitary Cancer Chemotherapy, Ed. S.B. Howel, Martinus Nijhoff Publishers, Boston, 1984. Regional Cancer Treatment, Eds. K.R. Aigner, Y.Z. Patt, K.H. Link, J. Kreidler, Karger, Munchen, 1988. M.C. Sheen, Intra-arterial Infusion Chemotherapy for Advanced Head and Neck Cancer, In: Advances in Regional Cancer Therapy, Eds. J. Kreidler, K.H. Link, K.R. Aigner, Karger, Munchen, 1988, 144-150. J. Kost, R. Langer, Equilibrium Swollen Hydrogels, In: Controlled Release Applications, in Hydrogels in Medicine and Pharmacy, Ed. N.A. Poppas, CRC Press, Boca Raton, 1987, 3, 95-108. W.R. Gombotz, A.S. Hoffman, Immobilization of Biomolecules and Cells on and within Synthetic Polymer Hydrogels, in Hydrogels in Medicine and Pharmacy, Ed. N.A. Poppas, CRC Press, Boca Raton, 1986, 1, 95-126. D. Horak, F. Svec, J. Kalal, A. Adamyan, Yu. Volynskii, O. Voronkova, L. Kokov, K. Gumargalieva, Hydrogels in Endovascular Embolization. II. Clinical Use of Spherical Particles, Biomaterials, 1986, 7, 467-470. D. Horak, F. Svec, A. Adamyan, M. Titova, O. Voronkova, N. Trostenyuk, N., Vishnevskii, V., Guseinov, E. and Gumargalieva, K., Poly[2-hydroxyethyl methacrylate) beads for the preoperative endovascular occlusion of branches of the hepatic artery in focal alterations of the liver, Call. Mater. 1990, 6, 287-297. D. Horak, F. Svec, J. Kalal, K. Gumargalieva, A. Adamyan, N. Skuba, M. Titova, N. Trostenyuk, Hydrogels in Endovascular Embolization. I. Spherical Particles of Poly(2hydroxyethyl methacrylate) and Their Medico-biological Properties, Biomaterials, 1986, 7, 188-192. D. Horak, F. Svec, J. Kalal, A. Adamyan, M. Titova, N. Trostenyuk, N. Skuba, V. Dan, O. Voronkova, K. Gumargalieva, Biologically Active Thrombin-containing Hydrogels Based on Poly(2-hydroxyethyl methacrylate) for Endovascular Occlusion, Polymers in Medicine, 1991, 21, 31-41. D. Horak, F. Svec, A. Adamyan, M. Titova, N. Skuba, O. Voronkova, N. Trostenyuk, V. Vishnevskli, K. Cumargalieva, Haemostatic Activity of Ethamsylate and Aminocaproic Acid Adsorbed Poly(2-hydroxyethyl methacrylate) Particles, Biomaterials. (in press) K. Motycka, K. Slavik, A. Kocovska, R. Cihar, P. Spacek, M. Kubin, Effect of Methotrexate Sorbed on Modified 2-Hydroxyethylmethacrylate Carriers in Mice of C3H Strain with a Solid Gardner Lymphosarcoma, Neoplasma, 1977, 24, 271-276. P. Spacek, M. Kubin, M. Bones, The Method of Production of Polymeric Esters of Methacrylic Acid and Multifunctional Alcohols, Czech. Pat. No. 191474, 1975. J. Drobnik, J. Labsky, H. Kudlvasrova, V. Saudek, F. Svec, The Activation of Hydroxy Groups of Carriers with 4-Nitrophenyl and N-Hydroxysuccinimidyl Chloroformates, Biotechnol. Bioeng., 1982, 24, 487-493.

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[14] J. Drobnik, J. Kalal, J. Labsky, V. Saudek, F. Svec, The Method of Activation of Insoluble Carriers Containing Hydroxy Groups, Czech. Pat. No. 204190, 1980. [15] LinShu Liu, G.E. Zaikov. Chemistry as music, New York: Nova Science Publ., 2008, 261 pp. [16] R.G. Makitra, A.A. Turovskii, G.E. Zaikov. Chemical thermodynamics and chemical kinetics. Step in liquid phase. Correlation analysis in chemistry of solution, New York: Nova Science Publ., 2008, 365 pp.

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In: Handbook of Chemistry, Biochemistry … Editors: L. N. Shishkina et al., pp. 51-58

ISBN: 978-1-61209-425-0 © 2010 Nova Science Publishers, Inc.

Chapter 6

HYDROGELS IN ENDOVASCULAR EMBOLIZATION PART VI. POLY(2-HYDROXYETHYL METHACRYLATE) WITH INTENSIFIED HAEMOSTATIC ACTIVITY AS A NEW EMBOLIC MATERIAL Daniel Horak1, K.Z. Gumargalieva2*, G.E. Zaikov**, L.A. Zimina3**, and N.N. Madyuskin**

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Institute of Macromolecular Chemistry, Czech Academy of Sciences, Prague, Czech Republic *N.N. Semenov Institute of Chemical Physics, Russian Academy of Sciences, Moscow, Russia **N.M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia

ABSTRACT Haemostatic properties of ethamsylate-sorbed hydrogel particles tested on normal healthy plasma were not very distinct. Tests carried out with pathologically changed plasma of the patients suffering from focal alteration of the liver showed a pronounced haemostatic effect of ethamsylate or aminocaproic acid-containing hydrogels. A comparison of haemostatic properties of drug-free PHEMA emboli with those of ethamsylate or aminocaproic acid-treated PHEMA indicate their potential in the treatment to prevent bleeding, particularly for those caused by a disturbed haemostasis system.

1

2 Heyrovskogo sq., Prague-6, Czech Republic. 4 Kosygin str., Moscow 119991, Russia 3 4 Kosygin str., Moscow 119334, Russia, [email protected]. 2

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Keywords: haemostatic activity, embolic materials, hydrogels, surgery, clinical oncology, tumor.

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6.1. INTRODUCTION Embolization of arterial vessels is increasingly used in surgery and clinical oncology [1]. It may be indicated by a threatening haemorrhage from a decomposing tumor, the necessity of creation of a “dry” operative field or the need to reduce the release of tumor cells into the bloodstream during surgical intervention. In the case of nonresectable tumors, embolization of the supply vessels often slows the growth of the neoplasm, postpones formation of metastases, removes painful paraneoplastic syndromes and provides favourable conditions for the subsequent radiation or drug treatment with palliative aims. Earlier, we demonstrated the prospects and justification of endovascular embolization in haemostasis and as a prophylactic measure for bleeding, using emboli made from poly(2hydroxyethyl methacrylate) (PHEMA) hydrogel [2, 3, 6], which is capable of primary activation of thrombocytic haemostasis, reduction in activity of fibrinolysis and secondary activition of the plasma haemostasis. The thrombogenic properties of PHEMA hydrogels were affected by three factors [4]: degradation products of the hydrogel, adsorption of plasma proteins by the hydrogel, and the structure of the implant surface. Endovascular embolization by means of PHEMA is justified as a preparatory step before surgical interventions in zones of increased vascularization in cases of hypocoagulation, where no immediate haemostasis is required. To achieve an immediate and efficient management of bleeding from a variety of origins, further development of new composition of the emboli, having increased haemostatic properties, is needed. This may be accomplished by immobilization or sorption of biologically active compounds. In an earlier study we reported the preparation and properties of thrombin-containing PHEMA hydrogel [5]. The immobilization or sorption of a coagulating enzyme (thrombin) to the PHEMA emboli strengthened their haemostatic properties and activated the coagulation system of the organism. However, the thrombin used in the preceding study showed several drawbacks: it may stimulate allergic reactions and, if applied in the wrong way, the activation of the whole coagulation system can be pathological. This is probably due to the fact that, because of the high costs of purified thrombin, an unpurified enzyme was used. For this reason, haemostatics, such as ethamsylate and aminocaproic acid, whose effect is not identical with that of thrombin, were used in this study to intensify the haemostatic properties of PHEMA emboli. Ethalnsylate (Dicynone in Switzerland and other countries) is a well-known synthetic water-soluble non-steroidal haemostatic agent (diethylammonium 1,4-dihydroxy-3benzenesulfonate) with a broad range of activity. Unlike thrombin, ethamsylate can be used in the treatment of capillary bleedings without stimulation of allergic reactions. It exerts its action on primary haemostasis and is effective in restoring platelet functions, e.g., reduces bleeding time in patients with thrombopathies (platelet disfunctions). Essentially, the effect of ethamsylate consists in that it helps: a) raise the number of thrombocytes, b) increase their adhesiveness, and c) increase capillary strength by reinforcement of the capillary basal membrane while shortening the bleeding time.

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Aminocaproic acid (6-aminobexanoic acid), as a synthetic inhibitor of proteolysis, stimulates the blockage of proteolytic enzymes while simultaneously inhibiting the effect of plasmin (fibrinolysin) on fibrinogen and coagulation factors. By blocking activators of plasminogen and partly suppressing the effect of plasmin, aminocaproic acid has a specific haemostatic effect in haemorrhages connected with increased fibrinolysis. Unlike ethamsylate, which intensifies the aggregation of thrombocytes, aminocaproic acid has no effect on their aggregation.

6.2. MATERIALS AND METHODS 6.2.1. Synthesis The synthesis of spherical PHEMA particles and the method of their purification have been described [2]. The beads used in this investigation were 0.4–0.6 mm in size in the dry state. A part (1 g) of these particles of medical grade purity was immersed in a 16 ml of a 0.125 wt.% aqueous solution of ethamsylate (Dicynone, Lek, Ljubljana, Yugoslavia) for 6 days. The amount of ethamsylate sorbed on 1 g of carrier amounted to 0.87 g, which represents 43.5% of the ethamsylate in the initial solution; this was determined from the weight balances before and after ethamsylate sorption. Another part, (1 g) of the abovementioned PHEMA particles, was immersed in 16 ml of a 5% aqueous solution of aminocaproic acid (Acikaprin, Polfa, Warsaw, Poland) and left there for three hours.

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6.2.2. Purification Methods In a 10 ml cell was placed 0.1 g of the spherical emboli that had been treated with ethamsylate. Water was changed after incubation times of 5, 15, 25, 40, 60 min. Washings took place under spectrophotometric control until the release of the drug had ceased. The spectroscopic measurements were carried out at 215 nm with a Pye-Unicam SP 100 apparatus. The kinetic curve of drug release from the spherical particles is obtained as a result of summation of the amount of drug released within each time interval. The amount of ethamsylate sorbed on 1 g of the hydrogel was determined from the weight balance of ethamsylate in solutions prior and after washing.

6.2.3. Biocompatibility Tests Analysis of the donor blood before and after contact with the polymeric materials in terms of number of leucocytes, erythrocytes and thrombocytes was used as a criterion of the quality of emboli purification. The morphology of thrombocytes and erythrocytes was also examined. Haemostatic properties of the emboli were characterized in terms of donor plasma clotting: the activated partial thromboplastin time (APTT), the thrombin time (TT) and the prothrombm time (FT). All of these indicators were determined using a Hyland coagulometer in the donor

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plasma, to which 0.1 ml of a suspension of hydrogel emboli in water (1 : 3) was added. This is a ratio analogous to that introduced into the vessels during the embolization. The control consisted in the addition of water (without particles) which diluted the plasma and thus prolonged both APTT and TT.

6.3. RESULTS AND DISCUSSION

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In an attempt to prepare an embolic material suitable for efficient management of massive bleeding, spherical PHEMA particles used in earlier endovascular occlusion studies [3] were used as the matrix for sorbing haemostatics (ethamsylate or aminocaproic acid). For any modification of PHEMA emboli, preservation of their biological compatibility must be examined. Investigation of the interaction between native blood and unwashed spherical hydrogel immediately after treatment with ethamsylate solution showed that this embolic material causes a substantial pathological change in blood (acute haemolysis, leucocytopenia, thrombocytopenia, anaemia and morphological damage to blood cells). This would indicate potential tissue incompatibility. Indeed, histomorphological investigation of excess of ethamsylate-containing emboli implanted in the rabbit’s femoral artery revealed necrosis of the infcima and of the muscular skeletal envelope of the vessel surrounding the hydrogen.

Figure 6.1. Desorption of redundant ethamsylate from spherical PHEMA particles as a function of time.

For the purpose of subsequent clinical application a washing of the emboli to achieve desorption of the excess of ethamsylate is required if medical grade particles are to be obtained. To remove excess ethamsylate, the ethamsylate-sorbed emboli were placed in distilled water at room temperature; the release rate was obtained by using the kinetics of desorption (Figure 6.1). The curve is characterized by a large initial slope corresponding to a fast release rate (50% of unbound ethamsylate is released within 10 min) and is followed by a

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stationary state, which should indicate the complete elimination of redundant ethamsylate. Redundant ethamsylate-release is virtually completed within 40 min. This is achieved by the four to five changes of water at 10 min intervals at room temperature. Desorption of the excess of ethamsylate shows that this ethamsylate is sorbed in a less stable way than is, e.g., methotrexate, which is released from the hydrogel at a concentration lower by one and a half orders of magnitude [7]. That is, in contrast to methotrexate, which is ionically bound to the aminohexyl derivative of PHEMA, ethamsylate appears to be attached to PHEMA via hydrophobic interactions. Calculations carried out using the kinetic curve data (Figure 6.1) showed that after purification by washing with distilled water the hydrogel contained 0.33 g of sorbed ethamsylate per g of the polymer. The efficiency of ethamsylate binding with the PHEMA hydrogel from the ethamsylate saturated solution is 16.5%. After purification of the polymer by washing with distilled water, the haemolysis of blood cells was retested and minimal damage was observed. All blood cells retained their natural character and no haemolysis took place. Thrombocytopenia was still observed with the washed ethamsylate-containing hydrogel; this was accompanied by the formation of thrombocytic aggregates (8–10 aggregates per 1000 erythrocytes). The aggregate of thrombocytes was bulkier than that seen with the ethamsylate-free PHEMA hydrogel, this indicates that ethamsylate is slowly released from the washed embolic material inducing aggregation of the thrombocytes. However, such material satisfies the toxicological requirements and thus can be used in the endovascular occlusion. The spectrophotometrically determined ethamsylate concentration in an external aqueous medium which does not stimulate haemolysis was 5×10-7 g ethamsylate/ml water. In order to determine the in vitro haemostatic properties of the purified ethamsylatecontaining PHEMA in contact with donor plasma, both thrombocytic and plasma haemostasis were characterized by determining the activated partial thromboplastin time (APTT, standard 30–35 s), thrombin time (TT, standard 16–21 s) and prothrombin time (PT, standard 12 s). The investigation concerned both normal plasma, of healthy donors and pathologically altered plasma of patients suffering from focal alterations of the liver (haemangiomas). In such patients the acute hypocoagulation reaction of blood is typical. Basically, this blood can be divided into two groups, namely, with less pronounced pathological changes (APTT 40–48 s, the standard being 30–35 s) and more pronounced pathological changes (APTT 69–90 s). The control consisted in the determination of indicators of plasma clotting, with distilled water added instead of the suspension of particles. The indicators did not exceed the standard very much, i.e., water has no haemostatic effect at all (Table 6.1). As can be seen, the values of indicators of coagulogram for a healthy plasma in contact with ethamsylate-free and ethamsylate-containmg hydrogel are close to each other. This suggests that in the case of normal healthy blood one could use the hydrogel without its modification with ethamsylate (or aminocaproic acid) if endovascular embolization is indicated. However, in the group of patients with more distinct blood changes the haemostatic properties of free hydrogel will probably be unsatisfactory. In the case of blood with more or less distinct pathological changes the ethamsylate-treated emboli possess stronger haemostatic properties than the control starting ethamsylate-free emboli. Such materials accelerate the coagulation (APTT and TT) of pathologically changed blood whereas the blood clotting time had been slowed down and is almost three times that of normal blood. It should be noted that, while the APTT and TT reduction is very pronounced, the PT shows rather minor changes. Such reduction in APTT and TT indicated that ethamsylate, which is a general haemostatic, is

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56

effective even after being sorbed into the PHEMA hydrogel. The haemostatic effect of PHEMA is thus intensified. Table 6.1. Indicators of normal and abnormal plasma in contact with ethamsylate or aminocaproic acid-containing hydrogels

APTT (sec) Healthy plasma Less pronounced changes More pronounced pathological changes

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TT (sec)

PT (sec)

Healthy plasma Less pronounced pathological changes More pronounced pathological changes Healthy plasma Less pronounced pathological changes

More pronounced pathological changes

Aminocaproic acid containing PHEMA

No. of donor 1 2 3

Standard 32 32 45

36 31 52

Ethamsylate PHEMA containing PHEMA 23 16 25 21 24 15

5 6 7 8 9

40 82 89 90 69

42 ⎯ ⎯ ⎯ ⎯

30 71 76 70 58

24 24 43 46 22

14

1 2 3

20 20 35

24 ⎯ 38

18 14 17

14 10 15

10 ⎯ 14

4

31

⎯

24

21

⎯

6 7 8 9 1 2 3

105 110 112 89 12 12 21

⎯ ⎯ ⎯ ⎯ 13 ⎯ 21

100 106 82 75 9 10 9

30 35 20 43 7 9 8

7 ⎯ 7

4

24

⎯

14

13

8

6 7 8 9

26 30 ⎯ 32

⎯ ⎯ ⎯ ⎯

⎯ 28 ⎯ 27

16 20 ⎯ 19

Water

8 16 5

APTT is the activated partial thromboplasmin time; TT the thrombin time; PT the prothrombin time. Abbreviated title: PHEMA is an embolic material.

The data shows the morphology of blood in contact with the hydrogel treated with a 5% aqueous solution of aminocaproic acid for 3 h. Since this solution is the same concentration as Handbook of Chemistry, Biochemistry and Biology : New Frontiers, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

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that used clinically in the intravenous infusion (and does not provoke haemolysis), the hydrogel treated with aminocaproic acid was not washed to remove any redundant acid. While the morphology shown by the data confirms that no haemolysis takes place, there is indication of a mild aggregation of thrombocytes (less than with ethamsylate) which can be explained by the effect of aminocaproic acid on blood plasma factors. The aminocaproic acid-treated hydrogel possesses a pronounced haemostatic effect (Table 6.1), although the latter was examined using only blood plasma with less pronounced pathological changes. Unlike the ethamsylate-treated hydrogel, not only the effect of decreased APTT, but also the effects of the other two decreased indicators of a coagulogram, TT and FT, can be seen. Thus, we recommend PHEMA as the carrier of two drugs: ethamsylate, which increases thrombocyte adhesiveness and capillary resistance while affecting the thrombocyte-vessel wall interaction, and aminocaproic acid which is a synthetic inhibitor of proteolysis and an antiallergic substance. The etham- sylate or aminocaproic acid-treated PHEMA hydrogel has a particularly strong effect on the pathologically abnormal blood (i.e., accelerates the clotting time) of patients suffering from focal alterations of the liver. These patients have a disturbed synthesis of proteins and blood coagulation factors. Thus, this biologically active hydrogel is recommended for the endovascular occlusion treatment of haemangiomas of the liver associated with acute hypocoagulation. The use of this emboli with intensified haemostatic properties will reduce intraoperational blood loss from the liver tissue in acute parenchymatic haemorrhages, not only due to the blockage of blood vessels with the emboli, but also because of the effect of sorbed aminocaproic acid. The rate of release of haemostatics from PHEMA is due to the porous structure of the hydrogel and to the gradual washing out of the haemostatics by tissue liquids. The haemostatics may be effective in restoring factors of the disturbed plasma and thrombocytic haemostasis, and can mediate activation of reparatory processes in the dysfunction of the third stage of coagulation (the stabilization of fibrin when fibrin S is transformed into fibrin I). This extends the application of endovascular occlusion in the urgent treatment of various forms of hypocoagulation bleedings (parenchymatous bleeding of the liver, pulmonary bleeding, bleeding from stomach ulcer, acute bleedings in childbirth, including haemangiomas of the small pelvis), as well as in the treatment of pronounced allergic reactions. The haemostatic properties of PHEMA emboli can be improved by sorption of ethamsylate or aminocaproic acid. The ethamsylate-sorbed emboli cause haemolysis and morphological changes of blood cells, unless they are purified to remove the redundant solution of ethamsylate. The threshold ethamsylate concentration, below which no haemolysis takes place, is 5×10-7 g/ml. An analysis of donor blood in contact with the material (change in the number of erythrocytes, leucocytes, thrombocytes and their morphological changes) as well as determination of indicators of coagulogram can be used for the evaluation of haemostatic properties of new materials intended for contact with blood. Haemostatic properties of ethamsylate-sorbed hydrogel particles tested on normal healthy plasma were not very distinct. Tests carried out with pathologically changed plasma of the patients suffering from focal alteration of the liver showed a pronounced haemostatic effect of ethamsylate or aminocaproic acid-containing hydrogels. A comparison of haemostatic properties of drug-free PHEMA emboli with those ofethamsylate or aminocaproic acid-treated PHEMA indicate their potential in the treatment to prevent bleeding, particularly for those caused by a disturbed haemostasis system [8-10].

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REFERENCES

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[1]

D.J. Allison, Therapeutic Embolization and Venous Sampling, In: Recent Advances in Surgery, Ed. Selwyn Taylor, Churchill Livingstone, 1980, 27-64. [2] D. Horak, F. Svec, J. Kalal, K. Gumargalieva, A. Adamyan, N. Skuba, M. Titova, N. Trostenyuk, Hydrogels in Endovascular Embolization. I. Spherical Particles of Poly(2hydroxyethyl methacrylate) and their Medico-biological Properties, Biomaterials, 1986, 7, 188-192. [3] D. Horak, F. Svec, J. Kalal, A. Adamyan, Yu. Volynskii, O. Voronkova, L. Kokov, K. Gumargalieva, Hydrogels in Endovascular Embolization. II. Clinical Use of Spherical Particles, Biomaterials, 1986, 7, 467-470. [4] D. Horak, F. Svec, J. Kalal, A. Adamyan, N. Skuba, M. Titova, V. Dan, B. Varava, N. Trostenyuk, O. Voronkova, K. Gumargalieva, V. Timochina, Hydrogels in Endovascular Embolization. IV. Effect of Radiopaque Spherical Particles on the Living Tissue, Biomaterials, 1988, 9, 367-371. [5] D. Horak, F. Svec, J. Kalal, A. Adamyan, M. Titova, N. Trostenyuk, N. Skuba, V. Dan, O. Voronkova, K. Gumargalieva, Biologically Active Thrombin-containing Hydrogels Based on Poly(2-hydroxyethyl methacrylate) for Endovascular Occlusion, Polymers in Medicine, 1991, 21, 33-43. [6] D. Horak, F. Svec, A. Adamyan, M. Titova, O. Voronkova, N. Trostenyuk, V. Vishnevskii, E. Guseinov, K. Gumargalieva, Poly(2-hydroxyethyl methacrylate) Beads for the Preoperative Endovascular Occlusion of Branches of the Hepatic Artery in Focal Alterations of the Liver, Can. Mater., 1990, 6, 287-297. [7] D. Horak, F. Svec, A. Adamyan, M. Titova, N. Skuba, O. Voronkova, N. Trostenyuk, V. Vishnevskii, K. Gumargalieva, Hydrogels in Endovascular Embolization. V. Antitumor Agent Methotrexate-containing PHEMA, Biomaterials. (in press) [8] G.E. Zaikov, G. Kirshenbaum. Chemical Physics and Physical Chemistry: Step into the Future, New York, Nova Science Publ., 2007, 158 pp. [9] Handbook in Polymers Research: monomers, oligomers, polymers and composites, Ed. by R.A. Pethrick, G.E. Zaikov, A. Ballada, New York, Nova Science Publ., 2007, 549 pp. [10] A. Ballada, G.E. Zaikov. Preparation and Properties of Monomers, Polymers and Composite Materials, New York, Nova Science Publ., 2007, 195 pp.

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Chapter 7

ON THE POSSIBILITY OF USING AN EMBOLIZING PREPARATION DERIVED FROM POLY(2HYDROXYETHYL METHACRYLATE) (POLY-HEMA) FOR CHEMOEMOBOLIZATION E.V. Koverzanova1, S.V. Usachev, K.Z. Gumargalieva, and L.V. Kokov*  N.N. Semenov Institute of Chemical Physics, RAS, Moscow , Russia *A.V. Vishnevsky Institute of Surgery, 27 Bol. Serpukhovskaya st., Moscow 115093, Russia 

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ABSTRACT The principal opportunity of uptake of weakly crosslinked hydrogel emboli is shown by Doxorubicin at different temperatures. Optimal time of process is 1.5–2.5 hours. It is revealed that Doxorubicin is able to diffuse from a polymeric matrix, having a targeted medical effect on surrounding tissue, reducing side impacts on other organs.

Keywords: 2-hydroxyethyl methacrylate, emboli, Doxorubicin, drug uptake, drug release.

INTRODUCTION Weakly crosslinked hydrogels derived from hydroxyethyl methacrylate (HEMA) are representatives of a broad class of biomedical polymers. Their basic advantages are: high porosity, swelling properties in aqueous solution (65%), elasticity and resistance to biological environment. These are the properties that assign high biocompatibility of the material to an organism’s tissues that allow performance of purposeful occlusive embolization of vessels in order to arrest bleeding or to reduce blood filling in pathologies of various origins.  1 4 Kosygin st., Moscow 11999, Russia. e-mail: [email protected].

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Now there is no doubt in the urgency of the embolization method, which allows us to selectively impact a damaged organ. However, in some cases (malignant neoplasms) this procedure requires additional application of medicinal preparations. As these preparations are injected in a traditional manner, they affect all organs in the organism, including damaged ones. This requires searching for a solution, which would allow for a targeted effect of cytostatics on the damaged organ and, therefore, reduction of adverse side effects. The studies performed in this direction indicate that in some cases, at simultaneous use of cytostatic preparations and embolization of vessels, greater effect than in cases of individual use of embolization method can be reached [1-4]. Although embolizing systems used in these works had different chemical and physical properties, all authors indicate a significant increase of curative antitumor effect without increasing total dose of the drug and reduction of total toxic load on the patient. The purpose of this work is the study in vitro of a possible cytostatic drug, doxorubicin hydrochloride, uptake and release by embologenic material (EM) derived from poly-HEMA with determination of the concentration dependences of these processes.

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MATERIALS AND METHODS In this work we used weakly crosslinked hydrogel emboli, cylindrically shaped (D = 1,000 μm, L = 1 cm), synthesized on the basis of 2-hydroxyethyl methacrylate (Figure 2A). As crosslinking agent, we used ethylene glycol dimethacrylate (EGDMA). In the uptake study, two types of emboli: swollen emboli (SE) and preliminarily dried emboli (DE) were used. Emboli were dried at room temperature up to an established constant mass; the water loss was 63.89 ± 0.89%. The surface and porosity of cylindrical emboli were studied using a scanning electron microscope by Jeol YSM-5300 LV Company (Japan). Samples were stained by gold sputtering on Jeol YFC-1100 E unit, called ‘Ion sputtering device’. The sputtering thickness is ~40A. Uptake of EM by doxorubicin hydrochloride (Doxorubicin-LENS by LENS-Farm) and release of it were controlled by changes in DR concentration in aqueous solutions on spectrophotometer Shimadzu UV-160A.  For studying the process of uptake with emboli (20 pieces) of both SE and DE were placed in DR aqueous solution (7 ml) with 0.1 mg/ml concentration (0.7 mg DR). Changes of concentration DR were controlled specrophotometrically every 30 min. For studying the process of release DR both SE and DE were withdrawn from the solution, dried slightly by blotting paper and placed in 5 ml of distilled water. Changes in DR concentration at release were measured analogously to uptake process control. DR uptake and release degrees were estimated by concentration changes in solution and calculated by the formula: R = (C0 - Ct)/C0×100, where C0 is the initial DR concentration; Ct is the current DR concentration. 

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DISCUSSION Synthesis and Structure of EM Synthesis of cylindrical emboli on the basis of weak cross-linked hydrogel was performed in water solution under the action of an oxidation-reduction catalyst - persulphate of ammonium (APS) - N, N, N ’, N ’-tetramethylethylenediamine (TMED) at 20ºС [5], unlike synthesis poly-HEMA used before a method at 80–90°С [6]. Polymerization was performed in tubes 12–15 cm long and 1 mm in diameter. By polymerization completion hydrogel preparations were cut into parts 1 cm long and washed at 80°C changing water regularly during several hours. Table 1. The composition of polymerization mixture Reagents 

H 2O

HEMA

EGDMA 

APS/TMED

Reagent ratio (%) 

67.21

31.43

1.12

0.23

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Linear macromolecules formed during polymerization of 2-hydroxyethyl methacrylate monomer possess hydrophilic properties. This is the property that allows them to be unlimitedly miscible with water, hence, forming viscous transparent gel (or hydrogel) “solutions”. At the polymerization stage, injection of some few (1 ± 0.5%) of ethyleneglycol dimethacrylate comonomer capable of embedding between two linear macromolecules forms the so-called cross-linked hydrogels (Figure 1), which are already insoluble in water. Inherently, all these systems are amorphous and are unable to form any regular structures.

Figure 1. The formation scheme of cross-linked poly-HEMA hydrogel.

However, it has been found that at polymerization carried out under the above conditions forms a polymeric structure having heterogeneous architecture different from hydrogel (Figure 2).

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A 

B

Figure 2. (A) A picture of embologenic material from poly-HEMA, cylinder shaped (D = 1000 μm, L = 1 cm); (B) microphotograph of cross-section of the same material (amplified ×2000).

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Figure 2B shows conglomerate polymeric structure consisting of globules sized 3–5 μm, and the gap between them filled by water. The water content is up to 65%. Hence, high porosity of hydrogels derived from poly-HEMA has a positive effect on the material miscibility to organism’s tissues and promotes growth of connective fibrous tissue in hydrogel pores during water substitution by blood. This leads to stable fixation in the blood vessel. Another definite advantage of the porous structure is a possibility to use hydrogel emboli as drug carriers with controlled drug release.

Quantitative Determination of DR in Aqueous Solutions  UV-spectrum of DR is characterized by several maxima: three in the UV region (231, 252 and 290 nm) and one in the visible region (480 nm). For determination of quantitative variations in DR concentration at uptake and release, we have chosen the wavelength of 480 nm. Linear dependence of optical density on the solution concentration is observed in a wide concentration range of 0.2 to 0.005 mg/ml with the regression coefficient R = 0.99968. Uptake of EM by Doxorubicin  Selection of the time period for emboli uptake (1.5, 2 and 2.5 hours at 20 or 40°С) was determined by a possibility of carrying out this procedure directly before embolization. Analysis of kinetic uptake curves (Figure 3) has shown that for SE the uptake process starts almost immediately. At the initial stages, the uptake rate at 40°C is higher that at 20°C (Table 1). Hence, DE uptake proceeds with some delay. It is noted, that in the initial 30 min there is an increase of cytostatic concentration in solution. This event may be explained by the fact that, primarily, DE is swollen and then DR uptake begins. Gradual penetration of water inside DE causes opening of pores “collapsed” at drying and promotes penetration of DR molecules inside the polymer matrix.

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0.125

2 0.100

3

Mt/M0

0.075

1 0.050

0.025

0.000 0.0

0.5

1.0

1.5

2.0

2.5

time, h Figure 3. DR uptake curves of emboli. 1 – DE, uptake at 20°С; 2 − SE, uptake at 20°C; 3 – SE, uptake at 40°C.

Table 2. Uptake degrees and rates of DR at different temperatures

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Uptake time (h)  0.5 1 1.5 2 2.5

Uptake degree (%) At 20°C  At 40°C  DE  SE 0 2,16 2,99 1,72 6,16 5,90 4,23 8,82 7,13 5,55 10,39 8,43 5,99 11,75 8,47

Uptake rate (mg/h)  At 20°C  At 40°C  DE SE  0 0,031 0,051 0,014 0,046 0,052 0,022 0,044 0,042 0,022 0,039 0,037 0,019 0,035 0,030

Table 2 shows that at different temperatures SE uptake degrees differ insignificantly. However, they are somewhat higher at 20°C rather than at 40°C. Thus, as is shown, DR uptake of cylindrical emboli may be performed at room temperature.

Doxorubicin Release from EM   The analysis of kinetic curves of DR release from emboli has determined the following regularities. For SE, at temperature 40°C (Figure 4, curve 3) an abrupt DR release is observed sharp excretion: up to 50% of the preparation is extracted during 2 hours. The equilibrium state is reached 2-2.5 h after the process initiation. DR release from SE at 20°C (Figure 4, curve 2) proceeds less intensively than at 40°C, without typical effect of explosion. Similar to uptake, half an hour delay of DR release from DE is observed. This may be explained by the fact that

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64

for reaching equilibrium swelling, 2.5 hours is not enough for uptake in aqueous solution, and swelling dominates over release.  0.6

3 0.5

Mt/M0

0.4 0.3

2 0.2

1 0.1 0.0 0.0

0.5

1.0

1.5

2.0

2.5

time, h Figure 4. DR release curves from emboli. 1 – DE, uptake at 20°С; 2 – SE, uptake at 20°C; 3 – SE, uptake at 40°C.

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Table 3. DR release degrees and rates from cylindrical emboli at different temperatures Uptake time (h)  0.5 1 1.5 2 2.5

Release degree (%) At 20°C  At 40°C  DE  SE 0.00 0.94 20.94 6.08 13.37 41.31 7.77 19.82 48.39 9.76 22.89 51.66 12.39 25.45 52.47

Release rate (mg/h)  At 20°C  At 40°C  DE SE  0.000 0.010 0.028 0.002 0.014 0.029 0.002 0.012 0.023 0.002 0.011 0.019 0.002 0.010 0.015

CONCLUSIONS Kinetic data on DR uptake of emboli allow a conclusion that optimal duration of the process is 1.5–2.5 hours. Hence, uptake of the drug from the solution is 11.5 ± 0.25% (regarding initial DR concentration of solution). Similar uptake degree may also be expected for higher DR concentrations.

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Experimental data on DR uptake of EM and DR release rate to the solution in vitro may be the model for estimation of medicinal preparations’ behavior in the organism after carrying out chemoembolization. At the temperatures approaching in vivo conditions, intensive release of the drug release is observed in the initial 2–2.5 hours. However, it should be taken into account that behavior of drugs in the organism (the rate of release from emboli, diffusion to the embolized organ) may differ from the results presented. Thus, application of embologenic preparations derived from poly-HEMA may be rather prospect in the course of chemoembolization. The high porosity of the preparation presents a possibility to make it uptake with water-soluble medicinal forms. It is shown that DR is able to diffuse from polymeric matrix and, as may be expected, will have the extended therapeutic effect on the surrounding tissue and minimize side impacts on other organs.

REFERENCES [1] [2] [3] [4]

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[5] [6]

A.V. Pavlovsky. Applied oncology // V. 4, No. 2, P. 108-114, 2004. (in Russian)  M. Jelinkova, J. Strohalm, T. Etrych, K. Ulbrich, B. Rihova. Pharmaceutical Research // V. 20, No. 10, P. 1557-1563, 2003.  D. Horak, K.Z. Gumargalieva, G.E. Zaikov, In: Chemical Reactions in Liquid and Solid Phase // Nova Science Publ., New York, P. 11-59, 2003. D. Horak, E. Guseinov, V. Vishnevskii, A. Adamyan, L. Kokov, V. Tsvirkun, A. Tchjao, M. Titova, N. Skuba, N. Trostenyuk, K. Gumargalieva. J. Biomed. Mater. Res. // V. 5, P. 184-190, 2000.  RF Patent No. 61120, 2007 (in Russian).  Patent of USSR No. 1403418, 1988 (in Russian). 

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In: Handbook of Chemistry, Biochemistry … Editors: L. N. Shishkina et al., pp. 67-79

ISBN: 978-1-61209-425-0 © 2010 Nova Science Publishers, Inc.

Chapter 8

A NEW MECHANISM OF E. COLI RESISTANCE TO ALKYLATION DAMAGE INDUCED BY NO-DONATING AGENT—A “QUASI -ADAPTIVE RESPONSE” Svetlana V. Vasilieva1, Elena Ju. Moschkovskaya, and Michael R.Volkert* N. M. Emanuel Institute of Biochemical Physics Russian Academy of Sciences, Moscow, 119334 Russia *Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, U.S.A.

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ABSTRACT All living organisms posses mechanisms protecting cells from toxic and genotoxic effects of alkylation damages, induced from endogenous and environmental sources. O6methylguanine is the major mutagenic base derivative, which strictly modifies base pairing and leads to mutations. To prevent the effects O6- alkylguanine - DNA alkyltransferase (Ada protein) directly dimethylates O6-meG in cellular DNA by transferring the methyl group onto one of their cysteine residues. In E. coli the protective mechanism involves the ada, alkA, alkB and aidB genes expression as well, which is positively controlled by the Ada protein. This DNA repair pathway (the Ada response) is well known as very specific and ubiquitous. Since cysteine methylation at SH groups is the crucial factor for the Ada activation, we assumed that the protein activity can be alternatively regulated by NO – containing agent, via the S - nitrosyl cysteine, functioning in place of S - methyl cysteine in key position. In the present work a new original mechanism of E. coli resistance to alkylation damages induced by NO-donating agent—a “quasi -adaptive response”—was verified experimentally.

Keywords: Adaptive response, quasi-adaptation, NO-donating agents, alkylation damage, E.coli, Ada protein, O6-alkylguanine-DNA alkyltransferase. 1

[email protected].

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Potentially mutagenic alkylating agents occur both in the environment and in the living cells as metabolic products. Suggested candidates for the endogenous DNA alkylating agents included nitrosated amines and amides, S-adenosylmethionine and lipid peroxidation products [1, 2]. An important source of endogenous alkylation is bacterial nitrosation of amino acids and peptides with endogenous nitric oxide (Taverna and Sedgwik, 1996; Garcia-Santos, Calle and Casado, 2001). All organisms—eubacteria, archaebacteria and eukaryotes—have mechanisms protecting cells from toxic and genotoxic effects of alkylation (Pegg, 2000). In E. coli the protective mechanisms involve tag and ogt genes, which are expressed constitutively, and ada, alkA, alkB and aidB genes, expression of which is associated with induction of the adaptive response (Ada –response) and positively controlled by the Ada protein [6]. Induction of the Ada response increases the cellular levels of the Ada and the Ada-regulating proteins which protect cells against the mutagenic and killing effects of DNA alkylating agents. The tag and the alkA gene products act as DNA glycosylases and both excise N3-meA from DNA to yield apurinic sites [7]. The alkB gene belongs to the same operon as the ada gene, and its expression is similarly controlled by the ada promoter [8]. The AlkB protein is iron-dependent and utilizes a unique mechanism of oxidative demethylation to direct repair N1-meA and N3meC, eliminating the methyl group in the form of formaldehyde [9]. The multifunctional AidB protein is regulated via two pathways: one is ada-dependent while the other is ada-independent and is activated when cells are grown in the absence of aeration in response to acidification of the medium [10]. The aidB genes are transcribed by a σs -directed RNA polymerase holoenzyme (rpoS gene) [11].The rpoS gene is required for the expression of a wide variety of genes expressed in stationary-phase cultures. The rpoS gene product is present at low levels in exponentially growing cells, as well. But as cells enter stationary phase, rpoS levels increase and expression of rpoS –dependent genes is activated at both the level of transcription and the level of translation. Since cysteine methylation at SH groups is the crucial factor of Ada activation, we assumed that the Ada activity can be alternatively regulated by NO –containing compounds, via the S- nitrosylcysteine functioning in place of S- methylcysteine in key position. This assumption was based on the data that within proteins, transcription factors and enzymes namely cysteine residues and the formation of disulfide bridges are frequently crucial for tertiary structure and function, and it was evident that S-nitrosylation was a valid mechanism for the signaling effects of NO and NO-containing signal molecules. Moreover this modification also fulfills the criteria of signal reversibility, which is essential for the signal transduction. In this work an original hypothesis of a “quasi-adaptive response” to N-methyl-Nnitrosourea (MNU) in E. coli cells was verified experimentally for the first time.

MATERIALS AND METHODS Chemicals Cysteine, reduced glutathione (GSH), o-nitrophenyl β-D-galactopyranoside (ONPG), o phenanthroline (OP) and HEPES were purchased from Sigma (U.S.A.), ferrous sulfate from

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Fluka (UK). Dinitrosyl iron complex with glutathione (DNICglu) was used in dimeric form and prepared according to Dr. Vanin methodology [12]—by treatment of 5.4 mM FeSO4 and 10.8 glutathione ( iron : thiol ratio= 1:2) with gaseous nitric oxide in Thunberg vessels ( pressure 200–300 mm Hg) in a solution (15 mM HEPES, pH 7.6) previously degassed by evacuation. S-nitrosoglutathione (GSNO) was synthesized in Thunberg vessels by treatment of 50 mM glutathione with a mixture of gaseous NO and air for 5 min with subsequent evacuation of excess NO2. The concentration of nitroso adducts was determined spectrophotometrically at 340 nm (molar extinction coefficient 980 M-1 cm -1 DNICglu was prepared just before the experiment. MNU was synthesized at the Institute of Chemical Physics RAS and dissolved in a phosphate buffer (pH 6.0).

Bacterial Strains We used E. coli strains MV1571( alkA51::Mu d1(lac bla), MV1601(alkB52::Mu d1(lac bla), and MN2176 (aidB1::Mu d1(lac bla) which were constructed by M. Volkert. Genetic characteristics of the strains were described in [13]. These strains contain the lacZ structural gene for β- galactosidase under the control of the ada promoter, while the chromosomal lac operon is deleted. Thus, expression of the genes under study was assayed indirectly by the βgalactosidase activity, which was measured colorimetrically at 420 nm on digital spectrophotometer PD-303 UV (Apel Co. Ltd, Japan).

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Gene Expression L medium and 63 buffer were used for growth, manipulation and storage of bacteria [14]. Gene expression was monitored as described by Quillardet et al. [15]. In brief, log-phase E. coli cells were treated with the appropriate inducers for 30 min, diluted 50 –fold with LB, supplemented with chromogen ONPG, and incubated at 37oC for 2 h. The buffer for βgalactosidase activity assays was as described in [15]. β- galactosidase units (E) were calculated from the equation : E =1000 х D420 /t where D420 is the absorbance at 420 nm and t is the incubation time with the chromogen. Each experiment was performed in triplicate. The (true) adaptive response to MNU was induced by treating log-phase cells with 0.01mM MNU (the sublethal concentration) in a liquid L medium with aeration at 37oC for 1 h. To induce a quasi –Ada response the exponentially growing E. coli cells were pretreated in aerobic conditions with DNICglu at a sublethal conc., in the dark. In some experiments (for the super-quasi-Ada induction) log-phase cells were treated with sublethal MNU conc. just before successive treatment with DNICglu. Incubation E. coli MV2176 aidB::lacZ under oxygen –limiting conditions. In our previous work it was demonstrated that lack of aeration provides culture conditions that are sufficiently anaerobic to cause expression of known anaerobically regulated gene aidB1, during stationary phase of growth [13]. So, oxygen-limiting conditions were provided by incubation of the culture in tubes without aeration during 1-5 h. Each hour the parameters of D600 and E were

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compared in the alternative experiments with/without aeration and the level of aidB1 expression was evaluated as the ratio E/ D600. Each experiment of aidB1 gene expression was repeated 5 times and the mean value in five repeats was used for the results evaluation and presentation. β- galactosidase activity assays with a cell-free system were performed with the chromogenic substrate ONPG in the presence of MNU and DNICglu. A sample (2 ml) was incubated at 37oC for 1 h, combined with 0, 4 ml of buffer B containing ONPG [14, 15] and then incubated for 20 min until it yellowed. The reaction was terminated by addition 1M Na2CO3 [15].All samples free of DNICglu were tested for D420 spectrophotometrically against the buffer. Samples containing DNICglu., which is yellowish in solution, were tested against 0,2 mM DNICglu.

Electron Paramagnetic Resonance (EPR) Study Cells were grown aerobically in LB medium to OD600 = 0, 4. To prepare of one EPR sample, 100 ml of culture was centrifuged at 7000 x g and concentrated to 5 ml prior to 30 min incubation with small aliquots of NO donor or an iron-chelating agent, o-phenanthroline (OP). The cells were then centrifuged, resuspended in 0, 3 ml of the medium and quickly frozen in calibrated tubes for EPR analyses. X – band EPR spectra were recorded on a Radiopan spectrometer (Poland) under the following conditions : temperature 77 K, microwave power 5 mW, modulation amplitude 0,5 mT.

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RESULTS The primary aim of this work was to examine experimentally the hypothesis of a“quasi Ada” response to MNU in E. coli. To compare the levels of the Ada-response genes expression in true (classical) and in quasi-Ada-response the exponentially growing E. coli mutants were treated with sublethal concentrations of MNU (0.01mM) or DNICgly. Our preliminary experiments showed that DNICgly possesed a relatively low cytotoxic effects to the E. coli mutants: the sublethal concentration after 30 min exposure was estimated at 0,1 mM for [alkA ::lacZ] mutant, 0.2 mM for [aidB::lacZ] mutant and 0.5 mM for [alkB ::lacZ] mutant. An analysis of MNU dose response –cell sensitivity showed that the level of the Ada- regulon gene expression increased considerably, both in true and in quasi-Ada responses. 1.0 mM MNU increased the level of alkA and alkB gene expression about twofold in true Ada response and 3–3.5 –fold in quasi-Ada response, resp. ( Figures 1,2).The highest level of quasi-Ada response was observed in the cells with a high content of the Ada protein - after preliminary adaptation of the experimental cells with sublethal dose of MNU (i.e., after two successive pretreatments of the cells : the first, with sublethal dose of MNU, and the second with DNICglu) just before the main treatment with 1.0 mM MNU. Level of alkB gene expression in such “super-quasi adapted” cells was more than fivefold higher than in intact cells (nonadaptive control) and more than twofold higher than in true Ada-response to MNU (Fig 2).

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Figure 1. Effect of MNU on induction of alkA :: lacZ in E. coli MV1571.Cells were grown to log phase of growth with aeration. After that the cells were tested before or after pretreatment with: (1) 0,01mM MNU; (2) 0,1mM DNICglu; (3) 0,01mMNU→ 0,1 mM DNICglu (the successive pretreatments) ; (4) 0,1 mM OP→ var.2,3.

Figure 2. Effect of MNU on induction of alkB :: lacZ in E. coli MV1601. Curves 1-4 significance is the same as in Figure1, but DNICglu conc. was 0.5 mM.

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Figure3. Effect of MNU on induction of aidB :: lacZ in E. coli MV2176. Curves 1-4 significance is the same as in Figure1, but DNICglu conc. was 0. 2 mM.

Regardless the adaptation the aidB gene is expressed normally to a far lower level than the alkA and alkB genes in the experiments with MNU [16]. In the present work the highest level of aidB expression (80-90% of the cell viability) was observed at 0.2 mM MNU in intact cells, opposite to 1 mM –a fivefold higher MNU concentration, in the adapted cells (Figure 3).An increased levels of the aidB expression were observed in all variants with the cell preadaptation. The phenotypic expression of the Ada response to alkylating agents involves an increasing resistance to toxic and mutagenic activity of the agents in the adapted E. coli cells. Our experiments with quasi- adaptation in E. coli alkB::lacZ revealed that the cell resistance to MNU increased more than in true Ada, at least in terms of the parameters under study (Table 1). Cytotoxic effects of NO and NO-containing agents are mostly associated with intracellular enzyme inactivation. As we used the methodology of inducible β - galactosidase activity for indirect characterization of lacZ and the fusion gene expression, we performed some additional control experiments to monitore the effects of MNU and DNICglu – separately and in complex (1:1)- on the enzyme activity in a cell-free system. β - galactosidase activity assays in a cell free system were performed according to “ Materials and methods”, and the Ada –inducers were tested at concentrations allowing the maximum levels of the experimental gene expression. Neither MNU nor DNICglu or their combined action affected β - galactosidase activity in vitro (Table 2). Similarly, present with the MNU in solution, DNICglu (1:1) had no effect on the UVabsorption spectrum of the mutagen in the region of 200–350 nm (data not shown) The primary aim of the next stage of our work was to elucidate the role of the Ada protein in the quasi-Ada response development. For this purpose we used the unique phenomenon –the double genetic control of the aidB gene expression and its relative independence on the Ada protein regulation through anaerobic cultivation.

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Table 1. Survival and mutation of E.coli alkB::lacZ as a function of MNU concentration and the pretreatment agents The rate of Arg+ revertants per 106 cell survival

Cell survival, % MNU concentra -tion (mM)

control

Agent for the adaptive pretreatment MNU

DNICglu

control

Agent for the adaptive pretreatment MNU

DNICglu

10,0

1,0

15,5

19,5

4,6

1,8

1,3

20,0

0,6

12,0

16,75

40

9,5

5,0

40,0

0,16

2,3

2,5

380

21

16,5

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Table 2. MNU and DNICglu influence on the β - galactosidase activity in vitro Sample composition

D420

Buffer B

0,25

Buffer B + β - galactosidase

1,8

Buffer B + β - galactosidase + 0,2 мМ DNICglu

1,6

Buffer B + β - galactosidase +1 mM MNU

1,9

Figures 4–5 present the kinetic curves of E. coli MV2176 aidB expression during 5-h anaerobic cultivation (curves 3–4) in comparison with the data of the aerobic growth and depending on the conditions of the cell pretreatment. According to our expectation no changes in the levels of the aidB expression were observed in the course of aerobic cultivation of the cells without any treatment, whereas anaerobic conditions caused more than two fold increase in the aidB expression in the same cells. During anaerobic incubation of the cells which were preincubated with the sublethal concentration of MNU(10 mM, Figure 4) or DNICglu (0.5 mM, Figure 5), the maximum levels of the aidB expression decreased nearly to the control level in both cases (Figures 4–5, curves 4). The EPR spectroscopy method was used to check the permeability of E. coli MV1571 alkA ::lacZ cells to 0.5 mM DNICglu and to study the changes in DNICglu concentration and structure in the cells, as well as the conversion of GSNO to DNIC inside the cels. DNICglu is converted from the dimeric into the monomeric form and displays paramagnetic properties in the presence of free thiols in solution or upon binding with SH groups of proteins. In our experiments in the cells incubated with DNICglu, a typical anisotropic EPR signal with the axially symmetric g-factor with g‫ =׀‬2, 03 and g‫ = ׀׀‬2,014 was observed (Figure 6, curve a). A single washing of the cells (Fig 6, curve b) halved the signal intensity, showing that a considerable dose of DNICglu was absorbed on the cell surface and lost during the cell washing.

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Figure4. Kinetics of aidB :: lacZ induction in E. coli MV2176 (stationary phase of growth) during 1-5 hours of cultivation in aerobic or anaerobic conditions and depending on the cells pretreatment with 0,01mM MNU: (1) no pretreatment, aerobic cultivation; (2) MNU pretreatment, aerobic cultivation; (3) no pretreatment, anaerobic cultivation; (4) MNU pretreatment, anaerobic cultivation.

Figure5. Kinetics of aidB :: lacZ induction in E. coli MV2176 (stationary phase of growth) during 1–5 hours of cultivation in aerobic or anaerobic conditions and depending on the cells pretreatment with 0.2 mM DNICglu. Curves 1-4 significance is the same as in Figure 4, but MNU pretreatment was substituted with 0.2 mM DNICglu pretreatment.

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Figure 6. EPR spectra of E. coli cells incubated with NO - donating agents. The samples were prepared, as described in “Materials and methods”. The EPR spectra of E. coli cells incubated with 1mM DNICglu (a) and washed once (b) after incubation, incubated with 2 mM GSNO (c) and treated with 0.1 mM OP before the addition of DNICglu (d), were recorded at 77 K, microwave power 5 mW, modulation amplitude 0.5 mT and gains of 0.5x 105 ( a and b), 1x 105 (c), and 2x105(d).

Incubation of the cells with 0.5 mM GSNO led to the appearance of a DNIC –type EPR signal, in agreement with the reverse reaction (1) in the scheme, which indicates the availability of iron inside the cells( Figure6, curve c). Preincubation of cells with 0.1 mM ironchelating agent OP reduced tenfold the intensity of the EPR signal at g‫= ׀‬2,03 in cells exposed to DNICglu , as compared with the initial level. The shape of the spectrum was also changed, suggesting decomposition of DNICglu. In the alkA mutants low concentration of OP (0.1 mM) already prevented quasi – adaptation in all variants with DNICglu pretreatment (Fig 1) because of NO-complex decomposition. Expression of the alkB gene (Fe2+- dependent AlkB protein) was completely suppressed by OP treatment (Fig 2). After 30-min aidB cells preincubation with DNICglu in aerobic conditions an appearance of EPR signal, typical of DNIC, was observed . The EPR signal increased 3 fold after 3-hour incubation of the culture with /or without aeration (the data not shown). So, in E. coli aidB cells treated with DNICglu, a typical anisotropic EPR signal with the axially symmetric g-factor was observed during 1–3 hour incubation of the cells in aerobic as well as in anaerobic conditions, but only the anaerobic conditions provided the significant inhibition of the aidB gene expression in the cells..

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DISCUSSION The adaptive response to alkylating agents is a unique in the complex set of DNA repair pathways in E. coli. On the one hand the Ada protein acts as a chemosensor in genetic signal transduction, with its cysteine residues accepting the alkyl groups from alkylated DNA. On the other hand, the Ada protein is involved directly in removing the alkyl group from O6-alkG, restoring the native DNA structure. When the molecular genetic models of O6-alkG repair by the Ada (the MGMT protein in mammals) were constructed and the Ada DNA repair process proved to be conserved in the course of biological evolution (23), studies were initiated to search for potential chemicals regulating the Ada (MGMT) protein activity as a perspective direction in chemotherapy with alkylating carcinolytics. Up-to-date the most potent inhibitor of human MGMT - O6-benzylG was constructed on the base of theoretical computation as a pseudosubstrate of MGMT (24, 25).This agent has come into clinical practice as the component of adjuvant chemotherapy, notwithstanding its high toxicity for marrow cells (23). As a rule such inhibitors are partly specific: the Ada protein of E. coli is far less sensitive to O6-benzylG than human MGMT. DNICglu was used in our experiments as a model for NO-donating agents because it generates NO in water solutions [20].Under physiological conditions the NO generated may form GSNO or DNIC in reactions with thiols (such as glutathione, cysteine or protein SH groups) and iron [21]. The chemical structures of the NO donor used and the reactions which can take place inside the cells in the present of DNICglu, iron and free thiols are given in the scheme below. RS- NO+ \ / redox reactions +

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Fe RS-NO + Fe

2+

-

-

+ NO + RS (1)

/ \ Fe 2+ ions RS- NO+ (DNIC) (GSNO ) DNIC + SH- protein or Fe-S- protein DNIC- protein + 2RS- (2) The EPR study showed that DNIC enters the cells because cells treated with the agent and then washed still exhibited a DNIC –type EPR signal ( Figure6). Influence of the iron – chelating agent OP on deleterious free radical effects in biological systems can be changed from protection to sensitization due to the significant role metal ions play in O2-. toxicity. In our case, however, it seems that OP decreased the effects of DNIC by extracting iron ions from the cells and preventing the back DNIC formation. No is produced by many (if not all) mammalian cells, as well as bу some prokaryotes, and fulfills a wide spectrum of signaling functions in genetic, physiological and pathophysiological processes. NO has multiple molecular targets and can not only directly influence the activity of transcription genes but also modulates upstream signaling cascades, mRNA stability and translation, as well as the processing of the primary gene products. For example, the newest data in E. coli cells have illustrated the regulatory functions of NO in DNA repair pathways,

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such as the SOS-, SoxRS-, OxyR-, Uvr-, etc. systems [12, 22-24]. The mechanisms of NO activation of the regulatory proteins are different. For example, NO- activation of SoxR protein proceeds through two steps and involves generation of low-molecular- weight “DNIC primers” [23], which interact with thiol groups of the SoxR protein as components of an iron-sulfur complex and facilitate disintegration of [2Fe-2S] clusters in the SoxR protein. Intramolecular S=S bond is formed between Cys-199 and Cys-208 of OxyR tetramer upon its NO oxidation [24] that underlies the control of cell sensitivity to NO-oxidative stress. In this work we have shown that NO-donating agent DNICglu induces a quasi-adaptive response to MNU in E. coli cells when used instead of MNU for the adaptive pretreatment of the cells. On the one side this finding means an additional function played by NO in genetic signal transduction in E. coli. On the other side it suggests a new mechanism functionally activating the Ada chemosensor protein to control the Ada- regulon gene expression and resistance to alkylating agents- the mechanism of S- nitrosyl- cysteine generation as compared with S-methyl –cysteine generation in the true Ada-response. We found that during stationary phase of growth nitrosylated Adaprotein, like meAda, suppressed the aidB1expression, due to inhibition of the initial binding of E. coli σs to aidB promoter, possibly by competition with E. coli σs for the same binding site [25]. Alternatively, it appeared, that binding of nitrosylated Ada- protein aidB promoter might alter the aidB1 conformation to make it less favorable for interaction with E. coli σs. Taken together these results demonstrate a new mechanism regulating the activity of the Ada sensory protein in the “quasi- adaptive” response by NO –containing complex of [Fe2+(NO2+)2] with glutathione. While Cys-69- meAda initiates adaptation in the true adaptive response, [(Cys-)2Fe+(NO+)2] complex with the Ada protein Cys - residues, plays a critical key role in the quasi-adaptive response. According to our recent work [12] namely DNICs ( no NO) play a key role as the universal signal molecule in genetic signal transduction. Indeed, the EPR study showed that OP pretreatment of E. coli cells can remove the iron accessible for DNIC formation after GSNO addition and can prevent of the SOS response induction by NO-donating agents [12]. In the present work even a low OP concentration prevented DNICglu induction of the Adadependent gene expression (Figure 1–3), as well as the typical DNIC signal appearance in the same cells (Fig 6). In the present study namely DNICglu efficiently stimulates DNA repair ability of the Adadependent proteins in E. coli and decrease sensitivity of the cells to cytotoxic and mutagenic potencies of MNU. According to Liu and coauthors [26] exposure to the alternative NO-donating agents Snitroso- N- acetylpenicillamine (SNAP) or GSNO - caused irreversible loss of DNA repair capacity for alkylating adducts by human MGMT (hAGT) in vivo and in vitro. It appeared that in these cases such NO-donating agents would increase the therapeutic efficiency of alkylating drugs. Indeed, a cytotoxic efficiency of some new crystal NO- donating agents in combined action with the well known alkylating drugs was demonstrated in vitro at the first time [27]. Our experiments with the series of NO - donating agents - as the quasi-Ada-response inducers in E. coli cells - are now in progress. This work was supported by the Russian Foundation for Basic Research.

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Lutz W.K. Endogenous genotoxic agents and processes as a basis of spontaneous carcinogenesis. Mutat. Res. 1990. V. 238. P. 287 - 295. Sedgwick B. Nitrosated peptides and polyamines as endogenous mutagens inO6 – alkylguanine -DNA alkyltransferase deficient cells. Carcinogenesis.1997. V. 18. N8. P. 1561-1567. Taverna P., Sedgwick B. Generation of an endogenous DNA- methylating agent by nitrosation in Escherichia coli. J.Bacteriol.1996.V178. P. 5105-5111. Garcia – Santos M.P., Calle E., Casado J. Amino acid nitrosation products as alkylating agents. J. Am. Chem. Soc. 2001. V.123. P 7506 – 7510. Pegg A.E. Repair of O6 - alkylguanine by alkyltransferases. Mutat. Res. 2000. V.462. P.83- 100. Samson I., Cairns J. A new pathway for DNA repair. Nature.1977.V. 267. P. 281- 283. Teo I., Sedgwick B., Kilpatrik M. et al., The intracellular signal for induction of resistance to alkylating agents in E.coli. Cell. 1986. V.45. P. 315 – 324. Lindahl T., Sedgwick B., Sekiguchi M. et al. Regulation and expression of the adaptive response to alkylating agents. Annu. Rev. Biochem. 1988.V.57. 133-157 Falnes P.O., Johansen R.F., Seeberg E. A third mechanism for DNA damage reversal catalysed by the AlkB protein in E.coli. Abstracts of the 32nd Annu. Meeting of EEMS. Poland. 2002. P.115. Smirnova G.V., Oktyabrsky O.N., Moshonkina E.V. et al. Induction of the alkylation – inducible aidB gene of Escherichia coli by cytoplasmic acidification and Nethylmaleimide. Mutat. Res.1994. V. 314. P. 51 – 56. Landini P., Hajec L.I., Volkert M.R. Structure and transcriptional regulation of the Escherichia coli adaptive response gene aidB. J. Bacteriol. 1994. V. 276. P. 6583 – 6589. Lobysheva I.I., Stupakova M.V., Mikoyan V.D., Vasilieva S.V., and Vanin A.F. Induction of the SOS DNA repair response in Escherichia coli by nitric oxide donating agents: dinitrosyl iron complexes with thiol – containing ligands and S –nitrosothiols. FEBS Lett. 1999. V. . 454. P.177 -180. Volkert M. Adaptive response of Escherichia coli to alkylation damage. Environ. Mol Mutagen. 1988. V. 11. P. 241 – 255. Miller J.H. Experiments in Molecular Genetics. N.Y.: Cold Spring Harbor Lab. Press, 1972. Quillardet P., Hofnung M. The SOS chromotest, a colorimetric bacterial assay for genotoxins: procedures. Mutat. Res. 1985. V. 147. P. 65 – 78. Vasilieva S.V., Makhova E.V., Moshkovskaya E.Y. Expression and functions of adaptive response genes in Escherichia coli treated with mono- and bifunctional alkylating agents: interference with the SOS response. Genetics.(Russ.). 1999. V. 35. № 4. P. 364-369.) Pegg A.E., Boosalis M., Samson L. et al. Mechanism of inactivation of human O6alkylguanine-DNA alkyltransferase by O6-benzylguanine. Cancer Res. 2002. V. 62. P. 3037-3043.

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[18] Dolan M.E., Chae M.Y., Pegg A.E. et al. Metabolism of O6-alkylguanine-DNA alkyltransferase. Cancer Res. 1994. V. 54. P. 5123-5130. [19] Dolan M.E., Pegg A.E. O6-benzylguanine and its role in chemotherapy. Clin. Cancer Res 1997. V. 3. P. 837-847. [20] Vasilieva S.V., Moschkovskaya E. Ju., Terekhov A.S., Sanina N.A., Aldoshin S.M. Genetic activity of structurally different NO-donating agents depends upon the cellular iron. Genetics (Russ.). 2006. V. 42.N.7. P. 1-7. [21] Vanin A.F. Dinitrosyl iron complexes and S-nitrosothiols as two possible forms for stabilization and transport of nitric oxide in biosystems. Biochemistry (Russ.).1998. V. 63 (7). P.924-938. [22] Gaudu P., Moon N., Weiss B. Regulation of the SoxRS oxidative stress regulon. J.Biol. Chem.1997. V. 272. № 8. P.5082-5086. [23] Vasilieva S.V., Stupakova M.V., Lobysheva I.I., Mikoyan V.D., Vanin A.F. Activation of the Escherichia coli SoxRS - regulon by nitric oxide and its physiological donors. Biochemistry (Moscow) 2001.V. 66.№ 9. P. 984-988. [24] Storz G., Imlay J.A. Oxidative stress. Current Opinion in Microbiol.1999.V.2. P.188194. [25] Landini P., Volkert M.R. Regulatory responses of the adaptive response to alkylation damage: a simple regulon with complex regulatory features. J. Bacteriol. 2000. V. 182. № 23. P.6543-6549 [26] Liu L., Xu- Welliver M., Kanugula S., Pegg A.E .Inactivation and degradation of O6 – alkylguanine - DNA alkyltransferase after reaction with nitric oxide. Cancer Res. 2002. V. 62. P. 3037 – 3043. [27] Zhukova O.S., Sanina N.A. Fetisova L.V., Gerassimova G.K. Cytotoxic effects of nitrosyl- iron complexes in human tumor cells in vitro. Russian J.Biotherapy.2006.V. 5(1). P.14 -17.

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Chapter 9

A KINETIC APPROACH TO EXPLAIN THE EFFECTS OF α-TOCOPHEROL AT THE PHYSIOLOGICAL AND ULTRA-LOW CONCENTRATIONS ON THE ACTIVITY OF PROTEIN KINASE C IN VITRO E.L. Maltsevau,1, K.G. Gurevich2 and N.P. Palminav,1 1

N.M.Emanuel Institute of Biochemical Physics of Russian Academy of Sciences, Moscow, Russia 2 Sub-Faculty of Pathological Physiology of Medical Department, Moscow State Medico-Dental University, Moscow, Russia

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ABSTRACT The effect of a natural antioxidant α-tocopherol (α-TL) (in concentrations from 10-2 to 10-17 M) on the activity of protein kinase C (PKC) isolated from rabbit hearts was studied. Subsequent modeling was performed in terms of kinetic methods. It was shown that α-TL inhibits PKC to a maximum of 80% by a non-competing mechanism. It was found that the dose dependence yields a bimodal curve with the maxima of inhibition at the α-TL concentrations 10-4 and 10-14 M. It was shown that the substrate (histone H1) dependences of the PKC activity in the absence and in the presence of high (10-4 M) and ultra-low (10-14 M) doses of α-TL exhibit maxima at the same concentration of histone H1 (1 μM). The effect can be described by a formal kinetic scheme of inhibition with an excess amount of the substrate. Identification of the parameters of the system was performed with a conjugate gradient technique; the approximation of the experimental results is 98%. A kinetic scheme of allosteric regulation of the PKC activity under the action of α-TL was suggested; the scheme adequately describes the bimodal dose—effect dependence. A good agreement between the experimental and theoretical constants was obtained.

Keywords: Protein kinase C (PKC), α-tocopherol (α-TL), enzyme kinetics, ultra-low doses. u

Corresponding Address to: Dr. E.L. Mal’tseva (Ph.D.), N.M. Emanuel Institute of Biochemical Physics of Russian Academy of Sciences, Kosygin str. 4, 119334 Moscow, RUSSIA. v [email protected]. Handbook of Chemistry, Biochemistry and Biology : New Frontiers, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

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AIMS AND BACKGROUND Protein kinase C (PKC) is the key enzyme of the phosphoinositol cycle. Its activity on the cell and membrane is interrelated with many physiological processes and pathologies that affect cell proliferation [1-3]. Protein kinase C belongs to membrane-bound (in the active form) and peroxilipid-dependent enzymes [4-6]. As known, peroxide oxidation of lipids (POL) in membranes is controlled by natural antioxidants; α-TL is the most important one [7-9]. On the other hand, antioxidants can interact directly with enzymes and modify their activity as effectors [10-13]. Of special interest is the effect of ultra-low doses (ULD) of modulators of the enzyme activity. In this dose range, the effects were observed for higher hierarchic levels: from organism to biological membranes [14-16]. Since the levels in vivo have cascade systems of secondary messengers capable of a multiple amplification of a signal, it was essential that the definition of this work would include detection of the ULD effect for a less complicated system in vitro; the system should contain a minimum number of components that are necessary for a PKC-involving enzyme reaction to take place. The aim of this work is to study the effect of α-TL on the PKC activity in a wide range of concentrations and to suggest a possible mechanism of the effect in terms of kinetic concepts of enzyme reactions.

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EXPERIMENTAL Protein kinase C was isolated from rabbit hearts (3–5 hearts for a specimen). The isolation procedure [17] included four stages: ammonium sulfate salting out, an ion-exchange chromatographic study on a DE-52 DEAE-cellulose (Sigma, USA), adsorption on porous glass, and gel filtration on S-200 sephacryl (Serva, Germany). The purification degree of the enzyme was controlled with an electrophoresis technique and subsequent scanning on a Chromoscan automatic densitometer (Joyce-Loeble, Great Britain). The purity of the enzyme fraction was up to 90%. The protein content was determined with brilliant indigo pigment (Sigma). The PKC activity was determined with radioisotope technique modifications [17,18]. The incubation medium (volume 5μl) contained 1.5 μmol Tris-HCl (pH 7.5), 0.5 μM MgCl2, 0.5 μmol CaCl2, 0.01 μmol EGTA, 1.5 nmol [γ-32P] ATP, (0.4–0.6)x106 imp/min (Amersham, Germany), 6 μg Sigma phosphatidylserine (activator), 0.02–20 μM histone H1 (substrate), 1017 –10-3 M α-TL (effector), and 2 μg PKC (enriched fraction). The reaction was performed at 25oC for 10 min and was stopped with addition of EDTA. No less than five replicated experiments were performed. Inclusion of radioactive phosphate was measured with a Unisolv-100 scintillator on a Beckman-9000 (Austria) counter. For the statistical data processing, average values were calculated within the ” 95% confidence interval and compared in terms of the Wilcoxon—Mann—Whitney U-criterion [19].

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RESULTS AND DISCUSSION The main objective of the work was to study the effect of α-TL (in concentrations from 10-2 to 10-17 M) on the activity of PKC; the obtained results are shown in Figure 1. It was found that α-TL inhibits the enzyme activity in concentrations up to 10-16 M. The concentration dependence of the effect is apparently bimodal in character and exhibits two maxima: sharp (at 10-4 M) and broad (in the region of super-low concentrations of α-TL from 10-13 to 10-15 M). The inhibition at the maxima is 60–80%; for the concentrations 10-4 and 1014 , the effect is almost equal. Between the maxima, a so-called “dead zone” is observed, which covers about five orders of α-TL concentrations; the inhibiting effect in this zone. Within this zone, the inhibiting effect of the antioxidant decreases to 20% of its value. Note that the inhibiting effect of α-TL in the dose 10-5 M on the PKC activity had been discovered by other investigators both for the isolated (from brain) enzyme and cellular culture [12, 20, 21]. However, we were the first who have determined the effect of α-TL on the activity of this enzyme in a wide range of concentrations and the second maximum in the range of ULD. Since the main substrate in the PKC-catalyzed enzyme reaction is histone H1, we studied the activity of the enzyme as a function of the substrate (in concentrations from 0.2 to 10 μM) for various α-TL contents in the sample. The first stage of the work included investigation of the effect of histone H1 on the PKC activity in the absence of α-TL (Figure 2). As seen from Figure 2, low concentrations of the substrate result in an increase in the rate of the enzyme reaction; the concentrations of the substrate above 1 μM result in a decrease of the rate. From the kinetic point of view, the process may be described by the following schemes [22, 23].

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1) Inhibition of the enzyme by the reaction product: E + S ↔ ES ES ↔ E + P E + P ↔ EP or ES + P ↔ ESP

(1)

2) Inhibition by an excess amount of the substrate: E + S ↔ ES ES ↔ E + P ES + S ↔ ES2,

(2)

where E is the enzyme (PKC); S is the substrate (histone H1); ES is the enzyme—substrate complex; P is the enzyme reaction product; EP, ESP, and ES2 are the non-reactive enzyme— product complexes and the double enzyme—substrate complex, respectively. If Scheme (1) was valid, the accumulation of the product could have resulted in a gradual shift of the maximum activity of the enzyme reaction to the region of lower concentrations of histone H1.

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Figure 1. The degree of inhibition (I, %) of PKC activity as a function of α-TL concentration (M).

Figure 2. Activity (A) of PKC (pmol/mg min) as a function of concentration of histone H1 (μM) in the absence of α-TL. Approximation of the experimental data by Scheme (2) including parameters K2E0 = 220 pmol/mg min, Km = 0.081 μM, K1 = 2.5 μM.

However, that was not the case. In addition, there are no reference data on inhibition of the PKC activity by any product of enzyme degradation of histone H1, whereas there is information that the substrate may play the role of a regulator of the enzyme activity [18, 24, 25]. Thus, Scheme (2) can be suggested as a more probable mechanism of the effect of histone H1 on the PKC activity, as compared with Scheme (1). In view of this fact, we performed the identification of the parameters of Scheme (2) in terms of the method of conjugate gradients. The obtained parameters are: k2E0 = 2200 pmol/mg/min, Km = 0.081 μM, and Ki =2.5 μM;

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the approximation level is 98% (Figure 2), where E0 is the α-TL initial concentration. As known, in schemes that include inhibition of a substrate, a point of the maximum enzyme activity corresponds to the relationship √KmK1 [23]. In our study, the relationship is equal to 0.5 μM, which is lower than the maximum value (1 μM). The observed (two-fold) differences are insignificant for the method applied; they can be attributed to errors in determining Km, because this value is much lower than Ki and the range of histone H1 concentrations studied (from 0.2 μM). The next task was to investigate the substrate dependence of the PKC activity with the addition of antioxidant concentrations that result in a maximum effect, i.e., 10-4 and 10-14 M α-TL. The obtained results are shown in Figure 3. In both cases, the substrate dependence is of nearly the same character; the maximum is observed for the same histone H1 concentration as that in the absence of α-TL (1 μM). After addition of SLD, the maximum activity of the enzyme decreased to about one-fifth; a higher α-TL concentration (10-4 M) decreased further the maximum PKC activity: to one-seventh of its value. Beginning with the substrate concentration - 5 μM, an insignificant increase in the PKC activity was observed; the increase was most pronounced at the α-TL concentration 10-4 M. In view of the fact that, regardless of the α-TL concentration, the point of the maximum activity of PKC depending on the substrate concentration did not vary, a conclusion may be drawn about a non-competing regulation of the PKC activity by D-TL with respect to histone H1. According to the published data [25-28], a PKC molecule contains spatially-independent binding sites for a substrate and effectors. Since the α-TL concentration 10-4 M results in not only the supression but also in a reiterated activation of the enzyme activity, we may suppose an allosteric regulation of the PKC activity by α-TL. A simple case of the regulation is described by the following scheme (3):

(3) where L is the ligand (α-TL). Calculations of kinetic parameters for the above scheme showed a good approximation of the experimental data (Figs 2, 3) with the coefficient of correlation 97-98% (see Table 1). As follows from Table 1, addition of α-TL scarcely alters the value of Km, which defines the affinity of the enzyme to the first molecule of the substrate.

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Figure 3. Activity (A) of PKC (pmol/mg min) as a function of concentration of histone H1 (μM) in the presence of α-TL at high (10-4 M) and ultra-low concentration (10-14 M). Approximation of the experimental data includes the parameters K2E0 = 368 pmol/mg min, Km = 0.061 μM, K1 = 11.7 μM for 10-14 M α-TL and 205, 0.086, 41.3 for 10-4 M α-TL, respectively.

Thus, we may state about a non-competing effect of α-TL on the PKC activity, which supports the validity of Scheme (3). Note, however, a decrease in the value of k2E0 by an order of magnitude, as a result of addition of α-TL, both in high and super-low doses. For a comprehensive identification of the scheme parameters, there were no sufficient experimental data; therefore, the parameters were determined in the following approximations: K1 = 9 x 10-13 M, K2 = 2 x 10-6 M, α = 0.2, and β = 0.6. With the above relationships between the parameters, the curve of the effect of α-TL on the activity of PKC as a function of dose should exhibit two maxima at the inhibitor concentrations corresponding to K1 and K2. Indeed, as follows from the experimental data (Figure 3), the maximum inhibition of the PKC activity by α-TL is observed for the range of its concentrations from 10-14 to 10-13 M and 5 x 10-5 M. Thus, as a result of the studies of the PKC activity as a function of concentrations of α-TL (the effector) in the range from 10-3 to 10-17 M and substrate (histone H1) in the range from 0.01 to 10 μM in the absence and in the presence of high and super-low doses of α-TL, the following conclusions may be drawn. (1) The natural antioxidant α-TL is an inhibitor of PKC as used in a wide range of concentrations from 10-2 to 10-16 M. (2) The inhibition is noncompeting in character. (3) The dose—effect curve is bimodal: it has two maxima at the α-TL concentrations 10-4 and 10-14 M. The degree of inhibition is 80%. (4) The identification of the system was performed with the approximation 98%. (5) A formal-kinetic scheme was suggested that describes satisfactorily the enzyme reaction under the action of the inhibitor in the given range of concentrations. The corresponding kinetic parameters were calculated; a good agreement between the experimental and theoretical constants was obtained. It should be noted that the suggested kinetic scheme is one of possible mechanisms of the effect of ULD and explanations of the bimodal character of the curve for the effect of α-TL on the activity of PKC in vitro. At present, there are several hypotheses that describe satisfactorily the experimentally observed effects of ULD in terms of physico-chemical concepts, e.g., the

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pioneering allosteric hypothesis proposed by Prof. Burlakova et al.[29], the concept of parametric resonance by Prof. Blumenfeld [30] and that of fluctuation including the Smoluchowski equation [31], the Zaitsev—Sazanov model [32] of receptive adaptation based on the Koshland “cell memory” principle, and the novel kinetic model of a ligand—receptor interaction involving the Markov chains [33]. The above hypotheses and models are not in conflict with each other; they describe consistently the bimodal dose—effect relationship and existence of the maximum for ULD. The allosteric hypothesis [29, 34] that includes the smallest number of assumptions and is based on the principle of allosteric regulation of the activity of enzymes containing sites of various affinities is best suited for our case of the effect of α-TL on the PKC activity. In particular, in terms of this hypothesis, a hypothetic situation was considered when the constants for two sites of binding of ligand (or substrate) of the enzyme reaction are 10-13 and 10-8 M and the difference between them is not less, than five orders of magnitude [34]. According to the calculations of these constants on the basis of the suggested scheme, K1 = 9 x 10-13 M and K2 = 2 x 10-6 M; the difference between these values is six orders of magnitude. Thus, kinetic scheme (3) may be regarded as a particular case of the allosteric hypothesis about the effect of ULD.

CONCLUSIONS

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1) The inhibition of PKC activity by natural antioxidant - α-TL in a wide range of concentration (10-2 -10-16 M) is characterized by the bimodal curve with two maxima at 10-4 and 10-14 M.. 2) The inhibition is described by complicated non-competing enzyme kinetics. 3) The identification of the system was performed with the approximation 98%. 4) The formal-kinetic scheme proposed is well described; the enzyme kinetic reactions under consideration and the corresponding kinetic parameters have been calculated.

REFERENCES [1] [2] [3] [4] [5] [6] [7]

Y. Nishizuka: Turnover of Inositol Phospholipids and Signal Transduction. Science, 225, 1365 (1984). J.-F. Kuo (Ed.). Protein Kinase C. New York - Oxford University Press, 1994, p.223. C.M. Gould, A.C. Newton. The Life and Death of Protein Kinase C. Curr. Drug Targets, 9, 614 (2008). R.R. Rando. Regulation of Protein Kinase C Activity by Lipids. FASEB J. 2, 2348 (1988). N.P. Palmina N.P., E.L.Maltseva, E.B. Burlakova. Protein Kinase C – a Peroxyl-Lipid Dependent Enzyme. Physical Reports, 14, 1753 (1995). E.L. Maltseva: The Role of Lipid Peroxidation Products in the Regulation of Protein Kinase C Activity in vitro. Adv. Exp. Med.and Biol., 400, 339 (1997). E.B Burlakova, S.A. Krashakov, N.G. Khrapova. A Role of Tocopherols in Lipid Peroxidation of Biomembranes. Membr. Cell. Biol. 12, 173 (1998).

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[17]

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E.L. Maltseva, K.G. Gurevich and N.P. Palmina V.E. Kagan, E.R. Kisin, K. Kawai, B.F. Serikan, A.N. Osipov, E.A. Serbinova, I.Wolinsky, A.A. Shvedova. Toward Mechanism-based Antioxidant Interventions: Lessons from Natural Antioxidants. Ann.NY Acad. Sci., 959, 188 (2002). M.G. Traber, J. Atkinson: Vitamin E, Antioxidant and Nothing More. Free Rad. Biol. Med. 43, 4 (2007) E.L. Maltseva, E.B. Burlakova: The Difference in Brain and Liver Cell Membrane Response to Antioxidant and Fatty Acid Effects In Vitro (Changes in Activity of Cyclases and Viscosity). Biol. Membr., 3, 769 (1986). M. Ruszene, A. Donella-Deana, A. Alexandere, M. Francesconi, and D.Renzo: The Antioxidant Butylated Hydroxyluene Stimulates Platelet Protein Kinase and Inhibits Subsequent Protein Phosphorylation Induced by Trombin. Biochem. Biophys. Res. Commun., 1094, 121 (1991). C.W. Mahoney, A. Azzi: Vitamin E Inhibits Protein Kinase C Activity. Biochem. Biophys. Res. Commun., 154, 694, (1988). E.L. Mal’tseva, N.P. Pal’mina, E.B. Burlakova: Natural (alfa-Tocopherol) and synthetic (Phenosan Potassium Salt) Antioxidants Regulate the Protein Kinase C Activity in a Broad Concentration Range (10-4 -10-20 M). Membr. Cell Biol., 12, 251 (1998). C. Doutremepuich C. (Ed.): Ultra Low Doses. Taylor and Francis, London, Washington, 1991, p.184. The Effects of Ultra-Low Doses of Biological Active Substances. Russian Chem. J, 18 (1999). E.B. Burlakova, A.A. Konradov, E.L. Mal’tseva: Effect of Supersmall Doses of Biologically Active Substances and Low-Intensity Physical Factors. J. Adv. Chem. Physics, 2, 140 (2003). B.C. Wise, R.L Raynor, and J.F. Kuo: Phospholipid-sensetive Ca2+-dependent Protein Kinase from Heart. I. Purification and General Properties. J. Biol. Chem., 257, 8481 (1982). E.L.Mal’tseva, N.V. Kurnakova, N.P. Pal’mina, E.B. Burlakova: Kinetic Behavior of Protein Kinase C as Dependent on the Extent of Effector Oxidation. J. Chem. Biochem. Kinetics, 1, 93 (1991). V.Y. Urbakh V.Y.: Statistic Analysis in Biological and Medicinal Studies. Nauka, Moscow,1975. D. Boscoboinik, A. Szewczyk, C. Hesley, and A.Azzi: Inhibition of Cell Proliferation by alfa-Tocopherol. Role of Protein Kinase C. (1991) J. Biol. Chem. 266, 6188, (1991). E. Chatelain, D.Boscoboinik, G. Bartoli, V. Kagan, F. Gey, L. Packer, and A. Azzi: Inhibition of Smooth Musle Cell Prolieration and Protein Kinase C Activity by Tocopherols and Tocotrienols, Biochim. Biophys. Acta, 1176, 83 (1993). S.D. Varfolomeev, K.G. Gurevich: Biokinetics. Fair-press, Moscow, 1999. A.A. Podkolsin, K.G. Gurevich: The Effect of Biologically Active Substances in Low Doses.KMK, Moscow, 2002. Hannun Y.A., and Bell R.M.: Rat brain Protein Kinase C. Kinetic Analysis of Substrate Dependence, Allosteric Regulation, and Autophosphorylation. J. Biol. Chem., 265, 2962 (1990). A.C. Newton: Protein Kinase C: Structural and Spatial Regulation by Phosphorylation, Cofactors, and Macromolecular Interactions. Chem.Rev., 101, 2353 (2001).

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[26] A.C. Newton, Koshland D.Jr.: High Cooperativity, Specificity, and Multiplicity in the Protein Kinase C - Lipid Interaction. J. Biol. Chem., 264, 14909 (1989). [27] P.M. Blumberg, N. Kedei, N.E. Lewin, D. Yang, G. Czifra, M.L. Peach, V.E. Marquez: Wealth of Opportunity – the C1 Domain as a Target for Drug Development. Curr. Drug Targets, 9, 641 (2008). [28] V. Kneifets, D. Mochly-Rosen: Insight into Intra- and Inter-Molecular Interactions of PKC: Design of Specific Modulators of Kinase Function. Pharmacol. Res. 55, 467 (2007). [29] E.B Burlakova, A.A. Konradov, I.V. Khudyakov: The Action of Chemical Agents in Ultra- Low Doses on the Biological Objects. Izves. Acad. Nauk SSSR, Biology, 2, 184 (1990) [30] L.A. Blumenfeld: A Conception of Structure in Biophysics. Question about the Mechanism of Action of Ultra-Low Doses. Russ. Chem. J., 18, 15 (1999). [31] L.A. Blumenfeld, A.Yu. Grosberg, A.N. Tikhonov: Fluctuations and Mass Action Law Breakdown in Statistical Thermodynamics of Small Systems. J. Chem Phys., 95, 7541 (1991). [32] L.A. Sazanov, S.V. Zaitsev: Effect of Superlow doses 10-18-10-14 M of Biologically Active Substances: General Rules, Features, and Possible Mechanisms. J. Biochem.Org., 1, 253, (1994). [33] K.G. Gurevich, S.D. Varfolomeev: Probable Description of the Ligand-Receptor Interaction. Estimation of Reliability of the Events with small and super-small doses. Biokhimiya, 64, 1038 (1999). [34] E.B. Burlakova: Specificity of Action of Super-Low Doses of Biologically Active Substances and Physical Factors at Low-Intensity. Russ. Chem. J. 18, 3 (1999).

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Chapter 10

PHARMACOLOGICAL PREMISES OF THE CREATION OF NEW ANTITUMOR PREPARATIONS OF THE CLASS OF NITROSOALKYLUREA J. A. Djamanbaev1a, Ch. Kamchybekovaa, J. A. Abdurashitovaa, and G. E. Zaikov2b a) Institute of Chemistry and Chemical Technology, Kyrgyz National Academy of Sciences, Bishkek, Kyrgyzstan b) N. M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia

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ABSTRACT Perspectives in the field of creation of highly effective anticancerogenic preparations have been evaluated. For their creation is offered a new regio-selective method of glycosylation of alkylurea in conditions of nucleophilic catalysis with some following nitrosing of glycosyl carbamides of the D- and L-rows. This method opens principally new possibilities for modification of compounds by means of glycosylamides bond allowing us to get preparations, possessing small toxicity and high selectivity.

Keywords: monosaccharides, glycosylureas, N-glycosyds.

1

2

267 Chui Prospect , 720 071 Bishkek, Kyrgyzstan, E-mail: [email protected]. Corresponding author: G. E. Zaikov, 4 Kosygin Street, 119 334 [email protected] .

Moscow,

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AIMS AND BACKGROUND In this work we tried to motivate searching and elaboration of the methods of the syntheses of the antitumor preparations among the derivatives of nitrosomethylurea with the purpose of obtaining new biologically active substances with high selective action, low toxicity by means of putting the carbamides fragment in to monosaccharides. These investigations are of great interest for the solution of practically important problems for physiologically active substances and medical preparations with known therapeutic action. The given direction provides working out of the recommendations on decrease of toxicity, change of water and lipid solubility preparations as well as obtaining derivatives with selective permeability through cell membranes1. A particular interest to nitrosoalkylurea, as potentially antitumor agents, was shown at the beginning of the 60s, after revealing the high antileukemic activity of N-methyl-Nnitrozoguanidin and N-methyl-N-nitrosourea2,3. These compounds have quickly increased the essential range of antitumor preparations of alkylation action4,5. However, a number of side effects of the compounds of this type, first of all high myelotoxicity, restrained their introduction to medical practice. Modification of preparations by introduction to the molecular structure of different substituents, although increasing the range of potentially active compounds, has not eliminated the undesirable influence of the preparations on an organism. The success reached in pharmacology of nitrosoalkylureas is described in particular in Refs. 2 and 6. First natural carbohydrates analogue of N-nitrosomethylurea the antibiotic — ‘streptosotocin’ got from cultural liquid — a streptomycin was found in the same years4,7. It was realised that ‘streptosotocin’ possesses a broad spectrum of actions—antidiabetic, diabetogenic, mutagenic and antitumor—and presents by itself a carbohydrate derivative of nitrosomethylurea from secondary carbon atom of glucoses. The multiple pharmacological studies carried out showed that though ‘streptosotocin’ has the above-mentioned side effects, its general toxicity is much below the most citotoxic fragment, connected with atom C2 D-glucopiranosyd ring. These observations stimulated studies on syntheses and tests on antitumor activity of carbohydrate derivatives of N-methylN-nitrosourea. Repeated suggestions that carbohydrates molecules are the transport carrier, relieving carrying the citostatic groups in the tumors fabric promoted the development of this work. In the process of pharmacological investigations, it was ascertained that glycosylation leads to the sharp decrease of toxicity of medicinal preparations (LD50 falls, as a rule, by two orders). In clinical oncology, of great importance is not only the high antitumor activity of preparations of the group of nitrosourea, but also their ability to run through the hematoencephalitic barrier that opens real possibilities for chemotherapy of metastasis and primary tumors of the cerebrum. The majority of known preparations do not get through the hematoencephalitic barrier and so do not warn of the spreading metastasis in the cerebrum, and they are not practically efficient at defeat of the central nervous system. As to the selectivity of action, it is known that permeability of some cell membranes of animals regarding to monosaccharides is different, and the degree of permeability correlates with the nucleophilic reaction ability of monosaccharides (Table 1)8.

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From that comes the principle possibility of processes regulation of the passing carbohydrates preparation through cell barriers by changing the nature of carbohydrates carrier, as well as their biological activity. It draws attention to the fact that a number of therapeutic characteristics greatly depend on the place of joining of a physiologically active fragment to the carbohydrates ring. Especially it is revealed in the process of study preparations of antitumor action. Table 1. The comparative dependence of the permeability of the monosaccharides from cell membranes from reaction ability of monosaccharides Monosaccharides Glucose Galactose Fructose Mannose Ksilose Arabinose Ribose Licsose

B1 1,0 1,1 0,4 0,2 0,15 0,09 -

B2 0 0,64 0,37 0,81 1,03 1,19 1.25

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B1 – the comparative permeability of the monosaccharides B2 - the comparative reaction ability of the monosaccharides

Rather perspective for practical application, in our opinion, is the usage as ‘blending links’ of more acid-stable amide bonds. What is the value of the amide bond formation? What are the prospects of their use in applied aspects of the chemistry of carbohydrates? It is enough to point to the broad presence of urea fragment amongst natural materials—the derivatives of pyramidine of the row: nucleosides, riboflavin, teobromin, caffeine and others—and development of the direction of heterocycles syntheses, including analogues of the nucleosides on the basis of carbohydrates derivatives of urea. The development of the work on specified directions greatly depends on elaboration of technically acceptable and economically profitable methods of putting the carbamides fragment into mono-, oligo- and polysaccharides.

EXPERIMENTAL RESULTS, DISCUSSION AND CONCLUSION As a result of searching conditions of the synthesis of N-nitroso derivatives of monosaccharides unprotected hydroxyl groups, providing the simplicity, effectivity of the process, it is perfected the general methods of getting N-alkyl-N-(β-D-glykopiranozyl) nitrosourea in which there were combined reactions: a) the interaction of monosaccharides with alkylurea under the condition of nucleophilic catalysis with addition of aryl amines and b) nitrozilation of N-alkylglycosylureas [9, 10, 11].

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Methods of investigation: paper chromatography, element analysis, IR, PMRspectroscopy Preparation of N-Methyl-N/-(β-D-glucosyl)-N-Nitrosourea A mixture of 1.98 g of D-glucose, 0.85 g of methylurea, 0.19 g of m-nitroaniline and 0.06 ml of a concentrated hydrochloric acid in 10 ml of methanol is heated at reflux for 20 minutes. The formed precipitate in the amount of 1.45 g is separated and added with 5 ml of glacial acetic acid, 1 .6 ml of distilled water, 0.83 g of sodium nitrite and stirred for 2 hours at the temperature of –20C. The solution is evaporated, the residue is recrystallized from ethanol. The product yield is 1.44 g (90% of the theoretical). M.p. 1800C with decomposition, [α]D =-190 (water), Rf=0.56. Found, % N - 15.62. Calculated, % N - 15.84. In this example and hereinafter the Rf is determined in the system: benzene-butanolpyridine-water 1:5:3:3.

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Preparation of N-Methyl-N/-(β-D-galactosyl)-N-Nitrosourea A mixture of 1.98 g of D-galactose, 0.85 g of methylurea, 0.19 g of m-nitroaniline and 0.07 ml of a concentrated hydrochloric acid in 10 ml of methanol is refuxed for 25 minutes. The resulting precipitate in the amount of 1.6 g is separated and added with 7.5 ml of glacial acetic acid, 1.5 ml of distilled water, 0.93 g of sodium nitrite and stirred for 2 hours at the temperature of –10C. The residue is filtered off, washed with an alcohol to give 1.26 g of the product (70% of the theoretical). M. p. 1210C., [α]D= +21.80 (water), Rf=0.53. Found, % N 15.50. Calculated, % N - 15.84. Preparation of N-Methyl-N/-(β-D-xylosyl)-N-nitrosourea A mixture of 1.5 g of D-xylose, 0.8 g of methylurea, 0.04 g of m-nitroaniline and 0.04 ml of a concentrated hydrochloric acid in 7 ml of ethanol is heated at reflux for 10 minutes. The resulting precipitate in the amount of 1.3 g is separated and added with 6 ml of glacial acetic acid, 1.2 ml of distilled water, 0.89 g of sodium nitrite and stirred at the temperature of –20C for 2 hours. The residue is filtered-off, recrystallized from an alcohol to give 0.9 g of the product (62% of the theoretical). M. p. 1090C. (decomposition), [α]D=-21.90 (water), Rf=0.66. Found, % N - 17.62. Calculated, % N – 17.87. The study of the reaction with the participation of glycosyl bonds is important not only for the theory of the carbohydrates structure and reaction ability of carbohydrates. They present also a significant interest for solving a number of actual problems of bioorganic chemistry as the glycosyl bond is one of the most important structural elements of many biologically active compounds. Studying of the reactions of glycosyl centre has allowed the determination of some typical peculiarities of this type of nucleophilic catalysis. In this connection, the attention was concentrated on synthetic aspects of the homogeneous catalysis and elaboration of methods of directing synthesis of carbohydrates to physiologically active substances. They have much significance for the creation of medical preparations, in particular antitumor preparations, possessing low toxic activity, high solubility in water and high selectivity.

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Thus, rather easy and mobile method of syntheses of a big group of carbohydrates derivatives of nitrosoalkylureas was worked out. On this basis it seems to be perspective the investigations of the biological activity of modified medical preparations with the help of the glycosylamides bond.

REFERENCES

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[1]

N. N. Nikoliskiy, A. S. Troshin: The Transport Sugar through Cellular Membrane. Science, Leningrad, 1973. [2] N. M. Emanuel, D. B. Corman, L. A. Ostrovskaya, L. B. Gorbacheva, N. P. Dementieva: Nitrozoalkylureas - A New Class of Antitumor Preparations. Moscow, 1978, p. 290. [3] M. O. Grenn, J. Greenderg: The Activity of Nitrozoguanidines against Ascites Tumors in Mice. Cancer. Res., 20 (8), 1166 (1960). [4] U. Ross: Biological Alkylation Substances. Medicine, Moscow, 1964. [5] V. A. Chernov: Citostatic Substances in Chemotherapies of Oncomas. Medicine, Moscow, 1964. [6] N. N. Blohina, C. H. Zubroda: The System of the Creation of Antitumor Preparations in USSR and USA. Medicine, Moscow, 1977. [7] R. R. Herr, H. K. Jahnke, A. D. Argoudelis: The Structure of Streptozotocin. J. Am. Chem. Soc., 89, 4808 (1967). [8] V. A. Afanasiev, I. F. Strelicova, et al.: Construction and Reaction ability of Nglycosydes. Ilim, Frunze, 1976, p. 221. [9] J. A. Djamanbaev, V. A. Afanasiev: The Method of the Synthesis of N-alkyl-(N-aryl)-N(β-D-glykosyl)urea. Avt sv. N772102 USSR. [10] V. A. Afanaciev, J. A. Djamanbaev: Patent USA. 4: 656.259. 1987. [11] V. A. Afanasiev, J. A. Dzhamanbaev, G. E. Zaikov: The Derivatives of Carbohydrate with Carbamides Fragments. Successes of Chemistry, 51 (4), 661 (1982).

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In: Handbook of Chemistry, Biochemistry … Editors: L. N. Shishkina et al., pp. 97-111

ISBN: 978-1-61209-425-0 © 2010 Nova Science Publishers, Inc.

Chapter 11

INFLUENCE ON THE OXIDATION PROCESSES REGULATION IS THE REASON FOR BIOLOGICAL ACTIVITY OF THE ECDYSTEROID-CONTAINING COMPOUNDS L.N. Shishkina1, O.G. Shevchenko2, and N.G. Zagorskaya2 1

N.M. Emanuel Institute of Biochemical Physics Russian Academy of Sciences, Moscow, Russia 2 Institute of Biology, Komi Scientific Center, Ural Division of Russian Academy of Sciences, Syktyvkar, Russia

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ABSTRACT Influence of serpisten and inokosterone on the phospholipids composition in liver and blood erythrocytes, intensity of lipid peroxidation in tissues (liver, spleen, blood plasma), catalase activity in the liver and general peroxidase activity of white outbreed mice has been studied. A biological activity of ecdysteroid-containing compounds is shown to be associated with an influence on the parameters of the physicochemical regulatory system of lipid peroxidation (LPO). Possessing pronounced membrane-tropic properties due to alterations in the exchange of predominantly choline-containing fractions of phospholipids, ecdysteroid-containing preparations are capable of modifying a cell membrane phase state. A substantial dependence of a biological effect of the compounds on a dose, duration of their application as well as on an intensity of the LPO processes in the tissues and an animal’s sex require a more detailed research on the properties of the given ecdysteroids.

Keywords: serpisten, inokosterone, lipid peroxidation, tissues of mice, catalase, peroxidase, phospholipids composition.

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AIMS AND BACKGROUND Well-known biological properties, in particular, tonic and adaptogenic qualities of either ecdysteroids or ecdysteroid-containing plant preparations [1–5] are similar to those of a number of natural and synthetic antioxidants (AO) [6–8]. This allows us to assume that a biological activity of ecdysteroids should be derived from their influence on the parameters of the lipid peroxidation (LPO) system in tissues of hematothermal animals. It is known, that regardless of a reason for LPO intensification, changes in the oxidation rate are associated both with a reduction in the amount of bioantioxidants and changes in the membrane phospholipid (PL) composition [9[ due to either a more rapid degradation of oxidized lipids or the acceleration of lipid transport by transferring proteins. Therein, the role of PL in the oxidative processes is multifunctional, since on the one hand they are substrates for oxidation, on the other hand PL are capable of modifying oxidative processes acting as antioxidants, their synergists or antagonists [9, 10]. LPO activation is associated with the vital physical and chemical membrane properties—penetrability, viscosity and phase state [9–12]. A state of the membrane lipid phase has an effect on the activity of membrane-associated enzymes and the cell sensitivity to hormonal and nervous regulation [10, 13, 14]. Phospholipids either maintain work of the vital cell mechanisms such as ion exchange, biological oxidation or influence both an activity of the mitochondria enzymes and the oxidative phosphorylation [15[. In biomembranes, a lipid component that is formed as a functionally active template integrates an external influence and participates in triggering the cell control programs [16–18]. A plasma membrane possesses the unique receptive and signaling functions of the vital cell processes regulation whose lesions could lead to a cell death. A mechanism of the origin and development of the majority of pathological states are due to the disturbances in a structure and properties of membranes [9, 10, 19]. The data on membrane-stabilizing effect of ecdysteroids are available in the current literature [5, 20, 21]. Noteworthy, that phytoecdysteroids were revealed to influence a lipid exchange, in particular, cholesterol synthesis and catabolism [22–24]. Moreover, hematoreologic activity of ecdysteroid-containing plants and ecdysterone which can be ascribed to a likely modification of an erythrocyte membrane lipid phase was shown previously [25]. A rising interest to a research on erythrocyte membranes has been caused by participation of these cells in the processes associated with maintaining homeostasis at the whole organism level. Regularities of changes in the erythrocyte membrane structure and functions can be extrapolated to other membrane systems with a definite correction due to the species specificity of cells [19, 26]. A visible simplicity of the erythrocyte structure enables one to investigate functional properties of a plasma membrane without any hindrances exerted by intracellular membrane formations or organelles [27[. Structural and functional peculiarities of the erythrocyte as well as its availability for investigations enable one to consider it a universal model for a research on the changes in cytoplasm membranes and cell metabolism of an organism The aim of the present research is an experimental verification of a hypothesis on a relation between a biological activity of ecdysteroid-containing preparations and their action on the parameters of the LPO physicochemical regulatory system in animal tissues.

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EXPERIMANTAL PART The experiments were carried out on males and females (2-3 months age) of white outbreed mice which lived at standard vivarium conditions. Since an antioxidant status of animal tissues substantially depends on a season [28], all the experiments were carried out from March to May. Serpisten and inokosterone were isolated by V.V. Volodin and coworker in their laboratory from Serratula coronata [29] and politely given to us for research. A range of the tested serpisten doses under a single per os preparation administration was from 10 to 3000 mg/kg of a body weight. A dose was calculated individually for each animal with regard to a body weight. The animals were decapitated 10 days after the preparation administration. To study a chronic serpisten action, a serpisten solution in distilled water was given to the animals instead of drinking water for 10 or 30 days. The concentrations were selected so that the total doses of serpisten were equal to 5; 50 and 500 mg/kg. For inokosterone, the total doses were 5 mg/kg of the body weight for 10 days or 5 and 15 mg/kg for 30 days. The calculation was performed taking into account animals’ weights and a volume of the consumed liquid. The control animals were given distilled water. Following decapitation of the mice, their organs were placed on ice. The blood was collected in test tubes treated by 5% solution of sodium citrate. The blood plasma was separated from the blood corpuscles by centrifugation. The content of the LPO secondary products reacting with 2-thiobarbituric acid (TBA-reactive substances, TBA-RS) was determined using the method described in [30]. The catalase activity in the liver was measured spectrophotometrically at the wavelength of 410 nm according to formation of a colored complex of ammonium molybdate in the presence of hydrogen peroxide [31]. The protein content was evaluated by the modified micro-biuretic method described in [32]. The total peroxidase activity (TPA) of blood which is characteristic of the intensity of LPO processes and reflects the ratio between pro-oxidative and antioxidative (AO) blood systems [33, 34] was determined by a photometrical registration of a drop in indigocarmine concentration that is oxidized by hydrogen peroxide in the presence of peroxidases [35]. Lipids from liver and erythrocytes were isolated by the method of Blay and Dyer in the Kates modification [36]. The qualitative and quantitative composition of phospholipids (PL) was analyzed by the thin-layer chromatography [37] with the use of silica gel of type G (Sigma, USA) and glass plates measuring 9 × 12 cm. We used a mixture of chloroform – methanol - glacial acetic acid - water in the ratio of 50:30:8:4 as the mobile phase. A quantitative analysis of the PL composition yielded after removal of pellets from a plate and burning them to an inorganic phosphate with perchloric acid was performed spectrophotometrically at the wavelength of 800 nm and according to formation of complex in the presence of ascorbic acid. A more detailed description of the PL composition determination is presented elsewhere [38]. In addition to a quantitative analysis of a ratio between the different PL fractions, generalized parameters of the lipid composition were evaluated: the PL amount in the total lipid composition (% PL); the phosphatidyl choline/phosphatidyl ethanolamine (PC/PE) ratio; the ratio of the sums of the more easily to the more poorly oxidizable PL fractions (ΣEOPL/POPL). The latter were calculated according to the formula [37]: ΣEOPL/POPL = (PI + PS + PE + CL + PA) (LPC + PC + SM), where PI is phosphatidyl inositol, PS is phosphatidyl serine, CL is cardiolipin, PA is phosphatidic acid,

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LPC are lysoforms of PL (mainly PC), SM is sphingomyelin. The LPC/PC ratio which is characteristic of a PC degradation rate and its lysoform utilization was also assessed [39, 40]. The obtained experimental data were processed with the commonly used methods of the variational statistics [41]. The experimental data are presented in tables and figures in form of arithmetic means with the indication of the mean square errors of the arithmetic means (M ± m).

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RESULTS AND DISCUSSIONS First of all, it is necessary to note that within the observed range of doses, serpisten possesses neither acute (10–3000 mg/kg) nor chronic (5–500 mg/kg for 30 days) toxicity [42]. An extreme low toxicity of other phytoecdysteroids even at very high doses was shown in the experiments on hematothermal animals by a number of authors [3, 42]. . LPO process regulation in cells and tissues is known to be realized either with participation of low molecular antioxidants and metabolites or due to functioning of enzymes of the antioxidant defence [9, 10, 17, 38, 43, 44]. Parameters for the physicochemical system of LPO regulation, that include the phospholipid composition directly associated with LPO intensity, as well as an activity of the antioxidant defence enzymes in murine liver and blood have been studied in the present work. According to our data (Table1), a single administration of serpisten at average and high doses (100–3000 mg/kg) led to no reliable changes in the quantitative composition of the male liver PL regarding most of the already investigated indicators 10 days after the preparation administration. Nevertheless, serpisten administration to an animal organism at a dose of 2000 mg/kg resulted in a reliable reduction of the PC share in liver PL. Moreover, a trend to an increase in the ratio LPC/PC was observed for phospholipids of all the groups of mice, the sum portion of CL and PA in the PL of animal liver rising upon introduction of the preparation at a dose of 3000 mg/kg (Table 1). Acid phospholipids (PS, PI, CL and PA) are known to regulate the activity of Ca+2- and Na+,K+ATPases and required for maintaining the cell ion balance [14, 45]. Modulation of the activity of these enzymes is a response of a cell to the factors disturbing its homeostasis. In addition, an increase in the CL share in liver PL is likely to be associated with the energy exchange activation, since the main portion of this PL synthesizes and localizes at mitochondria. A long-term (30 days) administration of low and average doses of serpisten (5-500 mg/kg) to an animal organism caused sufficient changes both in the quantitative ratio of different PL fractions and generalized indices for the liver PL composition (Tables 2 and 3). The analysis of the obtained data testifies to the fact that a long-term administration of high doses of serpisten causes a trend to a reduction of the PC proportion in liver PL in parallel to a growth of a relative LPC content both in males and females. Application of serpisten at a dose of 50 mg/kg evoked a decrease of the ratio PC/PE in the PL composition of liver both in males and females. That indicates a diminution in rigidity of the liver membrane structures. However, it must be noted, that murine females have a higher sensitivity to serpisten, which might be attributed to the existing in norm distinctions in the quantitative composition of the tissue membrane lipids with regard to a sex. In females, a long-term application of serpisten at a dose of 500 mg/kg led to a reliable growth of the ratio LPC/PC, the sum PC content and its lysoforms being unchanged (Table 3).

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Table 1.The phospholipid composition (%P) and the generalized parameters of the lipid composition (relative units) in liver of mice (males) after 10 days under a single administration of serpisten Phospholipid fraction, parameter LPC SM PC PC + PI PE CL+PA % PL PC/PE ΣEOPL/POPL LPC/PC

Dose, mg/kg 100 5.40±0.23 4.40±0.36 52.08±2.38 7.54±2.62 26.04±0.50 4.56±0.39 26.28±0.13 2.00±0.05 0.63±0.07 0.105±0.009

Control 5.05±0.11 2.09±0.96 53.84±0.51 3.68±1.53 30.56±3.29 4.79±0.40 22.91±1.20 1.86±0.22 0.65±0.04 0.094±0.003

2000 4.85±0.11 4.82±0.10 46.47±0.15*** 4.39±1.09 34.11±1.00 5.37±0.15 21.58±4.86 1.37±0.04 0.79±0.00 0.104±0.003

3000 5.07±0.24 3.97±0.10 49.84±2.03 5.00±0.81 27.52±0.04 8.61±1.60* 27.94±2.36 1.81±0.07 0.71±0.07 0.102±0.001*

Note: here and thereafter the significant differences from the control are * P < 0.05, ** P < 0.01, *** P < 0.001.

Table 2.The phospholipids composition (%P) and the generalized parameters of the lipid composition (relative units) in liver of mice (males) after 30 days under a chronic application of the serpisten solution (decapitation after 15 days under the preparation administration)

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Dose, mg/kg Phospholipid fraction, parameter

Control

5

50

500

LPC SM PC PI + PS PE CL + PA % PL PC/PE ΣEOPL/POPL LPC/PC

4.71±0.02 4.77±0.81 43.15±2.39 11.92±1.33 27.89±1.45 7.58±1.68 20.46±0.23 1.55±0.01 0.91±0.05 0.111±0.007

4.12±0.97 3.88±0.31 41.15±2.04 11.38±0.20 28.91±0.13 10.56±0.70 14.65±2.38 1.42±0.07 1.04±0.03 0.106±0.03

5.05±0.05*** 4.58±0.06 42.95±0.71 10.79±0.65 30.47±0.26 6.16±0.30 19.26±1.72 1.41±0.01*** 0.90±0.03 0.118±0.001

10.63±3.58 4.30±0.25 36.51±0.77 12.34±0.34 27.16±1.56 9.08±2.72 18.83±1.78 1.37±0.11 1.01±0.18 0.283±0.090

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Table 3.The phospholipid composition (%P) and the generalized parameters of the liver lipid composition (relative units) of the mice females under a chronic application of the serpisten solution for 30 days (decapitation 15 days after the preparation administration) Phospholipid fraction, parameter

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LPC SM PC PI + PS PE CL + PA % PL PC/PE ΣEOPL/POPL LPC?PC

Dose, mg/kg Control 5.65±1.60 10.63±0.37 37.27±2.31 9.96±0.03 25.35±0.76 11.14±1.12 46.61±0.37 1.47±0.05 0.86±0.02 0.175±0.050

5 8.70±0.24 2.47±0.19*** 33.21±3.47 17.74±2.68* 22.90±3.16 14.98±4.27 10.68±0.53*** 1.56±0.34 1.25±0.12* 0.271±0.030

50 6.40±3.17 1.07±0.54* 38.16±0.15 14.20±1.00** 29.67±0.42*** 10.51±2.21 27.18±2.26*** 1.29±0.01* 1.23±0.14 0.167±0.08

500 15.65±1.72*** 5.38±1.72** 30.07±2.95 14.30±1.24** 23.35±0.68 11.25±0.29 33.79±3.95** 1.28±0.09 0.96±0.03* 0.564±0.110**

In addition to an increase of a lysoform share in liver PL of the females that received serpisten we observed a reliable decrease in the relative content of SM that resulted in the total reduction of a share of choline-containing fractions, thus leading to a rise in the content of easily oxidizable PL fractions. A long-term administration to a female organism of serpisten at any dose also resulted doth in a reliable enhancement of a relative content PI+PS and decrease of the PL proportion in the total lipid composition of liver. The next step of our work was to evaluate how lower serpisten doses affect LPO parameters in murine tissues. Figures 1 and 2 present data on certain parameters of the lipid composition in liver and blood erythrocytes of the mice (males) which received serpisten at the doses of 5 and 50 mg/kg for 10 days. The animals were decapitated immediately after the preparation application had been stopped. Application of serpisten at either dose (especially at 5 mg/kg) caused a rise in the total PL content in lipids of blood erythrocytes (Figure 1). In liver lipids, an increase in the PL proportion was observed only in case of exposure to the preparation at a dose of 50 mg/kg. Noteworthy, that along with an increase of the total PL content in blood erythrocyte lipids, serpisten at a dose of 50 mg/kg causes a reliable reduction of lipid oxidizability that can be assessed with regard to the ratio of more easily to more poorly oxidizable PL fractions: ΣEOPL/ΣPOPL = 0.265 ± 0.020 in the experimental group and 0.372 ± 0.030 in the control. Also observed is a simultaneous enhancement of a rigidity of erythrocyte membranes that can be derived from a growth of the ratio PC/PE: 5.78 ± 1.00 in the experimental group and 3.22 ± 0.14 in the control. Application of serpisten at a dose of 50 mg/kg also resulted in a reliable diminution of the SM share in blood erythrocyte PL. Serpisten administration at a dose of 5 mg/kg led to a drastic rise in a share of lysoforms in PL of both blood erythrocytes and liver (Figure 2). However, a 10-fold increase in the preparation dose caused a reverse effect, mainly in erythrocytes (Figure 2). It is known LPC is required for the normal cell functioning, participates in the activity regulation of the majority of membrane-associate enzymes, and is a secondary mediator of trans-membrane transmission of a signal within a cell [46 - 49].

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LPC, % P

Figure 1. The content of phospholipids in the total lipid composition of the blood erythrocytes and liver of mice which received serpisten at the doses of 5 and 50 mg/kg for 10 days.

6

control 5 mg/kg

5

50 mg/kg

4 3 2 1 0 erythrocytes

liver

Figure 2. The share of lysoforms in phospholipids of the blood erythrocytes and liver of mice which received serpisten at the different doses for 10 days.

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At the same time, the LPC amphiphylicity and “wrong” structure underlie either its detergent, fluidizing action or ability to disturb the membrane topography, thus making it possible to employ a level of PL lysoforms for estimating an extent of pathological processes [46, 48]. The growth of the lysoform share in the phospholipids of the blood erythrocytes and liver of the animals which received serpisten represents a powerful factor of modifying the properties of either a lipid bilayer or integral membrane proteins and can result from both an rise of the phospholipase A2 activity and lysophospholipase and acyltransferase inhibition [46, 48]. Meanwhile, phospholipase A2 activation is accompanied by not only an increase in the amount of LPC, but also a growth of an intracellular pool of arachidonic acid [50]. Activation of an arachidonic cascade is considered by some authors [51] an integrated negative unit for autoregulating the activity of the phospholipid-depending signaling cell system, whereas ecdysteroids are believed to be possible effective modulators of intracellular eicosanoid pools in tissues of hematothermal animals. Modification of a phase state of the cell membrane system under administration of ecdysteroids can be conditioned not only by the PL lysoform action but also by a reduction in PL relative content of SM that along with cholesterol favors an enhancement of microviscosity and rigidity of the membrane lipid phase due to a predominant presence in its molecule of saturated fatty acid residues [52]. Metabolism of SM and cholesterol in cells is known to have an integrated character [53]. Cholesterol building in among PL molecules limits their mobility and significantly predetermines fluidity and viscosity of a red blood cell membrane, thus influencing on a lateral diffusion of receptors, ion transport, and penetrability for dissolved substances [52]. A phase state of the cell membrane exerts a remarkable influence on the processes of membrane transport, the systems of a trans-membrane information transmission, and the activity of membrane-bound enzymes. Changes of the viscous properties of membranes emerging as a result of alterations in the quantitative ratio of different fractions affect not only a form of the erythrocytes, but also their ability to be deformed. The changes in a relative content of PL lysoforms and SM are most likely to cause those changes of a shape and osmotic resistance of erythrocyte membranes that were detected after administration of serpisten and other ecdysteroid-containing compounds in the experiments on the laboratory animals [54, 55[; the observed by the authors effect depending to a considerable extent on an animal’s sex, compound dose, or a period of its application. It was shown [25] that ecdysteroid-containing extracts exhibited hematoreologic activity in model experiments at the concentrations under which ecdysterone constituting them displayed neither antiradical nor antioxidative activity. These authors later reported [56] that the extracts from Lychnis chalcedonica and Raponticum sarthamoides containing ecdysteroids actually prevented a pathological modification of erythrocytes shape under administration to animals through their influence on the content and ratio of different fractions of lipids and phospholipids in erythrocyte membranes. It is unanimously accepted that sphingolipids influence not only a structural state of biological membranes but also play a part of secondary messengers in the array of the most important cell processes and are the mediators of some extracellular stimuli [57 – 59]. SM exchange modulation is considered one of the effective mechanisms of an early (pre-genomic) action of ecdysteroids [60]. At the same time, there is evidence [61, 62] that ecdysteroids and ecdysteroid-containing plant extracts are able to influence a functional activity of the endocrinal system organs (pancreas and, most likely, adrenal gland and testicles).

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Table 4.The content of TBA- reactive substances (nmol/mg of protein) in the murine tissues after application of the inokosterone solution at different doses (decapitation after 5 days under finishing of the preparation administration) Variant of experiment Control Inokosterone 5 mg/kg, 10 days

Blood plasma 0.140±0.017 0.035±0.008***

Liver 0.152±0.017 0.175±0.031

Spleen 0.366±0.038 0.751±0.094***

Control Inokosterone 5 mg/kg, 30 days Inokosterone 15 mg/kg, 30 days

0.152±0.031 0.078±0.012* 0.200±0.010

0.101±0.031 0.171±0.017 0.122±0.041

0.472±0.061 0.541±0.035 0.435±0.035

Obviously, the revealed alterations in a relative SM content in the liver and blood erythrocyte PL are attributed to the changes in the thyroid gland functional activity, since the SM metabolism in different tissues is controlled by thyroid hormones [63] Indeed, that histomorphological study of thyroid gland of the mice which received serpisten for 10 days points to a diminution of the colloid volume density, that can testify to a diminution in the thyroid gland functional activity [64]. The fact that biological activity of serpisten depends in many respects on the presence in its content of inokosterone [42] caused a necessity of a more detailed research on the properties of the latter. In this connection, we studied the influence of inokosterone on the parameters reflecting an intensity of the LPO processes in murine tissues. Analysis of the LPO secondary product content showed a dependence of the obtained effects both on the preparation dose and the tested tissue (Table 4). Application of low doses of inokosterone (5 mg/kg) at both a short-termed (10 days, variant 1) and long-termed (30 days, variant 2) administration to the organism led to a reliable 2-4-fold diminution of the LPO intensity in the blood plasma. To the contrary, unidirectional changes in the TBA-reactive substances content were detected in liver and spleen: a growth of the LPO intensity at 5 mg/kg and the absence of reliable differences from the control at a dose of 15 mg/kg. It is significant that an increase of the inokosterone dose up to 15 mg/kg also resulted in normalization of this parameter in the murine blood plasma. In all variants of the experiments, no reliable differences in the activity of catalase in the liver of intact mice and animals which had received both serpisten and inokosterone were revealed due to a high individual variability. The total peroxidase activity (TPA) of blood of the animals that had received inokosterone was associated both with a scheme of the application of the preparation (for 10 or 30 days) and an initial level of activity in the control groups of animal (Figure 3). Interestingly, that under inokosterone application at a dose of 5 mg/kg a growth of the blood TPA was observed only in the group of mice with a low level of the enzymatic activity in the control. An analogous dependence of the influence on the antioxidative system is described for other biologically active substances in articles [65, 66]. Investigations of the blood erythrocyte lipids of the mice which received inokosterone according to different schemes have showed a sufficient influence of inokosterone on a exchange of choline-containing PL fractions.

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μ mole × min/ ml of blod

106

250

control

240

inokosterone 5 mg/kg

230

inokosterone 15 mg/kg

220 210 200 190 180 170 160 150

The first variant

The second variant

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Figure 3. The total peroxidase activity of blood of mice which received inokosterone at the doses of 5 and 15 mg/kg for 10 (the first variant) and 30 (the second variant) days.

The preparation was shown to change the LPC/PC ratio towards the intensification of the acylation reaction of LPC to PC (Figure 4.A), thus inducing a reduction in the lysoform proportion in PL of the blood erythrocyte. Some increase of a relative SM content has also been noted. Analysis of generalized parameters for the PL composition (PC/PE and ΣEOPL/ΣPOPL) shows that application of inokosterone at the investigated doses can evoke either some augmentation of the erythrocyte membrane rigidity or reduction of its lipid oxidizability (Figures 4.A and 4.B) thus interfering with a further LPO intensification. Sufficient differences in an individual reaction of animals to inokosterone administration causing a significant variability of parameters in the experimental group are in accord with the data of other researchers who tested this compound [42]. A high individual and tissue variability of the effects detected under inokosterone application to animals is likely to result from the fact that development of a cell response under administration of the biological active substances depends on the state of its antioxidant and pro-oxidant systems [66, 67].. Thus, a biological activity of the preparation significantly depended on the tested dose in the experiments with both inokosterone and serpisten. In a number of cases a 3-10-fold increase of a dose either led to an enhancement of the effect or induced a normalization of parameters and even changed the sign of the effect. The absence of the augmentation of the action with an increased dose of ecdysterone was also observed when studying haemoreological properties of ecdysteroid-containing extracts [25]. Evidence on a possible antiradical and antioxidative activity of ecdysteroids is rather contradictory [51, 68, 69]. Model experiments [70, 71] and tests on animals [51] established that ecdysteroids possess both pro-oxidant and antioxidant properties depending on their concentration and intensity of oxidative processes.

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0,7

107

control

0,6

inokosterone 5 mg/kg inokosterone 15 mg/kg

0,5 0,4 0,3 0,2 0,1 0

LPC/PC

4 3,5 3

a

ΣEOPL/ΣPOPL

control inokosterone 5 mg/kg inokosterone 15 mg/kg

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2,5 2 1,5 1 0,5 0

PC/PE b

Figure 4. The generalized parameters of the phospholipid composition of the blood erythrocytes of mice which received inokosterone at the different doses for 30 days: a - phospholipid lysoforms/phosphatidyl choline ((LPC/PC) ratio and the ratio between the sums of the more easily to the more poorly oxidizable phospholipid fractions (∑EOPL∑POPL); b – phosphanidyl choline/phosphatidyl ethanolamine (PC/PE) ratio.

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Such dualism of a response of the system of the organism antioxidant defense was recorded under administration of numerous biologically active compounds, including oxysterols, ά-tocopherol, carotinoids, ascorbic acid, and other natural and synthetic antioxidants [8, 10, 43, 67, 72, 73]. The total results of the study allow us to conclude that a biological activity of both serpisten and inokosterone is associated with the action on the parameters of the physicochemical system of LPO regulation. The most remarkable effect is observed under a long-term application into the organism of compounds at low doses. Possessing pronounced membrane-tropic properties, due to alterations in the exchange of, predominantly, cholinecontaining fractions of phospholipids, ecdysteroid-containing preparations are capable of modifying a phase state of the membrane system of tissues. A remarkable dependence of a biological effect of these preparations on a dose, duration of administration to the organism, as well as on the intensity of LPO processes in tissues and a sex of an animal require a more detailed research on the properties of the given ecdysteroid-containing compounds. The work was supported by the Program of Fundamental Research of Presidium of Ural Division of Russian Academy of Sciences “Fundamental Sciences for Medicine”.

REFERENCES V.N. Syrov, A.G. Kurmukov // Dokl. Akad. Nauk SSSR, N 12, pp. 27–30, 1977 (in Russian). [2] Yu.D..Kholodova // Biochemistry of Animals and Human, N 11, pp. 27–41, 1987; (in Russian). [3] A.A. Akhrem, N.V. Kovganko “Ecdysteroids. Chemistry and Biological Activity”, Minsk, Nauka and Tekhnika, 1989; 327 pp. (in Russian). [4] K. Koudela, J. Tenora, J. Bajer et al. // Eur. J. Entomol., V. 92, pp. 349–354, 1995. [5] Phitoecdysteroids. St.-Peterburg, Nauka, 2003. 293 pp. (in Russian). [6] E.Ya. Kaplan, O.D. Tsyrenzhapova, L.N. Shantanova. “Optimization of the organism adaptation processes” /Ed. S.M. Nikolaev. Moscow, Nauka, 1990; 94 pp. (in Russian). [7] K.M. Dyumaev, T.A. Voronina, L.D. Smirnov “Antioxidants in the Prophylactic and Therapy of the CNS Pathology. Moscow, Publisher of Institute of the biomedicine Chemistry of RAMS, 1995; 272 pp. (in Russian). [8] E.B. Burlakova. “Antioxidants: Yesterday, Today, Tomorrow” pp.1-33, in the book: “Chemical and Biological Kinetics. New Horizons” Vol. 2: Biological Kinetics / Eds. E.B. Burlakova, S.D.. Varfolomeev. Leden. Boston, VSP, 2005. [9] Ye.B.Burlakova., N.P. Pal’mina, Ye.L.Mal’tseva “A Physicochemical System Regulating Lipid Peroxidation in Biomembranes during Tumor Growth”, pp. 209–238, in the book Membrane Lipid Oxidation. Volume III / Ed. C. Vigo-Pelfrey. Boston: CRC Press, 1991. [10] E.B. Burlakova, A.V. Alesenko, E.M. Molochkina, N.P. Pal’mina, N.G. Khrapova, “Bioantoxidants in the Radiation Damage and the Tumor Growth”, Moscow, Nauka, 1975, 214 pp. ((in Russian). [11] Yu.A.Vladimirov // Pathol. Physiol. Exper. Therapy, N 4, pp. 7–19,1989 ((in Russian).

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[12] W.K. Marathe, K.A. Harrison, R.C. Murphy et al.// Free Radic. Biol. and Med., V. 28, pp. 1762 – 1770, 2000. [13] R.N. Farias, R.D. Morero, F.Sineriz, R.E. Fruco // Biochim. Biophys. Acta, V. 415, pp. 231 – 251,1975. [14] R.B. Gennis “Biomembrane: Molecular Structure and Function”, Moscow, Mir, 2001, 524 pp. (Russian version) [15] E.A. Stepanov, Yu.M. Krasnopol’srii, V.I. Shvets. Physiological Active Lipids, Moscow, Nauka, 1991, 136 pp. (in Russian) [16] V.A. Trofimov, R.E. Kiseleva, A.P. Vlasov et al. // Bull. Exterimentl. Biol. Med., V. 127, pp. 43 - 45, 1999 (in Russin). [17] K. Hensley, K.A. Robinson, S.P. Gabbita, S. Salsman, R.A. Floyd // Free Radic. Biol.and Med. V. 28, pp. 1456 - 1462, 2000. [18] V.E. Kagan, J.P. Fabisiak, A.A. Shvedova, Y.Y. et al. // FEBS Lett. V. 477, pp. 1 - 7, 2000. [19] V.V. Novitskii, N.V. Ryazantseva, E.A. Stwepovaya “Physiology and pathology of erythrocytes” Tomsk, Publishers of Tomsk University, 2004, 202 pp (in Russian) [20] V.N. Syrov, M.A. Tashmukhamedova, Z.A. Khushbaktova et al. // Ukr. Biochim. J., V. 64, pp. 61 – 67,1992 (in Russian). [21] V.N. Darmogray, V.K. Petrov, Yu.I. Ukhov “Plants-producers, api- and marine products as sources of pharmacologically active ecdysteroids”, pp. 143 - 144 in Abstracts of papers of Inter. Conf. “Search, Development and Adoption of new remedies and organization forms of the pharmaceutical work”, Tomsk, 2000.(in Russian). [22] V.N. Mironova, Yu.D. Kholodova, T.F. Skachkova et al. // Voprosy Med. Khim. N 3. pp. 101 – 104, 1982 (in Russian). [23] V.N. Syrov, A.N. Nabiev, M.B. Sultanov // Pharmacol. Toxocol., V. 49, pp. 100 – 103, 1986 (in Russian) [24] Jr. Gd. Schroepfer // Physiol. Rev. V. 80, pp.361 – 554, 2000. [25] M.B. Plotnikov, L.N. Zibareva, A.A. Koltunov et al. // Plant Resource, N 1, pp. 91 – 97, 1998 (in Russian). [26] V.V. Novitsky, N.V. Ryazantseva, E.A. Stwepovaya et al. // Bull. Sib. Med., N 2, pp. 62–69, 2006 (in Russian). [27] Yu.V. Postnov, S.N. Orlov “Primary hypertension as the pathology of cell membranes” Moscow, Meditsina, 1987, 192 pp. (in Russian). [28] L.N. Shishkina, E.B. Burlakova “The Value of Antioxidant Properties of Lipids in Radiation Damage and Membrane Repair”, pp. 334 – 364, in the book: Chemical and Biological Kinetics. New Horizons Vol. 2: Biological Kinetics / Eds. E.B. Burlakova, S.D.. Varfolomeev. Leden. Boston, VSP, 2005. [29] V.V. Volodin, S.O. Volodina Patent 2153346, Russia, MKIS A 61 K 35/78 “Method of the ecdysteroids production”, Institute of Biology Komi SC, Ural Division of Russian Academy of Sciences, N 99106351/14, Publ. 27.07.2000, BI N 21. [30] T. Asakawa, S. Matsushita // Lipids, V. 15, pp. 1137–1140, 1980. [31] M.A. Korolyk, L.I. Ivanova, I.G. Maiorova // Labor. Practice, N 1, pp.16–19, 1988 (in Russian). [32] R. Itzhaki, D.M. Gill // Anal. Biochem. V. 9, pp. 401–41`0, 1964. [33] I.A. Goroshinskaya, L.V. Mogil’nitskaya, L.A. Nemashkalova, A.A. Khodakova // Radiobiology, V. 12, pp. 232–235, 1972 (in Russian).

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[34] L.A. Tiunov, E.A. Zherbin, B.N. Zherbin. Radiation and poisons. Moscow, Atomizdat, 1977, 144 pp. (in Russian). [35] . T. Popov, L. Neikovskaya // Hygiiene and Sanitation, N 10, pp. 89-91, 1971 (in Russian). [36] M. Kates. The Technology of Lipidology. Moscow, Mir, 1975, 322 pp..(Russian version). [37] Biological membranes: A practice approach / Eds. J.B.R. Findlay, W.H. Evans. Moscow, Mir, 1990, 424 pp. (Russian version). [38] L.N. Shishkina, Ye.V. Kushnireva, M.A. Smotryaeva // Oxidation Commun., V. 24, pp. 276 – 286, 2001. [39] T.P. Kulagina, I.K. Kolomiitseva, V.I. Arkhipov // Bull. Exper. Biol. Med. V. 130. pp. 292 – 294, 2000 (in Russian). [40] A.E. Lychkova, V.M. Smirnov // Bull. Exper. Biol. Med., V. 133, pp. 364–366, 2005 (in Russian). [41] G.F. Lakin. Biometry. Moscow, Vysshaya shkola, 1990, 352 pp.(in Russian). [42] K. Slama, R. Lafon // Eur. J. Entomology, V. 92, pp. 355 – 377, 1995. [43] N.K. Zenkov, V.Z. Lankin, E.B. Men’shchikova. Oxidative Stress. Biochemical and Pathophysiological Aspects. MAIK-Interperiodika, 2001, 343 pp.(in Russian). [44] L.N. Shishkina, E.V. Kushnireva, M.A. Smotryaeva // Radiat. Biology. Radioecology, V. 44, pp. 289–295, 2004 (in Russian). [45] A.A. Boldyrev // Ukr. Biochem. J., V.64, pp. 5–10, 1992 (in Russian). [46] G.A. Gribanow // Voprosy Med. Khimii, V. 37, pp. 2–10, 1991 (in Russian). [47] N.V. Prokazova, N.D. Zvezdina, A.A. Korotaeva // Biochemistry, V. 63, pp. 38- 46, 1998 (in Russian). [48] T.I. Torkhovskaya, O.M. Ipatova, T.S. Zakharova et al // Biochemistry, V. 72, pp. 149– 157, 2007 (in Russian). [49] A.A. Kunshin, V.I. Tsyrkin, N.V. Prokazova // Bull. Exper. Biol. Med., V. 143, pp. 604 – 607, 2007 (in Russian). [50] M.G..Sergeeva, A.T. Varfolomeeva “Cascade of Arachidonic Acid”. Moscow, Narodnoye obrazovaniye, 2006, 256 pp.(in Russain). [51] A.V. Kotsyryba, O.N. Bukhnevich, S.S. Tarakanov // Ukr. Biochem. J., V.67, pp. 45-52, 1995 (in Russian). [52] E.V. Dyatlovitskaya // Biochemistry, V. 60, pp. 843 – 850, 1995 (in Russian). [53] H. Ohvo-Rekila, B. Ramstedt, P. Leppimaki, J.P. Slotte // Prog. Lipid Res., V. 41, pp. 66–97, 2002. [54] N.A. Moiseenko, Zh.E. Ivankova, A.S. Tsvetkova “Influence of 20-hydroxiecdydone on properties of the red blood components of rats during 24 hours after injection”, pp. 242256, In: Radioecological and biological consequences of low intensity actions / Ed. A.I. Taskaev. Syktyvkar, 2003 (in Russian). [55] Zh.E. Ivankova, N.A. Moiseenko, S.A. Moiseenko “Role of 20-hydroxiecdydone in the development of post-hemorrhagic anemia at rabbits”, pp. 257–273, in the book “Radioecological and biological consequences of low intensity actions” / Ed. A.I. Taskaev. Syktyvkar, 2003 (in Russian). [56] M.Ya. Plotnikov, O.I. Aliev, A.S. Vasil’eva et al. // Bull. Exper. Biol. Med., V. 146, pp. 50–53, 2006 (in Russian). [57] AV. Alesenko // Biochemistry, V. 63, pp. 75 – 82, 1998 (in Russian).

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[58] E.I. Ostashkin, Yu.B. Bespalova, I.M. Molotkovskaya et al. // Dokl. Acad. Nauk Russia, V. 371, pp.406 – 409, 2000 (in Russian). [59] . O.M. Ipatova, T.I. Torkhovskaya, T.S. Zakharova, E.M. Khalilova // Biochemistry, V. 71, pp. 882–893, 2006 (in Russian). [60] A.V. Kotsyryba, A.V. Tuganova, O.N. Bukhnevich, S.S. Tarakanov // Ukr. Bioch. J., V. 67, pp. 53-58, 1995 (in Russian). [61] I.N. Todorov, Yu.I. Mitrokhin, O.I. Efremova, L.I. Sidorenko // Chem.-Pharm. J., V. 34, pp. 3–9, 2000 (in Russian). [62] I.N. Todorov, G.I. Todorov “Stress, Aging and its biochemical correction” /Ed. S.M. Aldoshin. Moscow, Nauka, 2004, 479 pp. (in Russian). [63] N.A. Babenko, Yu.A. Natarova // Biochemistry, V. 64, pp. 1085–1089, 1999 (in Russian). [64] O.V. Raskosha, O.V. Ermakova, A.V. Selezneva, O.V. Strekalovskaya // Morphological Newspaper, N 1-2, Suppl. N 1,pp. 243 – 245, 2006 (in Russian). [65] E.V. Ryabikina, Z.I. Mikashinovich, V.N. Zhenilo, Yu.A. Kalmykova // BLOOD.RU, 2007 [66] L.N. Shishkina, Yu.P. Taran, S.V. Eliseeva, V.G. Bulgakov // Izv. Acad. Nauk SSSR, Ser. Biol., N 3, pp. 350–357, 1992 (in Russian). [67] E.M. Treshchalina “Antitumor activity of substances of the natural origin”, Moscow, Practicheskaya Meditsina, 2005, 270 pp. (in Russian). [68] L.F. Osinskaya, L.M. Saad, Yu.D. Kholodova // Ukr. Biochem. J., pp. 114–117, 1992 (in Russian). [69] A.I. Kuz’menko,R.N. Morozova, I.N. Nikolaenko et al. // Biochemistry, V. 62, pp. 712– 715, 1997 (in Russian). [70] L.N. Shishkina, Ye.V. Kushnireva, V.V. Volodin “The study of the 20-hydroxiecdyzone antioxidant properties in the model system”, p.125, in Abstracts of Workshop on Phytoecdysteroids, Syktyvkar, 1996. [71] L.N. Shishkina,, A.G. Kudyasheva, N.G. Zagorskaya et al. “Antioxidative Properties of ecdysteroids in systems in vitro and in vivo”, pp. 632 – 633 in Abstracts of papers of IV Inter. Conf. “Bioantioxidants”, Moscow, 2002 (in Russian). [72] L.N. Shishkina, E.B. Burlakova // Chem. Phys. Report., V. 15, pp. 43 – 53, 1996. [73] G.P. Zhizhina, T.M. Zavorykina, E.M. Mill’, E.B. Burlakova // Radiat. Biology. Radioecology, V. 47, pp. 414 – 422, 2007 (in Russian).

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Chapter 12

INFLUENCE OF THE COMPOSITION AND PHYSICOCHEMICAL PARAMETERS OF NATURAL LIPIDS ON PROPERTIES OF LIPOSOMES FORMED FROM THEM M.A. Klimovichaa,1, L.N. Shishkina1, D.V. Paramonovbb,2, and V.I. Trofimov2 1

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N.M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia 2 Scientific and Technological Centre “Lekbiotech”, Moscow, Russia

ABSTRACT The influence of composition and physicochemical parameters (the antiperoxide activity, the amount of the TBA-reactive substances, the content of diene conjugates and ketodienes) of lipids isolated from the liver and brain of outbreed mice on the characteristics of liposomes from these lipids has been studied. The data obtained make it possible to conclude that the phosphatidyl choline/phosphatidyl ethanolamine ratio and the diminution of the share of the more easily oxidizable phospholipids have an important role in the formation of liposomes from the natural lipids, and the [sterols] / [phospholipids] ratio in natural lipids has influenced the sizes formed from the liposomes.

Keywords: phospholipids composition, physicochemical parameters of lipids, TBA-reactive substances, liver, brain, liposome.

aa bb

4 Kosygin str., Moscow, 119334, Russia. 24 Kashirskoe shosse, Moscow, 115478, Russia.

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INTRODUCTION Surface active properties of phospholipids (PL) are widely used for the formation of liposomes, which are not only a model of cell membranes, but also a technique for the study of cells and exposure to them [1-3]. Structural heterogeneity of the eukaryotic cell membranes is common. Sphingomielin (SM) and cholesterol have a key role in maintenance of the structure of microdomains in the cell membrane [4-6]. It is shown that the conditions for the liposome formation (pH of medium, the lipid composition and the degree of their oxidation, the presence of antioxidants in the medium) have a significant effect on their physicochemical properties and structural characteristics [1, 2, 7-10]. Besides, physicochemical properties of surfactants in solution are due to the ratio of the sizes between their hydrophobic and polar groups [3]. The information described above suggests that the composition and physicochemical properties of liposomes formed from the mixture of natural lipids will depend on their physicochemical properties and composition. The aim of this work is to study the influence of composition and physicochemical parameters of lipids isolated from organs of laboratory mice on the characteristics of liposomes formed from them.

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EXPERIMENTAL Lipids were isolated from the liver and brain of mature outbreed mice (female, 3–3.5 months of age) by the Blay and Dyer method in the Kates modification [11]. Experiments were repeated two times: in September (the experiment № 1) and in May (the experiment № 2). The total number of animals in experiments is 60. The murine organs were placed in ace cooled weighing bottles immediately after decapitation. Qualitative and quantitative composition of phospholipids were analyzed by thin layer chromatography, as it was described in [12]. We used the type of silica gel G (Sigma, U.S.A.), glass plates 9 × 12 cm and mixture of solvents chloroform : methanol : glacial acetic acid : water (25 : 15 : 4 : 2) as a mobile phase. Spectrophotometrical measurements were carried out on KFK-3 (Russia) at the wavelength λ = 800 nm. This technique is described in detail in [13]. In addition to the quantitative analysis of different fractions of PL the following generalized parameters of the lipid composition were also evaluated: the PL proportion in the total lipid composition (% PL); the phosphatidyl choline to phosphatidyl ethanolamine (PC/PE) ratio in PL content; the lysoforms of PL to PC (LPC/PC) ratio in PL content; the ratio of the sums of the more easily oxidizable to the more poorly oxidizable PL (ΣEOPL/ΣPOPL). The ΣEOPL/ ΣPOPL value was calculated by the formula: ΣEOPL/ΣPOPL = (PS + PI + PE + PG + CL + PA)/(LPC + SM + PC), where PS is phosphatidyl serine, PI is phosphatidyl inositol, PG is phosphatidyl glycerol, CL is cardiolipin, and PA is phosphatidic acid. The sterol content in lipids was determined spectrophotometrically at 625 nm wavelength [14]. Serva Company (FRG) cholesterol was used for plotting the calibration curve. The antiperoxide activity (APA) of lipids, i.e., the ability of lipids to decompose peroxides, was assessed by the difference in the concentrations of peroxides in the oxidized methyl oleate and in the lipid solutions in this methyl oleate [15]. The content of diene conjugates (DC) and ketodienes (KD) were calculated from the ratio of the optical density of

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Influence of the Composition and Physicochemical Parameters of Natural Lipids… 115 the lipid solutions in hexane (0.1–0.3 mg/ml) at 228–232 nm and 268–272 nm to the main peak in the 202–205 nm, correspondingly, used spectrophotometer UV-3101 PC (Shimadzu, Japan). The amount of the lipid peroxidation products was estimated by their reaction with 2tiobarbituric acid (TBA-reactive substances, TBA-RS) [16]. The protein content was measured by the modified microbiuretic method [17]. The formation of liposomes was carried out by ultrasound dispersant UZDN-2T in 0,1 M C2H5OH aqueous solutions of lipids from the murine organs. The procedures used to prepare the liposomes dispersion and to control the average size were described previously [18, 19]. The conditions of the experiments were the following: a proceeding of cavitation by frequency of the emitter oscillation 22 ±1.65 kHz, the ultrasound action was 30 min under power 1 W/cm3, the concentration of the liver lipids was from 31 to 37 mg/ml and that for the brain lipids was from 17 to 21.5 mg/ml. Then, the dispersion of liposomes is centrifuged to increase the sedimentation rate of mechanical impurities (the result of erosion of the ultrasound emitter), to precipitate the large size liposomes and possibly liposomes which have not formed a lipid bilayer. The duration of centrifugation was 20 min by the rotation angular frequency of 4300–4800 R.P.M. (3000–3800 g). Measuring the pH of the medium is determined by widely known method. The liposome size was estimated by spectra turbidity. The experimental data were processed with a commonly used variational statistic method [20]. The experimental data are presented in the tables and figures in the form of arithmetic means with the indication of the mean square errors of the arithmetic means (M ± m).

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RESULTS AND DISCUSSION Earlier it was found that the PL composition from the liver of Balb/c mice significantly differs depending on the season [21]. Perhaps, there is the similar dependence of the PL composition for outbreed murine organs under proceeding of experiments during different seasons. Indeed, the analysis of the PL composition in the liver (Table 1) and brain (Table 2) of mice allows us to make this conclusion. In autumn we observed the higher share of SM and lysoforms in the PL composition of liver while the relative content of PE was about 23.8% higher in spring. SM is assumed to contribute maintaining lamellar structure of membrane system [6]. Besides, the proportion of PC, one of the major factions PL in the liver and brain, didn’t differ for certain in both experiments. However, in autumn there are slight amounts of an additional fraction in the PL composition of liver which can be characterized as oxidized PC. The significant differences are revealed between generalized parameters of the PL composition in the murine liver depending on season (Figures 1 and 2). So, the reduction of the PE proportion is due to increasing the PC/PE ratio 1.2 times in the experiment № 1 (Figure 2). It is believed that ratio of PC/PE is one of the most important indicators of structural state of the cell [4, 13]. The PL composition in the murine brain is more stable than that in the liver. The comparative analysis of data from Tables 1 and 2 has made it possible to make that assumption a conclusion. Nevertheless, in spring the PE proportion in the PL composition of the brain is for certain higher than in the autumn (Table 2).

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Figure 1. The ratio of sums of the more easily oxidizable and the more poorly oxidizable fractions (ΣEOPL/ΣPOPL) in the murine liver phospholipids and the liposomes formed from them in the different experiments.

Figure 2. The phosphatidyl choline (PC)/phosphatidyl ethanolamine (PE) ratio in the murine liver and the liposomes formed from them in the different experiments.

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Influence of the Composition and Physicochemical Parameters of Natural Lipids… 117 Table 1. The phospholipid composition (%P) of the murine liver and the liposomes formed from them in the different experiments experiment №1 Liver Fraction

n* = 10

Liposomes n = 10

LPC SM PC PC' PS PI PE PG CL PA

5,6 ± 0,2 4,6 ± 0,3 50,3 ± 0,9 0,06 ± 0,03 8,1 ± 0,3 4,0 ± 0,3 21,8 ± 0,7 1,0 ± 0,2 2,9± 0,2 1,6 ± 0,2

5,1 ± 0,7 2,7 ± 0,9 48,5 ± 1,9 1,20 ± 0,4 7,8 ± 1,2 5,9 ± 0,4 17,2 ± 1,0 2,8 ± 0,2 2,8 ± 0,6 6,0 ± 1,0

experiment №2 Liver n = 30 2,37 ± 0,2 2,7 ± 0,2 52,6 ± 1,7 0 10,5 ± 1,0 27,0 ± 0,9 1,4 ± 0,25 2,5 ± 0,25 0,93 ± 0,08

Liposomes n=5 5,1 ± 0,3 3,4 ± 0,2 52,7 ± 0,9 0 6,3 ± 0,5 3,08 ± 0,07 26,0 ± 1,0 0,59± 0,02 2,2 ± 0,2 0,5 ± 0,1

* - number of measurements.

Table 2. The phospholipids composition (%P) of the murine brain and the liposomes formed from them in the different experiments

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experiment №1 brain Fraction n*= 30 LPC SM PC PS PI PE PG CL PA

1,45 ± 0,12 3,6 ± 0,2 39,5 ± 0,6 4,1 ± 0,1 10,3 ± 0,3 36,0 ± 0,5 1,9 ± 0,3 2,6 ± 0,2 0,7 ± 0,1

Liposomes n=3

experiment №2 brain n = 28

Liposomes n=5

14,2 ± 0,8 7,3 ± 0,1 39,0 ± 3,6 4,7 ± 0,6 2,5 ± 1,0 22,0 ± 2,4 2,9 ± 0,4 6,1 ± 0,4 1,15 ± 0,02

0,9 ± 0,1 3,3 ± 0,1 38,4 ± 0,8 3,9 ± 0,2 7,8 ± 0,5 42,1 ± 0,8 0 2,1 ± 0,2 1,5 ± 0,1

2,6 ± 0,3 4,47 ± 0,04 48,0 ± 0,3 2,43 ± 0,02 6,4 ± 0,1 31,5 ± 0,6 0 2,53 ± 0,07 2,1 ± 0,1

* - number of measurements.

This is due to the change of the PC/PE ratio (Figure 3) although the ratio of the sums of the more easily oxidizable to the more poorly oxidizable PL has a similar value (Figure 4) in the brain PL composition in different experiments. The next stage of work was a comparative analysis of the PL composition in natural lipids and liposomes formed from them (Tables 1 and 2). It is necessary to note the relative stability of proportions of the more poorly oxidizable fractions in the PL composition of liposomes which are formed from the liver lipids (Table 1). Besides, the PL composition of liposomes from the brain lipids has reliable differences in shares of all fractions (Table 2).

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Figure 3. The phosphatidyl choline (PC)/phosphatidyl ethanolamine (PE) ratio in the murine brain and the liposomes formed from them in the different experiments.

Figure 4. The ratio of sums of the more easily oxidizable and the more poorly oxidizable fractions (ΣEOPL/ΣPOPL) in the murine brain phospholipids and the liposomes formed from them in the different experiments.

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Influence of the Composition and Physicochemical Parameters of Natural Lipids… 119 Besides, the proportion of the more easily oxidizable fractions in the PL liposomes from the brain lipids significantly decreases in comparison with that in the initial natural lipids (Figure 4).On the contrary, this parameter in the PL liposomes from the liver lipids increases for certain in the autumn experiment (Figure 1). The substantial growth the PC/PE ratio is found in the PL composition of liposomes in all cases except the PL liposomes from the liver lipids which are characterized by the most high proportion of both major PL fractions (Figures 2 and 3). Perhaps the PC/PE ratio and also the diminutions of share of the more easily oxidizable PL have an important role in the formation of liposomes from the natural lipids. In addition to significant changes in the PL composition there are differences between the lipid peroxidation intensity in liposomes formed from lipids of the murine organs (Table 3) which are the most substantial under proceeding of experiments in spring. The content of the oxidation products in brain homogenate is also 2 times lower in spring than that in autumn while the lipid peroxidation intensity in the liver homogenate is similar in both experiments (Table 3). Substantial changes of pH in medium are only observed by the liposome formation from the liver lipids: pH is equal 6.1 in the autumn experiment and 4.3 in the spring experiment. The pH value is equal 6.3–6.35 in medium of liposomes from the brain lipids. Perhaps, the pH value has an inverse dependence on the TBA-reactive substances content in the liposome dispersion and/or the share of the acidic fractions of phospholipids. There are the changes of another physicochemical characteristic of lipids under the formation of liposomes from natural lipids. So, it is revealed the diminution of the APA of the liposome lipids in comparison with the initial lipid which have APA in all cases. However, the direct correlation between the ratio of the sums of the more easily oxidizable to the more poorly oxidizable PL and the APA of lipids is only observed for brain lipids and liposomes with lipids formed from them (Figure 5). The diminution of the lipid APA in liposomes which are formed from the liver lipids is stronger if the initial APA of lipids is higher. It is necessary to note that the content of diene conjugates and ketodienes in the liver lipids are similar in both experiments (Table 4). These parameters in lipids of liposomes which are formed from them are also analogical (Table 4). On the contrary, although reliable differences between the degree of unsaturation and oxidation in the initial brain lipids are absent, the content of diene conjugates and ketodienes in liposomes formed from the brain lipids are different both in the spring and autumn experiments. The size of liposomes is due to different factors. We saw experimentally, too, tendencies to influence sizes of liposomes of both [sterols]/[PL] ratio and origin of the lipid isolation (brain or liver) as well as the season in which the experiment was carried out. Obviously, these facts should be important so they need more detailed investigations. Obviously, the [sterols]/[PL] ratio in natural lipids has influenced the size of the liposome formed from them. Indeed, in the liver lipids of mice which are decapitated in the different seasons, this ratio differs 1.8 times in N 1 (0.299 ± 0.023) and N 2 (0.165 ± 0.028) experiments. Besides, the average diameter of liposomes in the spring experiment is 131 nm while that parameter is lower in the autumn experiment (118 nm). However, the average diameter of liposomes which are formed from the murine brain lipids is increased by the growth of the [sterols]/[PL] ratio in the initial natural lipids. It is equal to 204 nm if this ratio in the brain lipids is 1.60 ± 0.095 and 62 nm if the [sterols]/[PL] ratio is 0.81 ± 0.12.

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Figure 5. Interrelation between the ratio of sums of the more easily oxidizable to the more poorly oxidizable phospholipids and the antiperoxide activity of lipids.

Thus, the data obtained allow us to conclude that the composition and physicochemical characteristics of the murine lipids have a significant influence on the composition and properties of the lipid bilayer of liposomes which are formed from these natural lipids. Table 3. The content of TBA-reactive substances [TBA-RS] in the homogenate of the murine organs and in the aqueous dispersion of liposomes formed from their lipids in the different experiments [TBA-RS] nmol/mg of proteins nmol/mg of lipids

Experiment liver (n*= 6)

1 0.018 ± 0.002

2 0.014 ± 0.001

brain (n = 6) liposome dispersion from the liver lipids liposome dispersion from the brain lipids

0.11 ± 0.01 7.7

0.052 ± 0.004 2.0

6.2

4.65

* - number of measurements.

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Influence of the Composition and Physicochemical Parameters of Natural Lipids… 121 Table 4. The content of diene conjugates (DC) and ketodienes (KD) in the lipids of the murine organs and the formed from them liposomes in the different experiments Experiment liver lipids (n*=6) brain lipids (n=6) liposome lipids from the liver lipids liposome lipids from the brain lipids

parameter DC KD DC KD DC KD DC KD

№1 0.031 ± 0.002 0.0098 ± 0.0013 0.062 ± 0.009 0.03 ± 0.004 0.061 0.015 0.044 0.022

№2 0.027 ± 0.002 0.0098 ± 0.0008 0.058 ± 0.009 0.028 ± 0.005 0.044 0.015 0.081 0.039

* - number of measurements.

REFERENCES [1] [2] [3]

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[4] [5] [6] [7] [8] [9] [10]

[11] [12] [13] [14]

L. B. Margolis, L.D. Bergel’son. Liposomes and their interaction with cells. Moscow, Nauka, 240. pp., 1986 (in Russian). G. Gregoriadis, A. C. Allison (eds.). Liposomes in biological systems, Wiley, 422 pp., 1980. K. Holmberg, B. Jönsson, B. Kronberg, B. Lindman. Surfactants and Polymers in Aqueous Solution, Moscow: BINOM, Laboratoriya znanii, 528 pp., 2007. (Russian version). R.C. Aloia (ed.). Membrane Fluidity in Biology V. 1. Concepts of membrane structure, Кiev: Naukova dumka, 312 pp., 1989. (Russian version). N.D. Radgway. Biochim. Biophys. Acta. V. 1484, pp.129 – 141, 2000. H. Ohvo - Rekila, B. Ramstedt, P. Leppimaki, J.P. Slotte. Prog. Lipid Res., V. 41, pp. 66 - 97, 2002. C.H. Hsiesh, S.C. Sue, P-C Lyu, W.-g. Wu. Biophys. J., V. 73, pp. 870 - 877, 1997. W. Van Klompenburg, I. Nilsson, G. von Heijne, B. de Kruijff. EMBO J., V. 16, pp. 4261 - 4266, 1997. Ju. Barauskas, C. Cervin, F. Tiberg, et al. Phys. Chem. Chem. Phys., V. 10 pp. 6483 6485, 2008. L.N. Shishkina, M.A. Klimovich, D.V. Paramonov, V.I. Trofimov “Influence of the forming conditions of liposomes on their physicochemical properties and lipid composition”, p. 40; In Program and Summaries ΙΙΙ International Conference on Colloid Chemistry and Physicochemical Mechanics, Moscow, 2008. M. Kates. The technique of Lipidology. Moscow: Mir, 322. pp., 1975 (Russian version). J.B.C. Findlay, W.H. Evans (eds.). Biological membranes: A practice approach. Moscow: Mir, 424 pp., 1990 (Russian version). L.N. Shishkina, E.V. Kushnireva, M.A. Smotrjaeva. Oxidation commun., V. 24, pp. 276 - 286, 2001. W. M. Sperry, M. Weeb. Biol. Chem.. V. 187, pp. 97- 106, 1950.

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[15] V.A. Menshov, L.N. Shishkina, Z.N. Kishkovsky. Appl. Biochem. Microbiology. V. 29, pp. 675 – 683, 1993. [16] T. Asakawa, S. Matsushita. Lipids. V. 15, pp. 137 - 140, 1980. [17] R. Itzhaki, D.M. Gill. Anal.Biochem V. 9, pp. 401 – 409, 1964. [18] Gregoriadis G.(ed.). Liposome Technology V.1. Preparation of Liposomes. Florida: CRC Press, Inc. Boca Raton, 268 pp., 1989. [19] D.V. Paramonov, E.A. Antonova, L.T. Bugaenko, V.I. Trofimov, V.M. Byakov. Radiolysis of 2,6-di-tert-butyl-4-methylphenol (ionol) in a lipid membrane in the presence of oxygen. Mendeleev Commun. pp.32-34, 1998. [20] G.F. Lakin. Biometry. 3rd edition, Moscow: Vysshaya shkola, 293 pp., 1990 (in Russian). [21] M.V. Kozlov, V.V. Urnisheva, L.N. Shishkina. J. Evol. Biochem. Physiology, V. 44, pp. 398 – 402, 2008 (in Russian).

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In: Handbook of Chemistry, Biochemistry … Editors: L. N. Shishkina et al., pp. 123-134

ISBN: 978-1-61209-425-0 © 2010 Nova Science Publishers, Inc.

Chapter 13

STATE OF LIPID COMPONENT OF SOYBEAN FLOUR ENZYMATIC HYDROLYZATES DURING STORAGE L.N. Shishkinacc,1, E.V. Miloradovadd,2, E.A. Badichko2 and S.E. Traubenberg2 1

N. M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia 2 Moscow State University of Food Production, Moscow, Russia

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ABSTRACT This chapter is a study of the influence of hydrolysis and centrifugation processes of soybean semifat flour on various indices of lipid components and dynamics of changes in the composition and characteristics in hydrolyzates within three months of storage. It was shown that processes of hydrolysis and centrifugation, as well as storage, cause reliable changes in the physical and chemical characteristics and lipid composition in hydrolyzates.

Keywords: soybean flour, hydrolysis, centrifugation, lipid peroxidation, phospholipid composition, storage

AIMS AND BACKGROUND The quality of the lipid content of a product is defined by the safety of its lipid components [1,2]. Many factors promote the oxidation of lipids, which involves a change in consistency, colour and taste of a product, as well as a loss of vitamins and essential fatty acids. Thus, not only is the oxidation of lipids accompanied by the loss of taste quality, aroma and nutritional value of a product, but it also leads to the formation of unhealthful compounds a b

4 Kosygin Str., Moscow 119334, Russia. Volokolamskoe Shosse, Moscow 125080, Russia.

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[1-3]. Because the accumulation of primary and secondary products of oxidation is due to the interaction of lipids with oxygen in the air, there is a need to monitor lipid components in the process of storage. It is necessary to note that the type of packing material and the storage period are of great importance to safeguarding the integrity of the quality of a product. Polyethylene film is one of the most widely-used packing material, as it helps maintain moisture and the appearance of products and interferes with and prevents oxidation [4]. Data have been published regarding significant changes in the lipid components of wheat flour [5], dry yeast envelopes [6], dried doughnut mixes [7], and lecithin-enriched soybean fat free flour in various types of packing [8] during storage which indicate complicated hydrolytic and oxidative processes in their lipid components during the storage of dry food items. However, the data regarding the stability of the lipid components of dried soybean enzymatic hydrolyzates are practically absent. Enzymes are used more often to yield hydrolyzates, the application of which allows creating and improving existing foodstuff [9]. Moreover, the process of centrifugation and drying are used, which also has a significant impact on the state of the lipid component of the initial sample [10-12]. The aim of this work is the study of the influence of hydrolysis and centrifugation of semifat soybean flour on various indices of lipid components and the dynamics of change in the composition and characteristics of its lipid hydrolyzates during storage.

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MATERIALS AND METHODS The objects of research were lipids isolated from soybean enzymatic hydrolyzates, received from soybean deodorized semifat flour (GOST 3898-56). Proteolytic enzyme Beerzym Chill was used for hydrolysis of soybean flour during 8 hours at its optimal condition before the study (temperature -50°С, рН = 8) [13]. The obtained liquid hydrolyzate was divided into two parts, one of which was clarified by centrifugation. The clarified part (supernatant) and nonclarified liquid hydrolyzate (partly hydrolyzed soybean flour) were dried with spray drying “mobile minor”. After that, samples were simultaneously tightly packed into single-layered transparent packing and kept in a dry place at room temperature. The analysis of samples was carried out before packing and later at 1, 1.5 and 3 months of storage. The content of products that interacted with 2-thiobarbituric acid (TBA-reactive substances, TBA-RS) in a suspension of hydrolyzates was determined by the method described in [14]. The amount of protein in soybean hydrolyzates was analyzed by using a modified microbiuretic method [15]. Lipids were isolated from the samples using the Blay and Dyer methods in the Kates modification [16]. The separated lipids were used for studying the content of peroxides and composition of lipids. The amount of peroxides in lipids was determined using the method of iodometric titration according to the procedure specified in GOST 26583-85. The ability of lipids to decompose peroxides (their antiperoxide activity, APA) was evaluated by method [17].

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State of Lipid Component of Soybean Flour Enzymatic Hydrolyzates during Storage 125 The qualitative and quantitative composition of phospholipids (PL) was determined using the thin-layer chromatography method with the use of silica gel, type G (Sigma, USA) and glass plates measuring 9×12 cm [18]. A chloroform–methanol–glacial acetic acid–water mixture in the ratio of 50:30:8:4 was used as the solvent system. The development of chromatograms was performed by iodine vapour. The quantitative analysis of the PL composition was determined after the removal of fractions from the plate and after PL perchloric acid digestion to inorganic phosphate. For the colour reaction to phosphorus, we used ammonium molybdate and ascorbic acid produced by Serva (Germany) and perchloric acid of chemically pure grade. The amount of inorganic phosphorus was judged according to the optical density of the solutions at the λ=800 nm wavelength as measured with spectrophotometer KFK-3 (Russia). Plotting of the calibration curve was carried out on the monosubstituted potassium phosphate of especially pure grade. In addition to the quantitative analysis of different fractions of PL, the generalized parameters of the lipid composition were evaluated: amount of PL of total lipid composition (%PL), the ratio between the sums of the more easily oxidizable to the more poorly oxidizable PL fractions (ΣEOPL/ΣPOPL) and the phosphatidyl choline/phosphatidyl ethanolamine (PC/PE) ratio. The value between ∑EOPL /∑POPL was calculated by the formula [19]:

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∑EOPL /∑POPL=(PI+PS+PE+PG+CL+PA)/(LPC+SM+PC), where PI is phosphatidyl inositol, PS is phosphatidyl serine, PG is phosphatidylglycerol, CL is cardiolipin, PA is phosphatidic acid, LPC are lysoforms of PL, and SM is sphingomyelin. The more easily oxidizable phospholipids are the fractions in the composition of which there are predominantly unsaturated fatty acids and the more poorly oxidizable phospholipids are the fractions in the composition of which there are predominantly saturated fatty acids in the hydrocarbon part of the molecules. That ratio makes it possible to judge the oxidizability of the lipids [19]. The content of sterols was determined spectrophotometrically at 625 nm according the methods described in [20]. The measurements in each from independent samples were made three to six times. The experimental data were processed with a commonly-used variational statistical method described in [21]. The experimental data are presented in the tables and figures in the form of arithmetic means with the indication of the mean square errors of the arithmetic mean (M ± m).

RESULTS AND DISCUSSION During the first stage of the work, the influence of technological process to the content of total lipids (TL), TBA-reactive substances, amount of PL and sterols in the composition of total lipids in the investigated samples were studied. The results are shown in the Table 1. Their analysis testifies that the reliable decrease of TL in 1 g of absolutely dry matter (ADM) is observed during the hydrolysis of flour and the following process of centrifugation and drying.

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126

In addition to the quantity of TBA-reactive substances in the samples, increases in inverse consequence by 1.6 times in non-clarified hydrolyzate and by 3.4 times in supernatant, correspondingly, were observed. This allows the assumption that the processes of hydrolysis and centrifugation intensify lipid peroxidation (LPO) because it is well known that LPO intensity is evaluated by the TBA-reactive substances’ content in a complex biological system [22]. Table 1. The influence of enzymatic hydrolysis and centrifugation processes on biochemical characteristics of soybean hydrolyzates Parameters

Soybean semi fat flour

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(n*=3)

Non-clarified hydrolyzate (partly hydrolyzed soybean flour) (n=6)

Clarified soybean hydrolyzate (n=6)

[TL]/ADM, mg/g

86.2 ±11.6

27.8±2.8

14.8± 0.7

[TBA-RS], nmole/mg of protein

0.109±0.023

0.179±0.029

0.370±0.071

0.257±0.034

0.049±0.007

0.0371±0.0024

[TL]/[protein]

0.544±0.027

0.70±0.13

3.08±0.79

[sterols]/[PL]

24.1±1.6

24.8±1.3

12.4±1.4

% PL

7.3±1.2

9.1±1.25

26.4±1.6

% sterols *n – number of independent measurements

Table 1 indicates that it is also possible to conclude that the process of centrifugation reduced the amount of PL in the total lipid composition by 2 times. However, the content of sterols after centrifugation, on the contrary, increased by 3 times. It is known that peroxide compounds are the primary products of lipid oxidation [1]. It is necessary to note the heterogeneity of the investigated samples in the given parameter. So, lipids in a soybean semifat flour mainly had the ability to compose peroxides, and peroxides have only been found in the amount of 1.8 μmol/g of lipids in one variant. The amount of peroxides is 4.9±0.8 μmol/g of lipids in clarified soybean hydrolyzate, and 6.8±2.8 μmol/g of lipids in non-clarified soybean hydrolyzate. However, lipids of hydrolyzates have APA in solitary cases. The composition of PL of soybean flour and its hydrolyzates are shown in Table 2. It is clear that the main fractions of PL are PC and PE in all samples. However, the processes of hydrolysis and centrifugation produce a significant change in quantitative ratio of PL fractions.

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State of Lipid Component of Soybean Flour Enzymatic Hydrolyzates during Storage 127 Thus, the reliable growth in the share of PC by 15.7% and the decrease of relative amount of CL+PA by 46.9% in PL of non-clarified hydrolyzate is observed compare to composition of PL in soybean flour. Besides, the appearance of the additional minor fraction is observed, which can be identify as oxidized PE (PE′). The portions of SM and CL+PA are reliably increased by 3.1 and 2.1 times, correspondingly, at reduction of the relative content of PC by 30.9% and PS by 1.9 times in PL composition of the clarified hydrolyzate compared with their amount in PL of non-clarified hydrolyzate. The amount of the oxidized PE′ showed the tendency for further growth.

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Table 2. Composition of phospholipids of soybean flour and its hydrolyzed products

Fraction, %Р

Soybean semifatty flour (n*=15)

Non-clarified hydrolyzate (partly hydrolyzated flour) (n*=29)

Clarified soybean hydrolyzate (n*=21)

LPC

3.9±0.6

3.55±0.50

6.60±1.85

SM

4.25±0.65

3.8±0.45

11.85±0.9

PC

31.4±1.2

36.3±0.9

25.1±1.45

PS

15.3±0.95

14.1±0.6

7.40±0.65

PI

5.2±0.85

5.65±0.45

6.3±0.70

PE

24.45±1.45

23.6±0.85

19.15±1.65

PE′

—

3.2±0.5

5.25 ±1.0

PG

3.6±0.4

3.5±0.3

4.8±0.8

CL+PA

12.0±1.8

6.35±0.8

13.55±1.8

* n – number of measurements.

The next step of this work was to study the lipid component stability during 3 months of storage. The dynamic of TBA-RS active substances in hydrolyzates is shown in Figure1. The data in Figure 1 show that these parameters in non-clarified hydrolyzates are quite stable, while the clarified hydrolyzates revealed only a trend of decline due to the high variability of the intensity of LPO in the initial sample. In addition, the content of oxidation products in the non-clarified hydrolyzates was significantly lower than in the clarified during the whole period of storage. Perhaps this is due to the presence of SH-containing amino acids which can inhibit the oxidation processes in non-clarified hydrolyzates [23]. Peroxides only were found in lipids of both hydrolyzates during storage. However, in nonclarified hydrolyzates the peroxide content was roughly decreased after 1 month from the

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beginning of the experiment, but increased during subsequent periods of storage. In the centrifuged hydrolyzates, the amount of peroxides in lipids changed at phases: a minimal degree of lipid oxidation was detected after 1 month and a maximal after 1.5 months of storage. The dynamics of the ratio [TL]/[protein] in both hydrolyzates during storage was similar: the maximal value of this parameter was found after 1 month of storage (Figure 2). Changes in the intensity of oxidative processes and the ratio [TL] / [protein] in dried hydrolyzates were due to quantitative change in the ratio of the various kinds of lipids. The analyses of the PL composition in hydrolyzates during storage are presented in Tables 3 and 4.

Figure1. Changes in the intensity of LPO in hydrolyzates of soybean flour during storage

The main fractions are still a fraction of PC and PE in both hydrolyzates during the whole experiment. The proportion of PC in the clarified hydrolyzates did not change significantly; on the contrary, this parameter increased in non-clarified hydrolyzates during 3 months of storage. Table 3 shows that the most significant changes in the ratio of PL fractions in clarified hydrolyzates were detected after 1.5 months of storage. Thus, a reliable drop in the relative content was found for the SM and PI, with a significant increase of PG and the sum of CL+ PA. Phase changes in the ratio of PS in PL of clarified soybean hydrolyzates revealed that their maximal value is detected after 1 and 3 months of storage. Changes in the PL composition of non-clarified hydrolyzate (Table 4) was different during storage. An authentic fall in the share of SM and PI was detected in 1.5 and 3 months of storage, but the most significant increase was found the relative amount of the CL+ PA during 1 month after the beginning of the experiment (Table 4). Moreover, low amounts of oxidized PE′ in the PL of both hydrolyzates were found only at the beginning and after 1.5 months.

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State of Lipid Component of Soybean Flour Enzymatic Hydrolyzates during Storage 129

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Figure 2. The ratio of the [total lipid]/protein in hydrolyzates during storage.

Such significant changes of quantitative ratio of different fractions within the PL composition cause the changes of relationship of generalized parameters of PL composition in hydrolyzates during storage. Interestingly, as in the original sample, within the first 1.5 months the value PC/PE (Figure 3) in PL of clarified hydrolyzates was also significantly lower than in non-clarified hydrolyzate. But at the end of the experiment, this ratio was almost identical in the PL of both hydrolyzates and significantly higher than in the initial samples. The ΣEOPL/ΣPOPL ratio characterizing the ability of lipids to oxidate was increased by 1.5 times in the clarified hydrolyzates during 1.5 months (Figure 4). This was due to the increased share of the more easily oxidizable fractions in PL composition. However, when stored for 3 months, there was a decrease in this ratio in the clarified hydrolyzates from the initial value, but it was significantly lower than the control in non-clarified hydrolyzates. Figure 5 presents the change of PL shares in the TL composition of hydrolyzates during storage. It is seen that the share of PL in non-clarified hydrolyzates during 1.5 months of storage was lower than in the initial samples. However, after 3.0 months, the share of PL was 1.4 times greater than in the control. The PL amount in the TL composition of clarified hydrolyzates varies within the limits of variability for the original sample. It may be noted that there was only a significant difference in value between this parameter after 1 and 1.5 months. The ratio [sterols]/[PL], in contrast, was nearly 4.4 times higher after centrifugation; this provides some evidence of the advantages of Pl absorption compared with sterol during the centrifugation process of hydrolyzed soybean flour (Figure 6). A higher ratio of [sterols]/[PL] in clarified hydrolyzates was retained during the entire period of storage. Besides, this parameter in the total lipids of non-clarified hydrolyzate remained unchanged during storage. The molar ratio [sterols]/[PL] in the lipid of clarified hydrolyzates changed at stages reaching a maximum after 1 and 3 months and decreased 2.3 times after 1.5 months of storage (Figure 6).

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130

Table 3. The phospholipid composition of clarified soybean hydrolyzate during storage Fraction, %Р

Duration of storage, month 0

1

1.5

3

LPC

6.60±1.85

6.77±0.40

9.55±1.00

6.75±1.05

SM

11.85±0.9

6.7±0.75

5.01±0.9

11.85±2.30

PC

25.1±1.45

24.85±1.7

21.35±1.45

25.3±3.6

PS

7.41±0.65

19.65±2.3

7.2±1.0

20.4±2.6

PI

6.3±0.70

6.6±0.6

4.13±0.47

2.6±0.8

PE

19.15±1.65

17.2±2.1

18.45±1.7

14.15±2.2

PE′

5.25 ±1.0

—

0.56±0.30

—

PG

4.8±0.8

2.35±1.15

12.4±0.8

5.55±1.2

CL+PA

13.55±1.8

15.9±2.5

20.45±2.9

13.4±3.3

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Table 4. The phospholipid composition of non-clarified hydrolyzate during storage Fraction, %Р

Duration of storage, month 0

1

1.5

3

LPC

3.55±0.50

2.75±0.55

3.9±0.60

5.85±0.85

SM

3.8±0.45

4.6±0.95

2.3±0.25

2.25±0.3

PC

36.3±0.9

36.7±1.3

40.25±1.1

41.35±0.95

PS

14.1±0.6

16.45±1.25

17.15±0.8

16.4±1.0

PI

5.65±0.45

3.2±0.45

1.57±0.30

2.3±0.35

PE

23.6±0.85

20.95±1.0

22.6±0.75

22.35±1.05

PE′

3.2±0.5

—

1.35±0.25

—

PG

3.5±0.3

4.5±0.9

2.85±0.35

2.25±0.65

CL+PA

6.35±0.8

10.9±1.35

8.0±1.1

7.3±0.90

The data obtained suggest that the high lability of a lipid component favours the proceeding of the hydrolytic processes. Handbook of Chemistry, Biochemistry and Biology : New Frontiers, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

State of Lipid Component of Soybean Flour Enzymatic Hydrolyzates during Storage 131 The results of the study also allow us to conclude that not only technological processes but also storage can cause reliable changes in lipid composition in both types of hydrolyzates. It sets up a need for finding a means of stabilizing and protecting the hydrolyzate lipids during storage in the event of their further application.

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Figure 3. The dynamic of changes of the PC/PE ratio in PL of soybean hydrolyzates during storage.

Figure 4. Changes in the ratio of sums of the more easily oxidizable to more poorly oxidizable phospholipid fractions of hydrolyzates during storage. Handbook of Chemistry, Biochemistry and Biology : New Frontiers, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

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L.N. Shishkina, E.V. Miloradova, E.A. Badichko et al.

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Figure 5. Changes of phospholipid content in the total lipid composition in hydrolyzates during storage.

Figure 6. The molar ratio of [sterols]/[PL] in lipid hydrolyzates during storage.

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State of Lipid Component of Soybean Flour Enzymatic Hydrolyzates during Storage 133

REFERENCES [1] [2] [3] [4]

[5] [6] [7] [8]

[9] [10] [11] [12]

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[13] [14] [15] [16] [17] [18] [19] [20] [21] [22]

[23]

I.M. Emanuel, J.N. Lyaskovskaya. Inhibition of Fat Oxidation. Moscow: Pishepromizdat, 1961, 355 pp. (in Russian). F.M. Rzhavskaya. Lipids of fishes and marine mammals. Moscow: Pishepromizdat, 1976, 470 pp. (in Russian). Min-Hsing Pan, Chi-Tang Ho. Chem. Soc. Rev., 2008. V. 37. pp. 2558-2574. V.B. Spirichev, L.N. Shatnyuk, V. M. Poznyakovskii. Enrichment of foodstuff by vitamins and mineral substances. Novosibirsk Publishers Sib. Universiti, 2004, 548 pp. (in Russian). V.L. Kretovitch. Biochemistry of grain and bread. Moscow: Nauka, 1991, 136 pp. (in Russian). V.A. Menshov, L.N. Shishkina, Z.N. Kishkovskii. Applied biochemistry and Microbiology, 1993, V. 29, No. 6, pp. 675-683. L.N. Shishkina, M.A. Klimova, G.F. Dremutcheva, S.E. Traubenberg. Applied biochemistry and microbiology, 2000, V. 36, No. 4, pp. 503-508. L.N. Shishkina, S.E Traubenberg, E.V. Miloradova, I.V Vialtseva, A.A. Kozlova. New Trends in Biochemical of Physics Research. Eds S.D. Varfolomeev at al. Nova Science Publishers: New York, 2007, рр. 101-109. M.L. Domoroshenkova. Food industry, 2001, No. 4, pp. 5-11. (in Russian). V.A. Menshov, L.N. Shishkina, E.B. Burlakova, Z.N. Kishkovskii, I.I. Samoiilenko, E.V. Idrisova. Applied biochemistry and microbiology, 1993, V. 29, No. 3, pp. 334-339. L.N. Shishkina, A.A. Kozlova, E.V. Miloradova. Storage and Processing of Farm Products, 2006, No. 1, pp. 25-27. (in Russian). L. N. Shishkina, S. E. Traubenberg, E. V. Miloradova, I. V. Vialtseva, A. A. Kozlova. Storage and Processing of Farm Products, 2006, No. 3, pp. 13 - 17. (in Russian). S.E. Traubenberg, E.V. Miloradova, E.V. Alekseenko, E.A. Badichko // Storage and Processing of Farm Products, 2007, No. 5, pp. 62 - 65. (in Russian). T. Asakawa, S. Matsushita. Lipids, 1980, V. 15, No. 3, pp. 137-140. R. Itzhaki, D.M. Gill. Anal.Biochem, 1964, V. 9, pp. 401-409. M. Kates. The technique of lipidology, Moscow: Mir, 1975, 322. pp. (Russian version). L.N. Shishkina, N.V. Khrustova. Biophisics, 2006, V. 51, No. 2, pp. 340-346. Biological membranes. A practice approach. Eds J.B.C. Findlay, W.H. Evans. Moscow: Mir, 1990. 424 pp. (Russian version). L.N. Shishkina, E.V. Kushnireva, M.A. Smotrjaeva. Radiat. biology. Radioecology, 2001, - V. 41, No. 3, pp. 301-306. (in Russian). W.M. Sperry, M. Weeb. Biol.Chem., 1950, V. 187, No. 1, pp. 97-106. [21] G.F. Lakin. Biometry. 3rd publication, Moscow: High School, 1990, 293 pp. (in Russian). V.E. Kagan, O.N. Orlov, L.L. Prilipko. Problems of Analysis of lipid Peroxidation Endogenous Produkcts, Itogi nauki I tekhniki VINITI Akad. Nauk SSSR. Ser. Biofizika, 1986, No.18, pp. 136. (in Russian). E.T. Denisov, T.J. Denisova Handbook of Antioxidants. Bond Dissociation Energy, Rate Constants, Activation Energy and Enthalpies, of Reactions (2nd ed.). Boca Raton, New York, Washington: CRC Press, 2000, 290 pр.

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In: Handbook of Chemistry, Biochemistry … Editors: L. N. Shishkina et al., pp. 135-145

ISBN: 978-1-61209-425-0 © 2010 Nova Science Publishers, Inc.

Chapter 14

XRD CHARACTERIZATION OF SUPERFINE FE POWDER AND EPR STUDY OF ITS INTERACTION WITH LIPID MEMBRANES Liudmila D. Fatkullina, Alexey V. Krivandin, Elena B. Burlakova and Alexander N. Goloschapov1 Emanuel Institute of Biochemical Physics, Russian Academy of Sciences; Moscow, Russia

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ABSTRACT The structural properties of the superfine iron powder and its interaction with lipid membranes of mice erythrocytes and lipid membranes of egg lecithin liposomes were studied by X-ray diffraction (XRD) and electron paramagnetic resonance (EPR) methods. The superfine iron powder was prepared by the method of heterophase interaction. It was shown by XRD analysis that this powder consisted predominantly of the crystalline iron in the α-form (α-Fe) with the crystal lattice parameter a = 0.2866 nm and the average crystal size about 30 nm. The microviscosity variation of liposome membranes and erythrocyte membranes under the action of the superfine iron powder in vitro was analyzed by EPRspectroscopy. The effect of the iron powder on the lipid membrane microviscosity depended on the powder concentration, the time of the powder interaction with membranes and the type of these membranes. It was shown that the superfine iron powder at the ultra low concentrations had a more pronounced effect on the lipid membrane microviscosity than this powder at high concentrations.

Keywords: superfine iron, nanodispersed iron, nanoparticles, microviscosity, EPR-spectroscopy, X-ray diffraction

1

lipid

membranes,

4 Kosygin Street, Moscow, 119334, Russia; Fax: +7 499 137 41 01; Tel: +7 495 939 71 81 Е-mail: [email protected].

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INTRODUCTION Metals in the nanodispersed state have been the object of intense research work in recent decades due to their high chemical activity, semiconductor type of conductivity, increased hardness and other outstanding properties [1, 2]. A range of metal nanoparticles exhibits biological activity and can be used in medicine or agriculture. That’s why so much attention is paid to research the effects of nanodispersed metals on humans and animals. Previously, a high biological activity of iron, zinc and copper nanopowders injected into the animal organism had been shown [3]. The cell membrane may be one of the main targets of the metal nanopowder activity. In such a way, the iron nanopowder inserted into an organism can influence the parameters of the oxidative stress in blood, e.g., lipid peroxide oxidation and the membrane physicochemical condition [4]. In this work we studied the influence of the superfine iron powder (nanopowder) on the structural state of lipid membranes of mice erythrocytes and egg lecithin liposomes in vitro. As far as the functional activity of metal nanoparticles may be dependent on their structure, we carried out X-ray diffraction (XRD) analysis of the iron nanopowder used in this study.

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MATERIAL AND METHODS The nanodispersed iron powder was obtained by the method of heterophase interaction [5]. The phase composition of this powder was analyzed by the Debye-Scherrer and BraggBrentano X-ray diffraction methods. In the Debye-Scherrer method, the IRIS-M X-ray generator (Nauchpribor, Russian Federation) with the fine focus molybdenum anode X-ray tube BSV25 (SvetlanaRentgen, Russian Federation) run at 40 kV / 5 mA and 20 μm Zr β-filter was used as an X-ray source. X-ray diffraction patterns were recorded with a cylindrical X-ray diffraction camera on the X-ray film RT-1 (Tasma, Russion Federation) and were digitized with the Umax Astra 4450 scanner at 16 bit black and white mode and 600 dpi resolution. One-dimensional profiles of intensity were obtained with the image analysis program ImageJ 1.34s (NIH, USA), corrected for background scattering and plotted as a function of I(S), where S=(2sinθ)/λ, θ is a half of scattering angle and λ is an X-ray wavelength equal on average to ~0.071 nm for Mo Kα1/Kα2 X-rays. The distances between crystal atomic layers were calculated according to the Wolf-Bragg formula as dhkl=(Shkl)-1=λ/(2sinθhkl), where θhkl is a Wolf-Bragg angle for an X-ray diffraction peak (reflection) corresponding to atomic layers with Miller indices (hkl). On the basis of dhkl values and intensities of X-ray reflections the phase analysis of the iron powder was performed. By means of the Debye-Scherrer method it was possible to record the X-ray diffraction pattern in the range of 3.5 nm-1 < S < 19.5 nm-1 that corresponds to the Wolf-Bragg distances dhkl from ~0.28 to ~0.05 nm. In order to investigate the presence of the higher values of dhkl and to determine dhkl values more accurately the X-ray diffraction patterns of the iron powder were obtained with the HZG4 X-ray diffractometer (Freiberger Prazisionsmechanik GmbH, Germany) in the Bragg-Brentano reflection mode. The IRIS-2 X-ray generator with the copper anode X-ray tube (40 kV, 20 mA) and Ni β-filter was used as an X-ray source. The X-ray diffraction patterns were recorded with the proportional gas-filled counter. The additional monochromatization was achieved with a pulse-height discriminator. This setup made it

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possible to analyze dhkl values from ~4 to ~0.08 nm. The background scattering was subtracted and dhkl values were calculated with the program package “Winscaler” written by Yu. V. Tashlanova. The average crystal size in the Fe powder was evaluated according to the SelyakovScherrer relation [6] as L = b-1, where b is the integral width of the Kα1 X-ray diffraction peak (the ratio of the peak integrated intensity to its height) corrected for the instrumental broadening and expressed in the units of S. For this purpose the profile of the X-ray diffraction peak was measured for a thin layer of Fe powder in the transmission geometry on the smallangle X-ray diffractometer (Mo X-ray tube BSV25, Zr β-filter). The primary X-ray beam was collimated with the 3-slit Rigaku small-angle scattering goniometer. The X-ray diffraction patterns were recorded with the one-dimensional proportional gas-filled (85% Xe, 15% Me) position-sensitive detector with the delay-line readout and a pulse-height discriminator [7]. The sample to detector distance was 420 mm. In order to diminish the instrumental broadening of the diffraction peak analyzed the detector was turned with goniometer at the angle approximately equal to the angle 2θhkl of this peak. In order to determine the instrumental broadening for the diffraction peak of the Fe powder the X-ray diffraction peak profile for thin aluminium foil was measured with this setup. The width of the X-ray diffraction peak for this aluminium foil gave the maximum possible value of the instrumental broadening for the Fe powder X-ray diffraction peak. The lowest possible value of this instrumental broadening was given by the width of the primary X-ray beam. The width of the primary X-ray beam was only slightly less than the width of the X-ray diffraction peak for the Al foil. Thus the uncertainty for the real instrumental broadening of the diffraction peak was rather small. The profile of X-ray diffraction peaks comprising Kα1 and Kα2 constituents was approximated with the expression

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I (S ) = 1+

k1 + ( S − S1 ) 2 b1

2

1+

k2 + c1 S + c 0 , (S − S 2 ) 2 b2

(1)

2

where k1, b1, S1, k2, b2, S2, c1, c0 are variables. In this expression the first and second terms are given by the Cauchy functions and approximate the Kα1/Kα2 doublet, the linear function c1S+c0 approximates the background scattering. The integral width of the Fe X-ray diffraction peak corrected for the instrumental broadening was determined as b = π(b1Fe – b1Al) and the average crystal size in the Fe powder was evaluated as L = b-1, where b1Fe and b1Al are the width parameters (half width at half maximum) of Kα1 diffraction peaks determined for the Fe powder and Al foil from approximation (1). In the biological experiments the mice red blood cell membranes and multilamellar egg lecithin liposome were used. Liposomes are microscopic spherical vesicles composed of lipid bilayers separated by the water phase. Due to the amphipathic nature of lipid molecules, liposomes can be prepared by blending lipids and water. In this work the liposome dispersion was prepared as in earlier work [8]. 200 mg of the egg lecithin (Sigma, USA) was dissolved in chloroform. Chloroform was evaporated under vacuum and 1 ml 40 mM phosphate buffer (pH-7.4) was added to the vial. Then the vial was heated up to ~55˚C and vigorously shaken for half an hour. The nanodispersed iron powder suspension was prepared by dispersion of the

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iron powder in the distilled water under ultrasonic disintegration at 44 kHz for 10 min with cooling on the ice. The iron suspension was mixed with the 5% erythrocyte suspension or the 0.01% liposome suspension and these mixtures were incubated for 1 or 24 hours at 4˚C. The concentration of the iron powder in the membrane suspension was varied from 10-9 to 10-1 mg/ml. Membrane suspensions without iron powder were used as a control. Each experiment was repeated at least 3 times and results were averaged. The structural characteristics of membranes were investigated with the ER-200D SRC EPR-spectrometer (Bruker, Germany) at room temperature using paramagnetic spin probes. The following radicals were used as probes: 2,2,6,6-tetramethyl-4-capryloyl-oxypiperidine-1oxyl, that localized mostly in the superficial part of the lipid membrane bilayer (probe I), and 5,6-benzo-2,2,6,6-tetramethyl-1,2,3,4-tetrahydro-γ-carboline-3-oxyl, which permeated into deep-located near-protein sites of the lipid membrane bilayer (probe II) [9]. These probes were added into membrane suspensions at final concentrations of 10-4 M. From the EPR-spectra obtained the correlation time for the probe rotation τс was calculated with the formulas for rapidly rotating probes. The correlation time τс approximately equals to the period of radical reorientation for the angle π/2 and denotes the membrane microviscosity [10]. The values of microviscosity (figs. 4 and 5) were expressed in relative units given by the ratio of τс obtained for membrane suspensions with the iron powder to the values of τс obtained for the control membrane suspensions.

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RESULTS AND DISCUSSION The nanodispersed iron powder was characterized by the X-ray diffraction method. The XRD patterns of the iron powder obtained by the Debye-Scherrer and Bragg-Brentano methods are shown in Figure 1 and 2. These patterns contain sharp X-ray diffraction peaks that indicate a crystalline structure of the Fe powder. Besides the X-ray diffraction peaks visible in figs. 1 and 2, the original X-ray diffraction patterns obtained by the Debye-Scherrer method (X-ray films) contain some weak diffraction peaks which are not resolved in the onedimensional X-ray diffraction profile depicted in Figure 1. Experimental values of the WolfBragg distances dhkl corresponding to all diffraction peaks recorded for the iron powder by the Debye-Scherrer and Bragg-Brentano methods are listed in the table 1. There are also given in this table the dhkl values calculated for the iron crystal structure in the α-form (α-Fe) which has the cubic volume-centered lattice with a lattice parameter a = 0.286645 nm (space group Im3m). It is evident from the table 1 that all diffraction peaks, except the first weak peak, can be assigned to the α-Fe phase. The lattice parameter a = dhkl(h2+k2+l2)1/2 calculated for α-Fe as the average for all diffraction peaks recorded was found to be 0.286-0.287 nm for the DebyeScherrer method and 0.2866 nm for the Bragg-Brentano method. These experimental values of the lattice parameter are in excellent agreement with the literary value of a = 0.286645 nm for the α-Fe lattice. The first diffraction peak (dhkl = 0.2524 nm) can be assigned to Fe3O4 which has the most intense diffraction line at dhkl = 0.253 nm. Intensity of this Fe3O4 diffraction peak is very low, it is less than 2 % of the intensity of the first diffraction peak of α-Fe (Figure 2). So the content of Fe3O4 in the iron powder investigated is negligible.

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Figure 1. X-ray diffraction pattern of the superfine iron powder obtained by the Debye-Scherrer method (MoKα, the background scattering was subtracted).

Figure 2. X-ray diffraction pattern of the superfine iron powder obtained by the Bragg-Brentano method (CuKα, the background scattering was subtracted).

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Table 1. The Wolf-Bragg distances dhkl obtained for the superfine iron powder by the Debye-Scherrer and the Bragg-Brentano X-ray diffraction methods and dhkl values calculated for α-Fe № of diffraction line

dhkl, nm

Phase identification

experimental

calculated for α-Fe

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1 2 3 4 5 6 7 8 9 10 11 12

Debye-Scherrer method (MoKα) – 0.2023 0.1428 0.1168 0.1001 0.0905 0.0827 0.0766 0.0709 0.0681 0.0643 0.0565

Bragg-Brentano method (CuKα) 0.2524 0.20293 0.14323 0.11695 0.10132 0.09058 – – – – – –

– 0.20269 0.14332 0.11702 0.10134 0.09065 0.08275 0.07661 0.07166 0.06756 0.06410 0.05622

Fe3O4 α-Fe α-Fe α-Fe α-Fe α-Fe α-Fe α-Fe α-Fe α-Fe α-Fe α-Fe

The profiles of X-ray diffraction peaks for the Fe powder and Al foil are sown in Figure 3. For the Al foil the Kα1/Kα2 peaks are quite narrow and well resolved (Figure 3b). At the same time, for the Fe powder these peaks are much more broad and merge (Figure 3a). It is well known that broadening of X-ray diffraction peaks arise in the case of small crystal size. Another reason of diffraction peak broadening may be distortions of a crystal lattice due to the residual stress. But such residual stress seems to be improbable for the Fe powder investigated. We assumed that all physical broadening of X-ray diffraction peaks for the Fe powder is associated with a small crystal size and used the width of the Al foil X-ray diffraction peak as instrumental broadening. Under these assumptions we found the average crystal size for the Fe powder to be about 30 nm. Thus, our X-ray diffraction study of the superfine Fe powder has shown that the predominant phase in this powder is the crystalline α-Fe with the average crystal size about 30 nm and the lattice parameter a = 0.2866 nm that coincides with the standard value of this parameter for α-Fe. As a minor constituent (1-2%) of the powder investigated the crystalline Fe3O4 was revealed. The data, shown in Figure 4, indicate that the superfine iron powder has an effect on the structural characteristics of the lipid membrane bilayers. One can see that incubation of the erythrocyte membranes with the iron powder during 1 hour lead to a noticeable decrease of the surface microviscosity of the lipid bilayers (probe I) at all iron concentrations used (Figure 4A). The maximum decrease of the surface microviscosity (about 20-30 percent as compared with the control) was revealed for the iron concentrations of 10–1, 10–2, 10–7 and 10–9 mg/ml.

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Figure 3. The profiles of the X-ray diffraction peaks for the superfine iron powder (A) and aluminium foil (B) recorded with the small-angle X-ray scattering diffractometer (MoKα). Dots – experimental values, solid lines – approximation with the expression (1).

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Microviscosity, relative units

142

1,8 1,6 1,4 1,2 1,0 0,8 0,6 0,4 0,2 0,0 -1

-2

-3

-5

-7

-9

lg [Fe powder], mg/ml suspensuon

A

Microviscosity, relative units

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1,6 1,4 1,2 1,0 0,8 0,6 0,4 0,2 0,0 -1

-2

-3

-5

-7

-9

lg [Fe powder], mg/ml suspension

B Figure 4. Alterations of the erythrocyte membrane microviscosity after incubation with the superfine iron powder for 1 hour (A) and 24 hours (B). Dark columns – probe I, light columns – probe II.

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XRD Characterization of Superfine Fe Powder and EPR Study of Its Interaction…

143

For the deep lying lipid membrane domains (probe II) the microviscosity increased after such incubation in the case of the iron concentrations of 10–1 and 10–9 mg/ml for 25 and 40 percent respectively. Quite another pattern was observed for the prolonged (24 hours) influence of the iron powder on erythrocytes (Figure 4B). In the sites of the localization of the probe I the microviscosity reduced for the Fe concentrations of 10–2 and 10-5 mg/ml, but increased for 20 percent in the case of the Fe concentration of 10–9 mg/ml. In the sites of the localization of the probe II the microviscosity increased at Fe concentrations of 10–3 and 10–7 mg/ml as much as for 25 and 20 percent. Results obtained showed that the iron nanoparticles had significantly changed the erythrocyte membrane microviscosity as early as in 1 hour of incubation (Figure 4). The increase of the incubation time up to 24 hours did not lead to the effect growth. That’s why we investigated the interaction of the iron nanopowder with liposome membranes only in 1 hour after incubation. It was concluded that in the sites of the localization of the probe I the microviscosity of liposome membranes increased for 20 percent at the Fe powder concentrations of 10–2 and 10–5 mg/ml but remained almost constant at other Fe concentrations (Figure 5). The more pronounced microviscosity changes were detected for the sites of the probe II. The microviscosity for these sites increased for 40 percent after addition of the iron powder at concentration of 10–2 mg/ml and reduced for 20-30 percent in the case of iron concentrations of 10–7-10–9 mg/ml. Earlier it was shown that in the medium containing glycine no detectible iron powder solubilization was observed in 12 hours and only 8 percent of inserted iron passed into the solution in 36 hours of incubation [11]. According to the work [12], the main parameters that influence the speed of cation transition from nanoparticles to the surrounding solution are the dimensions of nanoparticles and their composition. In this work it was shown that for the nanoparticles with dimensions in the range of 100-400 nm the amount and the speed of cation transition to the surrounding solution decreased with the decreasing of the particle dimensions. Taking into account the results of the work [12] and the small crystal size of the iron powder used in our study (~30 nm) it can be assumed that for this iron powder the cation transition to the solvent is negligibly small. Therefore, it seems to be very possible that mechanisms of interactions of this powder with lipid membranes are mainly associated with the whole iron nanoparticles but not with cations in the solution. This assumption is supported by the results of our EPR experiments in which we detected the interaction of the iron powder with lipid membranes in 1 hour of incubation of their mixture (Figs. 4A, 5). So, in our work the superfine iron powder was characterized by the X-ray diffraction method. It was shown that this powder consisted predominantly of the crystalline α-Fe phase with the crystall lattice parameter a = 0.2866 nm and the average crystal size ~30 nm. The influence of the superfine iron powder on the lipid bilayer microviscosity of erythrocyte membranes and liposome membranes in model experiments was revealed. It was found that the effect of the iron powder on the lipid membrane microviscosity depend on the powder concentration, the time of the powder interaction with membranes and the type of these lipid membranes. It was shown that the structural changes of erythrocyte membranes and liposome membranes took place under the action of the nanodispersed iron both in membrane superficial lipid sites and in deep-located near-protein membrane domains.

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Microviscosity, relative units

144

1,8 1,6 1,4 1,2 1,0 0,8 0,6 0,4 0,2 0,0 -1

-2

-3

-5

-7

-9

lg [Fe powder], mg/ml suspension

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Figure 5. Alterations of the liposome membrane microviscosity after incubation with the superfine iron powder for 1 hour. Dark columns – probe I, light columns – probe II.

It was concluded that the superfine iron powder at the ultra low concentrations had a more pronounced effect on the lipid membrane microviscosity than at high concentrations. Taking into account the expected widespread occurrence of nanomaterials and a risk for people of direct contact with superfine particles, a further detailed study of the mechanisms of lipid membranes interaction with nanoparticles seems to be extremely important for the development of the methods of estimation of the nanoparticle’s safety for living systems.

REFERENCES [1] [2] [3] [4] [5] [6]

Yu.I. Golovin. Introduction to nanotechnology. Moscow, Machinostroenie, 2007. 496 p. (in Russian). A.I. Gusev. Nanomaterials, nanostructures, nanotechnologies. Moscow, Fizmatlit, 2007. 416 p. (in Russian). Yu. I. Fedorov, E.B. Burlakova, I.P. Olichovskaya. DAN USSR, 1979. V. 248. P. 1277 (in Russian). L.D. Fatkullina, G.F. Ivanenko, L.A. Goncharov et al. Annual Proceed. 12 Internat. Plessk. Confer of Magnetic Fluid., Ples. 2006. P. 340 (in Russian). O.N. Leontyeva, I.V. Tregubova, M.I. Alumov. Physics and chemistry of methods of metal treatment. 1993. № 5. P. 156. (in Russian). R. W. James. Optical Principles of the Diffraction of X-rays. London, Bell and Hyman, 1948.

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Cheremukina GA, Chernenko SP, Ivanov AB, Pashekhonov VD, Smykov LP, Zanevsky YuV. Isotopenpraxis, 1990. V. 26. P. 547-549. [8] G.V. Archipova, E.B. Burlakova, A.V. Krivandin et al. Neurochimiya, 1996. V. 13. P. 128 (in Russian). [9] A.N. Kuznetsov. Method of spine probes. Moscow, Nauka, 1976. 209 p. (in Russian). [10] A.N. Goloschapov, E.B. Burlakova. Biofizica, 1975. V. 20. P. 816 (in Russian). [11] N.N. Gluschenko, I.P. Olichovskaya, T.V. Pleteneva et al. Izvestiya AN USSR, ser. biological, 1989. № 3. P. 415 (in Russian). [12] A.Yu. Godumchuk, K.I. Midander, A.A. Ladova. Proceedings of Conference «Nano 2007», Novosibirsk, 2007. P. 329 (in Russian).

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[7]

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In: Handbook of Chemistry, Biochemistry … Editors: L. N. Shishkina et al., pp. 147-156

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Chapter 15

THE QUENCHING OF INTRINSIC FLUORESCENCE OF SARCOPLASMIC RETICULUM FOR THE LIPIDPROTEIN INTERRELATIONSHIP DETERMINATION O.M. Alekseeva*,1, Yu.A. Kim2, V.A. Rykov2, and N.L.Vekshin2 1

Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, ul. Kosygina, 4, Moscow, 119334, Russia 2 Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Russia

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ABSTRACT This investigation deals with the structural properties of sarcoplasmic reticulum (SR) membranes. SR is the main Ca2+-pool in the rabbit skeletal muscle. The principal Ca2+pool functions of the vesicles of fragmented sarcoplasmic reticulum were greatly varied subject to the source of the vesicles’ origin. The heavy vesicles are the fragmented terminal cistern SR, which mainly released Ca2+. The light ones are the fragmented longitudinal tubules SR, which mainly pumped Ca2+. All tested vesicles have some similar and some different structural and functional characteristics that depend on the arrangements of their lipid and protein molecules in the membranes. The lipid-protein relationships were tested with the tryptophan fluorescence quenchers.

Keywords: sarcoplasmic reticulum; Ca2+-ATPase; ryanodine receptor; tryptophan; fluorescence; quenching.

ABBREVIATIONS DTT - dithiothreitol; PMSF - phenylmethylsulfonyl fluoride; *

Fax: (495) 137-41-01; E-mail: [email protected]

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EGTA - ethylene glycol-bis[β-aminoethylether]-N,N,N’,N’-tetraacetic acid; SR – sarcoplasmic reticulum; FSR - fragmented SR; TC – fragmented terminal cisterns of SR; LT – fragmented longitudinal tubules of SR; RyR - ryanodine receptor; ANS1anilinonaphtaline-8-sulphonate; Trp – tryptophan; λex, λem – wavelengths excitation and emission, respectively; PAAG – polyacrylamide gel. Ca/ATP- effect of Ca2+-accumulation to the ATP-hydrolysis.

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INTRODUCTION The membranes of the sarcoplasmic reticulum (SR) consist of the numerous cytoplasmic and luminal proteins, which are associated with membrane, and two large integral proteins that run through the membrane and formed a few transmembrane loops. These proteins are Ca2+ATPase that pumped Ca2+, and ryanodine receptor (RyR) that released Ca2+ (the marker of this function is the sensitivity to the plant methylxanthine—caffeine). Both of these proteins are characterized by large hydrophobic regions with hydrophobic amino acid residues. Ca2+ATPase has high content of tryptophan (Trp) – 18 [1]. 17 Trp faced to the lipid hydrophobic part of the membrane. Trp anchor the protein globule at the membrane. 1 Trp was found at the protein global interior. The ryanodine receptor contains 48 Trp [2]. But the relative content of RyR/Ca2+-ATPase at the SR is very small [3]. Thus the Trp of Ca2+-ATPase have the largest importance. We investigated the fragmented SR — FSR. FSR formed the closed vesicles with characteristic features. The efficiency of Ca2+-accumulation and Ca2+-releasing by the vesicles of the fragmented sarcoplasmic reticulum was greatly varied independent of source of the vesicles from which they originated. The heavy vesicles are the fragmented terminal cistern, light ones are the fragmented longitudinal tubules of the reticulum. The heavy vesicles have RyR and Ca2+-ATPase, light ones have Ca2+-ATPase only. We cleaned the heavy and light fractions by additional purification. Its functional properties and sensitivity or insensitivity to the caffeine were kept. Our fractions were called LT and TC. The active integral proteins with annular lipids were purificated from both factions. These preparations were represented by the closed vesicles, too. All four tested types of vesicles have some similar morphological characteristics. But the arrangements of its lipid and protein molecules in the membranes were significantly different. The lipid-protein relationships were tested with the tryptophan fluorescence quenchers: cesium, pyrene, anthracene, ANS, titanium yellow, trypan red, auramine-00, pyronine-B, coryphosphine, Dis-C3-5. These substances are localized at different loci of membrane and have varied mechanisms of action [4].

MATERIALS AND METHODS The materials: KCl, KH2PO4 (Merck); histidine, imidasol, caffeine (Merck); NaCl, MgCl2 (Merck); DTT (Serva); glycerol (Serva); PAAG (Serva); CaCl2 (Merck); sucrose (Merck);

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EGTA (Serva); PMSF (Helicon); cesium, pyrene, anthracene, ANS, titanium yellow, trypan red, auramine-00, pyronine-B, coryphosphine, Dis-C3-5 (Molecular probes). The standard methods of isolation and the purification of FSR were modified: the first step was realized in the presence of DTT, PMSF and 10 mM caffeine and with addition of aggregation stage in glycerine medium at the final one [5-7]. The protein concentration was determined by the fast method [8]. The protein content was determined by polyacrylamide gel electrophoresis method [9].The lipid content was determined by chromatographic method of extracted lipids [10]. The preparations of Ca2+ -ATPase from LT and TC FSR were resulted by [11]. Electron-microscope investigations were performed by ultrathin section and negative staining methods. Efficiency of Са2+-accumulation by FSR, and by preparations of Ca2+-ATPase from LT and TC FSR, and sensitivity to the caffeine were recorded by pH-metric method [12]. FSR vesicles (3–4 mkg protein/ml) were incubated in 4 ml medium, contained 5mM sodium oxalate, 0,1M NaCl, 4 mM MgCl2, 2,5 mM imidasol (pH 6,8) 37o C with intensive mixing. Reaction was stimulated by additions of 80 nmoles CaCl2. The function of Са2+-accumulation by Ca2+-ATPase preparations was reconstructed by method [13]. The conditions for measurements of fluorescence quenching: 1mkM of FSR protein, 4 mM MgCl2, 2,5 mM imidazole, 100 mM NaCl, pH 7,0, 20o C. The quartz cells (1 sm.) were used. “Perkin-Elmer MPF-44B”, “SLM-480” [14].

RESULTS AND DISCUSSION

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Electron-microscope investigation of 3 FSR fractions, which prepared with different degree of purification (Figure 1), showed that light fractions were represented by the smooth bubbles, heavy ones by the bubbles with the caps, and junctional bubbles—dyads, associated with fragments of T-tubules.

Figure 1. Ultrathin section of 3 types of FSR preparations: dyads FSR, heavy FSR, light FSR.

Our methodical task was the purification of crude fractions of vesicles from the mature mitochondrial units, and then purification of FSR from each other, and then from associated proteins. The main specific functions must be kept during these treatments. Dyad and heavy FSR had grateful sensitivity to the caffeine (60–80%). Light FSR had small sensitivity (10– 15%). We added the procedure of aggregation at the last stage of cleaning of LT and TC FSR from contaminations. As the result, we obtained two super cleaned fractions: LT without any fragments of TC, and TC without any fragments of T-tubules. TC FSR had big sensitivity to

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the caffeine (60–70%). LT FSR had no sensitivity to the caffeine. The next step was the purification of Ca2+-ATPase from fragmented terminal cisterns and longitudinal tubules. We used the method [15] with our modifications. The treatment FSR by the salt solutions with the high ionic strange under holeat allowed us to perforated the vesicles and shaded the luminal and cytoplasm surface of membrane from associated and peripheral proteins. Then, this solution was removed, and membranes were washed from holeat. Such preparations have integral proteins only in their lipid-protein vesicles. Preparation Ca2+ -ATPase from TC or LT have single ATPase activity. The Ca2+-accumulating and Ca2+-releasing activities were absent, because these vesicles had the high permeability for ions. We reconstructed Ca2+ accumulating activity by mixed phospholipids/hydrocarbon micelles adding to the vesicles at the medium before the activity recording. The best effect was under the dipalmitoillecithin + heptane additions under the relation protein/lipid/hydrocarbon = 1:0,45:0,18. The perforated vesicles were fusion with mixed phospholipids/hydrocarbon micelles. The high permeability of membranes for ions was decreased greatly. The Ca2+-accumulating function was formed. Ca/ATP = 0,55. And Ca2+-releasing function was formed too. If the procedures were carried out under the physiological temperature, the preparations of Ca2+-ATPase from LT had not some sensitivity to the caffeine, and Ca2+-ATPase preparations from TC FSR had small sensitivity to the caffeine (10–15%).

Figure 2. Negative staining of 4 types of preparations: TC - fragmented terminal cisterns; LT – fragmented longitudinal tubules; Ca2+ -ATPase TC Ca2+ -ATPase from fragmented terminal cisterns; Ca2+ -ATPase LT - Ca2+ -ATPase from fragmented longitudinal tubules.

The morphological characteristics of TC, LT and Ca2+-ATPase preparations from FSR are shown in Figure 2. All samples are represented by closed vesicles with bylayer circle

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membranes with luminal space, without any caps and dyads; crista-contained mitochondrial units are absent. The characteristic of its content was presented below. Lipid content was similar for PT and LT FSR (Figure 3). The phosphatidylcholine and phosphatidylethanolamine were the predominant phospholipids.

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Figure 3. Densitograms of chromatograms of lipids, extracted from FSR (TC) - terminal cisterns; (LT) longitudinal tubules; 1-Phosphatidylcholine; 2-Phosphatidylethanolamine.

Figure 4. Densitograms of polyacrylamide gel electrophoregrams of LT and TC FSR; 1 - Ca2+-ATPase; 2- calsequestrin.

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Protein content was standard for the FSR: TC contained the Ca2+ -ATPase and calsequestrin predominantly. LT contained the Ca2+ -ATPase predominantly and lesser amount of calsequestrin (Figure 4).

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Figure 5. Densitograms of polyacrylamide gel electrophoregrams of Ca2+-ATPase preparations from LT and TC FSR.

Purified preparations Ca2+ -ATPase from LT and TC FSR had Ca2+-ATPase protein only (Figure 5). The main protein content and functional characteristics of the TC and LT FSR and Ca2+ATPase preparations from TC and LT are show at the table 1. We see clear correlation between the Ca2+-ATPase specific activity and Ca2+-ATPase content for the fractions in dependence of purification degree. Thus we obtain pure preparations with specific properties for next spectral investigations. The spectral characteristics of 4 samples were practically identical. The spectral shapes of TC, PT and its Ca2+-ATPase preparations are similar. The data that characterized the adsorption specters of 4 samples are presented at the Table 2. At the base of these data we may conclude that the Ca2+-ATPase-molecules made the major contribution to the UV adsorption of the samples. Thus we can investigate the types of the Ca2+-ATPase-molecules organization at the LT and TC membranes by the fluorescence methods. As we can see at the figure 6, the fluorescent spectrum and lifetime of excited state of tryptophan fluorescence have identical shapes and values of the lifetime were closed for all 4 tested samples. This is the additional reason for the comparison of protein molecules organization at the FSR membranes. Early we obtained any suggestions to differences of organization: the polarization and viscosity the light and heavy FSR, were not similar. And the values for heavy FSR were larger then for light [16].

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Table 1. The protein content and functional characteristics of the FSR membranes TC and LT, and Ca2+-ATPase preparations from TC and LT Type of vesicles

Ca2+-ATPase content (%)

Ca2+-ATPase specific activity***

Area of 100kD peak (relative value LT/TC)

TC LT Ca2+-ATPase from TC Ca2+-ATPase from LT

37* 40-50** 66* 70-75** 60* 80-85**

11+-1 19+-1 18+-0,5

1,6

Ca2+-ATPase specific activity (relative value LT/TC) 1,8

1,1

1,7

100* 90-95**

30+-1

Key: (*) - Ca2+-ATPase content, which calculated with aid Ca2+-ATPase specific activity. (100% specific activity = 30 mkmol Pi/mg protein min);(**) –content of protein component 100kD, which calculated with aid PAAG densitograms. (***)-Ca2+-ATPase specific activity mkmol Pi/mg protein min.

Table 2. The spectrophotometric analysis of the membranes LT and TC FSR and Ca2+ATPase preparations from TC and LT Sample

the optical D278: D262 0,72 0,72 0,70 0,72

densities D278: D262 0,32 0,35 0,36 0,38

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Ca2+-ATPase from LT Ca2+-ATPase from TC LT TC

Relation of D278: D262 1,05 1,05 1,02 1,04

Figure 6. Fluorescent spectrum and lifetime of excited state of tryptophan at the membranes LT and TC FSR and Ca2+ -ATPase preparations from LT and TC FSR. 1 - Ca2+ -ATPase preparations from LT; 2 - Ca2+ -ATPase preparations from TC; 3 - LT FSR; 4 - TC FSR; 5 – DL- tryptophan.

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At the table 3 the efficacy of quenching of tryptophan fluorescence at the membranes LT and TC FSR (% from native fluorescence) are present. Some substances were embedded to the polar areas of membranes (cesium, calcium, trypan red) and anothers quenchers located at the hydrophobic regions (pyrene, anthracene), and others quenchers are appeared in both areas. The types of action are different for these quenchers: ANS quench the Trp fluorescence because it transfers the energy excitation itself from neighboring Trp. Cesium is dynamic quencher, it hits with Trp. Pyrene is dynamic and static quencher. Some substances may change the protein conformation, in addition. But all quenchers showed the largest efficacy of quenching for the LT FSR and minimal – for TC FSR. These data suggested to the smaller availability of Tpr for various quenchers. It was possible that the protein globules were masked the superficial Trp by oligomerization. The membranes of LT and TC FSR had the similar charges at its surface, because there are not any correlations between efficacy of quenching and charge of substance.

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Table 3. The efficacy of quenching of tryptophan fluorescence at the membranes LT and TC FSR (% from native fluorescence) Substance

Concentration of substance (mkM)

Charge of substance

Cesium Calcium Pyrene Anthracene ANS Titanium yellow Trypan red Auramine-00 Pyronine-B Coryphosphine Dis-C3-5 Caffeine

50 000 500 7 5 20 1 1 4 4 5 10 500

+1 +2 0 0 -1 -3 -5 +1 +1 +1 +1 0

Efficacy of quenching (%) for LT FSR 13 0 65 29 66 29 32 8 9 27 52 0

Efficacy of quenching (%) for TC FSR 9 0 54 21 55 28 29 7 9 18 42 0

As shown at the table 4, the most value of ANS quenching of tryptophan fluorescence was obtained for the Ca2+-ATPase preparations from LT FSR. This fact suggests that the availability of Tpr is maximal at these membranes. The Tpr of Ca2+-ATPase at membranes of Ca2+-ATPase preparation from TC FSR begin available for fluorescence by ANS due to the purification’s treatment. This efficacy of quenching becomes similar to efficacy of quenching of LT FSR. These facts support to the high probability of different structural organization Ca2+-ATPase molecules at the membranes of TC SR.

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Table 4. The efficacy of quenching of tryptophan fluorescence by ANS at the membranes LT, TC FSR and Ca2+-ATPase preparations from LT,TC FSR (% from native fluorescence) Type of vesicles

Efficacy of quenching (%)

Ca2+-ATPase from LT Ca2+-ATPase from TC LT TC

51 48 48 35

Conditions: 0,1 mg/ml of FSR, 10 mkM ANS, λex 286nm, λem 330nm.

CONCLUSION Based on data, obtained by methods of purifications and reconstruction of ATPase activity with our modifications, and spectral analysis, we may suggest that the lipid-protein relationships and quarter structure of Ca2+-ATPase molecules in SR membranes are different independent of cell compartment, from which FSR originated. The organization Ca2+-ATPase molecules at TC membranes is in oligomer form and at LT membranes is in monomer form. It is in agreement with literature [11]. The authors are grateful to doctors V.B.Ritov, P.G.Komarov, B.Galushenko, S.S.Husian for valuable contributions to performing of the experiments and discussion of the results.

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REFERENCES D.H MacLennan., C.J. Brandl, B. Korszak, N.M.Green. Nature, 1985, V.316, P. 696700. [2] F. Zorzato, J. Fuji, K. Otsu, M. Phillips, N.M. Green, F.A Lai, G.Meissner, and D.H MacLennam. J. Biol. Chem. 1990, V.265 N.4, P.2244-2256. [3] M. Inui, and S. Fleischer. Methods in Enzymology 1988, V.157, P.490-505. [4] J.R. Lakowicz. Principles of fluorescence Spectroscopy. New York: Plenum Press, (1983)P. 38-389. [5] V.B.Ritov, N.B.Budina, and O.M.Vekshina. Bulletin experimentalnoy biologii i medizini. 1985. V.1. P. 53-54. [6] O.M Alekseeva., and V.B. Ritov. Biochimia. 1979. V. 44. P. 1582-1593. [7] N.Ikemoto, D.H. Kim, and B. Antoniu. Methods Enzymol. 1988. V.157. P. 469-480. [8] N.L. Vekshin, Analyt. Chim. Acta 1989.V.227, P.291-295. [9] U.K.Laemmmli. Nature 1970 V.227, P.680-685. [10] J.Folch, M.Loes, G.H.S. Stanley. J. Biol. Chem. 1957, V.226, P.497-509. [11] V.B. Ritov. Biochimia. 1971. V.36. P.393-399. [12] V.B.Ritov, N.S.Scherbakova. Bulletin experimentalnoy biologii i medizini. 1982. V.4. P. 21-23. [1]

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[13] V.B.Ritov, M.K. Murzachmethova. Dokl.Akad.Nauk USSR .. 1979. V.246. P. 12461249. [14] N.L. Vekshin. Photonics of biopolimers. Springer: Biological and Medical Physics Series, 2002. [15] Meissner G., Corner G.E., Fleischer S. Biochem.et Biophys. Acta, 1973, V.298, P.264269. [16] O.Vekshina, Yu.Kim, N.Vekshin. “Magic” calcium gradient for the operation of the sarcoplasmic reticulum. In: Progress in Biochemical Physics, Kinetics and Thermodinamics. Nova Science Publishers, New York, 2008. Ed. by G.E.Zaikov. P. 141-155.

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Chapter 16

NEW EQUIPMENT TO FIGHT INDUSTRIAL EMISSIONS R.R. Usmanova1 and G.E. Zaikov2* Ufa State Technical University of Aviation, Ufa, Bashkortostan, Russia *N.M. Emanuel Institute of Biochemical Physics, Moscow, Russia

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ABSTRACT The problem of clearing of gas emissions is actual now. The efficiency of gas purification can be raised at the expense of working out new more perfect designs of dedusters. In this article, new designs of wet dedusters of centrifugal and inertial action are considered. Constructive schemes have resulted. We give a description of the principle of how the devices work and investigate their performance in industrial conditions for the clearing of gas emissions. Commercial operation has shown that the developed devices provide high degree of clearing of gas.

Keywords: equipment, emissions, gas purification, commercial operation, conductive schemes.

INTRODUCTION Rapid development of the industry has led now to serious deterioration of ecological conditions. One of the sharpest problems is pollution of air pool by gas emissions of the industrial enterprises. The problem of clearing of gas emissions from chips a dust is one of actual in gas purification and for a long time is pushed in the foreground of experimental and theoretical researches.

1 12 Karl Marx street, Ufa 450000 Bashkortostan, Russia. [email protected]. 2 4 Kosygin street, Moscow 119334 Russia. [email protected]. Handbook of Chemistry, Biochemistry and Biology : New Frontiers, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

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One of the most prospective methods of increasing the efficiency of dust separation is wet clearing. This method is more complex and expensive when compared to dry clearing, but also more effective. The process of wet clearing of gas can be realized in devices of centrifugal type. Centrifugal devices are characterized by high efficiency, simplicity of design and low metal consumption. The process of mass transfer due to increase in speed of movement of phases allows us to intensify applications of the given type of equipment also. Now, centrifugal devices are starting to be introduced actively in the manufacture of diversified chemical products, in metallurgy, and also for resolving environmental problems. For clearing and cooling of the smoke gases departing from furnaces in the roasting of burden—kiln shop, the device applied is the bubbling-vortical device with an axial sprinkler [1]. The device is made at the Yoint–Stok Company "Soda", Sterlitamak. The device for wet clearing gas (figure 1) contains a cyclone (1), the cylindrical chamber (2) with an entrance pipe (3), a pipe of an overflow slime (4), in a slimecollector (5). The cylindrical chamber is supplied by the axial sprinkler (6) punched on all lengths by apertures for submission of the irrigating liquid. In the cylindrical chamber is established swirler (7) gas streams, representing four blades rigidly fastened to a sprinkler (6). Fastening of the cylindrical chamber is carried out by means of flanges (8) owing to what extent the bubbling – vortical device can be installed in the vent dust removal system with the purpose of economy of material means and the areas of the industrial premises. Upon installation of the device as a preliminary step of clearing, a stopper must be put on the vent. Introduction of such a system of gas purification has allowed an increase in the efficiency of dust separation to from 53 to 95 % in comparison with only using a cyclone of dry clearing. For the purpose of evaluating the performance of the bubbling-vortical device, we compared the basic indicators by efficiency of clearing. Results of the work of the new device have compared with results of industrial tests of the centrifugal-bubbling device (working out “mechanical engineering”, Novosibirsk) established on a gas purification line. In Table 1 the technical characteristics established in the tests are shown. Table 1. Technical characteristics of the bubbling-vortical device and centrifugalbubbling device Parametre

centrifugal-bubbling

bubbling-vortical

Diameter, m Height, m Weight, kg Productivity on cleared air, m3/s Average speed of gas, m/s Pressure losses in the device, Pa Factor of hydraulic resistance. Depression, mm. water Efficiency of devices, % Power inputs on clearing, kilowatt * h on 1000 м3 air

1.5 2.0 175 11.11÷20.38 7.91 2200 88 540 91 0.64

1.0 1.0 60 6.94÷11.27 10.59 500 12.10 260 95 0.18

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The bubbling-vortical device has smaller overall dimensions under identical conditions, which allows us to increase the efficiency of clearing to 95%. Hydraulic resistance of the system does not exceed 500 Pa. Power inputs on gas clearing in 3 times was less than in the centrifugal-bubbling device. The time of recovery of outlay of capital expenses for the introduction of the bubbling– vortical device with an axial sprinkler takes less than three months from installation of the device in the vent dust removal system With initial concentration of a dust up to 50 g/m3 at the sizes of particles more than 2 microns, the recommendation is to apply dedusters with great dispatch-inertial action for clearing gases.

Figure 1. Bubbling – vortical device with an axial sprinkler It is possible to carry to such devices roto – clone with adjustable blades [2,10].

The roto – clone device (figure 2) is characterized by the presence of several slot-hole channels, forming the top (1) and the bottom (2) blades. Dusty gas acts in an entrance branch pipe (3) in the top part of the device. Hitting the surface of a liquid, it changes direction and passes in the slot-hole channel formed by the blades. Owing to high speed of the movement, cleared gas grasps the top layer of a liquid and splits it up into drops and foam. After consecutive passage of all slot-hole channels, the gas passes the drop-catcher (4) and through a target branch pipe (5) rises into the atmosphere. The caught dust settles in the bunker and is periodically removed from the device.

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Figure 2. Roto – clone with adjustable blades.

The optimum mode of dust separation is provided with regulation of position of the bottom blades in relation to the top, that allows an increased efficiency of gas purification in a wide range of dust content in a gas stream. Research has been carried out on such roto – clone devices installed to catch the dust of barite from smoke gases of burden—kiln shop.

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The roto – clone had 3 slot-hole channels, the speed of gas in which went up to 23 m/s; thus hydraulic resistance did not exceed 1000 Pа. Working in such mode, the roto – clone provided efficiency of catching dust from entrance concentration 0.32 g/nm3 at a level of 93.5 %. The roto – clone device has proved to be reliable enough. The level of a liquid is steadily supported by a regulator. However, the system of a conclusion slime demands completion by replacement of a manual periodic unloading by an automatic procedure. The bubbling – vortical device is applied to clearing technological gases where there is a need for low – head dedusters of the wet type [3]. Such devices are recommended to be applied for clearing gases with initial dust concentration of up to 50 g/m3 with particle sizes no more than 2 microns—except if the foam liquids of a dust are made moist, sticky. The device contains a cylindrical chamber which contains the swirler of the gas stream, represented by a pair of crossed planes forming four blades, forming the flowing section. In the device before the swirler is the central atomizer, and in each flowing section after the swirler are peripheral atomizers. Trial tests of the device have been conducted at the Joint-Stock Company " Caustik ", Sterlitamak, on a line of clearing of smoke gases. Table 2. The chemical compound of a dust contained in waste gases of the roasting furnace

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Component CO2 CO NO H 2S SO2

Before clearing, mg/m3 59.40 61.34 172.21 48.43 11865.54

After clearing, mg/m3 7.20 6.20 52.72 13.26 4412.76

According to the developed technological scheme (figure 3), departing from the furnace of roasting of (1) gas (productivity 15000÷28000 m3/h). At temperature 560 °C acts in the bubbling – vortical device (2). Here on an irrigation 1–3% a solution of limy milk (pH=11.5÷12.5) move. Separated slime acts in a drum – slaker (3). Clarification and cooling of limy milk occurs in the filter-sediment bowl (4) from which it again moves on an irrigation. The cleared gas smoke exhauster (7) is thrown out into the atmosphere. Research studies have shown that the admissible residual maintenance of a dust in gases (200 mg/m3) is provided at hydraulic resistance of the device nearby 400 Pа. Gas is cooled up to 65 °С. The bubbling – vortical device works with regeneration of flow, as all recirculation in system, the liquid is used in the technological process.

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1- furnace roasting; 2 – bubbling – vortical device; 3 – drum – slaker; 4 – filter – slime pond; 5 – reservoir; 6,7 – drawing fan; 8 – pump.

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Figure 3. the Technological scheme.

The magnetic hydrocatcher is intended for clearing industrial gases from ferromagnetik to a part [4,7]. Such device contains the case, in the bottom part the filled liquid, with a branch pipe of input of the gas (1), supplied confuser (2), coaxially to which is established diffuser (3). Thus from the external party of the case the electromagnetic system (4) representing magnetic coils with a winding is placed. From the interior of the case the restrictive ring five (5), interfering spread the magnetic liquid (6) which has been filled in the inside of the case (figure 4) is established. Increase of efficiency in dust separation is caused by action on a stream of two forces: the force of inertia arising at progress of a gas stream, and the centrifugal force arising at rotation of a stream in a magnetic field. The device works as follows: The Case is filled with a dust removal liquid to level of the bottom basis confuser (2). The electromagnetic system (4) is connected, in magnetic coils the alternating voltage is created, thus in the case there is a rotating magnetic field. The magnetic liquid (6) is drawn by a field to an internal wall of the case and results in rotation. The dusty gas stream under the influence of centrifugal forces also results in a rotary motion. Rotating gaseous liquid, the mix passes between walls confuser (2) and diffuser (3), the effect of a pipe of Venturi is thus formed.

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Figure 4. Magnetic hydrocatcher.

The work of such a magnetic system provides optimum efficiency dust separation (95– 98.5 %) at fluctuations of pressure dust – aerial a stream. It is especially important that such a hydrocatcher effectively catches finely dispersed the particles which are most hazardous to a person’s health. In foundry shop of Yoint – Stock Company "Machine-tool plant", Sterlitamak, one of the most harmful production factors is the raised dust content of air. In connection with continuous growth of capacities receipt of a dust in a working zone increases at manufacturing forming mixes, knockouts and clearing casting. For clearing air departing from shot - blasting of the chamber as second (wet) steps of the clearing after a dry cyclone, a dynamic gas washer [5,9] is used. Installation consists of the electric motor (1), the driving wheel (3), two snails (2) and (5), the directing device (4), air lines (6) (for tap of a dust and return of air), a cyclone (8), dynamic gas washer (7), the bunker for dust (9) and a sediment bowl (10) (figure 5).

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Figure 5. Installation for clearing air.

The demanded degree of clearing is reached by change of speed of gas in a zone of washing by its torch of a sprayed liquid at the second (wet) step dust separation. The irrigating liquid acts in the device on an axial branch pipe in the form of flat radial jets that causes intensive contact of phases. Air acts from the first step of clearing in the device on a tangential branch pipe and starts to rotate in a floor of centrifugal forces. The irrigating liquid circulates through a sediment bowl on the closed contour and at achievement of demanded concentration arresting a dust by means of the pump again moves on an irrigation. Dust sources in foundry shops are forming materials (sand, clay), containing particles of a dust in the size less than 160 microns. After pouring of the form by metal, not only the quantity of a dust is changed, but also its disperse and a chemical compound. So, the dust formed at clearing a casting by tumbling, contains 60–70 % of particles in the size less than 10 microns. The research studies conducted to catch a forming dust have shown that at initial concentration of a dust to 9 g/nm3 efficiency of clearing constitutes 97–98 %. The curve of fractional efficiency dynamic gas washer is shownin figure 6.

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Figure 6. Fractional efficiency.

The time of recovery of outlay of capital expenses for introduction of a dynamic gas washer takes less than 1.5 years. Application of a similar installation in the branch of clearing casting has shown its reliability and has allowed us to lower the concentration of dust in the air.

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CONCLUSIONS 1) The analysis of parameters of how various clearing equipment work has shown that, for clearing industrial enterprises’ gas effluent from an atmosphere, most prospective are the wet dedusters with centrifugal action. 2) Results of the carried out(spent) research studies were the basis for the design and the installation of devices for clearing industrial emissions of gaseous and firm impurities. 3) Industrial introductions confirm the high technical - operating parameters of the developed devices for clearing industrial emissions.

REFERENCES [1] [2] [3]

Usmanova R.R., Panov A.K. Bubbler–vortical the device with axial sprinkler//the Application for the invention №2006113869 from 24.04.06. Usmanova R.R., Panov A.K. Roto–clone with adjustable blades//the Application for the invention № 2006123585 from 09.08.06. Usmanova R.R., Panov A.K. Bubbler–vortical the device// the Patent № 2182843 from 27.05.02., Bull. № 15.

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R.R. Usmanova and G.E. Zaikov

Usmanova R.R. Magnetic hydrocatcher//the Application for the invention № 2007120000 from 29.05.07. [5] Usmanova R.R. Dynamic gas washer//the Application for the invention № 2007120001 from 29.05.07 [6] Lain S. Brxder D. Sommerfeld M. Experimental and numerical studies of the hydrodynamics in a bubble column. Chemical Engineering Science, 1999, Vol. 54, p.p. 4913-4920. [7] Patent USA № 6707362 (16.03.04)Way and the device for magnetic processing of a liquid. Inventors: Adam, Les, Harley, Inc. (USA). Intern’l Class: B 01D 035/06, H01F 007/00. [8] Patent USA № 6730236 (04.05.04) Way and the device for liquid division. Inventors: Kouba, Gene Edward, Inc. (U.S.A). Intern’l Class: B 01D 017/038. [9] Usmanova R.R., Zaikov G.E., Zaikov V.G. Calculation of dust separation efficiency of new design dynamic gas washer // Journal of the Balkan Tribological Association, 2008, vol. 14 № 2, p.p. 247-251. [10] Usmanova R.R., Zaikov G.E., Panov A.K. New design roto-clone for clearing air from dust // Journal of the Balkan Tribological Association, 2007, Vol. 13, № 2, p.p. 257-259.

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[4]

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In: Handbook of Chemistry, Biochemistry … Editors: L. N. Shishkina et al., pp. 167-178

ISBN: 978-1-61209-425-0 © 2010 Nova Science Publishers, Inc.

Chapter 17

SYNTHESIS OF PEROXY OLIGOMERS BASED ON EPOXY COMPOUNDS USING TERT-BUTYL PEROXYMETHANOL Michael Bratychak1, Olena Shyshchak1, Mikhailo Bratychak∗12 and Olena Astakhova1 1

2

Department of Chemistry and Technology of Petroleum Department of Chemical Technologies of Plastic Masses Processing Lviv Polytechnic National University, Lviv, Ukraine

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ABSTRACT The possibility of peroxy oligomers production has been examined. Chemical modification of epoxy resins or telomerization of diepoxy compounds with tert-butyl peroxymethanol have been used for the synthesis. Reaction conditions have been determined. The synthesis procedure has been developed. The structure of synthesized peroxy oligomers has been confirmed by chemical analysis as well as IR- and PMRspectroscopy.

Keywords: chemical modification, telomerization, peroxide, oligomer, resin, epoxide, glycol, trifluorine boron etherate.

1. INTRODUCTION The oligomeric compounds containing labile –O–O– bonds in their structure are the sources of free radicals. Therefore they are used as the initiators for polymerization reactions of unsaturated compounds or crosslinking agents of polymeric mixtures [1]. The advantage of peroxy oligomers (POs) over low-molecular compounds with –O–O– bonds is their higher ∗ 12 St. Bandera str., 79013 Lviv, Ukraine,[email protected].

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safety during storage and usage [2]. At the same time, in both processes of linear and crosslinked polymers production, POs are used as initiating and curing agent. The monograph [3] and series of scientific papers [4-6] deal with the problem of PO production on the basis of polycondensation resins. The initial reagents for PO synthesis are epoxy resins. Hydroperoxides of aliphatic, aromatic and alkylaromatic rows are used as modifiers. Inorganic hydroxides, Lewis acids, quaternary ammonium salts and Crown-ethers {1, 3-7] may be catalysts of the process. Tert-butyl peroxymethanol (TBPM) by following formula was used for the PO synthesis:

HOCH 2OOC(CH 3)3 The TBPM molecule contains two reactive groups in its structure: –O–O– bond and primary hydroxyl group. Under mild conditions, the TBPM molecule with the hydroxyl group is able to react with the epoxy or methylol [8] group saving the peroxy group in its structure. It is an advantage for PO synthesis. The synthesis of peroxy oligomers in the presence of TBPM may be carried out by the following reactions: -

chemical modification of the epoxy resins by TBPM; telomerization of diepoxy compounds with glycols using TBPM as a telogen.

2. EXPERIMENTAL 2.1. Starting Reagents and their Purification Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

TBPM used in this work was synthesized by the following reaction: (CH 3)3COOH + CH 2O

(CH 3)3COOCH 2OH

A three-necked reactor equipped with a mechanical stirrer, backflow condenser and thermometer was loaded with 79.0 g (38% aq.solution) of formalin, 90.1 g of tert-butyl hydroperoxide and 0.51 g of zinc oxide. The mixture was mixed at room temperature for 1 h. After the synthesis, the organic layer was separated from the aqueous one and dried by the waterless Na2SO4. Then it was distilled at 320 K and residual pressure of 10 gPa. 102.2 g 20

20

(yield is 85.2 %) of TBPM with the following characteristics was obtained: nD 1.4180, d 4

0.9684, MR 30.92, (MR ca. 30.79). ED-20 epoxy resin is a product of 4,4’-dihydroxy-2,2-diphenylpropane (diphenylolpropane), (DPhP) condensation with epichlorohydrin. MEG-1, DEG-1 and TEG-1 resins are obtained on the basis of epichlorohydrin and ethylene glycol (EG), diethylene glycol (DEG) and triethylene glycol (TEG), correspondingly. All used resins were commercial ones. EG, DEG and TEG were distilled under vacuum. The main fraction was dried with sodium sulphate and redistilled. The main physico-chemical constants of obtained products were in agreement with literature data [9].

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DPhP was purified by recrystallization with toluene. Its melting point was 429 K (lit. mp 429-430 K [10]). 2,2-di-[4-(2,3-epoxy-1-propoxy)phenyl]propane (DEPPhP) was synthesized by the procedure described in [11]. After distillation at 433 K and 1 Pa the product had the following 25

25

characteristics: n D 1.5690 (lit. n D 1.5690 [11]), epoxy number (e.n.) 25.30 % (theoretical e.n. 25.29 %). 1,2-di-(2,3-epoxy-1-propoxy)ethane (DEPE) was obtained by the procedure described in 30

[12]. Its characteristics: bp 401 K/13.3 gPa, n D 1.4498, e.n. 49.40 %. The same characteristics are in literature [12]. Trifluorine boron etherate [BF3⋅(C2H5)2O] was purified by distillation. Its bp 428 K [9]. Tin tetrachloride (SnCl4) P.A. was used without additional purification. Organic solvents were purified by the procedure described in [9] and their characteristics were similar to those in the literature.

2.2. Analytical Methods The average molecular masses Mn of the oligomers were determined by cryoscopic [13] or isopietic methods. The active oxygen content [O]act for the compounds or oligomers was determined by iodometry. The epoxy number was determined using back titration of hydrochloric acid acetone solution by 0.1 N alkali solution. Methylol groups (–CH2OH groups) and free formaldehyde were determined by the procedure described in [12].

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2.3. Spectral Methods Infrared spectra (IR) were obtained using a dispersive Perkin-Elmer apparatus with the relevant absorption range in 4000–400 cm–1 region. Proton magnetic resonance 1H-NMR spectra were recorded by the BS-487c spectrometer of Tesla, Brno, Czech Republic, at the frequency ν = 80 MHz in carbon tetrachloride. Hexamethyldisiloxane was used as an internal standard. The chemical displacements of group signals were determined by evaluating positions of symmetry centers of these signals.

2.4. Experimental Procedure 2.4.1. The Procedure for Determination of Synthesis Conditions for Peroxy Oligomers by Chemical Modification PO synthesis was investigated in the reactor equipped with mechanical stirrer, thermometer and reflux cooler filled with calcium chloride. Epoxy resin, TBPM and anhydrous benzene were loaded in such amounts that concentration of epoxy groups in the mixture was within the range of 0.4–0.6 g-eq/l. The reaction mass was heated till necessary temperature (303, 313 or 323 K) and trifluorine boron etherate was added while stirring. Samples by volume of 0.5 ml were withdrawn every definite intervals. The catalyst was

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neutralized by 1 ml of 0.1N solution of sodium hydroxide. 10 ml of acetone mixture (40 ml of acetone and 1 ml of hydrochloric acid) was added to the sample. The blank test was prepaid in a similar way. The catalyst concentration was determined by sample titration with 0.1N solution of sodium hydroxide. Obtained solutions were sustained for 2 h and concentration of epoxy groups were determined by following formula:

[C ]ep =

[Vx − (Vp + 1) − Vk ] ⋅ N ⋅ K Vs

(1)

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where Vx is a volume of 0.1 N solution of sodium hydroxide necessary for titration of blank test, ml; Vp is a volume of 0.1 N solution of sodium hydroxide necessary for titration of the sample, ml; Vk is a volume of 0.1 N solution of sodium hydroxide necessary for titration of the catalyst, ml; N is normality of the sodium hydroxide equals to 0.1; K is correction coefficient of the 0.1 N sodium hydroxide solution; Vs is a sample volume equals to 0.5 ml.

2.4.2. The Procedure for Determination of Synthesis Conditions for Peroxy Oligomers by Telomerization The synthesis of peroxy oligomers by telomerization was carried out in a three-necked reactor equipped with a mechanical stirrer, thermometer and funnel. Ethylene glycol, TBPM, anhydrous chloroform and catalyst were loaded into the reactor. The solution of 1,2-di (2,3 epoxy-1-propoxy)ethane in anhydrous chloroform was added to the mixture at 293–323 K during 1–4 hours. Then the mixture was sustained during 10–30 minutes. The reacting mass was cooled to the room temperature and neutralized by 5%-aqueous solution of the alkali. The obtained salt was separated, the organic layer was washed by water and vacuumized at 323– 328 K and residual pressure 1–2 gPa till the mass became constant. Synthesized oligomers were analyzed to determine the content of peroxy and epoxy groups. The molecular mass and functionality on the basis of end –O–O– bonds were determined. The functionality (f) of synthesized oligomers was calculated by the formula:

f =

M M eq

where M eq = M fg ⋅ 100

M

(2)

C fg

and M fg are the molecular masses of peroxy oligomer and functional group,

correspondingly; Cfg is the concentration (mass %) of the functional groups in oligomer.

3. RESULTS AND DISCUSSION 3.1. Obtaining of Peroxy Oligomers by Chemical Modification The synthesis of PO by chemical modification can be expressed by flowing equation:

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Synthesis of Peroxy Oligomers Based on Epoxy Compounds…

CH 2

CH 2 + HOCH 2OOC(CH 3)3

CHRCH

O

O

R' RCH

171

CH 2OCH 2OOC(CH 3)3

OH

(3)

where R is crosslinking fragment of the ED-20, MEG-1, DEG-1 or TEG-1

R' = CH 2 O

(CH 3)3COOCH 2CH

CH or

OH

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Investigations concerning the effect of initial reagents ratio, temperature and catalyst amount on the reaction (3) proceeding were carried out to determine the main kinetic regularities of PO obtaining on the basis of epoxy resins and TBPM in the presence of trifluorine boron etherate. The reaction was studied at 303, 313 and 323 K taking ED-20 resin in the anhydrous benzene as an example. The TBPM content was 0.5, 1.3 and 4.0 mol to calculate for one epoxy group. The catalyst concentration was 3.80⋅10-4, 5.63⋅10-4, 11.30⋅10-4 and 16.90⋅10-4 mol/l. The reaction rate was controlled using formula (1) by the change of epoxy group concentration. The procedure of kinetic investigations is represented in subsection 2.4.1 and obtained results – in Figs. 1–3. One can see from Figure1 that the increase of trifluorine boron etherate concentration ([Cat]) from 2.8⋅10-4 till 16.9⋅10-4 mol/l increases the reaction rate. At the same time the catalyst concentration more than 11.3⋅10-4 mol/l results in partial polymerization by epoxy groups. The precipitation of polymeric products indicates this fact.

Figure 1. Kinetic curves of epoxy groups concentration v. reaction time for the reaction of ED-20 epoxy resin with TBPM at 313 K and initial concentrations of: [EG]0 = 8.5⋅10-2 mol/l, [TBPM] = 54.7⋅10-2 mol/l and [Cat]0 = 2.82⋅10-4 (1), 5.63⋅10-4 (2), 11.3⋅10-4 (3) and 16.9⋅10-4 (4) mol/l.

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Figure 2. Kinetic curves of epoxy groups concentration v. reaction time for the reaction of ED-20 epoxy resin with TBPM at 313 K and initial concentrations of: [EG]0 = 8.5⋅10-2 mol/l, [Cat]0 = 11.3⋅10-4 mol/l and [TBPM]0 = 13.2⋅10-2 (1), 35.3⋅10-2 (2), 54.7⋅10-2 (3) and 67.6⋅10-2 (4) mol/l

Figure 3. Kinetic curves of epoxy groups concentration v. reaction time for the reaction of ED-20 epoxy resin with TBPM at initial concentrations of: [EG]0 = 8.5⋅10-2 mol/l, [TBPM] = 54.7⋅10-2 mol/l and [Cat]0 = 11.3⋅10-mol/l. Temperature: 303 (1), 313 (2) and 323 (3) K

The change of TBPM concentration from 13.2⋅10-2 to 67.6⋅10-2 mol/l also increases the reaction rate (Figure 2). Moreover, at the peroxide concentrations of 54.7⋅10-2 and 67.6⋅10-2 mol/l the change of epoxy groups concentration is practically the same. Therefore, the effect of temperature on the reaction proceeding was examined at TBPM concentration equals to 54.7⋅10-2 mol/l (Figure 3). As it was expected, the temperature growth increases the reaction rate. The temperature of 313 K was chosen as an optimal temperature. We have established following optimal conditions for the PO synthesis by chemical modification: process temperature is 313 K, reaction time is 2 h, TBPM amount is 6 moles and catalyst amount is 1.8⋅10-2 moles to calculate for 1 epoxy group of initial resin.

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.

Results so obtained were used for the synthesis of PO-I–PO-IV peroxy oligomers (see subsection 3.3.1). The characteristics of PO-I, PO-II, PO-III and PO-IV oligomers are represented in Table 1. Table 1. Characteristics of initial epoxy resins and PO on their bases synthesized by chemical modification Initial epoxy resin Resin Mn symbols ED-20 390 DEG-1 290 240 MEG-1 370 TEG-1

PO characteristics e.n., % 20.7 26.8 26.5 19.8

PO symbols PO-I PO-II PO-III PO-IV

Mn

[Oact], %

Yield, %

520 390 340 440

2.7 3.2 3.7 3.4

86.5 89.0 88.5 88.0

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Note: synthesized PO do not contain epoxy groups.

PO-I–PO-IV oligomers are stable, soluble in acetone, dioxane, chloroform and other organic solvents, viscous and light-yellow compounds. The structures of synthesized oligomers are confirmed by IR- and PMR-spectroscopy. Absorption bands at 910 cm-1 are absent in the IR-spectra of these oligomers. This fact indicates the absence of epoxy groups in the compounds that is adjusted with the results represented in Table 1. Data of PMRspectroscopy also indicate the absence of epoxy groups. Protons signals in the area of 2.75– 3.30 ppm were not found in the PMR-spectra of synthesized oligomers. At the same time there are weak absorption bands at 880–830 cm–1 in the IR-spectra, typical for the stretching vibrations of –O–O– bonds, there is also the doublet of gem-dimethyl vibrations at 1380 and 1360 cm–1 relating to the (CH3)3C-group and indicating the presence of peroxy groups in the oligomer molecules. The presence of –O–O– bonds in the compounds is also verified by PMR-spectroscopy. Protons signals of (CH3)3C-group introduced into oligomer molecule by the TBPM were detected in the area of 1.13–1.15 ppm. The presence of hydroxy groups formed by the opening of epoxy ring in the epoxy resins was confirmed by the IR- and PMR-spectroscopy. The wide absorption band at 3400–3300 cm–1 was detected in the IR-spectra and protons signals at 3.8– 4.0 ppm – in PMR-spectra, which are able to move towards upper field at the heating till 313 K. The presence of etheric bonds is confirmed by the protons signals in the area of 3.7– 4.3 ppm and absorption band in the IR-spectra at 1100 cm–1.

3.2. Obtaining of Peroxy Oligomers by Telomerization The synthesis of peroxy oligomers by telomerization using TBPM as a telogen may be expressed by following equation:

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Michael Bratychak, Olena Shyshchak, Mikhailo Bratychak et al. 2 CH 2 CHRCH CH 2 + HOR'OH + 2HOCH 2OOC(CH 3)3 O O (CH 3)3COOCH 2OCH 2CHRCHCH 2 OR'OCH 2CHRCHCH 2 OCH 2OOC(CH 3)3 OH OH

OH OH

n

(4)

where R = –CH2OCH2CH2OCH2– or –CH2OC6H4C(CH3)2C6H4OCH2–; R’ = –CH2CH2–, –CH2CH2OCH2CH2–, –CH2CH2OCH2CH2OCH2CH2– C6H4C(CH3)2C6H4; n = 0–3

or

–

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The initial reagents for PO synthesis were DEPE, DEPPhP, EG, DEG, TEG and DPhP. The optimal conditions were determined using interaction between DEPE and EG as an example. The effect of initial reagents ratio, catalyst nature and amount, temperature and reaction time on the characteristics of synthesized oligomers was studied at 293, 303, 313 and 323 K. DEPE:EG:TBPM molar ratio was 2.0:1.0: (1.5–4.0). Oligomers synthesis was carried out in the presence of 10 mas % of the catalyst, calculated from TBPM amount in the medium of anhydrous chloroform. The experimental methodic is described in subsection 2.4.2. Obtained results are represented in Figs. 4 and 5 and Tables 2 and 3. We can see from Figure 4 the greatest functionality is achieved at DEPE:EG:TBPM molar ratio equals to 2:1:2. The further increase of TBPM amount decreases the PO functionality, in spite of the general increase of active oxygen content.

Figure 4. Dependence of active oxygen content (1), molecular mass (2) and functionality (3) upon TBPM content in the initial mixture at 303 K and reaction time 1.5 h

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Figure 5. Dependence of active oxygen content (1-4) and epoxy groups (5-7) concentration in oligomer upon reaction time at 293 (1, 5), 303 (2, 6), 313 (3, 7) and 323 (4) K

Table 2. PO characteristics

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Temperature, K 293 303 313 323

Mn 920 1100 1400 960

[Oact], % 2.5 2.3 2.3 2.8

e.n., % 13.4 5.0 absent absent

Functionality 1.4 1.5 2.0 1.7

Table 3. The dependence of functional groups content in PO on the catalyst nature and amount Catalyst BF3(C2H5)2O BF3(C2H5)2O SnCl4 BF3(C2H5)2O BF3(C2H5)2O SnCl4

Catalyst amount (% from TBPM amount) 5 10 10 15 20 20

[Oact], %

e.n., %

2.2 2.3 1.8 2.2 2.3 1.7

1.4 absent 15.0 absent absent 5.0

Note: Reaction temperature is 313 K, reaction time is 1 h, DEPE:EG:TBPM ratio equals to 2:1:2.

Investigating the effect of temperature and reaction time on the oligomer characteristics we have found that formation of oligomeric chains at optimum ratio of initial reagents and 293 K (Figure 5 and Table 2) takes more than 4 h. At the same time at 303 K it lasts only 3 h. The increase of temperature prior to 313 K and higher considerably increases the reaction rate. The confirmative fact is absence of epoxy groups in oligomers synthesized for 1 h. At the same

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time epoxy groups concentration in PO synthesized for 0.5 h is 0.74 % at 313 K and 0.49 % at 323 K. The constancy of peroxy group content in oligomers (Figure 5) indicates the completion of oligomeric molecule formation at 313-323 K. Comparing the experimental results concerning the functionality of obtained oligomers one can see (Table 2) that bifunctional compounds may be obtained at 313 K. The increase of temperature prior to 323 K decreases the functionality due to side reactions, in particular the polymerization by epoxy groups in the presence of trifluorine boron etherate. As a result the ratio between functional groups participating in the PO formation failures and hydroxyl groups amount in the reaction mixture increases. Hence, PO molecular mass and functionality decrease as we can see from Figure 4. Taking into consideration the amount and nature of the catalyst it was established (Table 3) that trifluorine boron etherate has the highest catalytic activity in amount of 10 mas % from TBPM mass. Thus the optimal conditions for PO synthesis using TBPM as a telogen have been determined: DEPE:EG:TBPM ratio (mol) is 2:1:2, trifluorine boron etherate amount is 10 mas % from TBPM amount, reaction temperature is 313 K, reaction time is 1 h. Obtained results were the bases for the development of PO synthesis procedure using DEPE and DEPPhP. DPhP, DEG and TEG, as well as EG, were used for the synthesis as compounds containing mobile hydrogen atom. Synthesis procedures are presented in Subsection 3.4.2 and PO characteristics are in Table 4. Oligomers PO-V–PO-X are viscous colorless compounds which are stable at storing and soluble in organic solvents.

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Table 4.Characteristics of PO synthesized by telomerization Initial compounds Epoxy Monomer compound DEPE EG DEPE DPhP DEPPhP EG DEPPhP DPhP DEPE DEG DEPE TEG

PO symbols PO-V PO-VI PO-VII PO-VIII PO-IX PO-X

PO characteristics Mn

[Oact], %

Functionality

1400 770 880 930 1600 1000

2.3 1.8 2.4 2.0 1.8 1.9

2.0 0.9 1.3 1.1 1.9 1.2

Yield, % 91 85 89 83 90 87

Notes: 1. Epoxy groups in PO are absent. 2. Mn was determined by cryoscopy in dioxane

Taking into account IR- and PMR-spectra of PO-V–PO-X oligomers, it was established that absence of absorption band at 910 cm-1 in IR-spectra and proton signals in the area of 2.3– 3.1 ppm in PMR-spectra indicating absence of epoxy groups, analogously to the PO-I–PO-IV oligomers. The presence of peroxy groups in oligomers was confirmed by absorption bands at 870-880 cm-1 and doublet of gel-methyl vibrations at 1380 and 1360 cm-1, typical for (CH3)3COO– group. In PMR-spectra the presence of –O–O– bonds was confirmed by proton signals of (CH3)3COO– group, which was introduced into PO by TBPM telogen. The presence of etheric bonds was confirmed by proton signals in the area of 3.52–3.88 ppm in PMR-spectra and absorption band at 1110 cm-1 in IR-spectra. Hydrohyl groups in synthesized PO were formed due to the opening of epoxy ring and wide absorption band at 3400-3350 cm-1 in IR-

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spectra and proton signals at 4.88-5.55 ppm in PMR-spectra able to shift towards high field at the heating to 313 K confirmed this fact.

3.4. Synthesis of Peroxy Oligomers

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3.4.1. Synthesis by Chemical Modification PO-I was synthesized in a three-necked reactor equipped with a mechanical stirrer, thermometer and funnel. 2 mol of trifluorine boron etherate was added to 40.5 g of the mixture (0.33 mol of TBPM and 75 ml of anhydrous benzene) at 274–278 K and intensive stirring. Then mixture consisting of 22.0 g of ED-20 epoxy resin and 65 ml of anhydrous benzene was added dropwise for 1.5 h under stirring. The reacting mass was sustained for 0.5 h, cooled to the room temperature and washed by 5%-aqueous solution of sodium hydroxide. The solvent was toped and the residue was vacuumed at 333–338 K and residual pressure 1–3 gPa till the mass became constant. 30 g of peroxy oligomer was obtained. PO-II oligomer was synthesized in an analogous way as PO-I using 19.0 g (0.158 mol) of TBPM, 1.2 ml of the catalyst and 9.0 g of DEG-I epoxy resin. 30 g of peroxy oligomer was obtained. PO-III oligomer was obtained in an analogous way as PO-I and PO-II using 18.0 g (0.15 mol) of TBPM, 1.2 ml of trifluorine boron etherate and 10.0 g of MEG-I epoxy resin. 15.4 g of peroxy oligomer was obtained. PO-IV oligomer was synthesized in an analogous way as PO-I, PO-II and PO-III using 21.6 g (0.18 mol) of TBPM, 1.4 ml the catalyst and 12.0 g of TEG-I epoxy resin. 16.4 g of peroxy oligomer was obtained. 3.4.2. Synthesis by Telomerization PO-V was synthesized in a three-necked reactor equipped with a mechanical stirrer, thermometer and funnel. 7.8 g (0.125 mol) of EG, 30.0 g (0.25 mol) of TBPM, 250 ml of anhydrous chloroform and 3.0 g of trifluorine boron etherate were loaded into the reactor. Then mixture consisting of 43.5 g (0.25 mol) of DEPE and 125 ml of anhydrous chloroform was added dropwise at 313 K for 1.0 h. The reacting mass was sustained for 10 min, cooled to the room temperature and neutralized by 5%-aqueous alkali solution. The solvent was toped and the residue was vacuumed at 323–328 K and residual pressure 1–3 gPa till the mass became constant. PO-VI oligomer was synthesized analogously to PO-V. 28.5 g (0.125 mol) of DPhP was used instead of EG. PO-VII oligomer was synthesized analogously to PO-V and PO-VI using 85.0 g (0.25 mol) of DEPPhP instead of DEPE. PO-VIII oligomer was synthesized analogously to PO-V, PO-VI and PO-VII using 85.0 g (0.25 mol) of DEPPhP. DPHP was used as a monomer in amount of 28.5 g (0.125 mol). PO-IX oligomer was synthesized analogously to PO-V, PO-VI, PO-VII and PO-VIII. DEPE was used as diepoxy component in amount of 43.5 g (0.25 mol). DEG was used as a monomer in amount of 13.3 g (0.125 mol). PO-X oligomer was synthesized in a analogous way as PO-V–PO-IX using 43.5 g (0.25 mol) of DEPE and 18.8 g (0.125 mol) of TEG.

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4. CONCLUSIONS 1. The presence of primary hydroxyl and labile peroxy reactive groups in tert-butyl peroxymethanol molecule allows us to use this compound for the synthesis of oligomers with– O–O– bonds on the basis of epoxy compounds. 2. Tert-butyl peroxymethanol may be a modifier of epoxy resins with their chemical modification or be a telogen with cationic telomerization of diepoxy compounds with compounds containing primary hydroxyl groups. 3. In order to substitute epoxy groups for peroxy ones it is necessary to carry out the synthesis at 313 K during 1.0–1.5 h using trifluorine boron etherate as a catalyst. It is necessary to carry out the synthesis of peroxy oligomers at the temperatures below 323 K to preserve the labile –O–O– bonds.

REFERENCES [1] [2] [3] [4] [5] [6]

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[7] [8] [9] [10] [11] [12] [13] [14]

Bratychak M. and Brostow W.: Polym. Eng. and Sci., 1999, 39, 1541. Abdel Azim A.: Polym. Eng. and Sci., 1996, 36, 2973. Bratychak M., Bratychak M. (junior). Peroxydni pohidni epoksydnyh smol. Lviv Polytechnic National University, 2006, 236. Bazyliak L., Bratychak M. and Brostow W.: Mater. Res. Innovat., 1999, 3, 132. Bazyliak L., Bratychak M. and Brostow W.: Mater. Res. Innovat., 2000, 3, 218. Bratychak M., Bratychak M. (junior), Brostow W., and Shyshchak O.: Mater. Res. Innovat., 2002, 6, 24. Bratychak M., Chervinskyy T., Gagin M., Gevus O. And Kinash N.: Ukr.Khim.Zh., 2005, 71, 50. Ellis B.: Chemistry and technology of epoxy resins. Blackie, Glasgow 1994. Knunyanc I. (Ed.): Khimichaskij encyklopedicheskij slovar. Sovetskaya encyclopediya, Moskwa 1983. Cohen S. and Haas H.: J. Amer. Chem. Soc., 1953, 75, 733. Paken A.: Epoksidnye soedineniya i epoksidnye smoly. Goskhimizdat, Leningrad 1962. Suhanova N. and Shuvalova L.: Lakokrasochynye Materialy i Ih Primememie, 1981, 4, 47. Toropceva A., Belgorodskaya K. and Bondarenko V.: Laboratornyj praktikum po khimii vysokomolekulyarnyh soedinenij. Khimiya, Leningrad 1972. Ivanov V. (Ed.): Rukovodstvo k prakticheskim rabotam po khimii polimerov. Izd-vo Leningradskogo universiteta, Leningrad 1982.

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In: Handbook of Chemistry, Biochemistry … Editors: L. N. Shishkina et al., pp. 179-189

ISBN: 978-1-61209-425-0 © 2010 Nova Science Publishers, Inc.

Chapter 18

KINETICS OF THE FERMENTATIVE REACTION OF H2O2 DECOMPOSITION UNDER THE ACTION OF CATALASE IN THE PRESENCE OF BIOSAS FOR THE STATIONARY STATE *

A. A. Turovsky1, * R. O. Khvorostetsky, ** L. I.Bazylyak2, and *** G. E.Zaikov3

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*

Chemistry and Biotechnology of Combustible Minerals Division; Physical Chemistry of Combustible Minerals Department; Institute of Physical–Organic Chemistry and Coal Chemistry named after L. M. Lytvynenko; National Academy of Science of Ukraine, Lviv–60, Ukraine ** Chemistry of Oxidizing Processes Division; Physical Chemistry of Combustible Minerals Department; Institute of Physical–Organic Chemistry and Coal Chemistry named after L. M. Lytvynenko; National Academy of Science of Ukraine Lviv, Ukraine *** Kinetics of Chemical and Biological Processes Division; Institute of Biochemical Physics named after N. N. Emanuel; Russian Academy of Sciences Moscow, Russia

ABSTRACT The fermentative stationary kinetics of hydrogen peroxide decomposition under the action of catalase in the presence of bioSAS was investigated. We obtained the kinetic parameters of this process. It was shown that the bioSAS has an influence on the fermentative process, which can be explained by the change of the fermentative center activity or by the change of substrate concentration. It was determined that the temperature of a process has an insignificant influence on the value of kinetic parameters.

1 2

3а Naukova Str., Lviv–60, 79060, UKRAINE; e–mail: [email protected]. 3а Naukova Str., Lviv–60, 79060, UKRAINE; e–mail: [email protected].

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Keywords: fermentative catalysis, catalase, kinetics, bioSAS

I. INTRODUCTION Regulation of the ferment activity is a subject of great interest among scientists from the point of view both of fundamental and applied microbiology and biochemistry. Development of high–effective complex enzymatic preparations is the actual task of modern biotechnology. Among potential regulators of enzymatic activity, the surface–active substances (SAS) call a special attention, since they are characterized by unique physical–chemical properties. Thus, in the references, for example, there are data as to stimulative action of the separate synthetic SAS on the activity of the horse–radish peroxidase [1−3]. At the same time, biogenic SAS (or biosurfactants or bioSAS) of the high activity are characterized by a series of advantages in comparison with the synthetic SAS, in particular: they are non–toxic, biodegradable, characterized by high efficiency in a wide range of the temperatures and рН [4−8]. Since the references are practically absent of any data as to studies of bioSAS action on the activity of the ferments, we have studied an influence of the biosurfactants on fermentative activity with the following interpretation of obtained results. That is why, the aim of the presented work was to study the influence of bioSAS on kinetics of the fermentative catalysis of the hydrogen peroxide decomposition reaction under the action of catalase.

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II. EXPERIMENTAL PART Rhamnolipids and trihalosolipid [9] synthesized in our lab have been used in the presented work as bioSAS. The value of catalase activity was expressed in mCat/l [10], and the activity itself was determined spectrophotometrically with the use of a spectrophotometer of UV–visible diapason Uvmini–1240 (P/N 206–89175–92; P/N 206–89175–38; Shimadzu Corp., Kyoto, Japan) at the wave length λ = 410 nm. Catalase has been purified via the following stages: і) centrifugation of culture broth of bacteria by Bacillus Sp. ShR−05 strain; іі) precipitation of protein from supernathant with acetone and ііі) ion exchange chromatography. In order to carry out the ion exchange chromatography, the 50 mg of ferment preparation were re−dissolved in 5 ml of distilled water and after that were drifted on column (1,5 × 25 sm) with DEAE−Toyopearl 650 M (Toyo Soda MFG, Co. Japan). Washing out of proteins was carried out using the following buffers: (і) 20 mM tris−HCl, pH 7,0; (іі) 10 mM Na−acetate buffer, pH 5,5; (ііі) 1 M NaCl in Na− acetate buffer, рН 5,5. With the aim of carrying out more precise investigations, the eliminating and purification of catalase from the supernathant of culture broth of the Bacillus Sp. ShR−05 strain was done. Characteristics of separate stages of the ferment eliminating and also the efficiency of catalase purification are represented in Table 1 and Figure 1. Obtained in the above-mentioned purification method, catalase was used for carrying out the following experimental investigations with the use of bioSAS. 3 4 Kosygin Str., Moscow, 119991, RUSSIA; e–mail: [email protected]. Handbook of Chemistry, Biochemistry and Biology : New Frontiers, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

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Table 1. Characteristic of separate stages of the ferment eliminating and also the efficiency of catalase purification

Stage of purification

Volume, ml

General protein, mg

General activity, mCat/l

Supernathant Acetone DEAE−Toyopearl 650 M

250 80

400 48

79 47

Specific activity, mCat /l·mg 0,16 0,84

60

1,62

5,5

6

(1) 5,3

Yield upon activity, % (100) 60

36

6

Multiplicity purification

Figure 1. Ion exchange chromatography of catalase on DEAE−Toyopearl 650 M.

III. RESULTS AND DISCUSSIONS In a case when the concentration of substrate exceeds the concentration of the ferment [S0] >> E0, that is usually the condition for the study of fermentative reactions kinetics, the rate equation for these reactions can be written as

υ=

k 2 [ E0 ][ S ] K S + [S ]

(1)

where υ is the reaction rate; k2 is the rate constant of the ferment−substrate complex decomposition; [E0] is the concentration of ferment; [S] is the concentration of substrate; KS is the constant of ferment−substrate complex dissociation.

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For initial rates of the reaction, when the discharge of substrate can be neglected, that is, when [S] = [S0], we will obtain

υ=

k 2 [ E0 ][ S 0 ] K S + [S0 ]

(2)

Equation (2) describes the dependence of rate for the fermentative reaction, proceeding of which is ordered to the scheme (3): k1

kat E + S ⇔ ES ⎯k⎯→ E+P

k−1

(3)

on the initial concentration of substrate and permits practically to determine the constants k2 KS, which are very important characteristics of the enzymatic reactions. In a case, when k2 and KS are effective, in other words, they depend on рН of medium, presented in a system of inhibitor or activators, k2 is called the catalytic constant (kkat), and KS is the Michael’s constant (or КМ(im.)). In this case the equation (2) takes a form

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υ=

k kat [ E0 ][ S 0 ] K М ( уявн.) + [ S 0 ]

(4)

The equation (4) is called the Michael’s−Menten equation. Composition kkat[E0] is maximal rate (Vmax). At the great concentrations of the substrate S, (S0 >> KM), the ferment “saturated” by the substrate and, the rate of the catalytic process kinetically is controlled by chemical transformation of ferment−substrate complex

υ = k[ES ]

(5)

Or

kT − υ= e h

ΔG ≠ внутр . RT

[ ES ]

(6)

Thermodynamic efficiency of the fermentative catalysis is determined by a difference of free energies of external molecular (at the formation of Michael’s complex) and intermolecular ≠

[in the transitional state ( ΔG int . )] of bond formation between the groups of ferment and the substrate. In this case the motive force of the catalysis is a free energy of interaction between the groups of a ferment and the substrate into transition state of the reaction (but not into an intermediate complex).

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It is necessary to mention, that very often in biology references under the catalytic activity it is understood as the change of general reaction rate per some period of time. However, it should be remembered that the catalytic process foresees a decrease of the potential reaction barrier that is increasing of their rate constant (k). The reaction rate depends both on the value k, and on the concentrations of the reagents. The value k is accepted as a criterion of the catalytic reaction. In the presented case kkat places this role in biocatalysis. Typical figure in coordinates 1/V on 1/S is represented in Figure 2.

Figure 2. Dependence of the reaction rate on concentration of the substrate in coordinates of the Lainuiver−Berck’s equation.

Kinetic characteristics of the biocatalysis reaction of peroxide hydrogen by catalase (Vmax and kkat) under different conditions are represented in Tables 2.1 − 2.4. Table 2.1. Dependence of the biocatalysis reaction kinetics for peroxide hydrogen by catalase on concentration RL at t = 20 °C Concentration RL, ml/mg control 0.010 0.025 0.075 0.100 0.125

Vmax, mlg/ml·min. 2.35 3.07 4.00 5.00 6.66 5.71

k2, min.−1 0.94 1.22 1.60 2.00 2.66 2.28

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Table 2.2. Dependence of the biocatalysis reaction kinetics for peroxide hydrogen by catalase on concentration RL at t = 40 °C Concentration RL, ml/mg control 0.010 0.025 0.075 0.100 0.125

Vmax, mlg/ml·min. 2.55 2.95 4.12 5.40 6.52 5.90

k2, min.−1 1.02 1.18 1.64 2.16 2.60 2.36

Table 2.3. Dependence of the biocatalysis reaction kinetics for peroxide hydrogen by catalase on concentration RL at t = 60 0C Concentration RL, ml/mg control 0.010 0.025 0.075 0.100 0.125

Vmax, mlg/ml·min. 3.00 3.60 4.90 6.10 7.30 5.90

k2, min.−1 1.20 1.44 1.96 2.44 2.92 2.36

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Table 2.4. Dependence of the biocatalysis reaction kinetics for peroxide hydrogen by catalase on concentration RL at t = 80 0C Concentration RL, ml/mg control 0.010 0.025 0.075 0.100 0.125

Vmax, mlg/ml·min. 3.10 3.40 3.95 6.00 6.85 5.80

k2, min.−1 1.24 1.36 1.58 2.40 2.74 2.30

We can see from the presented Tables 2.1−2.4, that at the bioSAS [ramnolipid (RL)] concentration increasing, Vmax is increased up to some concentration RL. However, at the bioSAS concentration in a field of the micelle−formation (0.2 мг/мл) the reaction rate is decreased. The values kkat during the reactions proceeding under different concentrations of RL are also increasing; this fact proved some decrease of the process energy activation value depending on the concentration of RL. Tenuous decrease of Vmax at the change of RL concentration can be explained by micelles aggregation, that is, by decreasing the active surface on which the catalytic reaction takes place. Properly, as to mechanism of the catalysis it is enough in a complicated manner somewhat to affirm— this fact needs special studies of the role of a series of bioSAS by a different nature in catalysis.

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It is necessary to mention, that the Michael’s−Menten constant is insignificantly decreased depending on the concentration of RL and temperature. Since

K Мен. =

k −1 k kat + k1 k1

(7)

[see. eq. (3)], this means that at least the ratio of the constants

k −1 k1

and

k kat k1

without RL and

in the presence of RL is changed insignificantly, in other words, it can be assumed, that in reactions k1

kat E + S ⇔ ( ES ) ⎯k⎯→ E+P

(8)

k−1

k1

kat E sol + S sol ⇔ ( ES ) sol ⎯k⎯→ E sol + P

(9)

k −1

is the equality

k −1 k −' 1 ≈ ' k1 k1

і

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with taken into account that

' k kat k kat ≈ ' k1 k1

.

k k kat = K Міх . That is, k1 = kat K Міх k1

.

If the reaction practically displaced in a side of the Michael’s complex, then there is possibility enough simply to determine the constant rate k1. Interesting phenomena can be observed in the reactions of the peroxide H2O2 decomposition at the temperature variation. We can see from Tables 2.1−2.4, that Vmax. is somewhat increased upon the temperature increasing in the presence of high concentration of bioSAS. At low concentrations of bioSAS, the Vmax. is changed insignificantly. It can be concluded from the data of Tables 2.1−2.4, that the constants kkat. at the temperature variation is changed insignificantly. This means that the activation energy of a process is low and is neared to zero. We can see from Figure 3, that the Arrhenius’s law for above described systems is not realized.

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A. A. Turovsky, R. O. Khvorostetsky, L. I.Bazylyak et al. 1,2

lg kkat

1,0 0,8 0,6 0,4 0,2 0,0

0,0029

0,0030

0,0031

0,0032

1/T, K

-1

Figure 3. Dependence of kkat on temperature in Arrhenius’s coordinates.

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Among reasons for its omission is an increasing the ferment denaturations like to protein under higher temperatures. If we take into account the fact that the denaturation under low temperatures is insignificant and to calculate the activation energy of peroxide decomposition in absence of bioSAS in a range of temperatures 20−40 °С, then it can be obtained the value Е neared to 1 ccal/mole. If we take into account RL, then the value Е consists of not more than 0.1 ccal/mole, that is, practically, zero. Taking into account the described above circumstances, the reaction rate constant practically is equal to preexponent, which, in a practical, does not depend on temperature, that is:

KT e k =χ h where

χ

ΔS ≠ R

e

ΔH − RT

(10)

is the transmission coefficient; k is the rate constant; К is Boltzman’s constant; h is

Plank’s constant;

ΔS

KT k =χ e h

≠

is an activation entropy. Since the term

e

−

ΔH ≠ RT

≈ 0 , then

ΔS ≠ R

≠

(11)

Value ΔS for reactions at lower and higher temperatures is changed slightly. Rate constants at the lowest and at the highest temperatures are differed in 1.2 times:

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ΔS ≠

KT − χ 1e R k1 h = ≈ 1,2 ΔS ≠ k2 KT2 − R χ e h It is necessary to mention, that the rate of biocatalytic reaction of hydrogen peroxide decomposition depends on temperature, since the catalytic constant depends on temperature, however its role in the presented process is not dominating and increasing the rate with temperature of process increase can be conditioned by conformational transitions of ferment, complex−formation at the expense of hydrogen bonds “ferment−substrate−RL”, and also by a role hydrophobic interactions “ferment−H2O2−RL”. As to ferment denaturation at different temperatures it can be assumed that:

1) at low concentrations of SAS and low values of temperatures it will be dominated the

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process of hydrogen bonds formation H2O2−RL−E and denaturation of the ferment will be low; 2) at low concentrations of RL and high temperatures the ferment denaturation will be higher, the stabilization of the substrate will be less, and, respectively, the rate will be less; 3) at high concentrations of RL and low temperatures, the concentration of the ferment and the substrate centers is more; respectively, the denaturation is less and the rate is high; 4) at high concentrations of SAS and high temperatures the denaturation of the ferment is strong, the stabilization is less at the expense of hydrogen bonds, however, the high concentration of SAS prevails. The rate can be changed slightly or greatly enough. Kinetics of biocatalytic decomposition of H2O2 in the presence of trigalosolipide (TGL) practically is slightly differed from the kinetics of biocatalytic decomposition of H2O2 in the presence of rhamnolipide (see Tables 3.1−3.4). Table 3.1. Dependence of biocatalysis reaction kinetics of hydrogen peroxide by catalase on concentration of Th at t = 20 °C Concentration Th, ml/mg control 0.010 0.025 0.075 0.100 0.125

Vmax, mlg/ml·min. 2.35 3.07 4.00 5.00 6.66 5.71

k2, min.−1 0.94 1.22 1.60 2.00 2.66 2.28

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Table 3.2. Dependence of biocatalysis reaction kinetics of hydrogen peroxide by catalase on concentration of Th at t = 40 0C Concentration Th, ml/mg control 0.010 0.025 0.075 0.100 0.125

Vmax, mlg/ml·min. 2.55 2.95 4.12 5.40 6.52 5.90

k2, min.−1 1.02 1.18 1.64 2.16 2.60 2.36

Table 3.3. Dependence of biocatalysis reaction kinetics of hydrogen peroxide by catalase on concentration of Th at t = 60 0C Concentration Th, ml/mg control 0.010 0.025 0.075 0.100 0.125

Vmax, mlg/ml·min. 3.00 3.60 4.90 6.10 7.30 5.90

k2, min.−1 1.20 1.44 1.96 2.44 2.92 2.36

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Table 3.4. Dependence of biocatalysis reaction kinetics of hydrogen peroxide by catalase on concentration of Th at t = 80 0C Concentration Th, ml/mg control 0.010 0.025 0.075 0.100 0.125

Vmax, mlg/ml·min. 3.10 3.40 3.95 6.00 6.85 5.80

k2, min.−1 1.24 1.36 1.58 2.40 2.74 2.30

It can be seen from the presented Tables, that on all given intervals of the bioSAS concentrations the value Vmax is increased not more than 3 times. Decreasing the values Vmax and kkat at higher concentrations of bioSAS can be explained by aggregation of SAS micelles, and at decreasing the data of values at higher temperatures, possibly, it is necessary to take into account the denaturation of biocatalyst (catalase). Probably, such assumptions demand the carrying out of the additional investigations. Under the temperature reaction proceeding in the presence of TGL the values Vmax and kkat are changed insignificantly. Estimated value of the activation energy of catalytic reaction in absence of bioSAS consists of ~ 1 ccal/mole; in the presence of TGL this value consists of not more 0.1 ccal/mole. It can be considered, that Еact. ~ 0, that is the change of catalytic constants at different temperatures is conditioned by ratio

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Kinetics of the Fermentative Reaction of H2O2 Decomposition…

KT2 h = T2 KT χ 1 T1 h

189

χ

,

where Т2 is the highest temperature of the experiments being carried out; Т1 is the lowest temperature of the experiments being carried out. Constants rate at the highest and the lowest temperatures differed in 1.2 times. Generally, it is necessary to mention, that the nature of bioSAS (RL and TGL) in some manner has an influence on catalytic constant of the process, and its insignificant increase is explained by activity of the catalase centers. However, this assumption demands that future experiments be carried out.

REFERENCES: Eriomin A. N., Mietielitsa D. I., Smietan G. Biokhimiya, 1984, v. 49, № 6, p. p. 976−984 Helenius S., Simons K. Biochem. Biophys. Acta, 1975, v. 415 (1), p. p. 29−79 Nelson C. A. J. Biol. Chem., 1971, v. 246 (2), p. p. 3895−3901 Bognolo G. Physical Chemistry and Engineering, 1999, v. 152, p. p. 41−52 Eliora Z., R. and E. Rosenberg. Env. Microbiology, 2001, v. 3 (4), p. p. 229−236 Bezborodov A. M. Biotechnologiya produktov mikrobnogo sinteza М.: Agropromizdat, 1991, 235 p. [7] Karpenko E. V., Martynyuk N. B., Shulga A. N. Patent of Ukraine № 71222, bull. № 12, 2004 [8] Karpenko E. V., Vildanova R. I., Shcheglova N. S. Appl. Biochem. and Microbiology, 2006, v. 42, p. p. 156−59 [9] Shulga A. N., Karpenko E. V., Elysseev S. A., Vildanova−Martsishyn R. I., Patent of Ukraine № 10467 A, 1996 [10] Korolyuk M. A., Ivanova L. I., Mayorova I. G., Tokaryev V. E. Laboratornoye dielo, 1988, iss. 1, p. p. 16−19.

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[1] [2] [3] [4] [5] [6]

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In: Handbook of Chemistry, Biochemistry … Editors: L. N. Shishkina et al., pp. 191-196

ISBN: 978-1-61209-425-0 © 2010 Nova Science Publishers, Inc.

Chapter 19

KINETICS OF THE FERMENTATIVE PROCESS IN STATIONARY STATE FOR SUNFLOWER−SEED OIL HYDROLYSIS BY LIPASE IN THE PRESENCE OF BIOSAS *

A. A Turovsky1, * R. O. Khvorostetsky2, ** L. I. Bazylyak, and *** G. E. Zaikov3

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*

Chemistry and Biotechnology of Combustible Minerals Division; Physical Chemistry of Combustible Minerals Department; Institute of Physical–Organic Chemistry and Coal Chemistry named after L. M. Lytvynenko; National Academy of Science of Ukraine Lviv, Ukraine ** Chemistry of Oxidizing Processes Division; Physical Chemistry of Combustible Minerals Department; Institute of Physical–Organic Chemistry and Coal Chemistry named after L. M. Lytvynenko; National Academy of Science of Ukraine Lviv, Ukraine *** Kinetics of Chemical and Biological Processes Division; Institute of Biochemical Physics named after N. N. Emanuel; Russian Academy of Sciences Moscow, Russia

ABSTRACT The catalytic rate constants for the process in the presence of bioSAS by different concentrations was obtained. It was shown that some constants increase at bioSAS concentration increasing up to the beginning of their micelle−formation. The temperature has a slight influence on the value of catalysis constants, which can be explained by practically zero activation energies and depends on activation entropy.

1

3а Naukova Str., Lviv–60, 79060, UKRAINE; e–mail: [email protected]. 3а Naukova Str., Lviv–60, 79060, UKRAINE; e–mail: [email protected]. 3 4 Kosygin Str., Moscow, 119991, RUSSIA; e–mail: [email protected]. 2

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Keywords: fermentative catalysis, catalase, kinetics, bioSAS

1. INTRODUCTION

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One of the biggest problems in the fermentative catalysis is the problem of enzymes fermentative activity increasing depending on different physical−chemical factors. The aim of the presented work was to study the influence of the concentration and nature of the biosurface−active substances (bioSAS) on kinetics of fermentative catalysis of sunflower−seed oil hydrolysis reaction by lipase. Kinetics was studied in the stationary state accordingly to the Michael’s−Menten conception. Sometimes in the fermentative kinetics, biologists recognize as the criterion of fermentative activity the general rate of fermentative reaction that is not always true, since in accordance with the definition, the catalysis of chemical reaction is determined by decreasing the value of reaction potential barrier, in comparison with a non−catalytic reaction. That is why the catalytic constant, which is the function of activation energy and preexponential multiplier, should be accepted as the criterion of the catalytic fermentative reaction. If such constant is changed via the process of fermentative reaction under the action of some medium factor, etc., and concentration of the reagents via the process does not change, then the change of the general rate of fermentative reaction can be used as a criterion of fermentative activity. If via the process some factor is changed (for example, concentration of the reagents), then the general rate does not characterize the change of fermentative activity. In this case it is necessary to use the catalysis constant as a criterion of fermentative activity.

II. EXPERIMENTAL PART The value of lipase activity was expressed in mCat/l [1], and the activity itself was determined by spectrophotometrically with the use of spectrophotometer of UV–visible diapason Uvmini–1240 (P/N 206–89175–92; P/N 206–89175–38; Shimadzu Corp., Kyoto, Japan) at the wave length λ = 410 nm. Hydrolysis of sunflower−seed oil by lipase was carried out in accordance with the technique described in [2]. Sunflower−seed oil was used with characteristic described in [3].

III. RESULTS AND DISCUSSIONS At studying the initial reaction rates (when the substrate consumption can be disregarded), we assume, that [S] ≈ [S0] and

υ=

k 2 [ E 0 ][ S 0 ] K S + [S 0 ]

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(1)

Kinetics of the Fermentative Process in Stationary State for Sunflower−Seed Oil… 193 where S is the concentration of substrate; υ is the rate of fermentative reaction, with proceeds in accordance with scheme: KS

k2 E + S ⇔ ES ⎯⎯→ E+P

The equation (1) permits experimentally to determine the constants k2 and KS. In a case when k2 and Ks are effective values (that is, they depend on рН medium and also side reactions take place and etc.), they are «catalytic constants kcat.» and imagine Michael’s constant Кimag. Then the eq. (1) will be as follow

υ=

k kat [ E 0 ][ S 0 ] K imag . + [ S 0 ]

(2)

The equation (2) is the Michael’s−Menten equation. It characterizes the hyperbolic dependence of fermentative reaction rate on the initial concentration of substrate and linear rate on concentration of ferment. Composition of kкаt [E0], which has the dimensionality of reaction rate is denominated as «maximal reaction rate (Vm)». Linearization of the experimental data in coordinates

1 1 , , which are denominated as υ S0

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Lainuiver−Berck’s coordinates, permits to determinate the values υ max and Кimag. Kinetic regularities of catalysis under the conditions when the ferment «saturated» by substrate (that is, at S0 >> Ks), are some others. In this case the kinetics of fermentative process is determined by intermolecular chemical transformation of Michael’s complex ЕS in activated reaction complex [ES]#. The motive force of the catalysis is free energy of the interaction [ES]# in the transition state (but not into an intermediate complex ЕS). Thus, free activation energy is determined as a difference between free energy of activated complex [ES]# and free energy of Michael’s complex, that is, under such conditions the reaction of catalysis is «pseudomonomolecular». We can see from Tables 4.1−4.4, that Vmax. is increased till some concentration – 0,1 mgг/ml with the bioSAS concentration increasing. Following increase of the bioSAS concentration leads to the Vmax decreasing. This effect is explained by fact that the denoted range of bioSAS concentration is characterized by micelle−formation. Probably, that the decreasing of surface aggregations from the micelles leads to Vmax. reducing. As we can see from Tables 4.1−4.4, the value of catalytic constant k2 is increased less in twice with bioSAS concentration increasing. This points out some activation energy decreasing via the fermentative activity process. This process demands of separate detailed investigations; at the same time, it can be assumed the following scheme of reaction:

S + bioSAS ⇔ S ⋅ bioSAS

(3)

Е + bioSAS ⇔ Е ⋅ bioSAS

(4)

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A. A Turovsky, R.O. Khvorostetsky, L. I. Bazylyak et al.

S ⋅ bioSAS + E ⋅ bioSAS ⇔ S ⋅ E ⋅ bioSAS →

(5)

→ [S ⋅ E ⋅ bioSAS ] → E + P + bioSAS

(6)

≠

Activation energy is determined as the energies difference between activated complex [S E bioSAS]# and енергією Michael’s complex S E біоПАР. Probably, at bioSAS concentration increasing the Michael’s complex energy is rather increased, than the energy of activated complex that leads to decreasing the activation energy value and increase of the reaction rate constant. Increasing the energy of Michael’s complex can be explained at the expense of less thermodynamic probability of its formation. It’s not excepting that as a result of the fermentative reaction in the presence bioSAS it is important also the value of activation entropy ΔS#. In others words, generally the process should be characterized by value ΔF# of the reaction. It’s not excepting also is the process of the reagents concentration change under the action of bioSAS via fermentative reaction that is reflected on general rate of reaction and Vmax. This aspect also requires the additional experiments carrying out. We can see from Tables 4.1−4.4, that Vmax. and k2 in hydrolysis reaction are not much changed with variation of temperature. However, in the presence of enough great concentration of bioSAS it is observed some tendency to Vmax.і kкат. increasing. It is necessary to note, that the dependence of lgk2 on 1/Т is not rectilineal, in other words the Arrhenius’s equation is not fulfilled. The reason of this can be fact, that the constants are gross values. It’s not excepting, that at temperature increasing it is observed the reaction of protein (ferment) denaturation greatly. In such a case general rate of reaction will be consisted of V1 + V2, where V1 is the catalysis rate, and V2 is denaturation rate that leads to effective constants of hydrolysis.

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Table 4.1. Dependence of kinetic parameters of the fermentative catalysis on concentration of bioSAS at t = 20 °C Concentration RL, mgг/ml control 0.010 0.025 0.075 0.100 0.125

Vmax, mlg/ml·min. 2.35 3.07 4.00 5.00 6.66 5.71

k2, min.−1 0.94 1.22 1.60 2.00 2.66 2.28

Table 4.2. Dependence of kinetic parameters of the fermentative catalysis on concentration of bioSAS at t = 40 °C Concentration RL, mgг/ml control 0.010 0.025 0.075 0.100 0.125

Vmax, mlg/ml·min. 2.55 2.95 4.12 5.40 6.52 5.90

k2, min.−1 1.02 1.18 1.64 2.16 2.60 2.36

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Kinetics of the Fermentative Process in Stationary State for Sunflower−Seed Oil… 195 Table 4.3. Dependence of kinetic parameters of the fermentative catalysis on concentration of bioSAS at t = 60 0C Concentration RL, mgг/ml control 0.010 0.025 0.075 0.100 0.125

Vmax, mlg/ml·min. 3.00 3.60 4.90 6.10 7.30 5.90

k2, min.−1 1.20 1.44 1.96 2.44 2.92 2.36

Table 4.4. Dependence of kinetic parameters of the fermentative catalysis on concentration of bioSAS at t = 80 0C

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Concentration RL, mgг/ml control 0.010 0.025 0.075 0.100 0.125

Vmax, mlg/ml·min. 3.10 3.40 3.95 6.00 6.85 5.80

k2, min.−1 1.24 1.36 1.58 2.40 2.74 2.30

If assume, that at low temperatures the hydrolysis reaction is dominating (the range of temperatures 20−40 0С), then estimated activation energy of a process without bioSAS is equal to 1 ccal/mole. With taken into account the bioSAS in this range of temperatures, the activation energy is 0.1 ccal/mole (in other words, approximately in error limits). It can be considered that Еactiv. ≈ 0. The change of a rate at bioSAS concentration increasing proceeds at the expense of reagents concentration increasing; and kкат. increase takes place as a result of activation entropy increasing. As a result, the catalysis rate constant k2 is equal to preexponent, which weakly depends on temperature. Really,

kT k2 = e h

ΔS ≠ R

ΔH

e − RT

(7)

where k2 is constant of catalysis, k is Boltzman’s constant, h – is Plank’s constant, ΔS# is activation entropy of reaction, ΔН# is a heat of activation. When ΔН# ≈ 0 and ΔS≠ practically does not depend on temperature, we will obtained

kT k2 = e h

ΔS ≠ R

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196

A. A Turovsky, R.O. Khvorostetsky, L. I. Bazylyak et al. In such a case the rate constants at the highest and the lowest temperatures will be differed

kT2 as multipliers ratio h

kT1 h

, or as

T2

T1

; in other words the ratio is in ~ 1.2 times.

It is necessary to say some words as to Michael’s constants. Their values practically are not changed with the presence in reaction of bioSAS. Temperature has also an insignificant influence on their values. This fact is explained by fact that the ratio of elementary rate constants of fermentative catalysis of hydrolysis reaction both in absence of bioSAS, and also in their presence, are enough near. Michael’s constant

K imag .=

k1 + k 2 k1

(9)

or

K imag . = K eq. +

k2 k1

(10)

under condition, that k1 >> k-1

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K imag .≈

k2 k1

(11)

In other words, knowing k2 and Кimag., we can estimate k1. If for reaction k2 and Кimag. are near, then k1 will be near. However, since k2 and Кimag. in the presence of bioSAS some are differed, respectively k1 also will be differed. That is, reactive abilities of ferments and substrates will be in some manner differed.

REFERENCES [1] [2] [3]

Korolyuk M. A., Ivanova L. I., Mayorova I. G., Tokaryev V. E. Laboratornoye dielo, 1988, iss. 1, p. p. 16−19 Becker G., Berger V., Domshke G. Organicum. Practicum on Organic Chemistry. − 1979. − vol. 2. − 447 p. Sunflower−seed oil, Unrefined "First−class". GOST Р 52465−2005.

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In: Handbook of Chemistry, Biochemistry … Editors: L. N. Shishkina et al., pp. 197-223

ISBN: 978-1-61209-425-0 © 2010 Nova Science Publishers, Inc.

Chapter 20

THERMAL DEGRADATION AND COMBUSTION BEHAVIOR OF THE POLYETHYLENE/CLAY NANOCOMPOSITES PREPARED BY INTERCALATIVE POLYMERIZATION L.A. Novokshonovaoo1, S. M. Lomakin2, P.N. Brevnov1, A.N. Shchegolikhin2 and R. Kozlowskipp3 1

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N.N. Semenov Institute of Chemical Physics of Russian Academy of Sciences, Moscow, Russia 2 N.M. Emanuel Institute of Biochemical Physics of Russian Academy of Sciences, Moscow, Russia 3 Institute of Natural Fibres, Poznan, Poland

ABSTRACT A comparative study of thermal and thermal-oxidative degradation processes for polyethylene/organically modified montmorillonite (PE-MMT) nanocomposites, prepared by the ethylene intercalative polymerization in situ, with or without subsequent addition of an antioxidant, is reported in this chapter. The results of TGA and time/temperaturedependent FTIR spectroscopy experiments have provided evidence for an accelerated formation and decomposition of hydroperoxides during the thermal oxidative degradation tests of PE-MMT nanocomposites in the range of 170–200oC, as compared to the unfilled PE, thus indicating a catalytic action of MMT. It has been shown that effective formation of intermolecular chemical cross-links in the PE-MMT nanocomposite has ensued above 200oC as the result of recombination reactions involving the radical products of hydroperoxides decomposition. Apparently, this process is induced by the oxygen deficiency in the PE-MMT nanocomposite due to its lowered oxygen permeability. It is shown that the intermolecular cross-linking and dehydrogenation reactions followed by the shear carbonization lead to appreciable increase of thermal-oxidative stability of the PE nanocomposite as compared to that of pristine PE. Notably, the TGA traces for the a b

119991 Kosygin 4, Moscow, Russia ul. Wojska Polskiego 71 b, Poznan, Poland

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L.A. Novokshonova, S. M. Lomakin, P.N. Brevnov et al. antioxidant-stabilized PE-MMT nanocomposites recorded in air were quite similar to those obtainable for the non-stabilized PE-MMT nanocomposites in argon. The results of treatment of the experimentally acquired TGA data in frames of an advanced model kinetic analysis are reported and discussed. Significant decrease of the combustibility of the PE nanocomposite was shown by a cone calorimeter method.

Keywords Layered clay; catalysis; intercalative polymerization; nanocomposite; polyethylene; exfoliation; barrier properties; thermal and thermal-oxidative degradation; kinetics: combustibility.

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INTRODUCTION Polyethylene (PE), being the most commercially important thermoplastic commodity, is heavily used for consumer products in many applications, but in a number of cases general applicability of PE turns out to be undermined by its relatively low thermal stability and flame resistance. The concept of compounding polymer matrices with nanoscale fillers (in particular, clays or layered silicates) has already been proven to be an effective method of preparing nanocomposites with excellent physical and mechanical properties [1–11]. It is believed that, in the course of high temperature pyrolysis and/or combustion, clay nanoparticles are capable of promoting formation of protective clay-reinforced carbonaceous char which is responsible for the reduced mass loss rates, and hence the lower flammability. Accordingly, considerable attention has been paid also to polyolefin/layered silicate nanocomposites. Reportedly, the latter have exhibited improved mechanical properties, gas impermeability, thermal stability, and flame retardancy as compared with corresponding pristine polymers [4, 5, 9, 10, 12, 13]. This study deals with polyethylene/layered silicate nanocomposites prepared by an intercalative polymerization route [12, 13]. The method includes the intercalation of a metallorganic catalyst into the interlayer spacing of organically modified MMT and the subsequent polymerization of ethylene. As a result of polymer chains growing within the interlayer spacing of montmorillonite (MMT), the exfoliation of the original MMT particles to the nanoscale inorganic monolayers takes place. In order to clarify the mechanisms of the carbonaceous char formation, which may be responsible for the reduced mass loss rates and hence the lower flammability of the polymer matrices, a number of thermo-physical characteristics of the PE/MMT nanocomposites have been measured in comparison with those of the pristine PE (which by itself is not a char former) in both inert and oxidizing atmospheres. The evolution of the thermal and thermaloxidative degradation processes in these systems was followed dynamically with the aid of TGA and FTIR methods. The thermal-oxidative stability of PE nanocomposites in both the absence as well as in the presence of an antioxidant was investigated. Several sets of experimentally acquired TGA data have provided a basis for accomplishing thorough modelbased kinetic analyses of thermal and thermal-oxidative degradation of both pristine PE and PE-n-MMT nanocomposites prepared in this work.

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Thermal Degradation and Combustion Behavior…

199

1. EXPERIMENTAL 1.1. Materials A Cloisite 20A (purchased from Southern Clay Products, Inc.) has been used as the organically modified montmorillonite (MMT) to prepare PE/MMT nanocomposites throughout this study. The content of an organic cation-exchange modifier, N+2CH32HT (HT=hydrogenated tallow, C18≈65%; C16≈30%; C14≈5%; anion: Cl-), in the MMT was of 38% by weight. VCl4 (vacuum distilled at 40°С before use, TU 48-05-50-71) and Al(i-Bu)3 (Aldrich) have been used for catalytic activation of MMT. Ethylene monomer was of a standard polymerization grade.

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1.2. Procedure of Polyethylene Nanocomposite Synthesis Intercalation of the catalyst has been accomplished by treating the freshly dehydrated MMT with Al(i-Bu)3 and then with VCl4. The polymerization reaction was started by admitting ethylene into the reactor and was carried out until a PE nanocomposite (PE-n-MMT) of adjusted composition was obtained. The polymerization reaction was stopped by adding ethanolic HCl solution (5 wt.% HCl) to the reactor. The polymer composite product was filtered off, washed several times with ethanol and dried under vacuum at 60°C. The weight loads of MMT in the resulting composites were calculated by neglecting the contribution of the organic modifier in MMT. The sample of unfilled polyethylene was prepared by ethylene polymerization on VCl3 activated with Al(i-Bu)3 at the same conditions as applied to the nanocomposite synthesis. Stabilized samples of both the nanocomposites (st-PE-n-MMT) and pristine PE (st-PE) were prepared by treating them with synergetic composition of Topanol CA and di-lauryl-3,3’thiodipropionate (DLTDP)[14] solutions in heptane at 70°C, followed by drying in vacuum. The concentrations of Topanol and DLTDP in st-PE-n-MMT and st-PE comprised 0.3 and 0.5 wt.%, respectively. For further testing, the prepared materials were hot-pressed into films at applied pressure of 20 MPa and 160°С.

1.3. Characterization of Materials The structure of the composites was studied by SAXS using a KRM-1 camera (Cu Kα radiation, λ = 0.154 nm, Ni filter). The test samples were powders. The data collected were normalized with due regard to the concentration of MMT and the coefficients of attenuation. Very cold neutron (VCN) scattering was also used to study nanocomposite structures. In this method, scattering particles have average sizes of 2.5–50 nm. The method estimates the average size of scattering particles and their proportion in the overall MMT content of the polymer matrix; i.e., it quantifies the degree of exfoliation of precursor MMT particles in the polymer composite. A time-of-flight VCN spectrometer [15] was used.

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Micrograph of PE nanocomposite sample was obtained on a JEM-100B transmission electron microscope at an accelerating voltage of 80 kV. The sample of 70 nm thickness was cut with the aid of LKV-III ultramicrotome from the composite plate prepared by hot pressing. A Perkin-Elmer TGA-7 instrument calibrated by Curie points of several metal standards has been employed for non-isothermal thermogravimetric analysis. The measurements were carried out at a desired heating rate (in the range of 3 – 40 K/min) in both inert (argon) and oxidizing (oxygen) atmospheres, as appropriate. The N2, O2, and CO2 gas permeabilities were studied on a pressure setup [16] at 1–2 MPa and 20–22ºC. Composite samples of 70–80 μm thick were hot molded at 185ºC and 30 MPa. Infrared spectra of the investigated materials in their nascent form were acquired with the aid of a Perkin-Elmer 1725X FTIR instrument by using a Spectra-Tech "Collector" DRIFT accessory furnished with a heated sample post, embedded thermo couple and the corresponding external heater/controller providing temperature reading precision of ±1.0C. The series of FTIR spectra for the polymer samples have been recorded at systematically varied temperatures or over predetermined time intervals (in isothermal regimes) by employing a modified diffuse reflectance-absorbance Fourier Transform (DRAFT) spectroscopy technique published elsewhere [17]. All measurements were performed using the instrument DTGS detector and a 4cm-1 resolution. Kinetic analysis of PE compositions thermal degradation was carried out using Thermokinetics software by NETZSCH-Gerätebau GmbH. The combustibility of PE and PE–MMT was studied on a cone calorimeter with an external heat flow equal to 35 kW/m2 on samples with a standard surface area of 70×70 mm and an identical weight (13.5 ± 0.3 g).

Figure 1. SAXS patterns for the original C20A MMT (1) and PE nanocomposites with MMT content of 1.8 vol. % (2) and 6.5 vol. % (3).

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2. RESULTS AND DISCUSSION

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2.1. PE Nanocomposite Structure Small-angle X-ray scattering (SAXS) has been used to evaluate the degree of exfoliation of the organoclay particles in the polymer matrix [12, 13]. SAXS diffractograms of pristine C20A MMT and those of PE nanocomposites prepared by the intercalation polymerization route for MMT contents of 1.8 vol. % (2) and 6.5 vol. % (3) are shown in Figure 1. The SAXS curve for C20A MMT shows a reflection at around of 3.6o corresponding to the interlayer mean distance of 2.46 nm (Figure 1, 1). As can be seen from the same Figure 1 (2, 3), for the PE/clay nanocomposites having different MMT contents, the 3.6o reflection is absent. The full exfoliation of the MMT particles to the monolayers takes place under the action of PE forming in the course of polymerization in the interlayer spacing. Figure 2 shows TEM image of the PE nanocomposite containing 1 vol. % of MMT. The dark features in the micrograph correspond to the exfoliated monolayers and nanostacks of MMT distributed throughout the PE matrix. It can be seen that the nanoscale MMT layers lack any sort of orientation in the matrix of the pressed composite. The exfoliated MMT particles are characterized by very high aspect ratio (longitudinal size: thickness). Using VCN scattering for studying PE–MMT composite structures, we obtained another piece of evidence in favor of the exfoliation of precursor MMT particles into nanolayers during the synthesis of polymer composites. We used this method to estimate the proportion of scattering particles in composites and mean particle sizes [12, 13]. Table 1 shows the results obtained for PE nanocomposite samples with MMT concentrations in composites equal to 1– 1.8 vol %. Virtually complete exfoliation is achieved for these samples; it amounts to 95% as shown by VCN scattering. The absence of Bragg stripping on the full cross section versus VCN wavelength curve signifies the nonexistence of ordered structures with interplanar spacings of 2.5–50 nm.

Figure 2. TEM micrograph of the PE nanocomposite containing 1% by volume of MMT.

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L.A. Novokshonova, S. M. Lomakin, P.N. Brevnov et al. Table 1. VCN scattering data for PE–MMT composites

vol. % MMT in composite 1.0 1.8

VCN scattering data Mean size of scattering MMT particles, nm 13.3 8.7

Volume fraction, % 86 95

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2.2. Barrier Properties of PE Nanocomposites The gas permeability of polymer material is defined by its barrier properties and effects on the processes of thermal-oxidative degradation. To evaluate the barrier properties of prepared nanofilledd PE composites, we measured their N2, O2, and CO2 gas permeability. Table 2 lists the permeability factors P for nanocomposite films with various MMT percentages. For all three gases, the permeation decreases with increasing filler percentage. The greatest decrease is observed for small fillings (up to ~2 vol. %). The gas permeability decreases the 3-4 times for PE-nanocomposites at MMT content as low as 1.5 – 2 vol.% compared to the neat PE. Apparently, such a decrease in gas permeability is characteristic of nanocomposites containing high proportions of anisotropic particles (thin lamellae with high aspect ratios). The values obtained for Pc/P0, the ratio of the gas permeabilities of a composite and unfilled polymer, correlate with the theoretical curve for lamellar nanoparticles with a cross section 10×1500 Å, calculated according to the equation Pc/Pp = 1/ [1 + (L/2W) · Vf] [18] (Figure 3). In addition, the selectivity factors of composites remain virtually the same as for unfilled PE. All this demonstrates that the change in the properties of the polymer matrix is not responsible for the improvement of the barrier properties; the actual reason is the considerable increase in the effective free path of gas molecules during diffusion through a composite film containing lamellar MMT particles. Table 2. N2, O2, and CO2 gas permeability P and selectivity factors for nanocomposites with various MMT percentages vol % MMT 0 0.8 1.8 6.5

α (O2/N2)

Р×1014 cm3 cm/(cm2 s Pa) N2 8.0 6.1 2.2 1.3

O2 26.2 19.2 7.1 4.6

CO2 106 78.8 29.6 20.3

3.28 3.15 3.22 3.52

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Figure 3. Relative gas permeation of nanocomposites vs. MMT concentration for (1) N2, (2) O2, and (3) CO2. Pc and P0 are the permeabilities of the composite and unfilled PE, respectively. The dashed line is the theoretical curve for lamellar nanoparticles with a cross section 10×1500 Å.

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2.3. Thermal Degradation of PE Nanocomposite It is accepted that thermal stability of polymer nanocomposites is higher than that of pristine polymers, and this gain is explained by the presence of anisotropic clay layers hindering diffusion of volatile products through the nanocomposite material. It is important to note that the exfoliated nanocomposites, prepared and investigated in this work, have much lower gas permeability in comparison with that of pristine unfilled PE (Table 2, Figure 3). Thus, the study of purely thermal degradation process of PE nanocomposite seemed to be of interest in terms of estimation of the nanoclay barrier effects on thermal stability of polyolefin/clay nanocomposites. The radical mechanism of thermal degradation of pristine PE has been widely discussed in a framework of random scission type reactions [19-27]. It is known that PE decomposition products comprise a wide range of alkanes, alkenes, and dienes. The polymer matrix transformations, usually observed at lower temperatures and involving molecular weight alteration without formation of volatile products, are principally due to the scission of weak links, e.g., oxygen bridges, incorporated into the main chain as impurities. The kinetics of thermal degradation of PE is frequently described by a first-order model of mass conversion of the sample [27]. A broad variation in Arrhenius parameters can be found in literature, i. e., activation energy (E) ranging from 160 to 320 kJ/mol and pre-exponential factor (A) variations in the range of 1011 and 1021 s-1 [25-27] are not unusual. It is believed that the broad range of E values reported may be explained by the polymers molecular mass variations, by use of various additives, and by different experimental conditions [27] employed by different authors.

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Previously Bockhorn et al. have reported that thermal degradation of PE leads to a large number of paraffins, dienes and olefins without a residue formation [25]. In order to formulate a simple kinetic model adequately explaining the isothermic global kinetic data of the authors, a mechanism embracing only the main reactions including initiation by random scission of the polymer chains into primary radicals, β-scission of these radicals, hydrogen transfer (intra- and intermolecular) and recombination reactions has been proposed [25]. It is important that the change in the reaction order is dependent on the intermolecular hydrogen transfer in reaction leading to the alkanes. Thus, at high temperatures and at high degrees of conversion, the alkanes formation via reaction of intermolecular hydrogen transfer becomes favored and, therefore, the reaction order alters from 0.5 to 1.5 [25].

Figure 4. TGA thermograms for PE (firm lines) and PE-n-MMT (dotted lines) taken in Ar at the heating rates of: 3K/min - 1, 2 and 20K/min. - 3, 4.

In the present work, the processes of thermal degradation of both unstabilized PE and PEn-MMT nanocomposite with MMT content of 4.3 wt.% have been investigated by TGA in an inert (argon) atmosphere at the heating rates of 3, 5, 10, and 20 K/min. According to the dynamic TGA data, the polymer degradation starts at about 300°C and then, through a complex radical chain process, the material totally destructs and completely volatilizes in the range of 500-550oC (Figure 4). It is obvious that, taken at the same heating rates in argon, the thermograms for pristine PE and PE-n-MMT are practically identical, except that the solid silicate residue amounting to 4-5 % wt. can be seen on the curves for the nanocomposites (Figure4). This result suggests that the mechanisms of thermal degradation of PE and PE-nMMT nanocomposites, and hence the global kinetic parameters of their thermal degradation processes are rather similar.

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2.4. Kinetic Analysis of PE Nanocomposite Thermal Degradation Based on TGA Data Kinetic studies of material degradation have long history, and there exists a long list of data analysis techniques employed for the purpose. Often, TGA is the method of choice for acquiring experimental data for subsequent kinetic calculations, and namely this technique was employed here. It is commonly accepted that the degradation of materials follows the base equation (1) [19] dc/dt = - F(t,T co cf)

(1)

where: t - time, T - temperature, co - initial concentration of the reactant, and cf - concentration of the final product. The right-hand part of the equation F(t,T,co,cf) can be represented by the two separable functions, k(T) and f(co,cf): F(t,T,co,cf) = k[T(t)·f(co,cf)] (2) Arrhenius equation (4) will be assumed to be valid for the following: k(T) = A·exp(-E/RT)

(3)

Therefore,

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dc/dt= - A·exp(-E/RT)·f(co,cf )

(4)

All feasible reactions can be subdivided onto classic homogeneous reactions and typical solid-state reactions, which are listed in Table 3 [28]. The analytical output must provide good fit to measurements with different temperature profiles by means of a common kinetic model. Kinetic analysis of PE and PE-n-MMT thermal degradation at heating rates of 3, 5, 10 and 20K/min was accomplished by using a NETZSCH Thermokinetics software in accordance with a formalism we proposed earlier [7]. In order to assess the activation energy for development of a reasonable model for kinetic analysis of pristine PE and PE-n-MMT thermal degradation processes, a few evaluations by model-free Friedman analysis have been done as the starting point [29]. Further, nonlinear model fitting procedure for PE and PE-n-MMT TGA-curves has led to the following triple-stage model scheme of successive reactions (Figure 5): D1

A

B

Fn

C

Fn

D

(5)

Taking this as a reasonable approximation for PE and PE-n-MMT, the fits with the aid of nonlinear regression were attempted by the model (5), where an one-dimensional diffusion type reaction was used for the first step and the nth-order (Fn) reaction - for the two subsequent steps of the overall thermal degradation process (Figure 5, Table 4).

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Assuming a radical chain mechanism is operative in the process of PE and PE-n-MMT thermal degradation, the apparent activation energy and the pre-exponential factor values calculated in this work turned out to be in perfect match with the data from isothermal analysis and dynamic TGA published earlier (Ea = 268 ± 3 kJ/mol, logA = 17.7 ± 0.01 min-1 and Ea = 262.1 kJ/mol, log A = 18.09± 0.14 min-1 [30]).

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Table 3. Reaction types and corresponding reaction equations, dc/dt= - A·exp(E/RT)·f(co,cf ) Name F1 F2 Fn

f(co,cf ) c c2 cn

Reaction type first-order reaction second-order reaction nth-order reaction

R2 R3

2 · c1/2 3 · c2/3

two-dimensional phase boundary reaction three-dimensional phase boundary reaction

D1 D2 D3 D4

0.5/(1 - c) -1/ln(c) 1.5 · e1/3(c-1/3 - 1) 1.5/(c-1/3 - 1)

one-dimensional diffusion two-dimensional diffusion three-dimensional diffusion (Jander's type) three-dimensional diffusion (Ginstling-Brounstein type)

B1 Bna

co · cf con · cfa

simple Prout-Tompkins equation expanded Prout-Tompkins equation (na)

C1-X Cn-X

c · (1+Kcat · X) first-order reaction with autocatalysis through the reactants, X. X = cf. cn · (1+Kcat · X) nth-order reaction with autocatalysis through the reactants, X

A2 A3 An

2 · c · (-ln(c))1/2 two-dimensional nucleation 3 · c · (-ln(c))2/3 three-dimensional nucleation N · c · (-ln(c))(n- n-dimensional nucleation/nucleus growth according to Avrami/Erofeev 1)/n

Figure 5. Outcome of multiple models-based nonlinear fitting for pristine PE (a) and PEn-MMT (b). The experimental TGA-data (dots) in comparison with the model calculations results (firm lines) are shown for different heating rates: 3K/min – (1), 5K/min – (2), 10K/min – (3) and 20K/min – (4).

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The TGA data acquired for PE and PE-n-MMT in argon has not provided any evidence in favor of the hypothesis that the barrier effect, being clearly manifested in the gas permeability experiments with the same PE-n-MMT at room temperature (Table 2, Figure3), is operative also during thermally stimulated degradation of PE-n-MMT. It should be noted that, in an inert atmosphere, degradation/volatilization of both PE and PE-n-MMT starts at about 350°C and is totally completed up to 500-550oC, not taking into account a solid silicate residue amounting to 4-5 % wt. which remains in the case of the nanocomposites (Figure 4, 5). Based on TGA data, the first stage of the degradation process (1D-diffusion limiting stage) develops in the range of 350-410oC corresponding to the overall mass loss of 5-7%. The subsequent steps of the thermal degradation processes (410 - 500oC) for PE and PE-n-MMT proceed in the liquid melt of high molecular weight degradation products. In the light of the above findings, we believe that during the high-temperature degradation stages (above, e. g., 410oC) in an inert atmosphere, the barrier diffusion restrictions can become insignificant since the viscosity of the pyrolyzed polymer melt at these temperatures is rather low and, because of the intensified mobility of the clay layers in such melt, the overall ‘labyrinth effect’ normally provided by the clay particles in more viscous matrices may be considerably diminished. Table 4. The kinetic parameters for the three-step thermal degradation of PE and PE-nMMT as obtained by the multiple-curve analysis of the experimental TGA-data (heating rates 3, 5, 10 and 20 K/min) in frames of the reaction model Material

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PE

PE-n-MMT

Parameter

Value

logA1, s-1 E1, kJ/mol logA2, s-1 E2, kJ/mol n2 logA3, s-1 E3, kJ/mol n3 logA1, s-1 E1, kJ/mol logA2, s-1 E2, kJ/mol n2 logA3, s-1 E3, kJ/mol n3

11.7 197.7 15.5 253.1 0.50 16.6 268.1 1.50 10.3 186.3 14.5 237.5 0.50 17.6 274.3 1.50

Corr. Coeff.

0.9994

0.9992

2.5. Thermal-Oxidative Degradation of PE Nanocomposite Thermal oxidative degradation of PE and PE nanocomposites has been extensively studied over the past decades [31-35]. It has been reported that the main oxidation products of PE are aldehydes, ketons, carboxylic acids, esters, and lactones [31,32]. According to Lacoste and

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Carlsson [33], β-scission plays an important role in thermal oxidation of UHMWPE. Notably, the feasibility of intra-molecular hydrogen abstraction by the peroxy radicals for polyethylene has been questioned in frames of a thermal oxidation mechanism proposed by Gugumus [34, 35].

Scheme 1. A flow-chart of elementary steps constituting PE thermal-oxidative degradation process.

It is usually supposed that the reaction of hydrogen abstraction from an alkane molecule, R-H, may lead to hydroperoxides and alkyl radicals according to the overall reaction scheme (Scheme 1). A mechanism describing oxidation of organic molecules by virtue of complex chain reactions has been proposed earlier by Benson [36]. At temperature below 190oC, oxidation of organic compounds involves free-radical chain initiation and the main products are hydroperoxides and oxygenated species indicated in the routes A1 and A2 of Scheme 1. At temperatures below 200oC, the abstraction of H from R· resulting to HO2· + olefin (routes B1 and B2) proceeds at least 200 times slower than the addition of O2 to R· to give RO2·. Above 250oC, the route A2 becomes reversible, and the very slow step B2 becomes rate determining. As a consequence, at temperatures above 300oC, there is some retardation of the rate of oxidation of the polymer. The H2O2 can play the same role as ROOH in providing a secondary radical source just above 480oC, where the rate of oxidation picks up again. It is worth noting that a simple digital photo camera was of help for qualitative assessment of differences in the processes of thermal oxidation of neat PE as compared to PE-n-MMT. Figure 6 shows photographs of the neat PE (1) and PE-n-MMT (2) taken after both samples having been heated during 2 minutes at 180oC in air. It can be seen that coloration of the PE-n-

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MMT sample (2) is much darker than that of the neat PE (1), thus evidencing that MMT promotes the carbonization of the polymer. Figure 7 compares the TGA thermograms for neat PE and PE-n-MMT which has been acquired at a 10K/min heating rate in air. Obviously, under the thermal oxidative degradation conditions, these two materials demonstrate strikingly different behavior.

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Figure 6. TGA curves for PE (1) and PE-n-MMT (2) recorded in air at the heating rates of 10K/min.

The earliest stage of thermal oxidative degradation of unstabilized samples of PE-n-MMT and PE manifests itself as a clear weight gain feature emerging on the TGA curves well below 200oC and is attributed to the oxygen absorption followed by the hydroperoxides formation (Figure 7). Of importance, however, is the fact that for PE-n-MMT this process seems to be accelerated due to the presence of nanosilicate additive as compared with the pristine PE. Dependences of the hydroperoxides formation onset temperatures versus the heating rate, which have been derived from the TGA data for unstabilized PE-n-MMT and PE samples, are presented in Figure 8. We believe that O2 molecules, being adsorbed on the defect centers of MMT represented by the impurities, transform into more active species which are able to react with PE at lower temperatures, thus inducing formation of active centers on the hydrocarbons chains (Scheme 2). Apparently, this chain of events should result to accelerated formation of PE hydroperoxides.

Figure 7. TGA curves for PE (1) and PE-n-MMT (2) recorded in air at the heating rates of 10K/min.

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Figure 8. The onset temperatures of hydroperoxides formation vs. heating rate for: PE (1), PE-n-MMT (2).

Scheme 2. The earliest stages of the process of PE thermal oxidative degradation in the presence of exfoliated MMT nanoparticles.

Notably, while the hydroperoxides accumulation starts at lower temperatures in PE-nMMT than in the unfilled PE (cf. e. g., Figure8), the clearly visible mass loss of the nanocomposite (attributable solely to decomposition of the accumulated hydroperoxides) ensues at lower temperatures as well. It is reasonable to suggest that this effect is caused by a catalytic action of exfoliated MMT nanoparticles on the hydroperoxides decomposition. As it has been shown earlier [13], the treatment of PE-n-MMT with alcoholic HCl solution led to substitution of the major part of the organic modifier by acidic protonic centers. Moreover, it is widely accepted that MMT-type clay minerals always comprise a plenty of different catalytically capable sites, which may be represented by weakly acidic Brønsted-like Si-OH sites, by strongly acidic -OH groups localized at the edges of the silicate layers, by transition metal cations captured in the galleries, and by crystallographic defect sites within the layers [37, 38]. All these sites are able to trigger decomposition of hydroperoxides within the bulk of the PE-n-MMT. It has been shown that acid-catalyzed rearrangements of hydroperoxides can proceed in both polar and non-polar solvents. Hence, such rearrangements can be expected to occur also in PE-n-MMT. Acids can decompose primary and secondary hydroperoxides

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according to two different pathways [39]. Both these routes are depicted in Scheme 3 for the secondary hydroperoxides most probably present in PE. Since mobility of the methylene units in the PE backbone is rather limited, it is reasonable to assume that reaction (2) in Scheme 3 should be of minor importance. Then the main reaction [reaction (1) in Scheme 3] must lead to transformation of the hydroperoxide into the ketone group with elimination of water. The reaction might proceed according to the general mechanism or be simply dehydration [40].

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Scheme 3. Acid-catalyzed decomposition of PE hydroperoxides.

In addition to the accumulation and subsequent decomposition of the hydroperoxides on the earlier stage resulting to emergence of the oxygen-containing groupings, the process of thermal oxidative degradation of the nanocomposite involves the reactions of oxidative dehydrogenation and intermolecular cross-linking. It seems reasonable to suggest that namely at this step the thermally stable carbonized charred layer on the nanocomposite surface is formed and starts to hinder the diffusion transport of both the volatile degradation products (out of the polymer melt into the gas phase) and the oxygen (from the gas phase into the polymer). The above set of events results in actual increase of the nanocomposite thermal stability in the temperature range of 350-500°C, where normally a shear degradation of the main part of PE takes place. This point is illustrated by TGA and DTG plots presented in Figures 7, 9, and 10. The diverse behavior of stabilized and unstabilized samples (Figure9 and 10, curves 1,2 TG and DTG) shows that the addition of antioxidants has resulted to higher thermal-oxidative stability. It can be seen also that the overall thermal oxidative stability of PE-n-MMT independently of the antioxidant presence was higher than that of the pristine PE. Moreover, incorporation of the antioxidants in PE-n-MMT has led to a notable change in the character of the mass loss process (Figure10, curves 2, 3 of TG and DTG). It is quite probable that the antioxidant is able to “deactivate” the sites of MMT that have been occupied earlier with absorbed oxygen. In the result, the MMT nanolayers could become chemically inert in respect to the hydroperoxides formation and hence to further accelerated PE oxidation. It may be seen as well that, with the exception of the first thermal oxidation step, the TGA and DTG curves for st-PE-n-MMT taken in air become closely resembling those characteristic for PE-n-MMT run in argon. Having taken into account the above findings, it seems reasonable to explain the observed retardation of thermal oxidative degradation of st-PE-n-MMT by the capability of exfoliated MMT nanolayers to hinder the diffusion of oxygen throughout the partly cross-linked and carbonized nanocomposite matrix.

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Figure 9. Acquired at 10 K/min heating rate in air or argon TGA and DTG curves for: PE in air (1), stPE in air (2), PE in Ar (3).

Figure 10. Acquired at 10 K/min heating rate in air or argon TGA and DTG curves for: PE-n-MMT in air (1), st-PE-n-MMT in air (2), PE-n-MMT in Ar (3).

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2.6. Kinetic Analysis of PE-N-MMT Thermal Oxidative Degradation

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Kinetic analysis of thermal oxidative degradation of unstabilized PE and PE-n-MMT at the heating rates of 3, 5, 10, and 20K/min (as well as of the same samples stabilized with antioxidants) has been accomplished by using the aforesaid interactive model based nonlinear fitting approach (Figure 11 a, b).

Figure 11. Nonlinear kinetic modeling of PE-n-MMT (a) and st-PE-n-MMT (b) in air. Comparison between experimental TGA data (dots) and the model results (firm lines) at several heating rates: 3K/min (1), 5K/min (2) and 10K/min (3).

With best fidelity, the undertaken nonlinear model fitting for the stabilized samples of PE and PE-n-MMT has provided a triple-stage model scheme of successive reactions, wherein an nth-order autocatalysis reaction (Cn) was used at the first step, while a general nth-order (Fn)

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reaction was used for both the second and the third steps of the overall process of thermal oxidative degradation (Table 3): Cn

A

Fn

B

C

Fn

D

(8)

For unstabilized PE and PE-n-MMT at the beginning stage of degradation, the degree of conversion depends on the heating rate (Figure 11a), such dependence being a strong evidence in favor of a branched reaction path. For this case, the same approach has provided a dissimilar triple-stage model scheme comprising two competitive reactions: an n-th order autocatalytic reaction (Cn), for the first competing path, and two nth-order (Fn) successive reactions, for the second competing path (9). Cn

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A

Fn

B B

Fn

C

(9)

As data in Table 5 for the first stage of thermal oxidative degradation reaction show, the activation energies values for st-PE and st-PE-n-MMT amount to 74 and 96 kJ/mol, while for unstabilized materials those values are of 65 and 51 kJ/mol, correspondingly, thus indicating that the degradation of these samples is initiated by the similar oxygen induced reactions. At this stage, the lower activation energy of PE-n-MMT compared to that of PE may be related to the catalysis exerted by the ММТ during formation and decomposition of hydroperoxides. At the same time, the values of activation energy found at the second and the third stages of thermal-oxidative degradation for PE-n-MMT are higher than those for PE (Table 5). This difference might be attributed to a shift of the PE-n-MMT degradation process to a diffusionlimited mode owing to emergence in the system of a carbonized cross-linked material. The activation energy values of st-PE-n-MMT at the first and the last stages are higher than those of st-PE (Table 5). Actually, at the last stage, the activation energy of st-PE-n-MMT rises up to 274.8 kJ/mol, almost reaching the activation energy value found for degradation of PE-n-MMT in inert atmosphere (274.3 kJ/mol) (Table 4). This fact infers that the last stage of the st-PE-n-MMT degradation process is governed mainly by random scission of C-C bonds, rather than by an oxygen catalyzed reactions. On the other hand, these results are also consistent with the barrier model mechanism, which suggests that inorganic clay layers can play a role of barriers retarding the diffusion of oxygen from gas phase into the nanocomposite. Thus, from the TGA data for both antioxidant-stabilized and unstabilized PE and PEMMT nanocomposite it follows that the organoclay nanoparticles can exert two counteracting effects influencing the thermal-oxidative stability of the PE-MMT nanocomposite:

•

catalytically accelerated accumulation and decomposition of hydroperoxides observed at the earlier stages, thus in fact promoting degradation of the polymer matrix and hence impairing the thermal stability of PE-n-MMT;

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•

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promoting of the carbonization of the polymer matrix tending to improve the thermaloxidative stability of the nanocomposite.

Table 5. Results of the multiple-curve kinetic analyses for thermal-oxidative degradation of PE and PE-n-MMT in accordance with the reaction models 8 and 9. Material

st-PE

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PE

st-PE-n-MMT

PE-n-MMT

Parameter logA1, s-1 E1, kJ/mol n1 log Kcat 1 logA2, s-1 E2, kJ/mol n2 logA3, s-1 E3, kJ/mol n3 logA1, s-1 E1, kJ/mol n1 log Kcat 1 logA2, s-1 E2, kJ/mol n2 logA3, s-1 E3, kJ/mol n3 logA1, s-1 E1, kJ/mol n1 log Kcat 1 logA2, s-1 E2, kJ/mol n2 logA3, s-1 E3, kJ/mol n3 logA1, s-1 E1, kJ/mol n1 log Kcat 1 logA2, s-1 E2, kJ/mol n2 logA3 s-1 E3 kJ/mol n3

Value 3.6 74.9 0.79 0.59 14.9 225.9 0.51 16.1 254.4 1.79 3.6 65.3 1.62 0.14 5.1 120.2 0.55 13.7 219.7 1.37 6.3 96.3 2.4 0.15 13.8 230.2 0.64 16.8 274.8 1.66 2.2 51.5 2.81 0.12 6.8 146.2 0.53 14.7 238.2 1.69

Corr. Coeff.

0.9987

0.9953

0.9993

0.9993

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2.7. Dynamic FTIR Analysis of PE-N-MMT Thermal Oxidative Degradation

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Simultaneously to TGA measurements, thermal oxidative degradation of PE and PE-nMMT samples has been monitored in this work with the aid of a dynamic FTIR spectroscopy in a temperature range of 25–260oС. The overall evolution of the dynamic FTIR spectra in the course of thermal-oxidative degradation of PE and PE-n-MMT in the condensed phase is shown in Figure 12 (a, b). At room temperature (25oC), the FTIR spectra of both materials are typical for PE. The absorption bands at 2845-2960 cm-1 are assigned to -CH-, -CH2-, or -CH3 stretching vibration [41]. The absorption at 1472 cm-1 is due to the deformation vibration of -CH2- or -CH3 groups, while that at about 720 cm-1 is due to (CH2)n rock when n ≥ 4 [41, 42]. Beside these, the FTIR spectra of PE-n-MMT revealed the absorptions belonging to MMT (ν (Si–O) 1047 cm-1) [42]. Another MMT absorption band at 3630 cm-1 has been assigned to the structural hydroxyl groups, directed toward the vacant positions in the inner octahedral layer of montmorillonite. Else, a broad absorption band of hydroxyl groups was observed at 3400 cm-1 [43]. During the dynamic recording of the PE and PE-n-MMT spectra under the step-wise heating a sharp growth of the absorption in the range of 1700 - 1800 cm-1 was noted at temperatures above 200oC indicating the emergence and accumulation of carbonyl-containing products resulting from the thermal-oxidative degradation process (Figure 12, 13).

Figure 12. Dynamic FTIR analysis of PE – (a) and PE-n-MMT – (b).

Obviously, the complex band in the range of 1700-1800 cm-1 (Figure 13) comprises: a carbonyl absorption (shoulder at 1717 cm-1) belonging to ketone groups embedded into the polymer chain [42, 44], which are known to be the main oxidation products for the neat PE [42]; a shoulder peaking close to 1734 cm-1, which is attributed to aldehyde groups [45]; another shoulder with maxima in the vicinity of 1746 cm-1, which is normally assigned to ester groups vibrations [42, 44], and an absorption at 1790 cm-1 belonging to the carbonyl in γlactone moiety [46]. Gradual growth of concentration of the vinylene groups absorbing close to 1600 cm-1 [42] has also been noted (Figure 12). The dynamic FTIR spectra show that, while the fractional ratio of different carbonyl absorptions was almost the same for both PE and PEn-MMT, the apparent concentration of the carbonyl-containing products (overall intensity of

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the complex absorption band) in PE-n-MMT is considerably higher than in pristine PE (Figure 12, 13).

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Figure 13. FTIR spectra of PE (1) and of PE-n-MMT (2) taken at 220ºC in the course of the heating runs (excerpted from the corresponding dynamic FTIR spectra sets).

Figure14. The Carbonyl Index (CI) vs. temperature dependences for: (1) PE; (2) PE-n-MMT.

The temperature dependences of the Carbonyl Index (CI) for samples of PE and PE-nMMT are shown in Figure 14. The carbonyl index (CI) was defined to illustrate the formation of non-volatile carbonyl containing oxidation products:

where SC=O is peaks area of carbonyl containing groups at 1800-1700 cm-1, S2019 is internal reference at 2019 cm-1, CIo is initial carbonyl index.

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Figure 15. Comparative evolution of the IR absorption bands attributed to -СН2- units in: (a) neat PE, and (b) PE-n-MMT nanocomposite.

Along with the carbonyl absorptions growth, the FTIR spectra have revealed a clear decay of intensities of the bands belonging to -СН2- vibrations as the result of the PE chain scission. Both investigated materials experienced the chain scission during pyrolysis, but with drastically different rates. The relative rates of the decay are illustrated by the overlaid spectra in Figure 15 showing evolution of the symmetrical and asymmetrical –CH2- stretch absorption bands with the pyrolysis temperature for pristine PE as compared to PE-n-MMT. At any given temperature above 200oC, PE-n-MMT has higher content of intact CH2 units than the pristine PE sample, and the apparent rate of disappearance of the aliphatic units in the pyrolysis temperature range of 220-260oC is much higher for pristine PE than for the corresponding nanocomposite. Thus, the observed evolution of the spectra is an extra proof of the fact that, at temperatures above 220oC, PE-n-MMT nanocomposite undergoes thermal-oxidative degradation with a considerably slower rate than the neat PE does. The same conclusion has been derived in the preceding section based on the analysis of corresponding TGA data (Figure 7).

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Thermal Degradation and Combustion Behavior…  

(ROOH)

(RO )

OOH

O

C C H C H2 H2

(b)

PE

C H2

H2 C

(RH)

RO C H2

(a)

C H2

(c)

Chain scission (oxygen excess)

C C H C H2 H2

+ HO (d )

219

O C CH + H 2C H2

O2 / RH H C

C H2

RO

H2 C H C

(e)

O

(R )

C H2

x2

H2 C (ROR)

C H

C H2

Cross-linking (oxygen deficiency)

(f ) H2 C C H2

H C C H

H2 C (R-R) C H2

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Scheme 4. Alternative reactions of alkyl radicals during thermal-oxidative degradation of PE [31].

We explained this fact by the formation of chemical crosslinking between the polyethylene macromolecules in the nanocomposite. Really, Figures 15b and 16 show the medium absorption band at 1162 cm-1 which can be attributed to intermolecular esters groups (>С-О-С Pr4N+ > Bu4N+ > Hex4N+. The values of equilibrium constants of complexation between ROOH and Alk4NBr ions change similarly (Table 3). Isokinetic relationship between complexation parameters in the system leads to the insignificant changes in free energy of complexation for different alkyl substituent in ammonium cation. Considering the intermolecular bonds energy the strongest complex is formed between hydroperoxide and Et4NBr, the weakest - in the case of Hex4NBr (see the corresponding ΔHcom values in Table 3). The reactivity of complex-bonded ROOH also decreases with increasing of alkyl substituent size. Isokinetic relationship for the obtained

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Molecular Design and Reactivity of the 1-Hydroxycyclohexyl Hydroperoxide…

231

activation parameters points out upon the unified mechanism of ROOH - Alk4NBr complex decomposition. Table 3. Kinetic parameters of 1-hydroxycyclohexyl hydroperoxide decomposition in the presence of tetraalkylammonium bromides parameters kd·104, sec-1

KC, dm3mol-1

1

ΔScom, JmolK-1

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1

ROOH + Hex4NBr 0.34 ± 0.01 0.97 ± 0.02 2.40 ± 0.06 18 ± 3 16 ± 2

20 ± 1 81 ± 2 8.6 ± 0.3 -16 ± 2

18 ± 2 87 ± 3 9.4 ± 0.2 -12 ± 2

15 ± 2 96 ± 2 10.5 ± 0.3 -9 ±1

-21 ± 5

-10 ± 4

-3 ± 3

ROOH + Et4NBr

ROOH + Pr4NBr

333 343 353 333 343

1.14 ± 0.04 2.5 ± 0.1 5.1 ± 0.2 36 ± 2 29 ± 2

0.8 ± 0.1 1.80 ± 0.09 4.20 ± 0.09 28 ± 3

353

23 ± 1 73 ± 1 7.5 ± 0.6 -20 ± 1 -30 ± 4

-1

Ea, kJmol lgA, (A, c-1) ΔHcom, kJmol-

24 ± 1

ROOH + Bu4NBr 0.59 ± 0.06 1.51 ± 0.04 3.48 ± 0.08 23 ± 2 20 ± 1

T, K

333353

Changes of the peroxide bond activation in the complex in the case of different ammonium cations point out the role of steric factor at the stage of complex formation as well as at the formation of their decomposition transition state. In the simplest case the own volume of the investigated cations could describe the steric effect. A good correlation between activation parameters of the complex decomposition and calculated values of Van-der-Waals volumes of cations has been obtained (Figure 3). VVDW values for the tetraalkylammonium cations were calculated in HyperChem package. Calculated values are in agreement with corresponding own volumes of cations listed in [15]. Linear relationship has been observed between the complex heat of formation - ΔH0f and experimental activation parameters - ΔH≠ (Figure 4). Obtained experimental facts have shown that the salt cation participates both in complexation stage and in stage of complex ROOH - Alk4NBr decomposition. Ammonium cation has the regulating action upon the catalytic reactivity of the halide-anion in the reaction of catalytic decomposition of the 1-hydroxycyclohexyl hydroperoxide in the presence of Alk4NBr. Thus the cation structure influences on the reactivity of the hydroperoxide complex and on the extent of peroxide bond activation. The molecular modeling of the hydroperoxidecatalyst reactive complex can be used to preliminarily predict the reactivity of the system.

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Figure 3. Relationship between activation parameters of the complex decomposition and Van-der-Waals volume of the tetraalkylammonium cations.

Figure 4. Relationship between the complex heat of formation (ΔH0f) and experimental activation parameters (ΔH≠).

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CONCLUSIONS Summarizing, obtained experimental facts have shown that the chemical activation of the peroxide bond is observed in the presence of Alk4NBr. The kinetic parameters of the hydroperoxide catalytic decomposition have been obtained for the Et4NBr, Pr4NBr, Bu4NBr, and Hex4NBr. Catalysis of the 1-hydroxycyclohexyl hydroperoxide decomposition has been shown to occur through accompanied action of the ammonium salt cation and anion.

REFERENCES [1] [2] [3] [4] [5] [6]

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[7] [8] [9] [10]

[11] [12] [13] [14] [15]

E.T. Denisov, T.G. Denisova, T.S. Pokidova. Handbook of Free Radical Initiators. John Wiley and Sons Inc.: Hoboken, New Jersey, 2003. - 879 p. N.A. Turovskij, I.O. Opeida, O.V. Kush, E.L. Baranovskij. J. Appl. Chem. 77, 1887 (2004). C.J. Perez, E.M. Valles, M.D. Failla. Polymer, 46, 725 (2005). I.A. Opeida, N.M. Zalevskaya, E.N. Turovskaya. Kinetika i Kataliz, 45, 776 (2004). I.A. Opeida, N.M. Zalevskaya, E.N. Turovskaya, U.I. Sobka, Petroleum Chemistry, 42, № 6. 460 (2002). M.A. Turovskyj, I.A. Opeida, E.N. Turovskaya, O.V. Raksha, N.O. Kuznetsova, G.E. Zaikov. Oxid. Commun., 29, 249 (2006). N.A. Milas, S.A. Harris, P.C. Panagiotacos. J.Am.Chem.Soc., 61, No 9 2430 (1939). J.J.P. Stewart. MOPAC 2000.00 Manual; Fujitsu Limited: Tokyo: Japan, 1999. A.Klamt. J.Chem. Soc. Perkin Trans., №2 799 (1993). M.A. Тurovskyj, I.O. Оpeida O.M. Turovskaya, O.V. Raksha, N.O. Kuznetsova, and G.E. Zaikov. Order and Disorder in Polymer Reactivity. Howell New York: Nova Science Publishers, Inc.-2006. - pp. 37-51. N.A. Turovskij, S.Yu. Tselinskij, Yu.E. Shapiro, A.R. Kal’uskij. Teor. Eksp. Khim., 28, №4. 320 (1992). M.A. Turovskyj, A.M. Nikolayevskyj, I.A. Opeida, V.N. Shufletuk. Ukrainian Chem. Bull., 8, 151 (2000). L.M. Pisarenko, O.T. Kasaikina. Russ Chem Bull, 3, 419 (2002). L.M. Pisarenko, V.G. Kondratovich, O.T. Kasaikina. Russ Chem Bull, 10, 2110 (2004). Y.Marcus. Ion salvation. Chichester, etc.: Wiley. – 1985. – 306p.

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In: Handbook of Chemistry, Biochemistry … Editors: L. N. Shishkina et al., pp. 235-241

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Chapter 22

CHAR FORMATION FLAME RETARDANT OF PVC PLASTICATES N.A. Khalturinskiy1, D.D. Novikov, L.A. Zhorina, L.V. Kompaniets, T.A. Rudakova and S.L. Bobot’ko Semenov Institute of Chemical Physics, Russian Academy of Sciences

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ABSTRACT The effect of flame retardants on the combustibility and mechanical properties of PVC plasticates based on commercial materials is discussed. The smoke formation of the investigated samples was studied under pyrolysis and combustion modes. It was shown that, by addition of flame retardant to the PVC plasticate, its combustibility can be controlled with retention of the basic performance of the material. It should be noted that the studied method of the plasticate modification allows one to form a strong coke skeleton on the surface of the polymer composition and to avoid the flow of molten material upon burning and, as a result, to prevent flame propagation.

Keywords: polymers, combustion, smoke formation, char formation, reduction of combustibility

Most if not all of PVC-based materials and products contain plasticizers, which improve the elasticity and reduce the processing temperature. However, their addition to compositions results in an enhanced fire hazard of the materials. The presence of more than 38% dioctyl phthalate (DOP) in PVC compositions, for example, gives the oxygen index (LOI) of material corresponding to LOI - DOP [1]. This is the reason for the need to add flame retardants (FR) to reduce the combustibility of PVC plasticates.

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In NPO VNIIKP, the versatile combinations of flame retardants, smoke consumers, acceptors of hydrogen chloride of different nature with the contents varying from 0.1 to 350 pph PVC were studied [3]. The results obtained allowed one to develop PVC plasticates with a low combustibility for use as insulating materials. The works on the improvement of performance of flexible PVC plasticates, reduction of their combustibility, smoke formation, and toxic products evolution upon burning were recently carried out in Russia and other countries. For reducing the combustibility of PVC plasticates, along with antimonous oxide, aluminum oxide trihydrate, tin oxide, zinc stannates, zinc hydroxystannates, etc., were used [3, 4]; in this case LOI of plasticates varied from 25.6 to 34.3%. Regarding the use of solid solutions on the base of zinc, magnesium, and calcium oxides combined with antimonous oxide, the characteristic of the synergetic effect and significant enhancement of LOI is described in [5, 6]. The addition of tin-containing stabilizers and antimonous oxide for reducing the combustibility of PVC compositions with DOP plasticizer allowed one to obtain LOI = 34.1 [7]. Many researchers used well-known phosphorus-containing plasticizers (phosflex, tricresylphosphate, etc.) combined with antimonous oxide for reducing the combustibility of PVC. In the traditional technical literature, the following basic ways for decreasing the degree of inflammability of polymer materials are considered [8, 9].

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1) The use of different hydrates of salts and compounds, which evolve inert substances as a result of thermal degradation and thus reduce the flame temperature. 2) The use of different organic and inorganic flammable inhibitors, flame protectors changing the degradation rate. On the one hand, such inhibitors decrease the content of inflammable volatile degradation products and, on the other hand, the intumescent flame retardants provide the conditions of formation of a low-conductive coke layer on the burning surface. The latter process is realized via changes in the rheological properties of high-temperature pyrolysis products and the gas formation rate upon degradation. 3) The use of gas-phase flammable inhibitors, for example, halogenated compounds, which change the mechanism of reactions in flame and pre-ignition zone. Moreover, these inhibitors decrease the degree of combustion of the degradation products. On the one hand, by the aforementioned mechanisms, the heat evolution in flame decreases, on the other hand, the radiation heat losses enhance due to increased fraction of formed carbon black. 4) The filling of polymer matrix by inert materials to prolong the induction period and energy of inflammation. It is common knowledge that the formation of carbonization products results in the isolation of polymer from flame. As was shown [10], the combustion rate is determined by heat and mass transfer between the flame and polymer surface. The formation of inflammable gaseous products upon pyrolysis depends on the heat flux to the polymer surface from some source or flame. It was demonstrated that the lower concentration limit of the mixture of an oxidant and polymer

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Char Formation Flame Retardant of PVC Plasticates

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degradation products can be attained at the heat flux on the polymer surface not lower than 2–3 Wt/cm2. The effect of halogenated flame retardants are usually interpreted as their participation in gas-phase reactions similar to those that occur in the fuel--oxidant mixture prepared preliminarily. By contrast, many authors believe that phosphorus-containing compounds act in the condensed phase. For reducing the combustibility of polymers including PVC plasticates, the compounds, which decompose at temperatures below 400–500°C with heat absorption and evolution of carbon dioxide and/or water vapors, ammonia (hydroxides, carbonates, metal hydrocarbonates, phosphates, etc.), are mainly used [11]. The flame protection of PVC is performed by addition of aluminum and magnesium hydroxides. The comparative properties of hydroxides given in Table 1 are indicative of the predominant effect of magnesium hydroxide.

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Table 1. Comparative properties of magnesium and aluminum hydroxides Parameter

Mg(OH)2

AL(OH)3

Water content, % Density, g/cm3 Initial decomposition temperature,oC Enthalpy of decomposition, cal/g

31.0 2.36

34.6 2.42

330 328

250 280

Magnesium hydroxide is characteristic of higher thermal stability that is of great importance for the technological processing of compositions; its enthalpy of decomposition is higher by almost 20% due to the higher efficiency. The new line of modification of polymers to reduce their combustibility is the addition of intumescent (swelling) flame retardant systems, which promote the carbonization process in condensed phase. The foamed coke with low conductivity formed on the surface of burning polymer results in its isolation from flame accompanied by decreased rate of formation of inflammable volatile products of degradation, by disturbance of heat balance in the flame edge, and its extinction. In this work, the effect of coke-forming flame retardants FR 205 described in [12] on the combustibility and mechanical properties of PVC plasticates was studied.

EXPERIMENTAL PVC - FR blends were prepared in a Brabender mixer of the closed type at the rotor velocity of 100 rpm and 180°C for 10 min. The properties of the resulting compositions were measured using films 0.3, 1.0, and 2.0 mm thick prepared by pressing (180oC, 100 atm, 10 min) followed by cooling to room temperature under pressure.

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The mechanical tests were performed on an Instron-1122 test machine under tension mode at room temperature and the constant rate of the upper traverse motion of 50 mm/min. The samples for mechanical tests were prepared as dumbbells with the working length of 35 mm and 5 mm in width. From the strain diagrams, elastic modulus Ei (by the initial portion of curve), ultimate tensile strength σb, elongation at break εb, and stress at 100% elongation σ100 were determined. The results were averaged by 6–8 samples. Melt flow index (MFI) was measured on an IIRT-5 instrument by the standard method. The inflammability characteristics were determined by the method of oxygen index (LOI). The smoke formation and CO evolution were estimated by the ASTM E662 method. The samples were prepared as plates 65x55 mm, 2 mm thick. The calculation of results obtained to the GOST (State Standard) can be carried out by the following formula Dm(GOST) = S/M×Dm(ASTM), where S is the surface area of the sample tested (m2), and M is its weight (kg).

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RESULTS AND DISCUSSION The efficiency of FR 205 flame retardants was estimated with commercial OM-40 PVC plasticates and compared with the values for incombustible NGP 30-32 PVC plasticates tested under the same conditions. The results of testing of mechanical characteristics and combustibility of PVC compositions are given in Table 2. As can be seen from Table 2, the introduction of FR to OM-40 composition results in a monotonic change of properties. Based on the data listed in Table 2, the formulation of plasticates can be optimized for preparing compositions, which meet both the requirements on the properties and costs. Table 2. Characteristics of PVC-based blends

Material PVCOM-40 PVCNGP 30-32 PVCOM-40 +31%FR PVCOM-40 +15.5% FR PVCOM-40 +7.7% FR

Ei, MPa 16.8 50.1 24.4

σb, MPa

εb, %

σ100, MPa

14.2 16.4 9.4

248 162 195

20.5

10.5

18.0

11.6

LOI

7.9 12.9 6.4

MFI,g/10min 190oC, 2,16kg 7.0 1.2 3.4

30.8 37.1 39.9

179

7.4

6.0

35.7

228

7.2

7.8

33.2

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Char Formation Flame Retardant of PVC Plasticates

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Table 3. Smoke formation and toxicity parameters Gas concentration (upon burning), ppm

Smoke formation Material

Thickness, mm

Mass g

OM-40

2

14.08

ОМ-40 +31% FR

2

15.38

NGP 30-32

2

15.15

Test mode

burning pyrolysis burning pyrolysis burning pyrolysis

D2*

D4*

Dm**

205 270 263 107 290 76

379 355 604 320 624 278

488 372 653 586 656 572

τmax,s 375 345 440 530 335 535

CO 900 500 600 -

* - D2, D4 is smoke formation within 2 and 4 min, respectively. ** - Dm is the maximum smoke formation corresponding to the τmax time

The comparative data on smoke formation and the evolution of toxic products of burning are listed in Table 3. Figure 1 shows the data of LOI measurements for different plasticates. As can be seen from figure, LOI for NGP composition lies below the values obtained upon addition of FR to OM-40 plasticate.

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LOI 40 NGP 35

30

25 0

10

20

30

40

% FR Figure 1. LOI of OM-40 PVC plasticate vs. concentration of FR 205 flame retardant.

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The data on the combustibility of PVC plasticates presented in Table 2 and Figure 1 demonstrate that the combustibility characteristics (LOI) corresponding to the GOST can be attained by introduction of 10 wt % FR into OM-40 PVC composition. Figures 2 a–c show the photographic images of the sample structure after burning at the upper limit of LOI. It is evident that, upon OM-40 PVC burning, the melt flows along the surface from under flame (Figure 2a). Under real burning conditions, the burning melt will initiate the ignition of other incombustible materials in the vicinity. Upon burning of NGP 30-32 PVC samples, the melt does not flow off (Figure 2b); an instable (unbound) residue of mineral filler is formed, dispersed, and removed from the flame zone under the action of a convective flow. The burning of PVC plasticates with an intumescent flame retardant (FR 205) is accompanied by the formation of a stable char “cap” on the surface, which, on the one hand, diminishes the amount of volatile incombustible products of plasticate pyrolysis supplied to the flame zone and, on the other hand, changes the conditions of heat exchange between flame and the surface of burning material (Figure 2c).

a

b

c

Figure 2. Sample structure after burning: (a) original OM-40PVC, (b) incombustible NGP 30-32 and (c) composition based on OM-40 and FR.

Both effects result in an instable burning and flame breaking [10]. Thus, a conclusion can be made that the addition of flame retardants, which catalyze the condensation, crosslinking, and formation of a swollen coke layer with low heat conductivity on the burning surface decrease efficiently the combustibility of PVC plasticates. The data listed in Table 3 show that the smoke formation for the samples of PVC plasticates with FR is within the normal range and coincides practically with the value for

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Char Formation Flame Retardant of PVC Plasticates

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NGP PVC. However, it should be noted that the samples PVC + FR 205 are characteristic of significantly lower evolution of carbon monoxide.

ACKNOWLEDGMENTS The authors are grateful to D. V. Ptashinskii (the Energokabel’ plant) for kindly supplying samples of cable plasticate.

REFERENCES A. Shekov and V. V. Annenkov, Plast. Massy, No. 9, 42 (2007). V. G. Nikolaev, E. A. Kitaigora, N. I. Golovenko, et al., in Proceedings of 1 International Conference on Polymer Materials of Reduced Combustibility, 1990, Vol. 2, p. 135. [3] D. Chaplin, US Patent No. 5,342,874 (1994). [4] US Patent No. 3,928,502 (1994). [5] Tian, H. Wang, X. Liu, et al., J. Appl. Polym. Sci. 89, 3137 (2003). [6] H. Wang, Z. Guo, and S. Qi, J. Fire Sci. 24, 195 (2006). [7] R. L. Markezich, US Patent No. 6,448,310 (2002). [8] N. A. Khalturinskii and A. A. Berlin, “Polymer Combustion”, in Degradation and Stabilization of Polymers (Elsevier, New York, 1989), p. 145. [9] R. Aseeva and G. Zaikov, Combustion of Polymer Materials (Nauka, Moscow, 1980). [10] N. A. Khalturinskii and T. A. Rudakova, Khim. Fiz. 27 (6), 71 (2008). [11] N. A. Khalturinskii, V. M. Lalayan, and A. A. Berlin, Zh. Vses. Khim. O-va im. D.I. Mendeleeva 34, 560 (1989). [12] G. E. Zaikov, Polymer News 30 (7), 216 (2005).

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[1] [2]

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In: Handbook of Chemistry, Biochemistry … Editors: L. N. Shishkina et al., pp. 243-255

ISBN: 978-1-61209-425-0 © 2010 Nova Science Publishers, Inc.

Chapter 23

MOBILE STRUCTURAL DEFECTS AS CATALYST OF SOLID STATE POLYMERIZATION A.M. Kaplan* and N.I. Chekunaev Semenov Institute of Chemical Physics, Russian Academy of Sciences Kosygin Street 4, Moscow 119991, Russian Federation

ABSTRACT

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A model of the solid state polymerization based on the catalytic role of carriers of the surplus free volume, namely mobile structural defects, has been developed. This model succeeded for the first time to explain most of non-trivial kinetic peculiarities of solid state polymerization.

Keywords: kinetics of solid state polymerization, mobile structural defects, catalyst, “living active centres”.

1. INTRODUCTION A systematic investigation of the solid state polymerization was started in the 1950s. The main initial stimulus for such studies was expectation of stereo-regular polymer production of ordered monomer molecules in a crystal state. Unfortunately, most of the investigations of the solid state polymerization did not confirm the expected result. However, the investigations of such polymerization were highly important for fundamental science. Some abnormal kinetic phenomena have been observed during the course of these investigations. These are:

1) absence of any activation energy of some chemical reactions at low temperatures [1,2]; *

[email protected]

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2) realization of the non-activated high-rate solid state polymerization under shear stress in conjunction with high pressure [3,4]; 3) significant acceleration of the solid state polymerization down to explosive process in the samples of activated pure monomers or monomer-containing composites at temperatures near the phase transition temperatures of these objects [5-7]. To explain these anomalies of solid state polymerization kinetics, N.N.Semenov [8], V.A.Kargin and V.A.Kabanov [6], and A.A. Berlin [9] proposed different theories but with a common starting thesis. According to this thesis, the elementary step of polymer chain growth runs with the highest rate in the most well organized and ordered systems. The defects present in crystal structure are peculiar inhibitors of the process. Each of mentioned theories was designed to explain only peculiar groups of few in number experimental data of the solid state polymerization, obtained at that time. None of these theories was able to explain the totality of kinetic effects of the polymerization in solids, obtained at that time and later on. A critical analysis of experimental and theoretical results presented in [1-9] one can find in [10]. In the present study, experimental results of the most striking non-trivial kinetic effects of solid state polymerization are described and explained. This common standpoint of these explanations is the realization of the solid state polymerization process by means of carriers of surplus free volumes which are structural defects of matrix.

2. EXPERIMENTAL RESULTS

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Phenomenon of “Freezing” and “Reanimation” of Polymer Chains in Solids For the first time, non-trivial effect of “freezing” and “reanimation” of polymer chains in solids was observed in [11] during investigation of the radiation-induced post-polymerization in acrylonitrile crystalline modification AN(II), which is thermodynamically stable at temperatures T