Advances in Lipid Methodology. Volume 4 PDF
Preview Advances in Lipid Methodology. Volume 4
Woodhead Publishing in Food Science, Technology and Nutrition Advances in lipid methodology Volume4 Edited by William W. Christie The Scottish Crop Research Institute, Invergowrie, Dundee (DD2 5DA), Scotland WP WOODHEAD PUBLISHING ~ ~ ~ Oxford Cambridge Philadelphia New Delhi Published by Woodhead Publishing Limited, 80 High Street, Sawston, Cambridge CB22 3HJ, UK www.woodheadpublishing.com; www.woodheadpublishingonline.com Woodhead Publishing, 1518 Walnut Street, Suite 1100, Philadelphia, PA 19102-3406, USA Woodhead Publishing India Private Limited, G-2, Vardaan House, 7/28 Ansari Road, Daryaganj, New Delhi - 110002, India www.woodheadpublishingindia.com First published by The Oily Press, 1997 Reprinted by Woodhead Publishing Limited, 2013 ©The Oily Press Limited, 1997; ©Woodhead Publishing Limited, 2012 The authors have asserted their moral rights This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. Reasonable efforts have been made to publish reliable data and information, but the authors and the publisher cannot assume responsibility for the validity of all materials. Neither the authors nor the publisher, nor anyone else associated with this publication, shall be liable for any loss, damage or liability directly or indirectly caused or alleged to be caused by this book. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming and recording, or by any information storage or retrieval system, without permission in writing from Woodhead Publishing Limited. The consent of Woodhead Publishing Limited does not extend to copying for general distribution, for promotion, for creating new works, or for resale. Specific permission must be obtained in writing from Woodhead Publishing Limited for such copying. Trademark notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation, without intent to infringe. British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library ISBN 978-0-9514171-7-1 (print) ISBN 978-0-85709-799-6 (online) This book is Volume 8 in The Oily Press Lipid Library Printed by Lightning Source PREFACE This is the fourth volume of an occasional series of review volumes dealing with aspects of lipid methodology to be published by the Oily Press. As with the first three volumes, topics have been selected that have been developing rapidly in recent years and have some importance to lipid analysts. The authors are all lead ing international experts. For example, the first chapter by Hal Gardner deals with the analysis of plant lipoxygenase metabolites, the equivalent of eicosanoids in plants. These may pro found metabolic functions but have sensitive structural features that can trap the unwary. This could be the definitive account of the subject for some time to come. This volume has three chapters dealing with different aspects of high-perfor mance liquid chromatography (HPLC). The first by Mike Bell describes methods for separating molecular species of phospholipids, both in native form and follow ing derivatization. Theoretical aspects are thoroughly covered, but the author has also selected a wide range of practical examples from both the plant and animal world, and especially from the more challenging area of marine science. To my knowledge, no adequate review of preparative HPLC of lipids has been published to date, but Paul Van der Meeren and Jan Vanderdeelen remedy this deficiency in style here. I am confident that all lipid analysts will find something of immediate practical value to their work. The third HPLC review covers reversed-phase HPLC of fatty acids and triacylglycerols. Many previous reviews have dealt with this topic simply from a historic perspective -who did what and when. I was con fident that Boryana Nikolova-Damyanova would not produce a facile chapter along such lines and she has not disappointed. Here you will find a critical review that covers the principles of the technique showing all its strengths and weak nesses, as well as providing valuable practical guidance. My main contribution is a chapter dealing with modem methods for structural analysis of fatty acids. Here, I have not aimed for completeness, but have placed the emphasis on those methods that have developed most rapidly since the publi cation of my book "Gas Chromatography and Lipids" (Volume 1 in the Oily Press Lipid Library). The intention was again to lay emphasis on actual applications to show the strengths of the newer methodology. If I have not succeeded in my aim please write and tell me. Finally, Charlie Scrimgeour contributes a chapter describing the exciting new methodology for analysis of stable isotopes in lipids, giving examples of the many fascinating applications in fields as far apart as geochemistry and medicine. As an appendix, I have prepared literature searches on lipid methodology for the years 1995 and 1996, continuing a feature established in the first three vol umes. The objective of the Oily Press is to provide compact readable texts on all aspects of lipid chemistry and biochemistry, and many more books are in the pipe line for The Oily Press Lipid Library. If you have suggestions or comments, please let us know. By a careful choice of authors and topics, I trust that this vol ume will again prove to have met all our aims. My personal contributions to the book are published as part of a programme funded by the Scottish Office Agriculture, Environment and Fisheries Department William W. Christie Chapter 1 ANALYSIS OF PLANT LIPOXYGENASE METABOLITES Harold W. Gardner National Center for Agricultural UtiliZtltion Research, Mycotoxin Research, ARS, USDA, Peoria, Illinois 61604, U.S.A. A. Introduction B. Formation of Metabolites: the Pathways 1. Lipoxygenase (LOX) pathway 2. Allene oxide synthase and the jasmonates 3. Hydroperoxide lyase (HPLS). 4. Hydroperoxide peroxygenase (HPPR) and hydroperoxide isomerases 5. Alkoxyl radical rearrangement C. Preparation of Substrates and Metabolites 1. Fatty acid hydroperoxides: step-by-step preparation 2. 3Z-Alkenal preparation 3. Preparation of AOS products 4. Epoxyhydroxyene and trihydroxyene fatty acids D. Recovery and separation of metabolites I. Extraction 2. Separation strategies E. Structural Analysis. 1. Chiral methods 2. Preparation of derivatives 3. Spectral methods A. INTRODUCTION The lipoxygenase (LOX) or oxylipin pathway is receiving increasing attention, possibly because of its role in the physiological processes of plants, and particu larly as a defense against pathogens. At this time it seemed useful to review the methods available to analyse the various metabolites of the pathway. First, the 2 ANALYSIS OF PLANT LIPOXYGENASE METABOLITES pathway itself is reviewed briefly. Because most of the current interest has focused on the steps subsequent to LOX action, there is a section on substrate preparation, such as fatty acid hydroperoxides and other metabolites. Methods of recovery and separation are the next logical topic to review. This is followed by the preparation of useful derivatives, and often a series of derivatives can be a powerful tool if used in conjunction with spectral methods. In regard to spec troscopy, gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy are the most useful techniques, but other spectral methods can fill some gaps in structural analyses. The goal of preparing this chap ter is to provide an easy-to-use guide for investigators who need not be lipid chemists. B. FORMATION OF METABOLITES: THE PATHWAYS 1. Upoxygenase (LOX) Pathway LOX is a 90+ kDa non-heme iron enzyme that has been cloned and sequenced from many sources, and the tertiary structure of LOX-I from soybean has now been determined by X-ray analysis [I]. Details concerning LOX can be found in a number of reviews [2-7]. Here the emphasis is placed mainly on the formation of fatty acid metabolites. In this review scant attention is given to oxylipins of marine algae, but a recent review can be consulted [8]. i) Normal Aerobic Oxidation. Although plant LOXs are capable of oxygenating a variety of fatty acids with a Z,Z-pentadiene moiety, there are generally two pre dominant fatty acids in plants with this functionality, namely linoleic and linolenic acids, which are excellent substrates for plant LOX. Of all the octadeca dienoic acids with a Z,Z-pentadiene at various positions, 9Z,I2Z-octadecadienoic acid (linoleic acid) is the best substrate using the soybean LOX-I isozyme [9]. LOXs with differing oxidation specificities and pH optima are known, but soy bean LOX-I remains the principal model for an co6 oxygenating LOX operating at an alkaline pH optimum and giving an S-stereospecificity [IO]; that is the prod ucts of linoleic and linolenic acid are I3S-hydroperoxy-9Z,I IE-octadecadienoic acid (13S-HPODE) and I3S-hydroperoxy-9Z,11E,I5Z-octadecatrienoic acid (13S-HPOTE), respectively. However, at neutral pHs this enzyme is capable of forming a small percentage of 9S-hydroperoxy-l OE, l 2Z-octadecadienoic acid (9S-HPODE) in addition to the principal 13S-HPODE [I I]. Our lab was the first to report a neutral pH LOX from com germ that oxidized linoleic acid to 9S HPODE principally [I2], and similar LOXs were soon reported in potato tubers [I3], tomato fruit [I4], and others. Figure I.la summarizes the product formation by these two types of LOXs. A third type of LOX, like soybean LOX-3, seemed more peroxidative in nature [IS], and the products had a somewhat racemic char acter, much like hydroperoxides obtained from autoxidation [I6]. Recently, an unusualdioxygenase that oxygenates C-8 oflinoleic acid has been identified in the fungi, Gaeumannomyces graminis [17] and Laetisaria arvalis [I8]. ADVANCES IN LIPID METHODOLOGY - FOUR 3 ~~H H+ yF e3+. .._ 18:2 [RH] HOO".' \. " ' . - Fe~+R" FejRO:!-~ 135-H~ODE OH H '-Fe2+Ro · ff+ ' 2 ~.,, 95-HPODE H /Free radical products / H Pentadienyl radical "-Free radical products Fig. Ll. a. Aerobic oxidation of linoleic and linolenic acids by LOXs showing electron cycling by the iron active site. b. Oxygen-starved reaction of linoleic acid and its hydroperoxide by LOXs showing electron cycling by the iron active site. Other products of LOX have been described. For example, fatty acids with three or more double bonds are theoretically capable of being dioxygenated by LOX, and such dihydroperoxides have been identified [19,20]. Plant LOXs will also form leukotriene- and lipoxin-like fatty acids through abstraction of a bisal lylic hydrogen from the methylene of a hydroperoxy-octatriene moiety [21,22]. ii) Oxygen-starved reactions ofW X. In the normal functioning of LOX, the fer ric-form of the enzyme oxidizes a fatty acid to its pentadiene radical, which then combines with oxygen to form a fatty acid peroxyl radical (Figure l.la). The fer rous-form of LOX next reduces the peroxyl radical to the hydroperoxyl anion (essentially the hydroperoxide final product). If oxygen becomes insufficient, the ferrous form of LOX has no choice but to reduce the product hydroperoxide to a fatty acid alkoxyl radical. Now, both the alkoxyl radical and the previously formed pentadiene fatty acid radical are free to react in ways typical of these free 4 ANALYSIS OF PLANT LIPOXYGENASE METABOLITES radicals (Figure l.lb). Products arising from the oxygen-starved LOX reaction of linoleic acid [23,24] and linolenic acid [25] have been identified. 2. Allene Oxide Synthase and the Jasmonates Reviews are available that detail research on the allene oxide synthase (AOS) branch of the LOX pathway [26-29]. i) AOS. AOS is a 55 kDa cytochrome P450 [30] that metabolizes fatty acid hydroperoxides into unstable fatty acid allene oxides [31]. In the past AOS has also been named hydroperoxide isomerase [32] (not to be confused with the hydroperoxide isomerase described below), hydroperoxide cyclase [33], and hydroperoxide dehydrase/dehydratase [31]. The ostensible reason plants have this enzyme is the cyclization of the allene oxide into 12-oxo-phytodienoic acid (12- oxo-PDA), the precursor of the phytohormone, jasmonic acid [34]. However, it is not generally appreciated that the majority of the allene oxide hydrolyses to an a ketol and to a lesser extent r-ketol (Figure 1.2). If the enzyme, allene oxide cyclase, is present and the substrate concentration is low, then the formation of 12- oxo-PDA is favored over the production of ketols. By manipulation of the solvent in which the allene oxide is dissolved, a number of interesting substitution prod ucts are formed by allene oxide chemistry [26]. Until recently it was thought that only the allene oxide derived from 13S-HPOTE cyclized (Figure 1.2), but now it has been documented that 13S-HPODE forms the 15,16-dihydro equivalent of 12- oxo-PDA in much smaller yield [35]. ii) Jasmonic acid. Almost all of the physiological research completed with jas monic acid has been with the synthetic racemate comprised of about 47.5% each of the natural (3R,7R) and unnatural (3S,7S) jasmonic acids and 2.5% each of the natural (3R,7S) and unnatural (3S,7R) 7-iso-jasmonic acid (also known as 2-epi or 2-iso-jasmonic acid)[36]. It is the (+)-(3R,7S)-7-iso-jasmonic acid that is directly derived from natural (9S,13S)-12-oxo-PDA (Figure 1.2), and is also the only isomer (as the methyl ester) to display the characteristic pleasant odor of syn thetic methyl jasmonate [36]. Natural (-)-jasmonic acid is derived either from epimerization of the ring carbon binding the pentenyl side chain of either precur sor 12-oxo-PDA or (+)-7-iso-jasmonic acid. There is a large number ofjasmonate derivatives known in nature, including conjugates with glucose and amino acids [see reviews, 27,37]. Because of the recent discovery of the involvement of 13S HPODE in the formation of 15,16-dihydro-12-oxo-PDA, it seems certain that 9,10-dihydro-jasmonic acid has its origin from this precursor [35]. 3. Hydroperoxide Lyase (HPLS) Like AOS, HPLS is also a 55 kDa heme enzyme, but it has not yet shown evi dence of being a cytochrome P450 [38]. It is interesting that the product interme- ADVANCES IN LIPID METHODOLOGY - FOUR 5 Allene oxide synthase H Alk_~ oxide} Allene oxide intermediate cy~ °):);;:-----~ ~ ) / '!-- " A r' ) ../',, I 7-lso-jasmonic acid Jasmonic acid 12-0xo-PDA H Fig. 1.2. Conversion of 13S-HPOTE into ketols. 12-oxo-PDA andjasmonates by action of AOS and allene oxide cyclase. diate of both AOS and HPLS has been suggested to be a carbocation, which loses a proton (AOS) or gains a hydroxyl ion (HPLS) in product formation (Equation 1): / + ,;q R' ~ A -... + R' R/~ R~ /+oH· ) ~R' HPLS is an important hydroperoxide-metabolizing enzyme of plants leading to chain cleavage between the hydroperoxide-bearing carbon and the vicinal double bond. Thus, 13-hydroperoxides cleave into a C-6 aldehyde and a C-12 ce>-oxo-acid and 9-hydroperoxides give a C-9 aldehyde and a C-9 ce>-oxo-acid (Figure 1.3). The °' ANALYSIS OF PLANT LIPOXYGENASE METABOLITES H o y H es ts b HQC\ •. "· ~ ~# H 9S-HPODE 135-HPODE ~ /~~ __..-HEXAN-1-0L I ~ 3Z-NONEN-1-0L __ / 3Z-NONENAL HEXANAL 2E-NONENAL 4-HYDROXY-2E-NONENAL / O= 2E-NONEN-1-0L H 12-0X0-9Z-DODECENOIC ACID ~H \ 2E,6Z-NONADI EN-1-0L " 12-0X0-10E-DODECENOIC ACID 9-0XONONANOIC ACID 2E-HEXEN-1-0L 2E,6Z-NONADIENAL __..- 2E-HEXENAL ~ 3Z,6Z-NONADIEN-1-0L I -I ----4-HYDROXY-2E-HEXENAL ~------~~ 3Z-HEXENAL ---------3Z-HEXEN-1-0L 3Z,6Z-NONADIENAL HO'\ ~ ~# H 13S-HPOTE 9S-HPOTE 1.3. Formation of aldehydes by action of HPLS on HPODEs and HPOTEs. Subsequent enzymic steps show conversion of: aldehydFig. (I) by alcohol dehydrogenase, (2) 3(9)Z-alkenals to 2(1 O)E-alkenals by isomerase, and (3) 3Z-alkenals to 4-hydroxy-2£-alkenalalcohols 3Z-alkenal dioxygenase and HPPR.
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