Advances in lipid methodology. / 4

302 Pages·25.138 MB·English

by  edited by William W. Christie, ....

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
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First published by The Oily Press, 1997
Reprinted by Woodhead Publishing Limited, 2013
©The Oily Press Limited, 1997; ©Woodhead Publishing Limited, 2012
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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
CONTENTS
Preface
Chapter 1
Analysis of plant lipoxygenase metabolites.
Harold W. Gardner 1
Chapter2
Separations of molecular species of phospholipids by high-performance
liquid chromatography.
Michael V. Bell 45
Chapter3
Preparative high-performance liquid chromatography of lipids.
Paul Van der Meeren and Jan Vanderdeelen 83
Chapter4
Structural analysis of fatty acids.
William W. Christie 119
Chapters
Analysis of stable isotopes in lipids.
Charles M. Scrimgeour 171
Chapter6
Reversed-phase high-performance liquid chromatography: general
principles and application to the analysis of fatty acids and triacylglycerols.
Boryana Nikolova-Damyanova 193
Appendix
Some important references in lipid methodology -1995.
William W. Christie 253
Some important references in lipid methodology -1996.
William W. Christie 273
Index 293
Contents of Previous Volumes 299

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
(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0) 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
(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)b(cid:0)(cid:0)ean 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-OHH ~ODEH
'-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.
(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)(cid:0)
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