Advances in Lipid Methodology. Volume 3

359 Pages·1996·20.264 MB·English

by  W. W. Christie (Auth.)

Woodhead Publishing in Food Science, Technology and Nutrition
Advances in
lipid methodology
Volume3
Edited by
William W. Christie
The Scottish Crop Research Institute, Invergowrie,
Dundee (DD2 SDA), Scotland
WP
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First published by The Oily Press, 1996
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British Library Cataloguing in Publication Data
A catalogue record for this book is available from the British Library
ISBN 978-0-9514171-6-4 (print)
ISBN 978-0-85709-800-9 (online)
This book is Volume 7 in The Oily Press Lipid Library
Printed by Lightning Source
PREFACE
This is the third 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
two volumes, topics have been selected that have been developing rapidly in
recent years and have some importance to lipid analysts. For example, Amis
Kuksis presents a timely review of non-enzymatic methods for the determination
of positional isomers of glycerolipids; chiral chromatography is especially impor
tant to this but various spectrometric methods must also be considered.
I trust it will not be too difficult to convince readers of the importance of high
performance liquid chromatography for analysis of phospholipids, and I have
attempted to review the topic from the concept of selectivity in the choice of
mobile and stationary phases. However, most of our readers will be less aware of
the value of 31P nuclear magnetic resonance spectroscopy as a non-destructive
means for accurate analysis of phospholipids. Glonek and Merchant should con
vince you of the great utility and importance of this technique. I was greatly
impressed by the high accuracy of the technique and with the certainty of identifi
cation of so many phospholipids. In addition, 3'P NMR spectroscopy is· proving of
great value for preparation of phase diagrams of lipids; Goran Lindblom is an
internationally recognised expert in this methodology and presents an authorita
tive account of the topic.
One of the most difficult tasks facing lipid analysts is to isolate and quantify
long-chain acyl-CoA esters. It is evident from this account by Jens Knudsen and
colleagues that much remains to be done before the problem is truly solved,
although valuable data can be obtained with care. This chapter will be essential
reading for anyone interested in the problem.
The final chapter is an especially comprehensive account of the analysis of
plant glycolipids. These are vital components of all plant membranes, but they are
frequently ignored in many general review articles on the analysis of lipids. Ernst
Heinz leaves us no excuse to do so in future. Here, there are detailed descriptions
of structures, chromatographic separations, chemical degradation and spectromet
ric analyses of these fascinating compounds. This is certainly the last word on the
topic.
As an appendix, I have prepared literature searches on lipid methodology for
the years 1993 and 1994, continuing a feature established in the first two volumes.
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 own contributions to the book are published as part of a programme funded
by the Scottish Office Agriculture, Environment and Fisheries Dept.
William W. Christie
Chapter!
ANALYSIS OF POSITIONAL ISOMERS
OF GLYCEROLIPIDS BY
NON-ENZYMATIC METHODS
Amis Kuksis
Banting and Best Department ofM edical Research,
University of Toronto, Toronto, Canada, MSG IL6
A. Introduction
B. Prochiral Nature of Acylglycerols
C. Spectrometric Methods of Analysis
1. Optical rotatory dispersion
2. Nuclear magnetic resonance spectroscopy
3. Mass spectrometry
D. Chromatographic Resolution of Regioisomers
1. Adsorption chromatography
2. Silver ion chromatography
3. Reversed-phase chromatography
4. Gas-liquid chromatography
E. Chromatographic Resolution of Stereoisomers
1. Random generation of acylglycerols
2. Preparation of derivatives
3. Resolution of diastereomers
4. Resolution of enantiomers
5. Calculation of fatty acid distribution
F. Biological Significance
G. Conclusions and Future Prospects
A. INTRODUCTION
Natural acylglycerols exist in complex mixtures of molecular species, which
differ in composition, molecular association and positional distribution of fatty
2 ANALYSIS OF POSITIONAL ISOMERS OF GLYCEROLIPIDS
acids. Although the biological significance of the acylglycerol structure is not
well understood, there is evidence that it is generated and maintained by the con
certed action of acyltransferases and lipases of high positional and fatty acid
specificity [4 1 ]. Furthermore, there is evidence that structured acylglycerols pos
sess different metabolic and physiological properties [12,40,59,83 and references
therein]. As a result, there is growing interest in the determination of the regio
and stereospecific distribution of the fatty acids in the acylglycerol molecules, and
in the quantification of the enantiomer content of each molecular species.
Historically, lipolytic degradation was first used to determine the fatty acids in
the primary and secondary positions of the acylglycerol molecules [10] and to dis
tinguish between enantiomers [9]. More recently, non-enzymatic methods have
been developed for this purpose culminating in the separation of diastereomeric
and enantiomeric acylglycerol derivatives on chromatographic columns. The fol
lowing chapter discusses the positional analysis of natural acylglycerols using the
non-enzymatic methods along with the more general physico-chemical
approaches to characterizing the regio- and stereo-configuration of acylglycerol
molecules. The topic has been reviewed previously in part by Takagi [95] and
Christie [14].
B. PROCHIRAL NATURE OF GLYCEROLIPIDS
Acylglycerols, glycerophospholipids and glycoglycerophospholipids have
glycerol as a backbone and are widely distributed as components of living cells.
Glycerol possesses a plane of symmetry at C and by itself is achiral or optically
2
inactive. It becomes chiral by introduction of different substituents at C1 and C3•
Thus, sn-1-monoacylglycerol is chiral, as is its enantiomer, sn-3-monoacylglyc
erol. Similarly, sn-1,2-diacylglycerol is chiral, as is its enantiomer, sn-2,3-diacyl
glycerol. Natural glycerophospholipids are optically active because the prochiral
positions at C and C are occupied by different substituents. Triacylglycerols are
1 3
racemic, if both sn-1- and sn-3-positions contain identical fatty acids, or enan
tiomeric, if the primary positions are occupied by different fatty acids. The gen
eral structure and prochiral nature of acylglycerols has been discussed further by
Takagi [95] and Christie [14].
Specifically, natural triacylglycerols are mixtures of molecular species, which
possess one, two or three different fatty acids of varying chain length and number
and configuration of double bonds [60]. Unknown mixtures of triacylglycerols
must be checked for the presence of alkyl or alkenyl groups [5]. These mixtures
are generally too complex for a complete stereospecific analysis [9]. The triacyl
glycerols must be prefractionated on the basis of carbon number (molecular
weight), degree of unsaturation, or a combination of the two chromatographic
techniques before positional analysis, in order to simplify assignment of each
fatty acid to the sn-1-, sn-2-and sn-3-positions of specific acylglycerol molecules
[41].
ADVANCES IN LIPID METHODOLOGY -THREE 3
The chiral nature of natural triacylglycerols arises mainly via the sn-1,2-diacyl
glycerols with a characteristic placement of saturated acids in the sn-1-and unsat
urated acids in the sn-2-positions during the biosynthesis of phosphatidic acid as
an intermediate in triacylglycerol biogenesis [80]. The triacylglycerol biosynthe
sis in the intestine proceeds largely via the 2-monoacylglycerol pathway, which is
characterized by retention of the fatty acid composition of the secondary position
of dietary triacylglycerols and also exhibits some non-randomness in the reacyla
tion of the primary positions [58]. In addition, the sn-2-position contains largely
unsaturated fatty acids, while the polyunsaturated acids are mainly confined to the
sn-3-position of natural triacylglycerols, although there are important exceptions
[7,41 ]. During the subsequent transport in plasma and clearance by tissues the tri
acylglycerols undergo extensive transformation, which results in complex
exchanges of fatty acids, but considerable specificity is retained. Thus, lipoprotein
lipase [1,67] and hepatic lipase [1] are known to attack preferentially the sn-1-
position and lingual lipase [1] the sn-3-position of the triacylglycerol molecule.
These enzymes also exhibit specificity during the lipolysis of the sn-1,2- and sn-
2,3-diacylglycerol and 2-monoacylglycerol intermediates (e.g. [67]).
C. SPECTROMETRIC METHODS OF ANALYSIS
Physical methods for regio-and stereochemical characterization of triacylglyc
erols were first explored by Schlenk [88]. The methods tested included the mea
surement of optical rotation, determination of piezoelectric effect, melting point
depression and X-ray diffraction. Since that time optical rotatory dispersion, pro
ton and 13C nuclear magnetic resonance (NMR) spectroscopy and mass spectrom
etry have been extensively explored for this purpose. The success of these meth
ods has varied with the asymmetry of the molecules examined and the extent of
the prefractionation of the sample. The spectrometric methods are considered here
only to the extent to which they have contributed to practical positional analysis of
glycerolipids.
1. Optical Rotatory Dispersion
Schlenk [88] found that triacylglycerols in which the three acids differed
greatly in chain lengths show measurable optical rotation. The small differences
in the optical rotation of most asymmetrical natural triacylglycerols can be
detected best by the highly sensitive techniques of optical rotatory dispersion and
circular dichroism (CD), which allow measurements in the vacuum ultraviolet
region. By means of this technique Gronowitz et al. [24] observed that saturated
triacylglycerols with the greater chain length at sn-1-than at sn-3-position possess
negative rotation, while triacylglycerols with the greater chain length at sn-3-posi
tion possess positive rotation. For pure triacylglycerols, therefore, optical rotatory
dispersion can indicate the presence or absence of asymmetry in the molecule, as
well as the stereochemical configuration, if appropriate standards are available.
Natural triacylglycerols with fatty acids differing greatly in chain length also
4 ANALYSIS OF POSITIONAL ISOMERS OF GLYCEROLIPIDS
show readily measurable optical rotation, and optically active triacylglycerols
have been identified in the seed oil of Euonymus verrucosus [39), where an acetyl
group, and in bovine milk fat [33), where an acetyl and a butyryl group are located
specifically in the sn-3-position.
Uzawa et al. [104) have performed CD studies and NMR spectroscopy on a
series of chiral sn-1,2-dibenzoylglycerols, and have proposed a general method to
correlate the sign of the exciton CD curves with the absolute stereochemistry of
the chiral dibenzoylglycerols: sn-1,2-dibenzoylglycerols give positive exciton
CD, which is independent of the type of substituents at C-3. Therefore, it should
be possible to determine the optical purity and the absolute configuration of a dia
cylglycerol, if it can be converted to the chiral dibenzoate.
Uzawa et al. [105) have applied this CD method for determining the optical
purity and absolute configuration of sn-1,2-(or sn-2,3-)-dibenzoylglycerol via the
corresponding tert-butyldimethylsilyl (t-BDMS) ether. Initially, significant
racemization occurred during the conversion of the chiral diacylglycerol into the
intermediate silyl ether. A detailed study of the procedure revealed that migration
of the silyl group occurred during the NaOMe/MeOH deacylation. Subsequently,
Uzawa et al. [106) developed an improved method to determine the optical purity
of sn-1,2 (or sn-2,3)-diacylglycerols via the silyl ether intermediate. The chiral
diacylglycerols were first silylated and the acyl groups were removed by Grignard
degradation to yield the sn-3-or sn-1-t-BDMS ether and subsequent benzoylation
led to the corresponding dibenzoylsilyl ether without racemization. The optical
purity was determined from the strong exiton Cotton effect, which was positive
for sn-3-t-BDMS-1,2-dibenzoylglycerol and negative for the sn-1-t-BDMS-2,3-
dibenzoylglycerol at 238 nm at a concentration of about 1 mM. The t-BDMS ether
of sn-1,2-dipalmitoylglycerol, which had caused difficulty with alkaline deacyla
tion, was readily deacylated by ethyl or methylmagnesium bromide in diethyl
ether at room temperature in a few minutes, while the benzoylation was smoothly
achieved by treatment in situ with excess benzoyl chloride to give the sn-1,2-
dibenzoylglycerol-3-t-BDMS ether. To avoid isomerization during the initial sily
lation of the diacylglycerol, the diacylglycerols were added to the silylating
reagents already in The method was successfully applied to determine
the stereo-selectivities of lipases from three sources, Pseudomonas, porcine pan
creatin and Candida using tripalmitoylglycerol as substrate. Figure 1.1 shows the
CD and UV spectra of dibenzoyl-sn-glycerol t-BDMS ethers in methanol as
derived from lipolysis of an achiral triacylglycerol [105). The advantage of
Uzawa's method is the ability to determine the optical purity and the absolute con
figuration of diacylglycerols of different acyl groups by CD without authentic
samples of known configuration.
2. Nuclear Magnetic Resonance Spectroscopy
High-resolution proton magnetic resonance (PMR) spectroscopy has been used
only to a limited extent because of the small number of signals, which limits the
ADVANCES IN LIPID METHODOLOGY -THREE 5
[8]x10·4
2 .....
CD OBz
{
BzO
OSitBuMe2
i
.§ or
\
0
j·· ..
n•
200 ,'/ 250 275 300
• I•
\'-.- I
I . I
\ I
\/
Authentic
-·-------·
-1 PPL
-··-··--· cc
-----·- E
PS
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AP
UV 3
1. 5
0
200 225 250 275 300 n•
Fig. 1.1. CD and UV spectra (in methanol) of the t-BDMS ethers of sn-1,2-and sn-2,3-dibenzoylglyc
erols as obtained following lipolysis of tribenzoylglycerol [105]. PPL, porcine pancreatic lipase; CC,
Candida cylindracea lipase; PS, Pseudomonas lipase; and AP, Amano P lipase. (Reproduced by kind
permission of the authors and of Biochemical and Biophysical Research Communications).
6 ANALYSIS OF POSITIONAL ISOMERS OF GLYCEROLIPIDS
amount of information. Bus et al. [11] employed PMR spectroscopy in combina
tion with a chiral shift reagent to resolve the signals from ester groups near the
center of chirality of triacylglycerols. The chiral shift reagents form complexes
via the free electron pairs of the ester groups of triacylglycerols. The two enan
tiomers of a triacylglycerol molecule form diastereomeric associations, which
give basically different PMR spectra. Using synthetic model enantiomers to
assign the signals, the absolute configuration of the main triacylglycerol of the
seed oil Euonymus alatus was found to be 3-acetyl-1,2-distearoyl-sn-glycerol
[39] and that of a monobutyryl triacylglycerol fraction from hydrogenated bovine
butter fat was confirmed to be mainly l,2-diacyl-3-butyryl-sn-glycerol [8,79].
Other studies by means of PMR spectroscopy in combination with achiral and
chiral chemical shift reagents have been employed to verify the primary versus
secondary positioning of fatty acids in synthetic triacylglycerols that contain satu
rated chains in combination with either unsaturated [78] or branched [107] chain
fatty acids. Lok et al. [6 2] have shown, by PMR spectroscopy with a shift reagent,
that the predominant enantiomer of the diacylglycerols present in fresh milk has
the sn-1,2-configuration. Recently, Rogalska et al. [86] have demonstrated char
acteristic differences between the PMR spectra of sn-1,2-diacylglycerol (R,R-car
bamate) and sn-2,3-diacylglycerol (S,R-carbamate) derivatives, which are
depicted in Figure 1.2.
In contrast, high resolution 13C NMR spectroscopy is emerging as a powerful
technique for lipid analysis. Gunstone [25] has recently reviewed the application
of this methodology to lipids and has noted the potential for positional analyses.
Pfeffer et al. [78] were first to demonstrate the primary positioning of butyric acid
in unaltered bovine milk triacylglycerols by 13C NMR spectroscopy. The primary
positioning of butyric and caproic acids in butterfat was later confirmed by 13C
NMR spectroscopy by Gunstone [26]. Ng [73,74] has shown that the positional
distribution of acyl groups in palm oil can be defined from the carbonyl 13C NMR
spectra region, and the positions of the unsaturated fatty acids can be discerned
also [73,75]. Wollenberg [108,109] has discussed in detail the high resolution 13C
NMR location of the 1(3)-acyl and 2-acyl groups in vegetable oils. The positional
distribution data for corn, peanut, canola and sunflower oils indicated that polyun
saturates are replaced in the 1,3-glycerol position exclusively by saturates, while
the oleoyl distribution remains random. In a subsequent study, Wollenberg [109]
has shown that a distortionless enhancement by polarization transfer techniques
significantly improves sensitivity to the extent that whole rapeseeds can be exam
ined within an hour of acquisition time. Furthermore, some 1(3)-or 2-acyl groups
could be identified leading to a partial estimation of the positional distribution of
the fatty acids.
The above techniques have removed much of the uncertainty concerning the
isomeric positioning of fatty acids in natural triacylglycerols as derived by enzy
matic methods. The chemical methods for determination of the enantiomer con
tent and absolute configuration of natural triacylglycerols will hopefully show a
similar good agreement with the results of the physico-chemical approaches.