Advances in Conjugated Linoleic Acid Research, Volume 3

252 Pages·2006·5.407 MB·English

by  Yurawecz, Martin P.

Advances in Conjugated
Linoleic Acid Research
Volume 3
Editors
Martin P. Yurawecz
Food and Drug Administration
Center for Food Safety and Applied Nutrition
College Park, MD
John K.G. Kramer
Food Research Center
Agriculture and Agri-Food Canada
Guelph, Ontario, Canada
Ola Gudmundsen
LINK Medical Research
Oslo, Norway
Michael W. Pariza
Food Research Institute
Department of Food Microbiology and Toxicology
University of Wisconsin-Madison
Sebastiano Banni
Dipartimento di Biologia Sperimentale,
Università degli Studi di Cagliari, Italy
Champaign, Illinois
Copyright ©2006 by AOCS Press
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R. Adlof, USDA, ARS, NCAUR, Peoria, Illinois
P. Dutta, Swedish University of Agricultural Sciences, Uppsala, Sweden
T. Foglia, ARS, USDA, ERRC, Wyndmoor, Pennsylvania
V. Huang, Yuanpei University of Science and Technology, Taiwan
L. Johnson, Iowa State University, Ames, Iowa
H. Knapp, Deaconess Billings Clinic, Billings, Montana
D. Kodali, Global Agritech Inc, Minneapolis, Minnesota
T. McKeon, USDA, ARS, WRRC, Albany, California
R. Moreau, USDA, ARS, ERRC, Wyndoor, Pennsylvania
A. Sinclair, RMIT University, Melbourne, Victoria, Australia
P. White, Iowa State University, Ames, Iowa
R. Wilson, USDA, REE, ARS, NPS, CPPVS, Beltsville, Maryland
Copyright ©2006 by AOCS Press. All rights reserved. No part of this book may be
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ISBN 978-1-893997-87-5
Library of Congress Cataloging-in-Publication Data
Advances in conjugated linoleic acid research/editors,
Martin P. Yurawecz . . . [et al.].
p. cm.
Includes bibliographical references and index.
ISBN
1. Linoleic acid—Metabolism. I. Yurawecz, Martin P.
QP752.L5A38 1999
612.3’97—dc21
99-32986
CIP
Printed in the United States of America
Copyright ©2006 by AOCS Press
• l l •
Preface
Interest in the chemistry and biological properties of conjugated linoleic acid (CLA)
continues to grow. The number of peer reviewed publications on CLA that have
appeared in the literature since the publishing of the last volume in this series
(Advances in Conjugated Linoleic Acid Research, Vol.2) has more than doubled. CLA
can be a single isomer or a mixture of fatty acids (FA) that contain 18 carbons and at
least 2 double bonds separated by a single bond. The term CLA was originally coined
to reflect the origin of these FA from alkali isomerization of linoleic acid (cis-9,cis-
12-octadecadienoic acid, c9,c12-18:2) in which the double bonds of c9,c12-18:2 are
converted with an appropriate catalyst primarily to c9,t11-18:2 and t10,c12-18:2. The
solvent, temperature, time and catalyst will determine how many CLA isomers (and
other compounds) will be formed in the reaction mixture. These contrasts with the
biosynthesis of CLA in ruminants produced by enzymatic isomerization and reduction
of polyunsaturated FAs, desaturation of monounsaturated FAs, and elongation and
b-oxidation of CLA isomers. The main CLA isomer in ruminants is rumenic acid
(c9,t11-18:2), plus many other CLA isomers formed by different enzymatic processes
in numerous rumen bacteria. A summary of biosynthesis of CLA in ruminants is
outlined in Chapter 1, while the metabolic processes of CLA in animal and human
tissues are presented in Chapter 2.
The CLA and 18:1 isomer composition in the milk and meat fat of ruminants
is a mixture of numerous positional and geometric isomers, most of which are
generated by specific rumen bacteria, or subsequently re-synthesized in tissues by
specific enzymes. To understand their biosynthesis, with the aim of manipulating
these biochemical processes, requires appropriate techniques to determine each of
the individual isomers with confidence. Detailed chemical syntheses are presented
in Chapter 3 to prepare appropriate standards; Chapter 4 provides a summary of
complementary gas-, adsorption-, and argentation-chromatographic techniques
required for the analysis of all the CLA and trans- and cis-18:1 isomers; Chapter
5 presents improved separations of the CLA isomers using modified silver ion and
reverse phase HPLC techniques; while Chapter 6 is devoted to a complete structural
characterization of the methyl esters of CLA isomers using acetonitrile chemical
ionization tandem mass spectrometry.
The diversity of CLA structures is beginning to reflect marked differences between
the CLA isomers in their biological response, as pure isomers are becoming available
• iii •
Copyright ©2006 by AOCS Press
l Preface
for study. The multi-functional effect of the different CLA isomers is discussed in
Chapter 7, while Chapter 8 specifically deals with the immune properties of the CLA
isomers and their association with inflammation. The recently discovered effect of
CLA attenuating hypertension is discussed in Chapter 9. Chapter 10 explores the
potential role of CLA in cellular membranes by comparing the oxidative properties of
CLA isomers in liposomes using the electron spin resonance technique (ESR).
The remaining four chapters address our current understanding of the effects of
CLA isomers in humans. Chapter 11 summarizes the many attempts to determine
if dietary CLA affects body composition and body weight. A more precise location
of CLA in the organelles of healthy and cancerous human tissues is compared in
Chapter 12. The lipid-lowering actions of t10,c12-18:2, in primary cultures of
human (pre)adipocytes are discussed in Chapter 13. The final Chapter 14 presents an
overview of safety of CLA based on animal studies.
Research to determine the benefits of CLA for humans will present a
unique challenge in the next few years. It will certainly require the improved and
complementary methods of CLA and trans FA analysis to evaluate the biological
effects of dietary supplements to determine the true effects of CLA in humans. In
some studies the presence of CLA in human bodies and those produced by bacteria in
the gut may need to take into consideration. The CLA research to date in humans has
focused mainly on the effects of t10,c12-18:2 rather than on c9,t11-18:2, the major
CLA isomer present in the milk and meat of ruminants. This would appear to be due
to the readily available source of commercial CLA preparations compared to pure
c9,t11-18:2. Commercial CLA preparations consist of an equal mixture of t10,c12-
18:2 and c9,t11-18:2. On the other hand, the t10,c12-18:2 isomer is generally present
only in trace amounts in milk fats, while some ruminant fats may contain more of
the CLA isomers. The true response, if any, of c9,t11-18:2 in humans remains to be
determined. Several cohort studies have shown a significant reduction in risk factors
associated with the consumption of diets high in dairy products and certain types
of cancers but not with others. It remains to be seen if the benefits were due to
c9,t11-18:2, other components in dairy products, or synergistic processes. Regardless
of whether CLA’s apparent benefits can be translated to humans, it is likely that CLA,
as a model test object, will be used in the future in many more studies related to major
maladies such as cancer, atherosclerosis, diabetes, etc.
Martin P. Yurawecz
John K.G. Kramer
Ola Gudmundsen
Michael W. Pariza
Sebastiano Banni
iv
Copyright ©2006 by AOCS Press
Contents
Preface ..................................................................................................................... iii
Part 1 l Biosynthesis and Metabolic Processes
1 Conjugated Linoleic Acid Synthesis within the Gut Microbial
Ecosystem of Ruminants
Beverly B. Teter and Thomas C. Jenkins .....................................................................3
2 Detection of Partial b-Oxidation Products of Conjugated Linoleic Acid Isomers
and Their Metabolites in Animals and Humans
Gianfranca Carta, Maria Paola Melis, Elisabetta Murru, Lina Cordeddu,
Leonardo Lucchi and Sebastiano Banni....................................................................19
Part 2 l Techniques of Determination of Individual Isomers
3 Syntheses of Conjugated Linoleic Acids
Carolin Kellersmann, Lutz Lehmann, Wittko Francke, and Hans Steinhart ...............27
4 Systematic Analysis of trans and Conjugated Linoleic Acids in the Milk and
Meat of Ruminants
Cristina Cruz-Hernandez, John K.G. Kramer, Jana Kraft, Viviana Santercole, Mamun
Or-Rashid, Zeyuan Deng, Michael E.R. Dugan, Pierluigi Delmonte and
Martin P. Yurawecz ...............................................................................................45
5 New Developments in Silver Ion and Reverse Phase HPLC of
Conjugated Linoleic Acid
Pierluigi Delmonte, John K.G. Kramer, Sebastiano Banni, and
Martin P. Yurawecz ................................................................................................95
6 Structural Characterization of Conjugated Linoleic Acid Methyl Esters with
Acetonitrile Chemical Ionization Tandem Mass Spectrometry
Anthony L. Michaud and J. Thomas Brenna ..........................................................119
Part 3 l Diversity of Conjugated Linoleic Acid
7 Growth Inhibition and Apoptotic Cell Death of Cancer Cells
Induced by Conjugated Linoleic Acid
Masao Yamasaki, Hitomi Chujo-Miyazaki, and Koji Yamada .................................141
8 Modulatory Properties of Conjugated Linoleic Acid on Inflammation and
Immune Function: Cellular and Molecular Mechanisms
Josep Bassaganya-Riera and Raquel Hontecillas.......................................................153
9 Conjugated Linoleic Acid in Hypertension
Teruyoshi Yanagita, Koji Nagao, Nao Inoue, and Yu-Ming Wan .g............................171
v
Copyright ©2006 by AOCS Press
l Contents
10 A ntioxidative Activity of Conjugated Linoleic Acid Determined by ESR
Jun-Jie Yin, Liangping Yu, Martin P. Yurawecz, John A.G. Roach, Magdi M. Mossoba,
Liangli Yu, and John K.G. Kramer ......................................................................183
Part 4 l Effects of Conjugated Linoleic Acid Isomers in Humans
11 An Overview of the Effects of Conjugated Linoleic Acid on Body Weight and
Body Composition in Humans
Jean-Michel Gaullier and Ola Gudmundsen ........................................................203
12 Conjugated Linoleic Acid in Healthy and Cancerous Human Tissues
Kristina Hoffmann and Hans Steinhart ...............................................................209
13 Lipid-Lowering Actions of trans-10, cis-12 Conjugated Linoleic Acid in Pri-
mary Cultures of Human (Pre)Adipocytes
J. Mark Brown and Michael K. McIntosh ............................................................227
14 Safety Data on Conjugated Linoleic Acid from Animal Studies: An Overview
Margriet van der Zee, Sue O’Hagan, and Andreas Menzel ....................................239
vi
Copyright ©2006 by AOCS Press
2
• l l •
Detection of Partial
b-Oxidation Products of
Conjugated Linoleic Acid Isomers
and Their Metabolites in
Animals and Humans
Gianfranca Cartaa, Maria Paola Melisa, Elisabetta Murrua, Lina Cordeddua,
Leonardo Lucchia and Sebastiano Bannic
aUniversità degli Studi di Cagliari Dipartimento di Biologia Sperimentale Cittadella Universitaria 09042
Monserrato, Cagliari, Italy; b1 Dipartimento di Medicina Interna, Divisione di Nefrologia Dialisi e Trapianti,
Policlinico Unversitario di Modena, Modena I-41100 Italy; cDipartimento di Biologia Sperimentale,
Università degli Studi di Cagliari, Cagliari, Italy
Acknowledgments
This work was supported by a grant from EU Project N° QLK1-2002-02362.
The authors thank Prof. Benito Lombardi for revising the manuscript and Mr. Gia-
como Satta for technical assistance.
Introduction
The mechanisms of action by which CLA isomers exert beneficial activities have not
yet been fully clarified. Their metabolic pathways give hints of how they may interfere
with PUFA metabolism under a variety of experimental conditions and in different
pathological states. It has been shown that CLA isomers are metabolized to longer
chain PUFA that maintain the conjugated diene structure. Their metabolism shows
differences though, with t10,c12 yielding conjugated deiene 18:3 by D-6 desatura-
tion, but no detectable amounts of CD 20:3 (1). In contrast, c9,t11 CLA generates
less CD 18:3 but high amounts of CD 20:3 (1). In a few studies, detection of CD
20:4 has been reported in animal or human tissues; in all these cases, the level of di-
etary linoleic acid was low (2).
Conjugated diene fatty acids with 16 carbon atoms, probably derived from per-
• 19 •
Copyright ©2006 by AOCS Press
20 l G. Carta, et al.
oxisomal b-oxidation of CLA and its metabolites, have also been detected (2, 3).
These findings suggest that CLA and derivatives may be metabolized actively in per-
oxisomes. Therefore, CLA isomers yield both longer and shorter chain conjugated
PUFA. While longer chain PUFA probably interfere with LA metabolism, it is not
yet clear whether the formation of 16-carbon conjugated fatty acids contributes to the
biological activities of CLA. The longer chain conjugated fatty acids, as well as the
two main CLA isomers, show differences in the yield of conjugated 16-carbon fatty
acids (2).
Formation of CD 16-carbon fatty acids probably takes place in the peroxisomes
which are known to shorten fatty acids and to release them outside of peroxisomes
(4). Peroxisome metabolism is supported by the fact that CLA is a high-affinity ligand
for peroxisome proliferator-activated receptors (PPAR), a family of transcription fac-
tors known to affect gene expression and glucose and lipid metabolism. In this chap-
ter we will briefly review initial evidence of CLA isomer metabolism in peroxisomes
and its possible implications for CLA biological activities.
Peroxisomes
The peroxisome is a subcellular organelle present in most animal and plant tissues and
in unicellular eukariotes. Peroxisomes are most abundant and larger in size in liver
parenchymal cells than in most other mammalian cells. They are typically spherical
or slightly elliptical and consist of a unit membrane, a finely granular matrix, and a
dense paracrystalline core in some species. In rat liver, peroxisomes have a mean diam-
eter between 0.2 to 1 mm with an average of 0.7 mm. Normally, there are between 370
and 620 peroxisomes per parenchimal cell, and together they take up about 2% of
the cell volume. Peroxisomes have one-fourth to one-sixth the volume of the average
mitochondrion, and are four to two times less numerous than mitochondria. They
constitute 2.5% of total liver proteins or about 6.5 mg per g of liver. Catalase is the
most abundant protein of rat liver peroxisomes, contributing 16% to the total per-
oxisomal protein. The core contributes 10% protein and is mostly, if not exclusively,
urate oxidase. All other proteins are present in smaller amounts.
The phospholipids of the peroxisome membrane are the same as those found in
ER membranes, but the proportions are different. The peroxisomal membranes con-
tain pores permeable to NAD and CoA as well as smaller molecules such as sucrose,
lactate, urate, and amino acids.
Many of the biochemical reactions occurring in peroxisomes are oxidative and
function in catabolic pathways; however, some are anabolic, and some endproducts
of peroxisomal reactions are used as building blocks for anabolic reactions elsewhere
in the cell. Peroxisomal functions are essential to human life, as shown by often lethal
diseases involving malfunctions or the biogenesis of the organelles. In this chapter we
will mainly focus on peroxisomal b-oxidation.
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Detection of Partial b-Oxidation Products of CLA Isomers and Their Metabolites l 21
CLA Peroxisomal b-Oxidation in Rats
The detection of two fatty acids with characteristic conjugated diene UV spectra,
HPLC retention times, and MS spectra, identifying them as CD 16:2 and CD 16:3,
led us to hypothesize that they are degradation products of CLA and its metabolites
(3). Significantly higher levels of CD 16:2 than of CD 16:3 were detected in liver,
plasma, kidney and adipose tissue of rats fed CLA-enriched butter, which accounted
for about 0.8% of c9, t11 CLA present in the diet (3). Tissue distributions of CD 16:2
and CD 16:3 were similar, with plasma and adipose tissue showing the highest levels,
kidney the lowest and the liver an intermediate level. CD 16 fatty acids accounted
for about 20% of total CLA metabolites, but was only 11% in the kidneys. Interest-
ingly, kidney is known to be less responsive than liver to peroxisomal b-oxidation
induction (5). Analyses of liver lipid classes showed that CD 16:2 and CD 16:3 were
preferentially incorporated into neutral lipids (3). This preferential incorporation was
very similar to that of CLA, as previously shown (6). We hypothesize that CD 16:2
and CD 16:3 are derived from partial b-oxidation of CLA and its metabolites, re-
spectively. Incubation with c9, t11 CLA of skin human fibroblasts from patients with
X-linked adrenoleukodystrophy (ALD), a disease where peroxisomal b-oxidation is
compromised, showed that CD 16:2 formation was about 50% of that in control
cells (3). This result was taken as an indication that CD 16:2 is formed at least in
part in peroxisomes. Further evidence supporting this possibility was obtained very
recently in our laboratory from profiles of plasma and adipose tissue fatty acids of a
patient affected by long-chain CoA dehydrogenase (LCAD) deficiency. The ratio CD
16:2/CLA in the patient’s plasma was 10 times higher than in the plasma of control
subjects (manuscript in preparation). LCAD is a key enzyme for mitochondrial b-
oxidation of fatty acids, and its deficiency results in the accumulation of fatty acids
partially b-oxidized in peroxisomes, such as c16:1n9, c14:1n9, c16:2n6, and c14:2n6.
Accumulation of CD 16:2 from naturally occurring CLA should be also expected.
Sebedio et al. described the detection of t8,c10 16:2 in liver of rats fed t10,c12 (7).
Recently, we have shown that t10,c12 CLA results in the presence in most tissues of a
much higher level of CD 16:2 than of c9,t11 (manuscript in preparation). However, it
is unclear whether the CD 16 fatty acids are formed in the liver and then transported
to the other tissues by the lipoproteins in the bloodstream. Since they are mainly in-
corporated into liver neutral lipids, CD 16 fatty acids could very well be incorporated
into VLDL and then transported to extra-hepatic tissues, as evidenced by their high
concentration in plasma and adipose tissue. Further studies should be undertaken to
determine whether the flux from liver to extra-hepatic tissues is a major pathway or
whether CD 16 fatty acids are formed in situ in the various tissues. Recently we have
detected CD16 fatty acids in the brain of rats fed CLA (submitted for publication).
Interestingly, the ratio CD16:2/CLA was higher in the brain than in the other tissues.
In order to establish whether brain cells are able to produce CD16:2, or that found
therein derived from plasma, we incubated astrocytes with CLA; CD16:2 was found
to be formed at a rate similar to that observed in vivo in the brain. This may imply
Copyright ©2006 by AOCS Press
22 l G. Carta, et al.
that CLA is not only readily incorporated into brain lipids, but it is also efficiently b-
oxidized in peroxisomes, a possibility that could open new perspectives on potential
biological activities of CLA in the brain.
CLA Peroxisomal b-Oxidation in Humans
As mentioned above, the capacity of human cells to partially b oxidize CLA was
established by incubating human skin fibroblasts with a mixture of the two isomers;
both yielded CD 16:2 identified by HPLC as c7,t9 CD 16:2 and t8,c10 CD 16:2,
respectively (2). Interestingly, most of the CD 16:2 formed (about 70%) was found
in the medium length CD (2). Again, when human skin fibroblasts from an ALD
patient were used, the level of CD 16:2 generated from both isomers was much lower
than that found in control human skin fibroblasts.
We also checked whether CD 16:2 was formed in humans in vivo. In a pilot
trial where subjects with type 2 diabetes mellitus were supplemented with 6 g/day of
a CLA mixture, we detected CD 16:2 from both isomers, and again CD 16:2 from
t10,c12 was more abundant reflecting the data obtained in vitro with skin fibroblasts
(2) and in vivo in rats (manuscript in preparation). It appears therefore that both
isomers are partially b-oxidized in peroxisomes in both experimental animals and
humans, in vivo and in vitro. More studies are needed to find out whether the me-
tabolism of CLA is directly linked to its activity as a ligand of PPAR a. It has actually
been found that CLA is able to upregulate acyl CoA oxidase, the key enzyme of per-
oxisomal b-oxidation (8). If this link is definitively proven, CD 16:2 could be used as
a marker of PPAR a activation.
An altered ratio CD 16:2/CLA was also found in patients under peritoneal dia-
lytic treatment with high levels of C reactive protein (CRP) a marker of inflammation
(manuscript in preparation). It is tempting to speculate that a degree of inflammation
leading to enhanced production of eicosanoids may increase peroxisomal b-oxidation
as a mechanism to control eicosanoid concentration, by increasing their degradation.
The induction of peroxisomal b-oxidation by CLA could thus explain its antinflam-
matory activity. Furthermore, an energetically inefficient fatty acid b oxidation could
contribute also to the decrease of fat mass.
Conclusions
The metabolism of CLA in peroxisomes shows interesting aspects in terms of a pos-
sible role in the formation and concentration of eicosanoids, especially as effected in
different pathologic conditions. By either competing with linoleic acid for desatura-
tion and elongation, or via peroxisomal b-oxidation, CLA may interfere with eico-
sanoid production and degradation. It is likely however that its action may depend
on its incorporation and thereby its concentration in different tissues. The different
rate of peroxisomal b-oxidation between t10,c12 and c9,t11, may also explain the
different biological activities of the two isomers. Also, enhanced fatty acid oxidation
Copyright ©2006 by AOCS Press