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CONJUGATED LtNOLEIC ACID:
ANALYSIS, TISSUE DISTRIBUTION, METABOLISM, AND
ITS EFFECTS ON IMMUNE-INDUCED GROWTH DEPRESSION IN RATS
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy
in the Graduate School of The Ohio State University
By
Yiyu Fang, M.S.
******
The Ohio State University
1998
Dissertation Committee: Approved by
Professor David B. Min, Adviser
Professor Yung-Sheng Huang
Professor Ahmed E. Yousef Adviser
Professor Tammy M. Bray Food Science and Nutrition Graduate Program UMI Nimber: 9833974
UMI Microform 9833974 Copyright 1998, by UMI Company. All rights reserved.
This microform edition is protected against unauthorized copying under Title 17, United States Code.
UMI 300 North Zeeb Road Ann Arbor, MI 48103 ABSTRACT
Conjugated linoleic acid (CLA) has been reported to be a potential nutrient for
improving human health. However, detailed information on its effects in animal is not
available. This study examed the the effect of dietary CLA on the response of rats to the
imminu-induced growth depression caused by endotoxin injection and the posible mechanism involved in the effect. Four week old rats were fed either a 1% linoleic acid
(LA) or 1% CLA supplemented diet (30 rats in each dietary group) for 30 days. At day
31 of the experimental diet, 24 rats from each dietary group were injected with 2 mg
LPS/kg body weight. Body weight and feed consumption were monitored daily for 7 days after the endotoxin injection. Six rats from uach dietary group were sacrificed at 6, 24,
72, and 144 hour after the endotoxin injection. Plasma, liver, heart, lung, spleen, kidney, muscle and adipose tissue were collected for the lipid content analysis. Plasma prostaglandin 52 (PGE 2) and leukotriene B 4 (LTB4 ) were determined by enzyme immunoassay. The lipid contents of all tissues except heart in the CLA group were less than those in the LA group (p<0.05) while ihe organ weights in both groups were not significantly different. Rats in the CLA group experienced less body weight lose (p<0.05) and more food intake than the LA group after the endotoxin injection. Plasma LTB 4 in CLA group was significantly lower than in the LA group, but PGE 2 in both group were similar. Dietary CLA can effectively reduce lijiid contents of tissues and help to overcome the growth depression due to endotoxin injection.
UJ ACKNOWLE DGMENTS
I would like to thank my adviser, Dr. David B. Min, for his support, direction,
encouragement, and enthusiasm which made this dissertation possible.
I would like to thank Dr. Yung-Sheng Huang for his intellectual support, constant
encouragement, and his patience in correcting both my stylistic and scientific errors. I also would like to thank the members of my committee. Dr. Ahmed E. Yousef, Dr. Tammy
Bray, for their time and useful comments that ed to the improvement of this dissertation.
Special thanks is for Dr. Jim Liu and E. Bobik for their ideas, time, and superior
technical assistance. I am grateful for t ie financial support received from Ross
Laboratory.
My sincere appreciation is expressed to rny parents and my husband for their patient and constant support.
IV VITA
March 23, 1963 ...... Bom. Gangzhou, P.R. China
June, 1985 ...... B.S. Chemistry Department of Chemistry Harbin Institute of Technology Harbin, P. R. China
June, 1988 ...... M.S. Analytical Chemistry China University of Geosciences Beijing, P.R. China
July, 1988 - November, 1991 ...... Research Associate, Department of Chemistry, China University of Geosciences, Beijing, P.R. China
August, 1995 ...... M.S. Nutrition Department of Food Science & Human Nutrition Iowa State University.
January, 1992-July, 1995 ...... Research Assistant, Department of Food Science & Human Nutrition, Iowa State University.
October, 1995 - October, 1997 ...... Graduate Research Associate, Department of Food Science & Technology, The Ohio State University. PUBLICATIONS
1. Fang. Y., T.L. Li, D.B. Min, T. Bray, E. Bobik, and Y.S. Huang, Analysis of conjugated linoleic acid (CLA) in animal tissue samples. Abstract for 1997 IFT Annual Meeting, 1997.
2. Fang, Y., D.B. Min, T. Bray, and Y.S. Huang, Effeet of short-term feeding of conjugated linoleic acid (CLA) on fatty acid distribution in rat tissues. Abstract for 88 th ACCS Annual Meeting, 1997.
3. R. Serfass, Y. Fang and M. Wastncy, Zinc kinetics in weaned piglets fed marginal zinc intake: compartmental analysis of stable isotopic data. The Journal o f Trace Elements in Experimental Medicine, 9:73-86, 1996.
4. R. Serfass, Y. Fang and M. Wastncy, Compartmental modeling of stable isotopic zinc metabolism by piglets fed marginal zinc intakes. Abstract for Experimental Biology 95^^^. 5023, 1995.
5. Freeman, S.P.H.T., R E. Serfass, C. King, J R. Southon, Y. Fang, L.R. Woodhouse, G. S. Bench, and J.E. McAninch, Biological sample preparation and '^'Ca AMA measurement at LLNL. Nuclear Instruments and Methods in Physics Restarch, B99:557-561, 1995.
6 . R. E. Serfass, Y. Fang and J. C. King. Preparation of Urinary Calcium for ‘^^Ca/'^^Ca Determination b\" Accelerator Mass Spectrometry i AMS). Abstract for Experimental Biology 9 5 ™ , 4101,1994.
FIELDS 01' STUDY
Major Field: Food Science and Nutrition
VI TABLE OF CONTENTS
Page. A bstract ...... ii Acknowledgments ...... vi V ita ...... V Publications ...... vi Table of Contents ...... vii List of Tables ...... ix List o f Figures ...... x
Chapters
1. Introduction ...... l 1.1 Hypothesis ...... 4 1.2 Objectives ...... 7 1.3 Significance of this research ...... 8
2. Literature Review ...... 10 2.1 Conjugated linoleic acid ...... 10 2.1.1 Definition and chemical structures ...... 10 2.1.2 CLA analysis ...... 11 2.1.3 CLA in food ...... 13 2.1.4 CLA in biological materials ...... 17 2.1.5 CLA Origin in Animals and Humans ...... 20 2.1.6 CLA Intestinal absorption and metabolites ...... 24 2.2 Physiological properties of CLA ...... 28 2.2.1 CLA and cancer ...... 28 2.2.2 CLA and atherosclerosis ...... 39 2.2.3 CLA and immune functions ...... 42 2.2.4 CLA effect on growth and body composition ...... 46 2.3 Proposed mechanisms for CLA effects ...... 49 2.3.1 Antioxidant activity ...... 49 2.3.2 Prooxidant cytotoxicity ...... 52 2.3.3 Modulation of eicosanoid biosynthesis ...... 54 2.3.4 Inhibition of nucleotide synthesis and DNA-adduct formation 60 2.3.5 Modulation of signal transduction ...... 61
vii 3. Material and Methods ...... 64 3.1 Sources of materials ...... 64 3.2 General methods ...... 65 3.2.1 Tissue lipid analysis ...... 65 3.2.2 Plasma eicosanoid analysis ...... 70 3.3 Animals and experimental designs ...... 72 3.3.1 Short term animal experiment ...... 73 3.3.2 Long term animal experiment ...... 75 3.4 Statistic analysis ...... 76
4. Results and Discussion ...... 78 4 .1 Established analytical methods for tissue lipid analysis ...... 79 4.1.1 GC method for CLA quantification and fatty acid profile determination ...... 79 4.1.2 TLC separation of lipid fractions ...... 84 4.1.3 HPLC purification of CLA isomers ...... 87 4.1.4 GC method for CLA isomer analysis ...... 89 4 .1.5 Methods for plasma LTB 4 and PGE 2 analysis...... 102 4.2 Results from animal studies ...... 104 4.2.1 CLA tissue distribution and metabolism ...... 105 4.2.2 Growth of rats before and alter endotoxin injection ...... 117 4.2.3 Plasma eicosanoid concentrations before and after endotoxin injection ...... 125 4.2.4 Tissue fatty acid composition ...... 133 4.2.5 Tissue lipid contents ...... 137
5. Conclusions and Future W ork ...... 142 5.1 Conclusions ...... 142 5.2 Suggestions for future research ...... 145
List of References ...... 147
Appendix - A: Fatty acid composition of refeience standard 68 A ...... 162 Appendix - B: Fatty acid composition of reference standard RL 6 ...... 163 Appendix - C: Method o f lipid extraction ...... 164 Appendix - D: Method of sampnification and méthylation ...... 165 Appendix - E: Method for CLA synthesis and tlO,cl2-CLA isomer crystallization 166 Appendix - F: Method of enzyme immunoassay of leukotriene 84 (LTB4) ...... 169 Appendix - G; Method of enzyme immunoassay of prostaglandin E 2 (PGE2) ...... 173
V lli LIST OF TABLES
Table Page
1. CLA content in various food products ...... 14
2. Equivalent chain length (ECL) values of methyl CLA isomers and CLA isomer identification ...... 97
3. Major fatty acids in various tissues of rats fed with control linoleic acid diet or CLA supplemented diet for 4 weeks ...... 135
4. The major fatty acid contents in liver triacylglyceride, free fatty acids, and phospholipid fractions of rats treated with a signal CLA gavage dose. The liver samples were taken 8 hours after the gavage dose ...... 137
IX LIST OF FIGURES
Figure Page
1. Pathways of biosynthesis of eicosanoids from arachidonic acid ...... 5
2. Scheme of tissue sample preparation and analyses ...... 66
3. Scheme of plasma eicosanoids analysis ...... 71
4. Short term animal experiment design ...... 74
5. Long term animal experimental design ...... 76
6 . Capillary (SP2380) GC elution profile of various fatty acid methyl esters (standard 6 8 A) cochroinatographed with the CLA methyl ester m ixture...... 81
7. Capillary (SP2380) GC elution profile of CLA methyl ester mixture obtained from Sigma Chemical Company ...... 82
8 . Capillary (column SP2380) GC elution profile of fatty acid methyl esters from a liver sample ...... 84
9. TLC chromatogram for the separation of iiver lipid ...... 87
10. HPLC elution profile of CLA cochromatcgraphed with various fatty acids ...... 89 11. Capillary (Omegawax 320) GC elution profile of CLA methyl ester mixture (Sigma Chemical Com pany)...... 91
12. Capillary (Omegawax 320) GC elution profile of various methyl esters of saturated fatty acid standards cochromatographed with the methyl CLA isomer mixture from Sigma Chemical Company ...... 94 13. The calibration curve of equivalent chain length (ECL) for CLA isomers ...... 95
14. Capillary (Omegawax 320) GC elution profiles of the synthesized CLA isomer mixture ...... 99
15. Capillary (Omegawax 320) GC elution profiles of the crystal phase of synthesized CLA ...... 100
16. Capillary GC elution profiles (Omegawax 320) of the supernatant phase of the synthesized CLA ...... 101
17. CLA concentrations in different tissues of rats in control and CLA group 8 hours after the gavage dose ...... 107
18. CLA concentration in different tissues of rats in control and CLA groups 24 hours after the gavage dose ...... 108
19. CLA concentrations in various tissues of rats fed with control or 1% CLA diet for four weeks ...... 110
20. The CLA concentration changes in liver heart, liver, muscle, and kidney with time of CLA feeding ...... I ll
21. CLA concentrations as mg total CLA per g liver lipid in liver lipid fractions, triacylglycerid (TG), free fatty acid (FFA), and phospholipid(PL), for rats in control and CLA treatment groups 8 hours after the gavage dose ...... 115
22. CLA isomer concentration in liver triacylglyceride (TG), free fatty acids (FFA), and phospholipid (PL) (factions of rats in CLA group 8 hours after the gavage dose ...... 117
23. The average body weight of rats in control and CLA groups at the time of starting the experimental diets and 4 weeks after the experimental diets ...... 119
24. Body weight change of rats in control and CLA groups before and after the endotoxin Injection ...... 121
XI 25. Body weight gains relative to the body weigh before the endotoxin injection for rats in control and CLA groups before and after the endotoxin injection ...... 122
26. Feed intake for rats in control and CLA gi oups before and after the endotoxin injection ...... 124
27. Plasma leukotriene 84 concentrations in nits fed with 1% CLA supplemented diet or fed with control diei for 4 weeks ...... 127
28. Plasma LTB4 concentration changes for rats in the CLA group or in the control group before and after the endotoxin injection ...... 128
29. Plasma PGE2 concentrations in rats of control group and CLA group 4 weeks after the experimental diet and without endotoxin injection ...... 131
30. Plasma PGE2 concentration changes before and after the endotoxin injection for rats in control and CLA groups ...... 132
31. Lipid concentrations of liver, kidney, lung, spleen, heart, and muscle of rats in control and CLA groups ...... 139
32. Lipid concentration of adipose tissue of rats in control and CLA groups ...... 140
•M l CHAP! ER I
INTRODUCTION
Conjugated linoleic acid (CLA), a collective term for the group of positional and geometric isomers of linoleic acid, has been related to many physiological activities since its identification. The early finding of the correlation between increased CLA concentration in specific tissues and the occurrence of certain diseases have made it an important marker of free radical activity in many experimental and clinical pathology studies [Dormandy and Wickens, 1987]. The recent discoveries of its multi-modulatory roles in cancer inhibition, atherosclerosis prevention, overcoming immune-induced growth depression, and body fat reduction further indicated its potential role(s) in improving human health [Ha et al., 1990; Ip et al., 1991; Cook et al., 1993; Lee et al., 1994].
Synthetically prepared CLA has diverse physiological functions. Studies in several animal models have shown that low level of (CLA inhibited carcinogen-induced neoplasia in several animal models, including 7,12-dimethylbenz[a]anthracene (DMBA)-induced mouse epidermal tumors and rat mammary tumors[Ha et al., 1987; Ip et al., 1990; Belury et al., 1996], benzo[a]pyrene-induced mouse Ibrestomach neoplasia [Ha et al., 1990], and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-induced colon carcinogenesis [Liew et al.,
1997]. CLA has also been found to be antiliypercholesterolemic and antiatherogenic in
rabbits [Lee et al., 1994] and hamsters [Nicolosi et al., 1997]. Additionally, studies have
shown that CLA reduced tissue arachidonic acid levels and protected against the catabolic
effects of endotoxin administration in mice, rats, and chickens without adversely affecting
immune function [Cook et al., 1993; Miller ei al., 1994]. Recently it was established that
CLA effectively reduced body fat content in mice and in human [Park et al., 1997].
Just like its diverse functions, CLA has been suggested to influence its physiological
effectiveness through multi-mechanisms. Up to date, various mechanisms have been
proposed for CLA actions including antioxidant [Ha et al., 1990; Ip et al., 1991],
prooxidant cytotoxicity [ Schonberg and KroKan, 1995], inhibition of nucleotide synthesis
[Shultz et al., 1992], reduction of proliferative activity [Ip et al., 1994], inhibition of both
DNA-adduct formation [Zu and Schut, 1992] and carcinogen activation [Liew et al.,
1995], modulation of eicosanoid biosynthesis [Sugano et al., 1997; Cunningham et al.,
1997], regulation of signal transduction by hormone mediation [Durgam and Fernandes,
1997], alteration of lymphocyte functions [Wong et al., 1997], and regulation of energy
retention and metabolism [Park et al., 1997].
Even through great efforts have been made for CLA research, the studies on CLA are still very limited. There are more questions than answers remained. As mentioned earlier, many mechanisms have been proposed for CLA functions, however, a lot of them were proposed based on very limited data. In order to fully understand the CLA physiological
properties and possibly to explore some additional beneficial efFect(s) of CLA, more
research is needed to study the details of CLA mechanisms, especially to obtain the
information about the inter-relationship among these proposed mechanisms.
Among those proposed mechanisms, the modulatory role of CLA on eicosanoid
biosynthesis was of most interest to us in this research. Although the exact mechanism
whereby dietary fat illicits a response has not been elucidated, arachidonic-acid derived
eicosanoids are believed to play an important role in mediating several different processes
required for tumorigenesis, cell proliferation, and inflammation, including local and
systemic immune responses [Plescia and Racis, 1988]. Linoleic acid can be converted to
arachidonic acid, which gives rise to both the 2-series of prostaglandins and 4-series of
leukotrienes that are the two important eicosanoid groups in mediating tumorigenesis and
inflammation [Noguchi et al., 1995]. CLA as an isomeric derivative of linoleic acid,
therefore, was proposed to be able to compete with linoleic acid in the biosynthesis of
arachidonic acid and eicosanoids [Ha et al., 1987].
To date, only two CLA studies measured the parameters related to the biosynthesis of eicosanoids. In their study of immune-induced growth depression. Miller et al. [1997] have found that dietary CLA supplementation decreased muscle arachidonic acid concentrations in rats and chicks. However, they did not directly measure the eicosanoid changes in the body [ Miller et al., 1994]. Another study conducted by Sugano et al. [1997] measured the serum and spleen prostaglandin 2% content in rats fed with or without
CLA for two weeks. They found that CLA feeding resulted in a trend toward a decreasing concentration of PGE 2 in serum and spleen when compared with the control rats. Both studies indicated the possible effect of CLA on the biosynthesis of eicosanoids.
However, only the latter study measured one single parameter related to the biosynthesis of eicosanoids. Thus, to substantiate the potential modulatory role of CLA on eicosanoid biosynthesis, more studies and information in supporting this theory are needed.
1.1 Hypothesis
Arachidonic acid is the precursor for the biosynthesis of eicosanoids. By altering the metabolism of arachidonic acid, CLA may affect the in vivo eicosanoid production and in turn affect the function of various biological systems. The pathways for the biosynthesis of eicosanoids via linoleic and arachidonic aiids are shown in Fig. 1. As shown in the scheme, CLA may act at two different reaction steps where it causes the reduction of the availability of arachidonic acid as substrate for the enzymes of biosynthesis of eicosanoids.
First, CLA may be used as the substrate for the enzymes of elongase and desaturase to synthesize the conjugated arachidonic acid. As a result, it competes with linoleic acid for the enzymes of elongases and desaturase and decrease the in vivo arachidonic acid synthesis from linoleic acid. Secondarily, CLA itself also may compete with dietary or endogenous arachidonic acid for the incorporation into membrane phospholipids. A decreased incorporation of arachidonic acid in membrane phospholipids would reduce its Linoleic Acid (18:2) ♦CLA i S-6 desaturase Y-Linolenic Acid (18:3) \Elongase Diliomo-y-Linoienic Acid (20:3)
S-5 desaturase Dietary AA Arachidonic Acid (20:4) ♦CLA
Phospholipid with AA
Phospholipase
Arachidonic Acid Cyclo-oxygenase 5-iipoxygenase y \ Cyclic Endoperoxides 5-HPETE I / i i \\ LTA.-^LTC,-^ LTD.-^LTE. PGF:, PGEj pGDj PGIj i t XA j1 Ii 6-keto LTB. PGF,^ TXB,
Fig. I Pathways of biosynthesis of eicosanoids from arachidonic acid. availability for the enzyme of phospholypase Aj for the release of free arachidonic acid to
serve as the precursor for eicosanoid biosynthesis. In addition, the in vivo synthesized
conjugated arachidonic acid may compete with arachidonic acid for cyclooxygenease and
lipoxygenase to form metabolites which have different functions from the eicosanoids.
By measuring the changes of the eicosanoid production, as well as the arachidonic
acid content, in tissues and body fluids of testing animals one would be able to 6 nd out if
CLA indeed alters the metabolism of arachidonic acid.
One of the functions of eicosanoids, especially prostaglandins and leukotrienes, is to
affect the immune system to modulate cellular immunity, the antibody response, and the
inflammatory response. If CLA would affect the biosynthesis of eicosanoids in the body,
it would be able to affect many disease development, such as cancer and some
inflammatory diseases. Therefore, the find ng of the CLA effect on the eicosanoid
biosynthesis not only can confirm the mechanism of CLA in cancer prevention but may
also lead to further discovery of additional effects of CLA in some inflammatory diseases.
1.2 Objectives
As stated in the hypothesis, the goal of this research is to investigate the CLA effects on eicosanoid biosynthesis and the possible role(s) of CLA in inflammatory diseases.
Since this particular study was the initial reseaixh on CLA in this laboratory, it include two main parts; ( 1) to establish the analytical methods for measurement of the biological parameters; ( 2 ) to apply these analytical methods in animal study to obtain information about CLA metabolism and dietary CLA effects on arachidonic acid metabolism, eicosanoid production, and in responding tc the endotoxin injection. Two particular eicosanoids, leukotriene B4 (LTB4) and prostaglandin Ez (PGEz), are chosen in this study because LTB4 is one of the important metabolites of arachidonic acid by action of lipoxygenase while PGEz is the metabolite by the action of cycle-oxygenase. By monitering the changes of these two eicosanoids, the CLA effects on the enzymes lipoxygenase and cycle-oxygenase can be assayed. The more specific objectives for this study were:
( 1) To develop analytical methods for the quantification of CLA and its isomers, arachidonic acid, and other fatty acids in different tissues and in tissue lipid fractions, such as in triacylglycerids, phospholipids, and free fatty acids fractions.
(2) To develop methods for analysis of LTB4 and PGEz in plasma and other tissue samples.
(3) To examine tissue CLA (isomers) distributions in responding to signal dose and long term dietary supplementaion of CLA in order to obtain the basic information about CLA metabolism. The information expected to be obtained includ preference of different tissue in incorporation of CLA, the time of tissue to take up and metabolize CLA, and the limitation of tissue in taking up the CLA. (4) To study the effects of long term dietary CLA supplementation on body weight, feed consumption, tissue fatty acid composition ard plasma LTB4 and PGE 2 in responding to an endotoxin challenge. Injection of endotoxin, lipopolysaccharide (LPS), will elicit a systemic inflammation in rats. By monitoring both the physiological and biochemical parameters, the CLA effect on inflammation c;in be studied.
1.3 Significance of tliis researcli
Conjugated linoleic acid (CLA) has been recognized to be a potential nutrient for human health. Studies have shown its multi-functionality and effectiveness in many animal models [Belury, 1995; Haumann, 1996; P;irodi, 1997]. In spite of these, studies conducted up to now have not been able to provide detailed information to further understand the mechanisms of CLA effects.
Eicosanoids are known to play important roles in various physiological activities, such as cancer and imflammation [Chamber and Cohen, 1985; Bach, 1988; Evans and
Whicher, 1992]. Modulation of eicosanoid biosynthesis is one of the mechanisms proposed for CLA activity. However, no data which show the direct link between CLA and biosynthesis of eicosanoids is yet available. If CLA could indeed alter the biosynthesis of eicosanoids, it would be able to modulate the development of inflammatory diseases.
This dissertation was, therefore, designed to examine effects of CLA on eicosanoid biosynthesis and the possible role of CLA in inflammatory disease development in an animal model to examine the above discussed Iiypothesis. CHAFl ER 2
LITERATURE REVIEW
2.1 Conjugated Linoleic Acid
2.1.1 Definition and Chemical Structures
Conjugated linoleic acid (CLA) is a collective term for a mixture of octadecadienoic
fatty acid moieties containing two conjugated double bonds. In general, CLA refers to the
octadecadienoic fatty acid moieties that are formed when one of the two double bonds in
linoleic acid (LA), c9,c 12-octadecadienoic acid, is shifted in its positions between carbons
with or without a change in their cis/trans configurations.
Theoretically, eight possible geometric isomers of 9,11- and 10,12-octadecadienoic
acid (c9,cll; c9,tll; t9,cll; t9,tll; cl0,cl2; ol0,tl2; tl0,cl2; tl0,tl2) would form from the isomerization of c9,cl2-octadecadienoic acid. However, due to the difference in structure and thermodynamical preference the production of each individual isomer would not be equal. It has been found that c9,tl 1-, tI0.cl2-, t9,tll-, and tl0,tl2- octadecadienoic acids accounted for more than 90% of the isomers produced during the autoxidation or alkali isomerization of c9,cl2-linoleic acid [Ha et al., 1989]. Among these
four isomers, c9,tll- and tl0,cl2- isomers are predominant due to the coplanar characteristics of five carbon atoms around a conjugated double bond and special conflict of the resonance radical or anion [Nichols et al., 1951]. On the other hand, the relatively higher distribution of the t,t isomer of 9,11- or 10,12-octadecadienoic acid in some synthetically prepared CLA apparently resulted from the further stabilization of c-9,t-l 1 or t- l 0 ,c -l2 geometric isomers, which is thermodynamically preferred, during an extended processing time or long aging period [Ha et al., 1989]. Additionally, the t,t isomer of
9,11- or 10,12-octadecadienoic acid was found to be the predominant product of the isomerization of LA geometrical isomers, t9,tl2, c9,tl2, and t9,c 12-octadecadienoic acid,
[Nichols et al., 1951]. The remaining isomers are minor contributors of the CLA mixture.
2.1.2 CLA Analysis
Detection of CLA as fatty acid methyl esters (FAME) is usually accomplished by gas chromatography using a flame ionization or MS detector or by high performance liquid chromatography using a UV detector [Ha et al., 1989]. Isolation of lipid materials from various matrices is the same for CLA as any other lipid analysis except that the use of strong acids should be avoided, since the strong acid is suggested to cause artificial isomerization of the CLA [Yurawecz et al., 1997].
10 To achieve successful quantitative analysis of CLA, several factors have to be considered. First, high-purity reference materials for individual CLA isomer are generally not available, and the relative CLA isomer content in commercially available CLA mixture usually change from batch to batch. At present time, most CLA quantification was done based on the internal standard method using a reference CLA isomer mixture for the calibration of the correction factors [Ha et al.. 1989]. Second, the derivatization of fatty acid to FAME using strong acid catalysts may yield misleading results. It has been reported that derivatization of CLA fatty acid to FAME by using strong acid catalyst such as BFj/methanol or HCl/methanol could reduce yield of CLA isomers relative to other méthylation procedures [Shantha et al., 199.5b], create CLA artifacts[Yuraweca et al.,
1994], and drastic changes in the relative amounts of CLA isomers [Yuraweca et al.,
1997].
To date, an analytic technique for the separation and identification of individual CLA isomer has not been perfected because of the lack of high purity standard and the variations of the CLA isomer compositions in the reference CLA isomer mixture. Current identification for CLA isomers was based on the combination of GC-MS, GC-FT/ER. analysis with the help of measuring the equivalent chain length (ECL) value of each individual isomer [Ha et al., 1989].
11 2.1.3 CLA in Food
CLA is a minor constituent of a number of food products, including dairy products, meats and certain vegetable oils as shown in Table 1 [Chin et al., 1992].
In general food products originating from ruminants contained considerable more
CLA than those from nonruminants. Among the ruminants lamb showed the highest CLA content (5.6 mg CLA/g fat) and veal was the lowest in CLA content (2.7 mg CLA/g fat), while beef CLA content ranged from 2.9 to 4.1 mg CLA/g fat [Chin et al., 1994].
It is believed that microorganisms in the rumen are responsible for the relative high
CLA contents in food products originating Rom ruminants. Shorland et al. [1955] first demonstrated that CLA is produced from polyunsaturated fat by the ruminant microorganism. Kepler et al. [1966] obser/ed that CLA was an intermediate in the microbial biohydrogenation of LA to oleic acid by the rumen bacterium Butyrivibrio fibrisolvens. More recently, Chin et al. [1994b] observed that the intestinal flora of rats are also capable of converting free LA to CLA [Chin et al., 1994b]. Additionally, they found that a partially purified isomerase from B. fibrisolvens was able to convert the LA in hydrolyzed safiflower oil to CLA [Chin et al., 1991].
Dairy products, originating from ruminant animal, were also high in CLA content.
Cow’s milk, the starting material for dairy products, contained approximately 5.5 mg
12 Food Total CLA (mg/g fat) % c -9 ,t-ll Lamb 5.6 92 Ground beef 4.3 85 Veal 2.7 84 Ground turkey 2.5 76 Pork 0.6 82 Chicken 0.9 84 Egg yolk 0.6 82 Salmon 0.3 n.d* Shrimp 0.6 n.d* Homogenized milk 5.5 92 Butter 4.7 88 Cottage cheese 4.5 83 Blue cheese 5.7 90 Cheese whiz 6.4 48 Com oil 0.2 37
Table 1. CLA content in various food products [Chin et al., 1992].
13 CLA/g fat, while the reported CLA contents of most dairy products were ranging from 2.5
to 7,0 mg CLA/g fat [Lin et al., 1995; Chin et ai., 1994]. The CLA concentration in milk
and dairy products has been seen changing with different feeding and processing methods
[Jiang et al., 1996; Werner et al., 1992]. The amount of CLA in cow’s milk and butter has
been correlated positively with dietary intake of linoleic acid [Bartlet and Chapman, 1961;
Parodi, 1977]. In addition, milk from the cows fed with low ratio of forage to concentrate
showed significant higher CLA content than the milk from the cows fed with high ratio of
forage to concentrate ( 11.28 vs. 5.0 mg CLA/g fat) [Jiang et al., 1996].
CLA content of natural cheeses ranged from 2.9 to 7.1 mg CLA/g fat [Chin et al.,
1992]. Among them cheese such as Parmesan and Romano, which were aged or ripened
more than 10 months, were among the lowers in concentration, while bacterial surface
ripened cheeses such as Brick and Muenstre, aged 4 to 8 weeks, were among the highest
[Lin et al., 1995]. CLA concentration in processed cheese products varied from 3.9 to 8.9
mg CLA/g fat. It is found that CLA concentrations in all cheddar-based processed
cheeses were higher than in unprocessed checdar cheese. This increased level of CLA in
processed cheddar-based cheese was believed to be caused by a combination of processing
temperature and the presence of whey protein concentrate. The low molecular weight
compounds in the whey protein concentrate ai e considered as the primary component that caused the increased CLA formation [Shantha et al., 1992; Shantha et al., 1993a; Garcia-
Lopez et al., 1994]. In general, aging, heat treatment, and protein quality are among the
14 factors identified as contributing to the formation of CLA in the processed dairy products
[Shantha et al., 1992; Lin et al., 1995].
Pork and chicken were far lower in CLA content (0.6 - 0.9 mg CLA/g fat), same as
the sea foods with CLA content ranged from C.3 to 0.6 mg CLA/g fat. The CLA contents
in some meat products have been shown to be affected by cooking conditions [Shantha et
al., 1994; Ha et al., 1989]. Generally, the CLA concentration in cooked beef showed
higher than in raw beef [Shantha et al., 1994].
Plant oils contained less CLA than animal fats, ranging from 0.1 to 0.7 mg CLA/g fat
which was approximately four times less than in beef tallow [Chin et al. 1992].
When individual CLA isomers was considered, the c9,tl 1-18:2 CLA isomer was the
single major isomer detected in animal food products accounting for more than 90% of the
total CLA in the products. While the c9,tl 1- and tl0,cl2-18:2 CLA isomers were found to be the major CLA isomers in plant oils, accounting for 43 and 40%, respectively, of the total CLA in the products. [Chin et al., 1992].
It is believed that the rumenbacterial liiioleate isomerase produces exclusively the c9,tl 1-18:2 CLA isomer. Kepler and love 11967] showed that the first intermediate in the biohydrogenation of LA by rumen bacteria, B. fibrisolvens, is c9,tl 1-18:2. Therefore, microbial metabolism is believed to be the source of c9,tl 1-18:2 CLA isomer in meat and dairy products. The presence of CLA in plant oils was thought to be due to oxidation
15 and/or bleaching effects [Van den Bosch, 1973]. However, why plant oils have more
tlO,cI2-I8:2 and less c9,tl 1-18:2 CLA isomers than animal fat is unknown.
2.1.4 CLA in Biological Materials
In the 1930s, a polyunsaturated lipid with a light absorption peak around 230-235 nm
was identified as diene conjugation and found present in stored vegetable oil and various
biological materials [Gillam et al., 1931]. Laier in 1960s, it was found that over 95% of
the diene conjugation in human serum, tissue fluids and tissues was due to a single fatty
acid [Recknagel and Ghoshal, 1966]. In 1984 this single fatty acid (diene conjugation)
was identified to be octadeca-9,11-dienoic acid [Iversen et al., 1984]. Then in 1991, it
was further identified to be c9,tl 1-octadecadienoic acid [Smith et al., 1991]. Therefore,
the diene conjugation and c9,t 11-18:2 CLA basically are the two names for the same
compounds. In order to make the discussion easier, c9,tl 1-CLA will be used in place
where it might be “diene conjugation” in the original references.
Since its identification, the c9,tl 1-CLA has been found in a variety of biological
samples, including in human depot fat, bile, duodenal juice and blood serum [Dormandy
and Wickens, 1987]. The measurement of c9,tl 1-CLA in tissues has been used as free-
radical marker to monitor free-radical mediated changes in many medical researches. The concentration of CLA has also been correlated to many experimental and clinical pathology [Dormandy and Wickens, 1987].
16 In studying the possible role of free-radicals in ethanol toxicity, Di Luzio [1972] found that there was a considerable amount of c9,tl 1-CLA in human serum-lipid extracts and that both in chronic alcoholics and in controls this serum c9,tl 1-CLA concentration could be influenced by large doses of mixed tocopherols. Therefore, he suggested that serum c9,tl 1-CLA might reflect peroxidation in vivo. Later Shaw et al. [1981] found that the increased level of c9,tl 1-CLA paralleled with reduced glutathione concentration in chronically intoxicated baboons [Shaw et al., 1981] and in the biopsy material from patients with alcoholic liver disease [Shaw et al. 1983]. A significantly increased level of phospholipid-esterified c9,tl 1-CLA was also found by Fink et al. [1990] in serum of chronic alcoholics. Interestingly, they further demonstrated that the serum c9,tl 1-CLA level would rapidly return to normal on alcohol withdraws in those chronic alcoholics, by contrast, in the non-addicts the acute alcohol load had no significant effect on the level of
CLA in their serum. These alcohol-dependent increase in CLA level was suggested due to a two-stage abnormality in free-radical activity: first, the slow induction of a free-radical- dependent detoxifying mechanism during the development of the addiction, and second, the triggering of this mechanism by an acute alcohol load.
Beside the finding of abnormal c9,til-CLA level in alcoholics, Lunec et al. [ 1981,1982] measured the c9,tl 1-CLA both in the synovial fluid and in the serum of the patients with rheumatoid and degenerative arthritis. They found that there was more c9,tl 1-CLA in synovial fluid from inflammatory than from degenerative joints and in
17 serum the concentration of c9,tl I-CLA paralleled rheumatoid disease activity as measured by standard clinical and laboratory tests.
Significantly higher level of phospholipid-esterified c9,tll-CLA in bile and a corresponding increase in non-esterified c9,tl I-CLA in the duodenal juice have also been found by Braganza et al. [1983] in patients v/ith pancreatic disease compared to patients undergoing endoscopic examination for non-pancreatic disorders. This increased level of c9,tl 1-CLA in bile and duodenal juice of patients with pancreatic disease were suggested to related to the free-radical-dependent detoxiiying mechanism in the liver.
Increased concentration of c9,tl 1-CLA in biopsy material and exfoliated cells of patients with cervical precancer was observed as well. The molar ratio of c9,tl 1-CLA to
LA (c9,cl2) of the human biopsy material has been tested to be used as a diagnostic tool for cervical precancer [Fairbank et al., 1988; 989]. The concentration of LA provided a reference measurement which eliminates the need for a cell count or the chemical estimation of protein or DNA. The difference in the molar ratio between different groups of patients is, in fact, the function of the concentration of c9,tll-CLA. Results showed that the increasing in the molar ratio corresponded to the increasing in the accidents of the cervical precancer. The molar ratio of c9,tl 1-CLA to LA in the biopsy material was significantly higher for those patients diagnosed with cervical intraepithélial neoplasm than those diagnosed as normal [Dormandy and Wickens, 1987].
18 2.1.5 CLA Origin in Animals and Humans
As showed in previous sections, CLA has been detected not only in tissues of rumen
animal, but also detected in tissues of nonruminant animal and in tissues of normal persons
and patients in different pathological states [Braganza et al., 1983; Iversen et al., 1984;
Erskine et al., 1985; Fairbank et al. 1988; Situnayake et al., 1990; Banni et al., 1996].
However, the origin of CLA is still debated, especially its origin in nonruminant animals
and humans. In these regards three hypotheses have been proposed; a free radical attack
on polyunsaturated fatty acids [Situnayake et al., 1990; Recknagel et al., 1991; Halliwell
et al., 1993]; an endogenous synthesis by anaerobic bacteria [Kepler et al., 1966; Kepler et
al., 1966; 1969], and a dietary origin [Britton et al., 1992; Chin et al., 1992; Banni et al.,
1993; Huang et al., 1994].
CLA has been suggested to be a product of free radical-mediated degradation of its
parent compound, LA, because high blood levels of c9,tl 1-CLA isomer were found in a variety of inflammatory conditions in which f ee radical damage has been implicated. In addition, Cawood et al. [1983] observed that in vitro when LA was exposed to UV irradiation in the presence of human albumin or gamma globulin gave rise to a series of products that contain no additional oxygen but undergo double bond isomerization with or without conjugation and one of the compounds in the UV treated LA mixture was c9,tl 1-
CLA isomer. They emphasized that the c9,tl 1 -CLA can be generated in vitro by exposing
LA to free-radical activity only in the presence of a protein. Since protein and free radical
19 activity both present in the systems of nonru:ninant animals and humans, it is suggested that the tissues c9,tll-CLA of nonruminant mimais and humans may be a in vivo free- radical product of LA. However, direct obser/ation that the diene-conjugated compounds measured were in fact peroxides or peroxidation product was slender or non-existent. In addition, the observation that certain bacterial lung pathogens were capable of generating c9,t 11 CLA isomer but not able to elevate the thiobarbituric acid reactivity, which is a non-specific measure of lipid peroxidation, in vitro has cast doubt on this free radical theory [Jack et al., 1994].
The endogenous synthesis of CLA was supported by the factor that the c9,tl 1-CLA isomer was identified as an intermediate in the biohydrogenation of linoleic acid by the rumen microorganism Butyrivibrio fibrisolv^tts [Kepler et al., 1966; 1969], and the partially purified enzyme, linoleate isomerases, isolated from this mirooganism was observed to convert LA to CLA in vitro [Chin et al., 1991]. It is reasonable to believe that microbial metabolism is one of the source of CLA in tissues of rumen animals and in their dairy products.
In an efifort to find out whether the CLA in the tissues o f nonruminant animals is at least in part due to the conversion of LA to the CLA isomer by bacterial flaora. Chin et al.
[1994b] studied the effect of feeding free or esterified LA on the tissue CLA concentrations in conventional and germ-fiee rats. They observed that the CLA concentration in tissues (liver, lung, kidney, skeletal muscle, and abdominal adipose
20 tissues) of free LA-fed conventional rats were 5-10 times higher than those of the control conventional rats. However, the CLA concentration in the same tissues of germ-free rats were not affected by the different diets. These results indicated that the intestinal bacterial flora of rats are capable of converting free LA but not LA esterified in triglycerides, to
CLA isomers. Therefore, endogenous synthesis of CLA by intestinal flaora is believed to be a possible source of CLA in tissues of nonruminants and humans.
Additionally, in a cell culture study conducted by Jack et al. [1994], 180 strains o f common bacterial lung pathogens were examined to determine whether they could produce CLA isomers in vitro. The results showed that out of the 180 strains randomly sampled, 23 (12.8%) were found to produce elevated levels of the c9,tl 1-CLA isomer to more than the 99% confidence limit of control values when incubated with LA and tissue culture fluid. Their results also showed that this ability to produce the c9,tl 1-CLA isomer did not appear to be dependent on the morphology, staining and cultural characteristics of the bacteria. These results suggested that certain bacterial infections could cause elevated
CLA concentration in lungs. Another study conducted by Fairband et al. [1988] further showed that Corynebactehum andsp. Lactobacillus sp.from the female genital tract were capable of producing c9,tl 1-CLA isomers from nonesterified LA. All these results proved and supported the hypothesis of m vivo endogenous synthesis of CLA in nonruminants and humans.
21 The mechanism by which bacteria might produce c9,tIl-CLA isomer is unknown.
However, the ability of microorganism to generate this isomer might, in part, explain the rise in the diene conjugate in patients with certain lung diseases, even it cannot account for all of the elevated level of conjugated diene found in a large number of patients with no clinical or laboratory evidence of infection.
Despite the observation that microorganisms are capable of isomerizing free LA to
CLA, dietary CLA is still considered by far to be the predominant source in humans based on the relatively high concentration of c9,t 11-octadecadienoic acid in milk and other food products [Britton et al., 1992; Huang et al., 1994] and the factors that, consumption of the food containing CLA can significantly increase its concentration in the tissues of both human and experimental animals [Christie, 1979; Chin et al., 1992; Britton et al., 1992;
Huang et al., 1994; Banni et al., 1995; 1996b]
Huang et al. [1994] observed that increased Cheddar cheese consumption (112g/day) significantly increased plasma CLA concentrations (9.6 vs. 7.1 pmol/L) as well as plasma phospholipid-esterified CLA concentrations (5.9 vs. 4.4 pmol/mg phospholipids) in men, which indicated that the dietary CLA was readily absorbed.
In their studies to examine whether dietary CLA directly responsible for those detected in tissue lipids of rats. Banni et al. [1995, 1996b] fed rats with diets formulated com oil or with partially hydrogenated fat, which contains CLA and trans fatty acid. Their results showed that the CLA concentrations in the lipids of liver, adipose, as well as liver
22 nuclei were directly reflected the CLA contents in the diets formulated with partially hydrogenated fat. By contrast, no CLA were detected in lipids of same tissues of rats fed with diets formulated with only corn oil. The results further indicated that dietary CLA were readily absorbed and assimilated in rat tissue lipids [Banni et al., 1995; 1996b].
Although dietary CLA is considered to be an important source of CLA in animals and humans, in this connection one has to distinguish between base-line levels and abnormal variations as well as between the different molecular combinations in which c9,tll-CLA exist in the body. CLA as the isomers of LA is readily absorbed by the body and incorporated into tissues. It is unlikely that long-term dietary habits in different populations or even in different individuals are significant variables in determining the fasting level of phospholipid-esterified c9,tl 1-CLA in serum and could account for the precipitous changes which occur in chronic alcoholics on alcohol withdrawal. It is even more unlikely that they could explain the diiferences between the concentration of the compound in normal and in premalignant cervical cells. The origin of CLA in animals and humans remain to be unknown and more likely to origin from both dietary and in vivo production.
2.1.6 CLA Intestinal Absorption And Metabolites
Since dietary CLA is considered to be the predominant source in humans, the basic information about its absorption and distribution is valuable in understanding its physiological functions. Sugano et al. [19971 studied the model of intestinal absorption
23 and the tissue distribution of CLA. By measuring 3 and 24 hour lymphatic recovery of
CLA and LA, they observed that while the lymph flow rate was the same between the two groups, the apparent lymphatic recovery rate of CLA was slower than that of LA, and the cumulative recovery rate for CLA in 24 hours was significantly lower than that of LA
(78.5 vs. 53.4, P<0.05). They also observed tnat as CLA transported, approximately 80% of it was carried as chylomicrons and remaining 20% as very low-density lipoproteins. As it absorbed by the intestine, approximately 95% of CLA was incorporated into triacylglycerol and 5% incorporated into phospholipids. The distribution of individual
CLA isomers in the triglycerol was similar to that in the phospholipids. They further found that there were detectable differences in the compositions of CLA isomers between the gavage and lymph CLA. The analyses of lymph collected for 24 hour showed more tt-isomers while less ct- and tc-isomers in the lymph than in CLA given intragastrically.
Additionally, approximately the same proportion of tc- and ct-CLA isomers was incorporated into sn-2 and sn-1,3 positions of lymph triacylglycerol similar to the case of
LA, whereas tt-CLA isomers was exclusively distributed in sn-1,3 positions.
Tissue distribution results from their study showed that the incorporation of CLA depends on the tissues, and the adipose tissue and lung contained most and the brain the least. The composition of CLA isomers also was tissue-dependent, and it did not necessarily resemble that of dietary CLA. Although in general c9,tll-/t9,cl 1-CLA isomers were predominant component in both dietary and tissues CLA, the t9,tl 1- and tlO,tl2-CLA isomers were most preferentiall> absorbed. The incorporation of CLA and
24 the pattern of the incorporation of the individual CLA isomers was also not the same among the phospholipid species analyzed in this specific experiment, the order of the CLA incorporation was phophatidyllinositol > cardiolipin > phosphatidyl-ethanolamine > phosphatidylserine > phosphatidylcholine. This study for the first gave some very important detail information about CLA intestinal absorption and tissue distribution.
The metabolism of CLA was studied in several different ways[Banni et al., 1996;
Sebedio et al., 1997]. Banni et al. [1995; 1996] studied CLA metabolites on rats and lambs by characterize conjugated diene fatty acids in various tissues. In the rat study, a diet containing 0.04% CLA was fed to the rats for 1 week, conjugated linolenic acid
(C l8:3) and eicosatrienoic acid (C20:3), but not the conjugated arachidonic acid (C20:4) were then detected in the liver lipid of rats. Based on these results they suggested CLA in rat liver experienced the elongation and desaturation with the same fashion as in the case of LA and the elongation and desaturation processes do not affect the conjugated diene structure.
In the lamb study, the characteristic of the conjugated diene fatty acids were examined in liver and adipose tissues of the lambs. Results showed that the lamb liver total lipid and phospholipid contained CLA (C l8:2), conjugated linolenic acid (C l8:3), conjugated eicosatrienoic acid (C20:3), and conjugated arachidonic acids (C20:4), while the lamb liver neutral lipid contained all the conjugated diene fatty acids detected in the lamb liver total lipid and phospholipid but the conjugated arachidonic acid. Quantitative analyses of
25 lamb liver conjugated fatty acids showed that CLA was present in the highest
concentration, followed by conjugated linolenic, conjugated eicosatrienoic, and conjugated
arachidonic acid. Lamb total adipose tissue lipid showed the same profile of conjugated
fatty acids as in liver neutral lipids. Therefore, in addition to the two metabolites,
conjugated linolenic acid (C18;3) and eicosatnenoic acid (C20;3), found in the rats liver,
the final product, conjugated arachidonic acid, of the elongation and desaturation was
found in the lamb tissues. These results fiirther supported the suggestion that CLA
experienced the in vivo elongation and desaturation just like LA. Additionally, it also established that the conjugated diene intermediates were readily incorporated into tissues the same way as the LA metabolites.
Sebedio et al. [1997] studied CLA metabolites in rats by feeding high quantities of CLA
(180 mg/day) in the form of triglycerides for six days to rats that had been reared on a fat- free diet for two weeks, then analyses the possible metabolites of the CLA in the liver lipid. The metabolites identified in their experiment were conjugated eicosatrienoic acid
(C20:3 A8,12,14), conjugated arachidonic acids, C20:4 A5,8,12,14 and C20:4 A5,8,ll,13, which were assumed raised from the elongation and desaturation of C18;2 AID,12 and
C18:2 A9,l 1-CLA isomers.. Their results basically agreed with Banni et al.’s results and fiirther indicated that the elongation and desaturation may be carried out on all the CLA isomers. Thus far, the information on the CLA metabolites was very limited, the question of whether conjugated arachidonic acid can be metabolized as the arachidonic acid to the eicosanoids is widely open.
26 2.2 Physiological Properties Of CLA
2.2.1 CLA and Cancers
CLA and Skin Cancer
The anticarcinogenic property of conjugated linoleic acid was first identified in the mouse skin multistage carcinogenesis model [Ha et al., 1987]. In these early studies, a mixture of CLA isomers isolated from firied ground beef or synthesized by alkali- isomerization of LA in laboratory was topically applied to the dorsal area of mouse skin each time before the carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) was applied to the same area for the tumor initiation. LA, the parent compound of CLA, and acetone were used as controls in the experiments. Results showed that compared with LA and acetone controls, topical CLA treatment inhibited tumor incidence by approximately 20% and tumor yield by approximately 50% at 16 v/eeks post initiation [Ha et al., 1987].
The dietary CLA effect on skin tumor development was also tested by using the phorbol ester skin tumor promotion model [Belury et al., 1996]. This model was designed to test the CLA effect on the skin tumor promotion only, which means it was independent of the initiation. In this experiment, increasing levels of dietary CLA (0.0%; 0.5%; 1.0%;
1.5%) were started one week after the DMBA initiation and throughout the period of promotion in mice at weaning age. Tumor promotion was induced by 12-0- tetradecanoylphorbol-13-acetate (TPA) four weeks after the initiation. Results showed
27 that the tumor yield on the mice with high level CLA diet (1.0% and 1.5%) were approximately 65% of the yield in the control mice (with no CLA in their diet), while the tumor yield in the mice with low level CLA diet (0.5%) was 89% of the yield on the control mice at the time twenty-four weeks alter the tumor promotion was begun [Belury et al., 1996]. The reduction in tumor yield was significant even at the low level CLA supplementation. This results also clearly showed that CLA inhibited tumor promotion in a manner that was independent of its anti-initiator activity.
Because the mouse skin multistage carcinogenesis model allows for the initiation and promotion stages of carcinogenesis to be operationally and temporally separated
[DiGiovanni, 1992], these two studies uniquely showed the effectiveness of CLA on skin tumor inhibition at both initiation and promotion stages. The mechanisms of CLA in prevent skin tumor development are not cleai. Based on the factors that small doses of
CLA effectively inhibit the skin carcinogenesis during initiation and promotion, Belury et al. [1996] suggested that multiple anticarcinogenic mechanisms are involved.
CLA and Forestomach Cancer
CLA inhibition of carcinogenesis was also found in chemically induced mouse forestomach neoplasia [Ha et al., 1990]. When synthetic mixture of CLA isomers, LA, or olive oil were given introgastrically to mice prior to carcinogen benzo(a)pyrene treatment, the CLA treated mice showed approximately 50% reduction in development of neoplasia in their forestomachs relative to the LA and olive oil treated mice at the time 22 weeks
28 after the first carcinogen treatment. The tun;or incidence was also significantly reduced for the CLA treatment mice compared with the LA and olive oil treated mice. In an attempt to study the mechanisms of the inhibition effect of CLA on the mouse forestomach tumorigenesis, Ha et al. [1990] found that c9,tl 1-CLA isomer was preferentially incorporated into the forestomach membrane phospholipid. They suggested that c9,tl 1-CLA isomer may be the most biologically active isomer in preventing carcinogenesis.
CLA and Mammarv Cancer
The anticarcinogenetic property of CLA was further established by Ip et al. in a series studies on mammary carcinogenesis by using primarily the DMBA-induced mammary tumor model [Ip et al., 1991; 1994a,b,c; 1995; 1996; 1997a,b]. The protective roles of
CLA in mammary tumorigenesis were observed in various circumstances and in various stages of mammary carcinogenesis.
In their initial experiment [Ip et al., 1991], the effective dietary CLA levels and the protective role of CLA in initiation stages of mammary carcinogenesis were investigated.
Three levels of CLA, 0.5, I.O, and 1.5 % , were fed to rats started 2 weeks before DMBA carcinogen administration and continued for 6 months. All rats in study received ID mg of
DMBA (high dose) by gavage at 50 day age. Data from this experiment showed that the total number of mammary tumors in the 0.5, 1.0, and 1.5% CLA groups was reduced by
32, 56, and 60%, respectively, when compared to the control group. This study indicated
29 that the maximal inhibition of CLA on mammary tumorigenesis is for rats fed with 1%
CLA in their diet. Since the CLA diets wore started 2 weeks before the carcinogen administration, it is suggested that CLA has a anti-initiator effect on mammary carcinogenesis.
In an effort to expand the CLA efficacy curve below 0.5% , Ip et al. [1994b] carried out another experiment similar to the previous one with two modifications; animals were given 5 mg of DMBA (low dose) and the sample size per group was increased to 50. This increased sample size was done to make ensure adequate statistical power due to the reduced number of tumors produced per rat by the low dose of carcinogen. Rats were fed the diets containing CLA at 0.05, 0.1, 0.25, and 0.5% and diets were started 2 weeks before DMBA carcinogen administration and continuing for 9 months. Results showed a dose-dependent effect of CLA on mammary cancer inhibition. Total mammary tumor yield was reduced by 22, 36, 50, and 58% in the 0.05, 0.1 , 0.25, and 0.5% CLA diets, respectively, compared to the controls. Intergroup comparison showed that as little as
0.1% CLA was sufficient to cause a significant reduction in the number of tumors. This low does is close to the estimated levels of dietary CLA consumed by humans. The efficacy of such low dose of CLA in cancel prevention indicated that CLA is a more powerful anticarcinogen than any other fatty acid in modulating tumor development.
The third experiment carried out by Ip ct al. [1995] was designed to examine the effect of timing and duration of dietary CLA on mammary carcinogenesis prevention. In
30 addition, in this experiment a comparison was made between DMBA and methylnitrosourea (MNU)-induced carcinogenesis. DMBA is a procarcinogen requiring metabolic activation prior to becoming fully carcinogenic. MNU is a direct-acting carcinogen that requires no further metabolism for carcinogenic activity.
The first period they chose to start CLA supplementation was from weaning (21 days old of age) to the time of carcinogen administration (50 days old of age). This particular period corresponds to a time of active morphological development of the mammary gland to the mature state. Results showed that in this particular chosen period, 1% CLA supplementation significantly reduced total mammary tumor yield by 39 and 34 in rats treated with the DMBA and MNU, respectively, when compared to their respective controls. This results again proved that CLA is effective in prevent mammary tumorigenesis in the initiation stage. In addition, the similar extent of reduction in the
DMBA and MNU induced tumor yields indicated that CLA inhibited chemically induced mammary carcinogenesis via a mechanism that is independent of the type of chemical carcinogen present.
The second time period tested in this experiment was starting CLA supplementation immediately after the carcinogen administration then withdraw after 4 weeks, 8 weeks, or until the end of experiment (20 weeks). In contrast to the early postwearing exposure of
CLA, the short term (4 and 8 weeks) post initiation CLA (1%) supplementation showed relative ineflfective in reducing the mammary tumor yield (~6% reduction). However, the
31 uninterrupted supply of CLA (20 weeks) in the diet after the carcinogen administration significantly (P<0.05) inhibited the mammary carcinogenesis, as judged by a 48% reduction in tumor incidence and a 70% reduction in the total number of tumors. Theses data suggested that CLA may have an antipromoter effect independent of the anti-initiator effect, and a continuous intake of CLA was required for maximal inhibition of tumorigenesis when CLA feeding was started after carcinogen administration.
In summary, this study, agreed with the results fi'om skin cancer studies, indicated that CLA was effective to inhibit mammary tumorigenesis in both the initiation and the promotion stages. It also suggested that in order to achieve the maximal inhibition of carcinogenesis a life time continuous intake of CLA is important.
The effect of level and type of fat consumed by the host rats on the efficacy of CLA anticarcinogenic activity was also examined by Ip et al. [1996]. In this study, the efficacy of CLA anticarcinogenic activity was evaluated based on the same DMBA mammary tumorigenesis model used in previous studies. 1% CLA was supplemented to all tested diets (controls) and fed to rats 2 weeks before the DMBA carcinogen administration and through out the experiment (20 weeks). In the fat level experiment, a custom formulated fat blend that simulated fatty acid composition of the US diet was present at 6.7, 10, 13.3, or 20% (w/w) in the diets; while in the fat type experiment, a 20% fat diet containing either com oil or lard was used. Results showed that the magnitude of tumor inhibition by
1% CLA was not influenced by the level or P/pe of fat in the diet. That means the CLA
32 effect on mammary tumorigenesis is independent of the fatty composition and fat content
in the diet. The investigators, therefore, suggested that the cancer preventive activity of
CLA is likely due to the capacity of CLA to metabolized to some active metabolites which
are essential for cancer prevention.
Ip et al. [1997b] further studied the correlation between retention of CLA in
mammary tissue and the rate of occurrence of mammary carcinomas as a function of CLA
exposure/withdrawal. They found that the incorporation of CLA into mammary gland was
rapid once CLA was added to the diets and the level reached to maximum after 4 weeks of
the CLA diet. When the CLA was withdrawed from the diet, the rate of disappearance of
the CLA in mammary tissue was equally fast as the incorporation and the CLA
concentration return to basal value in about 4 weeks. Additionally, they found that CLA was incorporated more in the neutral lipids fraction than in the phospholipids fraction of the mammary tissue; and the rate of change ol‘ phospholipid CLA in either the upswing or downswing of the exposure/withdrawal curve was slower compared with that observed with neutral lipid CLA In studying the correlation between retention of CLA in mammary tissue and the rate of occurrence of mammary carcinomas, they found that the rate of disappearance of the neutral lipid-CLA, rather than phospholipid-CLA, subsequent to
CLA withdrawal paralleled more closely the rate of occurrence of new tumor in the mammary tissue. Therefore, they suggested that neutral lipid-CLA might be a more sensitive marker of tumor protection than phospholipid-CLA. Along with the factors that
CLA supplementation for 4 or 8 weeks had no protection effect on the occurrence of
33 mammary carcinomas, instead only the continuously CL A supplementation (20 weeks) had significant tumor inhibition effect, they suggested that continuous supply of CLA is necessary for effective tumor inhibition.
To further evaluate whether there might be an interaction between LA and CLA, Ip and Scimeca [1997b] conducted an experiment to examine the dose response of CLA (0.5,
1, 1.5, and 2%) in rats fed a 2% or 12 % LA diets to mammary carcinogenesis. The same
DMBA model was used for the mammary tumor initiation. The results showed that the efficacy of tumor suppression by CLA was similar as previous studies (35 to 63% inhibition at the different dietary CLA levels) and not affected by different level of LA in the diets. Additionally, with either LA diet, the maximal protection was found in the 1%
CLA supplementation. These findings suggest that there may be distinctive mechanisms in the modulation of tumor development by LA and CLA additional to, or other than, the competition between LA and CLA.
Beside the studies on chemically induced mammary carcinogenesis, recently a study conducted by Visonneau et al. [1997] showed that CLA suppressed the growth of human breast cancer cells in mice. This study used a human breast adenocarcinoma model
[Hillyard and Abraham, 1979; Ip et al., 1985] which was done by subcutaneous inoculation of MDA-MB468 cells into the mammary tissue of severe combined immunodeficient (SCID) mice. The subcutaneous inoculation of human breast cancer cells then resulted in the local growth of a primary tumor mass and systemic spread to the
34 lungs, peripheral blood and bone marrow of the mice. To test the CLA effect, the SCID mice were fed 1% CLA diet for two weeks prior to subcutaneous inoculation of 10^
MDA-MB468 cells and throughout the experiment. Results showed that dietary CLA significantly (P<0.05) inhibited local tumor growth (73% and 30% tumor growth inhibition in early and advanced stages, respectively) and completely abrogated the spread of breast cancer cells to lungs, peripheral blood, and bone marrow. Morever, the tumor histology was strikingly different in the CLA and control mice: the tumors in the CLA mice lacked the typical ductal organization of breast adenocarcinomas and presented significant fibrosis compared with the control mice. This results not only demonstrated and supported earlier observations on CLA antitumorigenesis activity, but for the first time showed the ability of dietary CLA to block both the local growth and systemic spread of human breast cancer, via mechanisms independent of the host immune system. The use in this study of an immunodeficient mouse model demonstrated that the CLA anticarcinogenic effects are independent of the host immune system, therefore, excluded hypothesis that the CLA anticarcinogenesic effects might occur through immunomodulatory mechanisms.
In studying the dietary CLA effects on transplantable mammary tumors growth,
Wong et al. [1997] infused 1x10* WAZ-2T metastatic mammary tumor cells into the right inguinal mammary gland of the mice. They found that the dietary CLA (0.3 and 0.9%) did not affect mammary tumor growth, tumor latency, tumor incidence and tumor lipid peroxidation activity in the infused mice. They concluded that dietary CLA had no effect
35 on the an established, aggressive growing mammary tumor. Therefore, CLA seems to
exert its tumor-inhibitory effect at a specific, early stage of carcinogenesis.
CLA and Colon Cancer
CLA has also been reported to inhibit the colon carcinogenesis [Zu and Schut, 1992;
Liew et al., 1995]. Results from study conducted by Liew et al. [1995] showed that
dietary CLA significantly reduced the total number of aberrant crytp foci (ACF) of colon
(4.0 vs. 14.3 ) and the number or ACF/colon (1.1 vs. 4.3) induced by a dietary
heterocyclic amine (2-amino-3 -methylimida:6o[4,5-f]quinoline (IQ)) in mice. This
anticarcinogenic effect of CLA on the IQ induced colon carcinogenesis was suggested to
due to the ability of CLA to inhibit the IQ-DN A adducts formation in the colon.
CLA and Cancer Cells
Additional evidence of CLA inhibition of carcinogenesis was found in the direct
inhibitory action of CLA on various cancer cells reported in several in vitro cell culture studies [Shultz et al., 1992a; 1992b; Schonber and Drokan, 1995; Liew et al., 1995;
Cunningham et al., 1997; Liu and Belury, 1997; Durgam and Fermamdes, 1997].
Shultz et al. [1992a] first studied the inhibition effects of physiologic concentrations of CLA (1.78 - 7.14 X 10"’ M) on human malignant melanoma (M21-HPB), Colorectal
(HT-29), and breast (MCF-7) cancer cells. Their results showed that culture media supplemented with CLA significantly retarded the growth of all those cancer cells (18% to
36 100% reductions in cell proliferation )as compared to control cultures following a 12 days
incubation. The CLA appeared to be cytostatic to M21-HPB and HT-29 cells at
concentration of as low as 1.78x 10'^ M, and to be cytotoxic for MCF-7 cells at
concentration of 3.57x 10’’ M. Therefore, CLA was proved to be a effective inhibitory
agent to prevent in vitro human cancer cell growth. Additionally, the MCF-7 breast
cancer cells was found to be the most sensitive to inhibition by CLA.
To compare the influence of LA and CLA on cancer cell growth, Shultz et al. [1992b] incubated human MCF-7 breast cancer cells with various concentrations (1.78 - 7.14 x 10'
* M ) of LA or CLA. They found that LA initially stimulated MCF-7 cell growth with an optimal effect at concentrations of 3.57 to 7.14 x 10'^ M, but was inhibitory at similar concentrations after 8 and 12 days of incubation. By contrast, CLA inhibited the growth of MCF-7 cells in a dose- and time- dependent manner with the CLA concentration of
1.78 X 10'^ M to be cytostatic and 3.57 -7.14>: 10'^ M to be cytotoxic to the MCF-7 cells.
These results further demonstrated and support the finding that CLA is associated with growth inhibition and mortality of cultured human breast cancer.
Other Factors Relate CLA to Cancer Inhibition
CLA also was suggested as a possible factor responsible for the inverse association between milk consumption and breast cancer risk in a recently reported Finnish prospective pathological study [Knekt et al., 1996]. In addition, it is reported that in
India, where ghee is often used, various religious communities have age-adjusted breast
37 cancer rates which vary up to three fold. This suggested an case-control study on the association between consumption of cow and buffalo ghee, other CLA-rich foods and incidence of breast cancer in Indian women[Jussawalla et al., 1985]. Interestingly,
Fogerty et al. [1988] found breast milk from women of the Hare Krishna religious sect contained twice as much CLA as milk from conventional Australian mothers (40.0 vs.
20.7 |imol/g). This was attributed to the butter and ghee diets consumed habitually by the
Hare Krishna women. Also in general, McGuire found that human milk from American women contained CLA range from 2.23 to 5.43 mg/g fat which were much higher than infant formula [McGuire et al., 1997]. Dietary CLA is suggested to be a potential nutrient in preventing cancer and other diseases.
2.2.2 CLA and Atherosclerosis
Besides its anticarcinogenic activity, CLA has also been observed to be hypocholesterolemic and antiatherogenic in rabbits and hamsters [Lee et al., 1994;
Nicolosi et al., 1997]. In the experiment conducted by Lee et al. [1994], a semi-synthetic atherogenic diet (14% fat and 0.1% cholesterol) supplemented with or without CLA (0.5 g CLA/rabbit/day) were fed to the rabbits. By 12 weeks after the atherogenic diet, they found that the plasma total and low density lipoprotein (LDL) cholesterol and triglycerides were significantly lower in the CLA treatment group than in the control group. They also found that the LDL cholesterol to HDL cholesterol ratio and total cholesterol to HDL cholesterol ratio were significantly reduced in the CLA treatment group relative to the
38 control group. From the histological examination of the aortas of the animals in both
groups, they observed that the same type of atherosclerotic lesion distributions,
characterized by an accumulation of lipids and matrix fibers in the hyperplasic areas of the
inner layer of the intima, were appeared in animals in both groups. However, the
percentage of total aortic surface covered by fatty lesions for CLA treatment group was
markedly reduced when compared to the control group (43% vs. 55%). Additionally,
CLA-fed rabbits exhibited less histological evidence of atherogenesis in lipid deposition
and in connective tissue development. Additionally, they found many correlations
between those variables used to assess atherosclerosis. In both groups, a correlation
between the aortic atherosclerosis involvement (%) and final LDL cholesterol (r=0.7626,
P<0.05) was found. The aortic atherosclerosis involvement (%) in the control group also
correlated with average LDL cholesterol (r= 0.9357, P<0.05) and with average plasma
total triglycerides (r=0.9577, P<0.05). Aortic cholesterol content was correlated with
final plasma total cholesterol (r=0.8071, P<0.01), and with average plasma total
cholesterol (r=0.71l5, P<0.05). There was a correlation between the abdominal plaque to
wall volume ratio and average plasma total cliolesterol/HDL cholesterol ratio (r=0.9938,
P<0.01) in the CLA group. Average LDL cholesterol/HDL cholesterol ratio and hepatic cholesterol content was found negatively correlated (r=-0.8854, P<0.05) in the CLA group. Therefore they suggested that the retardation of atherosclerosis by CLA is at least
in part due to the changes in lipoprotein metabolism. This study is the first to show the inhibition efifect of CLA on atherosclerosis de\ elopment.
39 Nicolosi et al. [1997] further examined the CLA effect on atherosclerosis development by using a hamster model. Hamster was chosen as the model for this study is because the established responsiveness to plasma cholesterol lowering and anti atherogenic interventions of hamster [Spady and Dietschy, 1985]. In this experiment, hamsters were fed a hyper-cholesterolemic control diet ( 10% coconut oil, 1% safflower oil, and 0.12% cholesterol) supplemented with either CLA or LA. Results showed that the plasma total cholesterol and non-HDL(conibined very low and low density lipoprotein) cholestrol in CLA group were reduced by approximately 23% and 25%, respectively, relative to the control group. While when compared to the LA group, the total cholesterol and non-HDL cholestrol in CLA group were reduced by approximately 11% and 12%, respectively. Plasma total triglycerides in CLA and LA group were also significantly reduce compared to the control. However, no significantly difference was found for plasma HDL cholesterol in all groups. Histological examination of the aortas of the animals showed that fatty streak area of the aortic arch was reduced by 25% and 26% in the CLA and LA group, respectively, compaied to the control group. That means less early atherosclerosis in the CLA and LA-fed hamsters compared to the control. However, in this experiment no significant correlations between plasma lipids, non-HDL cholesterol and early atherosclerosis were observed. This study proved and supported the finding that
CLA reduces plasma lipoproteins and aortic atherosclerosis.
The observations from these two studies agreed with each other in many ways and provided strong evidence indicated that dietary CLA can modify plasma lipids and
40 lipoprotein cholesterol levels. However, the mechanism(s) of the potential anti atherogenic effect of CLA were not studied in detail in these two studies. Both antioxidant and mediating lipoprotein metabolism were suggested to be the possible mechanisms of CLA effect, but more studies are need to elucidate them.
2.2.3 CLA and Immune Functions
The ability of CLA overcome the immune induced growth suppression was first demonstrated by Cook et al. [1993] in their studies on nutritional control of immune- induced growth depression. They carried out cwo chick trials and one rat trial to study the ability of CLA to prevent reduced growth rate following endotoxin injection.
In the chick trails, 1 day-old chicks were started with a basal (control) diet or the basal diet amended with 0.5% CLA. When the chicks were 3 weeks of age, they were given a i.p. injection of endotoxin, Escherichia coli lipopolysaccharide (LPS). Results showed that chicks fed with CLA and injected with the endotoxin continued to grow, whereas those fed with control diet either failed to grow or lost their body weight following the endotoxin injection. In addition, they found that the CLA feeding had no adverse eflfects on any immune variable measured the chicks.
In the rat trail, rats were fed a control diet or the control diet supplemented with 0.5%
CLA. The i.p. endotoxin (LPS) injection was given 4 weeks after starting the experimental diets. Results in this experiment showed that over the 24-hour period after
41 the endotoxin injection both the control and CLA-fed rats lost their body weight, however, the loss of their body weight in CLA-fed rats was only half of the weight loss of the control rats. In addition to the reduced body weight loss the CLA-fed rats also showed enhanced phutohemagglutin response and macrophage phagocytosis.
Therefore, studies on both chicks and rats demonstrated that CLA was effective in the prevention of growth depression induced by immune stimulation (endotoxin injection).
These findings also suggested that the CLA eflfects are not species-limited and the biological mechanism appeared conserved across animal species.
In attempt to compare fish oil and CLA in response to the same immune stimulation.
Miller et al. [1994] studied the effect of fish oil and CLA on endotoxin induced growth inhibition and food intake depression. In this experiment, mice were fed a semi-purified basal diet, the basal diet with 0.5% added fish oil, or the basal diet with 0.5% added CLA, and the endotoxin (LPS) or the vehicle injections (i.p.) were given 2 weeks after starting the experimental diets. Results showed that by 24 hours after the injection, body weight losses in basal and fish oil fed mice were similar but twice that of CLA-fed mice, which was twice of the body weight loss compared to vehicle injected basal fed control. By 72 hours post the injection, mice fed CLA diet and injected with the endotoxin had body weights similar to vehicle injected controls, however, body weights of basal and fish oil fed mice with endotoxin injection were still reduced. The results of feed consumption were parallel to the body weight loss results. Mice injected with buffer consumed
42 significantly more feed at ail time periods than mice fed either the basal diet, fish oil, or
CLA with the endotoxin injection, however, mice fed CLA consumed significantly more feed than mice fed fish oil or the basal diet with endotoxin injection. In addition, this experiment also showed that CLA fed mice had a 1.5 fold increase in phytohemagglutinin-
P induced spleen lymphocyte blastogenesis over basal fed mice and displayed greater immune responses than fish oil fed mice. Tliis study further proved and supported the finding that CLA is effective in the prevention of growth depression induced by immune stimulation. In addition, this study indicated that considerable less CLA is needed than fish oil (typical studies use 8 %) to achieve the effectiveness.
Although CLA was shown to be effective in preventing of growth depression induced by immune stimulation, thus far, only some limited number of studies are available describing the effect of dietary CLA on immune function and on the interaction between immune function and cancer or other CLA related disease. Michal et al. [1992] first reported that porcine blood lymphocytes cultured with 1.78 -7.14x10*^ M CLA displayed a dose-dependent enhancement of podeweed migogen-induced lymphocyte blastogenesis, and increased concanavalin A- and phytohemagglutinin-induced lymphocyte proliferation.
Supplementation suppressed EL-2 production. Cook et al. [1993] then showed with a foot pad assay that rats fed 0.5% CLA for 4 weeks elicited enhanced phytohemagglutinin- induced lymphocyte blastogenesis. Miller et al. [1994] also reported that CLA fed increased phytohemagglutinin-indeced lymphocyte proliferation and the phagocytosis of fluorescein isothiocyanate-labeled yeast cells by macrophages. However, dietary CLA
43 showed no effect on antibody production in response to sheep red blood cells in chicks or rats [Cook et al., 1993].
A resent study conducted by Wong et al. [1997] examined, in more detail, the effects of dietary CLA on lymphocyte function in vivo by using a mice model. In this study, different levels of CLA (0.1, 0.3, or 0.9%) were fed to mice for 3 or 6 weeks. The lymphocyte function was examined by assessing the lymphocyte proliferation, IL-2 production, and lymphocyte cytotoxicity of the spleenic lymphocytes. Data showed at week three, phytohemagglutinin-induced lymphocyte blastogenesis in mice fed CLA was stimulated (P<0.05) in a dose-dependent manner, and lymphocyte proliferation increased
146% and 192% in mice fed 0.3% and 0.9% ('LA respectively as compared to the control mice. Production of IL-2 also was stimulated by CLA feeding. However, the stimulatory effect observed with CLA at week three was not evident at week six. In addition, the dietary CLA did not show significant influence on Concanavalin A- or LPS-induced lymphocyte proliferation and did not effect lymphocyte cytotoxicity both at week three and six. This result clearly showed that dietary CLA could modulate certain aspects of the immune defense and in part agreed with other studies.
2.2.4 CLA Effect on Growth and Body Composition
Studies conducted on CLA research so far have shown no evidences of CLA directly improving growth or helping loss body weight. However, there are studies shown that dietary CLA acted as a growth factor for young rats in improving their body mass and
44 feed efficiency [Chin et al., 1994a], and dietary CLA changed body composition in
reducing total body fat and increasing body protein and water content, while kept body
weight unchanged [Park et al., 1997].
Chin et al. [1994a] studied the effect of dietary CLA on young rat development and
growth. In their study, female rats were fed control or CLA-supplemented diets during
gestation and lactation. By measuring feed consumption, body weight change, and litter
sizes, they found that consumption of CLA during gestation and lactation did not affect
the food intake or body weights of the dams and also did not affect the litter sizes.
However, the postnatal body weight gain of pups, measured on day 10 of lactation, was
significantly improved by the CLA feeding (12.8 vs. 14 g). Moreover, pups that continued
to receive the CLA-supplemented diet after weaning had significantly greater total body
weight gain (134.9 vs. 126.8 g) and improved feed efficiency (0.381 vs. 0.362 g /g of
food intake) relative to control animals. Additionally, when measured the milk
composition, they observed that there was more protein in milk from dams fed diet containing CLA (114.1 vs. 95.15 g protein/L) when compare to the control. This finding
seemed to indicate that a complex mechanism of action for CLA was involved to promote the growth and feeding efficiency in the pups. The actual mechanism for CLA action in the growth promotion is not clear. Chin et al. suggested that CLA may a growth factor for at least some animal species.
45 Although CLA showed no effect on body weight change for various animal species,
the effects of CLA on body composition were observed in several animal species including
in humans. Park et al. [1997] investigated the dietary CLA effects on body composition
on mice. In two separated experiments 0.5% CLA was fed to female and male mice for 4-
5 weeks. The body composition analyses, which were conducted by measuring total empty carcasses weight, total body water, toial body fat, and total nitrogen in the body,
showed that the percentage body fat in CLA-fed mice was reduced by 57% (males) and
60% (females) relative to their respective controls. By contrast, the percentages of whole body protein was significantly enhanced for CLA-fed mice by 5% (males) and 14%
(females), while total carcass water also enhanced by 7% (males) and 11% (female) relative to the controls. These data established that CLA is a potent regulator of body fat accumulation and retention.
Pariza et al. [1996] reported the same pattern of effects of CLA in decreasing body fat and increasing lean body mass or carcass water on several other animal models including mice, rats, chicks, and pigs. Among the tested animal species, they found that mice exhibited the most reduction in body fat (57-70% for mice vs. 22-25% for other animal species) and increasing in lean body mass (5-14% for mice vs. 3-4% for other animal species). Their results further establish the CLA functions as the a body composition regulator.
46 The results of the first CLA clinical trial on human subjects was announced in July
1997 at the National Foods Association’s Marketplace’97 trade show [PhannaNutrients,
1997]. This human clinical trial involved 20 men and women in a 90-day study with a double-blind, placebo-controlled experiment design. The subjects involved in this study had the average body weight and normal body mass index relative to the American population. CLA was given at 3 g/day dose to the subjects while kept them in their normal diet habits and exercised patterns. Results after 90 days of the CLA supplementation showed that the body fat percentage of the CLA group subjects reduced
20% (from 21.3% to 17.5%), which was a average 7 pounds fat reduction, when compared to the placebo group. In addition to the reduced body fat content, a slight, but insignificant, drop in the body weight was observed in the CLA group subjects. In order to explain the balance between the relative unchanged body weight and the reduced body fat content, the investigator suggested that the body lean mass or muscle increased to compensate for the body fat loss. This study actually was the first CLA study conducted on humans. The agreement of this results with the results from animal studies indicated the great potential of the CLA in improving human health.
2.3 Proposed Mechanisms For CLA Effects
While it is well established that the CLA is elective in inhibition of carcinogenesis, prevention of atherosclerosis and other diseases, the mechanisms by which CLA influences carcinogenesis and other disease development, although widely studied, are largely
47 unresolved. Various studies suggest that CLA may act by multimechanisms in response to its devised effects. As to date, several mechanisms have been proposed to explain its multi-effects and the proposed mechanisms include antioxidant activity [Ha et al., 1990; Ip et al., 1991], prooxidant cytotoxicity [Schonberg and Drokan, 1995], inhibition of nucleotide synthesis [Shultz et al., 1992], interruption of signal transduction [Ito and
Hirose, 1989], alteration of eicosanoid synthesis [Cunningham et al., 1997], reduction of proliferative activity [Ip et al., 1994], inhibition of both DNA-adduct formation [Zu and
Schut, 1992] and carcinogen activation [Liew et al., 1995], and interfering with the hormone regulated mitogenic pathway [Durgam and Fernandes, 1997].
2.3.1 Antioxidant Activity
Ha et al. [1990] and Ip et al. [1991] first proposed that the anticarcinogenic effect of
CLA was due to its antioxidant activity. There is large and growing body of evidence indicating that free radicals and radical-mediated oxidation processes play a role in carcinogenesis and atherosclerosis and variety of antioxidants have been shown to be protective in several experimental carcinogenesis and atherosclerosis models [Ito and
Hirose, 1989; Bjorkhem et al., 1990]. The antioxidant activity of CLA was first demonstrated by Ha et al [Ha et al., 1990]. In their in vitro culture experiment, the CLA antioxidant activity based on the peroxide value of LA oxidation was compared with ascorbic acid, a-tocopherol and butylated hydroxytoluene (BHT). They showed that at a molar ratio of 1 part CLA to 1000 parts LA, the peroxide formation from LA was
48 inhibited by more than 90%. The antioxidant activity of CLA was comparable to that of
BHT but more effective than that of a-tocopherol. When the UV spectra of the hexane extract of the reaction mixture containing CLA was examined, they found a decreased absorption at 234 nm and an increased ab.sorption at 268 nm, which suggested the incorporation of a ketone group into the CLA molecule [Allen et al., 1949; Sephton and
Sutton, 1956; Lundberg and Chipault, 1947]. Based on these findings, they hypothesized that an oxidized derivative of CLA with a P-hydroxy acrolein moiety, rather than CLA itself, is the active antioxidant species, and that this P-hydroxy acrolein moiety can either chelate iron or form resonance enolization to function as an antioxidant [Ha et al., 1990].
In a separated experiment. Ip et al. [1991] measured the amount of thiobarbituric acid-reactive (TBAR) substances, a biomarker often used to assess oxidation in biological systems, in liver and mammary gland homogenates from rats that had been fed diets containing either different levels of CLA (0.25, 0.5, 1.0, or 1.5%), 0.05% vitamin E, or
0.1% butylated hydroxyanisole (BHA) for one month but were not treated with carcinogen. Their results showed that dietary CLA had no effect on the amount of TBAR substances in the liver, but caused a significant reduction of that in the mammary gland
(17.9 vs. 27.0 nmol/g tissue), while vitamin E and BHA significantly reduced the TBAR substances in both liver and mammary. Interestingly, they also observed that the antioxidant activity of CLA was independent of the dose. The maximal antioxidant activity of CLA could be achieved by feeding rats with 0.25% CLA in the diet. However, the maximal tumor suppression was observed when about 1% CLA was used in diet, there
49 is clearly a discrepancy between antioxidant efficacy and anticarcinogenic activity, which suggests that some other mechanisms are likely involved in the anticarcinogenesis of CLA
[Ip et al., 1991].
By the structure of CLA itself it is no: apparent that these compounds could be effective antioxidants. CLA does not have an automatic form nor does it have the functional group required to chelate metal ions. Ha et ai. [1990] has speculated that CLA exhibits its antioxidant activity through its oxidation products but they were not able to identify the oxidation products. Yurawecz et ;il. [1995] have identified the following furan fatty acids (FFA) as the oxidation products of CLA: 8 ,11-epoxy- 8 ,10-octadecadienoic;
9,12-epocy-9,l 1-octadecadienoic; 10,13-epoxy-lO, 12-octadecadienoic; and 11,14-epoxy-
11,13-octadecadienoic. They further observed that CLA and the FF As were very stable in triacylglycerols and in methanol or borate buffer solution even subjected to heat and aeration. They attributed this greatly improved stability of CLA and its derivatives to their antioxidant properties, i.e., readily to react, when solvated, with oxidants that are more reactive than those found in ambient air.
However, this explanation was question by other researchers. Van den Berg et al.
[1995] examined whether CLA could protect membranes composed of l-palmitoyl-2- linoleoyl phosphatidylcholine (PLPC) from oxidative modification under conditions of metal ion-dependent or -independent oxidative stress. They monitored the progress of oxidation by direct spectrophotometric measurement of conjugated diene formation and
50 by gas chromatographic/mass spectrometric analysis of fatty acids. Their results showed that the oxidative susceptibility of CLA was comparable to arachidonic acid but higher than that of LA. In addition, CLA did not have any significant effect on PLPC oxidation.
This is in contrasting with vitamin E and BHT which can effectively inhibit PLPC oxidation. Thus, they concluded that CLA does not act as an efficient radical scavenger in a way comparable to vitamin E or BHT and that CLA does not appear to be converted into a metal chelator under metal-ion dependent oxidative stress.
2..3.2 Prooxidant Cytotoxicity
Shultz et al. [1992a; 1992b] proposed prooxidant activity as an alternative hypothesis for the involvement of CLA in the induction of cytotoxicity in cancer cell. This proposed mechanism is based on findings that addition of relatively high concentrations of various polyunsaturated fatty acids (PUFAs) to cultures of malignant cells increased the secretion of TBAR products and in paralleled the cancer cell death when compared with normal, nontransformed cells [Begin et al., 1988]. Shultz et al. [1992a] observed that CLA at concentration of 3.57 - 7.14 x 10*’ M could induce cytotoxicity to human M2I-HPB malignant melanoma, HT-29 colorectal, and MCF-7 breast cancer cells, and that CLA was a more potent c>'totoxic agent than linoleate or P-carotene in MCF-7 breast cancer. Thus they suggested that the lipid peroxidation of the unsaturated CLA may give rise to a variety of oxidation products with the potential of interfering with cell replication and /or cell survival.
51 CLA-induced cytotoxicity may be due to the nature of the conjugated double-bond
system, since it is suggested that the conjugated double bond system can allow for more
efficient trapping of electrons [Cornelius et al., 1991]. In the study conducted by
Cornelius et al. [1991], another conjugated fatty acid, parinaric acid (C l8:4, 9,11,13,15),
was found to be incorporated into undifferentiated cells (HL-60, Y-791, and U937 cells)
at approximately one sixth of the dose required for similar incorporation into differentiated
cells. In addition, parinaric acid was more cytotoxic in these same undifferentiated cells
than in the undifferentiated cells. This selective uptake and cytotoxicity of parinaric acid
in undifferentiated cells suggested that conjugated double-bonded PUFAs might be
protective against carcinogenesis by a cancer cell-specific effect. Additionally, parinaric
acid was also observed to be more cytotoxic than several nonconjugated unsaturated fatty
acids including oleate (18:ln-9), linoleate (C18:2n-9), and moroctate (18:4n-3) in several
tumor cell lines. Since the cytotoxicity of parinaric acid could be blocked by the addition of BHT, Cornelius et al. [1991] suggested that the more efficient trapping of electrons within the double-bond system enhanced the likelihood of superoxide anion generation, which led to cytotoxicity of cancer cells. Similarly, the cytotoxications of CLA may also be due to its conjugated double system and more potent in cancer cells than in differentiated cells.
52 2.3.3 Modulation of Eicosanoid Biosynthesis
Eicosanoids are hormonelike compounds derived from the oxygenation of arachidonic
acid. There are several different families of eicosanoids and each of them play different
orals in the biological system. Some cause platelets to aggregate and blood vessels to
constrict; others dilate blood vessels and increase blood flow; some induce powerful
muscle contraction; others are strong chemotactic agents, attracting leukocytes to sites of
inflammation; eicosanoids also modulate hypothalamic and pituitary release of hormones
[Nelson et ai., 1982; Lands, 1992]. Due to their diverse biological functions, eicosanoids
have involved in many physiological conditions including atherosclerosis, carcinogenesis,
diabetes, and various inflammatory disease [Simopoulos et al., 1991].
The involvement of eicosanoids in carcinogenesis is well established. Eicosanoids have been shown to be associated with the proliferation of cultured cells derived from normal breast epithelium and fibroadenomas /Blalkrishnan et al., 1989], breast [Noguchi et al., 1995], colon [Bull et al., 1989], and skin [Fischer et al., 1989] cancer cells. Both the cyclooxygenase metabolite, prostaglandin Ez and the lipoxygenase metabolite, leukotriene B 4 have been observed to directly stimulate the growth of malignant cells [Lee and Ip, 1992; Snyder et al., 1989; Chambers and Cohen, 1990]. Studies have also shown that modulation of eicosanoid biosynthesis through the inhibition of various pathways of arachidonic acid metabolism result in the supj)ression of tumor growth and the inhibition of mammary carcinogenesis [Lee and Ip, 1992; Noguchi et al., 1995].
53 Eicosanoids are synthesized from arachidonic acid by the actions of enzymes
cyclooxygenase and lipoxygenase. Since arachidonic acid is the precursor of eicosanoids,
anything that influences the availability of this fatty acid affects eicosanoid production.
Conversion of LA to arachidonic acid is an important pathway for the in vivo biosynthesis
of arachidonic acid. Alteration of this pathway can, therefore, affect the production of
eicosanoids. CLA as the isomeric derivative of LA has been proposed to be able to
compete with LA for the biosynthesis of aracliidonic acid and eicosanoids which intern to
cause the effects on carcinogenesis, atherosclerosis, inflammation, and other physiological
conditions [Ha et al., 1991; Lee et al., 1994; Cunningham et al., 1997; Liu and Belury,
1997; Sugano et ai., 1997].
Early studies showed that the anticarcinogenic effect of CLA was associated with
incorporation into rat mammary tumor and mouse forestomach phospholipids [Ip et al.,
1991; Ha et al., 1990]. This is believed to be significant in the development of cancer because arachidonic acid is predominantly presented in membrane phospholipids and the phospholipid arachidonate can be metabolized to form eicosanoids only after being released fi*om phospholipids via activation of phospholipase Az. The incorporated CLA may compete with LA in the phospholipids for the biosynthesis of arachidonic acid or it may compete with the arachidonic acid for the enzyme phospholipase A 2 for the releasing.
Miller et al. [1994] further showed that the CLA effect on endotoxin induced weight loss was related to the factor that dietary CL.\ (0.5%) partially displaced arachidonate in
54 fat pads and caused significant muscle arachidonic acid decrease. These decrease in
muscle arachidonic acid was suggested to cause depressed eicosanoids (PGEz) production
which then mediate to decrease muscle catabolism, therefore, prevent the endotoxin
induced weight loss.
The impact of dietary CLA on the relative composition and interconversion of
nonconjugated fatty acid in neutral and phospholipids of tissue lipids is also considered to
be an important measure in studying the efifect of CLA on the biosynthesis of eicosanoids.
Belury and Dempa-Steczko [1997] examined the effect of increasing level of dietary CLA
(0.5-1.5%) on the composition of neutral and phospholipids of mouse liver. They found
that increasing levels of dietary CLA exhibited dose-responsive eflfects on the content of
several fatty acids in neutral lipids. Significant elevated CLA and oleate (C18:l) along
with reduced linoleate (C18;2) and arachidonate (C20:4) were observed in liver neutral
lipids o f mice fed high levels (1,1.5%) CLA diets. However, these dose-responsive eflfects
were not seen on the liver phoshpolipids. Only significantly elevated CLA and reduced
linoleate were observed in the liver phospholijiids of mice fed with 1.5% CLA diet. These
findings suggested that CLA may affect metabolic interconversion of fatty acid in liver that
may ultimately result in modified fatty acid composition and arachidonate-derived
eicosanoid production in extrahepatic tissues.
Sugano et al. [1997] further studied the impact of dietary CLA on the relative composition of liver individual phospholipids. Although no significant differences in the
55 compositions of major fatty acids in each phospholipid between the control and CLA groups were observed, the ratio of(C20:3n-6 + C20:4n-6)/C18:2n-6, an index for linoleic acid desaturation, were seen significant higher in phosphatidylinositol and cardiolipin of rats fed CLA diet relative to the controls. They also observed that the incorporation of
CLA was not the same among the phospholipid species, a increased incorporation of CLA was shown in order of phosphatidyllinositol > cardinolipin > phosphatidyl ethanolamine > phosphatidylserine > phosphatidylcholine. These different patterns of incorporation of
CLA in liver phospholipid species may suggest the specific role of CLA in the metabolic function when considering different functions of individual phospholipid [ Ansel and
Spanner, 1982].
To investigate the cause of CLA impact on tissue fatty acid composition, Belury and
Dempa-Steczko [1997] tested the ability of CLA to act as a substrate for A 6 desaturase by using radio labeled [I-‘‘*C]-CLA and [l-"C]-linoleate in an in vitro assay. They observed that CLA was desaturated to an unidentified C18:3 product to a similar extent as linoleat conversion to y-linoleanate (9.88 and 13.63%, respectively), therefore, demonstrated that
CLA is a suitable substrate for A6 desaturase and the A 6 desaturase has no preference to utilize either CLA or linoleate. Banni et al. [1995] and Sebedio et al. [1997] have detected conjugated C l8:3, C20:3, C20:4 as the CLA metabolites in liver of rats and lambs. Which clearly supported the finding of CLA as a substrate for A 6 desaturase.
56 Based on these findings, three lines of e\ idence can be seen in suggestion that CLA
may compete with linoleate for the in vivo biosynthesis of arachidonic acid. First, when available as a sole substrate, ‘‘‘C -CLA was desaturated to a similar extent as "C-LA by
A6 desaturase, the rate-limiting step in the conversion of linoleate to form arachidonate.
Second, dietary CLA was associated with reduced arachidonate in liver lipid, suggesting less linoleate was converted to arachidonate. Finally, because oleate is also a substrate for
A6 desaturase, dietary CLA induction of oleate accumulation in total and neutral lipids suggests CLA was desaturated at the expense of oleate. Together, these findings suggest that dietary CLA modulation of A 6 desaturase activity to reduce phospholipid associated arachidonate, resulting in an overall decrease in arachidonate-derived eicosanoids.
The direct relationship between CLA and eicosanoids biosynthesis was also investigated [Liu and Belury, 1997; Sugano et al., 1997; Cummingham et al., 1997]. Liu and Belury [1997] studied the eicosanoids (POE) production in skin keratinocyte cell line
(HEL-30) prelabeled with radioactive LA, CLA, or AA. They observed that '^C- prostanglandin E release in ^^C-CLA prelabeled cultures was 6 and 13 times lower than cultures treated with ‘‘‘C-LA and ^'*C-AA, respectively. In addition, they found the phorbol ester-induced release of "C-CLA was 1.5 times higher than ‘‘‘C-LA and ‘‘‘C-AA.
These data indicated that the inhibition of CLA in eicosanoids production was associated with both CLA releasing and conversion.
57 The in vivo eicosanoid production was examined by Sugano et al. [1997]. After fed rats with 1% CLA for 2 weeks, they found that the serum prostanglandin Ez concentrations of rats in CLA group were significantly lower than that of rats in control group. Their results agreed with Liu and Belury’s finding. Therefore, evidences from both in vitro and vivo studies suggest that CLA is able to reduce the eicosanoid production.
By using eicosanoid synthesis inhibitors Cunningham et al. [1997] further proved the modulation effect of CLA on eicosanoid biosynthesis. They incubated normal human mammary epithelial cells (HMEC) and MCF-7 breast cancer cells with or without the eicosanoid synthesis inhibitors (indomethacin, INDO the cyclooxygenase inhibitor; or nordihydroguaiaretic acid, NDGA, the lipcxygenas inhibitor) in serum-free medium supplemented with LA or CLA. They found that growth medium supplemented with LA significantly stimulated the growth of both normal HMEC and MCF-7 breast cancer cells, however, that the addition of INDO or NDGA to the medium inhibited the growth of both cells. These results indicated that the conversion of LA to arachidonic acid and to various prostaglandins was essential for the observed stimulatory growth effect. They also observed that CLA supplemented medium significantly inhibited the growth of both
HMEC and MCF-7 breast cancer cells, and the addition of NDGA resulted in synergistic growth inhibition for MCF-7 breast cancer cells, which suggested that the growth suppression was augmented by CLA through inhibition of leukotriene synthesis. These evidences further confirmed the modulation efrect of CLA on eicosanoid biosynthesis.
58 2.3.4 Inhibition of Nucleotide Synthesis and DNA-adduct Formation
Shultz et al. [1992a] first observed when MCF-7 breast cancer cells preincubated in
CLA supplemented media incorporated significantly less [^Hjleucine (45%), [^H]uridine
(63%), and [^Hjthymidine (46%) than did the control cultures. While when M21-HPB malignant melanoma and HT-29 colorectal cells were similarly incubated with CLA, the cells incorporated significantly less [^H]leucine (25% and 30% respectively) than control cultures. Therefore, they proposed that growth inhibition of cancer cells by CLA may be due to the ability of CLA to inhibit protein and nucleotide biosynthesis.
Cunningham et al. examined the effects of LA and CLA on isotope incorporation of amino acid in normal human mammary epithelial cells (HMEC) and MCF-7 breast cancer cells. They found that both cells preincubated in media supplemented with LA incorporated 10% and 15% more [^H]thymidine than did control culture receptively, however, when the cells preincubated in media supplemented with CLA, the cells incorporated significantly less [^H]thymldine (27% and 82% respectively). These results further supported the hypothesis that the inhibition effect of CLA on cancer cell growth may be due to the ability of CLA to inhibit protein and nucleotide biosynthesis in cells.
The inhibitory effect of CLA against the formation of 2-amino-3-methylimidazo[4,5-
/]quinoline-DNA adducts in the liver, lung, kidney and colon of mice and rats was observed in two separated studies conducted by Zu et al [1992] and Liew et al[ 1992].
The 2-amino-3-methylimidazo[4,5-/]quinoline is a mutagenic heterocyclic amine which
59 has been shown to produce tumors at several sites including the liver, skin, small intestine
and colon [Takayama et al., 1984]. In both of these studies, CLA was administered during the initiation phase of carcinogenesis, therefore, indicated that the CLA inhibitor could act as a blocking agent to limit carcinogen activation [Zu et al., 1992; Liew et al.,
1992].
2.3.5 Modulation of Signal Transduction
Modulation of signal transduction pathways is another hypothesis for CLA anticarcinogenesis and has received some attentions in the past few years. Signal transduction describes the events that occur within a cell to send messages from outside of the cell through the plasma membrane inwaid to the nucleus. Stimulation of the cell membrane triggers a cascade of enzymatic and biochemical events that regulate a variety of cellular processes, including cell differentiation and division [Huang, 1989; Nishizuka,
1992].
One family o f structurally homologous enzymes known to be involved in signal transduction is protein kinase C. Once activated, protein kinase C initiates a cascade of events within the cell leading to regulation of a host of cellular processes. Because phospholipid is required for activation of protein kinase C, research efforts have been directed toward defining whether fatty acid composition of phospholipids could alter protein kinase C activation. Several studies have shown that many species of polyunsaturated fatty acids activate protein kinase C with differing potencies [Craven et
60 al., 1987; Merili, 1993; Belury et al., 1993; Lo et al., 1994]. CLA has been observed to
alter the composition of phospholipid composition [Sugano et al., 1997; Belury and
Dempa-Steczko, 1997], therefore, it may affect the activation of protein kinase C. Lo et al. [1994] reported that CLA was able to inhibit the stimulation of phospholipase C in
Swiss-mouse 3T3 cells by the tumor promoter, 12-0-tetradecanoylphorbol-13-acetate.
This CLA inhibition of phospholipase C may be relevant to the modulation of carcinogenesis, since CLA and LA may directly interact with the phospholipase C signaling pathway, acting to inhibit or stimulate the breakdown of inositol phosphate products and influence phosphatidylinositol turnover.
Hormones as the signal transduction mediator in regulating gene expression is well established. MCF-7 estrogen responsive and MDA-MB-231 unresponsive human breast cancer cell lines were extensively investigated in defining the mechanism by which hormones affect breast tumor cell proliferation [Brooks et al., 1993; Weigel and deConinck, 1993]. MCF-7 contains both estrogen receptor (ER) and progesterone receptor (PR), while MDA-MB-231 do not respond to estrogen and the ER gene is controlled transcriptionally in the ER negative breast cancer cell line. Since CLA has been shown to have a direct inhibitory action on MCF-7 human breast cancer cells in culture
[Schultz et al., 1992a], Durgam and Fernandes [1997] further tested whether the CLA inhibitory action is related to the estrogen response system of MCF-7 cells. They measured the rate of proliferation of estrogen responsive MCF-7 cells and unresponsive
MDA-MB-231 cells cocultured with CLA and LA and compared them with the cells
61 grown on normal media without adding any fatty acids. Their results showed that CLA was effective in inhibiting hormone responsive MCF-7 cell growth (75-80% decrease by viable cell count and 65-70% decrease by thymidine incorporation assay) as compared to
MDA-MB-231 cells (5-10% decrease by viable cell count and 10-15% decrease by thymidine incorporation assay). Their results indicated a possible role for CLA in interference with the estrogen response system.
The modulatory role of CLA in signal transduction was suggested to be an important mechanism in carcinogenic processes. At present time limited study has been done in this area and further investigation is required for the fully understanding of this mechanism.
62 CHAPIER 3
M ATERIA L AND M E T H O D S
3.1 Sources of Materials
The isomer mixture of free fatty acid-CLA and methyl esters-CLA (total CLA
isomers content ~ 99%) were purchased from Sigma Chemical Co. (St. Louis, MO USA).
Other fatty acids and/or their methyl esters, including myristate (C14:0), pentadecanoate
(Cl5:0), palmitate (C16:0), palmitoleate (C16:l), heptadecanoate (Cl7:0), stearate
(C l8:0), oleate (C18:l), linoleate (C18:2n-6), linolenate (C18:3n-3), y-linolenate (C18:3n-
6 ), homogama linolenate (C20:3n-6), arachidonate (C20:4n-6), behenate (C22:0),
lignocerate (C24:0), as well as two gas chromatography (GC) reference standard
mixtures, 6 8 A and RL 6 [Appendix - A and - B for their compositions], were obtained from Nu Chek Prep, Inc. (Elysian, MN USA). Linoleic acid (~ 95% pure) was purchased from Sigma Chemical Co. for CLA synthesis and animal feeding study. The LT 84 and
PGE2 standards and their enzyme immunoassay kits were purchased from Cayman
63 Chemical Comp. (Ann Arbor, MI USA). Organic solvents were purchased from Allied
Signal Inc. (Muskegon, MI USA). All other reagents and chemicals were of highest
available purity.
Sep-Pak C18 Cartridges for solid phase e.xtraction was purchased from Waters Corp.
(Milford, MC USA). Thin layer chromatography (TLC) plates were purchased from
Whatman Inc. (Clifton, NJ USA).
The basal diet AIN93G was purchased fiom DYETS, Inc. ( Bethlehem, PA USA ).
Com oil used in animal studies was obtained h orn local grocery store.
3.2 General Methods
3.2.1 Tissue lipid analysis
In this study total tissue lipid, total CLA concentration in tissue lipid, fatty acid
profiles of tissue lipid, CLA concentrations and fatty acid profiles of liver lipid fractions (
triacylgiycerol, phospholipids, and free fatty acids), and CLA isomer compositions in tissue lipid and in liver lipid fractions were determined. The scheme for the sample
preparation and parameter measurement is showed in Fig. 2.
64 Tissue
Lipid extraction (2 1 chloroform:methanoi)
Total tissue lipid
GC analysis TLC separation
CLA and fatty acid profile Phospholipids Triglycerid Free fatiy acid in total tissue lipid (PL) (TG) (FFA)
GC analysis HPLC separation
CLA and fatty acid profile Purified CLA in lipid fractions GC analysis
CLA isomers
Fig. 2 Scheme of tissue sample preparation and analyses.
65 Lipid Extraction and Fattv Acid Methyl Ester Preparation
Tissue total lipids were extracted according to Folch procedure [Folch et al., 1957].
Briefly, the weighed tissue samples were homogenized in 2:1 chloroform:methanol solution for 1 minute using a Polytron homogenlzer (Polytron Dispersing & Technology,
Kinematica). To ensure complete extraction all the tissue homogenates, which were in 2:1 chloroform/methanol ( 2 0 x sample weight), were hold at 4“C over night. The extracted lipids in chloroform phase were separated irom the aqueous phase by adding of 0.2 volume of saline. Trace water was removed by anhydrous sodium sulfate. Chloroform was removed by a stream of nitrogen [Appendix - 3]. The total extracted lipid of known amount tissue was measured by weighing the solvent free lipid. The concentrations, mg extracted lipid/g tissue, of the extracted lipid were used as tissue lipid concentration.
Aliquots of the extracted tissue lipids with added known amount of internal standard, heptadecanoic acid (C l7:0), were treated with 1 N KOH in methanol for 15 min at 90°C followed by reaction with boron trifluoride-methanol complex (BF 3/CH3OH) at 90°C for
15 min for the preparation of fatty acid methjl esters . The fatty acid methyl esters were then extracted by hexane and used for GC analysis [Appendix - C].
Gas Chromatographic Analvsis Of Total CLA Content And Fattv Acid Profile
The quantification of CLA and other fatty acid methyl esters was performed in a
Hewlett-Packard 5890 gas chromatography(GC) equipped with an on-column automatic
66 injector and flame Ionization detector and controlled by a Hewlett-Packard Chemstation
(Hewlett-Packard, Avondale, PA USA) The operation conditions were set as following:
SP2380 capillary column, 30 m x 0.25 mm i d. and 0.25 |.im phase thickness (Supelco
Inc., Bellefonte, PA); helium carrier gas; temperatures, injector 250“C, detector 280°C, oven start at 155 °C raised at 2 "C/min to 220 "C and held for 6 min. The CLA and other fatty acid peaks were identified by comparison with the retention time of the reference standard and the quantification was determined based on the internal standard. The CLA contents of the tissues were expressed as mg CLA/ g of total extracted lipid.
TLC Separation Of Lipid Fractions
The TLC separation was performed on a 20 x 10 cm (layer thickness, 200 nm)
LHPK silica gel 60A plates ( Wathman Inc. Clifton, NJ USA). Aliquots of tissue lipid dissolved in 2:1 chloroformimethanol were applied on the bottom edge of the plates. The plates were developed in the TLC developing tank with mobile solution of 70:30:1 (v/v/v) hexane:diethyl ether:acetic acid. The bands corresponding to different lipid fractions were visualized by spraying the plates with 2,7-dichlorofluorescein methanol solution. The bands of interest were then outlined with pencil under UV light and scraped oflf individually from the plates. Lipids in the bands were extracted into 2:1 chloroform:methanol solution for further use.
67 High Performance Liquid Chromatographic (HPLC) Purification Of CLA Isomers
HPLC purification of CLA isomers was performed in a Hewlett-Packard 1090 Series
II liquid chromatography equipped with an autoinjector and a dual-channel UV detector
and controlled by a Hewlett-Packard Chemstation (Hewlett-Packard, Palo Alto, CA
USA). CLA was separated from other major tatty acids on a 4.6 x 250 mm reverse-phase
Spherisorb DOS-5250 column (5 jim particle size) eluted with an isocratic phase of
70:30:0.2 CH3CN:H20:CH3C00H at a flow rate of 2 ml/min. The UV detector was set
to monitor the eluent at 205 nm to detect nonconjugated diene fatty acids and 235 nm to
detect conjugated diene fatty acid. The eluented CLA isomers were collected in to a glass
vial, and the solvent was removed under a stream of nitrogen to obtain more concentrated
CLA isomers. The HPLC system was reequilibrated at least 10 minute between two
consecutive injections.
CLA Isomer Separation And Identification
The same Hewlett-Packard 5890 series II GC was used for CLA isomer separation
and identification. However, the column used for this specific separation were three 30 m
X 0.32 mm (i.d ), 0.25 |tm film thickness, Omegawax 320 capillary columns connected to form a 90 m long column. GC operating conditions consisted of an on-column injection system with helium as the carrier gas at 0.5 ml'min linear gas flow rate. Oven temperature was set isothermally at 175°C, while injector and detector temperatures were set as 250°C and 280"C, respectively. Samples were run in both split and splitless modes.
68 In an effort to identify individual CLA isomers, the equivalent chain length (ECL) values of CLA isomers for this particular column were measured. A mixture of methyl esters of saturated fatty acid standards, contJiining C14:0, CI5:0, C16:0, C17:0, C18:0,
C20;0, C22;0, plus the methyl ester-CLA (from Sigma) in hexane was injected into the long column and run under the previous described conditions. ECL values were determined by plotting carbon numbers vs. retention times on semilog paper as described by Miwa et al. [1960] and Scholfield [1981].
For the CLA isomer identification, one of the CLA isomers, t-10,c-12- octadecadienoate, was synthesized following the procedure of Scholfield and Koritala
[1970]. Fifty grams of methyl linoleate was added to 100 g of ethylene glycol and 26 g of potassium hydroxide which had been heated under nitrogen to 180®C. Heating was continued for 30 minutes and the isomerizcd acids were recovered from the reaction mixture by extraction with hexane. The acids were esterified with 4% HCl in methanol at
60°C for 30 minute. The methyl esters of the synthesized CLA were then dissolved in acetone. The solution was cooled to -57 to -59°C to obtain the crystalline fraction, methyl t-lO,c-12 octadecadienoate [Appendix - D].
3.2.2 Plasma Eicosanoids Analysis
Blood samples were collected by cardiac punctuation and collected into plastic centrifuge tubes containing sodium citrate as anticoagulant agent and indomethacin as eicosanoid synthesis inhibitor. Plasma was separated from the red blood cells by
69 Plasma
Protein precipitation (15% ethanol)
Supernatant
Eicosanoid purification (C-18 cartridge)
Ethyl acetate eluate
PGE2 LTB4
(Monoclonal Enzyme Immunoassay) (Enzyme Immunoassay)
Fig. 3 Scheme of plasma eicosanoids analysis.
70 centrifugation at 15,000xg for 20 minutes at 4 C and stored frozen at -80 °C until analysis.
Plasma was used for measurement of LTB4 and PGEz Since the contents of these two eicosanoids were low, plasma samples from :wo rats in the same treatment group were pooled prior to the analysis. The scheme for the basic analytical procedures of plasma eicosanoids is shown in Fig. 2.
Plasma samples were first treated with ethanol to precipitate protein. The precipitated protein in the samples was then removed by centrifugation at 1 SOOxg at 4 “C.
After adjusting pH to approximately 4 with 0.1 N HCl and phosphate buffer (pH 4), the supernatants were passed through the pre-activated C-18 reverse phase cartridge (Sep-
Pak, Waters Corp. Milford, MC USA). The cartridges were first washed with ultrapure water followed by petroleum ether. The eicosanoids were then eluted with 1% methanol ethyl in acetate solution. After removing methanol and ethyl acetate from the eluents, the residuals were dissolved in the EIA buffer for PGE 2 and LTB4 measurement. The enzymatic immunoassay kits (LTB4 EIA Kit and PGE 2 Monoclonal EIA Kit, Cayman
Chemical, Ann arbor, MI USA) were used. Detailed procedures were shown in Appendix
- E and - F.
3.3 Animals And Experimental Designs
Two animal experiments (short term and long term CLA feeding) were conducted in this study. The short term experiment was aimed to study the CLA metabolism and tissue
71 distribution after one single oral dose of CLA. The long term animal experiment was designed to study the effects of long term CL.\ feeding on the changes of tissue CLA and other fatty acids distribution and the correlation between the changes and possible physiological responses. In addition, the effect of dietary CLA on the possible physiological and biological parameters changes of the animals during the endotoxin elicited systemic inflammation was also examined. The schemes in Fig. 3 and Fig. 4 outlined the two experimental design.
3.3.1 Short Term Animal Experiment
Female Sprague-Dawley rats at 7 weeks of age were purchased from Harlan Sprague-
Dauley (Indianapolis, IN, USA) and housed in stainless steel cages, 3 per cage, in a room with temperature- and humidity-control and a 12 hour light/12 hour dark cycle. All animals had free access to water and food (rats chaw) upon arriving. After five days acclimatizing period, food was removed for an over night fasting (water was still available
). In the morning of the sixth day, the rats were weighed and randomly divided into two treatment groups; (I) com oil control group (n=8) and (2) CLA treatment group (n=12), and a 0.8 ml com oil or a 0.8 ml CLA com oil solution (I mg CLA/ml) were given by gavage to each rat in the respective groups. Eight hours after the gavage dose, half of the rats in each group were scarified by CO 2 exposure, and tissues of liver, heart, lung, spleen, kidney, muscle, and adipose tissue were remo\ ed and stored frozen at -80°C until analysis.
72 0.8 ml cam (givagt)
control I
I2H R Fuung Cofiirol 8H R Remove Control U 24 HR liver heart 7 weeks old 4d«ys lung Rats fit chow spleen (20) kidney pancreases CLA CLA II 24 HR muscle ( 12) I2H R 8H R adipose Fasting
CLA I 0.8 mg CLA (gavage)
Fig. 4 Short term animal experiment design.
73 Twenty-four hours after the dose, the remaining rats in each group were scarified and following the same procedure as previous desc ribed.
Total lipid, CLA contents, and fatty acid profiles were determined in all the obtained tissues, liver, heart, lung, spleen, kidney, muscle, and adipose tissue. But the CLA content and distribution of CLA isomer in triacylgiycerid, phospholipids, and free fatty acid fractions were measured only in liver lipids.
3.3.2 Long Tenn Animal Experiment
In this experiment, female Sprague-Dawley rats at age of 4 weeks old were used.
Upon arrival, they were weighed and housed in stainless steel cages (2 rats/cage) in an temperature-, humidity-, and light cycle-controlled room. They were given free access to the basal AIN 930 diet (powder) and water for the first 4 days adjustment period. The basal AIN 930 diet contained in g/kg, casein 200, L-cystine 3.0, choline bitartrate 2.5, sucrose 100, com starch 397, dextranized com starch 132, com oil 60, cellulose 50, vitamin mixture 10, and mineral mixture 35 (DYETS, Inc., Bethlehem, PA USA). After the acclimatization period, the rats were randomized by allocating into 2 dififerent dietary groups (30 rats/group): (1) basal AIN 930 diet with 1% LA; and (2) basal AIN 930 diet with 1% CLA. The experimental diets were fed ad libitum and freshly provided every day. LA and CLA were mixed into the basal diet following the protocol of Frische and
74 LAconirol LA 24 HR LA 72 HR
B ual (IM 7il«y» 1% LA DM 4 nrcclu LA 144 HR (30) Remove PUunia 4 weeks old Heart Rats Eiidatoiin LPS 2m g(l«BW Liver (60) Kidney Spleen Musck 1%CLA Die* 4 weeks Adipose CLA 144 HR (30)
CLA 72 HR CLA 24 HR CLA control CLA 6H R
Fig. 5 Long term animal experimental design.
75 Johnston [Frische and Johnston, 1988]. To minimize autooxidation, the experimental diets were prepared twice a week and stored under nitrogen at 4“C.
Four weeks after the start of the experimental diets, 24 out of the 30 rats from each dietary group were weighed and injected intraperitoneally with lipopolysaccharide (E. Coli
055:85, Sigma chemical co., St. Louis, MO) at a dose of 2mg/kg body weight in pyrogen- free saline. The remaining 6 rats from each dietary group were scarified by exsanguination by heart puncture under light diethyl ether anesthesia. Tissues of liver, heart, lung, spleen, kidney, muscle, and adipose tissue from each rat were then excised and immediately stored frozen at -80°C until analysis.
The rats in the endotoxin group were continuously fed with the corresponding experimental diets, and their body weight and feed intake recorded daily. At different intervals, 9, 24, 72, and 144 hour post injeciion, six rats from each dietary group were scarified. Blood and tissues samples were collected and stored frozen at -80“C for analysis.
3.4 Statistical Analysis
The data are presented as mean ± standard error of mean (SEM) for at least three animals. An unpaired (two tailed) t test was applied to the comparison between the CLA treated rats and the control rats. The percent of control was calculated for tissue CLA
76 concentrations, plasma LTB4 and PGE 2 concentration, and some major fatty acids contents.
77 CHAP1 ER 4
RESULTS AND DISCUSSION
The purposes of this study were to study the CLA tissue distribution, metabolism, and
its effects on animal growth, plasma eicosanoids, and tissue fatty acid compositions before
and after an endotoxin challenge. In order to carry out this study, the analytical methods
for tissue lipid analysis, including quantification of CLA and its major isomers in various
tissue samples and quantification of LTB4 and PGE 2 in plasma were first established. On
this aspect, two GC methods have been developed. One was specific designed for total
CLA quantification and fatty acid profile determination, while the second one was for
CLA isomer analysis and quantification. The enzyme immunoassay methods were also
established for measurement of LTB4 and PGE 2 in plasma samples. Two animal
experiments, short and long term animal experiments, were conducted in this study. An endotoxin injection was given in the long term animal experiment. Tissue CLA distributions, fatty acid compositions, body weight, feed consumption, and plasma LTB4 and PGE 2 were determined for the animal experiments. Analysis and discussion of the results are given in this section.
78 4.1 Analytical Methods For Tissue Lipid Analysis
4.1.1 GC Method for CLA Quantification and Fatty Acid Profile Determination
Separation and detection of CLA as well as other fatty acids in the form of fatty acid
methyl esters were accomplished by GC using a flame ionization detector. The column
used for this separation was a Supelco SP 2380 capillary column (30m, 0.25 mm ID, 0.25
film thickness). This column was coated with poly (90%-biscyanopropyl-10%-
cyanopropylphenylsiloxane), a high polarity station phase, and recommended by the
manufacturer for the separation of conjugated fatty acids from nonconjugated fatty acids
[Supelco, 1997]. Fig. 6 shows the capillary GC chromatogram of various fatty acid
methyl esters (standard 68 A) plus the methylated CLA. The identification of the fatty acid
methyl esters in the chromatogram was made by comparing the retention times of
individual fatty acid standard run at identical conditions and by spectral analyses according
to structure properties of the fatty acids and the characteristics of the column. The elution of different fatty acid methyl esters basically followed the order of short chain to long chain and less saturated to more saturated fatty acids. The GC elution profile of the CLA methyl ester mixture obtained from Sigma Chemical Comp, was shown in Fig. 7. In this separation, the CLA methyl ester mixture showed 6 peaks between methyl 11-eicosenoate and methyl 11,14-eicosadienoate. For the analysis of total CLA content in tissue samples, individual CLA isomer identification was not made, instead all CLA peaks were summed up as total CLA.
79 I "4
16 I Cl . I-
9 0 0 0 .10 ‘2 Il4 17 18
8 0 0 0 - II 19
VOOO -
6 0 0 0
OOO iJUJ
1-000
o 1 o 20 3 0
Fig. 6 Capillary (SP2380) GC elution profile of various fatty acid methyl esters (standard
68A) cochromatographed with the CLA methyl ester mixture.
Peak identification: 1. - C14:0, methyl myristate; 2. - C14:ln5, methyl myristoleate; 3. -
Cl6:0, methyl palmitate; 4. - C16:ln7, methyl palmitoleate; 5. - Cl7:0, methyl heptadecanate; 6. - C l8:0, methyl stearate; 7. - C18:ln9, methyl oleate; 8. - C18:2n6, methyl linoleate; 9. - C 20:0, methyl arachidate; 10. - C18:3n3, methyl linolenate; 11. -
C20:l, methyl 11-eicosadienoate; 12. - CLA methy ester mixture; 13. - C20:2n6, methyl
11,14-eicosadienoate; 14. - C22:0,methyl behenate; 15. - C20:3n3, methyl homogamma linolenate; 16. - C20:4n6, methyl arachidonate; 17. - C24:0, methyl lignocerate; 18. -
C24:l, methyl nervonate; 19. - C22:6n3, methyl docosahexaenoate.
81 Fig. 8 shows a typical GC elution profile of fatty acid methyl esters in liver lipid extracted from rat fed with CLA for 4 weeks. The CLA content in comparison with other major fatty acids in liver lipid was very low. Injection of relative high concentration methyl ester solution of tissue sample is necessary for the CLA analysis. This GC method has been used for tissue CLA and fatty acid profile analysis during the entire study. In addition to the effective separation each GC run by this separation required only 36.8 minutes to finish. By using autoinjector, the time used between two runs was about 2 minutes, therefore, approximately 30 samples could be analyzed daily.
Quantification of CLA and other fatty ac;ds in the samples was based on the internal standard method. To obtain the correction factors (CF) for each individual fatty acids, a known amount of internal standard, heptadecanoic acid (C17:0), was added to the standard mixture (standard 68A) and subjected to lipid extraction, saponification, and méthylation procedures. After the méthylation, the extracted methyl esters of the standard mixture and the internal standard were chromatographed on the capillary GC column at the designed operation conditions. Then the CF for the individual fatty acids was calculated as follows;
CFx = (Areais/Weightis) x (Weight^/Area*)
Where the subscript IS refers to internal standard and subscript x refers to a given fatty acid. The amount of each fatty in the sample was then calculated as:
82 o o o o -
a o o o
6 0 0 0 -
6 0 0 0 U a
1 a 2 0 2 4
Fig. 7 Capillary (SP2380) GC elution profile of CLA methyl ester mixture obtained from
Sigma Chemical Company.
8 2 Fatty acid (mg/lOOmg lipid)
= [(Areax/Areais) x Weighty (mg)]/Extracted lipid (mg) x CFx x 100
To calculate the CFx value for CLA isomer mixture, a known amount of CLA mixture and the C l7:0 internal standard was subjec.ed to lipid extraction, saponification, and méthylation. Thereafter, the methyl esters from the mixture were chromatographed on the
GC column. The weight of each CLA isomer peak was calculated by multiply the total mixture weight by the area percentage of each particular peak in the sum of area of all the isomer peaks. Then the CFx values for each isomer peak were calculated as described for other fatty acid in previous section. Total CLA content in the sample was calculated by adding up each of the individual isomer peaks.
In tissue analysis, the internal standard (C17:0 fatty acid) was added to the sample homogenate period to carry out the whole preparation procures. CLA content was calculated as mg/g extract lipid, while the fatty acid profile was expressed as area % of each fatty acid relative to total fatty acids in the sample.
4.1.2 TLC Separation of Lipid Fractions
TLC is a type of liquid chromatography in which the stationary phase is in the form of a thin layer on a flat surface rather than packed into a tube. Preparative thin-layer chromatography is an important tool in lipid analysis due to its non-destructive of the sample, methodological simplicity and ease of sample visualization.
84 0 O O O
a o o o
* 7 0 0 0
e o o o
s o o o
4 0 0 0
3 0 0 0
2000 •
1 o o o IjjÜ JL, J LL ivdi/ /vjj >
- 1 o o o
— P— ------I------' — - : ' ' I----- lO 20 30 40
Fig. 8 Capillary (column SP2380) GC elution profile of fatty acid methyl esters from a liver sample.
84 Lipids extracted from animal tissue or other biological sample contains phospholipids
(which may be further separated to different phospholipid fractions), cholesterol, free fatty acids, mono-, die- and tri-glycerids, and cholesterol esters fractions. Each of the lipid fractions plays different roles in the biological system. The analyses of the composition of the lipid fractions are important steps in understanding the lipid metabolism and their functions.
Preparative TLC provides a convenient way to separate the different lipid fractions without damage the molecule structure of the lipid fractions, which is the key for subsequently study the compositions or functionality of each of the fractions. In preparative TLC separation, lipids, usually as a 5-10% solution in a volatile organic solvent, are applied as a band along one edge of the plate. The plate then is developed in the developing chambers with specifically designed mobile solvent. The separated bands then are visualized and the separated zones carefully marked for removal. Each of the lipid fraction can be extracted from the adsorbent material, which was scraped off from the plate, for further use.
Since information on distribution of CLA and its isomer in tissue lipid fractions is important in understanding the whole metabolism of CLA and its biological activities, we established a TLC separation of major lipid fractions, namely, phospholipid, triacylglycerid, and free fatty acids, of liver/tissue lipids. Fig. 9 shows the TLC chromatogram of the liver lipid fractions from livers of rats fed with or without CLA. For
85 :' ■»<’Ji-n*
- '* ' V . .. - Y% # B S «■'-■ .-^*ü
kSt":-
Fig. 9 TLC chromatogram for the separation o f liver lipid fractions.
Spots arrangement; A - TLC sandard mixture 18-5-A (containing: lecithin, oleic acid, triolein, cholesteryl oleate, and cholesterol); B - TLC standard mixture 18-5-A plus methyl oleate; C - cholesterol; D - methyl oleate; E - triglyceride (18:2); F linoleic acid, G - 1,3-diglyceride (16:0); H - 1,3 diglycerid (18:0); I - monoglyceride (18:1); J - monoglyceride (16:0); K to N - saponified and methylated liver lipid samples; O to R - liver lipid samples.
Band identification of liver lipid samples: 1 - phospholipids (PL); 2 - cholesterol; 3 - free fatty acids (FFA); 4 - triacylglyceride (TO); 5 - cholesterol ester ((TE). the identification of separation bands, the standard solutions (1-10) were applied on the left side of the plate, while saponified and methylated liver lipids (11-14) and untreated liver lipids (15-18) were applied on the right side of the plate (Fig.9). By comparing the band positions with the standard, the phospholipids, cholesterol, free fatty acids, triglycerids, and cholesterol esters were identified in the liver lipid samples.
This TLC separation method was used in the short term animal experiment for liver lipid separation. The sample loading capacity of the TLC plate (20 x 10 cm, 250 mm thickness) for liver lipid was tested in order to achieve maximum sample loading without compromising the proper separation. In this study the maximum loading for liver lipids used was estimated to be 40 mg lipid per plate. After development of the plate, the phospholipid, triacylglycerid, and free fatty acid bands were visualized by spraying of 2,7- dichlorofluorescein, carefully marked and scraped off from the plate. Each lipid fraction was then re-extracted with 2:1 chloform:methanol solution for further use. Due to the relative large loading limit, the TLC method is more efficient in separating lipid fractions than HPLC method.
4.1.3 HPLC Purification of CLA Isomers
The CLA content as compared with other major fatty acids is very low in tissue and other biological samples. To determine the CLA isomer distribution in liver lipid fractions, the very low CLA isomer content forced us to raise the injection volume on the GC
87 0004.D: wro, Mavalens soo.
4S0J
3S0.
300
2S0.
200.
150
100 .
50.
—
1C. 000 20.000 30.000
Fig. 10 HPLC elution profile of CLA cochromatographed with various fàtty acids.
89 analysis. The increased injection volume, however, caused over-load of other fatty acids on the column and the separation of CLA isomers could not be achieved. In order to pre
purify and concentrate the CLA mixture from the liver lipid fractions, a HPLC purification and concentration procedure was added to the lipid analysis. Fig. 10 showed the elution profile of CLA isomer mixture with some other major fatty acids which were most likely to interfere with the CLA separation. CLA mixture was eluted right after linoleic acid
(18:2) and before homo-y-linolenic acid (C20 3). The elution of CLA was monitored by
UV detector at wavelength of 232 nm wldle the elution of other fatty acids were monitored at wavelength 190 nm. The CLA eluent was collected from several injection to achieve the purpose of concentration. For real tissue samples, due to the relative much higher C18:2 linoleic acid content, the two peaks or C18:2 and CLA often partially over lap. However, the small amount of linoleic acid collected with CLA eluate was not a problem for the GC analysis of the CLA isomers because of the far apart of this two compounds in the GC separation.
4.1.4 GC Method For CLA Isomer Analysis
In order to achieve a better separation of the positional and geometrical isomers of
CLA, a modified GC method has been established. In this method, a 90 meter long column was used and the oven temperature was set at 175“C for the entire run. Fig. 11 showed the elution profile for the mixture of CLA methyl esters separated on the 90 meter
Omegawax 320 (0.32 mm (i d ), 0.25 |im film thickness). Five major peaks and three
89 s o o o 1
s o o o -
4 - 0 0 0 -
3 0 0 0 -
S O O O -
1 O O O o S O 4 06 0 8 0 1 O O 1
Fig. 11 Capillary (Omegawax 320) GC elution profile of CLA methyl ester mixture
(Sigma Chemical Company).
Peak identification: 1 - c9,tll-CLA 2 - clO,tl2-CLA; 3 - tl0,cl2 CLA; 4 -not identified;
5 - c9,cl 1-CLA; 6 - clO,cl2-CLA 7 - not identified; 8 - t9,tl 1-CLA; 9 - tlO,tl2-CLA
91 minor peaks were found in the separation. This method was used for the CLA isomer identification and quantification for this study.
Generally, identification of components on a gas chromatogram is established by comparing the retention characteristics, which include retention times, volumes, or ratios, with those of standards. And quantification of individual components is achieved by first determining the correction factor (CF) on known amount of standards and then applying the correction factors to the corresponding components for sample measurement. Since individual standard is needed for component identification and the exact amount of standard has to be known for the quantification , high-pure standard for each of the components which are subjects for identification and quantification are crucial during the whole processes.
Unfortunately, high-purity reference materials for CLA isomers were not commercially available, the identification and quantification of individual CLA isomers become quite difficult. Up to date there was only one published study done on the identification and quantification of individual CLA isomers. In that study the identification was based on determination of equivalent chain length (ECL) values of CLA isomers and spectral analyses of the CLA isolated from food sample or alkali-isomerized linoleic acid.
The quantification was done by using few self-synthesized CLA isomers as standard.
However, details on how the isomer standards were prepared and the actual correction factors for each of the isomers were not provided [Ha et al., 1989]. Most other studies on
91 CLA research simply imported the CLA isomer identification results from the Ha’s study,
despite that some of the column and operation conditions used were different from that in
Ha’s study. It is not clear how much this praczice effects interpreting the CLA results.
The measurement of equivalent chain length (ECL) value was based on the factor that
for a specified column packing and carrier gas, the relationship between molecular weight
of the reference saturated straight-chain monocarboxylic methyl esters and logarithm of
their retention times was linear at certain range. In practice, a reference curve was
established by plotting on a semilog graph the retention times (log-scale) of two or more
known, normal, saturated monocarboxylic methyl esters against their chain lengths
(number of carbons in the acid). The ECL values of components in subsequent samples were then read from the curve using observed retention times. Although the slopes of the curves varied with changes in column temperature, the ECL remained constant. Thus, the
ECL value was independent of operating conditions, such as column temperature, carrier gas flow rate, and column dimensions, but dependent on the column packing and carrier gas [Miwa et al., 1969]. The ECL values of CLA isomers have been determined on several different stationary phases and on dififerent columns [Miwa et al., I960; Scholfield and Dutton, 1971; Scholfield , 1981; Ha et al., 1989]. At the time when individual CLA isomer standards are lacking, the ECL values are useful parameters for the CLA isomer identification, since the ELC values are independent of GC operation conditions.
The chromatogram shown in Fig. 12 presents the GC elution profile (Omegawax 320) of the methyl esters of saturated fatty acid standards (CI4:0, Cl 5:0, C16:0, C17:0, C18:0,
92 8 0 0 0 -
a o o o -
8 0 0 0 -
5 0 0 0
4 0 0 0
3 0 0 0
JWjiL 2000 -
1 O O O -
O SO 1 O O 1 5 0 200
Fig. 12 Capillary (Omegawax 320) GC elution profile of various methyl esters of saturated fatty acid standards cochromatographed with the methyl CLA isomer mixture fi*om Sigma Chemical Company.
94 ECL Calibration Curve
10 1
y =0.1271%-0.5388 R*= 0.9945
# Log RT — Linear (Log RT)
Carbon Numbei^
Fig. 13 The calibration curve of equivalent chain length (ECL) for CLA isomers.
94 C20;0, C22;0) plus the methyl ester of CLA isomer mixture obtained from Sigma
Chemical Company. Fig. 13 shows the reference curve for the calculation of ECL values
of CLA isomers, which was obtained by plotting carbon numbers vs. retention times of the
elution of methyl ester of saturated fatty acid standard mixture on semilog paper.
The measured ECL values and identification of CLA isomers were shown in Table 2.
The reference ECL values and identification reported by Ha et al. on a Supelcowax-10
fused silica capillary column (60 m x 0.32 mm (i d.), 0.25 pm film thickness) and the ECL
values measured by Scholfield et al. on a Silai IOC Quadrex glass capillary column (50 m
X 0.25 mm i d.) and on a polyphenyl ether stainless-steel capillary column (150m x 0.01 in.
i d) were also shown in Table 2. The ECL vtJues for peak one to peak nine measured in
this study range from 19.42 to 20.08. The dilTerence in ECL (AECL) values between the
measurements of this study and the reference values reported by Ha et al. remained
constant within 0.05 and 0.11 units for the isomers tested by both columns. Two columns
used in present study (Omegawax 320) and in Ha’s study (Supelcowax 10) had same
bonded poly(ethylene glycol) stationary phases, therefore, they had the same colunm
polarity. However, the Omegawax column was developed for highly reproducible
analyses of fatty acid methyl esters, especially the omega 3 and 6 fatty acid, while the
Supelcowax 10 was more widely used foi separation and purity analyses of polar compounds such as alcohol, aromatics and flavor compounds. Since the ECL values are
95 P eak # ' EÇL Isomer identification Omagcowax Supelcowax- Plyphenyl Silar IOC*" Present study Ha study Scholfield study 320*’ 10** ether** 1 19.42 19.49 19.04 20.72 c9,tll- c9.tll-/t9,cll- c 9 ,tll- 2 19.45 19.53 19.07 c l 0 ,tl 2 - c l 0 ,t l 2 - c l 0 ,tl 2 - 3 19.53 19.62 19.16 2 0 .8 6 t l 0 ,c l2 - t l 0 ,c l2 - t l 0 ,c l2 - 4 19.56 19.67 c ll,c l3 - 5 19.72 19.80 19.31 c9,cll- c-9,cl 1- c 9 ,c ll- 6 19.75 19.82 19.32 21.23 c l 0 ,c l2 - c l 0 ,c l2 - c l 0 ,c l2- 7 19.79 8 20.06 20.01 19.60 21.22 t9 ,tll- t9,tll-/tl0,tl2- t9 ,tll- 9 2 0 .8 19.61 t l 0 , tl 2 - t l 0 .tl 2 - s Table 2. Equivalent chani length (ECL) values of methyl CLA isomers and CLA isomer identification.
*-the peaks in Fig. 10. the columns from which the ECL values were obtained. dependent on the column polarity but independent on other GC operation conditions, the
ECL values measured in these two studies wei e comparable.
When the chromatograms of the CLA isomer methyl ester mixture from this study and from Ha’s study were compared, the elution patterns for the CLA isomers were similar. Basically, they both contained three groups of peaks. The first group contained the same four isomer peaks in both chromatograms. The second group contained two isomer peaks in the Ha’s chromatogram but three isomer peaks in our chromatogram.
The third group contained one isomer peak for the Ha’s study and two isomer peaks in present study. The intensities of the peaks in these two chromatograms were different.
Since the CLA isomer mixture standards used in these two studies were not certified, the composition of the isomers in the mixtures could be different depending on how the isomer mixtures were prepared. Nonetheless, the elution orders and the identification results from Ha’s study can be used as reference. According to the reference identification and the ECL results, the five major peaks (1-4 and 8 ) in Fig. 10 were identified as methyl esters of c9,tl 1-, cl0,tl2-, tl0,cl2-, t9,cl 1- and t9,tl 1-octadecadienoate, respectively.
In an effort to obtain the correct factors and to confirm the identification for the major CLA isomers, we synthesized our own CLA isomer mixture and prepared pure methyl t-10,c-12-octadecadienoate as an isomer standard. The CLA isomer mixture was synthesized by alkali-isomerization of linoleic acid (>95% pure. Sigma Chemical
Company), (see Appendix -D for detailed procures) Fig. 14 showed the GC elution profile of the synthesized CLA mixture meth>l esters. Two major peaks and three minor
97 1 . O e 4 n
8 0 0 0
a o o o
- ? o o o -
8 0 0 0 -
s o o o
4 - 0 0 0 -
3 0 0 0 -
a o o o - JAuj .1 Lw 1
1 o o o a o 4 0 e o a o 1 O O
Fig. 14 Capillary (Omegawax 320) GC elution profiles of the synthesized CLA isomer mixture.
99 9 0 0 0
a o o o
e o o o -
s o o o -
4 0 0 0 -
3 0 0 0 -
a o o o -
1 o o o -
o -
a oo a o a o 1 O O 1 2
Fig. IS Capillary (Omegawax 320) GC elution profiles of the crystal phase of synthesized
CLA.
100 s o o o
a o o o -
e o o o -
■ 4 0 0 0 -
3 0 0 0 -
3 0 0 0 -
1 oo o -
o 3 0 8 0 8 0 1 O O 1
Fig. 16 Capillary GC elution profiles (Omegawax 320) of the supernatant phase of the synthesized CLA.
101 peaks beside the methylated linoleic acid peak appeared. The conversion of the linoleic to
CLA was approximately 95% calculated by area percent. We synthesized total 4 batches
of the CLA isomer mixtures intended to use as dietary supplement in animal studies. The
CLA isomer patterns in all these four batches as determined by the GC analysis were
identical (as showed in Fig. 14), although, the conversion rates were slightly different.
When compared the isomer patterns of oui synthesized CLA mixture with the CLA
mixture obtained from Sigma Chemical Company, which was used as the standard CLA
mixture for our method development study, the two major peaks in our CLA mixture
matched with the peak 1 and peak 3 in the (CLA mixture from Sigma based on the GC
retention time. Accordingly, the two peaks were considered as CLA isomer c9,t 11 - and
110 ,c 12 -octadecadienoate respectively.
From our synthesized CLA methyl ester mixture, the t-10,c-12-octadicadienoate was
further purified by crystallization. The CLA methyl ester mixture was dissolved in acetone solution and cooled to 57-59“C. This process crystallizes out the tl0,cl2- octadicadienoate isomer. The tlO,cI2-octacicadienoate crystal was then separated by centrifugation at low temperature. Fig. 15 and 16 showed the GC elution profiles of the crystal (Fig. 15) and the supernatant (Fig. 16) phases respectively. When the two GC elution profiles (Fig. 15 and Fig. 16) were compared with the GC elution profile of our synthesized CLA mixture in Fig. 14, the crystal phase (Fig. 15) showed one major isomer peak which matched with tlO,cl2-CLA peak and one minor peak which matched with the
101 c9,tIl-CLA isomer peak in Fig. 14. Meanwhile, the supernatant phase (Fig. 16) also showed one major isomer peak which matched with the c9,tl 1-CLA isomer peak and two minor peaks matched with the linoleate and f O,cl2-CLA isomer peaks in Fig. 14. Since the crystal prepared here is the clO,tl 2-octadicadienoate, by comparing the retention time the peak number three in GC elution profile cf the CLA isomer mixture was identified as tlO,cl2-CLA. This method gives the main basis for our CLA isomer identification.
By using the purified 110,c 12-octadicadienote isomer, the correction factor of the tlO,cl2-CLA as determined for the two GC methods used in this research. The known amount of tlO,cl2-CLA was cochromatographed with C17:0 standard and the CF values were calculated according to the method described in previous section. The newly calculated CF values were similar to the one determined by using the CLA isomer mixture, therefore, we gained our confident in the CLA isomer quantification.
4.1.5 Methods for Plasma LTB^ and PGEi Analyses
Currently, enzyme immunoassays are the most widely used methods in eicosanoids measurement. These methods provide an easy and reproducible biochemical measurement for various eicosanoids in very complex media, such as biological fluids (blood, urine) and cellular or tissue extracts due to the rem:irkable ability of antibodies to recognize molecules with high aflRnity and specificity. I'he enzyme immunoassays were used in this study to determine the plasma LTB4 and PGEz for rats in the long term CLA feeding study.
102 The LTB 4 enzyme immunoassay is based on the competition between free LTB 4
and a LTB 4-acetylcholinesterase conjugate (I-TB 4 tracer) for a limited number of LTB 4
specific rabbit antiserum binding sites. The concentration of LTB4 tracer is held constant
while the concentration of free LTB 4 varies. Thus, the amount of LTB 4 tracer that is able
to bind to the rabbit antiserum will be inversely proportional to the concentration of free
LTB4 in the sample. The enzymatic reaction of the LTB 4-acetycholinesterase tracer and
the substrate (Ellman’s reagent) of acetylcholinesterase gives a distinct yellow color and
absorbs for the quantitative determination of LTB 4 in the sample [Maclout et al., 1987].
The principle of PGE 2 enzyme immunoassay is the same as for LTB4 assay. Free
PGE2 and a PGE 2 tracer (PGE2-acetylcholinesterase conjugate) compete with each other for a limited number of PGE 2 monoclonal antibody. The amount of PGE 2 in sample were determined in same way as for LTB4 [Hamberg and Samuelsson, 1971].
The methods established in this study for plasma LTB4 and PGE 2 analyses was based on procedures provided by Cayman Chemical Company, who supplied the reagents for the
LTB4 and PGE 2 analyses. Detailed procedures for the assays were listed in Appendix -6 and -7. The LTB4 and PGE 2 assays were performed separately on aliquot of purified plasma samples without separating LTB4 and PGE 2 from each other. The cross-reaction between LTB4 and PGE 2 were tested on known amount of standard LTB4 and PGE 2 No significant interactions were observed for both LTB4 and PGE 2 measurements.
10;: Since eicosanoids are polyunsaturated molecules that require some special attention
to avoid artifactual degradation and organic contamination. Therefore, proper sample
storage and preparation are essential for consistent and accurate results. Indomethacin (5
pg/ml blood) was added to the blood sample during sample collection to prevent the
possible eicosanoids production during sample storage. The plasma samples obtained
from the rats were stored immediately at -SO°C until LTB4 and PGE 2 analyses. The
plasma samples were purified by first removing proteins by ethanol precipitation and then
separating other interference lipids by solid phase extraction [Powell, 1980]. The detailed
procedure can be seen in Appendix-7. The recovery of LTB4 and PGEz were tested by the cold spike method. Two identical aliquots from one sample were prepared and one of the two aliquots was added with precisely know n quantity of LTB4 or PGEz These two samples were then carried individually through the full procedures of purification and enzyme immunoassays. The average recoveries tested on four samples was 89.6% for
LTB4 and 87.9% for PGEz.
4.2 Results From Animal Studies
Two animal experiments have been carried out in this study. The first animal experiment, short term study, was designed ( 1) to examine the applicability of the analytical methods in real samples; (2) to obtain basic information about CLA metabolism in answering questions like: where does CLA go? How long it will take for CLA to be metabolized in the body? Do different tissues in the body have preference in taking up
104 CLA? The purpose of the second animal experimental, the long term study, were: (1) to obtain general information about the effects of long term dietary CLA supplementation on animal growth and tissue CLA distribution; (2) to examine the effects of long term dietary
CLA on tissue fatty acid composition on the production of eicosanoids; (3) to test the dietary CLA effects on animals in responding to endotoxin challenge. The questions that we expected to be answered from the long term feeding experiment were: Does dietary
CLA supplementation affect arachidonic acid metabolism and eicosanoid production?
Dose dietary CLA supplementation affect how the animal responds to the endotoxin challenge? Could this endotoxin stimulation model provide us information and warrant for further study the effect of CLA on inflammatory diseases.
4.2.1 CLA Tissue Distribution and Metabolism
Tissue CLA concentrations in responding to a single CLA dose
The CLA concentrations (mg CLA/g tissue lipid) in various tissues were examined 8 hours after the gavage CLA or com oil. Fig. 17 showed the tissue CLA concentrations in both control (com oil) and CLA groups 8 hours after the gavage doses. The CLA concentrations in liver, heart, kidney, spleen, lung, and muscle were significantly increased
(P<0.05) in the CLA treated rats as compared to those in control rats. Liver and heart of the CLA treated rats showed the highest increases in CLA concentrations with an increases of 6 folds in liver and 4 folds in heart. The CLA concentration in adipose tissue in the CLA group was also increased but the increase was not statistically significant.
105 CLA Concentrations (XDilTerent Tissues 8 Hours After The Gavage Dose
10
I Control I CIA
IJLiver J Heart J Kidney SpleenJ Lung J Muscle JI Adipose Tissues
Fig. 17 CLA concentrations in different tissues of rats in control and CLA group 8 hours after the gavage dose.
10(> CXA Concentrations ()f DilTerent Tissues 24 Hours After Tlie Gavage Dose
10 J
9 ■■
I 7 M 6 I Control L I CLA 2 ■■ ii É J ii J 1 Liver J Heart Kidney Spleen Lung Muscle Adipose Tissues
Fig. 18 CLA concentration in different tissues of rats in control and CLA groups 24 hours after the gavage dose.
10^ Eight hours after the treatments, liver and heart had the highest CLA concentrations in
CLA group (6.74 ± 1.66 and 6.34 ± 1.24, respectively), however, kidney and adipose
tissue showed the highest CLA concentrations in control group ( 1.8 ± 0.81 and 2.33 ±
0.26, respectively). The observations that CLA was incorporated rapidly into various
tissues suggested that CLA is absorbed efficiently like other common fatty acids.
Twenty-four hours after the CLA gavage dose, the CLA concentrations of liver,
kidney, spleen, lung, muscle, and adipose tissue in the CLA group fell back to the levels
comparable with those in the control group as shown in Fig. 18. Therefore, the absorbed
CLA was likely metabolized within twenty-four hours. The only exception was heart in
which the CLA concentration remained significantly (P<0.05) higher in the CLA group
relative to the control group. Interestingly, 24 hours after the CLA gavage dose, the CLA
concentration in liver of the CLA group was the lowest among all the examined tissues in
the CLA group.
Tissue CLA concentrations in responding to long term dietarv CLA supplementation
Tissue CLA concentrations were measured for rats fed with control (1% LA) or CLA
diet (1%) up to four weeks. Fig. 19 showed the CLA concentrations in various tissues
four weeks after the experimental diets. The CLA concentrations in all the examined
tissues in CLA group were significantly higher (P<0.01) than that in the control group.
The increase in CLA concentration in adipose tissue was the highest, a 17 folds increase.
los CLA Concentrations In Tissues After 4 Weeks Of Experimental Diets 60
50
40
30
I Control 20 I CLA
10
0 Liver Heart KidneySpleen Lung Muscle Adipose Tissues
Fig. 19 CLA concentrations in various tissues of rats fed with control or 1% CLA diet for four weeks.
lov Tissue CLA Concentration Changes
25 •o
20 ■ — « — Heart ai> — A — Liver 15 •• * • • A * ■ • Mjscle — • • Kidney 10 ••
26 28 30 32 34 36 Time (days)
Fig. 20 The CLA concentration changes in liver heart, liver, muscle, and kidney with time of CLA feeding.
11(1 followed by liver, a 16 folds increase, and heart, a 11 folds increase. Within the CLA
group, the adipose tissue had the highest CLA concentration (47.2 ± 7.4 mg/g lipid), while
the kidney had the lowest CLA concentration (5.9 ± 1.2 mg/g lipid). In between were
heart (26.8 ± 6.7 mg/g lipid) and liver (19.1 ± 4.0 mg/g lipid).
Tissue CLA concentrations were not further increased after 28 days on the CLA diet.
Fig. 20 showed CLA concentration changes with the time of CLA feeding in liver, heart,
kidney, and muscle. Within the seven days when the tissue CLA concentrations were
measured, each tissue had a constant CLA concentration. This results suggest that there
were limits on the amount of CLA might be incorporated into tissues.
Overall, the response of tissue CLA concentrations versus time to a single oral CLA
dose showed that CLA was taken up and metabolized rapidly by the tissues. The pattern
of responses resembles to the metabolism of other long chain fatty acids [Linscheer and
Vergroesen, 1994]. The absorption of long chain fatty acids mostly takes place in the
small intestine. Studies by Borgstrom, using a high-fat liquid test meal and an intubation technique, demonstrated lipid absorption is practically completed in the first 120 cm of the small bowel in the healthy men [Borgstrom ct al., 1957]. The absorbed lipids are then transported in water-soluble form, the lipoprotein particles, from the small intestine through lymph and blood circulation to the liver, fat depots, muscle, and other tissues. In these tissues the lipids will be metabolized for use as energy and other biological functions.
It is likely that CLA is metabolized in the body in way similar to other long chain fatty
H i acids: CLA is rapidly absorbed into blood stream and taken up by tissues. In tissues CLA
is metabolized through p-oxidation and used as fuel to support their biological activities.
Some of the CLA may be metabolized by :he actions of enzymes, such as elongase,
desaturase, or subjected to oxidation to become a specific group of conjugated
metabolites. CLA may also be reesterified and stored in certain tissues [Wardlay and
Insel, 1992].
Liver plays a central role in lipid transport and metabolism. It has active enzyme
systems for synthesizing and oxidizing fatty acids and synthesizing triacylglycerols and
phospholipids [Mayes, 1993]. The dramatic and rapid increase and decrease in liver CLA
concentration in responding to the CLA administration suggests that liver is the main site
where CLA is transported and metabolized. It is likely that when large amount of CLA
was introduced into the system, liver took up most of the CLA, as shown in a rapid
increasing in liver CLA concentration 8 hours after the CLA gavage dose. The CLA taken
up by the liver was then metabolized by the active enzymatic actions of the liver or
transported to other tissues as shown in a dramatic decreasing in liver CLA concentration
24 hours after the CLA dose.
Heart is another important tissue for lipid metabolism. It takes up the fatty acids in chylomicron or in triacylglyceride of very low density lipoprotein from the circulation and uses them for energy production. Fatty acids are the major fuel for energy production in heart [Linscheer and Vergroesen, 1994]. Since heart is the center for blood circulation, it
112 is likely that when CLA is increased in the circulation heart CLA concentration will also be
increased as observed in 8 hours after the CLA dose. However, the reason for the
consistent high concentration of CLA in heart 24 hours after the single CLA dose and
after continuously CLA feeding is not clear. \Vhen linoleic acid concentrations in different
tissues four weeks after CLA feeding were ex.imined, it showed that heart had the highest
concentration of linoleic acid in the linoleic acid feeding group. Since the CLA and
linoleic acid were fed to the rats as free fatty acids, the significant increased free fatty acids
in the diet might increase the free fatty acids in blood circulation and then preferably taken
up by heart.
Incorporation of CLA and its isomers in liver lipid fractions
To further examine the mode of the incorporation of CLA and its individual isomers
into different lipid fractions of the tissues, we measured the CLA isomer content in liver
triacylglyceride, phospholipids, and free fatt} acids fractions. Fig. 21 shows the CLA
concentrations of liver triacylglyceride, phospholipids, and free fatty acids fractions 8
hours after gavage of either corn oil or CLA. CLA was incorporated into these three mantioned lipid fractions. Among them, the highest CLA concentration was found in the triacylglyceride fractions (3.87 ± 0.5 mg/g liver lipid in CLA group and 0.72 ± 0.1 mg/g liver lipid in control group) and the lowest CLA concentration was found in phospholipid fraction (0.6 ± 0.1 mg/g liver lipid in CLA group and 0.1 ± 0.03 mg/g liver lipid in control group). CLA feeding raised the CLA concentrations by 437%, 358%, and 83% in
11:. CLA Concentrations in Uver Lipid Fractions
4.50 4.00
^ 3.50 ■ Control !§• 3.00 ■ CLA 50 2 .1 O - 2.00 1.50
^ 1.00 0.50 0.00
Liver Lipid Fractions
Fig. 21 CLA concentrations as mg total Cl-A per g liver lipid in liver lipid fractions, triacylglycerid (TG), free fatty acid (FFA), and phospholipid(PL), for rats in control and
CLA treatment groups 8 hours after the gavage dose.
114 triacylglyceride, free fatty acid, and phospholipid fractions, respectively, relative to the controls. Since triacylglyceride is the most abundant lipid fraction in liver, it is evident that most of the absorbed CLA was incorporated into the triacylglyceride fraction of the liver lipids.
Eight hours after the CLA administration most of CLA isomers were found in the liver lipids. Fig. 22 showed the major CLA isomer concentrations in the three liver lipid fractions. The extents of the incorporation of each individual CLA isomers to the three liver lipid fractions were different. In the triacylglyceride fraction, 47% of the total incorporated CLA was c9,tl 1-CLA, while 13.S, 24.5, and 11.7% of the total incorporated
CLA were cl0,tl2-, tlO,cl2-, and t9,tl 1-CLA, respectively. The percentages of c9,tl 1-, cl0,tl2-, tl0,cl2- and t9,tl 1-CLA of the total CLA were 39.2, 20, 21.7, and 15% in the free fatty acid fraction and 27.9, 32.6, 2.5.3, and 16.3% in phospholipid fraction, respectively. In comparison, the percentages of c9,tl 1-, cl0,tl2-, tl0,cl2-, c9,cl 1-, clO,cl2-, and t9,tl 1-CLA of the total CLA m gavage CLA were 30.4, 20.3, 32.3, 1.24,
0.68, and 17.1%, respectively. Although the incorporation of different CLA isomers in liver lipid fractions varied somewhat, by and large the composition of CLA isomers in the tissues still reflected to the composition of gavage CLA isomers.
Sugano et al. [ 1997] have reported that after 2 weeks of CLA supplementation not only the magnitude of deposition, but also the composition of CLA isomers diftered among tissues. They observed that most predominant CLA isomer was t9,cl 1-CLA
115 Major CLA Isomer Concentrations in Liver Lipid Fraction
2.50 T ITG 2.00 IPL IFFA k 1.50 .
U = Ofi 1.00 ■ M 0.50 •
0.00 c9,t11-CLA c10,t12-CLA t10,c12-CüA t9,t11-CLA
CLA Isomen
Fig. 22 CLA isomer concentration in liver triacylglyceride (TG), free fatty acids (FFA), and phospholipid (PL) fractions of rats in CLA group 8 hours afrer the gavage dose.
ilo followed by the tt-CLA isomers in most of the tissues. They concluded that it is difficult to predict which isomer(s) is the putative candidate for CLA biological/physiological activities, even though c9,tl 1-CLA has been postulated as the most biologically active form of CLA [Belury, 1995]. Our results showed that after a single CLA dose the composition of CLA isomers in tissue lipids was changed but lagerly resembled to the isomer composition of dietary CLA. Different from Sugano’s observation, our finding suggests that there is no differential uptake of CLA isomers by tissues.
4.2.2 Growth Of Rats Before And After Endotoxin Injection
Growth before endotoxin iniection
During the 4 weeks period of experimental diet feeding, rats in both the CLA and the control groups grew well and no abnormal behaviors towards their daily activities and attitude to the food were observed. The average growth rates for rats in these two dietary groups were practically the same, which was about 4.1 g/day per rats. Fig. 23 showed average body weight of rats in both dietary groups at time starting the experimental diets and at the day when rats were on the diets for 4 weeks and before the endotoxin was injected. There were no differences in body w eight between the two dietary groups at the time of starting the experimental diets. Four weeks after the experimental diet, the rats in the CLA group appeared to exhibited slightly reduced body weight as compared to control rats, although the apparent reduction was not statistically significant.
11' 200 180
160 140 "02 120 I Control 100 .2f> I CLA « 80
60 40
20 0 Day 1 Day 28 Time
Fig. 23 The average body weight of rats iii control and CLA groups at the time of starting the experimental diets and 4 weeks after the experimental diets.
us Our findings were consistent with that reported by Park et al. [1997]. They have observed that feeding with 0.5% CLA supplemented diet for 30 days female mice exhibited slightly reduced body weight. However, the male mice fed the same CLA supplemented diet showed indistinguishable body weights from their controls. On the other hand. Chin et al. [1994] have found that feeding CLA to rat dams during gestation and lactation enhanced pup growth. They also showed that the pups received the CLA- supplemented diet afrer weaning had further extended body weight gain and improved feed efficiency. Therefore, CLA was suggested to be a growth factor for young rats.
Growth after endotoxin injection
The endotoxin injection was given afrer the rats were on the experimental diets for four weeks. Fig. 24 showed the body weight of rats in the CLA and the control groups prior to and 1,2,3, 4, 5, and 6 days afrer the endotoxin injection. Within the first day afrer the endotoxin injection, rats in both dietary groups lost their body weight. However, the body weight lost was less in the CLA group than that in the control group. Twenty- four hours afrer the endotoxin injection, rats in the CLA group lost an average of 6.2 g of their body. By contrast, rats in the control group lost an average of 13.6 g of their body, twice of that in the CLA group. From the second day on rats in both dietary group progressingly regained their body weight. Since the rates of body weight gain were similar in both groups, the actual body weights remained higher in the CLA group than in the control group.
llv Body Weight Be lore And After The Endotoxin Injection
196 T
190 ■ Endotoxin Iniectvion 185 ■■ 01 ) ■CLA ■Si 180 - • D • - Control
170 • .O'
cr ■ 165 0 1 2 34 5 6 Time (days)
Fig. 24 Body weight change of rats in control and CLA groups before and after the endotoxin injection.
120 Body Weight Gain After The Endotoxin Injection
15 T
10 ■
c r
I -5 •• X ■CLA — "O ' — Control -10
Time (days)
Fig. 25 Body weight gains in control and CLA groups after the endotoxin injection relative to the body weigh before the endotoxin injection.
12! When the relative body weight gains, as compared to the body weight previous to the endotoxin injection, versus time were plotted in Fig.25, it was evident that rats in these two dietary groups responded to the endotoxin injection differently only in the initial body weight lose. Due to the differences in the initial body weight lose, it took approximately two and half days for the CLA group rats to return to their initial body weight, while it took more than four days for the control group rats to do just the same. Earlier, we have shown that dietary CLA did not effect the growth of the rats at their normal health state
(before the endotoxin injection). The result here showed that after the endotoxin injection, the growth rates were also same in the two groups. This results suggests that dietary CLA only prevented the body weight loss due to the endotoxin injection but did not affect their normal growth.
Feed consumption before and after the endotoxin iniection
Feed consumption for rats in both control and CLA group were recorded for the day prior to and 1, 2, 3, 4, 5, and 6 days after :he endotoxin injection. The average food intake for rats in control or CLA group at the day prior to and 1,2,3, 4, 5, and 6 days after the endotoxin injection were plotted in Fig. 26. Before the endotoxin injection, the rats in CLA group ate slightly less than the rats in the control group. In the first day after the endotoxin injection, feed consumption in both dietary groups was less than their usual food intake. However, rats in the CLA group ate about double the amount of the controls in the first day after the endotoxin injection. In the second day, rats in both dietary groups
I2i: Feed Consumption Before And After The Endotoxin Injection
18
16 o- - a
14 a 12 — ■— CLA I 10 “ • D * — Control 8 1 6 4
2 0 0 1 2 3 4 5 6 Time (days)
Fig. 26 Feed intake for rats in control and CLA groups before and after the endotoxin injection.
1 2 .1 started to eat their food, except the control group rats ate slightly less than the CLA group
rats. In day three or four, the food intakes were same in both dietary groups and similar to
their food intake before the endotoxin injection.
The endotoxin (LPS) is a potent immunomodulator. It elicits systemic
inflammation and causes sickness of fever, lethargy, anorexia, and loss of body weight
(cachexia) [Kozak et al., 1997]. These symptoms also are the common nonspecific clinical
manifestations of disease resulting from injury and trauma, infection and malignant and
inflammatory disorders [Kent et al., 1996; Kluger, 1991]. Data presented in this study
demonstrated that dietary CLA supplementation can substantially alleviate the endotoxin
induced anorexia (loss of appetite) and cachexia (loss of body weight). In addition, the
inhibition of loss of body weight and appetite by dietary CLA supplementation in this
study were accompanied by the depression in LTBa, which is a key mediator in immune and inflammatory responses.
Miller et al. [1993] have also reported that dietary CLA supplementation could reduce the body weight lose and increase feed consumption in rats and chicken. Our results are in agreement with their findings and further show that CLA enhanced resistance to endotoxin may be related to the eicosanoid production.
1 2 4 4.2.3 Plasma Eicosanoid Concentrations Bcibre and After Endotoxin Injection
Plasma LTBj concentrations
The plasma LTB, concentration were measured in rats (n= 6 ) in the long term animal experiment. Due to the low concentration of these two eicosanoids, every two plasma samples from the same treatment group were pooled (n=3) for the LTB4 measurement. Fig. 23 showed the concentration of plasma LTB, in rats fed with either
CLA supplemented diet or the control diet for 4 weeks. CLA feeding lowered the plasma
LTB4 concentration by 5%.
The plasma LTB4 concentration changes before and after the endotoxin injection were measured. Fig. 28. showed that the concentrations of plasma LTB4 in both dietary groups responded similarly to the endotoxin injection with the plasma LTB4 concentrations maintained lower in CLA group than in the control group. Within eight hours after the endotoxin injection, the plasma LTB4 concentrations increased from 37.8 pg/ml to 38.9 pg/ml in the control group and from 35.8 pg/ml to 37.8 pg/ml in the CLA group. Twenty- four hours after the endotoxin injection, the plasma LTB4 concentrations fell below the initial level to 35.7 pg/ml in control group and 33.4 pg/ml in CLA group. Six days after the endotoxin injection, the plasma LTB4 concentrations returned to the level before the endotoxin injection, 37.1 pg/ml in control group and 35.9 pg/ml in CLA group. Thus dietary CLA significantly (P<0.05) reduced plasma LTB4 concentrations during the endotoxin stimulation.
125 Plasma LTBw Concentrations (4 weeks after the experiental diets)
40 T 39 38 37 I 36 35 I Control 34 I CLA 33 32 31 30 Control CLA
Dietaiy Ti-eatment
Fig. 27 Plasma leukotriene B 4 concentrations in rats fed with 1% CLA supplemented diet or fed with control diet for 4 weeks.
I2(i Plasma LTB4 Concentrations Before And After The Endotoxin Injection 45 43 Endotoxin Injection 41 ^ 39
Î - 1 a, ■ -D- - Control w 35 - a — CLA è 33 ^ 31 29 27 25 ■ -20 20 40 60 80 100 120 140 160 Time (hr)
Fig. 28 Plasma LTB4 concentration changes for rats in the CLA group or in the control group before and after the endotoxin injection.
1 2 '’ Leukotriene B 4, one of the arachidonic acid metabolites resulted from the action of
enzyme lipoxygenases, is known to play an role as an important mediator in
vasoregulation, neurotransmission, hormonal regulation, inflammation immune responses,
and a host of diseases [Landauer et al., 1990]. LTB 4 is a potent pro-inflammatory agent
[Brain and Williams, 1990; Ford-Hutchinson, 1990] and is produced mainly by
polymorphonuclear leukocytes (PMN or neutrophils), macrophages, and monocytes. It
enhances immune responses by attracting infuction/antigen-fighting cells (leukocytes and
macrophages) and stimulating their activity at the host site of injury or invasion. LTB 4 is
also know to stimulate the production of cytokines, the key substances that help regulate
immune responses [Rola-Pleszcaynski and Lemair, 1985; Rola-Pleszczynski et al., 1986].
LTB4 has been observed in excess in many chronic inflammatory diseases such as
rheumatoid arthritis and psoriasis [Klickstein et al., 1980; Davidson et al., 1983]. An
increase in plasma LTB 4 concentration in rats right after the endotoxin injection and later
recovery to normal level indicated that LTB 4 acted as a mediator in the endotoxin elicited systemic inflammation.
Since LTB 4 is a derivative of fatty acids, it is subject to dietary manipulation. The consumption of eicosapentaenoic acid (EPA) iind docosahexaenoic acid (DHA) in fish oils has been reported to suppress the formation of LTB 4 in neutrophils, monocytes, and macrophages [Lee et al., 1985; Lokesh et al., 1988]. The reduction as great as 75% in
LTB4 formation has also been reported [Broughton et al., 1991]. As a consequence,
1 2 S DHAÆPA feeding geadily diminished immune and inflammatory responses. This effect was attributed to the inhibitory effect of EPA and DHA on the enzyme lipoxygenase
[Jakschik et al„ 1980].
CLA has been hypothesized to alter the eicosanoid, including LTB4, production by interfering with linoleic and arachidonic acids metabolism, therefore reduce the availability of arachidonic acid for the synthesis of eicosanoid [Ha et al., 1992; Ip et al.,
1990]. However, Up to date, no direct link has been made between the dietary CLA and the production of LTB,. Our observation that long term CLA supplementation reduced the plasma LTB4 concentration showed that dietary CLA effect on the LTB4 synthesis. As can be seen in next section, our data also showed that dietary CLA reduced the linoleic acid levels in liver and spleen and arachidonic acid level in heart. Based on previous mentioned hypothesis the decrease in plasma LTB4 concentration might due to the decreased availability of arachidonic acid for the biosynthesis of eicosanoids.
Plasma PGE; concentrations
Plasma PGE2 concentrations were also measured in rats from the long term animal experiment. After four weeks on the experimental diets without the endotoxin injection, CLA feeding reduced the plasma PGEz concentration by 6% from 145 pg/ml to
145 pg/ml (Fig. 29).
1 2 " Plasma PGE% Concentrations 4 Weeks After The Expérimental Diets 165 T
160 •
155 ■ Control S 150 -• ■ CLA 145 -
Ü 140
135 -•
Control CLA Dietaiy Treatments
Fig. 29 Plasma PGEz concentrations in rats of control group and CLA group 4 weeks after the experimental diet and without endotoxin injection.
130 Plasma PGEz Concentratioiis Be lore and After the Encotoxin Injection 200
180 ■ Endotoxin 160 ■ Iniection
140 ■
120 ■ • - Cortrol I 100 (3 — CLA g 80 t 60 ■ Î 40 •
20 •
-20 20 40 60 80 100 120 140 160 Time (hr)
Fig. 30 Plasma PGEz concentration changes before and after the endotoxin injection for rats in control and CLA groups.
13; The responses of plasma PGE 2 concentrations to the endotoxin injection were similar in both dietary groups (Fig. 30). Within twenty-four hours after the endotoxin injection, plasma PGE 2 concentrations fell in both CLA and control group. However, the drop in the plasma PGE 2 concentration in the CLA group (from 145 pg/ml to 48 pg/ml) was not as sever as in the control group (from 154 pg/ml to 36 pg/ml). Twenty-four hours after the endotoxin injection, the plasma PGE 2 concentrations in both dietary groups started to increase. The rates of plasma PGE; concentration increase were similar in both dietary groups. Hence, during recovery from endotoxin injection, the plasma PGE 2 concentrations in control group remain lower than that in the CLA group.
PGE2 is also a arachidonic acid metabolite via the action of enzyme cyclooxygenase. It has been suggested to be involved in the mediating sickness behavior during infection/inflammation [Kent et al., 1996]. PGE 2 has been found to be an important mediator of fever [Milton, 1989]. However, studies on the involvement of
PGE2 in the generation of anorexia and cachexia suggested that anorexia and weight loss during infection/inflammation was not mediated by PGE 2 [Mahoney and Tisdale, 1989;
Uehara et al., 1989].
Sugano et al [1997] has reported that dietary CLA supplementation decreased the concentrations of PGE 2 in serum and spleen in health rats. This effect was also observed in this study.
13: 4.2.4 Tissue Fatty Acid Composition
After four weeks of dietary CLA supplementation, tissue fatty acid profiles were
different between the CLA group and control group. Table 3 showed the composition of
major fatty acids, CI6;0, CI8:0, C18;I, CI8:2n-6, C20:4n-6, C22:5n-6, and C22:6n-3, (%
of the total fatty acids) in various tissues, e.g., liver, heart, lung, spleen, kidney, muscle,
and adipose tissue in both CLA and control group.
The tissue levels of C l6:0 and C22:5n-6 were significantly higher (P<0.05) in the
CLA group than in the control group. Compared with the control, the increase in C16:0
was greatest in adipose tissue (12%) and lung (12%), followed by heart (10%) and muscle
(10%), kidney (7%), liver (5%). Spleen had the smallest increase (3%) in content of
C l6:0. The percentage of increase in C22:5n-6 for liver, heart, lung, spleen, kidney, and
muscle were 11, 14, 30, 45, 20, and 26%, respectively.
By contrast, CLA feeding significantly reduced (P<0.05) the levels of C18:l in all tissues exempted. Among them, muscle showed the biggest decrease (31%), while liver showed the smallest decrease (15%). The decreases in C l8:1 in spleen, adipose tissue, heart, kidney, and lung were 29,24, 20, 18, and 17%, respectively.
CLA feeding significantly (P<0.03) increased C l8:0 level in liver (15%), whereas it decreased significantly (P<0.03) the C18:0 le\ els in heart (4%) and adipose tissue (10%).
However, CLA feeding had no significant effect on the C l8:0 levels in other tissues.
13:. Tissue Diet C16.0 C18:0 C18.1 C18;2n6 C20.4n6 C22:5n6 C22:6n3
Liver Control 21.0±1.2 14.411.8 14.312.0 23.112.8 14.612.1 0.910.2 6.510.9 CLA 22.111.1* 16.511.6* 12.110.7* 20.112.8* 13.910.8 1.010.05* 7.410.6
Heart Control I2.6±0.4 21.210.7 9.110.6 23.011.2 19.110.9* 2.110.1 10.010.9 CLA 13.9±0.7* 20.410.6* 7.310.2* 22.610.5 17.510.9 2.410.2* 10.210.6
Lung Control 31.810.8 10.710.5 18.911.9 15.111.6 10.411.3 1.010.1 1.810.2 CLA 35.511.2* 10.610.6 15.710.5* 14.011.4 10.210.7 1.310.1* 1.410.1
Spleen Control 24.710.5 15.511.1 15.611.0 16.611.3 17.111.8 1.110.1 1.510.2 CLA 25.410.8* 16.210.5 11.110.6* 15.410.4* 18.810.7* 1.610.1* 1.810.07 w v$ Kidney Control 23.110.5 20.910.5 13.810.7 14.510.8 22.511.0 0.510.03 1.110.08 CLA 24.810.8* 20.611.5 11.310.5* 15.210.8 21.910.6 0.610.06* 1.310.1
Muscle Control 23.210.8 11.411.5 15.013.7 21.911.3 12.512.2 2.310.4 8.712.1 CLA 25.511.1* 11.710.8 10.311.3* 20.911.1 11.811.5 2.910.5* 9.111.1
Adipose Control 25.510.54 4.710.7 32.610.6 27.410.4 n.d. trace trace CLA 28.511.4* 4.210.7 24.711.0* 28.312.5 n.d. trace trace
Table. 3 Major fattay acids content (% of total extracted fatty acid)ln various tissues of rats fed with control linoleic acid diet or CLA supplemented diet for 4 weeks. ( n = 6 )
Data are presented as mean ± standard error of mean. * means significant at P ^ 0.05 when compared to control groups. CI8;2n-6 level were also significantly (P<0.05) decreased in liver and spleen, by 17%
and 7%, respectively. The Cl8:2n6 contents in other tissues were not different between
the two dietary groups.
In case of C20:4n-6, CLA feeding caused a 8% decrease (P<0.01) in heart but a 10%
increase (P<0.05) in spleen. The levels of C20:4n-6 in other tissues were also decreased,
however, the decreases were not statistically significant.
Overall, the long term dietary CLA supplementation affected the distribution of major fatty acids in tissues.
The fatty acid compositions of tissues from rats 8 hours after administration by gavage of a single dose of CLA or com oil were also examined. There were no significant difference between the CLA and control groups. The fatty acid compositions in three liver lipid fractions, triacylglycerid, free fatty acids, and phospholipid fractions, were examined.
Table 4 shows the major fatty acid composition as % of total fatty acids in liver triacylglycerid, free fatty acids, and phospholipid fi-actions from rats treated with the single
CLA gavage dose. The liver samples were taken 8 hours after the gavage dose. CLA treatment reduced the levels of C16:0 and C2C;4n-6 but increased those of C18:2 in all the three liver lipid fractions. These differences between the CLA treatment and control did not reach statistically significance. It should be note, however, that only three rats was used in the control group and six in the treatment group in this short term animal experiment.
135 Triacvlglvcerid Free Fatty Acids Phospholipid
Control CLA Control CLA Control CLA C16:0 16.5+1.6 14.810.8* 19.211.3 18.211.2* 20.311.4 18.512.2*
C18.0 3.510.3 3.610.4 14.611.0 14.011.92 42.111.5 44.710.7*
C18:l 23.2+0.5 23.710.9 15.910.8 16.011.0 5.610.5 5.110.4
C I8:2 40.611.7 42.811.7* 28.512.6 31.611.9* 12.810.5 12.810.3
C20;4 7.6710.9 6.7210.5* 13.811.3 12.310.6* 12.811.3 12.511.4 w C22:5 1.710.09 1.310.07* 1.210.09 1.010.1* 0.510.1 0.510.05
C22:6 2.010.05 1.4410.1 2.610.4 2.210.2 3.610.8 3.010.8
Table 4 The major fatty acid contents (% of total extracted fatty acids) in liver triacylglyceride, free fatty acids, and phospholipid fractions of rats treated with a singal CLA gavage dose. The liver samples were taken 8 hours after the gavage dose. (n=3 for control group and n=6 for CLA group)
Data are presented as mean ± standard error of mean. * means significant at P 0.05 when compared to the control group. Prior to this study, little information regarding the effect of dietary CLA on tissue
fatty acid composition is available. Miller et al. [1994] have reported that muscle
arachidonic acid (C20:4n-6) level decreased significantly in rats (39.9 vs. 60.8 mg/g fat)
and chicks (38.8 vs. 43.6 mg/g fat) fed 15 days of CLA as compared to the basal diet. It
was suggested that the decrease in arachidonic acid levels was due to competition between
the CLA and LA (C18:2n-6) for the in vivo biosynthesis of arachidonic acid and between
the CLA and the dietary arachidonic acid for the incorporation into the membrane
phospholipid [Ip et al., 1990]. In this study, the significant increase in C16;0 and decrease
in C l8:1 in CLA fed rats as compared to their controls seems to suggest that dietary CLA
also affected the availability of these two fatty acids. Thus, dietary CLA not only
competes with linoleic or arachidonic acid, but also affect the absorption, transportation,
and possibly metabolism of other fatty acids.
4.2.5 Tissue Lipid Contents
The lipid concentrations were measured in all the examined tissues. Fig. 31
showed the lipid concentrations (mg extracted lipid/g tissue) of liver, kidney, lung, spleen,
heart, and muscle in rats fed with either control or CLA diets for 4 weeks. Since the lipid
concentrations of adipose tissue was much higher than that of other tissues, the lipid concentrations of adipose tissue in both dietary groups were shown in Fig. 32. Significant
reduction (P<0.05) in lipid concentrations were observed in liver, kidney, lung, spleen, and adipose tissue in the CLA group in compaiison to those in the control group. The
13' Tissue Lipid Concentrations (4 weeks after the experimental diets) 70 T
60
50
40 a Control ÎIw ^ ■ CLA 2 ^ 30
20
10
Liver Kidney Lung IfciM Spleen Heart Muscle
Tissues
Fig. 31 Lipid concentrations of liver, kidnej , lung, spleen, heart, and muscle of rats in control and CLA groups.
13S Adipose Tissue Lipid Concentration (4 weeks after the experimental diet) 1000 900 600 700 'S' tns 600 .52 I Control 500 u I CLA 400 M s M 300 200 100 • 0 Control CLA Dietary Group
Fig. 32 Lipid concentration of adipose tissue of rats in control and CLA groups.
139 greatest reduction was seen in rat spleen, which was a 53% reduction in the lipid
concentration. The reductions of the lipid concentrations in liver, kidney, lung, and
adipose tissue were 15, 12, 9, and 7%, respectively. The lipid concentrations in muscle
was not significantly different between the two dietary groups. But the heart lipid
concentration in the CLA group was slightly higher than that in the control group.
Park et al. [1997] reported that mice fed 0.5% CLA supplemented diet for 30 days
exhibited 57% (males) and 60% (females) low er body fat, 5% (males) and 14% (females)
increased body protein, and 9% (males) and 14% (females) increased body water content
relative to the controls. In studying the mechanism of CLA action on body composition
changes. Park et al. found that dietary CLA supplementation increased total carnitine
palmitoyltransferase activity, which is the rate-limiting factor for fatty acid |3-oxidation, for
fat pad of fed mice and for skeletal muscle of fasted mice. They also found that in
cultured 3T3-L1 adipocyte, CLA treatment significantly reduced heparin-releasable
lipoprotein lipase activity (-66%) and the intracellular concentrations of tracylglyceride (-
8%) and glycerol (-15%), but significantly increased free glycerol in the culture medium
(+22%) when compared to controls. Therefore, they suggested that the efifect of CLA on body composition was due to reduced fat deposition and increased lipolysis in adipocytes, possibly coupled with enhanced fatty acid oxidation in both muscle cells and adipocytes.
Results from our study showed the decreased lipid content in most tissues examined.
Our results are basically in agreement with Pai k’s finding. One of the mechanisms Park et
140 al. proposed for the efifect of CLA on body fat reduction was the possible enhanced fatty acid oxidation in muscle cells. However, the (XA-feeding induced lipid content reduction in muscle was not found in our study.
141 CHAP1 ER 5
CONCLUSIONS AND FUTURE WORK
5.1 Conclusions
Conjugated linoleic acid (CLA) was readily absorbed and incorporated into tissues of the rats, which was demonstrated by the fact that eight hours after a single CLA gavage dose, the CLA concentrations in various tissues increased significantly relative to the controls. The absorbed CLA in the tissues was then rapidly metabolized. As observed that the CLA concentrations in various tissues returned to the control levels 24 hours after the CLA gavage dose. These responses were similar to the metabolism of other long chain fatty acids. Liver showed to be the active site of CLA metabolism. CLA concentration in liver changed dramatically as it to be the highest S and lowest 24 hours after the CLA dose among the examined tissues. Heart was also active in incorporating
CLA, however, it was not as effective as livers to transport and metabolize CLA. CLA concentrations in hearts remained significantly higher as compared to the control 24 hours after the CLA dose.
14?. When CLA was fed to the rats on a continuous basis, it was found to accumulate in
the tissues. CLA concentrations in all the examined tissues were significantly higher in
rats fed with CLA diet than in those fed with control diet for 4 weeks. They were also
higher than rats received the single CLA dose These elevated tissue CLA concentrations
are suggested to be necessary for the effectiveness of CLA effects. In agreement with the
responses to the single CLA dose, liver and heart had the highest CLA concentrations among the examined tissues 4 weeks on the ('LA diet. This could indicate that liver and heart are the important sites for CLA metabolism.
Dietary CLA supplementation showed no significant effect on animal growth. The growth rates for rats in both CLA and control groups were comparable during the four weeks on the experiment diets and the recovery ( one day after the endotoxin injection) from the endotoxin challenge. The weights of liver, heart, kidney, lung, and spleen were also comparable between the CLA and the control group rats. Therefore, CLA is likely to be as effective as linoleic acid (other long chain fatty acids) in providing energy for animal growth.
The body weight loss caused by endotoxin injection was significantly reduced by the long term dietary CLA supplementation. Twenty-four hours after the endotoxin injection, the body weight loss for rats in CLA group was half of that for rats in the control group.
This difference in body weight loss between the CLA and control rats remained during the time of recovery. Further more, feed consumption showed the same response as the body
14;: weight loss. In the first twenty-four hours post endotoxin injection, rats in the CLA group
ate double the amount of the fed as rats in the control group. Started at the second day
after the endotoxin injection, the feed consumption was similar for rats in both groups.
These results demonstrated that dietary CLA substantially modified the sickness behavior
(body weight loss and anorexia) caused by the endotoxin injection induced systemic
inflammation.
As expected in the stated hypothesis, the modification of endotoxin induced sickness
behavior were accompanied by the depression in LTB4 production. The plasma LTB4
concentration in both CLA and control group increased at the first 8 hours post endotoxin
injection and returned to the normal level (before the endotoxin injection) in about 36
hours afier the endotoxin injection. The plasma LTB4 concentration in the CLA groups
was significantly lower than that in the control at all time. However, no significant
different was observed in the plasma PGE 2 concentration between the two dietary groups.
LTB4 is believed to play a key role in mediating immune and inflammatory responses. The
finding that dietary CLA depressed LTB4 production indicates that CLA may play a role in
modifying the eicosanoid biosynthesis during inflammation.
In addition to the modification of eicosanoid production, long term dietary CLA
supplementation affected the tissue fatty acid compositions. Significant changes on fatty
acid C16;0, CIS: I, and C22:5 contents were observed in all examined tissues between the
CLA group and control group after 4 weeks on the experimental diets. However, the
144 expected changes in fatty acid C l8:2 content were only found significant in liver and
spleen, while the significant changes in C20:4 contents were found in heart and spleen
between the two dietary groups. Since CLA showed a preference in incorporating into
liver and heart, the selected modification of the C l8:2 and C20:4 in liver and heart may
indicate that sufficient amount of CLA in tissues is necessary for CLA to compete with
linoleic (C l8:2) and arachidonic (C20:4) acids
Long term dietary CLA supplementation reduced tissue lipid content. Tissue lipid concentrations (mg extracted lipid/g tissue) in liver, kidney, lung, spleen, and adipose tissue were significantly reduced in CLA group compared to the control 4 weeks on the experimental diets. However, this reduced tissue lipid concentrations were not found in heart and muscle.
5.2 Suggestions For Future Research
Since this study is the initial research on C LA in this lab, the focus o f the study was to obtain basic information about CLA and to justify the hypothesis we want to investigate.
Data from this study indicated that dietary CLA could alter the arachidonic acid metabolism and have some beneficial efifect on inflammatory disease. However, this study is far from detail, more work is expected to confirm some of the results and further study the detail of the CLA effects.
I4.S In order to better understand relationship between CLA and arachidonic acid
metabolism, cell culture experiments are needed to examine the ability of CLA in utilize
the enzymes elongase and desaturase. In addition, the relationship between CLA and enzymes cyclooxygenase and lipoxygenas is a good area to investigate.
Since the plasma eicosanoid concentration is not a very reliable index to evaluate the in vivo production of eicosanoid, the measurement of urinary metabolites of eicosanoid or measurement of eicosanoid in some specific type of cells may be useful to confirm the finding of CLA effect on the eicosanoid biosynthesis.
The protective effect of CLA has been seen in the endotoxin induced systemic inflammation model. The potential of CI-A in prevent or reduce the severity of inflammatory disease is great. Therefore, studies to examine the effect of dietary CLA on inflammatory disease such as arthritis are useful to investigate the CLA potential in modulation of inflammatory diseases.
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16. Appendix - A: Fatty Acid Composition of Reference standard 68 A
FAME ID Actual Weight (g) Calculated Wt/Wt % C I4;0 5.0 3.0 C I4;ln5 5.0 1.0 C I6:0 10.0 10.0 C l6 ;ln 7 5.0 2.0 C18:0 5.0 15.0 C I8:In9 5.0 25.0 C l8;2n6 5.0 10.0 C18;3n3 5.0 4.0 C20:0 5.0 2.0 C20:ln9 5.0 2.0 C20:2n6 5.0 2.0 C20:3nn6 5.0 4.0 C20:4n6 5.0 4.0 C22:0 5.0 4.0 C22:ln9 5.0 2.0 C22;6n3 5.0 4.0 C24;0 5.0 2.0 C24;l 5.0 4.0
162 Appendix - B: Fatty Acid Composition of Reference Standard RL 6
FAME ID Actual Weight (g) Calculated Wt/Wt % C6:0 0.0998 0.498 C8:0 2.4012 11.984 C10;0 1.1945 5.962 C l 2:0 0.8052 4.019 C I3:0 1.0103 5.042 C I4:0 0.4007 2.000 C14:In9 0.1033 0.516 015:0 0.1029 0.514 016:0 1.0007 4.995 016: In? 0.1149 0.573 017:0 0.2070 1.033 018:0 0.4021 2.007 C 18:ln9 5.6013 27.956 018:2n6 2.7995 13.972 018:3n6 0.2999 1.497 018:3n3 0.6015 3.002 0 2 0 :0 0.2057 1.027 020: ln9 0.2038 1.017 0 2 0 :2 n 6 0.2032 1.014 C20:3n6 0.1573 0.785 020:4n6 0.3066 1.530 020:5n3 0.3023 1.509 0 2 2 :0 0.2024 1.010 0 2 2 :1n9 0.2001 0.999 C22:4n6 0.1977 0.987 C22:5n3 0.1989 0.993 024:0 0.2061 1.029 022:6n3 0.2991 1.493 024:1 0.2077 1.037 Total 20.0357 100.000
1 6 3 Appendix - C: Method of Lipid Extraction
1. Weigh ~ 2 g of tissue sample and cut into small pieces in a 50 ml glass tube.
2. Add 20 ml chloroform/methanol (2;l,v/v) to the tube and ground the tissue sample with a homogenizer for 1 minute.
3. Add another 20 ml chloroform/methanol (2:1) to the tube and cap the homogenate under nitrogen then allow to set for 2 hours at room temperature (or overnight at 4°C).
4. Filter the extract with Whatman #41 filter paper to 250 ml separation funnel. Rinse the residue in the test tube and on the filter paper with the chloroform/methanol solution
(2: 1).
5. Add 10 ml saline to the extract, vetax for 30 seconds and set to settle.
6 . Let the bottom phase (chloroform phase) go through a filter with anhydrous sodium sulfate into a 250 ml flask. Evaporate the solvent on a rotavapor.
7. Transfer the dried extract to a weighted test tube with ~5 ml chloroform/methanol solution. Remove the solvent under nitrogen then weigh the extracted lipid.
1 6 4 Appendix - D; Method of Saponification and Methvlation
1. Add 1.5 ml methanol and 1.5 mi IN KOH in methanol to the test tube with extracted lipid. Flash with nitrogen and screw-cap with Teflon-lined cap.
2 . boil gently at 95°C for 15 minutes then cool to room temperature.
3. Add 3 ml of boron trifluoride-methanol (BF3CH3OH). Cap the tube and then boil at
95“C for 15 minutes.
4. Cool the tube to room temperature then add 2 ml twice to extract the methylated fatty a cid s.
165 Appendix - E: Method for CL A Synthesis and tl0.cl2-CLA Isomer CrvstalllTatinn
1. Weigh 100 g ethylene glycol into a 500 ml three-neck round bottom flask equipped with a branched-hollow stopper.
2. Place the flask in an oil bath and bubble nitrogen through the ethylene glycol. Raise the oil bath temperature to 180°C and hold for 10 minutes.
3. Reduce the oil bath temperature to 160®C and remove the flask from the oil bath.
4. Add 26 g KOH to the flask and return the flask to the oil bath. Raise the oil bath temperature again to 180“C and hold for 10 m nutes.
5. While remove the flask from the oil bath add 50 g linoleic acid as the mixture is swirled.
6 . Replace the flask to the oil bath and maintain the temperature at 180°C for 2 hours.
7. Remove the flask from the oil bath and coo to room temperature with cold tap water.
8 . Add 200 ml methanol and transfer the solution to a 1000 ml separatory funnel then add 250 ml 6 N HCl to acidify the solution to pH < 2.
I6
10. Wash the hexane extract with 200 ml 30% methanol in water three times, then with
200 ml double distilled water for another three times.
11. Let the hexane extract go through a funnel with anhydrous sodium to remove residual water.
12. Remove the hexane under vacuum rotoevaporator. Store the synthesized CLA under nitrogen at -80°C.
13. To prepare the tlO,cl2-CLA isomer, add 400 ml 4% HCl to the synthesized CLA in a
500 ml flask. Cap the solution under nitrogen and place on a water bath of 60®C for 30 minutes to methylate the CLA acid to CLA methyl ester.
14. Cool the solution to room temperature then transfer to a 500 ml separatory funnel.
Extract the CLA methyl ester with 100 ml hexane twice.
15. Wash the hexane extract twice with 50 ml double distilled water. Remove trace water by anhydrous sodium sulfate. Remove the hexane under vacuum rotoevaporator.
16. Dissolve the CLA methyl ester in 500 ml acetone and transfer the solution to several centrifuge tubes. Place centrifuge tubes with the solution into a freezer set at temperature of-60“C.
16' 17. After crystallization centrifuge at 2000g for 5 minutes in a temperature controllable centrifuge. Separate the crystalline from the supernatant.
18. Redissolve the crystalline fraction in 200 ml acetone and recrystallize at the same temperature. Separate the crystalline again and save as the 110,c 12-octadecadienoate.
16S Appendix-F: Method oFEnzvme Immunoassay of Leukotriene fLTBA
Preparation of Reagents:
1. El A Buffer: dilute the concentrate to a final 100 ml in ultrapure water.
2. Wash Buffer: add I ml Tween 20 then dilute the concentrate to a final 2000 ml in ultrapure water.
3. LTB4 Standard: add 6 ml o f BOA Bufifer to the vial with the standard.
4 . LTB4 Acetylcholinesterase Tracer; add 6 ml El A Buffer to the vial with the tracer.
5. LTB4 Antiserum: add 6 ml EIA Buffer to the vial with the antiserum.
6 . Ellman’s Reagent: dilute the concentrate to a final 20 ml in ultrapure water.
Standard Curve:
1. Obtain 8 clean plastic tubes and number them #1 through # 8 .
2. Add 900 pi EIA Buffer to tuve #1 and 500 pi EIA Buffer to tube #2 to # 8 .
3. Transfer 100 pi of the bulk standard (5 ng/ml) to tube #1 and mix thoroughly.
16î> 4. Serially dilute the standard by removing 500 \i[ from tube #1 and placing in tube #2
and mix thoroughly.
5. Repeat the procedure to the tube # 8 .
Plate Set Up;
1. Rinse every well of the plate once with Wash Buffer and aspirate the buffer from the plate.
2. Invert and blot the plate on paper towel after aspiration and be sure all of buffer is removed from the wells.
3. The plate is seted as:
1 2 3 4 5 6 7 8 9 10 11 12
AB S8 S4
BB S8 S4
C NSB S7 S3
DNSB S7 S3
E Bo S6 82
17(1 F Bo S6 S2
G Bo S5 SI
HTA S5 SI
B - Blank; NSB - Non-Specific Binding; TA - total Activity; Bo - Maximum Binding; SI
- S8 - Standard 1 - 8 .
4. Add 100 pi EIA Bufifer to NSB wells, and 50 pi EIA Bufifer to Bo wells.
5. Add 50 pi different concentrations of diluted LTB 4 Standard to standard wells (S 8 -
Sl).
6 . Add 50 pi of sample per well in the wells not oqupied.
7. Add 50 pi LTB 4 Tracer to each well exceot TA and B wells.
8 . Add 50 pi LTB 4 Antiserum to each well except TA, NSB, and B wells.
9. Cover the wells with film and incubate for 18 hours at room temperature.
Deverlop the Plate:
1. Empty the wells by aspiration.
2. Rinse the plate five times with Wash Buflfor.
171 3. Add 200 |il Ellman’s Reagent to each well and 5 ml Tracer to the TA well.
4. Cover the plate with the plastic film and develop in dark for 60 to 90 minute.
5. Read the plate at 405 nm on KC3.
172 Appendix - G: Method of Enzyme Immunoassay of Prostaglandin fPGE^)
Preparation of Reagents:
1. EIA Buffer: dilute the concentrate to a final 100 ml in ultrapure water.
2. Wash Buffer; add I ml Tween 20 then dilute the concentrate to a final 2000 ml in ultrapure water.
3. PGEj Standard: add 6 ml of EOA Buffer to the vial with the standard (bulk standard).
4. PGEa Acetylcholinesterase Tracer: add 6 ml EIA Buffer to the vial with the tracer.
5. PGEz Monoclonal Antibody: add 6 ml EIA Buffer to the vial with the antiserum.
6 . Ellman’s Reagent: dilute the concentrate to a final 20 ml in ultrapure water.
Standard Curve:
1. Obtain 8 clean plastic tubes and number them #1 through # 8 .
2. Add 900 |il EIA Buffer to tuve #1 and 500 pi EIA Buffer to tube #2 to # 8 .
3. Transfer 100 pi of the bulk standard (10 ng/ml) to tube #1 and mix thoroughly.
17:; . 4. Serially dilute the standard by removing 500 )il from tube #1 and placing in tube #2 and mix thoroughly.5. Repeat the procedure to the tube #8.
Plate Set Up:
1. Rinse every well of the plate once with Wash Buffer and aspirate the buffer from the plate.
2. Invert and blot the plate on paper towel after aspiration and be sure all of buffer is removed from the wells.
3. The plate is seted as:
1 2 3 4 5 6 7 8 9 10 11 12
AB S8 S4
B B S8 S4
C NSB S7 S3
DNSB S7 S3
E Bo S6 S2
F Bo S6 S2
1 7 4 G Bo S5 SI
HTA S5 SI
B - Blank; NSB - Non-Specific Binding; TA - total Activity; Bo - Maximum Binding; Si
- S8 - Standard 1-8.
4. Add 100 til EIA Buffer to NSB wells, and 50 tU EIA Buffer to Bo wells.
5. Add 50 til different concentrations of diluted PGEj Standard to standard wells (S8-
Sl).
6. Add 50 til of sample per well in the wells not oqupied.
7. Add 50 til PGE 2 Tracer to each well except TA and B wells.
8. Add 50 til PGE 2 Antiserum to each well except TA, NSB, and B wells.
9. Cover the wells with film and incubate for 18 hours at room temperature.
Deverlop the Plate:
1. Empty the wells by aspiration.
2. Rinse the plate five times with Wash Buffer.
3. Add 200 til Ellman’s Reagent to each well and 5 ml Tracer to the TA well.
17.-Î 4. Cover the plate with the plastic film and develop in dark for 60 to 90 minute.
5. Read the plate at 405 nm on KC3.
17(i IMAGE EVALUATION TEST TARGET (QA—'ô)
% %
1.0
c 1^ 4 .0 Li 2.0 l.l 1.8
1.25 1.4 1.6
150mm
V
«9
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