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Dietary lipids and in vivo antioxidant status in atherosclerosis resistant (rat) and sensitive (quail)… Yuan, Yvonne Veronica 1995

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DIETARY LIPIDS AND IN VIVO ANTIOXIDANT STATUS IN ATHEROSCLEROSIS RESISTANT (RAT) AND SENSITIVE (QUAIL) ANIMALS.  By  YVONNE VERONICA YUAN  B.Sc., The University of British Columbia, 1987 M.Sc., The University of British Columbia, 1990  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Food Science)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA September 1995 ©  Yvonne Veronica Yuan, 1 995  _____________________  In presenting this  thesis in  partial fulfilment of the requirements for an advanced  degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or  by  his  or  her  representatives.  It  is  understood  that  copying  or  publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature)  Department of  OcXi  Sc  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  OCk.L3  Ifc1(  ABSTRACT Studies were performed in two animal models known to be susceptible to (i) age-dependent hypertension and (ii)  atherosclerosis, for the purpose of examining the role of specific dietary fat  sources and dietary cholesterol intake in modifying hypertension and atherosclerosis, two well defined risk factors in the development of cardiovascular disease. spontaneously hypertensive rat (SHR) had lower (p  Despite exhibiting hypertension, the  0.05) plasma cholesterol and triacyiglyceride  concentrations than normotensive Wistar Kyoto (WKY) controls.  SHR and WKY animals exhibited  tissue- and enzyme-specific strain differences in antioxidant status. Systolic blood pressure (SBP) was positively (r  =  0.709, p  =  0.049)  correlated with RBC superoxide dismutase (SOD) activity and  negatively correlated with liver glutathione reductase (GSSG-Red; r  =  -0.798, p  =  0.018). Dietary  fat source or the proportion of saturated to unsaturated (n-6 or n-3) fatty acids fed at a moderate level of fat intake (e.g. 22% calories)  did not have an effect on plasma lipids, with the exception of  menhaden oil which significantly reduced both plasma cholesterol and triacylglyceride levels. There was no effect of dietary fat source or cholesterol on SBP of SHR or WKY animals. Menhaden oil fed to SHR and WKY animals resulted in decreases in RBC, heart and liver glutathione peroxidase (GSH-Px) and SOD activities as well as reduced tissue susceptibility to in vitro lipid peroxidation in these same animals. High levels of dietary cholesterol resulted in reduced (p  0.05) hepatic CAT, SOD and GSH  Px activities as well as greater resistance to in vitro lipid peroxidation than in counterparts fed low cholesterol diets. Despite significant effects of dietary cholesterol and fat source on plasma lipids and antioxidant parameters in hypertensive SHR, changes to SBP were not observed in the present study involving relatively young animals. Similar studies examining the effects of dietary cholesterol intake and fat source or level on plasma lipids, antioxidant status and aortic plaque were conducted in the atherosclerosis-susceptible Japanese quail.  Diet-induced hypercholesterolemia in quail fed 0.5%  cholesterol-O.25% cholic acid diets for 9 weeks was associated with the presence of aortic plaque containing cholesterol and cholesterol oxides.  Aortic atherosclerosis was not associated with  alterations in the antioxidant status of these quail. Moderate dietary levels (22% calories from fat) of saturated fat (e.g. butter, beef tallow) or unsaturated fat (e.g. soybean) did not alter plasma lipids, or severity of aortic plaque in birds, although activity of GSH-Px in RBCs and heart were increased and  decreased, respectively, in soybean oil-fed birds. In the final study, an increase in total calories from saturated fat (e.g. beef tallow) together with different levels of dietary cholesterol were found to have interactive effects on plasma triacylglycerides, and aortic plaque scores of atherosclerosis-susceptible Japanese quail. These dietary treatments did not have any effect on antioxidant enzymes of heart or aortic tissue. Dietary cholesterol and the higher level of saturated fat did, however, influence hepatic CAT and SOD activities, as well as reducing the susceptibility of liver and heart tissues to in vitro lipid oxidation. It was noteworthy that a significant (p  0.05) interaction was observed between dietary  cholesterol and fat level for plasma triacylglycerides but not plasma cholesterol. Elevations in plasma cholesterol and triacylglycerides in atherosclerotic quail paralleled the severity of aortic lesions, as determined by plaque score, cholesterol content and presence of cholesterol oxides in aortic tissue from these birds. The presence of plasma hypercholesterolemia and hypertriacylglyceridemia in the atherosclerosis-susceptible Japanese quail appeared to play a greater role in determining susceptibility to the development of aortic plaque than tissue antioxidant status under the experimental conditions employed herein. In summary, specific differences in tissue antioxidant enzymes and susceptibility to lipid peroxidation were more evident in the genetically hypertensive SHR rat model, than in diet-induced atherosclerosis in the atherosclerosis-susceptible Japanese quail.  III  TABLE OF CONTENTS Page TITLE PAGE ABSTRACT TABLE OF CONTENTS  iv  LIST OF TABLES  vii  LIST OF FIGURES LIST OF APPENDICES  xii  LIST OF ABBREVIATIONS  xiii  ACKNOWLEDGEMENTS  xvi  INTRODUCTION  1  LITERATURE REVIEW  2  1.  a. b. c. d. e.  Lipid oxidation: reaction mechanism and products Factors influencing lipid oxidation Antioxidants Antioxidants in foods Lipid autoxidation interactions with other nutrients or cell constituents  2 4 6 8 11  2.  a. b. c. d. e. f. g.  Digestion and absorption of dietary lipids Dietary lipids and dyslipidemias Endogenous antioxidants in vivo Lipid peroxidation in vivo Lipid oxidation in heart disease Oxidized lipid species in plasma Influence of dietary fatty acids on LDL oxidative stability  13 17 26 30 32 35 35  CHAPTER 1: Antioxidant status and plasma lipid levels in spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats Introduction Hypothesis, Objective and Specific Aims for Chapter 1 Materials and Methods Blood pressure measurement Tissue sample preparation Tissue antioxidant analysis i. Tissue glutathione (GSH; sulfhydryl group) content ii. Tissue susceptibility to in vitro forced peroxidation iii. Catalase (CAT) activity iv. Glutathione peroxidase (GSH-Px) activity v. Glutathione reductase (GSSG-Red) activity vi. Superoxide dismutase (SOD) activity vii. Correction of enzyme activities for blood contamination Statistics  40 40 43 44 44 44 45 45 46 47 48 48 49 50 50 iv  Page Results Discussion Conclusion  •  .  .  .  •  .  .  .  CHAPTER 2: Effect of saturated and polyunsaturated dietary fat sources on systolic blood pressure, plasma lipids and antioxidant status in spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats Introduction Hypotheses, Objective and Specific Aims for Chapter 2 Materials and Methods Diet gross energy determination Dietary fatty acid analysis Statistics Results Discussion Conclusion  51 63 68  69 69 71 72 74 74 76 77 101 112  .  CHAPTER 3: Species-related differences in plasma lipids and susceptibility to atherosclerosis between atherosclerosis-resistant (rat) and -susceptible (quail) animals fed diets supplemented with cholesterol Introduction Hypothesis, Objective and Specific Aims for Chapter 3 Materials and Methods Animals and diets Experimental procedures Determination of aortic plaque score Analysis of cholesterol and cholesterol oxides by gas chromatography! mass spectrometry Results Discussion Conclusion  119 123 129 132  CHAPTER 4: Effect of dietary fat source on aortic plaque, plasma lipids and antioxidant status of atherosclerosis-susceptible Japanese quail Introduction Hypotheses, Objective and Specific Aims for Chapter 4 Materials and Methods Scanning electron microscopy Results Discussion Conclusion  133 133 135 136 140 141 160 169  CHAPTER 5: Influence of increased caloric intake from beef tallow on plasma lipids, antioxidant status and diet-induced atherosclerosis in atherosclerosis-susceptible Japanese quail Introduction Hypothesis, Objective and Specific Aims for Chapter 5 Materials and Methods Mixing of diets Results  114 114 116 117 117 117 117  •  170 .170 • .172 • .173 .173 .177 .  V  Page Discussion Conclusion  196 202  SUMMARY AND GENERAL CONCLUSIONS  203  REFERENCES  210  APPENDIX  228  vi  LIST OF TABLES Table 1.1  Body weight gain and systolic blood pressure of SHR and WKY rats fed a standard chow diet  Page  52  1 .2 Plasma lipids of SHR and WKY rats fed a standard chow diet  53  1 .3 Antioxidant enzyme activities in red blood cells of SHR and WKY rats fed a standard chow diet  54  1 .4 Antioxidant enzyme activities in heart tissue of SHR and WKY rats fed a standard chow diet  55  1 .5 Antioxidant enzyme activities in liver tissue of SHR and WKY rats fed a standard chow diet  57  1 .6 Tissue glutathione levels of SHR and WKY rats fed a standard chow diet  58  2.1  73  Composition of diets fed to SHR and WKY rats  2.2 Fatty acid composition of diets fed to SHR and WKY rats  75  2.3 Body weight gain and systolic blood pressure of SHR and WKY fed experimental diets  78  2.4 Plasma lipids of SHR and WKY fed experimental diets  80  2.5 Antioxidant enzyme activities in red blood cells of SHR and WKY fed experimental diets  82  2.6 Antioxidant enzyme activities in heart of SHR and WKY fed experimental diets  84  2.7a Reactive oxygen species metabolizing antioxidant enzyme activities in liver of SHR and WKY fed experimental diets  86  2.7b Glutathione metabolizing antioxidant enzyme activities in liver of SHR and WKY fed experimental diets  87  2.8 Basal glutathione levels in heart and liver tissue and red blood cells of SHR and WKY fed experimental diets  89  2.9 Red blood cell susceptibility to H -induced GSH depletion and MDA production 0 2 in SHR and WKY fed experimental diets  90  -induced GSH depletion and TBARs production 0 2 2.10 Heart homogenate susceptibility to H in SHR and WKY fed experimental diets  95  -induced GSH depletion and TBARs production 0 2 2.11 Liver homogenate susceptibility to H in SHR and WKY fed experimental diets  97  vii  Table  Page  3.1 Composition of diets fed to Wistar rats and atherosclerosis-susceptible Japanese quail  118  3.2 Plasma lipids of Wistar rats and atherosclerosis-susceptible Japanese quail fed tallow diets containing low and high levels of cholesterol  1 24  3.3 Aortic plaque score and area covered in Wistar rats and atherosclerosis-susceptible Japanese quail fed tallow diets containing low and high levels of cholesterol  1 25  3.4 GC quantitation of cholesterol and cholesterol oxidation products in aortic tissue from Wistar rats and atherosclerosis-susceptible Japanese quail fed tallow diets containing low and high levels of cholesterol  1 26  4.1 Composition of diets fed to atherosclerosis-susceptible Japanese quail  137  4.2 Fatty acid composition of diets fed to atherosclerosis-susceptible Japanese quail  139  4.3 Plasma lipids of atherosclerosis-susceptible Japanese quail fed experimental diets  142  4.4 Antioxidant enzyme activities in red blood cells of atherosclerosis-susceptible Japanese quail fed experimental diets  144  4.5 Antioxidant enzyme activities in heart of atherosclerosis-susceptible Japanese quail fed experimental diets  145  4.6a Reactive oxygen species metabolizing antioxidant enzyme activities in liver of atherosclerosis-susceptible Japanese quail fed experimental diets  146  4.6b Glutathione metabolizing antioxidant enzyme activities in liver of atherosclerosissusceptible Japanese quail fed experimental diets  147  4.7 Antioxidant enzyme activities in aorta of atherosclerosis-susceptible Japanese quail fed experimental diets  148  -induced GSH depletion and TBARs 0 2 4.8 Heart homogenate susceptibility to H production in atherosclerosis-susceptible Japanese quail fed experimental diets  151  -induced GSH depletion and TBARs 0 2 4.9 Liver homogenate susceptibility to H production in atherosclerosis-susceptible Japanese quail fed experimental diets  1 53  4.10 Aortic plaque score and area covered in atherosclerosis-susceptible Japanese quail fed experimental diets  155  4.11 GC quantitation of cholesterol and cholesterol oxide content of aortic tissue from atherosclerosis-susceptible Japanese quail fed experimental diets  1 59  5.1  Composition and energy content of diets fed to quail  5.2 Fatty acid profile of diets fed to quail  174 175  VIII  Table  Page  5.3 Plasma cholesterol and triacyiglyceride concentrations in atherosclerosissusceptible Japanese quail fed experimental diets  1 78  5.4 Antioxidant enzyme activities in red blood cells of atherosclerosissusceptible Japanese quail fed experimental diets  180  5.5 Antioxidant enzyme activities in heart tissue of atherosclerosissusceptible Japanese quail fed experimental diets  181  5.6 Antioxidant enzyme activities in liver tissue of atherosclerosissusceptible Japanese quail fed experimental diets  1 82  5.7 Antioxidant enzyme activities in aortic tissue of atherosclerosissusceptible Japanese quail fed experimental diets  1 84  5.8 Basal glutathione content of heart and liver homogenate and red blood cells of atherosclerosis-susceptible Japanese quail  1 85  5.9 Heart and liver homogenate susceptibility to H -induced GSH depletion 0 2 and TBARs production in atherosclerosis-susceptible Japanese quail fed experimental diets  1 86  5.10 Aortic plaque score and area covered in atherosclerosis-susceptible Japanese quail fed experimental diets  190  5.11  GC quantitation of cholesterol and cholesterol oxide content of aortic tissue from atherosclerosis-susceptible Japanese quail fed experimental diets  1 95  ix  LIST OF FIGURES Figure  Page  1.1 Susceptibility of SHR and WKY red blood cells to in vitro -induced oxidative challenge 0 2 H  60  1 .2 Susceptibility of SHR and WKY heart tissue homogenate to in vitro -induced oxidative challenge 0 2 H  61  1 .3 Susceptibility of SHR and WKY liver tissue homogenate to in vitro -induced oxidative challenge 0 2 H  62  2.1  Susceptibility of red blood cells from SHR and WKY animals fed diets varying in dietary fat source and cholesterol intake level to in vitro H -induced glutathione (GSH) depletion 0 2  91  2.2 Susceptibility of red blood cells from SHR and WKY animals fed diets varying in dietary fat source and cholesterol intake level to in vitro H -induced malondialdehyde (MDA) production 0 2  93  2.3 Susceptibility of heart tissue from SHR and WKY animals fed diets varying in dietary fat source and cholesterol intake level to -induced glutathione (GSH) depletion 0 2 in vitro H  94  2.4 Susceptibility of heart tissue from SHR and WKY animals fed diets varying in dietary fat source and cholesterol intake level to in vitro H -induced thiobarbituric acid reactive substances (TBARS) production 0 2  96  2.5 Susceptibility of liver tissue from SHR and WKY animals fed diets varying in dietary fat source and cholesterol intake level to -induced glutathione (GSH) depletion 0 2 in vitro H  99  2.6 Susceptibility of liver tissue from SHR and WKY animals fed diets varying in dietary fat source and cholesterol intake level to in vitro H -induced thiobarbituric acid reactive substances (TBARS) production 0 2  100  3.1 A representative GC-FID chromatogram of derivatized cholesterol oxide standards with internal standard  120  3.2 A representative standard curve depicting the response-linearity of derivatized cholesterol oxide standards using GC-FID  1 22  3.3 GC-FID chromatogram of atherosclerosis-susceptible Japanese quail aortic tissue derivatized nonsaponifiables  1 27  3.4 GC-FID chromatogram of Wistar rat aortic tissue derivatized nonsaponifiables  1 28  4.1  Susceptibility of heart tissue from atherosclerosis-susceptible Japanese quail fed diets varying in dietary fat source and cholesterol intake level to -induced depletion of glutathione (GSH) and production 0 2 in vitro H of thiobarbituric acid reactive substances (TBARs)  1 50  x  Figure  Page  4.2 Susceptibility of liver tissue from atherosclerosis-susceptible Japanese quail fed diets varying in dietary fat source and cholesterol intake level to in vitro H -induced depletion of glutathione (GSH) and production 0 2 of thiobarbituric acid reactive substances (TBARs)  1 52  4.3 A representative scanning electron micrograph of aortic tissue from atherosclerosis-susceptible Japanese quail fed a low (0.05%) cholesterol diet  156  4.4 Representative scanning electron micrographs of aortic tissue from atherosclerosis-susceptible Japanese quail fed a high (0.5%) cholesterol diet  157  5.1  Susceptibility of heart tissue from atherosclerosis-susceptible Japanese quail fed a reference Turkey Starter (TS) diet alone, or TS diets supplemented with varying levels of beef tallow and cholesterol to in vitro H -induced depletion of glutathione (GSH) and production of 0 2 thiobarbituric acid reactive substances (TBAR5) 1 87  5.2 Susceptibility of liver tissue from atherosclerosis-susceptible Japanese quail fed a reference Turkey Starter (TS) diet alone, or TS diets supplemented with varying levels of beef tallow -induced depletion of glutathione (GSH) and production of 0 2 and cholesterol to in vitro H thiobarbituric acid reactive substances (TBAR5) 1 88 5.3 A representative scanning electron micrograph of aortic tissue from atherosclerosissusceptible Japanese quail fed the reference Turkey Starter (TS) diet alone  191  5.4 A representative scanning electron micrograph of aortic tissue from atherosclerosis susceptible Japanese quail fed a Turkey Starter (TS) diet supplemented with a high (0.5%) level of cholesterol and a minor amount of additional beef tallow (0.6%)  192  5.5 Representative scanning electron micrographs of aortic tissue from atherosclerosis susceptible Japanese quail fed a Turkey Starter (TS) diet supplemented with a high (0.5%) level of cholesterol and a higher level of beef tallow (6.6%)  193  xi  LIST OF APPENDICES Table  Page  1.  Relative retention times of TMS-sterol derivatives  229  2.  Response linearity of derivatized cholesterol and oxides  230  xii  LIST OF ABBREVIATIONS a  Water activity  ANOVA  Analysis of variance  CAD  Coronary artery disease  CAT  Catalase  CHD  Coronary heart disease  COP  Cholesterol oxidation product  CVD  Cardiovascular disease  DHA  Docosahexaenoic acid  DTNB  5,5’-dithiobis-(2-nitrobenzoic acid)  EDTA  Ethylenediamine tetraacetic acid  EPA  Eicosapentaenoic acid  FER  Feed efficiency ratio  FFA  Free fatty acid  GC  Gas chromatography  GC-FID  Gas chromatography with flame ionization detector  GC-MS  Gas chromatography with mass spectrometry  GSH  Glutathione (reduced form)  GSH-Px  Selenium-dependent glutathione peroxidase  GSSG  Glutathione (oxidized form)  GSSG-Red  Glutathione reductase  O 2 H  Hydrogen peroxide  Hb  Hemoglobin  HDL  High density lipoprotein  hr  Hour  I DL  Intermediate density lipoprotein  IHD  Ischemic heart disease  LDL  Low density lipoprotein XIII  MANOVA  Multiple analysis of variance  MDA  Malondialdehyde  mm  Minute  mLDL  Modified low density lipoprotein  mM  Millimolar  mmHg  Millimetre of mercury  mmol  Millimole  MUFA  Monounsaturated fatty acid  NADPH  Nicotinamide adenine dinucleotide phosphate (reduced form)  nmole  Nanomole  102  Singlet oxygen  ‘OH  Hydroxyl radical  02’  Superoxide radical  P/S  Polyunsaturated to saturated fatty acid ratio  PUFA  Polyunsaturated fatty acid  R’  Lipid free radical  RBC  Red blood cell  RH  Lipid molecule  ROOH  Lipid hydroperoxide  ROO’  Lipid peroxy radical  SBP  Systolic blood pressure  sec  Second  SFA  Saturated fatty acid  SH R  Spontaneously hypertensive rat  SOD  Cu/Zn Superoxide dismutase  TBARs  Thiobarbituric acid reactive substances  TCA  Trichloroacetic acid  TG  Triacyiglyceride xiv  TMS  Trimethyl-silyl  TS  Turkey Starter  pL  Microlitre  VLDL  Very low density lipoprotein  WKY  Wistar Kyoto (rat)  wt  Weight  xv  ACKNOWLEDGEMENTS I would like to express my gratitude to the many individuals who assisted me during the course of this study. In particular, I would like to acknowledge the Department of Food Science for the use of the animal and laboratory facilities. I wish to express my sincere gratitude to my thesis advisor, Dr. David D. Kitts, Department of Food Science, for his dedicated participation in the animal surgeries as well as in the completion of this thesis. I am also grateful for his neverending support and encouragement throughout the course of my studies. His friendship and unique sense of humour have made my years in Food Science at U.B.C. most memorable. I would also like to thank Dr. David Godin, Department of Pharmacology and Therapeutics for allowing me the use of his laboratory facilities to conduct the biochemical antioxidant enzyme analyses of my experiments. I am also grateful for his untiring editorial assistance in the preparation of manuscripts as well as during the preparation of this thesis. His friendship and comraderie outside of the lab have been an added bonus to my years at U.B.C. Thanks are also given to the members of my thesis committee, Drs. Eunice Li-Chan and John Vanderstoep of the Department of Food Science and Dr. Jiri Frohlich, Department of Pathology for their suggestions and help during the preparation of this thesis. I would also like to acknowlege the assistance of Ms. Lana Fukumoto and Ms. Donna Smith, Department of Food Science, with some of the laboratory analyses during my experiments. I would like to thank Ms. Maureen Garnett and Dr. Philip Toleikis, Department of Pharmacology and Therapeutics, for helping me to learn the biochemical assays utilized in my experimental methodologies. Ms. Cathleen Nichols of the Quail Genetic Resource Centre, at U.B.C. deserves recognition for her help in planning the animal studies using the atherosclerosis-susceptible Japanese quail in my experiments. I am also indebted to Dr. Guenter Eigendorf, Director of the Mass Spectrometry Centre, Department of Chemistry, and Ms. Lina Madilao for allowing me the use of the gas chromatography and mass spectrometry facilities as well as their invaluable help with some of trouble-shooting aspects of this portion of my experiments. Thanks are also given to Dr. Tom Scott, Agriculture Canada Research Station, Agassiz, B.C. for his assistance with the pelleting of the formulated semi-synthetic quail diets used in my experiments. I would like to acknowledge the Dairy Farmers of Canada for their support and funding of this project. Finally, I would like to thank my sisters Bernadette (a.k.a. Cha-cha) and Trinie and their families, and especially my mother, Mrs. Janet M. Yuan, for their good natured support and understanding during the course of my studies.  xvi  INTRODUCTION Cardiovascular disease (CVD) is the greatest cause of mortalities in Canada, accounting for 39% of all deaths (Heart and Stroke Foundation, 1993). Moreover, 73% of males and 90% of females who die of CVD are 65 years of age and over.  Risk factors for the development of CVD and  atherosclerosis include: hyperlipidemia, obesity, age, hypertension, diabetes, smoking as well as a heritable genetic component. Dietary fat intake, more specifically, the relative ratio of saturated versus unsaturated fatty acids contained in fat sources, has been a primary focus in the risk for developing hypercholesterolemia and atherosclerosis by proponents of the “Lipid Hypothesis” (Keys eta!., 1957). Thus, foods with a lipid component composed primarily of saturated fatty acids, such as those of animal origin which also contain cholesterol (beef tallow, milk and butter), have been labelled as hypercholesterolemic. Dietary lipids from vegetable and marine sources containing a high proportion of polyunsaturated fatty acids (PUFA)  have been identified as hypocholesterolemic (Ahrens et a!.,  1957; Mattson and Grundy, 1985).  Of particular interest is the finding that the incidence of  atherosclerosis is not necessarily correlated with level of hypercholesterolemia, but rather, can occur at any given level of hypercholesterolemia (Steinberg eta!., 1989).  It is noteworthy that levels of  oxidized lipids in plasma lipoproteins have been reported to increase with age (Hagihara eta!., 1984). Moreover, lipid peroxides have been observed to be present in not only plasma lipoproteins, but also atherosclerotic plaque from subjects with CVD (Stringer et a!., 1989; Rosenfeld et a!., 1990). Cytoxicity of lipid peroxides and sterol oxides demonstrated in vitro with tissue cultures may have a role in atherogenesis (Addis, 1990). This thesis will address various factors involved in the link between dietary lipid source, cholesterol level and antioxidant status in animal models which are susceptible to the development of hypertension and atherosclerosis.  1  LITERATURE REVIEW la. Lipid oxidation: reaction mechanism and products. Lipid oxidation is characterized by a self-catalytic free radical mechanism (autoxidation; Bolland and Gee, 1 946a), so that the reaction is self-renewing once it has been initiated (Bateman eta!., 1953; Bolland and Gee, 1946a).  Primary products of the reaction of lipid species (RH)  with molecular  oxygen are lipid hydroperoxides (ROOH; Farmer eta!., 1942). Hydroperoxides are short-lived, unstable molecules which undergo further isomerization and decomposition to form a variety of breakdown products characteristic of the specific hydroperoxide, including aldehydes, ketones, alcohols, hydrocarbons, acids, esters and epoxides (Chan and Levett, 1977a,b; Labuza, 1971; Bateman eta!., 1953; Farmer eta!., 1942). These initial breakdown products can then participate in further oxidation reactions, resulting in a diverse pool of end products (Bolland and Gee, 1946a; Farmer eta!., 1942). The generation of volatile oxidation products and reactive intermediates in the cascade of free radical reactions is associated with the production of desirable or undesirable flavours and odours in processed foods (Ladikos and Lougovois, 1990; Finley and Given, 1 986), as well as reduced availability of certain co-nutrients (Nielsen et a!., 1985a,b; Cuq et a!., 1973).  !n vivo, lipid peroxides of dietary or  endogenous origin have been implicated in various physiological processes from aging (Halliwell and Gutteridge, 1984) 1986).  to chronic diseases such as atherosclerosis and cancer (Pryor, 1986; Diplock,  These effects of ROOH may be mediated, in part, by oxidative damage to membranes in  subcellular compartments, resulting in tissue damage. Oxidation of unsaturated fatty acids via interaction of molecular oxygen with the methylenic carbon (carbon atom neighbour to a double bond or bonds; Farmer eta!., 1942) in a PUFA follows a three-step free radical chain reaction. The process consists of initiation, propagation and termination stages, as follows (Nawar, 1985; Bateman eta!., 1953; Bolland and Gee, 1946a,b): catalysis Initiation:  RH +  Propagation:  R’ + R00  02  °2  + RH  > R  ,  R00  > R00  > ROOH + R  2  Termination:  R  +  R  >  R  +  ROO’  >  R00  +  R00  Nonradical products  >  The interaction of unsaturated lipids with molecular oxygen tends to be quite slow for monoenes, with the rate of reaction increasing rapidly with an increase in unsaturation (Farmer eta!., 1942). Initiation of lipid autoxidation requires catalysis to generate the initial free radical species.  Catalysis occurs  through energy provided from radiant heat, transition metal ions or complexes, photochemical reactions with sensitizer molecules or ionizing radiation (Jung eta!., 1991; Foote, 1985; Farmer etal., 1942). Singlet oxygen,  102,  a highly electrophilic reactive oxygen species, can insert at either end of a double  bond (-C=C-) to yield hydroperoxides (ROOH; Labuza, 1971). Once the initial ROOH is formed, the autocatalytic free radical chain reaction proceeds due to the reactivity of peroxyl (R00)  radicals  (Foote, 1985; Bateman eta!., 1953). Propagation of lipid autoxidation is facilitated by the production of reactive intermediates from reactions such as peroxide decay and chain scission (e.g. at double bonds; Farmer et  8/.,  1942). The production of more stable chemical forms resulting from the decay  of hydroperoxides (see page 2)  occurs as well at this stage of the reaction.  Termination of lipid  autoxidation commonly involves two lipid free radicals as illustrated above (Bolland and Gee, 1946a). Products from the termination reactions of free radical chain carriers include the formation of dimers, alcohols and peroxides (Labuza, 1971; Bateman eta!., 1953). The vast array of lipid autoxidation products is due in part to the shift in double bond position which occurs with resonance stabilization of lipid radicals from PUFA (Chan and Levett, 1977a,b; Bolland and Gee, 1946a).  This phenomenon can result in the formation of several positional and  geometrical isomers depending on the level of unsaturation of the native fatty acid (Chan and Levett, 1977a,b).  Several characteristic products of lipid autoxidation reactions are used as indicator  substances to determine the extent of lipid oxidation in food systems as well as in vivo. The most prevalent of these methods is the determination of malondialdehyde, a dialdehyde product of PUFA autoxidation, via the 2-thiobarbituric acid (TBA) reaction using spectrophotometric (Botsoglou et a!., 1994; Buege and Aust, 1978) or chromatographic techniques (Draper eta!., 1993). Determination  3  of short chain alkanes, namely ethane and pentane in the breath, can also be used as an indicator of lipid oxidation in vivo (Dillard and Tappel, 1989). Lipid oxidation products are not restricted to fatty acids and triacylglycerides, but can also include sterol oxides present in the lipid system. Various cholesterol oxidation products (COPs) have been quantified in spray-dried eggs (Pizzoferrato eta!., 1993; Fontana eta!., 1992; Emanuel eta!., 1991), potatoes french-fried in tallow (Zhang eta!., 1991), light-abused butter (Luby eta!., 1 986a,b), butter oil and cheese (Nourooz-Zadeh and Appelqvist, 1988; Finocchiaro et a!., 1984)  and  temperature-abused tallow (Park and Addis, 1986a,b). Conversely, the analysis of fresh egg yolk or eggs and freshly prepared freeze-dried egg yolk powder did not indicate the presence of significant levels of COPs (Pizzoferrato eta!., 1993; Emanuel eta!., 1991; Nourooz-Zadeh and Appelqvist, 1987). However, cholesterol is known to oxidize much less readily than PUFA.  Oxidation of cholesterol  (cholest-5-en-3f-ol) can be catalyzed by exposure to a variety of reactive oxygen species, such as singlet oxygen (102), peroxides, and the hydroxy radical (0H; Smith and Johnson, 1989). Interestingly, the superoxide radical (O2) does not oxidize cholesterol (Smith eta!., 1977). Exposure of cholesterol to lipid autoxidation intermediates during oxidizing conditions, such as extensive heating of fats, may also be involved in the production of COPs (Park and Addis, 1986a,b). Ingestion of lipid oxidation products is a regular occurrence in the North American diet through the processing of foods. The commonplace heating of cooking oils and lipid-containing foods as well as the fact that food lipids, especially PUFA, are in close contact with trace metal catalyst complexes (e.g. hemoproteins, namely hemoglobin, myoglobin and cytochromes) can easily result in the formation of lipid peroxides consumed in the diet. Numerous lipid oxides such as secondary lipid oxidation and co-oxidation products have been identified in heated and stored foods, particularly muscle foods (Ladikos and Lougovois, 1990). lb. Factors influencing lipid oxidation. The rate of lipid autoxidation reactions can be influenced by a number of factors, including lipid composition, environmental factors, catalysts, inhibitors (i.e. transition metal chelators, antioxidants) and co-nutrients. The relative degree of unsaturation of a fatty acid can greatly influence its rate and susceptibility to oxidation, polyunsaturated fatty acids being many times more susceptible than fatty 4  acids with two or one double bonds (Farmer eta!., 1942). Thus, linolenic acid (C18:3,n-3) contains two doubly activated methylenic carbons (located between the three double bonds) at positions 11 and 14 as sites for initiation of autoxidation, compared to linoleic acid (C18:2,n-6)  which has one  doubly activated methylenic carbon at position 11 and oleic acid (C18:1,n-9) with its single double bond (Chan and Levett, 1977a,b; Farmer eta!., 1942).  Fatty acids with conjugated double bonds  (double bonds adjacent to each other; -C=C-C=C-) are more susceptible to autoxidation by oxygen addition at the double bond site than are their nonconjugated counterparts (double bonds separated by one carbon; -C=C-C-C=C-; Bolland and Gee, 1946b). Furthermore, the cis form of fatty acids, which is the predominant form in nature, have been reported to be more prone to autoxidation than the trans isomers, which have a more thermodynamically favoured conformation (Labuza, 1971). The fact that fatty acid esters, such as the triacyiglycerides, are more stable to autoxidation than free fatty acid molecules is important to the stability of edible oils (Nawar, 1990).  Other forms of fatty acid  esters, such as those contained in phospholipids, can also influence the rate of lipid autoxidation. Phospholipids contained within fats (Sherwin, 1978) and membrane systems, such as the milk fat globule membrane (MFGM; Chen and Nawar, 1991), can increase the rate of autoxidation due to their characteristic content of polyunsaturated fatty acids. Environmental conditions, such as those present during storage and processing, also have an effect on the reactivity and stability of lipids to oxidation. The availability of oxygen as a participant in the reaction under normal conditions is not limiting, but at low oxygen pressures, such as under vacuum, the rate increases in proportion to oxygen pressure (Nawar, 1990). Temperature can also play a role in determining oxygen availability. Generally, as the temperature of the lipid autoxidation reaction medium increases, so does the rate of the reaction (Farmer et a!., 1942). However, at high temperatures, the solubility of oxygen begins to decrease, such that any increase in rate of reaction due to oxygen pressure is offset by the reduction in oxygen solubility due to temperature (Nawar, 1985). The presence of moisture in lipid systems has a variable effect on lipid autoxidation, depending on the water activity (a)  of the system (Finley and Given, 1986).  Thus, in dehydrated foods  characterized by a very low a value, lipid autoxidation occurs at an accelerated rate.  However,  increasing the a to an intermediate value effectively reduces autoxidation by quenching free radicals 5  and inactivating any metallic catalysts which may be present.  Raising the a of a system further  increases the mobility of reactants, resulting in an increase in the rate of lipid oxidation (Finley and Given, 1986). Catalysts  of lipid autoxidation include transition metal  ions (e.g.  iron and copper),  photosensitizers (e.g. chlorophyll and heme) and radiant energy (e.g. heat, visible and U.V. light, and ionizing radiation). Transition metal elements have three or more electrons available for interaction, depending on their state of valency (Chang, 1984). Transition metals are present in edible oils as well as biological tissues in both a free ion or complexed form. Lipid autoxidation reactions are catalysed by these metals through interaction with the fatty acids, hydroperoxides or by activation of molecular oxygen to yield oxygen radicals (Sherwin, 1978; Farmer eta!., 1942). The catalysis of the formation of the highly reactive hydroxyl radical (0H) by iron in the Haber-Weiss cycle (3) plays an important role in the initiation of lipid autoxidation in model and biological systems (Graf et a!., 1984): 1. Fe +  2. 202  °2  + 2H  3. Fe + H 0 2  > Fe 3 + > H O 2 >  °2  °2  0H + OH- + Fe 3  Photosensitizing molecules such as hemoglobin or chlorophyll also contain transition metal ions within their larger complexes. These trace metal complexes frequently do not occupy all the coordination sites of the metal ion, thereby allowing the metal to act as a catalyst of lipid autoxidation (Mahoney and Graf, 1986; Graf eta?., 1984). These reactions are involved in the autoxidation of lipids contained in biological tissues (e.g. meat muscle; Ladikos and Lougovois, 1990; Buckley eta!., 1989) as well as oils (e.g. soybean oil; Jung eta!., 1991). Exposure of lipids to light energy can have significant surface effects by catalyzing oxidation of lipids, such as on the exposed surfaces of butter (Luby et a?., 1986a,b).  ic. Antioxidants. Control or retardation of the rate of lipid autoxidation can be achieved through the action of antioxidants. Lipid autoxidation antioxidants include both those naturally occurring in lipid systems such as the tocopherols, B-carotene, ascorbic acid and citric acid, as well as synthetic compounds which are added to foods to maintain the stability of the lipid component.  Synthetic antioxidants 6  include the phenolic compounds butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tertiary butyl hydroxyquinone (TBHQ), and propyl gallate.  Metal chelating agents such as  ethylenediamine tetraacetic acid (EDTA) and phytic acid can also act as antioxidants by binding and effectively removing pro-oxidant metals from the reaction medium (Empson eta!., 1991; Graf and Eaton, 1990).  Other types of natural, food-derived antioxidant molecules include the phenolic  compounds in the grains, oilseeds and plants, namely the flavonoids and phenolic acids (Kitts, 1 994; Thompson, 1994). The structure of flavonoids is characterized by two aromatic rings linked by a 3carbon aliphatic chain which may be condensed to form a further pyran or furan ring structure (Thompson, 1994). The polyphenolic character of these compounds as well as their ability to chelate metals make them ideal antioxidants. Thus, flavonoid compounds exhibit antioxidant activity by acting as free radical acceptors and as chain-breakers. The limited lipid solubility of plant phenols can be improved through alkylation or esterification to long chain fatty acids or alcohols (Namiki, 1990). The natural and synthetic phenolic antioxidants have in common an aromatic carbon ring structure with one or more substituent hydroxyl groups which act as H donors in breaking free radical chain reactions. The resonance stabilization associated with the unsaturated carbon ring structure of these compounds enables them to be effective antioxidants without acting as radicals themselves. Antioxidants act at the initiation and propagation stages of lipid autoxidation (Jung  et a!.,  1991; Burton, 1989). The induction of lipid autoxidation can be delayed by metal catalyst chelators (e.g. EDTA, citric and phosphoric acids), as well as singlet oxygen quenchers (e.g. a-tocopherol, 1.carotene and ascorbic acid; Burton, 1989; KahI and Hildebrandt, 1986). Free radical chain inhibitors, such as H donors (e.g. a-tocopherol, BHA, BHT, TBHQ and propyl gallate) or free radical scavengers (e.g. a-tocopherol, 1-carotene, BHA, BHT and TBHQ) interrupt the self-catalytic chain reaction during the propagation stage of lipid autoxidation. The free radical chain inhibitors are able to successfully compete with the autoxidizable lipid substrates for peroxy radicals, for example, due to the ready abstractability of the phenolic hydrogen atom: Phenolic-OH + R00  > Phenoxy-O’ + ROOH.  The phenoxy radical can either go on to be consumed in a subsequent reaction with another R00, or be regenerated to its active form by a reducing agent (e.g. ascorbic acid). The regeneration of the 7  phenolic antioxidants by reducing agents illustrates the “sparing effect” that one antioxidant can have on another, leading to a synergism, as seen in the example of a-tocopherol and ascorbic acid (KahI and Hildebrandt, 1986).  An additional example of synergism displayed by the various classes of  antioxidants occurs with the phenolic compounds and metal chelating agents.  Chelators act to  decrease the concentration of available transition metal ions, effectively removing the catalysts of free radical production (initiation) and hydroperoxide decomposition (propagation; KahI and Hildebrandt, 1986). However, in order to be effective antioxidants, chelates formed must occupy all coordination sites of a trace metal. Any coordination sites which are left open or that are hydrated can be involved in oxygen radical production (Graf eta?., 1984). Thus, phytic acid and large chelating organic acids such as diethylenetriamine pentaacetic acid have been reported to successfully prevent Fe 3 from acting as a pro-oxidant (Empson et a?., 1991; Graf et a?., 1984). In the case of vegetable oils which are already rich in naturally occurring tocopherols, the addition of synthetic antioxidants will provide little added antioxidant protection (KahI and Hildebrandt, 1986). id. Antioxidants in foods. Foods generally contain an endogenous complement of both pro-oxidant as well as antioxidant molecules. Some of these compounds (e.g. a-tocopherol and ascorbic acid; Mahoney and Graf, 1986) can exhibit anti- or pro-oxidant activity depending on the reaction conditions (reactant concentration, time, temperature, pH and a) as well as the presence of phospholipids and amino acids. In studies using model systems, Mahoney and Graf (1986)  demonstrated that ascorbic acid exhibited both  antioxidant and pro-oxidant activity for Cu -mediated 2  0H production.  Ascorbate enhanced  0H  formation when present at low concentrations, by reducing the cuprous ion, similar to the 2 /Fe 3 Fe pathway above.  At higher concentrations, ascorbate acted as an antioxidant by scavenging free  radicals. Generally, a-tocopherol acts as a very efficient radical scavenger, especially in lipid systems, such as a linoleic acid model system (Mahoney and Graf, 1986). However, high concentrations of a tocopherol can reduce transition metal ions, thereby enhancing the Haber-Weiss cycle (see above, page 6) and acting as a pro-oxidant (Mahoney and Graf, 1986). Despite the natural presence of antioxidants, it is frequently necessary to stabilize foods with added or supplemental antioxidants in order to extend the shelf-life or storage-life of commodities. For 8  fresh muscle tissue, membrane lipids are generally rich in phospholipid and consequently have a high PUFA content, which is highly susceptible to oxidative attack (Ladikos and Lougovois, 1990; Buckley  et a!., 1989). Different sources of muscle foods can also vary in their sensitivity for development of lipid autoxidation depending upon their relative content of PUFA. Thus, beef is far less susceptible to lipid autoxidation than is chicken meat, which in turn is less sensitive than turkey meat (Ladikos and Lougovois, 1990).  Living tissues are usually well protected against free radical damage by in vivo  enzyme systems and tissue a-tocopherol. However, once meat tissue is damaged, such as by cutting and  slicing, the  protection  against oxidative stress  provided  by intact  muscle fibres and  compartmentalization of in vivo systems is lost. Differences in stability even exist between intact cuts of meat versus ground tissue which makes lean tissue lipids accessible to contact with heme catalysts of oxidative rancidity.  For example, incorporation of a-tocopherol into cellular and subcellular  membranes should influence the stability of meat during storage and processing.  Post-mortem  application of vitamin E, on the other hand, has had only variable success for the inhibition of lipid autoxidation in meat tissue (Buckley eta!., 1989). Supplemental dietary a-tocopherol in the feed of animals (swine) for a 10 week period significantly stabilized mitochondrial and microsomal lipids of pork (Buckley et a!., 1989). Oil seeds and grains are also good sources of antioxidants, including the lipid soluble constituents (e.g. tocopherols, tocotrienols and B-carotene), trace minerals as cofactors of antioxidant enzymes (e.g. Se, Cu, Zn, Mn), phenolic compounds (e.g. phenolic acids, lignans, isoflavonoids, phytoestrogens)  and finally, phytic acid as a metal sequestrant (Thompson, 1994).  The lipid-rich  fractions of grains contain a variety of compounds with vitamin E activity notably the a, B, y and ó tocopherols and tocotrienols.  The mineral content of seeds and grains depends somewhat on the  mineral content of the soil they are grown on. Phenolic acids are especially concentrated in the bran fraction of whole grain; examples include ferulic, vanillic, caffeic and chlorogenic acids (Thompson, 1994). While commonly considered an anti-nutrient, phytic acid also has antioxidant activity due to its potential for binding transition metals. The bran of whole wheat is particularly rich in phytates. Protection against autoxidation of lipids during oil seed processing is necessary because oxidative damage can result in decreased yields, rancid off-flavours, discolouration, reduced vitamin and nutrient 9  content, polymerization, as well as reduced stability of the refined product (Nawar, 1990). Thus, the “carry-through” of naturally occurring tocopherols during the processing of vegetable oils is important to the oxidative stability of the oil during purification, as well as in the finished product. In fact, the concentration of tocopherols in finished oils (e.g. soybean oil; 1,100 ppm tocopherol)  is often  sufficient to confer considerable shelf-stability (Jung eta!., 1991). However, with processing such as extensive heating (>70 hours, 135°C), the activity of antioxidants such as a-tocopherol or ascorbyl palmitate is lost. In studies with heated tallow, Park and Addis (1986b) suggested that antioxidants in heated oils are lost either due to their consumption by excess free radical production, or to an inability to retain functionality at elevated temperatures over a long period of time. This lability of antioxidants in frying oils no doubt contributes to the presence of lipid oxides in heated frying fats and oils, and consequently in their fried products. Commercially prepared french-fried potatoes from fast food restaurants have been reported to contain substantial amounts of lipid oxidation products, including cholesterol oxides (Zhang eta!., 1991). Dairy products are unique in their susceptibility to deterioration associated with oxidative lipid rancidity due to their mild, delicate flavour, as in fluid milk and butter, as well as the composition of the lipid system.  Milk fat is composed of 97-98% triacylglycerides with small amounts of sterols  (0.22-0.41 %) and phospholipid (0.2-1.0%). Further, milk fat is contained within globules which are surrounded by membrane (milk fat globule membrane, MFGM; Jensen and Clark, 1988). Milk fat is unusual in its composition, in that short and medium-chain fatty acids (C4:0 to C14:0)  make up  approximately 23% of triacylglyceride fatty acid residues and saturated fatty acids make up 61 .6%, monounsaturates (C16:1, C18:1), 27.3%, and polyunsaturates (C18:2 mainly), 2.1% of total fatty acid residues (Jensen and Clark, 1988). Adding to the complexity of milk fat are significant amounts of odd-numbered carbon and branched-chain fatty acids.  Oxidation of milk fat can be initiated by  MFGM components, whose composition reflects its plasma membrane origins, containing neutral lipid, phospholipid and proteins (Keenan eta!., 1988). Thus, oxidative attack of fat globule triacylglycerides may be preceded by the oxidation of MFGM phospholipid (Sherwin, 1978). The MFGM contains both antioxidant and pro-oxidant factors, with some components having a dual character depending on environmental conditions. The protein component of the MFGM includes various metalloproteins, some 10  of which have oxidoreductase activity, such as xanthine oxidase, catalase and cytochrome C red uctase (Keenan eta!., 1988). These enzymes contribute to the trace metal content (mainly iron and copper) of the MFGM involved in the catalytic initiation of lipid peroxidation in milk and dairy products. Activity of xanthine oxidase may be partially responsible for oxidative attack of milk lipids due to its catalytic production of hydrogen peroxide 2 (H ) 0 , and superoxide anion (02; Jensen and Clark, 1988).  In  contrast, aqueous phase proteins adsorbed onto the surface of the MFGM, such as superoxide dismutases, behave as antioxidants to inhibit milk fat oxidation by dismutating superoxide anions to produce 02 and H 0 (Jensen and Clark, 1988). 2  Acceleration of milk fat lipid peroxidation by the  MFGM was demonstrated by Chen and Nawar (1991) using cream incubated at 50°C. Conversely, at 95°C oxidation was not different between samples with, or without MFGM, which was attributed to antioxidative effects of membrane protein -SH groups, or the presence of Maillard reaction products which overcame the pro-oxidative activity of MFGM components. Other dairy products can be seen to be sensitive to the photochemical catalysis of lipid autoxidation, as demonstrated by the presence of carbonyl oxidation products contributing to ‘cardboardy” off-flavours in fluid milk (Labuza, 1971). Photo-oxidation resulted in the production of cholesterol oxides at the surface of butter exposed to different light sources (Luby et a!., 1 986a,b). Also, oxidation of cholesterol in cheese lipid has been attributed to the catalytic effects of milk trace metals present as protein-metal complexes (Finocchiaro  eta!., 1984). In contrast, the stability of butter oil (milk fat without MFGM) to oxidative damage, while not fully defined as yet, is not solely attributed to the relative saturation of the triacylglyceride fatty acids, but may involve a recently identified conjugated linoleic acid isomer, . 11 This ’ 9 C18:24 conjugated linoleic acid has been reported to possess potent antioxidative activity, similar to BHT (Jensen eta!., 1991; Ha eta!., 1990). While this isomer has been isolated from bovine milk, further studies are required to evaluate its efficacy in in vitro systems as well as in dairy products. 1 e. Lipid autoxidation interactions with other nutrients or cell constituents. Co-nutrients in food systems as well as biological macromolecules are not immune to the effects of lipid autoxidation. Much of the research investigating the reaction rates and products of lipid autoxidation has been performed using model lipid systems, or purified oils high in unsaturated fatty acid content.  This work allowed the elucidation of the mechanisms involved in lipid peroxidation; 11  however, in the majority of foods and indeed biological systems, lipid exists as either fat depots (e.g. animal adipose tissue, oil glands of plants) or constituent fats (free lipids in cell protoplasm; cell wall components; membrane-bound lipids such as phospholipids). Thus, a variety of co-nutrients exist within the same environment which can influence both the rate as well as the products of lipid free radical interactions with oxidizable substrates. Examples of co-nutrients include proteins and amino acids, carbohydrates, salts as well as lipid- and water-soluble vitamins.  The basic side-chains of  protein amino acid residues may be lost in aldol condensation reactions with carbonyl lipid autoxidation products (Nielsen et a!, 1985a,b).  For example, casein has been described as having antioxidant  activity due to the fact that sulphur-containing amino acids and proteins are oxidizable by lipid hydroperoxides (McGookin and Augustin, 1991). Polymerization of proteins mediated by lipid peroxide free radicals has also been described by Roubel and Tappel (1966). Taken together, these reactions of lipid autoxidation free radicals and hydroperoxide breakdown products with the protein and amino acid components of food can result in reduced protein functionality and decreased availability of essential amino acids. The carbohydrate component of foods can have a variety of roles, ranging from catalysis of hydroperoxide decomposition (Labuza, 1971) to exhibiting antioxidant effects through the formation of Maillard reaction products (MRP) with strong reducing properties (McGookin and Augustin, 1991). In studies using amino acids, protein hydrolysates or dipeptides reacted with various monosaccharides, Lingnert and Eriksson (1 980a,b) reported greatest antioxidant effect with MRP from basic amino acids or peptides reacted with reducing sugars. The presence of salts such as NaCl in food systems can result in the acceleration of the oxidation of triacylglycerides, depending on the level of free moisture present in the system (Love and Pearson, 1971). The fat soluble vitamins A, 0, E (tocopherols) and K as well as provitamin A (F-carotene) act as antioxidants by being oxidized by lipid hydroperoxides and their breakdown products (Burton, 1989; Park and Addis, 1 986b). In addition, the water soluble vitamins C (ascorbic acid and its lipid soluble form, ascorbyl palmitate) and folate are also susceptible to oxidative attack (Cort, 1974). These interactions in foods are virtually unavoidable, since they will occur to some extent at all levels of processing and storage of foods, from harvesting to consumption. Reactions such as these are responsible not only for the development of desirable flavours, odours and 12  even colours of cooked and cured meats, french-fried potatoes, heated milk, aged cheeses, and the undesirable volatiles associated with oxidative rancidity, but also for alterations to the nutritional value and functionality of food constituents. Oxidative rancidity of lipid-containing foods is characterized by very low detection thresholds, in the parts per billion (ppb) range. Therefore, only a very small fraction of the lipid component of foods needs to be oxidized in order to produce off-odours and flavours (0.00002%; Labuza, 1971). The organoleptic threshold for lipid autoxidation volatiles and the relative extent of these lipid oxidation interactions suggests that the palatability and functionality of lipid-containing foods will render them unusable before significant decreases in nutritional value are reached (Finley and Given, 1 986; Nielsen  eta?., 1985a). 2a. Digestion and absorption of dietary lipids. Digestion and absorption of dietary fat (e.g. triacylglycerides, phospholipids, cholesterol esters and lipid-soluble vitamins) occurs primarily within the lumen of the small intestine (Guyton, 1977). The majority of dietary lipid is freed from other dietary constituents (i.e. protein and carbohydrate) through the action of salivary amylases and gastrointestinal proteases. Minor amounts of fat are digested by lingual and gastric lipases; however, 95-99% of fat digestion occurs in the small intestine (Fernando Warnakulasuriya eta?., 1981). Ingested dietary fat released into the upper small intestine (duodenum) is mixed with the intestinal chyme (i.e. pancreatic lipases) and bile salts within the intestinal lumen. An emulsion is formed with lipid droplets emulsified by the amphipathic bile acid molecules within the aqueous intestinal contents. Gastrointestinal peristaltic movement enhances the breakdown of the fat droplets, effectively increasing the surface area available for enzymatic hydrolysis. The pancreatic lipases are responsible for the hydrolysis of the triacylglyceride molecules at positions one and three of the molecule, yielding diacylglycerides (1 ,2-diacylglycerides)  as well as monoacylglycerides (2-  monoacylglycerides) and free fatty acid molecules. The hydrolysis of triacylglycerides is subject to feedback inhibition from the accumulation of digestion products. Thus, bile salts play an important role in the removal of these products via the formation of bile salt micelles. These micelles consist of a nonpolar sterol core surrounded by a highly negatively charged polar surface. Monoacyiglycerides and free fatty acids are miscible within the nonpolar core of the micelles, thus facilitating their removal 13  from the site of enzymatic hydrolysis.  The rate of hydrolysis by pancreatic lipases is somewhat  dependent on chain length and degree of saturation. Unsaturated fatty acids are hydrolysed at a faster rate than are saturated fatty acids (SFA).  The bile salt micelles containing hydrolysis products of  triacylglycerides migrate to the intestinal mucosa for absorption of the monoacylglycerides, free fatty acids and glycerol across the epithelial cell brush border membrane. The bile salts are not absorbed through the mucosa, but instead act as a type of “ferrying” system and are recycled back into the intestinal contents to form new micelles. The bile salts undergo enterohepatic circulation by being recirculated to the liver for release back into the intestinal contents. Also, a certain amount of bile acid salt is lost in the excreta. The monoacylglycerides and free fatty acids (FFA) from the bile salt micelles become dissolved in the epithelial cell membrane and immediately diffuse across into the cytosol. Epithelial cell lipase further hydrolyses the monoacylglycerides into constituent molecules. When the longer chain fatty acids are absorbed into the mucosal cells, they are re-esterified once more into triacylglyceride molecules. These esterification reactions occur within the endoplasmic reticulum of the intestinal cells. The glycerol used in these reactions is synthesized de novo from a-glycerophosphate (Guyton, 1977). Only a small amount of the original glycerol is used in the synthesis of new triacylglycerides. The newly synthesized triacylglycerides (TGs)  coalesce into globules along with absorbed cholesterol,  phospholipid and newly synthesized phospholipid. These globules, surrounded by a membrane from the endoplasmic reticulum, are then extruded into the intercellular spaces as chylomicrons. Shorter chain fatty acids, however, are transported to the liver from the small intestine via the portal vein. Fatty acids with chain lengths of C4:O to C1O:O are targeted for B-oxidation by hepatic cell mitochondria for energy. The chylomicrons travel along with the lymph via the lymphatic pump upward to the thoracic duct to be emptied finally into the main circulation.  Thus, the chylomicrons are emptied into the  venous blood supply at the juncture of the jugular and subclavian veins. Chylomicrons have a relatively short residence time in the plasma, being removed in about one hour, primarily through the capillaries of the adipose tissue. Adipose cells contain high amounts of lipoprotein lipase activity to hydrolyse triacylglycerides to their constituent free fatty acids and glycerol. The FFA diffuse into the adipose 14  cells where they are re-synthesized into triacylglycerides for storage, until needed elsewhere. When the TG lipids are required, they are once more transported as FFA via the action of cellular lipases. When transported, the FFA are in an ionized state bound to plasma albumin. In the post-absorption state, or under fasting conditions, chylomicrons are absent from the blood with the majority of plasma lipids existing as lipoproteins.  Lipoproteins are composed of  approximately 25-33% protein with the remainder as lipid species. The lipoprotein classes consist of the TG-rich very low density lipoproteins (VLDL; containing high concentrations of TG with moderate amounts of PL and cholesterol), low density lipoproteins (LDL; consisting of little TG with a high amount of cholesterol) and the high density lipoproteins (HDL; containing approximately 50% protein with smaller concentrations of TG and cholesterol). The lipoproteins are synthesized mostly by the liver, with some species also originating from the intestine. They function in the transport of lipid species among the tissues, primarily from the liver to other tissues.  The protein content of the  lipoprotein particles consists of specific classes of apolipoproteins. These apolipoproteins in large part dictate the metabolic fate of these particles.  When they are initially secreted by the intestine,  chylomicrons contain apolipoprotein A-I, A-Il, C, and B-48 (Weisgraber and Mahley, 1983). The VLDL synthesized by the liver contain apolipoprotein B-i 00, C and E. Apolipoprotein B-48 is synthesized by the intestine and is required for chylomicron secretion. On the other hand, apolipoprotein B-100 is synthesized only by the liver and is necessary for VLDL secretion. The primary role of apolipoprotein B-100 is in the recognition of LDL particles by the LDL receptor protein of hepatocytes. Thus, LDL particles contain only a single apolipoprotein, B-100. HDL lipoprotein particles contain apolipoproteins A-I, A-Il, A-lV, C and E (depending upon the species of donor). For example, rat HDL contain all five of the above apolipoproteins, whereas human HDL lack apolipoproteins E and C (Weisgraber and Mahley, 1983). Apolipoprotein A-I is the main activator of lecithin:cholesterol acyltransferase (LCAT), the enzyme responsible for esterification of cholesterol within the plasma and thus, the HDL particles (Frohlich and McLeod, 1987). The synthesis of apolipoprotein A-I occurs in the liver and the intestine. The role of apolipoprotein A-Il has not been elucidated as yet; however, this protein is synthesized by both the intestine and the liver. Apolipoprotein C consists of a complex of three polypeptides, C-I, C-Il and C-Ill, each with their own activity. Apolipoprotein C-I has activity as another activator of LCAT. 15  Apolipoprotein C-Il is an activator of lipoprotein lipase (LPL), whereas C-Ill acts as an inhibitor of this enzyme activity (Norum, 1992; Weisgraber and Mahley, 1983).  Finally, apolipoprotein E acts as  another ligand for the LDL receptor protein in hepatocytes. When the chylomicron has been stripped of its triacyiglyceride content through the activity of LPL, the remaining material (consisting of surface unesterified cholesterol, phospholipids and apolipoproteins)  is removed from the circulation by the liver (Norum, 1992).  This removal of  chylomicron remnants from the plasma is facilitated by receptors (i.e. apolipoprotein E receptor or LDL receptor) on the surface of hepatocytes. The formation of the TG-rich VLDL by the liver is dependent on the synthesis or availability of TG in hepatocytes.  Thus, VLDL production and secretion is a  function of the balance between the influx of FFA to the liver and their oxidation for energy. The VLDL lipoprotein particles are once more subject to the hydrolysis of their triacylglycerides by LPL of adipose and muscle tissue to yield remnant particles known as intermediate density lipoproteins, or IDL. Lipoprotein remnants are either taken up by LDL receptors of the liver or directly synthesized into LDL particles from the VLDL remnants in the plasma. This conversion of IDL particles into the cholesteryl ester-rich LDL lipoproteins is facilitated by the transfer of cholesteryl esters to the precursor VLDL from circulating HDL particles. Some LDL cholesteryl ester may originate from direct action of LCAT without cholesteryl ester transfer (Frohlich and McLeod, 1987). This process allows the LDL particles to deliver their cholesterol load to hepatocytes as required (i.e. for the biosynthesis of bile acids). Low density lipoproteins are taken up by hepatocytes via the LDL receptor pathway which recognizes the apolipoprotein B on the surface of the LDL. This receptor population for uptake of LDL is subject to down-regulation by a saturated fat diet (Woollett eta!., 1992; Fernandez and McNamara, 1991). A diet high in saturated fatty acids can result in reduced numbers of LDL receptors, resulting in hypercholesterolemia.  Newly formed HDL particles can vary in their composition and morphology,  depending on their origin from either of two pathways. Nascent HDL lipoproteins can either be directly secreted from the liver or intestine as small sized spheres, or form within the plasma as discoidal particles developed from VLDL remnants (Norum, 1992; Weisgraber and Mahley, 1983). The newly secreted HDL particles contain primarily apolipoproteins A-I and A-Il, phospholipids and unesterified cholesterol. Once in the plasma, HDL become more spherical in shape due to the catalytic activity of 16  LCAT to esterify cholesterol, resulting in the transfer of the apolar cholesteryl esters into the HDL core. Transfer of the apolar cholesteryl esters from HDL to the lower density lipoprotein fractions occurs by a specific protein-mediated pathway involving the cholesteryl ester transfer protein (CETP; Norum, 1992; Frohlich and McLeod, 1987). This process involves the transfer of cholesteryl esters to VLDL in order to reconstitute their cores which have been depleted through the action of LPL. The transfer of cholesteryl esters to this lipoprotein fraction is reciprocated by an equimolar transfer of TG to the HDL lipoproteins (Frohlich and McLeod, 1987). 2b. Dietary lipids and dyslipidemias. The development of hypercholesterolemia cannot be solely linked to the consumption of diets high in animal fat, which have been traditionally labelled as saturated fat diets. A considerable body of evidence exists to indicate that not all saturated fatty acids (SFA) have equivalent cholesterolemic effects (Denke and Grundy, 1992; Hayes eta!., 1991; Bonanome and Grundy, 1988; Hegsted et a!., 1965; Keys at al., 1957).  Certain saturated fatty acids as well as monounsaturated (MUFA) fatty  acids do not contribute to plasma cholesterol equally. Saturated fatty acids of chain length from C4:O to C10:0 do not have any plasma cholesterol raising effect, as shown in humans fed formula diets containing either butter or a short chain triacylglyceride preparation isolated from coconut oil (Hashim et a!., 1960).  Similarly, when Toda and Oku (1995)  fed a diet, containing shorter chain  triacylglycerides derived from coconut oil (C8:0, 26%; C 10:0, 20%) with 2% cholesterol, to Japanese quail, plasma total cholesterol and development of aortic lesions were reduced compared to birds fed corn oil and palmitic acid. This may explain the intermediate effect of diets containing 14% butter fat on plasma total cholesterol and TG in rats compared to counterparts fed palm oil, beef tallow, coconut oil or corn oil (Ney eta!., 1991). On the other hand, longer chain saturated fatty acids, namely lauric (C12:O) and myristic (C14:0) acids which are present in relatively high proportions in palm kernel oil (C12:O, 52% and C14:0, 15%)  and coconut oil (48% and 17%, respectively), both can produce  elevated plasma cholesterol levels in humans and animal models fed diets containing sources of these fatty acids (Denke and Grundy, 1992; Hayes eta!., 1991). Palmitic acid (C16:0) has intermediate effects on plasma cholesterol concentrations, being hypercholesterolemic in comparison to diets high in stearic or oleic acids in studies with humans fed liquid formula diets (Bonanome and Grundy, 1988). 17  However, in studies with baboons fed formula diets containing palmitic acid, plasma total and LDL cholesterol were lower than in counterparts fed diets containing lauric and myristic acids (Hayes eta!., 1991).  In this same experiment, the palmitic acid-fed animals were slightly hypercholesterolemic  compared to those fed linolenic acid (Cl 8:2,n-3). Stearic acid (Cl 8:0), a principal saturated fatty acid in animal fats (i.e. beef tallow), was found to reduce human serum cholesterol levels when substituted for palmitic acid (C16:0)  in formulated liquid diets (Bonanome and Grundy, 1988).  The neutral  character of stearic acid for plasma lipoprotein composition was further demonstrated in studies with pigs fed 20% or 40% of dietary energy as beef tallow, soybean oil or a 50:50 blend of these oils (Luhman eta!., 1992; Faidley eta!., 1990). dietary treatment  In these studies, there were no differences between  groups for either fasting or postprandial concentrations of cholesterol or  triacylglyceride in total plasma or in the major lipoprotein fractions. Beef tallow not only contains a substantial amount of the longer chain saturated fatty acids, palmitic acid (Ca. 35%) and stearic acid (ca. 16%), but also the monounsaturated oleic acid (Ca. 44%). The lack of effect of beef tallow diets on cholesterolemia can be attributed to the relative activities of two of the enzyme-mediated mechanisms of longer chain saturated fatty acid metabolism. The first pathway involves the elongation of palmitic acid to stearic acid via fatty acid elongase activity and in the second, the desaturation of -desaturase enzyme. Thus, the conversion of dietary fatty acids in 9 stearic acid to oleic acid by the A vivo to less cholesterolemic species may play a significant role in the overall effect of dietary fat composition on tissue fatty acid composition and plasma cholesterol concentrations. For example, the high ratio of palmitic acid to stearic acid in the plasma TG of humans may be due to the rate-limiting nature of the elongation step in the elongation and desaturation conversion of C16:0 to C18:1 in fatty acid metabolism. While plasma TG levels of stearic acid did not become elevated in studies with a high stearic acid diet, levels of oleic acid did increase in plasma TG as well as cholesteryl esters in human studies (Bonanome and Grundy, 1988). Thus, the efficiency of the metabolic conversion of individual longer chain SFA may influence their effect on plasma lipids. Differences in plasma LDL and HDL concentrations due to saturated fatty acid diets may include effects on LDL receptor populations (Fernandez and McNamara, 1991; Fernandez eta!., 1992a,b) or apolipoprotein A-I secretion (Stucchi eta!., 1 991), respectively. In studies with guinea pigs, Fernandez 18  and McNamara (1991) indicated that animals fed lard-based diets had higher plasma LDL levels than their counterparts fed PUFA corn oil. Moreover, the LDL from lard-fed animals had both lower flotation densities as well as an increased core-to-surface component ratio, the latter parameter being a reflection of greater amounts of cholesteryl ester and TG in the LDL core compared to surface protein and phospholipid components. These alterations in LDL composition coincided with a reduction in apolipoprotein B receptor-mediated LDL binding activity of hepatic membranes from lard-fed animals. The hypercholesterolemic response to the lard diet was mediated by both dietary fat-induced changes to LDL cholesterol concentrations and biochemical composition, as well as changes to the apolipoprotein BIE receptor population resulting in decreased catabolism of LDL cholesterol in these SFA-fed animals (Fernandez and McNamara, 1991). Similarly, when Woollett and coworkers (1992) fed saturated triacylglyceride diets (hydrogenated coconut oil) to hamsters, LDL receptor activity was reduced compared to animals fed either a control chow diet or PUFA safflower oil. Animals fed the SEA diet exhibited both increased LDL cholesterol production rates and, consequently, increased plasma LDL cholesterol levels (Woollett eta!., 1992). Further work investigating the effect of dietary SFA on LDL composition and catabolism has focused on the turnover kinetics of this lipoprotein class (Fernandez et a!., 1992a,b).  Low density lipoproteins from animals fed SFA lard diets have an  increased LDL apolipoprotein B pool size associated with a reduced LDL fractional catabolic rate and an increased flux rate. Catabolism of LDL was related to the apolipoprotein BIE receptor population as well as radiolabelled plasma LDL disappearance rates (Fernandez eta!., 1992a). Studies with diets containing different sources of SEA confirmed that these LDL metabolic parameters were influenced by SFA of differing chain lengths (Fernandez eta!., 1992b). Palm kernel oil (C12:O, 52%; C14:O, 18%)  diets resulted in the greatest LDL cholesterol levels, reduced apolipoprotein B/E receptor  population and higher LDL apolipoprotein B flux rates in comparison to palm oil (Cl 6:0, 43%; Cl 8:0, 4%) and beef tallow (C16:0, 23%; C18:0, 14%; Fernandez eta!., 1992b). In studies to determine the effect of SFA diets on HDL metabolism in cebus monkeys fed coconut oil diets, Stucchi and coworkers (1991) reported that plasma total, VLDL, LDL and HDL cholesterol levels were increased compared to PUFA-fed counterparts.  Alteration in the composition of HDL particles from SFA-fed  animals included not only greater amounts of cholesteryl ester and phospholipid, but also decreased 19  levels of TG and protein. These compositional changes in lipoproteins resulted in an increased core lipid to surface component ratio for HDL from SFA-fed animals. Increased HDL cholesterol was also accompanied by an increase in the apolipoprotein A-I plasma pool due to increased production of apolipoprotein A-I, as well as a decrease in the apolipoprotein A-I fractional catabolic rate. Elevations in hepatic tissue levels of apolipoprotein A-I mRNA were also observed in SEA-fed animals (Stucchi et a!., 1991). Thus, dietary SFA can be seen to increase plasma cholesterol concentrations by influencing the metabolism of more than a single lipoprotein class. Monounsaturated oleic acid (C18:1) is as effective as PUFA at lowering plasma cholesterol when substituted for saturates in a liquid formula diet (Mattson and Grundy, 1985). This change in plasma lipid profile was observed to occur without the undesirable concomitant lowering of HDL cholesterol often seen with PUFA diets (Mott eta!., 1992; Vega eta!., 1982).  Other studies with  whole food diets fed to humans reported a hypocholesterolemic response with olive oil, but the plasma cholesterol lowering activity of the MUFA olive oil diet was less than that associated with a PUFA corn oil diet (Kris-Etherton et a!., 1993). Plasma cholesterolemic responses to diets high in oleic acid can vary, however, between human studies and those carried out with animal models.  Bonanome and  Grundy (1988) reported that when humans were fed liquid formula diets high in oleic acid (derived from high-oleic safflower oil; C18:1, 80%)  plasma total and LDL cholesterol levels were reduced,  compared to those on diets rich in SFA. Similar results were obtained by Denke and Grundy (1992) in a study comparing liquid formula diets containing either oleic acid or saturated fatty acid sources such as lauric and palmitic acids. However, in studies with guinea pigs fed olive oil at either 7.5% or 1 5% of diet (wt/wt), plasma total and LDL cholesterol concentrations were increased compared to corn oil-fed animals (Fernandez and McNamara, 1994; Fernandez eta!., 1992a; Fernandez and McNamara, 1991). This response to the MUFA olive oil diet was associated with a reduction in the number of hepatic apolipoprotein B receptors and a reduced LDL catabolic rate. These workers suggested that this response to the MUFA olive oil diets by guinea pigs and other animal models (e.g. rats and rabbits) may be unique to olive oil, or the high levels of oleic acid consumed (Fernandez and McNamara, 1994). The plasma cholesterol-lowering ability of the longer chain PUFA has been well documented in numerous studies in both humans (Kris-Etherton eta!., 1993; Mattson and Grundy, 1985; Vega et 20  at., 1982)  and animal models (Fernandez and McNamara, 1994; Woollett et a!., 1992; Ney et al.,  1991; Fernandez and McNamara, 1991; Garg eta!., 1989). Early studies using corn oil as the dietary source of PUFA reported that plasma concentrations of total cholesterol were reduced in humans fed liquid formula diets (Hashim eta!., 1960). The hypocholesterolemic effect of diets high in PUFA was demonstrated by Vega and coworkers (1982) in humans fed liquid formula diets with 40% of calories provided by either safflower oil or lard.  In subjects fed the PUFA diet, plasma total, LDL and HDL  cholesterol levels were decreased in comparison to SEA-fed patients (Vega eta!., 1982). Further, the ratios of VLDL, LDL and HDL lipoprotein cholesterol to apolipoproteins B (VLDL and LDL) and A-I (HDL) were not altered by dietary treatment. Thus, the hypocholesterolemic effect of PUFA was observed in all lipoprotein classes and was associated with a concomitant decrease in apolipoprotein levels. Similar results were obtained when baboons were fed diets with PUFA/SFA (P/S) ratios varying from 0.37 to 2.1 (Mott et at., 1992).  The plasma total, LDL and HDL cholesterol concentrations were  reduced by the PUFA diet in non-human primates fed 40% of energy as fat (Mott eta!., 1992). These changes in lipoprotein cholesterol concentrations were associated with decreases in the levels of apolipoproteins B, A-I and E in PUEA-fed groups. Mattson and Grundy (1985) reported that plasma total cholesterol, LDL and HDL cholesterol concentrations were reduced in patients fed high-linoleic acid safflower oil in the diet. The percentage contributions of cholesterol and protein to lipoprotein composition were not influenced by dietary treatment. Thus, all plasma lipoprotein constituents could be seen to be reduced by the PUFA dietary fat in this study (Mattson and Grundy, 1 985). When Ney and coworkers (1991) fed diets containing 16% (wt/wt) corn oil to rats, plasma total, LDL and HDL cholesterol concentrations were reduced compared to counterparts fed SFA diets.  However, the  plasma concentration of apolipoprotein A-I was greater in corn oil-fed animals. The reduced ratio of HDL triacylglycerides plus cholesterol to protein in PUFA-fed animals compared to SEA-fed counterparts indicate the presence of less lipid-rich HDL particles in the PUFA-fed group (Ney eta!., 1991). All of the above work was performed using semi-synthetic formulated diets; however, studies with complex whole food diets high in linoleic acid from soybean oil also significantly decreased plasma total and LDL cholesterol concentrations (Kris-Etherton eta!., 1993). This study also reported that apolipoprotein B levels were reduced by the soybean oil diet. However, neither HDL cholesterol nor 21  apolipoprotein A-I levels were affected by the dietary treatment in this study. Similarly, other workers have not reported a decrease in HDL cholesterol when subjects were fed PUFA diets (McDonald et a?., 1989). When humans were fed 36% of energy as canola oil (high in oleic acid) or sunflower oil (high in linoleic acid), plasma total, VLDL and LDL cholesterol levels were reduced by both of these PUFA dietary lipid sources (McDonald eta?., 1989).  On the other hand, HDL cholesterol levels were not  affected by the PUFA dietary treatments in this study. While studies investigating the hypocholesterolemic response to vegetable and oil seed lipids have primarily focused on the C18 series of PUFA (i.e. C18:2,n-6, linoleic acid, and C18:3,n-3, linolenic acid), other longer chain PUFA, namely the marine lipids, are also potent plasma lipid-lowering agents (Levy and Herzberg, 1995; Ikeda et a?., 1994; Garg et a?., 1988).  Consumption of large  amounts of marine oils in the diet has been linked to decreased risk of cardiovascular disease associated with reduced levels of plasma cholesterol, triacylglycerides and decreased platelet aggregating activity (Drevon, 1992; Garg eta?., 1988). marine  oils  are  the  longer  chain  n-3  PUFA,  The primary fatty acids of interest in the  eicosapentaenoic  acid  (EPA), C20:5,n-3  and  docosahexaenoic acid (DHA), C22:6,n-3. Through metabolism, the essential fatty acids (EFA) linoleic, Cl 8:2,n-6 and linolenic, Cl 8:3,n-3 acid are successively desaturated and elongated to yield the longer chain n-6 and n-3 EFA, respectively (Drevon, 1992). These transformations yield products specific to the starting precursor since mammalian cells are incapable of transforming n-3 and n-6 fatty acids into one another. Thus, Cl 8:2,n-6 is biotransformed into y-linolenic acid, Cl 8:3,n-6 via the t -desaturase 6 enzyme and then elongated to dihomo-y-linolenic acid, C20:3,n-6 which is desaturated by t desaturase to yield arachidonic acid (AA), C20:4,n-6 which is elongated to C22:4,n-6 before being -desaturase to yield C22:5,n-6. On the other hand, C18:3,n-3 is desaturated by acted on by 4 desaturase to C18:4,n-3, elongated to C20:4,n-3, desaturated to EPA, C20:5,n-3 via A -desaturase 5 and finally elongated to C22:5,n-3 before desaturation to DHA, C22:6,n-3 by -4 desaturase (Drevon, 1992).  From these pathways, it can be seen that the ratio of n-6 to n-3 fatty acids in the diet can  influence the biosynthesis of the eicosanoid precursor fatty acids (Garg et a?., 1989; Garg et a?., 1988). Indeed, the relative abundance of n-3 and n-6 fatty acids in the diet as influenced by feeding SEA or C18:2,n-6 with marine lipids (i.e. EPA, DHA) can result in alterations in the fatty acid profile 22  of plasma and tissue TG and phospholipids (SküladOttir eta?., 1994; L’Abbé eta!., 1991; Nalbone et a?., 1989; Garg et al., 1988). In studies with rats fed diets containing beef tallow or safflower oil, both supplemented with fish oil, Garg and coworkers (1988) reported that plasma lipoprotein and hepatic triacylglyceride, phospholipid and cholesteryl ester contents of arachidonic acid were reduced in the SFA + fish oil group only. Animals fed safflower + fish oil in the diet had increased levels of Cl 8:2,n6 and AA in these same plasma and hepatic lipid fractions (Garg eta?., 1988). These alterations in the composition of plasma and tissue fatty acid profiles when marine oils are consumed in the diet are due to the greater affinity of the n-3 fatty acids for the elongation and desaturation enzymes (Drevon, 1992). The competition of the longer chain marine fatty acids at the level of  and 5 -desaturase  activity effectively reduces the biotransformation of C18:2,n-6 to C20:4,n-6 (Drevon, 1992; Garg et a?., 1988). Once they are synthesized or provided in the diet, the fatty acids dihomo-y-linolenic acid, AA, and EPA can subsequently be utilized in the biosynthesis of eicosanoids, such as the leukotrienes, thromboxanes and prostaglandins (PG). Specific eicosanoids, depending on the precursor (n-3 or n-6) involved, have characteristic effects on platelet aggregation and vasoconstriction or vasodilatation. Thus, thromboxane A 2 which is derived from n-6 precursors, such as AA, has platelet aggregating activity and is vasoconstrictive.  Conversely, products from EPA, namely thromboxane A 3 and PGI 3  have an anti-aggregating effect on platelets and a vasodilating effect on blood vessels, respectively. Many of the positive effects of fish oil fatty acids on plasma lipid metabolism may be linked to their capacity to alter triacylglyceride and cholesterol metabolism (Coniglio, 1992; Garg et a?., 1989). The hypotriacylglyceridemic effect of marine lipids is thought to occur due to a number of effects on lipid metabolism in general, and more specifically, lipoprotein composition (Coniglio, 1992). Thus, marine lipid fatty acids reduce plasma triacylglyceride concentrations by not only having an inhibitory effect on hepatic TG synthesis, but also by increasing hepatic mitochondrial and peroxisomal 1-oxidation of PUFA (Coniglio, 1992; Halminski eta?., 1991). Moreover, Drevon and coworkers have observed that postprandial concentrations of free fatty acids were reduced in subjects fed marine oil diets (Rustan eta!., 1993).  This situation would, in turn, reduce the availability of fatty acids for  hepatic triacylglyceride synthesis. Hypocholesterolemic effects of marine lipids may involve a reduction 23  in the amount of cholesteryl ester in newly secreted VLDL due to the presence of EPA (Drevon, 1992). Small increases in the concentration of HDL cholesterol in association with lower plasma VLDL concentrations may be due to the reduced levels of plasma free fatty acids causing a reduction in the transfer of cholesteryl ester from HDL to LDL and VLDL particles (Drevon, 1992). Central to the effects of marine oil fatty acids on plasma cholesterol concentrations is their influence on hepatic metabolism of this sterol (Levy and Herzberg, 1995; Smit eta!., 1994). When Garg and coworkers (1988)  fed rats diets containing 20% (wt/wt)  fat provided by either SFA or  Cl 8:2,n-6 PUFA, both supplemented with fish oil, plasma cholesterol levels were reduced in the PUFA + fish oil group but not altered by the SEA + fish oil diet. These authors also reported that hepatic  cholesterol concentrations were reduced by the addition of fish oil to both diets, although the effect with the PUFA-based diet was greater than that seen with the SFA-based diet. Short-term feeding studies in rats fed fish oil diets to investigate hepatic bile flow and composition reported reduced plasma cholesterol and TG concentrations; however, liver cholesterol levels were not influenced in these studies, possibly due to the short time course (14 days) involved (Smit eta!., 1994). These studies in rats with chronically catheterized bile ducts reported that biliary cholesterol secretion was greatly increased in fish oil-fed rats (400%) compared to their corn oil-fed counterparts (Smit et a!., 1994). Biliary secretion of bile acid and phospholipid were also increased, but to a much smaller extent in fish oil-fed animals.  More recently, Levy and Herzberg (1995)  reported that in acute bile duct  cannulation studies with rats fed fish oil diets not only was the volume of bile secreted increased, but the total amounts of bile acid, cholesterol and phospholipid secreted were also elevated in animals fed fish oil compared to a corn oil diet. Thus, one of the mechanisms by which dietary fish oil acts to reduce plasma levels of cholesterol is by increasing the diversion of hepatic cholesterol into the bile, through bile-acid dependent cholesterol secretion (Smit eta!., 1994). Eurther efforts to elucidate the relative influence of the two major marine oil fatty acids, EPA and DHA, on lipid metabolism have yielded some evidence of differential effects between these two longer chain n-3 PUFA (Ikeda eta!., 1994).  In rats fed 10% (wt/wt) fat supplemented with equal  proportions of EPA or DHA at 1 % (wt/wt)  of the diet, plasma and hepatic cholesterol levels were  reduced to a greater extent by DHA than by EPA (Ikeda et a!., 1994).  Conversely, plasma 24  concentrations of TG were decreased to a greater extent in EPA-fed animals than in those fed DHA in the diet. Reduction of hepatic cholesterol levels may reflect an inhibitory effect of dietary fish oil on the rate-limiting enzyme involved in cholesterol synthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-C0A) reductase, possibly mediated by DHA (Ikeda eta!., 1994). The ability of n-3 and n-6 PUFA to produce changes in plasma lipid concentrations may relate to their effects on lipoprotein composition and size as well as tissue lipoprotein metabolism (Woollett eta!., 1992; Fernandez eta!., 1992a; Fernandez and McNamara, 1991).  Woollett and coworkers  (1992) examined LDL cholesterol levels in hamsters fed SFA or PUFA (high in C18:2,n-6) diets. In PUFA-fed animals, reduced plasma LDL cholesterol concentrations were associated with a reduced LDL cholesterol production rate and an increase in hepatic LDL receptor activity, and thus receptordependent LDL transport. Moreover, these workers reported that substituting PUFA for SFA in the diets increased LDL receptor activity more than did merely reducing the amount of SEA in the diet (Woollett et a!., 1 992).  In guinea pigs fed a corn oil-based diet, plasma total and LDL cholesterol  concentrations were reduced compared to SFA-fed counterparts (Fernandez and McNamara, 1991). Examination of LDL particles from PUFA-fed animals revealed higher densities and decreased core-tosurface component ratios, reflecting decreased LDL cholesteryl ester and increased phospholipid and protein content of these LDL. Receptor-mediated binding of LDL to apolipoprotein BIE receptors of hepatic membranes was two-fold greater in PUFA-fed animals compared with SEA-fed animals. These workers demonstrated that this increased receptor-mediated binding was associated with an increase in the hepatic apolipoprotein B!E receptor number (Fernandez and McNamara, 1991). In further studies to investigate the effect of dietary PUFA on lipoprotein kinetics, Fernandez and coworkers (1992a) reported that a corn oil PUFA diet fed to guinea pigs resulted in a 50% reduction in LDL apolipoprotein B pool size, and thereby a reduced plasma LDL cholesterol concentration in these animals.  These  observations were accompanied by an increased hepatic receptor-mediated LDL fractional catabolic rate and a reduced LDL cholesterol flux rate (Fernandez et a!., 1992a).  Moreover, there was a  significant correlation between hepatic apolipoprotein B/E receptor number and receptor-mediated LDL fractional catabolic rate in vivo. Thus, PUFA diets modulate plasma LDL cholesterol levels in part by altering lipoprotein composition and receptor-mediated catabolism. Studies have also indicated that 25  the effect of certain PUFA, e.g. the n-3 fatty acids, on lowering plasma cholesterol is related to the ratio of linoleic acid to saturates consumed (Garg eta!., 1989; Garg et a/., 1988). These workers demonstrated that the beneficial effects of n-3 fatty acids were greater in subjects consuming mainly animal fats, as opposed to vegetable oils. Thus, a modest intake of n-3 fatty acids in combination with animal fats, such as butter, may be of greater benefit than switching to mainly polyunsaturated sources of dietary fat (Dairy Bureau of Canada, 1990). 2c. Endogenous antioxidants in vivo. The potentially damaging effects of reactive lipid peroxides in vivo are controlled by endogenous antioxidant defenses. components.  These cellular defenses include enzymatic and non-enzymatic  Enzymes involved in the detoxification of lipid and oxygen radicals include Cu/Zn  superoxide dismutase (SOD; cytoplasm), Mn-SOD (mitochondria), catalase (peroxisomes), the selenium glutathione peroxidase/reductase redox cycle enzymes (cytoplasm and mitochondria) and finally, the non-selenium glutathione-S-transferases (cytoplasm). Non-enzymatic cellular antioxidants active in vivo include a-tocopherol, B-carotene, ascorbyl palmitate present in membranes as well as the tripeptide glutathione present in the cytoplasm (Reed, 1989).  Extracellular antioxidants include plasma  components such as uric acid, bilirubin and carnosine (Harris, 1992a; Reed, 1989). In the absence of adequate endogenous antioxidant defenses, the propagation of free radical reactions can lead to the oxidation of nucleophilic cellular constituents as well as the reaction of secondary lipid autoxidation products with nucleophilic macromolecules, such as membrane constituents, enzymes or DNA (Fraga  eta!., 1989; Reed, 1989). These events can result in the disruption of cellular membranes and cell death (cytotoxicity), ultimately leading to tissue damage.  The pathological significance of lipid  peroxides and free radical damage in man continues to be demonstrated and investigated. Cu/Zn-SOD and Mn-SOD catalyze the dismutation of the superoxide radical in two ways, both yielding hydrogen peroxide and oxygen as products (Remade eta!., 1992). SOD  2O2  +  2H  2 + HO H0 2  > H 0 + 02 2 >  O + 02 2 H  The structure of bovine red blood cell Cu/Zn-SOD has been characterized as consisting of two identical 151 amino acid residue subunits with two active sites on opposite sides of the enzyme (Harris, 1 992b). 26  The copper ion which is situated within a deep cleft is easily displaced, whereas zinc is buried inside the enzyme structure. Studies have shown that zinc can be replaced by other metallic cations such as cadmium, mercury or cobalt, with only slight decreases in enzyme activity (Harris, 1992b). On the other hand, copper cannot be replaced with another cofactor. Thus, zinc deficiency has little effect on Cu/Zn-SOD activity, whereas copper deficiency results in reduced enzyme activity (Harris, 1 992b; L’Abbé and Fischer, 1984). During enzyme catalysis, the copper ion is reduced by 02  to yield  °2’  whereupon the copper is then reoxidized to produce H . The SOD enzymes are sensitive to the 0 2 inhibitory effects of H 0 concentrations in the reaction environment (Remade et al., 1992). Some 2 interaction between these two forms of SOD exist, in that the Cu/Zn- and Mn-SOD forms can compensate for each other in certain deficiency states (Harris, 1992b). Catalase (CAT) is located intracellularly within mitochondria and peroxisomes. This enzyme catalyses two types of reactions. The first involves the decomposition of 2 H to water and oxygen, 0 while the second involves the conversion of a hydroperoxide to the corresponding alcohol in the ; Aebi, 1974). 2 presence of a hydrogen donor (AH CAT  0 2 2H 2 ROOH + AH  >  2HO + 02 > H 0 + ROH + A 2  In the first reaction, the second molecule of H 0 acts as a hydrogen donor for the deactivation of the 2 first H . O 2  Each enzyme molecule contains four ferriprotoporphyrin groups in its structure (MW  240,000). The activity of CAT varies between tissues, with the liver and kidney having the greatest amounts, and connective tissue the least. The enzyme is mainly particle-bound within organelles, but is free within red blood cells (Aebi, 1974). Furthermore, species differences have been found to exist for red blood cell (RBC) CAT activity (Godin and Garnett, 1992). For example, while human RBCs are rich in CAT activity, those of avian species (e.g. duck and quail) are almost devoid of CAT activity (Godin and Garnett, 1992; Aebi, 1974). Thiol (-SH)  groups of proteins have antioxidant activity as seen in studies with casein  (McGookin and Augustin, 1991). Similarly, intracellular thiols, such as glutathione (GSH; L-y-glutamyl L-cysteinyl-glycine), possess strong antioxidant activity (Reed, 1989). The y-glutamyl linkage renders the GSH molecule resistant to proteases. The main role of GSH in vivo is to serve as a primary agent 27  for deactivating electrophilic free radicals. The antioxidant capacity of thiol groups is a function of the percentage of the thiol groups in the anion form (Reed, 1989). Thus, while the amino acid cysteine has a greater number of charged moieties than GSH at pH 7.5, the antioxidant capacity of GSH dominates due to its greater concentration (liver tissue ratio is approximately 1:50; Reed, 1989). Approximately 90% of intracellular nonprotein thiol content is GSH. In the hepatocyte, 85-90% of GSH is present in the cytoplasm, with the remaining 10-15% being localized within the mitochondria. In addition to acting as a coenzyme or substrate for antioxidant enzymes (i.e. GSH-peroxidase and GSH-S-transferase), cellular GSH also has a protective role to protect protein thiol groups from oxidation, as an intracellular redox buffer (Moron et a!., 1979).  The GSH tripeptide also acts as a  cysteine reserve when required. The mammalian liver normally has a high concentration of reduced GSH.  Approximately 0-8% of the GSH content in rat liver tissue is in the oxidized (GSSG)  form;  hence, 0-16% of total GSH may exist in the disulfide form (Moron et a!., 1979). Glutathione has been shown to undergo inter-organ circulation via translocation from cells into the plasma (Anderson and Meister, 1980). Metabolism of GSH within the blood is thought to involve the reduction of disulfide bonds of plasma constituents and mobilization of compounds bound by disulfide bridges to plasma proteins. These products, in turn, are used to synthesize GSSG and low molecular weight derivatives of GSH, such as disulfides. Levels of GSH in rat plasma have been reported to range from 22-27 pM. Approximately 85% of plasma GSH is in the reduced form (Anderson and Meister, 1980).  Cellular  levels of GSH are dependent on the growth, nutritional status and hormone balance of the subject (Moron eta!., 1979). Pélissier and coworkers (1993) demonstrated that with a protein deficient diet (6% casein), reduced levels of GSH were observed in the small intestine and liver of rats. As an index of in vivo susceptibility to lipid autoxidation, these workers reported increased levels of thiobarbituric acid reactive substances in the intestinal mucosa of experimental animals.  In studies varying the  protein content of diets fed to rats, Hum and coworkers (1992) reported an increase in hepatic GSH concentration and total liver GSH content when dietary casein was increased from zero to twenty percent. A further increase in dietary protein to forty percent did not result in an additional increase in liver GSH content, but did reduce the relative concentration of GSH due to increased liver tissue weight (Hum et a!., 1992).  These workers demonstrated a strong correlation between liver GSH 28  concentration and total GSH content with sulphur amino acid intake.  In this study, the plasma  concentration of free cysteine was a better predictor of liver GSH content than was plasma GSH content (Hum et al., 1992). This observation may be due to the sigmoidal relation between plasma GSH turnover and liver GSH content (Hum eta!., 1992). Moreover, the plasma GSH concentration was strongly associated with the absolute turnover rate of plasma GSH. Fasting for 24 to 48 hours can reduce liver GSH in the rat to 50-70% of that in fed counterparts (Reed, 1989). Liver GSH content can be altered by variations in diurnal/circadian rhythms (Reed, 1989). This observation may be linked to the diurnal variation in GSH metabolism (Hum et a!., 1992).  Tissue concentrations of GSH are  greater at night and early morning, compared to lower concentrations measured in the late afternoon (Reed, 1989). The enzymes of the glutathione peroxidase/reductase redox cycle are comprised of a seleniumdependent glutathione peroxidase (GSH-Px) and glutathione reductase (GSSG-Red). These enzymes occur in the plasma as well as intracellularly (Harris, 1992a; Paglia and Valentine, 1967). Glutathione peroxidase catalyzes the deactivation of hydrogen peroxide or hydroperoxides using the reducing power of glutathione (GSH). GSH-Px  0 + 2GSH 2 H ROOH + 2GSH  > 2H 0 + GSSG 2 > ROH + H 0 + GSSG 2  These reactions yield oxidized glutathione (GSSG) as one of their products. The GSSG, in turn, is reduced back to its native form via catalysis by GSSG-Red and nicotinamide adenine dinucleotide phosphate (NADPH; Moron eta!., 1979; Paglia and Valentine, 1967). GSSG-Red  GSSG + NADPH  > 2GSH + NADP  Glutathione peroxidase has been considered as a key enzyme of the in vivo antioxidant syste.m under both normal conditions as well as oxidative stress (Remade eta!., 1992).  In studies with cultured  fibroblasts and human endothelial cells lower amounts of GSH-Px were required, compared to CAT or SOD, for the same level of antioxidant protection (Remade eta!., 1992). reactive oxygen species such as 02  Inhibition of GSH-Px by  and hydroperoxides is possible. Enzyme activity was reduced  50% in the presence of 50 pM tertiary butylhydroperoxide (Remade et a!., 1992). The enzyme is directly inactivated by the superoxide radical (Turrens, 1991).  The activity of GSSG-Red can be 29  inhibited by the phenylalanine chain of insulin (Long and Carson, 1961), and increased levels of GSSG Red activity have been observed in hemolysates of diabetic patients’ RBCs (Long and Carson, 1961). The non-selenium dependent glutathione-S-transferases are involved in the biotransformation (detoxification)  of xenobiotics (Moron et a!., 1979).  For example, GSH-S-transferases detoxify  potential alkylating agents (Habig eta!., 1974). Glutathione-S-transferases have a high specificity for GSH as reflected by the finding that this substrate is bound with a high affinity to the active site, thus providing an enzyme-bound glutathione thiolate anion (GS1, which is very effective for conjugate formation (Reed, 1989). These enzymes mediate the conjugation of reactive metabolic intermediates of xenobiotics with GSH.  The reaction of the -SH group of GSH with a xenobiotic to neutralize  electrophilic sites yielding a more water-soluble product, is catalyzed by GSH-S-transferase (Habig et a!., 1974). The GSH conjugates can be metabolized further by cleavage of the glutamate and glycine residues, followed by acetylation of the resulting free amino group of the cysteinyl residue to yield a mercapturic acid for excretion (Moron eta!., 1979). Further, the glutathione-S-transferases also have GSH-Px activity (Moron et a!., 1979). Thus, the formation of GSH adducts or other biotransformed products generally yields much less reactive or toxic products. 2d. Lipid peroxidation in vivo. Many of the mechanisms involved in the initiation and propagation of lipid peroxidation in model and food systems are also common to the progression of lipid peroxidation in vivo. Thus, similar to the situation in food systems, the in vivo concentration of free radicals is proportional to the balance between their formation and elimination or deactivation by enzymatic and non-enzymatic scavengers. Free radical production intracellularly can occur through a number of biochemical pathways, including oxidases (e.g. both xanthine oxidase and glucose oxidase produce  °2),  flavoprotein dehydrogenases,  450 enzymes, as well as autoxidation P mitochondrial electron transport proteins, cytochrome -dependent of thiols and  hydroquinones (Remade eta!., 1992). The production of reactive oxygen species is  facilitated by the addition of single electrons to molecular oxygen during reduction of 1992b).  °2  to H 0 (Harris, 2  For example, the superoxide radical is formed through the addition of a single electron to  molecular oxygen (Diplock, 1986). 02 + le  >  °2  30  Another pathway for superoxide radical formation is the reaction of molecular oxygen with the ferrous ion in the Haber-Weiss cycle (see above, page 6; Graf eta!., 1984). The superoxide radical itself has limited reactivity, only abstracting hydrogen atoms from very strong donors such as ascorbic acid (Foote, 1985). Dismutation of the superoxide radical, either spontaneously or catalyzed by SOD, yields hydrogen peroxide.  0 interacts with ferrous ion, the reaction yields the highly reactive 2 When H  hydroxyl radical (OH).  The ‘OH radical can then abstract another electron from a neighbouring  compound, such as an unsaturated lipid, to initiate a cascade of lipid autoxidation reactions. Singlet oxygen  (102),  another reactive form of oxygen is produced when  °2  absorbs energy, such as in  photochemical reactions involving photons of light. This species of oxygen radical is more selective in further reactions than 0H and R00, but is less selective than 02’ (Foote, 1985). Dietary deficiencies of enzymatic trace metal cofactors (Cu, Mn, Se, Zn) or vitamins (A, C, E) may lead to the accumulation of radical 0H  °2  and H , favouring the generation of the highly reactive hydroxyl O 2  (Zamora eta!., 1991; Leibovitz eta!., 1990; Fraga eta!., 1989). Other sources of lipid  autoxidation products occurring in vivo include various exogenous contributions, namely the diet, air pollution and cigarette smoke. These are examples of agents which can exert oxidative stresses on biological systems.  Normally, the compartmentalization of cellular oxidative and antioxidant  components within organelles in vivo facilitates the control of a balance between these two events. However, the formation of reactive lipid free radicals and hydroperoxides may result in the disruption of cell membranes due to the increased polarity of lipid autoxidation products.  Thus, membrane  phospholipid components will undergo degradation due to the polar -OOH group of lipid hydroperoxides moving out of the relatively nonpolar environment within the fluid membrane structure. detrimental  effects  of  oxidative  stress in  vivo  may  involve  specific  biological  Other  processes.  Malondialdehyde 2 (OHC-CH CHO), a secondary product of PUFA autoxidation, is capable of crosslinking proteins, inactivating enzymes and interacting with cellular DNA (Addis, 1986). Fraga and coworkers (1989)  demonstrated decreased protein synthesis in rat liver tissue exposed to oxidative stress  mediated by halogenated compounds. Furthermore, aging may be the result of cumulative oxidative events throughout the lifetime (Simic, 1991; Diplock, 1986). Development of lipofuscin age-pigments in older animals is due to the condensation of carbonyl lipid autoxidation products with basic protein 31  amino acid residues, similar to Maillard reaction products (Pryor, 1986; Reddy etal., 1973). Damage to DNA by 0H or lipid free radicals resulting in alterations to bases, single or double strand breakage, crosslinking or chromosomal aberrations may initiate carcinogenesis (Thompson, 1994; Simic, 1991). The involvement of lipid autoxidation products in the pathogenesis of cardiovascular disease has also been suggested in reference to the elucidation of disease mechanisms (Steinberg et al., 1989). The link between age and the development of chronic diseases such as cancer and atherosclerosis is reflected by the fact that the progression of these biological outcomes occurs over several years. 2e. Lipid oxidation in heart disease. In elucidating the mechanism of the development of CVD, plasma hypercholesterolemia was hypothesized to play a major role in the etiology of atherosclerosis (Nordoy and Goodnight, 1990; Lipid Research Clinic Program, 1984). One of the hallmark events in atherogenesis is the appearance of lipid-laden foam cells containing cholesteryl esters beneath the arterial endothelium (Steinberg et al., 1989).  However, the presence of elevated levels of plasma cholesterol can be seen to be only one  factor of many, as there is considerable variation in the expression of atherosclerosis at any given level of hypercholesterolemia. Moreover, considerable evidence exists to suggest that cholesterol oxides, rather than native cholesterol, are responsible for the initiation of atheroma (Addis, 1990). The role of native cholesterol perse in atherogenesis has come into question, in large part due to studies performed with cell cultures of foam cell precursors.  Monocytes and macrophages  incubated in the presence of high concentrations of native (unoxidized) LDL fail to transform into foam cells (Goldstein et a!., 1979).  Moreover, the macrophage LDL receptors are down-regulated in the  presence of elevated levels of LDL (Goldstein et a!., 1979).  As well, endogenously induced  hypercholesterolemia was only minimally atherogenic, compared to studies in which commercial cholesterol was fed to subjects resulting in severe atherosclerosis (Addis and Park, 1989). Studies on the acute response of rabbits to the intravenous administration of various COPs indicated the presence of arterial lumen damage similar to that seen in the early stages of atherogenesis (Peng etal., 1985). The development of atherosclerosis can be broken down into three phases, namely initiation, propagation and termination as follows (Addis, 1990). Phase 1. Initiation: arterial endothelial injury; 32  Phase 2. Propagation: accumulation of plaque material; Phase 3. Termination: thrombosis or spasm leading to myocardial infarction, cardiac muscle damage, or death. From the above mechanism, the progression of atherosclerosis-related events appears to be similar to those of lipid autoxidation outlined previously (see page 2). Thus, it may be more than coincidental that lipid autoxidation is thought to be involved in the progression of this disease process. The primary event in the development of atherosclerosis as defined by this pathway is a “response to injury.” This is in contrast to the “cause and effect” hypothesis which focuses on the role of dietary fat in disease development. The nature of the initial injury to the arterial endothelium has yet to be clearly defined, but cytotoxic, oxidatively modified LDL and COPs likely play a role. containing COPs or other lipid oxidation products (LOPs)  Low density lipoproteins  are referred to as modified LDL (mLDL).  Oxidized lipid species have been identified in the plasma lipoproteins and aortic plaque of atherosclerotic patients (Rosenfeld eta!., 1990; Stringer eta!., 1989). Moreover, several cholesterol oxides have been identified as having cytotoxic, angiotoxic, carcinogenic and mutagenic activity (Smith and Johnson, 1989; Peng eta!., 1992).  In contrast, native cholesterol has not been found to be  angiotoxic, as have some COPs (Hessler et a!., 1979).  Specifically, 25-hydroxycholesterol and  cholestane-3I,5a,6f-triol have been found to be particularly toxic to cultured rabbit aortic smooth muscle cells (Peng eta!., 1978). The COPs may be exerting an effect in vivo through inhibition of protein (Fraga et al., 1989) or cholesterol synthesis (Addis and Park, 1989). The sequence of events following arterial injury is dominated by the role of monocytes and other leucocytes at the site of injury (Addis, 1990). Circulating monocytes are mobilized to these sites due to the bioactivity of mLDLs as strong chemoattractants (Quinn eta!., 1987). The chemotactic activity of the mLDL has been found to reside in the lipid component, namely lysolecithin (Steinberg eta!., 1989). Once the monocytes have arrived at, and adhered to, the damaged tissue site of the arterial endothelium, they begin to migrate into the arterial intima (the layer of cells below the endothelium).  At this time, the monocytes are converted into phagocytic macrophages.  The  mechanism of this transformation is believed to involve the stimulative activity of the highly immunogenic mLDL (Steinberg et a!., 1989).  The transformation of macrophages into foam cells 33  involves the uptake of lipid and cholesteryl esters.  Macrophages within the arterial walls can  internalize mLDL, and thus their constituent COPs, by two mechanisms.  The first involves the  engulfment via phagocytosis of immune complexes of autoantibodies binding mLDL (Steinbrecher and Lougheed, 1992).  The second route consists of the “Scavenger Pathway” involving an acetyl LDL  receptor which recognizes the modified apolipoprotein B moiety of mLDL molecules (Steinbrecher, 1993). This alternative LDL removal pathway is not subject to down-regulation, as are the native LDL receptors of macrophages. Proliferation of foam cells derived from macrophages and smooth muscle cells of the arterial media (medial cells are located below the intima), results in the development of “fatty streaks”  in the subendothelial tissues.  This progression of infiltration of tissue by lipid  contributes to the progressive tissue damage in atherogenesis.  Response to tissue injury involves  platelet activity stimulated by lipid oxides, such as linoleic hydroperoxide (Ross and Glomset, 1976). Narrowing of the arterial lumen occurs due to the promotion of smooth muscle cell growth by the release of platelet-derived growth factor (Addis, 1990).  Finally, extensively raised plaques which  occlude the arterial lumen can be observed. At this stage of the disease, complications can arise, such as arterial thrombosis and spasm, ultimately leading to myocardial infarction, or death (Addis, 1990). Pathological consequences of myocardial infarction or tissue ischemia may not be related to the blockage of blood flow during the period of ischemia itself, but rather, the reperfusion phase that follows (Bulkley and Morris, 1986). During reperfusion, the tissue undergoes oxidative stress induced by the sudden restoration of blood circulation and thus, availability of molecular oxygen. It has been suggested that ischemic injury is a result of the rapid proteolytic conversion of xanthine dehydrogenase to xanthine oxidase (XOD) at the beginning of the ischemic period (Bulkley and Morris, 1986). An accumulation of the enzyme’s substrate, hypoxanthine, via adenine nucleotide degradation also occurs at this time. Thus, production of an excess of reactive oxygen species, namely  °2’  0 and ‘OH. 2 H  can occur upon the restoration of oxygen during reperfusion. These cytotoxic oxygen metabolites can promote tissue damage through oxidative attack of membrane lipids and other cellular constituents. This cytotoxic damage was shown to be preventable with the administration of SOD prior to reperfusion (Bulkley and Morris, 1986). Similarly, the size of myocardial infarcts was shown to be decreased in canines treated with allopurinol (a specific inhibitor of XOD; Bulkley and Morris, 1986). 34  2f. Oxidized lipid species in plasma. Low levels of lipid peroxides have been reported in human plasma lipoproteins of children, with increasing levels to 70 years of age, followed by declining levels thereafter (Hagihara eta!., 1984). The source of these plasma lipid peroxides likely involves both in vivo oxidation as well as absorption of lipid oxides from the diet (Emanuel eta!., 1991; Steinberg eta!., 1989). Absorption of dietary lipid oxides has been demonstrated in both animal studies (Peng et a!., 1 987; Peng et a!., 1982), as well as in human trials (Emanuel eta!., 1991). Dietary lipid oxides absorbed across the intestinal mucosa are then incorporated into chylomicrons for transport via the lymph to peripheral tissues as are native dietary lipids. The profile of post-prandial plasma COPs will reflect those contained in the diet. The greater polarity of COPs, relative to sterol esters or triacylglycerides, suggests that they are likely to be situated within the surface monolayer of plasma lipoproteins. This would allow rapid transfer of COPs out of the chylomicrons, and rapid exchange between lipoproteins (Emanuel et al., 1991). Moreover, COPs appear to be rapidly cleared from the plasma, compared to the more lengthy temporal pattern of absorption for other dietary lipids (Addis, 1990). The in vivo oxidation of lipoprotein lipids, e.g. low-density lipoproteins, requires the initiation of peroxidation of the constituent PUFA. This can be precipitated by the formation of lysolecithin , and the 2 (lecithin from which a fatty acid moiety has been cleaved) by the action of phospholipase A subsequent formation of LDL fatty acid peroxides (Steinberg eta!., 1989).  Fragments produced from  the scission of lipid peroxides can then covalently attach to apolipoprotein B, thereby masking the  E  amino group of lysine residues, and possibly other sites as well, resulting in another form of mLDL (Steinberg eta!., 1989). 2g. Influence of dietary fatty acids on LDL oxidative stability. The oxidative instability of PUFA, which has been well demonstrated in both in vitro model systems and various food systems, has resulted in queries as to the appropriateness of promoting the consumption of these lipid sources in modulating the development of atherosclerosis. This is especially true given the concern towards the involvement of lipoprotein oxidized lipid species in the genesis of atherosclerosis. Enhanced in vivo and in vitro tissue lipid peroxidation has been reported in animals fed PUFA diets, particularly those containing marine oil PUFA (SküladOttir eta!., 1994; De Schrijver 35  et a?., 1992; L’Abbé et a!., 1991; Hu et a!., 1989). The majority of these studies evaluated in vivo and tissue levels of thiobarbituric acid reactive substances (TBARs). This assay, although widely used as an index of lipid peroxidation in animal studies, is regarded as being relatively non-specific in nature since not only does malondialdehyde (a lipid peroxidation product of PUFA) react with TBA, but other aldehydes (e.g. 4-hydroxynonenal, hexanal, propanal) will also react with the TBA reagent (Draper et a!., 1993).  In an attempt to circumvent this problem, Hu and coworkers (1989)  quantitated  conjugated dienes, hexanal and total volatiles released by tissue homogenates from treated animals. Rats fed fish oil diets (high in C20:5,n-3 and C22:6,n-3) had greater levels of TBARs and conjugated dienes in both liver and kidney tissue compared to corn oil-lard (high in C18:2,n-6)-fed animals. Conversely, greater levels of hexanal and total volatiles were reported from induced lipid peroxidation in liver tissue from corn oil-lard-fed rats compared to the fish oil group of animals. These results clearly reflect the influence of dietary lipid treatment on tissue fatty acid content, and thereby, products of lipid peroxidation.  For example, the production of hexanal results only from the breakdown of lipid  peroxides derived from n-6 fatty acids such as those from the corn oil-lard diet. On the other hand, the polyunsaturated marine oil PUFA are good substrates for the formation of TBARs, as these fatty acids have multiple nonconjugated double bonds. Further analysis of data from this study revealed a significant inverse correlation of tissue TBARs, conjugated clienes and total volatiles with the log of dietary vitamin E content (Hu eta!., 1989). Dietary PUFA and vitamin E contents can influence/n vitro estimates of lipid peroxidation in animal models fed different lipid sources.  When L’Abbé and  coworkers (1991) fed menhaden oil to rats over 16 weeks, while total and HDL cholesterol levels were reduced, indices of in vivo lipid peroxidation such as urinary and tissue TBARs were increased and tissue antioxidant enzymes (e.g. superoxide dismutase) were reduced compared to groups fed diets high in oleic or linoleic acid. In studies with rats fed beef tallow and fish oil blends, De Schrijver and coworkers (1992)  reported that excretion of urinary malondialdehyde, assayed as TBARs, was  elevated when diets containing greater than 1 .8% fish oil were fed to rats. Highly polyunsaturated dietary fatty acids may contribute to oxidative stress in vivo not only due to their relative instability to oxidation, but also to effects on endogenous antioxidant enzyme systems. This effect of PUFA in vivo is even more evident when systems are challenged with oxidizing agents (Sküladóttir et a!., 1 994) 36  or cancer inducing agents (Kuratko eta!., 1994). When animals fed fish oil diets were challenged with a free radical generator (methyl ethyl ketone peroxide) in vivo, tissue lipid peroxides and hepatic GSH levels were reduced compared to counterparts fed corn oil diets (SkiiladOttir eta!., 1994). Moreover, injection of the oxidizing agent in this experiment could be seen to reduce tissue levels of the longer chain PUFA from fish oil-fed rats. Administering an inducer of colon carcinogenesis to rats fed fish oil diets increased liver microsomal levels of TBARs in young animals only (Kuratko eta!., 1994). In this experiment, the liver showed susceptibility to microsomal lipid peroxidation which was influenced by dietary lipid source and animal age (Kuratko eta!., 1994). In contrast to the above reports of consistently increased in vivo susceptibility to oxidation from dietary longer chain PUFA, such as those from marine oils, data relating to the oxidative susceptibility of plasma lipoproteins are considerably more variable (Frankel eta!., 1994; Kleinveld eta!., 1993). Kleinveld and coworkers (1993) found that LDL susceptibility to oxidation, as assessed by rate and extent of oxidative changes, was inversely correlated with the ratio of oleic to linoleic acid content of these particles.  Conversely, this ratio was positively correlated with the lag period preceding lipid  peroxidation of these LDL particles. Interestingly, despite the fact that these LDL were collected from vitamin E deficient subjects, they were less susceptible to oxidation than control LDL. The LDL from vitamin E deficient patients not only contained less vitamin E, but also lower amounts of cholesteryl esters and increased levels of TG compared to LDL from control patients (Kleinveld et a!., 1993). However, these LDL also contained a greater ratio of oleic to linoleic acid in the TG fraction. Thus, both the fatty acid content as well as antioxidant content can influence the susceptibility of LDL to oxidation. When Frankel and coworkers (1994) examined the effect of dietary fish oil supplementation on the oxidative stability of LDL from hypertriacylglyceridemic humans, despite differences in the profiles of volatiles released from samples from subjects fed fish oil or control diets, the total amount of volatiles was not different.  The two groups of LDL samples did not differ in susceptibility to  oxidation, although oxidation products were distinct between the two groups of samples (Frankel et a!., 1994). Oxidation of LDL in vivo has been investigated by a variety of methods, including immunoassays (Palinski eta!., 1990; Rosenfeld eta!., 1990; Palinski eta!., 1989) and electrophoresis 37  with chromatographic techniques (Jialal et a!., 1991). Antibodies generated against lipid oxidation product-LDL component conjugates (Palinski et al., 1 990) recognize and bind to atherosclerotic lesions from tissue obtained from diseased rabbits (Rosenfeld eta!., 1990; Palinski eta!., 1989). Also, LDL extracted from atherosclerotic lesions was recognized by an antiserum against malondialdehyde conjugated LDL. Moreover, autoantibodies against MDA-LDL conjugates have been observed in serum from humans and rabbit animal models (Palinski eta!., 1989). Further evidence of a function for these autoantibodies against LDL conjugates was reported by Steinbrecher and Lougheed (1992) in studies investigating the degree of modification of LDL isolated from normal and diseased human aortic intima. Electron microscopy of LDL isolated by these authors from aortic plaque revealed the presence of large aggregates, which appeared to be clusters of LDL particles (Steinbrecher and Lougheed, 1992). Interestingly, the LDL isolated by these authors from diseased aorta appeared to be quite variable as to the extent of oxidation (assessed by degree of apolipoprotein B fragmentation). In association with the variation in degree of LDL oxidation, uptake by cultured macrophages appeared to be scavenger receptor-independent (Steinbrecher and Lougheed, 1992).  Thus, uptake of mLDL by cells of the  arterial intima can also be attributed to phagocytosis of autoantibody-mediated immune complexes of mLDL. In studies to investigate the susceptibility of LDL to in vitro copper-catalyzed oxidation, Jialal and coworkers (1991)  reported that electrophoretic mobility and oxysterol content (as COPs)  increased in a parallel temporal pattern. The major oxysterol generated in the oxidized LDL in this experiment by in vitro or macrophage modification was 7-ketocholesterol. Preparations of LDL from different normolipidemic individuals proved to be variable in their susceptibility to induced oxidation. Thus, while a considerable amount of information has been obtained from investigating the susceptibility of LDL to in vitro transition metal-catalyzed oxidation, several of the parameters measured are applicable to cell culture or tissue samples from humans and animal models. In summary, while the susceptibility of polyunsaturated fatty acids to oxidation in model lipid systems is well recognized, the oxidative stability of PUFA in food systems and in vivo is considerably more variable due to the presence of pro-oxidants, antioxidant mechanisms and co-nutrients or other biomolecules.  A large amount of work has been performed over the years to investigate the link  between dietary lipid source and plasma lipid profiles involved in the genesis and progression of 38  cardiovascular disease. In recent years, it has become increasingly clear that while dietary fat source may influence plasma cholesterol concentrations, the latter is not the sole determinant of the development of atherosclerosis. Rather, in vivo oxidative status as related to the presence of mLDL and oxysterols within plasma and aortic lesions may be more relevant to the risk of development of heart disease. Thus, the influence of oxidative stress (e.g. from the diet) on endogenous antioxidant status (enzymatic and non-enzymatic mechanisms) may play a role in the disease process. Previous workers have attempted to elucidate the impact of PUFA and cholesterol from dietary lipid sources on indices of in vivo oxidation and susceptibility to atherosclerosis; however, data generated from these studies remains somewhat equivocal. The overall purpose of this thesis is to examine the effect of dietary fat sources varying in proportions of saturated (short-chain versus long-chain) and polyunsaturated (n-6 versus n-3) fatty acids and dietary cholesterol level on plasma lipids and antioxidant status in animal models known to be susceptible to the development of hypertension (spontaneously hypertensive (SHR) rat)  and  atherosclerosis (atherosclerosis-susceptible Japanese quail).  39  CHAPTER 1 Antioxidant status and plasma lipid levels in spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats. Introduction: Despite a declining rate in deaths from cardiovascular disease (CVD) in Canada since the mid 1960’s, CVD (ischemic heart disease and stroke) remains the number one cause of mortalities among Canadian adults (Heart and Stroke Foundation (HSF), 1993). In 1990, approximately 40% of deaths in Canada were due to CVD. Of these deaths, 58% were caused by ischemic heart disease (IHD) associated with atherosclerosis (HSF, 1993).  Elevated blood pressure is considered to be an  independent risk factor for CVD (HSF, 1993), along with other factors such as smoking, dyslipidemia, diabetes and obesity. In many cases, these risk factors tend to co-exist within the population, acting synergistically to increase the risk of atherogenesis. For example, the combination of hypertension and hyperlipidemia is known to potentiate the risk for development of CVD in humans (Dzau, 1 990) and experimental animal models (Yamori eta!., 1976, 1975). The spontaneously hypertensive rat (SHR) developed by Okamoto and Aoki (1963) has been used as an animal model for human essential hypertension.  Efforts to elucidate the underlying  biochemical mechanisms for the development of hypertension in the SHR have included the examination of endothelial membrane abnormalities and associated disturbances in ion transport across cell membranes (Sagar eta!., 1992; Wu eta!., 1 990). Disturbances in mineral metabolism (i.e. calcium and magnesium) have also been reported to be associated with the development of hypertension in the SHR (Kitts et a!., 1992; Jones et a!., 1988). While atherosclerosis is difficult to induce through dietary means in the rat, studies conducted with the SHR have reported the appearance of ring-like aortic lipid deposits in hyperlipidemic animals fed a highly atherogenic diet (Yamori eta!., 1975). These lipid deposits within the arteries of hyperlipidemic SHR were observed to occur at branch points of the vessels and were associated with increased endothelial permeability (Yamori et al., 1975). Increasing clinical and epidemiological evidence indicates that the association between hypertension and atherosclerosis may be related to the in vivo oxidative status of plasma lipid components and tissues (Gey eta!., 1 991; Stringer et al., 1989). Several studies investigating the role 40  of oxygen-derived free radicals in CVD have reported enhanced lipid peroxidation and reduced endogenous antioxidant capacity in tissues from patients exhibiting various CVD risk factors (Buczynski eta!., 1993; Jayakumari eta!., 1992; Sagar eta!., 1992; Hunter eta!., 1991). Elevated levels of lipid peroxides have been observed in platelets and red blood cells (RBCs) from patients with coronary heart disease (CHD; Buczynski eta!., 1993; Jayakumari eta!., 1992).  Moreover, these products of lipid  oxidation are associated with decreased activities of tissue antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) in CHD patients (Buczynski et a!., 1993; Jayakumari eta!., 1992). Also, hypertension has been reported to coincide with increased production of reactive oxygen species (ROS) as well as decreased levels of the antioxidant tripeptide glutathione (GSH)  and reduced SOD activity in polymorphonucleocytes (PMNs; Sagar eta!., 1992).  Thus, these reports are suggestive of a role for oxidative processes in the pathogenesis of hypertension and atherosclerosis. Rodent animal models used in the study of the influence of CVD risk factors such as hypertension and hyperlipidemia on the development of atherosclerosis have included rabbits (Mantha et a!., 1 993), mice (Capel and Dorrell, 1 984) and various strains of rats (Mon eta!., 1 993; Yamori et a!., 1975). Rabbits fed an atherogenic, high cholesterol diet exhibited reduced RBC SOD and GSH-Px activities and an increase in RBC CAT activity (Mantha eta!., 1993). On the other hand, activities of SOD, CAT and GSH-Px were increased in the aorta from high cholesterol-fed rabbits. Some of these changes in tissue antioxidant activities in rabbits fed the atherogenic diet were subsequently observed to be reversed with the addition of vitamin E to diets, indicating a protective effect of this nonenzymatic antioxidant against in vivo oxidative injury (Mantha eta!., 1993). The SHR has been reported to exhibit myocardial hypertrophy relative to its normotensive counterpart, the Wistar Kyoto (WKY) rat (Kashii eta!., 1977). This condition is similar to the ventricular hypertrophy considered to be a risk factor associated with atherosclerosis in humans (Dzau, 1990). Furthermore, the heart of the SHR shows greater vulnerability to membrane lipid peroxidation (Toni eta!., 1992). These reports indicate that animal model studies are of value for investigating the interaction of endogenous antioxidant status with CVD risk factors, namely hypertension and dyslipidemia, in the development of atherosclerosis. However, the majority of the preceding studies were performed with 41  animals fed highly atherogenic diets without a control group to indicate the antioxidant status of the animal model on a standard, basal non-atherogenic diet. Thus, further characterization of the in vivo endogenous antioxidant capacity of the SHR on a standard chow diet could potentially contribute to the elucidation of the role of oxidative status in the development of hypertension, as one risk factor of CVD.  42  Hypothesis for Chapter 1: Animal models with genotypic differences in CVD risk factors exhibit characteristic differences in plasma lipid profile and endogenous antioxidant status. Obiective for Chapter 1: To determine whether differences in plasma lipids and endogenous antioxidant status exist between the spontaneously hypertensive rat (SHR) and its normotensive counterpart, the Wistar Kyoto (WKY) rat. Specific Aims for Chapter 1: i. The endogenous antioxidant status of SHR and WKY rats will be characterized by measuring specific tissue antioxidant enzymes and susceptibility of tissues to in vitro oxidative challenge. ii. Plasma lipid parameters will be used as indicators of differences associated with lipid metabolism between SHR and WKY rats.  43  Materials and Methods: Animals: Six-week  old,  male spontaneously hypertensive rats  (SHR)  and their normotensive  counterparts, Wistar Kyoto (WKY) rats (Charles River, Montreal, PQ) were fed a standard commercial chow diet (Ralston Purina) for an eight week period. Bomb calorimetry (Miller and Payne, 1959) of the chow diet indicated a gross energy content of 16.19 kJIg.  All animals were individually housed  in stainless steel cages under controlled temperature (25°C)  and lighting (14:10 Iight:dark cycle)  conditions. Animals were trained to meal-feed from 09:00 to 16:00 hr, after 1 week of adilbitum feeding (Kitts eta!., 1992). This meal-feeding schedule ensures a similar postprandial time period in animals before blood pressure measurements are performed. Animals had access to distilled deionized water ad ilbitum.  Daily feed intake and weekly body weight gain of animals were routinely recorded  throughout the experimental period. Animals were cared for in accordance with the principles of the Guide to the Care and Use of Experimental Animals, VoL 1 of the Canadian Council of Animal Care (1993). Blood pressure measurement: Systolic blood pressure (SBP) recordings were obtained on animals at 1 3 weeks of age after a 1-week training period, to confirm the presence of hypertension in the SHR animals. Measurements were taken between 1 3:00 and 16:00 hr in conscious rats using an indirect tail-cuff method (Harvard Apparatus Ltd., South Natick, MA; Kitts eta!., 1992). Each recorded value represents the mean of three successive determinations over a period of 10-15 mm. Tissue sample preparation: At the end of the experiment, animals (14 weeks of age) were sacrificed at 09:00 hr after an overnight fast, by exsanguination under halothane anaesthesia. Blood was collected into heparinized tubes for plasma separation at 1000 x g for 5 mm, 4°C. The aortic tree (the brachiocephalic arteries to their bifurcations and the aorta to the iliac branching) was dissected out, opened longitudinally and examined under a 10-30X dissecting microscope to confirm the absence of lesions in animals fed a basal non-atherogenic diet. Tissues (heart and liver) were collected into chilled 50 mM Tris 0.1 mM 44  EDTA, pH 7.6 homogenizing buffer. Aliquots of plasma were analyzed for total cholesterol (Siedel et a!., 1983), triacylglyceride (Ziegenhorn, 1975), and phospholipid (Takayama etal., 1977; Boehringer Mannheim, Laval, P0). Red blood cells (RBCs) were washed twice with isotonic (0.9%) saline for use in biochemical assays. Hemolysates were prepared by diluting RBCs 1:10 with double distilled H 0 2 and freeze-thawing 3X in dry-ice/acetone to ensure complete cell disruption. Hemoglobin content of RBC hemolysates was assayed according to the method of Drabkin and Austin (1935). Heart and liver were blotted dry, weighed and prepared as 10% homogenates in fresh, chilled homogenizing buffer using a Polytron homogenizer (Brinkmann Instruments, Inc., Westbury, NY) at 25% maximum speed, for 30 seconds (2 X 15 sec). Tissue cytosolic fractions used in enzymatic assays were prepared by ultracentrifugation at 105,000 x g, 1 5 mm, at 4°C using a Beckman L2-65 ultracentrifuge with an SW 4OTi rotor. Tissue cytosolic fractions were assayed for protein content according to Bradford (1976). Enzyme  activity  determinations  were  carried  out using  a  Perkin-Elmer model  Lambda  6B  spectrophotometer (Perkin-Elmer, Norwalk, CT) with temperature control set for 25°C. Tissue antioxidant analysis: i. Tissue glutathione (GSH) sulfhydryl group content: Tissue (heart and liver) and RBC sulfhydryl group content (an indirect measure of GSH) was measured according to the method of Moron and coworkers (1979) Aliquots (200 pL) of tissue homogenate diluted 1:2 with cold 0.9% NaCI  with minor modifications. -  2 mM NaN 3 were treated  with lOOpL ice-cold 25% Trichloroacetic acid (TCA; Sigma, St. Louis, MO) followed by centrifugation at 1 2,000 x g, 4°C, for 5 mm. The supernatant was assayed for acid-soluble sulfhydryl groups at 41 2 nm using 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB; Sigma)  in 0.1 M phosphate buffer, pH 8.0.  Briefly, 200 pL cold supernatant was assayed in 0.96 mL 0.1 M phosphate buffer, pH 8.0 and the reaction started with 4OpL cold 3mM DTNBin 0.1 M phosphate buffer, pH 8.0. The absorbance was read at 412 nm after 10 minutes. RBC acid-soluble sulfhydryl groups were assayed in packed RBCs (50 pL) diluted into a 10% suspension in 0.9% NaCI wash with 0.9% NaCl  -  -  2mM NaN 3 and centrifuged (12,000 x g, 4°C, 5 mm) followed by a second  2 mM NaN . 3  The pelleted RBCs were then lysed with 50 pL cold double  distilled 2 H 0 , followed by the addition of 325 pL cold 5% TCA  -  1 mM Na -ethylenediamine tetraacetic 2 45  acid (EDTA), vortexed and centrifuged as above. The cold supernatant, 120 yL, was assayed in 1 .04 mL 0.1 M phosphate buffer, pH 8.0 and the reaction started with 40 pL cold 3 mM DTNB.  The  absorbance was read at 41 2 nm after 5 minutes. RBC and tissue GSH contents were calculated from standard curves using GSH (reduced form, Sigma) as a standard. ii. Tissue susceptibility to in vitro forced peroxidation: Tissue (heart and liver) susceptibility to in vitro forced peroxidation was determined on tissue homogenates incubated with various concentrations of hydrogen peroxide (H ) prepared in 0.9% 0 2 NaCI  -  , followed by determination of acid-soluble sulfhydryl groups as well as 23 2 mM NaN  thiobarbituric acid reactive substances (TBARs), an indirect measurement of lipid peroxidation. Aliquots (200 pL)  of tissue homogenates were incubated with an equal volume of various  0 (0, 0.05, 0.1, 0.2, 0.3, 0.5 and 1 .0 mM H 2 concentrations of H 0 in 0.9% NaCI 2 30 minutes at 37°C.  -  2 mM NaN ) for 3  The reaction was terminated by the addition of 100 pL cold 25% TCA and  centrifuged (12,000 x g, 4°C, for 5 mm) to obtain supernatant for use in the determination of acidsoluble sulfhydryl groups as above. Production of TBARs in tissue homogenates following incubation with H 0 was determined 2 according to Buege and Aust (1978)  with modifications. Aliquots of homogenate (400 pL) were  incubated with an equal volume of various concentrations of H 0 (1.0, 1 .5, 2.0, 3.0 and 5.0 mM 2 0 for heart and 1 .0, 5.0, 10.0, 15.0, 20.0, 30.0 and 40.0 mM H 2 H 0 for liver, in 0.9% NaCI 2  -  2  mM NaN ) for 30 minutes at 37°C. The reaction was terminated by 400pL cold 28% TCA -0.1 M 3 Na-arsenite followed by centrifugation at 1 2,000 x g, 4°C for 5 minutes.  An 800 pL aliquot of  supernatant was mixed with 400pL 0.5% 2-thiobarbituric acid (TBA; Sigma) in 0.025 M NaOH and heated in a boiling water bath for 1 5 minutes. When the tubes were cooled, the absorbance was read at 532 nm. The oxidation of intracellular RBC acid-soluble sulfhydryl group content in response to 0 was measured as follows: aliquots of packed RBCs (50 pL) were 2 increasing concentrations of H preincubated as 10% suspensions in 0.9% NaCI  -  2 mM NaN 3 for 5 mm  at 37°C. At the end of the  0 (0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 2 preincubation period, 500 pL of various concentrations of cold H and 1 .0 mM H 0 in 0.9% NaCI 2  -  2 mM NaN ) were added to the RBCs, followed by incubation at 3 46  37°C for 30 mm.  The reaction was terminated by centrifugation (12,000 x g, 4°C, 5 mm) and the  RBCs washed with fresh 0.9% NaCI  -  2 mM NaN 3 and centrifuged again as before. The packed RBCs  were lysed with 50 pL double distilled H 0 for determination of acid-soluble sulfhydryl groups using 2 DTNB as described above. Susceptibility of RBCs to in vitro peroxidation was determined by the production of malondialdehyde (MDA) and measured according to the method of Stocks and Dormandy (1971) with modifications as reported by Gilbert and coworkers (1984). Aliquots of packed RBCs (5OpL) were preincubated as 1 0% suspensions in 0.9% NaCI  -  2 mM NaN 3 for 5 mm  at 37°C. At the end of the  preincubation period, 500pL of various concentrations of cold H 0 (1.0, 2.0, 3.0, 4.0, 5.0, 10.0 and 2 20.0 mM H 0 in 0.9% NaCI 2 mm.  -  2 mM NaN ) were added to the RBCs and incubated at 37°C for 30 3  The reaction was terminated by the addition of 500 pL cold 28% TCA  -  0.1 M Na-arsenite.  Following centrifugation (12,000 x g, 4°C, 5 mm), a 1 .0 mL aliquot was removed and combined with 500pL 0.5% TBA in 0.025 M NaOH and heated in a boiling water bath for 15 mm. When the tubes were cooled, absorbances were read at 532 and 453 nm.  MDA content of RBCs was calculated  according to the method of Gilbert and coworkers (1984).  This method corrected the MDA  thiobarbituric acid derivative absorbance at 532 nm for interfering absorbance due to the presence of RBC hemolysate and reduced glutathione by subtracting 20% of the absorbance intensity at 453 nm. iii. Catalase (CAT) activity: CAT (EC.1 .11.1.6) activity in tissues and RBCs was assayed according to the method of Aebi (1974)  which measures absorbance at 240 nm.  where k is the first-order rate constant (sec  1)  Enzyme activity was expressed as k/mg protein,  Cytosolic CAT activity was determined by incubating  0.9 mL of tissue supernatant with 1 8 pL 50% diluted ethanol for 30 mm  on ice. To this mixture was  then added lOOpL 10% Triton-X100 in homogenizing buffer before vortexing thoroughly. From this solution, aliquots of samples (500 pL for heart and 50 pL for liver) were diluted to 10 mL with 50 mM phosphate buffer, pH 7.0 immediately prior to assaying. To perform the assay, 2.0 mL of diluted sample was added to a 3.0 mL cuvette and the reaction started by adding 1 .0 mL of freshly prepared 30 mM H 0 in 50 mM phosphate buffer, pH 7.0. After rapid mixing, the rate of decomposition of 2  47  0 was determined from the change of absorbance at 240 nm. The blank for this assay consisted 2 H of 2.0 mL diluted sample with 1 .0 mL 50 mM phosphate buffer, pH 7.0. Red blood cell CAT activity was determined on RBC hemolysate prepared as above. Sample aliquots (20 pL)  were diluted to 10 mL with 50 mM phosphate buffer, pH 7.0 and assayed as  described above.  Enzyme activity was expressed as kI g hemoglobin (Hb).  iv. Glutathione peroxidase (GSH-Px) activity: Tissue GSH-Px (EC.1.11.1.9) Valentine (1967)  activity was assayed according the method of Paglia and  and expressed on the basis of pmoles of nicotinamide adenine dinucleotide  phosphate (NADPH) oxidized to NADP per minute per mg protein using the extinction coefficient of NADPH at 340 nm of 6.22 x 106 pmole’ cm . Tissue cytosolic GSH-Px was assayed in a 1.5 mL 1 cuvette containing 800 pL 75 mM phosphate buffer, pH 7.0, to which was added 20 pL 60 mM reduced glutathione (GSH), 40 yL of 30 units/mL glutathione reductase (GSSG-Red) in 3 M ammonium , 4OpL 15mM Na 3 EDTA and 4OpL 3mM NADPH. Aliquots of cytosolic 2 sulphate, 2OpL 0.12 M NaN supernatants of samples diluted 1:2 with double strength Drabkin’s reagent (0.001 6 M KCN M 6 Fe(CN) 3 K  -  -  0.001 2  0.0238 M NaHCO ) were added to the cuvette (20, 40 and 60 pL) along with double 3  distilled H 0 to make up a final volume of 1 .1 6 mL. The reaction was started with 40 pL 7.5 mM H 2 0 2 in double distilled H 0 and the conversion of NADPH to NADP was monitored by continuous recording 2 of the absorbance change at 340 nm over 5 mm. Red blood cell GSH-Px activity was determined on RBC hemolysate diluted 1:10 with double distilled H 0. Diluted RBC hemolysate was then further diluted 1:2 with double strength Drabkin’s 2 reagent and assayed as above, with enzyme activity expressed as nmoles NADPH oxidized to NADP per minute per g Hb. v. Glutathione reductase (GSSG-Red) activity: Tissue GSSG-Red (EC.1 .6.4.2) activity was assayed by the method of Long and Carson (1961) and expressed as nmoles of NADPH oxidized to NADP per minute per mg protein. Tissue cytosolic GSSG-Red was determined in a 1.5 mL cuvette containing 400pL 0.45 M Tris -90 mM EDTA, pH 7.6, 200 pL 18 mM oxidized glutathione (GSSG) and tissue cytosolic supernatant (160 pL for heart and 0 to make up the volume to 1.16 mL. The reaction was started 2 4OpL for liver) plus double distilled H 48  with 40 pL 3 mM NADPH and the absorbance at 340 nm was recorded over 5 mm  to monitor the  conversion of NADPH to NADP. Red blood cell GSSG-Red activity could not be determined due to previously noted limitations encountered using this assay methodology (unpublished observation). vi. Superoxide dismutase (SOD) activity: Tissue SOD (EC. 1 .1 5.1 .1) activity was assayed according to the method of Winterbourn and coworkers (1975) and expressed as units of SOD per mg protein. One unit of SOD activity is defined as the amount of enzyme activity that causes 50% inhibition of nitroblue tetrazolium (NBT) reduction. Aliquots of tissue cytosolic supernatants (1 .5 mL for heart and 0.5 mL for liver) were extracted by adding double distilled H O (0.5 mL for heart and 3.5 mL for liver), 95% ethanol (0.5 mL for heart and 2 1 .0 mL for liver)  and chloroform (0.3 mL for heart and 0.6 mL for liver) to tissue supernatant and  vortexed vigorously after sitting on ice.  Once the supernatants were decolourized, the extraction  mixtures were centrifuged at 3,000 x g, 4°C for 5 mm. The resulting supernatant was then further centrifuged at 1 2,000 x g, 4°C for 5 mm to remove any sediment. The assay mixture consisted of 1 .0 mL 75mM phosphate buffer, pH 7.8, 200pLO.1 M Na EDTA- 1.5 mg% NaCN, lOOpL 1.5mM NBT 2 (stored in a dark bottle, 4°C) and various aliquots of prepared sample (0, 25, 50, 75, 100, 1 50, 200 and 500 pL for both heart and liver) plus double distilled H O to make up a final volume of 2.95 mL. 2 The reaction was started by the addition of 50 pL 0.12 mM riboflavin (stored in a dark bottle, 4°C) to the tubes and placement of the rack of tubes in a light box for illumination twice for 2.5 mm. The light box consisted of a five-sided wooden box painted inside with white paint with the open end of the same dimensions as the light fixture used to illuminate the test tubes. The tubes were vortexed vigorously before each illumination and at the end of the illumination with fluorescent light at a constant distance and protected from any extraneous room light.  The rate of inhibition of NBT  reduction by superoxide generated by photoreduction of riboflavin was determined by measuring the absorbance at 560 nm. Red blood cell SOD activity was determined on 500 pL RBC hemolysate to which was added O, 1.0 mL 95% ethanol and 0.6 mL chloroform for extraction of 2 3.5 mL double distilled H contaminating colour due to the presence of Hb. Once clarified, the extract was centrifuged (3,000 49  x g, 4°C for 5 mi n) and the supernatant used in the assay as above. Enzyme activity was expressed as units of SOD enzyme activity per g Hb. vii. Correction of enzyme activities for blood contamination: Since RBCs contain various amounts of activity of the antioxidant enzymes assayed herein, it was necessary to correct tissue cytosolic enzyme activity values for any contribution due to the presence of contaminating RBCs. For the determination of Hb content, RBC hemolysate and tissue cytosolic fractions were assayed for Hb by the method of Drabkin and Austin (1935). RBC hemolysate 0 before assaying for Hb. The assay tubes contained 1 .0 mL 2 was diluted 1 :10 with double distilled H each of 2.4 mM KCN and 1 .8 mM 6 Fe(CN) and the reaction was started by the addition of 0.5 mL 3 K diluted RBC hemolysate (plus 0.5 mL double distilled H 0) or 1.0 mL tissue supernatant for a final 2 volume of 3.0 ml. Absorbance was read at 540 nm after 30 mm. Statistics: All data are expressed as mean ± SEM. Student’s t-test for independent samples (SPSS Inc.) was used to test for differences between animal strains at a p regression analysis (SPSS)  0.05 significance level.  Linear  was performed to examine associations between tissue antioxidant  parameters.  50  Results: Animal growth and systolic blood pressure: Initial body weights of SHR animals (range 11 6 ± 1 g) were lower (p < 0.05) than for WKY counterparts (range 1 34 ± 6 g). Amount of body weight gained throughout the experimental period, however, was greater (p < 0.05) in SHR compared to WKY animals (Table 1.1), resulting in greater final body weights for SHR (data not shown).  Systolic blood pressure of animals measured at 13  weeks of age indicated that SHR were hypertensive compared to WKY animals (Table 1.1).  As  expected, there were no visible signs of atherosclerosis in aorta from SHR or WKY rats when examined using a dissecting microscope. Plasma lipid profiles: Both plasma total cholesterol and triacylglyceride concentrations were lower (p < 0.05) in SHR animals  compared to WKY counterparts  However,  plasma  phospholipid  In the RBCs, the activities of CAT and SOD were both greater (p < 0.05)  in the SHR  (Table  1.2).  concentrations were not different between animal strains. Red blood cell and tissue antioxidant enzymes: i. Red blood cell (RBC) antioxidant enzymes:  compared to WKY animals (Table 1 .3). The activity of a glutathione metabolizing enzyme, GSH-Px, in RBCs was not different between the SHR and WKY animals (Table 1 .3). RBC CAT activity was positively correlated with RBC SOD activity (r  =  0.636, p  to be positively correlated with systolic blood pressure (r  =  =  0.026) and RBC SOD activity was found 0.709, p  =  0.049) for SHR and WKY  animals. ii. Heart antioxidant enzymes: Heart tissue from both the SHR and WKY animals had very low levels of CAT activity. When the heart CAT activity was subsequently corrected for the contribution due to presence of contaminating RBCs in the cytosol prepared from heart tissue homogenate, CAT activity associated with heart cytosol alone was negligible (values not reported). Values for SOD activity in the heart were greater (p < 0.05) in SHR compared to WKY counterparts (Table 1 .4). Activities of GSSG-Red and GSH-Px in the heart were lower (p  0.05) and unchanged, respectively, in SHR compared to WKY 51  ai  *  0.133±0.004  0.199 ±0.003*  Feed efficiency 3 ratio  132± 14  197 ± 12*  Systolic blood pressure (mm Hg)  Values represent mean ± SEM, n = 8. Body weight gained from 6 to 14 weeks of age. Feed efficiency ratio = body weight gained/feed consumed from 6 to 14 weeks of age. SHR = Spontaneously hypertensive rat, WKY = Wistar Kyoto rat. denotes a significant (p 0.05) animal strain difference within a column.  97±6  WKY  2  155 ± 2*  Body wt. gain 2 (g)  SHR  Animal strain : 4  Parameter:  Table 1.1 Body weight gain and systolic blood pressure of SHR and WKY rats fed a standard chow diet . 1  0  01  *  0.640±0.031  0.335 ± 0.032*  Triacylglycerides  Values represent mean ± SEM, n = 8. SHR = Spontaneously hypertensive rat, WKY Wistar Kyoto rat. denotes a significant (p 0.05) animal strain difference within a column.  1.75±0.08  WKY  2  1.46 ± 0.03*  Total cholesterol  SHR  : 2 Animal strain  Plasma lipids: (mmol/L)  Table 1.2 Plasma lipids of SHR and WKY rats fed a standard chow diet . 1  1.28±0.06  1.17 ± 0.06  Phospholipids  C,,  *  2  4.20 ± 0.23  5.64 ± 0.34*  SOD (U/mg Hb)  Values represent mean ± SEM, n = 8. CAT = catalase, k = first-order rate constant (sec ); GSH-Px = glutathione peroxidase; 1 SOD = superoxide dismutase. SHR = Spontaneously hypertensive rat, WKY = Wistar Kyoto rat. denotes a significant (p 0.05) animal strain difference within a column.  52.0 ± 4.0  0.052 ± 0.002  WKY  1  60.0 ± 1.6  GSH-Px (nmoles NADPH/min/mg Hb)  0.061 ± 0.002*  CAT (k/mg Hb)  SHR  : 3 Animal strain  Antioxidant Enzyme : 2  Table 1.3 Antioxidant enzyme activities in red blood cells of SHR and WKY rats fed a standard chow diet.’  01  *  2  110 ± 5  114±8  GSH-Px (nmoles NADPH/ mm/mg protein)  Values represent mean ± SEMI n = 8. GSSG-Red = glutathione reductase; GSH-Px = glutathione peroxidase; SOD SHR = Spontaneously hypertensive rat, WKY = Wistar Kyoto rat. denotes a significant (p 0.05) animal strain difference within a column.  30.5 ± 0.2  WKY  1  19.6 ±0.6*  GSSG-Red (nmoles NADPH/ mm/mg protein)  SHR  Animal strain : 3  Antioxidant enzyme : 2  =  superoxide dismutase.  20.8 ± 1.1  28.1 ±0.9*  SOD (U/mg protein)  Table 1.4 Antioxidant enzyme activities in heart tissue of SHR and WKY rats fed a standard chow diet. 1  animals (Table 1 .4). Heart SOD activity was positively correlated with systolic blood pressure in SHR and WKY animals (r  0.727, p  =  0.041).  iii. Liver antioxidant enzymes: Liver tissue activities of CAT and SOD were greater (p 0.05) and not different, respectively, for SHR compared to WKY counterparts (Table 1 .5). Activities of GSSG-Red and GSH-Px in liver tissue were greater (p  0.05) in the SHR compared to WKY animals (Table 1 .5). Liver GSH-Px and GSSG  Red activities were found to be negatively correlated (r  -0.682, p  =  =  animals. Liver CAT and GSSG-Red activities were found to be positively (r negatively (r  =  -0.798, p  =  for SHR and WKY  0.015) =  0.781, p  =  0.022) and  0.018) correlated, respectively, with systolic blood pressure in SHR and  WKY animals. iv. Tissue and RBC glutathione (GSH) levels: Tissue and RBC levels of GSH are reported in Table 1 .6. For RBCs, heart and liver, the levels of acid-soluble sulfhydryl groups as GSH were consistently greater (p < 0.05) in tissue collected from SHR animals when compared to WKY counterparts (Table 1.6). Heart GSH was found to be negatively correlated with heart GSSG-Red activity (r correlated with liver GSH-Px activity (r  =  =  -0.801, p  -0.708, p  =  =  0.002).  0.010).  correlated with systolic blood pressure in SHR and WKY animals (r  Liver GSH was negatively  Also, liver GSH was positively =  0.796, p  =  0.018).  Tissue and RBC susceptibility to in vitro oxidative challenge: In order to determine tissue and RBC susceptibility to oxidative challenge, tissues (heart and liver homogenates) and RBCs were exposed to varying concentrations of hydrogen peroxide (H ) 0 2  in vitro and the depletion of GSH recorded. In addition, heart and liver TBARs and red blood cell MDA production was also evaluated as an indicator of susceptibility to in vitro lipid peroxidation.  The  profiles of the in vitro oxidative challenge of RBCs, heart and liver tissue are presented in Figures 1 .1, 1 .2 and 1 .3, respectively. i. RBC susceptibility to in vitro oxidative challenge: Susceptibility of RBCs to depletion of GSH was lower (p < 0.05) in SHR compared to WKY animals at concentrations of 0.4 and 0.5 mM H O (Figure 1.1A). 2  However, at the higher  concentrations of H 0 used to evaluate the production of MDA as an indicator of lipid peroxidation 2 56  C,,  *  2  93.9±8.3  69.3±6.0*  GSSG-Red (nmoles NADPH/ mm/mg protein)  202±24  118±11*  GSH-Px (nmoles NADPH/ mm/mg protein)  8. Values represent mean ± SEM, n CAT = catalase, k = first-order rate constant (sec’); GSSG-Red = glutathione reductase; GSH-Px = glutathione peroxidase; SOD = superoxide dismutase. SHR = Spontaneously hypertensive rat, WKY = Wistar Kyoto rat. denotes a significant (p 0.05) animal strain difference within a column.  0.318±0.040  WKY  1  0.426±0.040*  CAT (k/mg protein)  SHR  Animal strain : 3  .  Antioxidant enzyme : 2  236±24  278±19  SOD (U/mg protein)  Table 1.5 Antioxidant enzyme activities in liver tissue of SHR and WKY rats fed a standard chow diet . 1  01  *  2  1.76±0.09  1.98 ± 0.02*  Heart (nmoles/mg tissue)  3.94±0.31  5.36 ± 0.12*  Liver (nmoles/mg tissue)  8. Values are mean ± SEM, n GSH = glutathione Spontaneously hypertensive rat, WKY = Wistar Kyoto rat. SHR denotes a significant (p 0.05) animal strain difference within a column.  1.28±0.03  WKY  1  1.47 ± 0.04*  Red blood cells (nmoles/mg RBC)  SHR  : 3 Animal strain  : 2 Tissue GSH  Table 1.6 Tissue glutathione levels in SHR and WKY rats fed a standard chow diet . 1  in RBCs, the hypertensive rats exhibited greater (p  0.05)  MDA production at the added  concentrations of 3-5mM (Figure 1.1B). O 2 H ii. Tissue susceptibility to in vitro oxidative challenge: Depletion of GSH in heart tissue homogenate was not different between animal strains (Figure 1.2A). On the other hand, SHR animals had greater (ps. 0.05) levels of induced TBARs production 0 concentrations of 1 .5 2 compared to WKY counterparts at added H tissue homogenate from SHR animals had a greater (p  .  0.05)  -  3.0 mM (Figure 1 .2B). Liver  susceptibility to GSH depletion at  0 of 1 .0 and 5.0 mM compared to WKY animals (Figure 1 .3A). 2 levels of added H  However,  production of TBARs in liver tissue homogenates exposed to varying concentrations of H 0 was not 2 different between SHR and WKY animal strains (Figure 1.38).  59  0 0.00  20  40  60  0.10 0.20 0.40  mM H202 added  0.30  0.50 0.60  S  0, 4, 0  0  0 0.00  100  200  300  400  500  600  700  5.00  0) 0  15.00 mM H202 added  10.00  Figure 1 .1 Susceptibility of SHR and WKY red blood cells to in vitro H -iriduced oxidative challenge. 0 2 (A) Depletion of glutathione (GSH); (B) Production of malondialdehyde (MDA). * indicates a significant (p 0.05) animal strain difference. = SHR; A = WKY.  4,  4,  C 0  80  100  20.00  25.00  C 0  0 0.00  20  40  60  0.25 0.75  mM H202 added  0.50 1.00 1.25  LO  C’,  0.00 0.00  0.13  0.25  0.38  0.50 B  1.00  -  0)  *  3.00 mM H202 added  2.00  -induced oxidative challenge. 0 2 Figure 1 .2 Susceptibility of SHR and WKY heart tissue homogenate to in vitro H thiobarbituric reactive Production acid of substances (TBARs). (GSH); glutathione of (B) (A) Depletion * difference. A = strain SHR; = WKY. animal (p 0.05) indicates a significant  .  0. 4,  4,  4-  80  100  4.00  C 0  0 0.00  20  40  60  1.00 3.00 4.00  mM H202 added  2.00 5.00  6.00  to  (.4  0.00 0.00  0.50  1.00  1.50  2.00  10.00  0) F’.)  30.00 mM H202 added  20.00  Figure 1.3 Susceptibility of SHR and WKY liver tissue homogenate to in vitro H -induced oxidative challenge. 0 2 (A) Depletion of glutathione (GSH); (B) Production of thiobarbituric acid reactive substances (TBARs). * indicates a significant (p 0.05) animal strain difference. A = SHR; A = WKY.  0 0. C V  4-  80  100  40.00  50.00  Discussion: Clinical and epidemiological studies investigating the interaction of CVD risk factors such as hypertension, smoking and dyslipidemia are suggestive of a role for in vivo lipid peroxidation in the initiation of atherosclerosis (Buczynski et al., 1993; Jayakumari et a!., 1992; Sagar et a!., 1992; Gey eta)., 1991). Lipid peroxidation is involved not only in oxidation of plasma components such as LDL, but also may be involved in damage resulting in increased permeability of the aortic endothelium to macromolecules such as oxidized LDL (Wu eta!., 1990). The SHR exhibits several characteristics that suggest the involvement of reactive oxygen species (ROS) and lipid peroxidation in the development of hypertension in this animal model (Ito et a!., 1992; Toni eta!., 1 992; Janero and Burghardt, 1988, 1989). As yet, an analysis of the/n vivo antioxidant capacity of SHR tissues (i.e. heart and liver) and red blood cells has not previously been performed with animals fed a basal, nonatherogenic diet. Animal growth, systolic blood pressure and plasma lipids of SHR and WKY animals: Spontaneously hypertensive rats exhibited greater body weight gain and feed efficiency on a standard chow diet compared to normotensive Wistar Kyoto (WKY) rats in the present experiment, despite having lower initial body weights. The hypertension observed in the SHR animals at 13 weeks of age is consistent with previous findings in both our laboratory (Kitts et a!., 1992) (Janero and Burghardt, 1988; Jones et a!., 1988). triacylglycerides)  and others  Plasma lipids (i.e. total cholesterol and  were lower in chow-fed SHR animals compared to WKY counterparts.  Mon and  coworkers (1993) reported a similar result with SHR and WKY fed extremely high levels of saturated dietary fat (25% suet)  and cholesterol (5% cholesterol, 2% cholate) in the diet.  Elevated plasma  triacylglycerides with no difference in cholesterol levels have been observed in hypertensive, streptozotocin (STZ) -induced diabetic SHR relative to diabetic WKY animals (Rodrigues and McNeilI, 1986). The differences in plasma triacylglyceride and cholesterol levels observed between SHR and WKY rats in the present study likely represent differences in lipid metabolism between these two inbred strains of rat. It is generally recognized that the initiation of atherosclerosis as a result of cholesterol deposition into aortic luminal tissue is potentiated by hypertension (Sowers, 1992; Dzau, 1990). Thus, the absence of any visible signs of atherosclerosis in hypertensive SHRs in the present study is consistent with the relative resistance of this animal species to atherosclerosis. 63  RBC and tissue antioxidant status: Previous workers have reported several differences in the endogenous antioxidant status between the SHR and its normotensive counterpart, the WKY rat (Ito et a!., 1992; Toni et al., 1 992; Janero and Burghardt, 1 989, 1988). In the present study, SHRs exhibited elevated levels of CAT and SOD activities in RBCs compared to WKY animals, while the activity of the glutathione metabolizing enzyme, GSH-Px, was not different between animal strains. The increased activity of CAT in SHR RBCs may indicate up-regulation of this enzyme’s activity, possibly to compensate for the increased 0 generated from SOD activity. The positive correlation of the activities of these two 2 production of H enzymes in the RBCs would be consistent with such a possibility.  Increased RBC SOD activity  suggests a greater requirement for antioxidant enzyme protection against the production of ROS and in vivo lipid peroxidation in SHR animals. The antioxidant capacity of the SHR myocardium is of special interest, given the previous reports of hypertrophy of this tissue in the SHR (Kashli et a!., 1977). While heart tissue activities of CAT were below the level of detection, SOD activity was greater in SHR than WKY. paralleled the situation in SHR RBCs above.  This result  This finding may indicate a generalized compensatory  antioxidant enzyme response to enhanced levels of ROS production in SHR tissues. Previous workers have reported greater heart GSH-Px activity in SHR at 6 weeks of age and lower activity in SHR at 1 6 weeks of age (Ito eta!., 1992). The results of the present study in 14 week old animals are likely intermediate in the temporal pattern of GSH-Px activity. While the activity of heart tissue GSH-Px was not different between the two strains of rat herein, GSSG-Red activity was reduced in the SHR. Moreover, the activity of GSSG-Red was found to be inversely related to heart GSH content, suggesting an adaptive relationship, wherein when tissue GSH levels are high, less GSSG-Red activity is required, but if tissue GSH content is reduced, an adaptive increase in GSSG-Red activity may occur. The liver exhibited an antioxidant enzyme status quite different from the heart and RBCs. Antioxidant enzyme activities in general were much greater in the liver compared to the heart and RBCs. Liver tissue activities of CAT were greater in SHR compared to WKY, while the activity of SOD was not different. Both liver GSH-Px and GSSG-Red activities were reduced in SHR compared to WKY animals, with GSH-Px activity negatively correlated with liver tissue GSH content, suggesting a 64  protective effect of the greater levels of nonenzymatic antioxidant GSH content against tissue lipid peroxidation in the liver. When RBCs and heart and liver tissues were subjected to in vitro peroxidative challenge, marked differences in tissue responses were evident. Susceptibility of heart tissue to GSH depletion did not differ between animal strains, but, production of TBARs was greater in SHR animal heart tissue compared to WKY. Similar findings of enhanced susceptibility of SHR myocardium to lipid peroxidation have been noted by others using a variety of in vitro oxidation systems, such as Fe /ascorbate (Ito 2 eta!., 1992), xanthine oxidase (superoxide radical production; Janero and Burghardt, 1989) or in vivo administration of doxorubicin, a cardiotoxic drug (Toni eta!., 1992). In all these cases, formation of lipid peroxidation products (i.e. TBARs or MDA) membrane preparations.  was enhanced in SHR heart tissue or isolated  The antioxidant enzyme analyses in the present study indicate that the  greater susceptibility of the SHR myocardium to oxidative damage may be due to the presence of higher levels of H 0 resulting from the greater SOD activity in the SHR heart, in the presence of 2 vanishingly low levels of CAT activity. When RBCs were subjected to similar in vitro forced peroxidation conditions, depletion of GSH was lower in SHR animals, while production of MDA, at higher levels of pro-oxidant was enhanced in SHR RBCs. These seemingly contradictory results can be explained on the basis that at the lower 0 used in the GSH depletion assay, the elevated levels of GSH in the RBCs of the 2 concentrations of H SHR were sufficient to inactivate the peroxidizing agent. The greater SOD activity in RBCs from SHR animals (as an in vivo source of 2 H ) 0 , could contribute to the greater production of MDA in RBCs from SHR animals. While both the heart and RBCs from SHR demonstrate a greater susceptibility of these tissues to lipid peroxidation, in vitro oxidative challenge of liver tissue was quite different from these two tissues. Depletion of liver tissue GSH was greater in SHR animals than WKY, despite the greater level of tissue GSH in SHR animals. On the other hand, production of TBARs from liver tissue was not different between these two animal strains. The lower activities of the GSH metabolizing enzymes in SHR liver tissue may be responsible for the enhanced depletion of GSH observed in the SHR. The reduced activity of GSH-Px for inactivating peroxides, in association with reduced regeneration of GSH 65  reducing equivalents by GSSG-Red enzyme activity, would be consistent with the enhanced GSH depletion observed in the liver tissue of SHR animals. In contrast, the higher concentrations of H 0 2 used in the TBARs assay not only exhausted the supply of GSH in the liver tissue, but may also have inactivated antioxidant enzymes by binding to enzyme protein moieties resulting in similar production of TBARs in liver tissue from SHR and WKY. The  endogenous  antioxidant status  has  been  similarly  investigated  in  clinical  and  epidemiological studies with subjects exhibiting various risk factors of CVD, including hypertension and atherosclerosis. Hypertensive patients have been reported to have reduced levels of ascorbic acid as well as SOD activity in diseased aortae compared to normal tissue from normotensive controls, and it has been suggested that the reduced antioxidant capacity of aortic tissue in hypertensive subjects may increase the susceptibility of aortic tissue to lipid peroxidation (Hunter at a!., 1991). Interestingly, in the present study increased SOD activity was noted in the RBCs and heart of the SHR, indicating possible tissue- or species-related differences between these results and those of other workers. Studies which have investigated the role of oxygen-derived free radicals in CVD reported that PMN chemiluminescence (a measure of ROS production) was increased in hypertensive patients (Sagar et a!., 1992).  These workers also showed that levels of GSH and SOD activity in PMN were both  inversely correlated with the degree of hypertension in these patients.  Conversely, in the present  study, RBC and heart SOD activities showed a positive correlation with systolic blood pressure, possibly reflecting a compensatory response to a hypertension-related increase in oxidative stress in the SHR. More recently, Buczynski and coworkers (1993)  reported that platelets from patients with  coronary artery disease (CAD) exhibited increased aggregability (which may predispose to atheroma formation)  in association with increased levels of cellular TBARs, while platelet activities of the  antioxidant enzymes SOD, CAT and GSH-Px were reduced. Furthermore, Jayakumari and coworkers (1992)  found that lipid peroxidation products (i.e. MDA and conjugated dienes)  were elevated in  patients with CAD, and the extent of elevation of plasma lipid peroxides appeared to increase with increasing severity of CAD. Although RBC activities of CAT and GSH-Px in these patients with CAD were normal, SOD activity was decreased (Jayakumari eta!., 1992). Those patients with CAD who 66  were also diabetic exhibited the greatest levels of plasma lipid peroxides compared to smokers and hypertensive patients. Taken together, these findings suggest that subjects which have several risk factors for CVD and exhibit atherosclerotic vascular changes have a reduced tissue antioxidant capacity and thereby, increased levels of tissue lipid peroxides. In the present study, while the SHR animal model exhibited hypertension, there were no visible signs of atherosclerosis. This finding is consistent with the observation that rats generally do not develop atherosclerosis easily (Bishop, 1980) and in some cases only when fed a highly atherogenic diet (Yamori eta!., 1975). Other considerations of hypertension in SHR compared to WKY: The SHR has also been reported to be a model of non-diabetic insulin resistance with secondary hyperinsulinemia compared to its WKY counterpart (Hulman et a!., 1993; Finch et a!., 1990).  In  studies investigating the effect of insulin on in viva antioxidant enzyme activity, Long and Carson (1961)  demonstrated that insulin was inhibitory to GSSG-Red activity of RBCs.  Patients with  insufficient plasma insulin levels exhibited elevated GSSG-Red activity in red blood cells (Long and Carson, 1961).  Hypertensive SHR animals exhibited reduced levels of GSSG-Red activity in heart  tissue in the present study, which may be related to their relative insulin resistance and hyperinsulinemia (Hulman eta!., 1993).  67  Conclusion: In summary, hypertension in the SHR fed a normal (commercial rat chow) diet was associated with alterations in antioxidant profiles of RBC, liver and heart tissues, the latter also showing an increased susceptibility to in vitro lipid peroxidation.  The plasma cholesterol and triacylglyceride  concentrations of the SHR were lower in comparison to WKY counterparts.  Underlying metabolic  factors that may directly or indirectly regulate in vivo antioxidant status and lipid metabolism appear to reflect the genotype of the hypertensive SHR animal model, compared to its normotensive WKY counterpart.  Further experiments will explore the influence of dietary lipid source and cholesterol  content on the antioxidant status and lipidemia in hypertensive SHR and normotensive WKY rats.  68  CHAPTER 2 Effect of saturated and polyunsaturated dietary fat sources on systolic blood pressure, plasma lipids and antioxidant status in spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats. Introduction: Hyperlipidemia, characterized by elevated levels of plasma cholesterol and/or triacylglyceride, is a primary risk factor in the development of atherosclerosis. It is recognized that a 1 % decrease in plasma cholesterol is associated with a 2% decrease in the incidence of IHD in middle aged men (Lipid Research Clinic Program, 1 984). Therefore, much research has investigated the role of dietary fat intake in modulating dyslipidemias in humans (Kris-Etherton eta!., 1993; Denke and Grundy, 1992; Bonanome and Grundy, 1988; Mattson and Grundy, 1985; Hegsted eta!., 1965; Ahrens eta!., 1957) as well as animal studies (Levy and Herzberg, 1995; Toda and Oku, 1 995; Fernandez and McNamara, 1994, 1991; De Schrijver eta!., 1992; Mott eta!., 1992; Hayes eta!., 1991; Faidley eta!., 1990). The intake of saturated versus polyunsaturated (n-6 and n-3)  fat sources has been linked to the  development of hypercholesterolemia (Keys eta!., 1957). Diets high in monounsaturated fatty acids and marine oil  long-chain PUFA have been found to be relatively hypocholesterolemic and  hypotriacylglyceridemic, respectively (Bairati eta?., 1992; Mattson and Grundy, 1985). As well, the role of dietary cholesterol intake and its interaction with dietary fat source in determining plasma lipid levels have been investigated (Liu et a?., 1 995; Smit et a?., 1994; Fungwe et a!., 1993; Mott et a!., 1992; Mahley and Holcombe, 1977). In addition to control of plasma lipid levels, dietary fat source has also been found to influence blood pressure in the treatment of cardiovascular disease (H SF, 1993). The influence of dietary fatty acid composition on blood pressure consists primarily of effects on the biosynthesis of vasoactive agents following the elongation and desaturation of essential fatty acids. Thus, consumption of marine oils has been found to be effective in reducing blood pressure in hypertensive patients (Bairati et a!., 1992). However, the consumption of such highly unsaturated fatty acids in the diet may increase the oxidative load of plasma lipid constituents and membrane lipids in vivo. Studies have indicated not only that lipid oxidation cannot be completely inhibited by the addition of antioxidants to fish oil diets 69  (Gonzalez et a!., 1992), but also that tissue levels of lipid oxidation products are enhanced in animals fed diets containing marine oil (SküladOttir et a!., 1994; L’Abbé at at., 1991; Nalbone et a!., 1989). In addition, some controversy exists as to whether or not the incorporation of marine oil n-3 PUFA into plasma LDL increases the susceptibility of these particles to lipid peroxidation (Suzukawa et at., 1995; Frankel at a!., 1994). This issue is especially important from the standpoint that oxidation of LDL is thought to play a significant role in the development of atherosclerosis.  The susceptibility of  membrane lipids and plasma lipoproteins to lipid oxidation will depend on in vivo antioxidant status as influenced by dietary fatty acid composition and overall nutrition (Xia et at., 1 995; Hum et a!., 1 992; Bauman eta!., 1988a,b). The spontaneously hypertensive rat (SHR) developed by Okamoto and Aoki (1963) has been used in a variety of metabolic studies as an animal model of human essential hypertension. Previously in Chapter 1, the SHR was demonstrated to differ from the normotensive WKY rat in the activity of several tissue antioxidant enzymes, as well as in the susceptibility of tissues to in vitro oxidative challenge. Moreover, plasma lipid profiles of the chow-fed SHR were observed to differ from those of WKY counterparts.  The influence of dietary fat source and cholesterol level on plasma lipids,  systolic blood pressure and antioxidant status of the SHR compared to WKY animals may provide further insight into the effects of dietary fatty acid composition on cholesterolemia, hypertension and susceptibility of tissues to lipid oxidation.  70  Hypotheses for Chapter 2: i. Genetic predisposition for a risk factor of atherosclerosis, namely hypertension, can be modulated by diet. ii. Dietary fat source and level of cholesterol may have independent, or possibly interactive effects on hyperlipidemia and associated changes in endogenous antioxidant status in hypertensive animals. Obiective for Chapter 2: To determine whether dietary fat source (i.e. saturated versus n-6 or n-3 polyunsaturated lipids) and level of cholesterol act independently, or interact to alter the plasma lipid profile and endogenous antioxidant status of spontaneously hypertensive (SHR) and normotensive, Wistar Kyoto (WKY) rats. Specific Aims for Chapter 2: i. SHR and WKY rats will be used to investigate the effects of different dietary fat sources and levels of cholesterol intake on plasma lipid profiles. ii. The relative effect of atherogenic diets on specific tissue antioxidant enzymes and susceptibility of tissues to H -induced glutathione depletion and lipid peroxidation in vitro will be 0 2 determined in SHR and WKY rats.  71  Materials and Methods: Animals: Sixty-four, six week old, male spontaneously hypertensive rats (SHR)  and sixty-four  normotensive Wistar Kyoto (WKY) rats (Charles River, Montreal, PQ) were randomly divided into eight dietary treatment groups differing in dietary fat source (i.e. butter, beef tallow, soybean oil, and menhaden oil) and cholesterol level (i.e. low, 0.05% (wt/wt) and high, 0.5% (wt/wt) cholesterol). Formulation of semi-synthetic diets: The compositions of semi-synthetic diets used in this study varied in both lipid source and cholesterol content and are detailed in Table 2.1. Each dietary fat source was obtained in sufficient quantities of single batches from respective suppliers to ensure consistency of fatty acid composition in the present, and later experiments. A basal diet, containing 3% canola oil (Neptune Food Services, Richmond, B.C.) to provide an adequate supply of essential fatty acids was formulated with thorough mixing of ingredients prior to the addition of experimental dietary fat sources and sterols. Dietary fat sources consisted of non-salted butter (Dairyworld Foods, Burnaby, B.C.), beef tallow (Cargill Foods, High River, AB), soybean oil (Bioforce Canada, Burnaby, B.C.)  or menhaden oil (Zapata Haynie,  Reedville, VA; Table 2.1). No additional antioxidants were added to diets, with the exception of the vitamin E that was present as a component of the vitamin mixture. Both the butter and tallow fats were liquefied using a short time mild heat (10-15 mm at 45-50°C) treatment to facilitate the uniform -  distribution of cholesterol and cholic acid (2:1 ratio, to ensure maximal absorption of dietary cholesterol) into diets. No heat treatment was necessary for the soybean or menhaden oils. Sterols were added slowly to the liquefied experimental fat sources and thoroughly mixed to ensure uniform incorporation. Dietary fats containing sterols at levels reported in Table 2.1 were slowly added to the basal diet during reblending and mixed in completely using a Hobart mixer with an aluminum bowl over a period of approximately 20-25 minutes. After mixing, individual diets were stored in double, dark plastic bags in a walk-in freezer (-15°C)  throughout the experimental study.  A sample of each  experimental diet was removed for analysis of fatty acid, gross energy and dry matter content.  72  Table 2.1 Composition of diets fed to SHR and WKY rats . 1  Cholesterol level (% by weight)  0.05  0.5  Dietary component (gil OOg) 2 Casein Ca-free mineral mix 2 3 CaCO Vitamin mix 2 4 DL-methionine Choline chloride 5 Cornstarch 6 6 Sucrose 2 Alphacel 5 Monofos Canola oil 6 Dietary fats: Butter, beef tallow, 7 soybean or menhaden oil 2 Cholesterol Cholic acid 2  1  25.0  25.0  3.5 2.0 3.0 0.3 0.2  3.5 2.0 3.0 0.3 0.2  47.0 3.0 5.0 3.0  47.0 3.0 5.0 3.0  3.0  3.0  5.0 0.05 0.025  5.0 0.50 0.25  Gross energy (kJig) of diets: low cholesterol, butter (16.84), tallow (16.88), soybean (16.97) and menhaden oil (17.65); high cholesterol, butter (17.13), tallow (17.21), soybean (17.09) and menhaden oil (17.58). Dry matter content of all diets ranged from 89 93%. ICN Biochemicals Inc., Cleveland, OH, USA. BDH Chemicals, Toronto, ON, Canada. United States Biochemical Co., Cleveland, OH, USA. Van Waters & Rogers, Abbotsford, B.C., Canada. Neptune Food Services, Richmond, B.C., Canada. Butter (Dairyworld Foods, Burnaby, B.C.); beef tallow (Cargill Foods, High River, AB); soybean oil (Bioforce Canada, Burnaby, B.C.); menhaden oil (Zapata Haynie, Reedville, VA). -  2  6  73  Experimental fats were added to the basal diet at a level of 5% to make a final calculated fat content of 8% dietary fat. This level of fat in the diets meets the nutritional recommendations for the laboratory rat (eg. 5-10%) as outlined by the National Academy of Sciences (1978) and the Canadian Council on Animal Care (Vol.2, 1984).  The amount of butter fat added to experimental diets was  calculated to take into account the amount of moisture naturally present in processed butter (approximately 20% moisture). The levels of cholesterol incorporated into diets were 0.05% and 0.5% (wt/wt)  for the low cholesterol and high cholesterol diets, respectively.  The level of 0.05%  cholesterol was chosen to equalize all low cholesterol diets for the sterol content naturally present in butter (approx. 0.2%; Jensen and Clark, 1988) and beef tallow (approx. 0.1 %) sources. Analysis of the diets for cholesterol confirmed the expected uniform content of sterol in both individual low cholesterol and high cholesterol diets.  Experimental diets were isonitrogenous and contained a  comparable level of energy (e.g. 16.84- 17.67 kJ/g; Table 2.1). Diet gross energy determination: Energy content of all experimental diets was determined using a bomb calorimeter and was corrected for dry weight of diet (Miller and Payne, 1959). The energy value represents the amount of heat, measured in calories, released upon complete oxidation of the sample. Dietary fatty acid analysis: Samples were extracted with Folch’s reagent (Folch et a!., 1957), methylated with BF 3 (Nwokolo et a!., 1988)  and analyzed for component fatty acids using a Varian Model 3700 gas  chromatograph equipped with a 60 m X 0.53 mm i.d. column coated with 0.25 p Supelcowax 10 (Supelco, Bellefont, PA).  The internal standard included in these fatty acid analyses was C17:0  (Supelco). Fatty acid content of diets is summarized in Table 2.2. Animal feeding: The meal feeding protocol used in Chapter 1 and in previous studies from this laboratory (Yuan and Kitts, 1 994; Kitts et a!., 1 992)  was also used in this experiment. The meal feeding schedule  ensures a similar postprandial time period in animals before blood pressure measurements are performed. All diets were replaced daily to minimize autoxidation of dietary lipids during the 6 hour feeding period. 74  Table 2.2 Fatty acid composition of diets fed to SHR and WKY rats. Diets Fatty acid  Butter  Beef tallow  Soybean oil  Menhaden oil  Area % 14:0 16:0 16:1(n-7)  8.4 25.2 1.2  2.9 21.0 2.5  0.3 9.1 0.2  4.6 13.4 6.7  18:0 18:1(n-9) 1 8:1 (isomers) 1 8:2(n-6) 1 8:3(n-3)  9.0 34.6 1.7 8.6 3.1  12.9 47.8 2.3 7.5 2.7  4.3 47.6 3.4 15.3 2.7  3.2 33.1  0.4 0.8  0.4 1.6 0.5 7.3  20:0 20:1 20:4(n-6) 20:5(n-3) 22:0 22: 5(n-3) 22:6(n-3)  13.3 4.7  0.9 1.2 5.5 42.7 37.5 11.7  36.8 52.7 10.2  14.1 52.1 17.9  22.5 41.4 32.8  n-6 n-3  8.6 3.1  7.5 2.7  15.3 2.7  13.8 18.7  P/Si  0.3  0.3  1 .3  1.5  Saturates Monounsaturates Polyunsaturates  1 -  P/s = polyunsaturated fatty acid! saturated fatty acid ratio. denotes not detected.  75  Blood pressure measurements: Systolic blood pressure recordings were taken using a tail-cuff sphyngomanometer (Harvard Apparatus) between 15:00  -  17:00 hr in SHR and WKY rats fed semi-synthetic diets, as reported in  Chapter 1. Experimental procedures: After 8 weeks on the experimental diets, non-fasted animals were sacrificed by exsanguination under halothane anaesthesia at 1 3:00 hr. Animals were sacrificed in a non-fasted state to minimize changes to antioxidant status due to possible stresses of fasting as well as any uncontrolled nutritional effect on antioxidant levels attributed to diet withdrawal. Blood and tissues (i.e. heart and liver) were collected and prepared for plasma lipid analysis and antioxidant status, respectively, as previously described in Chapter 1. Analytical methods: Plasma lipids were quantified using the procedures given in Chapter 1. Similarly, all enzymatic and nonenzymatic antioxidant measurements made in RBC, heart and liver tissues were performed according to the methods described in Chapter 1. Statistics: All data are expressed as mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA; SPSS Inc.)  was used to test for differences between experimental treatments.  Where differences did exist, the source of the differences at a p  0.05 significance level was  identified by the Student-Newman-Keuls multiple range test. Three-way multiple analysis of variance (MANOVA) was used to identify any interactions between animal strain, dietary cholesterol level and dietary fat source. In order to separate treatment differences due to animal strain, dietary fat source or dietary cholesterol level effects, treatment groups were pooled and subjected to a two-tailed, Student’s t-test for independent samples for animal strain (SHR versus WKY) and dietary cholesterol level (0.05% versus 0.5%) or one-way ANOVA for dietary fat source (butter, tallow, soybean oil and menhaden oil) differences. Linear regression analysis (SPSS) was performed to examine associations between tissue antioxidant parameters.  76  Results: Fatty acid content of semi-synthetic diets: Fatty acids with chain lengths of less than 14 were not determined in the present analysis (Table 2.2). The absence of this information is important only for butter, which is noted to contain approximately 15% of total fatty acids as chain lengths of C:4 to C:1 2 (Kitts and Jones, 1995; Jensen and Clark, 1988). The weight percentage of myristic (C14:O) acid was greater in butter diets than in diets containing beef tallow, soybean or menhaden oils. Butter and beef tallow diets contained similar amounts of palmitic (C16:0) and stearic (C18:O) acids, which were greater than those found in soybean and menhaden oil diets. Beef tallow and menhaden oil diets contained very similar amounts of oleic (C18:1,n-9) acid, which were greater than those found in butter and soybean oil diets; these latter two diets also contained very similar amounts of Cl 8:1.  Soybean and menhaden oil diets  contained similar quantities of linoleic (Cl 8:2,n-6) acid which, in turn, were greater than in butter and beef tallow diets.  The content of linolenic (C18:3,n-3)  acid was greater in menhaden oil diets  compared to butter, beef tallow and soybean oil diets. The menhaden oil diets were unique in their content of eicosapentaenoic (C20:5,n-3; EPA) and docosahexaenoic (C22:6,n-3; DHA) acids. On the basis of the fatty acid analyses performed, the calculated polyunsaturated to saturated fatty acid (PIS) ratios for butter and beef tallow diets were both 0.3, while that of soybean oil diets was 1 .3 and menhaden oil diets was 1 .5. The ratio of n-6 to n-3 fatty acids was 2.8 for butter and beef tallow diets, 5.7 for soybean oil diets and 0.86 for menhaden oil diets (Table 2.2). Animal growth when fed semi-synthetic diets: Body weight gain and systolic blood pressure measurements for SHR and WKY rats are summarized in Table 2.3. Initial body weights of animals were not different between animal strains (SHR range 156 ± 2 g; WKY range 150 ± 2 g). Body weight gain during the experimental period was not influenced by animal strain or dietary cholesterol level. However, body weight gain was influenced by dietary fat source (p < 0.05), as animals fed menhaden oil diets gained less weight than other dietary fat groups (Table 2.3).  The feed efficiency ratio (FER)  of SHR and WKY animals was  influenced (p < 0.05) by both animal strain and dietary fat source as demonstrated by the interaction recorded. SHR animals had lower (p < 0.05) FER values than WKY counterparts. Animals fed 77  03  3  2  133± 9  131 ± 5  Body wt gain  N.S.  N.S. N.S. <0.001  137±11  126 ± 8  167± 7  156± 7  0.162±.009  0.157 ± .004  0.176±.010  0.167±.008  0.198±.008  0.190 ± .007  0.198±.008  0.204 ± .008  SHR WKY  0.190±.011  0.175 ± .012  0.228±.009  0.228±.013  0.236±.008  0.220 ± .008  0.220±.006  0.224 ± .008  0.023  <0.001 N.S. <0.001  FER  FER  =  140± 7  Feed efficiency ratio  N.S.  <0.001 N.S. N.S.  =  feed  133 ± 10  134± 8  128± 7  140± 6  125 ± 6  131 ± 5  Systolic blood pressure  190± 6  194 ± 6  187± 8  180± 6  186± 8  187 ± 10  174± 4  147 ± 7  Systolic blood 2 (mmHg) pressure SHR WKY  197 ± 10  Values represent mean ± SEM, n = 8. SHR = Spontaneously hypertensive rat, WKY = Wistar Kyoto rat; FER intake (g)/body weight gained (g) during 8 week feeding study. Systolic blood pressure measured in animals at 1 3 weeks of age. A = animal strain effect, C = cholesterol intake level effect, F = dietary fat source effect by 3-way MANOVA; treatment interactions by 3-way MANOVA.  A x F  MANOVA p-value 3  A C F  3 ANOVA p-value  0.5%chol.  Menhaden 0.05% chol.  0.5% chol.  164± 7 163±10  167± 8  0.5% chol.  154 ± 7  154± 9  157 ± 10  WKY  154± 4  166 ± 3  172± 7  0.5% chol.  0.05% chol.  168 ± 5  0.05% chol.  Soybean 0.05% chol.  Tallow  Butter  Dietary Treatment:  SHR  Body wt gain (9)  Table 2.3 Body weight gain and systolic blood pressure of SHR and WKY fed experimental diets. 1  menhaden oil diets exhibited reduced (p  0.05) FER values compared to other dietary fat groups and  this was especially apparent in WKY animals fed menhaden oil. Liver and heart organ weights: Liver weights of SHR were greater (p  0.05)  than in WKY animals for both the low  cholesterol diets (range 12.1 ± 0.1 versus 10.1 j 0.1 g) and high cholesterol diets (19.0± 0.1  versus 1 5.4  ± 0.1 g). Dietary cholesterol level, but not fat source, had a significant (p < 0.05) effect  on liver weight. Heart weights of SHR (range 0.9 ± 0.1 g) and WKY (range 1.0 ± 0.1 g) were not significantly different and were not affected by dietary fat source or cholesterol content. Systolic blood pressure of SHR and WKY animals: Systolic blood pressure measured at 13 weeks of age confirmed that SHR animals were hypertensive (p < 0.05) compared to WKY counterparts. Systolic blood pressure was not affected by dietary fat source or cholesterol level (Table 2.3). Plasma lipid profiles: Plasma lipids of SHR and WKY rats are presented in Table 2.4. Both plasma free and total cholesterol concentrations were significantly (p < 0.05) affected by animal strain, dietary cholesterol intake level as well as dietary fat source (Table 2.4). SHR animals had lower (p and total cholesterol levels compared to WKY counterparts (Table 2.4). containing 0.5% cholesterol exhibited increased (p  0.05) plasma free  Also, animals fed diets  0.05) levels of plasma free and total cholesterol,  compared to counterparts fed diets containing 0.05% cholesterol, as expected. Animals fed menhaden oil diets had reduced (p  0.05)  levels of plasma free cholesterol compared to other dietary fat  groups, regardless of dietary cholesterol intake level, as demonstrated by the interactions recorded (Table 2.4). Plasma total cholesterol concentrations of SHR animals were not affected by dietary fat source. Also, plasma total cholesterol levels of WKY animals fed 0.05% cholesterol diets were not affected by dietary fat source.  However, in WKY rats fed 0.5% cholesterol diets, plasma total  cholesterol was greatest in tallow-fed animals and lowest in the menhaden oil-fed group.  These  observations demonstrate the interaction recorded between animal strain, dietary fat source and cholesterol level for plasma total cholesterol concentrations. significantly (p  Plasma triacylglyceride levels were  0.05) influenced by dietary cholesterol intake level and dietary fat source (Table 79  C  0.11  0.94 +  0.56 ± 0.04  2  WKY  3.68 +  3.04 +  4.14 +  2.96 +  6.04 +  2.98 +  4.90±  2.82±  <0.001 <0.001 <0.001 <0.00 1  <0.001 <0.001 <0.001  Total cholesterol  1.92 ± 0.13  1.68 ± 0.05  1.89 ± 0.09  1.75 ± 0.09  2.01 ± 0.11  1.84 ± 0.07  1.98 ± 0.05  1.71 ± 0.05  SHR  Total cholesterol  0.34  0.08  0.25  0.09  0.34  0.09  0.20  0.10.  0.43 +  0.46 +  1.07 +  1.11 +  1.03 +  1.38 +  0.96±  1.33 ±  SHR  0.41 +  0.52 +  0.96 +  1.50 +  0.88 +  1.27 +  0.78±  1.29 ±  0.034 N.S. 0.022 N.S.  N.S. <0.001 <0.001  Triacylglycerides  0.06  0.05  0.11  0.10  0.08  0.12  0.06  0.07  WKY  Triacyiglycerides  0.05  0.05  0.06  0.09  0.08  0.15  0.11  0.09  Values represent mean ± SEM, n = 8. SHR = Spontaneously hypertensive rat, WKY = Wistar Kyoto rat. A = animal strain effect, C = cholesterol intake level effect, F = dietary fat source effect by 3-way MANOVA; treatment interactions by 3-way MANOVA.  0.007 0.023 N.S. N.S.  2 MANOVA p-value AxC AxF CxF AxCxF  1  <0.001 <0.001 <0.00 1  Free cholesterol  0.04  0.70 +  0.31  0.13  0.44  0.20  0.38  0.19  0.40 ± 0.03  1.95 +  0.98 ± 0.14  2 ANOVA p-value A C F  Plasma lipids  0.5% chol.  Menhaden 0.05% chol.  0.5% chol.  1.30 +  2.76 +  1.05 ± 0.11  0.5% chol.  0.68 ± 0.06  1.43 +  2.35 ±  1.09 ± 0.12  0.80 ± 0.07  1.40±  WKY  0.76 ± 0.06  SHR  Free cholesterol  0.05% chol.  Soybean 0.05% chol.  Tallow  0.5% chol.  Dietary Treatment: Butter 0.05% chol.  Plasma lipids: (mmol/L)  Table 2.4 Plasma lipids of SHR and WKY fed experimental diets.’  2.4).  However, animal strain did not influence plasma triacylglyceride levels.  containing 0.5% cholesterol exhibited reduced (p  Animals fed diets  0.05) levels of plasma triacylglycerides compared  to those fed 0.05% cholesterol diets (Table 2.4). Also, animals fed menhaden oil diets had reduced plasma triacylglyceride concentrations in both SHR and WKY compared to other dietary fat groups (Table 2.4). It is of interest to note that plasma cholesterol values for SHR and WKY animals fed the semisynthetic diets containing 0.05% cholesterol were elevated in comparison to values reported in chowfed animals in Chapter 1. Moreover, both SHR and WKY animals fed low cholesterol semi-synthetic diets containing butter, beef tallow, and soybean oil as principal fat sources, exhibited relatively greater plasma triacylglyceride levels than chow-fed animals. The exceptions to this observation were both the SHR and WKY animals fed the menhaden oil diets. Both animal strains exhibited uniformly reduced plasma triacylglyceride levels relative to those observed in chow-fed animals in Chapter 1. Tissue antioxidant status: i. Red blood cell (RBC) antioxidant enzymes: Activities of the RBC antioxidant enzymes, catalase (CAT) and superoxide dismutase (SOD) were higher (p  0.05)  and lower (p  0.05), respectively, in SHR animals compared to WKY  counterparts when treatment groups were pooled for dietary fat source and cholesterol level (Table 2.5). RBC CAT activities were not influenced by either dietary fat source or cholesterol intake level. RBC SOD activity was significantly (p < 0.05) affected by dietary fat source (Table 2.5). Soybean oil-fed animals had higher (p . 0.05)  RBC SOD activity than menhaden oil-fed counterparts when  treatment groups were pooled for animal strain and dietary cholesterol level.  Animals fed diets  containing 0.5% cholesterol exhibited higher (p< 0.05) activities of RBC SOD when treatments were pooled for animal strain and dietary fat source. Activity of RBC glutathione peroxidase (GSH-Px) was significantly (p  0.05) affected by animal strain and dietary fat source but was not influenced by  dietary cholesterol intake level (Table 2.5). SHR animals exhibited greater (p<0.05) activities of RBC GSH-Px than WKY animals when treatment groups were pooled for dietary fat source and cholesterol level. Animals fed menhaden oil diets exhibited lower (p  0.05)  RBC GSH-Px activity than other  dietary fat groups (Table 2.5). Other dietary fat source treatment effects on RBC GSH-Px activity 81  2  1  CAT  0.056 ± .004  0.066 ± .004  <0.001 N.S. N.S.  0.054±.002  0.065±.003  0.050±.002  0.066±.003  58.0 ± 3.3  56.0 ±3.7  64.8±2.3  65.6 ± 4.7  64.9 ± 3.2  66.1 ±1.1  57.9 ± 1.5  62.9 ± 4.9  0.002 N.S. <0.001  GSH-Px  46.3 ± 2.7  49.2±3.8  55.4+2.5  56.6 ± 2.4  58.5 ± 4.0  69.2±5.8  61 .1 ± 5.1  56.6 ± 2.1  GSH-Px (nmoles NADPH/min/mg Hb) SHR WKY  6.85 ± 0.42  6.13 ±0.44  6.80±0.17  6.47 ± 0.24  7.43 ± 0.86  6.00±0.33  6.67 ± 0.44  WKY  <0.001 0.044 0.029  SOD  6.75 ± 0.26  6.34±0.30  8.47±0.26  7.66 ± 0.38  7.65 ± 0.39  7.15±0.27  7.33 ± 0.29  7.91 ± 0.55  SOD (U/mg Hb)  6.94 ± 0.48  SHR  Values represent mean ± SEM, n = 8. SHR = Spontaneously hypertensive rat, WKY = Wistar Kyoto rat. CAT = catalase, k = first-order rate constant (sec ); GSH-Px = glutathione peroxidase; SOD = superoxide dismutase. 1 A = animal strain effect, C = cholesterol intake level effect, F = dietary fat source effect by 3-way MANOVA.  3 ANOVA p-value A C F  Antioxidant Enzyme  0.5% chol.  Menhaden 0.05% chol.  0.5% chol.  0.048 ± .001  0.048 ± .001  0.062 ± .003  0.5% chol.  0.064 ± .002  0.054±.005  0.052 ± .004  0.064 ± .005 0.055±.003  0.051 ± .005  WKY  0.058 ± .004  SHR  CAT (k/mg Hb)  0.05% chol.  Soybean 0.05% chol.  Tallow  0.5% chol.  Dietary Treatment: Butter 0.05% chol.  Antioxidant Enzyme : 2  Table 2.5 Antioxidant enzyme activities in red blood cells of SHR and WKY fed experimental diets. 1  included differences between animals fed butter and tallow diets, as well as differences between animals fed tallow and soybean oil diets. ii. Heart antioxidant enzymes: Similar to the results reported in Chapter 1, both SHR and WKY animals had negligible levels of heart tissue CAT activity (values not reported) after correction for enzyme activity attributed to the presence of contaminating RBCs in the heart tissue homogenate preparation. Heart SOD activity was significantly (p  0.05) influenced by dietary fat source but not by animal strain or dietary cholesterol  intake level (Table 2.6). Animals fed butter and soybean oil diets exhibited greater (p  0.05) heart  SOD activities than those fed menhaden oil diets when treatment groups were pooled for animal strain and dietary cholesterol intake level (Table 2.6).  The activities of heart GSH-Px and glutathione  reductase (GSSG-Red) were both significantly (p  0.05) influenced by animal strain and dietary fat  source. Dietary cholesterol intake did not affect either heart GSH-Px or GSSG-Red activities. Heart GSH-Px activity was lower (p  0.05)  in SHR animals compared to WKY animals when treatment  groups were pooled for dietary fat source and cholesterol level.  Animals fed menhaden oil diets  exhibited reduced (pO.O5) heart GSH-Px activities compared to other dietary fat groups (Table 2.6). Heart GSSG-Red activities were greater (p  0.05) in SHR animals compared to WKY animals when  treatment groups were pooled for dietary fat source and cholesterol level (Table 2.6). Also, butterand tallow-fed animals had greater (p < 0.05) activities of heart GSSG-Red than those fed menhaden oil diets when treatment groups were pooled for animal source and cholesterol intake level. iii. Liver antioxidant enzymes: Liver CAT activity was significantly (p  0.05)  influenced by animal strain and dietary  cholesterol intake level but was not affected by dietary fat source (Table 2.7a).  When treatment  groups were pooled for dietary fat source and cholesterol intake level, SHR animals had greater (p < 0.05)  levels of liver CAT activity than WKY counterparts.  cholesterol exhibited reduced (p  0.05) liver CAT activities when treatment groups were pooled for  animal strain and dietary fat source. significant (p  Animals fed diets containing 0.5%  Dietary fat source and cholesterol intake level both had a  0.05) influence on liver SOD activities (Table 2.7a). However, liver SOD activities  were not different between the two animal strains. Animals fed menhaden oil diets were observed to 83  2  1  17.3±2.1  1 7.2 ± 1.0  18.1 ±1.1  0.014 N.S. 0.001  GSSG-Red  69.0 ± 6.2 81.6± 16.7  15.0± 1.6  108 ± 14.0  97.9± 11.5  108 ± 11.7  1 5.5 ± 1.8  17.2 ±0.4  17.2 ±0.7  16.9 ± 0.8  95.8± 7.4  114 ± 8.0  110 ± 7.5  0.024 N.S. <0.001  GSH-Px  91.7± 18.8  74.3 ± 2.2  119 ± 7.2  114 ± 4.9  114 ± 8.2  112 ± 8.1  110 ±5.8  120 ±6.5  GSH-Px (nmoles NADPH/min/mg protein) SHR WKY  40.8±4.1  38.8 ± 2.9  48.1 ±2.9  47.5 ±2.5  45.9 ± 2.8  47.6±4.7  51.7±7.2  N.S. N.S. 0.014  SOD  44.1 ±2.5  44.7 ± 3.6  56.9 ±5.3  48.4± 2.6  45.2 ± 1.5  47.5±4.1  49.4±2.9  52.0±5.1  SOD (U/mg protein) SHR WKY  47.9 ±4.1  Values represent mean .±. SEM, n = 8. SHR = Spontaneously hypertensive rat, WKY = Wistar Kyoto rat. GSSG-Red = glutathione reductase; GSH-Px = glutathione peroxidase; SOD = superoxide dismutase. A = animal strain effect, C = cholesterol intake level effect, F = dietary fat source effect by 3-way MANOVA.  3 ANOVA p-value A C F  Antioxidant Enzyme  0.5% chol.  Menhaden 0.05% chol.  0.5% chol.  18.5± 1.6  20.5 ± 0.6  0.5% chol.  18.6± 1.6  20.0± 1.7  19.2± 1.0  20.2±0.6  19.2 ±0.9  20.1 ±0.6  GSSG-Red (nmoles NADPH/min/mg protein) WKY SHR  0.05% chol.  Soybean 0.05% chol.  Tallow  0.5% chol.  Dietary Treatment: Butter 0.05% chol.  : 2 Antioxidant Enzyme  Table 2.6 Antioxidant enzyme activities in heart of SHR and WKY fed experimental diets. 1  have reduced (p  0.05) liver SOD activities compared to other dietary fat groups (Table 2.7a). Also,  liver SOD activities were reduced (p  .  0.05) in animals fed diets containing 0.5% cholesterol. Liver  GSH-Px and GSSG-Red activities were both significantly (p < 0.05)  influenced by animal strain,  dietary fat source and cholesterol intake level (Table 2.7b). SHR animals had reduced (p  0.05) liver  GSH-Px activities compared to WKY counterparts when treatment groups were pooled for dietary fat source and cholesterol intake level. Also, liver GSH-Px activities were reduced (p < 0.05) in animals fed 0.5% dietary cholesterol when treatment groups were pooled for animal strain and dietary fat source.  WKY animals fed soybean oil diets had greater (p < 0.05)  liver GSH-Px activities than  menhaden oil-fed counterparts (Table 2.7b). Activity of liver GSSG-Red was greater (p  0.05) in  SHR animals compared to WKY animals (Table 2.7b). When animals were fed diets containing 0.5% cholesterol, liver GSSG-Red activity was increased (p  0.05)  compared to those fed 0.05%  cholesterol in the diet. Liver GSSG-Red activities were higher (p< 0.05) in animals fed soybean oil and menhaden oil diets, compared to butter- and tallow-fed counterparts (Table 2.7b). iv. Differences in antioxidant enzyme activities compared to chow-fed animals: The antioxidant status of rats fed commercial rat chow in Chapter 1 and those fed semisynthetic diets in the present study showed similarities as well as some differences, which resulted in considering the chow-fed SHR and WKY animals from Chapter 1 as a reference group of animals, but not as an adequate control group for animals fed semi-synthetic diets herein. Similar rat strain differences in RBC CAT activity were obtained in animals fed either rat chow in Chapter 1, or semisynthetic diets in the present study. RBC GSH-Px and SOD activities were relatively higher in chowfed WKY than SHR in Chapter 1, but these trends were reversed in animals fed the semi-synthetic, low cholesterol diets in the present study.  Activity of heart GSSG-Red was greater in SHR than WKY  animals fed the semi-synthetic diets which was opposite to the results obtained Chapter 1 with chow fed animals. Animals fed semi-synthetic diets in the present study, did not exhibit strain differences for heart SOD activities which contrasted with the result obtained in Chapter 1 with chow-fed animals, in which heart SOD activity was greater in SHR. GSH-Px activities, while not different in chow-fed SHR and WKY animals, was lower in SHR fed semi-synthetic diets in the present study. Differences between animal strains for liver CAT, SOD and GSH-Px activities in SHR and WKY animals fed semi 85  2  CAT  .300±.013  .332±.017  <0.001 0.045 N.S.  .308 ± .01 1  .356 ± .023  SOD  N.S. 0.04 <0.001  73.2+ 4.0  87.0 ± 4.7  1.1  95.7±  .248±.017  .366±.031  78.1 ± 4.2  79.6 ± 3.0  93.9± 1.0  102 ± 5.6  96.0 ± 10.7  99.6 ± 4.6  88.3 ± 3.8  96.8 ± 4.5  WKY  Values represent mean ± SEMI n = 8. CAT = catalase, k = first-order rate constant (sec); SOD = superoxide dismutase. A = animal strain effect, C = cholesterol intake level effect, F = dietary fat source effect by 3-way MANOVA.  3 ANOVA p-value A C F  Antioxidant Enzyme  0.5% chol.  Menhaden 0.05% chol.  0.5% chol.  92.8 ± 1 1.0  91.7 ± 7.0  .348 ± .026  .279 ± .022  .332 ± .041  0.5% chol.  88.7 ± 3.9  85.2 ± 4.4  99.2 ± 5.4  SHR  SOD (U/mg protein)  .300 ± .01 1  .320 ± .038  .225 ± .035  .367 ± .019 .375 ± .018  .283 ± .027  WKY  .359 ± .004  SHR  CAT (k/mg protein)  0.05% chol.  Soybean 0.05% chol.  Tallow  0.5% chol.  Dietary Treatment: Butter 0.05% chol.  Antioxidant Enzyme : 2  Table 27a Reactive oxygen species metabolizing antioxidant enzyme activities in liver of SHR and WKY fed experimental diets. 1  72.8 ± 8.5  84.1 ± 6.0  2  N.S. N.S. N.S. N.S.  MANOVA p-value 3 AxC A x F CxF AxCxF  174 ± 16  201 ± 12  192 ± 1  170 ± 9  156 ± 3  148± 4  135 ± 3  131 ± 18  0.014 <0.001 N.S. .005  <0.001 0.022 0.001  GSH-Px  194 ± 14  187 ± 1 1  234 ± 41  357 ± 14  282 ± 8  281 ± 12  195 ± 32  271 ± 25  GSH-Px (nmoles NADPH/min/mg protein) SHR WKY  Values represent mean ± SEM, n = 8. SHR = Spontaneously hypertensive rat, WKY = Wistar Kyoto rat. GSSG-Red = glutathione reductase; GSH-Px = glutathione peroxidase. A = animal strain effect, C = cholesterol intake level effect, F = dietary fat source effect; treatment interactions by 3-way MANOVA.  <0.001 <0.001 0.049  GSSG-Red  58.0 ± 5.0  72.8 ± 4.3  60.6 ± 1.2  89.2 ± 2.0  3 ANOVA p-value A C F  Antioxidant Enzyme  0.5% chol.  Menhaden 0.05% chol.  0.5% chol.  56.7 ± 1.5  58.6 ± 3.6  76.3 ± 3.2  0.5% chol. 61.0 ± 3.3  51.0± 1.7  58.4 ± 3.2  52.9 ± 1.6  60.2 ±0.9  75.9 ± 0.6  61.3 ±2.6  GSSG-Red (nmoles NADPH/min/mg protein) SHR WKY  0.05% chol.  Soybean 0.05% chol.  Tallow  0.5% chol.  Dietary Treatment: Butter 0.05% chol.  : 2 Antioxidant Enzyme  Table 2.7b Glutathione metabolizing antioxidant enzyme activities in liver of SHR and WKY fed experimental diets. 1  synthetic diets were consistent with the results obtained in Chapter 1 with chow-fed animals. However, GSSG-Red activities recorded in SHR fed semi-synthetic diets was greater than WKY and opposite to the results obtained for chow-fed animals in Chapter 1. Despite these alterations in the specific strain-related differences in antioxidant enzyme activities found between these two studies, the relative range of enzyme activity values was similar. v. Tissue and RBC glutathione content: Both heart and liver GSH levels were significantly (p < 0.05) affected by animal strain. Heart and liver tissue concentrations of GSH were greater (p  0.05)  in SHR animals compared to WKY  counterparts (Table 2.8). Liver GSH was not affected by dietary fat source or cholesterol intake level. However, heart tissue GSH was reduced (p  0.05) in animals fed menhaden oil diets compared to  other dietary fat groups when treatment groups were pooled for animal strain and dietary cholesterol intake level (Table 2.8). RBC GSH concentrations were significantly (p < 0.05) reduced in animals fed menhaden oil diets compared to counterparts fed the other dietary fats when treatment groups were pooled for animal strain and dietary cholesterol intake level (Table 2.8). Animal strain and dietary cholesterol intake level did not influence RBC GSH content. Tissue and RBC susceptibility to forced peroxidation: Tissue and RBC susceptibility to oxidative challenge was examined by subjecting RBCs as well as heart and liver homogenates to increasing concentrations of hydrogen peroxide (H ) in vitro, and 0 2 monitoring the depletion of GSH.  Heart and liver TBARs and red blood cell MDA production were  evaluated as further indicators of in vitro lipid peroxidation.  The profiles of the in vitro forced  peroxidation of RBCs, heart and liver tissues are presented in Figures 2.1 to 2.6. As well, the results from the oxidative challenge of RBCs, heart and liver at a single concentration of H , with treatment 0 2 differences identified, are presented in Tables 2.9 to 2.11. i. Red blood cell GSH depletion and MDA production: Depletion of GSH in RBCs was significantly (p  0.05) lower in menhaden oil-fed animals  compared to other dietary fat groups (Table 2.9, Figure 2.1A, B, C, D). However, neither animal strain nor dietary cholesterol level influenced RBC GSH depletion. Production of MDA in RBCs was influenced  88  CD  2  1  A C F  1.70 ± .137  1.72 ±.076  Heart  <0.001 N.S. 0.049  1.59 ± .060  1.54 ±.078  1.72 ± .052  1.86 ± .054  1.06 ± .036  1.16 ±.180  1.17 ± .038  1.19 ± .052  1.16 ± .043  1.07 +.033  1.04 +.062  1.06 ±.037  N.S. N.S. 0.025  RBC  .991 ± .053  .960±.041  1.17 ± .034  1.20 ± .031  1.16 ± .052  1.09±062  1.13 ±.064  1.07 ±.022  RBC (nmoles GSH/mg RBC) SHR WKY  .c 62  4.43 ± .172  5.24 ±.193  4.53 ± .121  5.70 ± .280  4.72 ± .205  4.98 +.394  5.35 +.188  5.06±125  0.014 N.S. N.S.  Liver  4.93 ± .438  5.68 ±.348  6.03 ± .014  5.70 ±  6.19 ± .016  5.10 ±.144  5.36±.285  5.76 ±.404  Liver (nmoles GSH/mg tissue wet wt.) SHR WKY  Values represent mean ± SEM, n = 8. SHR = Spontaneously hypertensive rat, WKY = Wistar Kyoto rat. GSH = glutathione. A = animal strain effect, C = cholesterol intake level effect, F = dietary fat source effect by 3-way MANOVA.  ANOVA p-value 2  0.5% chol.  Menhaden 0.05% chol.  0.5% chol.  1.66 ± .025  1.63 ± .053  1.74 ± .107  0.5% chol.  1.96 ± .094  1.60 ±.066  1.51 ±.073  1.57 ±.081  1.80 ±.071  1.82 ±.068  1.76 ±.032  0.05% chol.  Soybean 0.05% chol.  Tallow  0.5% chol.  Dietary Treatment: Butter 0.05% chol.  Heart (nmoles GSHImg tissue wet wt.) SHR WKY  Table 2.8 Basal glutathione levels in heart and liver tissue and red blood cells of SHR and WKY fed experimental diets. 1  0  2  1  N.S.  N.S. N.S. <0.001  122 ± 21.4  181 ± 31.4  0.026  0.001 <0.001 0.003  MDA (nmolesf g RBC)  107 ± 20.4  49.8 ± 1.79  48.2 ± 2.16 GSH depletion (%)  136±33.1  53.4 ±4.76  50.0 ±4.25  127± 13.7  171 ±3.82  60.1 ± 1.74  55.3± 1.33  Values represent mean ± SEM, n = 8. SHR = Spontaneously hypertensive rat, WKY = Wistar Kyoto rat. GSH = glutathione; MDA = malondialdehyde. A = animal strain effect, C = cholesterol intake level effect, F = dietary fat source effect by 3-way MANOVA; treatment interactions by 3-way MANOVA.  AxF  2 MANOVA p-value  A C F  2 ANOVA p-value  0.5% chol.  Menhaden 0.05% chol.  0.5% chol.  184 ± 1 2.0  222 ± 7.00  104 ± 1 1.1  62.2 ± 1 .25  176 ± 16.8  63.9 ± 5.82  65.8 ± 5.44  0.5% chol.  204 ± 8.40  148 ± 10.3  186 ± 6.49  54.6 ± 4.82  220 ± 5.25  75.2 ± 7.45  180 ± 29.9  70.3 ± 5.09  48.0 ± 15.0 64.8 ± 4.38  183 ± 1 1.7  68.4 ± 3.39  54.9 ± 3.88  MDA (nmoles/ g RBC) 0 2 5.0 mM H SHR WKY  0.05% chol.  Soybean 0.05% chol.  Tallow  0.5% chol.  Dietary Treatment: Butter 0.05% chol.  GSH depletion (%) 0 2 0.5 mM H WKY SHR  -induced GSH depletion and MDA production in SHR and WKY fed 0 2 Table 2.9 Red blood cell susceptibility to H experimental diets. 1  g  0.00  100  0.25  0.75  mM H202 added  0.50  mM H202 added  1.00  1.25  V  g  V  g  0.00 0,25  0.75  mM H202 added  mM H202 added  0.50  1.00  1.25  1.25  CD  Figure 2.1 Susceptibility of red blood cells from SHR and WKY animals fed diets varying in dietary fat source and cholesterol intake level -induced glutathione (GSH) depletion. (A) SHR, low cholesterol diets; (B) SHR, high cholesterol diets; 0 2 to in vitro H (C) WKY, low cholesterol diets; (0) WKY, high cholesterol diets. * indicates a significant (p .. 0.05) dietary fat source difference. = butter; A = beef tallow; 0 = soybean oil; • = menhaden oil.  C  V  ‘C  C  100  (p  0.05) by animal strain, dietary fat source and cholesterol level (Table 2.9, Figure 2.2A, B, C, D).  SHR animals exhibited greater (p  .. 0.05) levels of RBC MDA production than WKY animals when  treatment groups were pooled for dietary fat source and cholesterol level. Animals fed menhaden oil diets had lower (p  0.05) levels of MDA production in RBCs than other dietary fat groups (Table 2.9;  Figure 2.2A, B, C, D). Also, MDA production in RBCs was reduced (p < 0.05) in animals fed 0.5% cholesterol diets compared to those fed 0.05% cholesterol diets, regardless of dietary fat source (Table 2.9). ii. Heart GSH derletion and TBARs production: Heart tissue GSH depletion was significantly (p  0.05) influenced by animal strain and dietary  fat source at the lower concentrations of added H 0 (0.05 and 0.1 mM H 2 ; Figure 2.3A, B). SHR 0 2 animals exhibited lower (p  0.05) depletion of heart tissue GSH than WKY counterparts at 0.05 and  0.1 mM added H 0 (Table 2.10; Figure 2.3A, B, C, D). 2 menhaden oil diets exhibited reduced (p . 0.05)  Also, SHR animals fed soybean oil and  depletion of heart GSH at 0.1 mM added H , 0 2  compared to WKY counterparts (Table 2.10; Figure 2.3A, B, C, D).  However, these treatment  differences were not maintained at the higher concentrations of added peroxidizing agent (Figure 2.3A, B, C, D). 0.05)  Production of lipid peroxidation products (TBARs) in heart tissue was significantly (p  influenced by dietary fat source (Table 2.10; Figure 2.4A, B).  However, animal strain and  dietary cholesterol level did not affect heart tissue TBARs production (Table 2.10; Figure 2.4A, B, C, D). When treatment groups were pooled for animal strain and dietary cholesterol level, heart TBARs of animals fed menhaden oil diets were significantly (p < 0.05) lower than for other dietary fat groups at 1-3mM of added H 0 (Table 2.10). However, when the animal strains were examined individually, 2 the dietary fat source effect was significant only in SHR animals (Figure 2.4A, B, C, D). observation confirms the significant (p  This  0.05) interaction recorded between animal strain and dietary  fat source for heart tissue TBARs production (Table 2.10; Figure 2.4A, B, C, D). iii. Liver GSH depletion and TBARs production: Susceptibility of liver tissue to GSH depletion was significantly (p  0.05) influenced by animal  strain, dietary fat source and cholesterol intake level (Table 2.11). SHR animals had lower (p . 0.05) levels of GSH depletion in liver tissue compared to WKY counterparts when treatment groups were 92  0  100  200  300  400  15  mM H202 added  10 20  20  25  25  C)  0  2  I  0  100  200  300  400  500  0  15  mM H202 added  10  500  6  600  0  100  200  600  0  100  2  0  0  300  300 200  400  400  I  600  500  6  5 16  15 mM H202 added  10  mM H202 added  10  20  20  25  25  CD C,)  .  Figure 2.2 Susceptibility of red blood cells from SHR and WKY animals fed diets varying in dietary fat source and cholesterol intake level -induced malondialdehyde (MDA) production. (A) SHR, low cholesterol diets; (B) SHR, high cholesterol diets; 0 2 to in vitro H (C) WKY, low cholesterol diets; (D) WKY, high cholesterol diets. * indicates a significant (p 0.05) dietary fat source difference. = butter; A = beef tallow; 0 = soybean oil; • = menhaden oil.  2  I  600  600  0 0.00  20  40  60  80  100  0 0.00  20  40  60  0.25  0.25  0.76  1.00  0.76  1,00  mM H202 added  0.50  mM ff202 added  0.50  1.25  1.25  1.60  1.50  C 0. C  C  0.00  100  0,00  100  0.25  0.25  0.75  1.00  0.75  1.00 mM ff202 added  0.50  mM H202 added  0.50  1.25  1.25  1.50  1.50  CD  Figure 2.3 Susceptibility of heart tissue from SHR and WKY animals fed diets varying in dietary fat source and cholesterol intake level -induced glutathione (GSH) depletion. (A) SHR, low cholesterol diets; (B) SHR, high cholesterol diets; 0 2 to in vitro H (C) WKY, low cholesterol diets; (D) WKY, high cholesterol diets. * indicates a significant (p = butter; A = beef tallow; 0 = soybean oil; • = menhaden oil. 0.05) dietary fat source difference.  C 0.  C 0  C  80  100  Ca C,’  2  1  46.4± 1.52  38.9 ±2.74  N.S.  <0.001 N.S. <0.001  GSH depletion (%)  45.9 ± 3.25  40.4± 3.74  35.8 ± 2.82  36.4± 3.63  WKY  0.132±.031  0.166 ± .024  0.184±.021  0.179 ± .019  0.160±.008  0.150±.013  0.172 ± .017  0.156±.018  <0.001  N.S. N.S. <0.001  TBARs (A532)  0.088±.019  0.109 ± .019  0.182±.037  0.161 ± .007  0.231 ±.021  0.174±.011  0.200 ± .024  0.187±.009  SHR  TBARs (A532) 2.0 mM H 0 2  Values represent mean ± SEM, n = 8. SHR = Spontaneously hypertensive rat, WKY = Wistar Kyoto rat. GSH = glutathione; TBARs = 2-thiobarbituric acid reactive substances. A = animal strain effect, C = cholesterol intake level effect, F = dietary fat source effect by 3-way MANOVA; treatment interactions by 3-way MANOVA.  A x F  MANOVA p-value 2  2 ANOVA p-value A C F  0.5% chol.  Menhaden 0.05% chol.  0.5% chol.  52.2 ± 3.73  53.2±4.09  48.7 ±4.26  0.5% chol. 40.5 ± 3.00  57.8± 5.08  56.8 ± 5.86  52.2 ± 3.41 43.9 ±3.58  50.0±6.84  46.7±5.29  0.05% chol.  Soybean 0.05% chol.  Tallow  0.5% chol.  Dietary Treatment: Butter 0.05% chol.  SHR  GSH depletion (%) 0.1 mM H 0 2 WKY  -induced GSH depletion and TBARs production in SHR and WKY 0 2 Table 2.10 Heart homogenate susceptibility to H fed experimental diets.’  C  I  mM 11202 added  0.00 0  1  4  mM H202 added  I  5  I  3  I  2  0.00  0.10  0.10  0.30  0.20  S  N 0 In  0,40  0.50  0.20  0.30  0.40  0.50  0.00 0  0.00  0.20  0.30  0.10  I-  In  N  0.40  0.50  0.10  0.20  0.30  A  0  D  •  0  I  1  1  3  4  I  3  I  4 mM H202 added  2  mM 11202 added  2  5  I  T  5  6  6  CD 0)  Figure 2.4 Susceptibility of heart tissue from SHR and WKY animals fed diets varying in dietary fat source and cholesterol intake level -induced thiobarbituric acid reactive substances (TBARS) production. (A) SHR, low cholesterol diets; (B) SHR, high cholesterol 0 2 to in vitro H diets; (C) WKY, low cholesterol diets; (D) WKY, high cholesterol diets. * = butter; A = beef tallow; 0 = soybean oil; • = menhaden oil. 0.05) dietary fat source difference. indicates a significant (p  I  N  I.  0)  S  N  0.40  0.50  2  1  46.3±2.4  32.1 ± 2.0  N.S. N.S.  0.011 <0.001 0.041  GSH depletion (%)  34.0±3.5  25.5 ± 2.2  60.5 ± 4.6  48.2 ± 0.8  0.156±.032  0.296 ± .056  0.303 ± .052  0.005 0.006  0.014 <0.001 <0.001  TBARs (A532)  0.154±.027  0.240 ± .070  0.536 ± .041  0.525 ± .038  0.214 ± .027  0.776 ± .058  0.289+.019  0.545 ± .010  WKY  Values represent mean ± SEM, n = 8. SHR = Spontaneously hypertensive rat, WKY = Wistar Kyoto rat. GSH = glutathione; TBARs = 2-thiobarbituric acid reactive substances. A = animal strain effect, C = cholesterol intake level effect, F = dietary fat source effect by 3-way MANOVA; treatment interactions by 3-way MANOVA.  A xC C x F  2 MANOVA p-value  A C F  ANOVA p-value 2  0.5% chol.  Menhaden 0.05% chol.  0.5% chol.  0.654 ± .046  0.436 ± .026  64.2 ± 0.4 39.2 ± 4.5  37.4 ± 8.9  0.5% chol.  0.666 ± .162  0.490±.004  0.531 ± .009  SHR  TBARs (A532) 40.0 mM H 0 2  31.3 ± 5.6  41.6±5.1  39.9 ± 2.8  36.0 ± 5.8  33.0 ± 2.1  27.2±3.0  32.8 ± 1.0  0.05% chol.  Soybean 0.05% chol.  Tallow  0.5% chol.  Dietary Treatment: Butter 0.05% chol.  GSH depletion (%) 0.5 mM H 0 2 SHR WKY  -induced GSH depletion and TBARs production in SHR and WKY 0 2 Table 2.11 Liver homogenate susceptibility to H 1 fed experimental diets.  pooled for dietary fat source and cholesterol intake level (Table 2.11). Animals fed diets containing 0.5% cholesterol exhibited increased (p  0.05) liver GSH depletion compared to those fed 0.05%  cholesterol diets (Table 2.11; Figure 2.5A, B, C, D). Animals fed menhaden oil diets exhibited reduced (p  0.05) depletion of liver GSH compared to other dietary fat treatment groups when treatment  groups were pooled for animal strain and dietary cholesterol level (Table 2.11; Figure 2.5A, B, C, D). Production of TBARs lipid peroxidation products in liver tissue was significantly (p < 0.05) influenced by dietary fat source and cholesterol intake level (Table 2.11; Figure 2.6A, B, C, D). Animals fed butter, tallow and soybean oil diets exhibited greater (p < 0.05) levels of TBARs when liver tissue was incubated with 1-40 mM added H , compared to those fed menhaden oil diets (Table 2.11; 0 2 Figure 2.6A, B, C, D).  Animal strain did not have an effect on liver tissue TBARs production.  Production of TBARs was reduced (p  0.05) in liver tissue from animals fed 0.5% cholesterol in the  diet (Table 2.11). There tended to be a relatively greater influence of dietary cholesterol intake level on liver TBARs production in WKY animals compared to their SHR counterparts as demonstrated by the interaction (p  0.05) recorded between these two variables (Table 2.11).  98  g  0  0  1  1 3 4  3 4  mM H202 added  2  mM H202 added  2  5  5 6  C C  C C  0  0  1  1  3  4 mM H202 added  2  3  4  mM H202 added  2  5  5  6  6  CD CD  Figure 2.5 Susceptibility of liver tissue from SHR and WKY animals fed diets varying in dietary fat source and cholesterol intake level to in vitro H -induced glutathione (GSH) depletion. (A) SHR, low cholesterol diets; (B) SHR, high cholesterol diets; 0 2 (C) WKY, low cholesterol diets; (D) WKY, high cholesterol diets. * indicates a significant (p 0.05) dietary fat source difference. = butter; A beef tallow; 0 = soybean oil; • = menhaden oil.  C  100  100  10 20 30 40 50  20 30  40  60  mM H202 added  0.00 10  0,00  0.40  0.20  C,  0.60  0.20  0.40  0  I’,  0.80  0.80  0.60  1.00  1.00  mM H202 added  0  0  0.00 0  0.00  0.40  0.60  0.20  I-  0,  N 4., In  0.20  0.40  0.60  0.80  0.80  10  10  30  30 mM H202 added  20  mM H202 added  20  40  40  50  50  0 0  -  Figure 2.6 Susceptibility of liver tissue from SHR and WKY animals fed diets varying in dietary fat source and cholesterol intake level to in vitro H -induced thiobarbituric acid reactive substances (TBARS) production. (A) SHR, low cholesterol diets; (B) SHR, high cholesterol 0 2 diets; (C) WKY, low cholesterol diets; (D) WKY, high cholesterol diets. * indicates a significant (p 0.05) dietary fat source difference. = butter; A = beef tallow; 0 = soybean oil; • = menhaden oil.  I-  C,  N C.,  I-  C,  In  1.00  1.00  Discussion: The role of dietary fat as a source of saturated, monounsaturated, or polyunsaturated n-6 and long-chain n-3 fatty acids in determining plasma lipid concentrations has been the subject of extensive study. It is now well recognized that while medium-chain saturated fatty acids (i.e. myristic and Iauric acids) have a relatively hypercholesterolemic effect, longer chain saturated acids (e.g. stearic acid) have a neutral or hypocholesterolemic effect (Kritchevsky, 1 995; Denke and Grundy, 1992; Bonanome and Grundy, 1988).  Dietary fat sources which contain primarily monounsaturated fatty acids (e.g.  oleic acid) are reported to be relatively hypocholesterolemic in nature (Mattson and Grundy, 1985). Moreover, monounsaturated dietary fat sources are not noted to reduce HDL cholesterol, in contrast to the effects of n-6 polyunsaturated fats present in vegetable oils (Mott et a!., 1 992; Vega et a!., 1982).  Marine oil sources which are characterized by a relatively high content of longer chain  polyunsaturated fatty acids (e.g. eicospentaenoic (EPA) and docosahexaenoic acids (DHA)) are able to reduce plasma cholesterol levels as well as having beneficial effects on hypertension and blood clotting time (Smit eta!., 1994; Bairati eta!., 1992; Coniglio, 1992; Drevon, 1992; Garg etal., 1988). These effects of marine oil n-3 fatty acids are attributed to various bloactive agents synthesized from various essential  fatty  acids,  including thromboxanes  prostaglandins involved in vasodilatation (Drevon, 1992).  involved  in platelet  aggregation  and  However, the increased incorporation of  polyunsaturated fatty acids from marine or vegetable oil dietary fat sources into plasma lipoprotein and cell membrane phospholipids may increase tissue susceptibility to lipid peroxidation (Suzukawa et al., 1 995; Frankel et a!., 1 994; Sküladóttir et a!., 1 994; De Schrijver et at., 1992; L’Abbé et at., 1991; Nalbone et a!., 1989; Hu et a!., 1989).  An increase in the in vivo requirement for antioxidant  molecules, such as tocopherols, has been demonstrated in animals fed fish oil diets (Gonzalez et a!., 1992; Hu et a!., 1989; Reddy et a!., 1973). The present experiment describes the effects of diets containing dietary fat sources varying in degree of saturation or unsaturation  (n-6 or n-3  polyunsaturated fatty acids) as well as the level of cholesterol intake on the plasma lipid profile and antioxidant status of SHR and WKY rats.  101  Feed efficiency and plasma lipids: The consistently reduced FER in SHR fed semi-synthetic diets is similar to previous studies from this laboratory and other investigations in which SHR were fed semi-synthetic diets (Yuan and Kitts, 1992; Jones eta!., 1988). The insulin-resistance reported in the SHR (Hulman eta!., 1993) would likely result in reduced energy accretion and feed efficiency similar to that previously reported in diabetic animal models (Wohaieb and Godin, 1987a,b). A considerable body of evidence indicates that dietary fatty acid composition can influence the partitioning of fatty acids into oxidation for energy purposes or storage in viva (Clandinin eta!., 1 995; Takeuchi eta?., 1995; Su and Jones, 1993; Shimomura eta?., 1990; Leyton eta!., 1987). It is now well recognized that polyunsaturated fatty acids are oxidized more rapidly for energy than long-chain saturated fatty acids (Takeuchi eta!., 1995; Shimomura eta?., 1990; Leyton eta!., 1987). Moreover, the oxidation rates of individual saturated fatty acids and PUFA vary according to chain length and degree of unsaturation (Leyton et a?., 1987).  The short- and medium-chain fatty acids which  predominate in butterfat (C:4to C:14, approx. 25% of total fatty acids; Jensen and Clark, 1988) are much more rapidly oxidized than palmitic or stearic acids and hence are neither incorporated into plasma lipoproteins nor deposited into adipose tissue (Kitts and Jones, 1995; Leyton eta!., 1987). Oxidation of unsaturated fatty acids varies considerably, with oleic (C18:1) and linolenic (C18:3,n-3) acids being oxidized more rapidly than linoleic (C18:2)  acid (Leyton eta!., 1987).  In general, the  unsaturated C18 fatty acids were oxidized more rapidly than the C20 metabolites of linoleic acid (Leyton eta!., 1987). The rate of oxidation of dietary fatty acids influences the thermogenic effect of experimental diets due to variations in substrate utilization for energy or deposition in body stores and thereby energy balance in studies using different dietary fat sources (Takeuchi eta?., 1995; Su and Jones, 1993; Shimomura eta?., 1990). While Su and Jones (1993) reported that whole body energy gains differed between animals fed high-fat vegetable, animal or marine oil based diets, neither body weight gained nor energy expenditure differed between treatment groups.  Similarly, in the  present study, while the oxidation of the individual dietary fatty acids would differ between animals fed the butter, tallow and soybean oil diets, the substrate utilization and energy accretion from these diets might not be different, as demonstrated by the similar body weight gain, FER and plasma total 102  cholesterol and triacylglyceride concentrations between these three dietary fat sources. The decrease in body weight gained and FER of animals fed menhaden oil diets in the present study is consistent with previous reports of reduced energy gain and energy efficiency in animals fed fish oil diets (Su and Jones, 1993), albeit these workers did not observe a concomitant reduction in body weight gained. The lack of difference in body weight gained between animals fed fish oil or other dietary fat sources in studies from Jones and coworkers (Jones eta!., 1995; Su and Jones, 1 993) likely reflects the high fat content (22%) of the diets fed to animals in these studies, in contrast to the nutritionally adequate level of dietary fat (8%) for laboratory rats used in the present study. In other studies when rats were fed long-chain PUFAs characteristic of fish oil (i.e. EPA, DHA) in the diet, lipid utilization for energy was decreased (Rustan eta!., 1993). Rustan and coworkers (1993) demonstrated that reduced oxidation of long-chain n-3 fatty acids for energy was associated with reduced plasma concentrations of triacylglyceride, phospholipids and free fatty acids. Taken together, these studies indicate that through effects on substrate utilization for oxidation (i.e. energy) or deposition, dietary fatty acid composition can influence energy accretion and animal growth characteristics. The lower concentrations of plasma lipids observed in SHR animals compared to WKY animals fed formulated semi-synthetic diets in the present study are consistent with the results from Chapter 1 in chow-fed animals and those of others (Mon eta!., 1993; McGregor eta!., 1981). These strainrelated differences in relative plasma lipid concentrations persisted in the present study, regardless of the dietary fat source or cholesterol level fed to the animals.  Differences in cholesterol metabolism  between SHR and WKY animals have been demonstrated in vitro using radiolabelled acetate precursor incorporation into cholesterol as an index of hepatic cholesterol synthesis (Iritani et al., 1977). These workers reported a reduced incorporation of acetate into cholesterol in liver tissue slices from SHR animals compared to their WKY counterparts. The relative metabolic differences underlying plasma lipid levels between SHR and WKY did not appear to be altered by feeding the formulated, semi synthetic diets in the present study as compared to the complex, commercial chow diet fed to animals in Chapter 1. However, it is interesting to note that animals fed semi-synthetic diets containing the low (0.05%) level of cholesterol in the present study exhibited higher plasma lipid levels as well as differences in tissue antioxidant status compared to those fed chow diets in Chapter 1.  These 103  differences further substantiate the argument that the SHR and WKY animals fed chow diets in Chapter 1 should only be used as reference groups for the qualitative comparison of results, rather than as true dietary treatment controls in the present study. For the purposes of the present study with SHR and WKY animals fed semi-synthetic diets containing a low or high level of cholesterol, the groups fed 0.05% cholesterol in the diet are considered as controls for those fed the high (0.5%) level of dietary cholesterol. While the hypotriacylglyceridemic effect of dietary fish oil is well recognized, the effect on plasma cholesterol concentrations can be variable (Ikeda eta!., 1 994; Bairati eta!., 1992; De Schrijver eta!., 1992; Coniglio, 1992; L’Abbé eta!., 1991; Garg eta!., 1988). Mechanisms whereby dietary fish oil exerts a plasma lipid-lowering effect include the inhibition of the synthesis and secretion of triacylglyceride-rich VLDL particles as well as effects on fatty acid oxidation and esterification (Rustan eta!., 1993; Coniglio, 1992; Halminski eta!., 1991).  The n-3 PUFA of dietary fish oil influenced  triacylglyceride metabolism via decreased mobilization of fatty acids from adipose tissue as substrates for triacylglyceride synthesis, as well as inhibitory effects on enzymes involved in fatty acid esterification (Rustan eta!., 1993; Halminski eta!., 1991). Moreover, increased hepatic mitochondrial and peroxisomal fatty acid B-oxidation is known to occur in animals fed fish oil diets (Rustan et a!., 1993; Halminski et al., 1991; Mohan et a!., 1991).  These mechanisms reduce the availability of  triacylglycerides for incorporation into chylomicron and VLDL particles, hence lowering postprandial levels of plasma triacylglycerides in animals fed fish oil compared to other saturated or n-6 PUFA dietary fat sources as observed in the present study. Previous workers who have reported reduced plasma cholesterol levels when feeding fish oil diets to animals have generally used high levels of fat in the experimental diets (i.e. 13  -  20% wt/wt;  De Schrijver eta!., 1992; L’Abbé eta!., 1991; Garg eta!., 1989). However, in studies where lower levels of fat were fed to animals, as in the present experiment, plasma total cholesterol levels were not reduced by dietary fish oil (McGregor eta!., 1981). The plasma cholesterol lowering effects of dietary fish oil likely involve a combination of effects related to the decreased synthesis and secretion of VLDL, leading to decreased levels of LDL cholesterol (Drevon, 1992; Rustan eta!., 1988), inhibition of HMG CoA reductase activity (Ikeda etal., 1994), as well as effects on bile acid flow and composition (Levy 104  and Herzberg, 1995; Smit et a/., 1994). In studies to compare the effects of individual fish oil n-3 fatty acids on lipid metabolism, Ikeda and coworkers (1994)  reported that plasma cholesterol  concentrations were reduced in rats fed DHA, whereas EPA diets did not yield a similar result. However, EPA has been reported to reduce cholesterol esterification and, thereby, the amount of VLDL cholesteryl ester (Rustan et a!., 1988).  Dietary fish oil has been demonstrated to enhance bile  secretion (Levy and Herzberg, 1995) as well as the amount of cholesterol in bile secreted by rats (Smit et a!., 1994; De Schrijver et a!., 1992).  It is plausible that fish oil n-3 fatty acids influence plasma  cholesterol levels through effects on hepatic cholesterol handling as well as plasma lipoprotein composition. Systolic blood pressure: The hypertension observed in SHR animals at 1 3 weeks of age is consistent with the results from Chapter 1 as well as previous reports from this laboratory (Kitts et a!., 1992), but differs from other workers who reported a hypotensive effect of dietary fish oil and butterfat compared to corn oil in older (26 weeks of age) SHRs (Karanja eta!., 1989). However, these workers did not begin to observe a significant hypotensive effect of 1 8% menhaden oil or butterfat diets until animals were 1 6 weeks of age (Karanja eta!., 1989). Thus, the results of thepresent study in 14-week old animals are likely intermediate in the temporal lowering of blood pressure by dietary fish oil, as well as reflecting the lower fat content of the diets used herein. In studies with stroke-prone SHR fed diets containing 20% milk fat (butterfat), the incidence of cerebrovascular disease was lowered in the absence of a reduction in blood pressure (Ikeda eta!., 1987). The content of short-chain saturated fatty acids and the relatively high level of palmitoleic acid (C16:1) unique to butterfat was suggested to have a role in the effects of butterfat diets on these vascular diseases (Karanja et a!., 1989; Ikeda et a!., 1987). McGregor and coworkers (1981) reported that systolic blood pressure of SHR fed an n-6 PUFA diet for 1 6 weeks was not reduced compared to animals fed a highly saturated fat diet. These workers also demonstrated that SHR animals had greater platelet aggregation activity than their WKY counterparts. Saturated fat diets elicited a similar platelet response as did hypertension in animals and this effect was not significantly reduced by the PUFA diet in the SHR (McGregor eta!., 1981).  105  Animal strain differences in tissue antioxidant status: The insulin-resistance of the SHR reported by others (Hulman et a!., 1993; Finch eta!., 1990) may be a key underlying mechanism for the difference in antioxidant status between SHR and WKY animals.  Studies with diabetic rats and food-deprived rats have reported alterations in tissue  antioxidant enzyme activity and susceptibility to induced oxidation (Wohaieb and Godin, 1987a,b). More recently, rats restricted to 60% of normal feed intake exhibited tissue-specific and enzymespecific changes in antioxidant status (Xia et a!., 1995). It is important to recognize that despite an increase or decrease in the activity of an individual antioxidant enzyme, the susceptibility of a tissue to oxidative stress may not be altered. Tissue antioxidant status usually reflects a balance between enzymatic and nonenzymatic components. Differences in antioxidant status between animals fed chow diets or semi-synthetic diets: The semi-synthetic diets fed to animals in the present study appeared to have a greater effect on animal strain differences for the glutathione metabolizing enzymes than those involved in metabolizing reactive oxygen species. observed to be organ-specific.  These diet-induced effects on enzyme activity were also  Tissue antioxidant status is known to be sensitive to changes in  nutrition which may involve energy restriction (Xia et a!., 1995), level of protein intake (Hum et a!., 1992; Bauman et a!., 1988a,b), or dietary lipid source (L’Abbé et a!., 1991).  By comparing the  antioxidant enzyme activities of animals fed semi-synthetic diets in the present study to those fed a commercial chow in Chapter 1, it is evident that various antioxidant enzyme activities can also be altered by subtle changes in dietary composition, including the source of dietary fibre or the presence of sterols, such as cholesterol.  Evidence indicates that both soluble and insoluble non-starch  polysaccharides reduce plasma lipids to an extent that is related to the viscosity of the fibre in the gastrointestinal tract (Abbey et a!., 1993). These effects of dietary fibre on plasma lipid disposition may have had an indirect effect on tissue antioxidant enzyme activities as well, since there were considerable differences in fibre sources between the chow and semi-synthetic diets used in Chapter 1 and the present study, respectively. Another important factor to consider was the absolute amount of lipid included in the two types of diet. The chow diet used in Chapter 1 contained tallow as the principal fat in addition to lipids contributed by wheat and soybean meals included in the complex diet, 106  resulting in approximately 5% total crude lipid.  In contrast, the semi-synthetic diets used herein  contained 8% dietary lipid, derived from the experimental fats (5%) and canola oil (3%), present to provide adequate essential fatty acids. In addition, the low cholesterol semi-synthetic diets contained 0.05% cholesterol to balance all diets for the cholesterol contribution from the animal fat sources. Total unsaponifiable matter in the chow diet (e.g. plant sterols, tocopherols) was not quantitated, but could also be a factor in determining antioxidant status.  It is known that the functional state of  specific enzymes (e.g. glucose-6-phosphate dehydrogenase; G6PDH) can be subject to modulation by changes in dietary lipid composition (Mohan eta!., 1991). Also, tissue antioxidant enzyme activity and susceptibility to lipid oxidation can be influenced by factors that alter membrane lipid composition (L’Abbé eta!., 1991; Nalbone eta!., 1989).  Moreover, cholesterol has been shown to influence  membrane fluidity and stability (Kirby eta!., 1980)  and therefore could also represent a source of  differences between animals fed the two types of diet. Despite the similar gross energy content of the chow and semi-synthetic diets, the different fibre and lipid sources, as well as the presence of a small amount of cholesterol in the low cholesterol diet, should not be overlooked in attempting to explain the differences between the results obtained herein and in Chapter 1. Dietary fat source and cholesterol effects on antioxidant status: Several studies have reported alterations in the endogenous antioxidant status of rats fed diets varying in n-6 and n-3 PUFA composition (Kuratko eta!., 1994; SkiladOttir eta!., 1994; De Schrijver eta!., 1992; Gonzalez eta!., 1992; L’Abbé eta!., 1991; Mohan eta!., 1991; Huetal., 1989; Nalbone eta!., 1989). Studies with rats fed diets varying in n-6 and n-3 PUFA content from corn and olive oils have reported that RBC membrane fluidity and stability can be greatly altered by dietary fatty acid composition (Periago eta!., 1990). Moreover, various in vivo biochemical parameters involving plasma membrane fluidity can be altered by membrane fatty acid composition, such as insulin receptor binding and uptake (Storlien eta!., 1987) and cation flux (Schedl eta!., 1989).  In the present study, RBC  SOD and GSH-Px activities were both decreased in animals fed menhaden oil diets, while RBC CAT activity was not influenced by dietary fat source. A positive correlation between RBC CAT and SOD activities was previously observed in chow-fed SHR and WKY in Chapter 1, suggesting a compensatory mechanism for elevated SOD activity. L’Abbé and coworkers (1991) reported an inverse correlation 107  between the ratio of tissue SOD/GSH-Px activities with urinary TBARs. These workers suggested that the balance in activity between these two enzymes is a factor in tissue susceptibility to lipid peroxidation.  Moreover, despite the decrease in RBC SOD and GSH-Px activities in animals fed  menhaden oil in the present study, both the in vitro H -induced production of MDA as well as GSH 0 2 depletion in RBCs were actually decreased. The reduced susceptibility of RBCs from menhaden oil-fed animals to induced oxidative stress suggests that dietary n-3 PUFA affected RBC membrane fluidity and stability (Periago eta!., 1990).  Increased incorporation of n-3 PUFA into RBC membranes can  result in changes to the membrane cholesterol/phospholipid ratio to stabilize membrane fluidity (Periago  eta!., 1990). Similarly, animals fed the high cholesterol diets exhibited reduced susceptibility to H 0 2 induced MDA production in RBCs, suggesting that increased membrane stability due to cholesterol incorporation (Kirby eta!., 1980) reduced oxidation of membrane n-3 PUFA. The greater RBC GSH-Px activity observed in tallow-fed rats compared to those fed butter or soybean oil diets suggests that the stability of RBC membrane fatty acids to lipid oxidation can be altered by the ratio of C18:2,n-6 to C18:3,n-3 in the dietary fat. The competition for 6 -desaturase activity by linoleic and linolenic acids could be responsible for an improved linolenate desaturation in the presence of reduced quantities of linoleic acid, such as may be the case for both butter and tallow diets compared to soybean oil diets used in the present study. This effect of dietary fatty acid composition could conceivably result in the increased incorporation of linolenic acid and its metabolites into RBC membranes.  These n-3  polyunsaturated fatty acids are known to exhibit enhanced susceptibility to lipid peroxidation, thereby possibly contributing to an increased requirement for intracellular RBC GSH-Px activity to inactivate lipid peroxides formed in vivo. As a muscular tissue primarily involved in oxygen exchange, the heart uses circulating free fatty acids as an energy source as opposed to lipoprotein sources of lipid. This may explain the relative insensitivity of heart tissue fatty acid composition to dietary lipid manipulation (Jones et a!., 1 995; Nalbone et a!., 1989).  For example, while dietary fat source did not affect heart cholesterol or  triacylglyceride concentrations in the aforementioned studies, myocardial phospholipid fatty acids were found to reflect dietary fatty acid composition. In the present study, activities of heart SOD, GSH-Px and GSSG-Red were reduced in SHR and WKY animals fed menhaden oil.  L’Abbé and coworkers 108  (1991) also reported that heart SOD activity was reduced in rats fed diets containing 20% menhaden oil. Other workers have reported that rats restricted to 60% of normal energy intake exhibited reduced heart SOD activity (Xia eta!., 1995). The fact that animals fed menhaden oil diets exhibited reduced body weight gain and FER values could suggest that diet-induced changes in fatty acid substrate availability from adipose tissue are a factor in modulating SOD, GSH-Px and GSSG-Red activity in vivo. Interestingly, the reduced antioxidant enzyme activities observed in the hearts of menhaden oil-fed animals did not result in enhanced susceptibility to H -induced lipid oxidation. O 2  The decreased  susceptibility of heart tissue from animals fed menhaden oil diets to in vitro GSH depletion and TBARs formation agrees with the results in RBCs above. Therefore, despite the decreased activities of the antioxidant enzymes measured in heart tissue from animals fed menhaden oil diets, susceptibility to lipid oxidation appeared to be lower in heart tissue from these animals. Dietary fatty acid composition conceivably influences hepatic membrane lipid composition via triacylglyceride synthesis and secretion. Several reports have demonstrated an influence of dietary fat source on hepatic membrane fatty acid profiles (Jones et a!., 1 995; De Schrijver et a!., 1992) and susceptibility to lipid peroxidation (L’Abbé eta!., 1991; Hu eta!., 1989). It is well recognized that the relatively high content of n-3 PUFA provided by marine oil diets can result in the replacement of arachidonic acid (C20:4,n-6)  in membrane phospholipids by EPA and DHA (L’Abbé eta!., 1991;  Kinsella, 1988). This change in membrane PUPA content results in changes not only to membrane fluidity, but also enzymatic activity, namely mixed function oxidases (Saito et a!., 1 990)  and  cyclooxygenases and lipooxygenases involved in eicosanoid synthesis from n-6 and n-3 precursors (Kinsella, 1988). The replacement of membrane arachidonic acid by n-3 PUFA supplied in the diet may conceivably reduce the activity of cyclooxygenase and lipooxygenase enzymes for which arachidonic acid is a substrate, thereby reducing the load of reactive oxygen species (ROS) produced from these biosynthetic pathways. Decreased production of ROS in vivo would result in a reduced necessity for tissue antioxidant enzyme activity. This hypothesis is supported by the fact that liver GSH-Px and SOD activities were reduced in both SHR and WKY animals fed menhaden oil diets in the present study, although liver GSSG-Red activity was increased in the same animals. L’Abbé and coworkers (1991) also reported that liver GSH-Px and SOD activities were decreased in animals fed 20% menhaden diets. 109  An alternative explanation for the decreased hepatic GSH-Px activity in menhaden oil-fed animals is the association between hepatic GSH-Px activity and tissue G6PDH activity described by others (Mohan eta!., 1991). These workers reported that reduced activity of hepatic G6PDH in animals fed 18% menhaden oil diets was observed to coincide with reduced liver GSH-Px activity. Another factor in the effect of dietary fatty acid content on tissue antioxidant status is the oxidative stability of fatty acids incorporated into membrane phospholipids (De Schrijver et a!., 1 992; Hu eta!., 1989). Dietary n-3 fatty acids from marine oils are associated with increased levels of tissue and urinary TBARs as well as tissue lipid peroxides (Sküladóttir eta!., 1994; De Schrijver eta!., 1992; L’Abbé eta!., 1991). Moreover, increased levels of fluorescent pigments derived from the reaction of lipid peroxidation products with membrane proteins have been identified in tissues from animals fed fish oil diets (Nalbone eta!., 1989; Reddy eta!., 1973). The increased peroxisomal li-oxidation of long-chain n-3 PUFA from dietary marine oils suggests a potential increase in the production of H 0 2 in vivo (Mohan et a!., 1991). Thus, reactive oxygen species as well as products of lipid peroxidation may be present in vivo, necessitating their detoxification by CAT and GSH-Px.  However, excess  amounts of these metabolic products are known to have inhibitory effects on antioxidant enzymes (Remade at a!., 1992).  For example, SOD and GSH-Px are known to be inhibited by elevated  concentrations of H 0 and lipid hydroperoxides (ROOH), respectively (Remade et a!., 1992). Thus, 2 the reduced activities of SOD and GSH-Px in tissues from animals fed diets containing menhaden oil may be due to enzyme inhibition from excess generation of reactive oxygen-derived substances. However, while this mechanism may apply to studies using a high level (12.5  -  20%) of marine oil in  the diet, it does not appear to be valid for the present study with animals fed a 5% menhaden oil diet, since peroxide-induced liver TBARs production was actually reduced in these animals.  Rather, the  reduced susceptibility to H -induced GSH depletion and TBARs production of liver tissue from SHR 0 2 and WKY fed menhaden oil may be related to the lower body weight gain and FER of these animals and possibly to a relative reduction in available energy stores associated with the marine oil diets (Xia eta!., 1995; Su and Jones, 1993). While neither heart antioxidant enzyme activities nor susceptibility to H -induced oxidative 0 2 stress were affected by the level of dietary cholesterol, animals fed the high cholesterol diets exhibited 110  reduced hepatic CAT, SOD and GSH-Px activities, and increased hepatic GSSG-Red activity. It has been hypothesized by Smith (1991)  that cholesterol may itself act as an antioxidant molecule by  stabilizing cell membranes and preventing peroxidation of membrane fatty acids.  The presence of  increased membrane cholesterol due to dietary cholesterol supplementation could reduce the requirement for antioxidant enzyme activity due to increased membrane lipid stability associated with the replacement of phospholipid polyunsaturated fatty acids with cholesterol in tissue membranes. The reduced susceptibility to H -induced TBARs production of liver tissue from animals fed high O 2 cholesterol diets supports this concept of increased  membrane stability due to cholesterol  incorporation. Feeding rats cholesterol results in a fatty liver as evidenced by the greater liver weights and abnormal macroscopic appearance as noted in the present study. The increased susceptibility to -induced GSH depletion of liver from animals fed high cholesterol diets, therefore, suggests that O 2 H the fatty liver observed in these animals may be regarded as an additional factor contributing to increased GSH depletion. Other considerations: It has been shown that the development of insulin resistance in rats fed high fat diets (Barnard et al., 1993) can be prevented by replacing a portion of dietary fat with fish oil (Storlien eta!., 1987). Even though plasma insulin levels were not measured in this study, the activity of GSSG-Red, which has been reported to be inhibited in the presence of high levels of insulin (Long and Carson, 1961), may be an indirect marker of the insulin status of SHR compared to WKY rats. Previous workers examining the antioxidant status of animals fed fish diets have considered the possibility of the effect of a relative selenium (Se) deficiency on antioxidant enzyme activity in these animals (L’Abbé eta!., 1991; Nalbone eta!., 1989). Feeding animals either 20% menhaden oil or 12.5% salmon oil in the diet did not result in a functional Se deficiency as demonstrated by the tissue activities of GSH-Px in these studies (L’Abbé eta!., 1991; Nalbone eta!., 1989). Similarly, in the present study, while GSH-Px activities were reduced in RBCs and heart of SHR and WKY animals fed menhaden oil diets, the variable effect on hepatic GSH-Px activities in SHR compared to WKY animals indicates that functional Se deficiency did not occur in menhaden oil-fed animals in the present study. 111  Conclusion: In conclusion, hypertensive SHR exhibited reduced plasma lipids and FER values in combination with tissue-specific and enzyme-specific alterations in antioxidant status compared to normotensive WKY animals. The differences in heart and liver tissue GSH levels and GSH depletion paralleled the activity of GSSG-Red in these tissues and may indicate an up-regulation of this enzyme activity to maintain GSH levels against possible oxidative stress in hypertension. The fact that the level of GSH, as well as depletion of GSH from RBCs were not different in hypertensive SHR may indicate a compensatory regulation of antioxidant enzyme activities to maintain cellular levels of GSH in RBCs. On the other hand, MDA production in RBCs from hypertensive SHR was elevated, consistent with the previous results with chow-fed animals in Chapter 1, suggesting that this may be a useful marker of RBCs in the SHR. Diets containing a high concentration of long chain n-3 fatty acids, namely menhaden oil, reduced body weight gain and FER values in both SHR and WKY animals, suggesting potential differences in fatty acid substrate availability in animals fed these diets. The reduced plasma lipid concentrations in animals fed menhaden oil diets are consistent with the effects of long-chain n-3 PUFA on lipoprotein metabolism and fatty acid peroxisomal B-oxidation. Both the decreased tissue GSH-Px and SOD activities in RBCs, heart and liver tissue from animals fed menhaden oil diets, in addition to the reduction in tissue susceptibility to in vitro H -induced GSH depletion and lipid 0 2 peroxidation in these same animals, may be related to the influence of dietary fatty acids on membrane fatty acid composition. Diet-induced alterations to cell membrane fatty acid stability to lipid oxidation may be due to the replacement of n-6 (i.e. arachidonic acid) by n-3 fatty acids (eicosapentaenoic and docosahexaenoic acids) in membrane phospholipids. Diets containing a high level of cholesterol increased plasma cholesterol levels as expected, as well as inducing pathophysiological changes to the liver, as reflected by liver size and weight. These effects of feeding high cholesterol diets to both SHR and WKY animals were associated with a reduction in the activity of hepatic CAT, SOD and GSH-Px, possibly indicating a reduced requirement for antioxidant enzyme activity due to increased membrane fatty acid stability to lipid oxidation resulting from the potential incorporation of cholesterol into cell membranes. This result was further 112  substantiated by the reduced susceptibility to in vitro 2 H 0 induced lipid peroxidation of liver tissue, and also RBCs from animals fed high cholesterol diets. However, the increased susceptibility to GSH depletion of liver tissue from these same animals is evidence for a pathophysiological effect of the high cholesterol diets on liver tissue. In summary, dietary fat source and dietary cholesterol content had a greater influence on animal strain differences in plasma lipid levels as demonstrated by the experimental treatment interactions observed than on animal strain differences in tissue antioxidant status parameters. The one exception to this was the activity of liver GSH-Px, suggesting that this liver antioxidant enzyme was particularly sensitive to tissue compositional changes induced by dietary treatment interactions. Finally, it was noteworthy that semi-synthetic diets fed to the SHR and WKY animals in the present study resulted in alterations in antioxidant status parameters when compared to the chow diet in Chapter 1, demonstrating the subtle effects of non-purified dietary components on  in vivo antioxidant status.  113  CHAPTER 3 Species-related differences in plasma lipids and susceptibility to atherosclerosis between atherosclerosis-resistant (rat) and -susceptible (quail) animals fed diets supplemented with cholesterol. Introduction: Numerous animal models have been utilized to study the effects of dietary fat source and cholesterol intake level on atherosclerosis. Through the use of genetic selection for specific traits of interest, models for elevated plasma lipids (Watanabe Heritable Hyperlipidemic (WHHL) rabbit; Bilheimer eta!., 1982; Kita eta!., 1981), arterial plaque deposition (atherosclerosis-susceptible Japanese quail; Shih eta!., 1983; Smith and Hilker, 1973) or elevated blood pressure (spontaneously hypertensive rat (SHR); Okamoto and Aoki, 1963) have been developed for research purposes.  Additional animal  models used include various species of rodents (Woollett eta!., 1992; Fernandez and McNamara, 1991), swine (Faidley eta!., 1990) and non-human primates (Mott eta?., 1992). Overall, the ability of these animal models to reproduce a disease state that is similar to the human situation is variable. Certain animal models can be useful in studying specific dyslipidemias, such as the use of the WHHL rabbit as a model for human familial hyperlipidemia (Kita et a!., 1981).  Often, the interaction of  experimental treatment effects with the lipid and lipoprotein metabolism of animals does not mimic that of humans, so that extrapolation of results obtained from animal studies to the human is difficult. Variations in feeding behaviour and dietary composition between rodent or avian animal models versus that of humans (Chapman, 1 980), differences in lipoprotein composition (Terpstra et a!., 1982; Mills and Taylaur, 1971), and susceptibility to atherosclerosis (Godin eta?  .,  1994; Godin and Dahlman,  1993; Bishop, 1980) are some examples of species-related differences. The use of experimental diets with atypical lipid and cholesterol levels in animal models has been successful in accelerating the rate of atherogenesis, in comparison to the natural, but lengthy development of the disease in these animals (Masuda and Ross, 1990a).  Studies in non-human  primates have indicated that the cellular events and tissue morphological changes which occur during the progression of atherosclerosis are similar whether animals are fed diets inducing modest levels of hypercholesterolemia or diets inducing extremely high levels of cholesterolemia (Masuda and Ross, 1 990a,b). Studies to characterize events involved in the initiation of atherogenesis have suggested 114  the possible involvement of cytotoxic cholesterol oxides in this disease process (Peng et a?., 1 985, 1978). Moreover, cholesterol and its oxide derivatives have been identified as significant components of arterial plaque composition in humans (Steinbrecher and Lougheed, 1992)  and various animal  species (Morin and Peng, 1992). While the rat (Rattus norvegicus) is noted to be resistant to the development of atherosclerosis (Bishop, 1980), the plasma lipid profiles of this species are well characterized for nutritional biochemistry studies. When high levels of cholesterol are fed to rats, changes in plasma lipid profiles are similar to those observed in human type Ill hyperlipoproteinemia (Mahley and Holcombe, 1977). Also, several avian species (e.g. pigeon, Japanese quail) have been used as experimental models in atherosclerosis research (Stewart-Phillips et a?., 1992; Shih eta?., 1983). Japanese quail (Coturnix japonica) made hyperlipidemic by dietary cholesterol supplementation develop atherosclerotic lesions in the aorta in a dose-dependent manner (Radcliffe eta?., 1982). Moreover, studies feeding oxidized dietary cholesterol to Japanese quail resulted in greater severity of aortic lesions than was observed with purified cholesterol (Donaldson, 1982).  115  Hypothesis for Chapter 3: Differences in the susceptibility of animal models to diet-induced hyperlipidemia contribute to species-specific variations in the development of atherosclerosis. Obiective for Chapter 3: To link the relative differences in susceptibility to diet-induced atherosclerosis in the atherosclerosis-susceptible Japanese quail  and -resistant rat to species-related  variations in  hyperlipidemia and aortic sterol content. Specific Aims for Chapter 3: i. A comparison between species for susceptibility to diet-induced hyperlipidemia will be made by feeding diets varying in cholesterol content with a constant level of fat for a 9 week period to the Wistar rat (a species showing a high degree of resistance to the development of atherosclerosis) and the atherosclerosis-susceptible Japanese quail (known to develop aortic atherosclerotic plaques when fed cholesterol-rich diets). ii. Gas chromatography combined with mass spectrometry (GC-MS) will be used to identify and quantitate the cholesterol and cholesterol oxide content in aortic tissue samples.  116  Materials and Methods: Animals and diets: Sixteen male Wistar rats (5 weeks of age; Charles River, Montreal, PQ) and 1 6 male Japanese quail (Coturnixjaponica; 6 weeks of age; University of British Columbia Quail Genetic Resource Centre, Vancouver, B.C.)  were used in this study.  The Japanese quail used in these experiments were  selected for susceptibility to cholesterol-induced atherosclerosis (Shih et a!., 1983). Animals (8 per group for rats and quail) were fed semi-synthetic diets (Table 3.1) varying in protein source (casein and soy protein for rat and quail diets, respectively) and containing 3% canola oil (to provide essential fatty acids) and 5% beef tallow. Quail diets were mixed, pelleted and crumbled at the Agriculture Canada research station at Agassiz, B.C. Cholesterol was added to the diets to provide either a low (0.0264 mg/kJ) or high (0.264 mg/kJ) level of cholesterol for rats, and a low (0.0274 mg/kJ) or high (0.274 mg/kJ) level of cholesterol for quail. Diets were fed to the animals for a 9 week experimental period. Animals had access to distilled deionized water at all times. Animals were separately housed with a 14-hr light/i 0-hr dark cycle and ambient temperature. Birds were housed in brooder cages in a separate room with lighting and temperature controls. Experimental procedures: At the end of the experimental feeding period blood was collected into heparinized tubes from rats by exsanguination under halothane anaesthesia (4% halothane at a flow rate of 4L/ mm  for  induction, and 2.5% at a flow rate of 2 L/min for maintenance), while quail were decapitated and trunk blood collected into heparinized tubes. Plasma was isolated by low speed centrifugation (1000 x g, 5 mm, 4°C) for determination of total and free cholesterol as well as triacylglycerides as described in Chapter 1 (Boehringer Mannheim, Laval, PQ). Determination of aortic placiue score: In both the rat and quail, the aortic tree (the brachiocephalic arteries to their bifurcations and the aorta to the iliac branching) was dissected out, opened longitudinally and examined under a iO-30X dissecting microscope for lesions on the inner wall. Aortic lesion scores from 0 to 4 were assigned according to Shih et a!. (1983) and Godin eta!. (1994). Scoring was performed by two independent investigators (in a blinded fashion) as follows: 0  =  clean surface; 1  =  < 5 plaques; 2  =  6-20 plaques 117  Table 3.1. Composition of diets fed to Wistar rats and atherosclerosis-susceptible Japanese quail.  Rat diet Cholesterol level (% by weight)  0.05  0.5  Quail diet  0.05  0.5  Dietary component (gil OOg) Casein’ 2 Soy protein meal Ca-free mineral mix 1 3 CaCO Vitamin mix 1 4 DL-methionine Choline chloride 2 Cornstarch 5 5 Sucrose Alphacel’ 2 Monofos 5 Canola oil Beef tallow 6 Cholesterol’ Cholic acid 1  1 2  6  25.0 -  25.0 -  -  -  34.0  34.0  3.5 2.0 3.0 0.3 0.2  3.5 2.0 3.0 0.3 0.2  2.0 5.0 0.3 0.4 0.3  2.0 5.0 0.3 0.4 0.3  47.0 3.0 5.0 3.0  47.0 3.0 5.0 3.0  39.5 2.5 5.0 3.0  39.5 2.5 5.0 3.0  3.0 5.0 0.05 0.025  3.0 5.0 0.50 0.25  3.0 5.0 0.05 0.025  3.0 5.0 0.50 0.25  ICN Biochemicals Inc., Cleveland, OH, USA Van Waters & Rogers, Abbotsford, B.C., Canada BDH Chemicals, Toronto, ON, Canada United States Biochemical Co., Cleveland, OH, USA Neptune Food Services, Richmond, B.C., Canada Cargill Foods, High River, AB, Canada  118  and an affected area less than 50%; 3  =  > 20 plaques with an affected area greater than 50%; 4  =  massive atheromas present. Immediately after scoring was completed, the aortic tree was frozen at -35°C. Analysis of cholesterol and cholesterol oxides by gas chromatography/mass spectrometry: For analysis of aortic cholesterol oxidation products (COPs) by gas chromatography (GC) with confirmation by mass spectrometry (GC-MS), frozen aortic tissues from rats and quail were thawed, cleaned of adhering tissue, weighed and lipids extracted according to Folch eta!. (1957). An internal standard (5a-cholestane, lOOpg) was added to samples before lipid extraction. The extracted lipids were evaporated to dryness under a stream of N 2 and subjected to a cold saponification with 1 N KOH (in methanol) overnight at room temperature (25°C). The saponified samples were extracted with diethyl ether (3X)  and washed with 0.5 N KOH (1X) and distilled deionized water (2X).  Nonsaponifiables were dried using anhydrous 4 SO before reducing the sample volume with a N 2 Na 2 stream for transfer to Reacti-Vials (Pierce Chemical Co., Rockford, IL).  Samples were dried under  vacuum to remove traces of moisture, before solubilization in 200 p1 dry pyridine. A 100 p1 aliquot of sample was derivatized with 50 p1 Sylon BTZ (Supelco, Inc., Oakville, ON) and the reaction allowed to proceed to completion (30 mm)  at room temperature.  After derivatization, standards (5a-  cholestane, cholesta-3,5-dien-7-one, cholest-5-en-3f-ol (cholesterol), cholest-5-ene-31,4B-diol (4lhydroxycholesterol), cholest-5-ene- 3R, 7a-diol (7a-hydroxycholesterol), cholest-5-ene-3I&71-diol (7Ihydroxycholesterol), cholest-5ene-31,25-diol (25-hydroxycholesterol), 5 ,6a-epoxy-5a-cholestan-31-ol (cholesterol 5a,6a-epoxide), 5a-cholestan-3R,5,61-triol (cholestane-triol),and 3I-hydroxycholest-5-ene7-one (7-ketocholesterol); Steraloids, Inc., Wilton, N.H., USA) and samples were analyzed using a DB 1 column (15 m X 0.25 mm i.d., 0.1 p film thickness; J & W Scientific, Inc., Folsom, CA, USA) on a Carlo Erba gas chromatograph (Carlo Erba Strumentazione, Italy)  equipped with a flame ionization  detector (GC-FID). A representative chromatogram of derivatized cholesterol oxide standards is shown in Figure 3.1.  Carrier gas used was He with N 2 as the make-up gas.  Injector and detector  temperatures were 250°C and 280°C, respectively, oven temperature was programmed from 1 80°C to 250°C at 3C° per minute, final temperature was held for 1 5 minutes. During GC-FID analysis,  119  Is  5  LJiL 4 L Figure 3.1 A representative GC-FID chromatogram of derivatized cholesterol oxide standards with internal standard (IS) = 5a-cholestane. 1 = cholesta-3,5-dien-7-one; 2 = cholesterol; 3 = 7ahydroxycholesterol; 4 = 5,6a-epoxy-5a-cholesterol; 5 = 7f-hydroxycholesterol; 6 = 4Bhydroxycholesterol; 7 = cholestane-triol; 8 = 7-ketocholesterol; 9 = 25-hydroxycholesterol.  120  chromatograms were stored and analyzed using a Hewlett-Packard HP 3393A integrator (Hewlett Packard Inc., Avondale, PA). Confirmation of identity of COPs was performed using a Kratos MS8O mass spectrometer (Ramsey, N.J.) coupled to a Carlo Erba gas chromatograph as above. between mass range m/e  =  Mass spectra were scanned  60-700 using DS 55 software on a Data General Eclipse S/i 20 computer  (Data General Corp., Westborough, MA). Corrections for background were performed as required. Quantitation of COPs was performed after determining response linearity of each derivatized sterol.  Response linearity of GC-FID to trimethyl silyl (TMS)  ether sterols was confirmed by  constructing a calibration curve of the response to mixtures of varying amounts of sterols with a constant amount of internal standard (IS; 5a-cholestane). Mixtures of standards were prepared such that concentrations of sterols ranged from 10 nglpL to 1 .Opg/pL with the ratio of IS:standard ranging from 1:1 to 100:1. Response linearity was analyzed by plotting the area response ratio of each sterol to IS versus the weight ratio of the sterol to IS (Figure 3.2). Refer to Appendix Tables 1 and 2 for additional information regarding relative retention times and response linearity of derivatized sterols determined by GC-FID. Statistics: All data are expressed as the mean ± SEM. Statistical analysis was performed using Student’s t-test at a significance level of 0.05.  121  Calibration curve for standard #5: 7 B hydroxycholesterol —  1.25  1.00 0 .  4.’  0  0.75  0 4.’ I  0.50 I  0.25  0.00 0.00  0.25  0.50  0.75  1.00  1.25  Weight ratio, Std.IIS  Figure 3.2 A representative standard curve depicting the response linearity of derivatized cholesterol oxide standards using GC-FID. Standard curve is that of 7-hydroxychoIesterol, r = 0.995.  122  Results: Plasma lipid profiles of rat and quail: Plasma lipid profiles of Wistar rats and atherosclerosis-susceptible Japanese quail are summarized in Table 3.2.  Species differences in plasma lipid response to feeding cholesterol-  supplemented semi-synthetic diets were apparent at both 0.05% and 0.5% cholesterol levels, with quail exhibiting a much greater increase (ca. lOX) in plasma total cholesterol between low and high cholesterol-fed birds, as compared to rats (ca. 2X). Aortic tissue atherosclerotic plaque of rat and quail: Aortic plaque scores reflected the plasma lipid profiles of the atherosclerosis-susceptible Japanese quail (Table 3.3). The severe hyperlipidemia of high cholesterol-fed quail was associated with severe atherosclerotic plaque (reflected in the high plaque score and high percentage of lumen area covered). No visible atherogenesis was observed by dissecting microscope in aortae from quail fed low dietary cholesterol or from rats fed either diet.  Aortic tissue cholesterol and cholesterol  oxidation product content (COPs; Table 3.4) coincided with the plaque scores obtained from visual microscopic evaluation of the vessel lumen walls. Aortic tissue cholesterol content was greater in quail than in rats regardless of dietary cholesterol content.  Feeding high levels of cholesterol to animals  resulted in an increase in aortic cholesterol content in quail only. Rat aortic tissue cholesterol content was not altered by cholesterol feeding. Combined gas chromatography and mass spectrometry (GC MS)  was used to identify and quantitate two COPs (7R-hydroxycholesterol and 7-ketocholesterol)  present in the aortic tissue from quail fed the high cholesterol diet (Figure 3.3b).  COPs were not  detected in the nonsaponifiable extracts from aortae of low cholesterol-fed quail (Figure 3.3a) or from either dietary groups of rats (Fig. 3.4a,b).  123  2  1.40 ± 0.20  6.22 ± 0.46*  3.06 ± 0.12  *  3.55 ± 0.21  1.47 ± 0.12  0.05  53.1 ± 2.5*  0.5  0.99 ± 0.07*  4.12 ± O.36  0.5  Triacyiglycerides  597 ± 0.25  0.05  Total cholesterol  9.08 ± 0.17*  0.5  Free cholesterol  Values represent mean ± SEMI n = 8. * denotes a significant (p 0.05) difference between cholesterol levels across a row.  1.82 ± 0.08  Rat  1  1.55 ± 0.09  0.05  Quail  Animal species:  Cholesterol level (% by weight):  : 2 Plasma lipids (mmol/L)  Table 3.2. Plasma lipids of Wistar rats and atherosclerosis-susceptible Japanese quail fed tallow diets containing low 1 and high levels of cholesterol.  c,1  -  2  N.D.  N.D.  0.05  (%)3  N.D.  61 ± 10  0.5  Area covered  Values represent mean ± SEM, n = 8. Plaque score based on scale of 0 (N.D.) = clean surface; 1 = 5 plaques; 2 = 6-20 plaques; 3 = > 20 plaques; 4 = massive atheromas observed. Values represent two judges evaluating in a blinded protocol. Area covered (%) = percent of aortic epithelium covered by plaque, range 0 (N.D.)-100%.  N.D.  N.D.  Rat  1  3.7 ± 0.2  0.5  N.D.  0.05  Quail  Animal species:  Cholesterol level (% by weight):  Plaque score 2  Table 3.3. Aortic plaque score and area covered in Wistar rats and atherosclerosis-susceptible Japanese quail fed tallow 1 diets containing low and high levels of cholesterol.  C)  r%)  2  N.D.  N.D.  0.05  N.D.  0.24±0.03  0.5  7R-hydroxycholesterol  Values represent mean ± SEM, n = 8. Values expressed on basis of tissue wet weight. N.D. = none detected. * denotes a significant (p 0.05) difference between cholesterol levels across a row.  0.91 ± 0.49  0.93 ± 0.04  Rat  1  11.48±2.13*  0.5  3.19±0.91  0.05  Cholesterol  Quail  Animal species:  Cholesterol level (% by weight):  (mglg)  : 2 Sterol  N.D.  N.D.  0.05  N.D.  0.27±0.04  0.5  7-ketocholesterol  Table 3.4. GC quantitation of cholesterol and cholesterol oxidation products in aortic tissue from Wistar rats and atherosclerosis-susceptible Japanese quail fed tallow diets containing low and high levels of cholesterol. 1  A 1  2  B  3  -j  15  20  time  25  30  (mm.)  Figure 3.3 GC-Fl 0 chromatogram of atherosclerosis-susceptible Japanese quail aortic tissue dervatized nonsaponifiables. (A) 0.05% cholesterol diet, (B) 0.5% cholesterol diet. 1 = internal standard, 5acholestane; 2 = cholesterol; 3 = 7B-hydroxycholesterol; 4 = 7-ketocholesterol.  127  A  30  time (mm.)  Figure 3.4 GC-FID chromatogram of Wistar rat aortic tissue derivatized nonsaponifiables. (A) 0.05% cholesterol diet, (B) 0.5% cholesterol diet. 1 = internal standard, a-cholestane; 2 = cholesterol.  128  Discussion: While both the quail and the rat are HDL predominant animals, their individual responses to feeding semi-purified diets containing either a low or high level of cholesterol were quite different. Quail exhibited plasma total cholesterol and triacylglyceride (TG) concentrations which were greater than their rat counterparts at both the low and high levels of dietary cholesterol.  This species  difference has been attributed to the presence of the large, absorptive TG-rich portomicrons (large VLDL) in the plasma of birds during the initial stages of dietary lipid absorption (Ho and Taylor, 1 983). Lipid metabolism of birds is unique in that fat is absorbed from the GI tract via the portal circulation, as portomicron particles, compared to the lymphatic pathway of mammals (Chapman, 1980). Species differences were more pronounced when the high (0.5%) cholesterol diet was fed to animals, with the quail showing dramatic increases in both plasma total cholesterol and TG concentrations. The rat also exhibited increased plasma cholesterol levels, but to a much lesser extent, reflecting the known resistance of this species to the induction of hypercholesterolemia via cholesterol feeding (Bishop, 1980). When Smith and Hilker (1973) fed Japanese quail diets containing 40-50% of calories from beef tallow with 0.5% cholesterol, plasma cholesterol levels approached those observed in the present study only after 9 months on these experimental diets. The large increase in plasma total (free and esterified) cholesterol concentration observed in cholesterol-fed quail reflects both the increase in plasma free cholesterol levels as well as the increase in cholesteryl ester levels (an increase from 74% to 83% esterified cholesterol in plasma from low and high cholesterol-fed quail, respectively). In studies using atherosclerosis-susceptible Japanese quail fed diets containing a 1.5% cholesterol:cholic acid (2:1) supplement, Radcliffe and coworkers (1982) reported that both plasma total and esterified cholesterol were increased, but no change was observed in the relative proportion of esterified cholesterol (ca. 70%). However, these workers did observe a dose-dependent elevation in arterial tissue cholesterol with a greater percentage in the esterified form when birds were fed high cholesterol diets (Radcliffe eta!., 1982). In the present study, greater levels of aortic cholesterol were observed in the quail in comparison to the rat at both the low and high levels of dietary cholesterol. The fact that the rat is generally extremely resistant to the development of atherosclerosis was demonstrated a9ain in the present study by the absence of atherosclerotic plaque and the considerably 129  lower aortic cholesterol content in the normotensive Wistar rat fed a hypercholesterolemic diet.  In  marked contrast, several avian species (chicken, pigeon and quail) do develop atherosclerotic plaque in the aorta when fed dietary cholesterol (Stewart-Phillips et a!., 1992). Several workers (Shih eta!., 1983; Donaldson, 1982; Radcliffe eta!., 1982) using similar or higher levels of dietary cholesterol supplementation (0.75%, 1 .5%)  than those used in the present study, have also reported the  development of macroscopic atherosclerotic lesions in the aorta of Japanese quail. One of the risk factors for the development of atherosclerosis is the requirement for a state of hypercholesterolemia. However, it is clear that hypercholesterolemia alone is not sufficient to induce atherosclerotic plaque, as seen with the rat model used herein and other resistant animal lines used in previous studies, such as the resistant pigeon and quail strains (Godin eta!., 1994; Chapman, 1980). Modified (oxidized)  lipid species have been identified in the plasma lipoproteins and aortic  plaque of atherosclerotic humans (Steinbrecher and Lougheed, 1992; Stringer eta!., 1 989) and animal models (Rosenfeld eta!., 1990; Palinski eta!., 1989). Furthermore, the presence of COPs in circulating lipoprotein has been demonstrated in healthy humans (Dzeletovic et a!., 1995) and monkeys (Peng  eta!., 1982). The atherogenic potential of COPs has been demonstrated by in vitro cell culture (Peng eta!., 1985, 1978)  as well as in animal feeding studies (Donaldson, 1982).  In a study wherein  Japanese quail were fed either purified cholesterol or oxidized cholesterol, those birds fed the latter diets exhibited greater plasma and liver cholesterol concentrations in association with increased severity of atherosclerotic lesions as compared to animals fed purified cholesterol (Donaldson, 1982). Differences in aortic lesion severity were also observed between animals fed purified cholesterol compared to USP cholesterol diets.  The latter diet resulted in greater lesion severity than purified  cholesterol, but less so than oxidized cholesterol feeding. The supplementation of oxidized cholesterol diets with antioxidants (synthetic phenolics or a-tocopherol) reduced severity of aortic lesions, but did not completely prevent the development of atherosclerosis in those animals (Donaldson, 1982). While it is well known that atherosclerotic plaque contains large amounts of cholesterol, tissue culture studies with human fibroblasts and macrophages have indicated that native (unoxidized) cholesterol is not internalized by these cell lines; however, oxidized cholesterol is taken up in a nonsaturable fashion (Steinberg eta!., 1989). Several COPs have been identified as having cytotoxic, 130  angiotoxic, carcinogenic and mutagenic bioactivities (Peng et a!., 1992; Smith and Johnson, 1989), all or some of which may play a role in the initiation and/or proliferation of atherosclerotic plaque. Specifically, 25-hydroxycholesterol and cholestane-3I,5a,6I.-trioI are particularly toxic to cultured rabbit aortic smooth muscle cells (Peng eta!., 1978). Thus, potentially angiotoxic cholesterol oxides may be present in the lower density lipoprotein fractions of animals which develop atherosclerotic lesions. In other studies, human plasma has been reported to contain detectable amounts of COPs, namely the cholesterol-5,6a- and f.-epoxides, the 7-hydroxycholesterol isomers, and 7-ketocholesterol (Dzeletovic eta!., 1995; Morin and Peng, 1992).  Steinbrecher and Lougheed (1992) reported the  presence of these same COPs in the LDL extracted from human atherosclerotic plaques. The presence of 7l.-hydroxychoIesterol in tissues may be due to the autoxidation of cholesterol, whereas its 7ahydroxycholesterol isomer is known to be a product of in viva oxidation in bile acid synthesis (Smith and Johnson, 1989). Other workers have been able to confirm the identity of several COPs (cholest 3,5-diene-7-one, cholestane-triol, 7-hydroxycholesterols, 7-ketocholesterol, 24-hydroxy-, 25-hydroxyand 26-hydroxycholesterol) in human aortic specimens (Morin and Peng, 1992). Detectable amounts of 7-ketocholesterol have also been reported in aortic tissue from rat, cat, bovine, horse and baboon samples (Morin and Peng, 1992). The present study represents the first time that COPs have been measured in aortic plaque material collected from atherosclerosis-susceptible Japanese quail. In the present study, precautions were taken to minimize exposure of sample lipid extracts to excess oxygen. One indicator of breakdown of cholesterol oxides during sample preparation is the presence of cholest-3,5-diene-7-one due to the instability of 7-ketocholesterol through thermal dehydration (Park and Addis, 1985). None of the experimental samples had detectable amounts of this compound as determined by GC-MS analysis.  Using GC-MS, 7l-hydroxycholesterol and 7-  ketocholesterol were identified and quantitated in quail aortic tissue exhibiting plaque formation. These COPs are identical to those reported from aortic plaques from both humans as well as other animal models, thereby further validating the use of the atherosclerosis-susceptible Japanese quail for research in the area of experimental atherogenesis.  131  Conclusion: While both the rat and the Japanese quail are HDL predominant species, atherosclerosis was inducible only in the latter animal model. This result was associated with species differences in plasma lipid profiles as observed with the greater response to cholesterol feeding in the quail. Aortic tissue sterol content was also shown to vary between animal models.  Quail aortae exhibited greater  cholesterol contents than corresponding tissues from similarly treated rats, which could be associated with differences in plasma cholesterol concentrations between species. Moreover, the presence of the cholesterol oxides (7B-hydroxycholesterol and 7-ketocholesterol) in aortic plaque from atherosclerosis susceptible Japanese quail similar to those observed in human aortic plaque were identified using GC MS. These findings further substantiate the use of the atherosclerosis-susceptible Japanese quail in experimental atherosclerosis research.  132  CHAPTER 4 Effect of dietary fat source on aortic plaque, plasma lipids and antioxidant status of atherosclerosis-susceptible Japanese quail. Introduction: Studies conducted with tissues collected from patients with coronary heart disease suggest an association between increased susceptibility to lipid peroxidation and reduced levels of specific antioxidant enzymes in the plasma and red blood cells (Jayakumari eta!., 1 992), platelets (Buczynski  eta!., 1993) and diseased aortic tissue (Hunter eta!., 1991).  Further evidence of a role for lipid  oxidation and in vivo antioxidant status in atherosclerosis is the presence of oxidized LDL in aortic plaque material from humans and animal models (Steinbrecher and Lougheed, 1 992; Rosenfeld eta!., 1990; Palinski eta!., 1990). The Japanese quail (Coturnix japonica) has been used by several groups of investigators to study diet-induced atherosclerosis (Toda and Oku, 1995; Toda et a!., 1988; Donaldson, 1982; McCormick etal., 1982; Smith and Hilker, 1973). Histological evaluation of the aortic plaque which develops when diets containing a high level of cholesterol are fed to these birds indicates that the cell populations involved in the initiation and propagation phases of plaque development are very similar between the quail and human atherosclerosis (Shih, 1983; Shih eta!., 1983; McCormick eta!., 1982). The characterization of atherosclerosis-susceptible and -resistant strains of Japanese quail has contributed to the use of this animal as a model for human atherosclerotic disease (Shih eta!., 1983). In studies examining the role of endogenous antioxidant status in the development of atherosclerosis in Japanese quail, Godin and coworkers (1994) reported that differences between atherosclerosissusceptible and -resistant strains of Japanese quail for activity of antioxidant enzymes in aortic tissue, red blood cells and plasma were tissue- and enzyme-specific. Previously in Chapter 3, the atherosclerosis-susceptible Japanese quail was demonstrated to exhibit a much greater increase in plasma lipid concentrations in response to feeding a high level of cholesterol in the diet than the rat, the latter species generally being considered to be resistant to the development of atherosclerosis (Bishop, 1980).  The greatly increased plasma lipid concentrations  133  observed in these quail coincided with the appearance of severe atherosclerotic plaque in the aortae of birds fed semi-synthetic diets containing 0.5% cholesterol (with 0.25% cholic acid). A considerable body of evidence has accumulated examining the histology and temporal development of aortic plaque in relation to plasma cholesterol and triacylglyceride concentrations in the Japanese quail (Godin et a!., 1 994; Wang et a!., 1 989; Shih eta!., 1983; McCormick eta!., 1982; Radcliffe et a!., 1982). However, relatively less attention has been focused on the effect of dietary fat sources on plasma lipid concentrations and the severity of aortic plaque development in the Japanese quail in light of the association between dietary fatty acid composition and hyperlipidemia as a risk factor for cardiovascular disease (Toda and Oku, 1995; Toda et a!., 1 988; Smith and Hilker, 1973). The purpose of the present study was to examine the influence of different dietary fat sources and cholesterol intake levels on plasma lipid response, aortic plaque deposition as well as aortic cholesterol  and cholesterol  oxide composition in  atherosclerosis-susceptible Japanese quail.  Furthermore, the effect of dietary fat source on endogenous antioxidant status was studied to determine the potential association between severity of aortic plaque deposition and the antioxidant status of selected tissues of these birds.  134  Hypotheses for Chapter 4: i. fatty acids)  Dietary fat sources differing in fatty acid composition (i.e. saturated versus unsaturated and cholesterol intake level have independent or interactive effects on plasma lipid  response and aortic plaque development in atherosclerosis-susceptible Japanese quail. ii. The endogenous antioxidant status of atherosclerosis-susceptible Japanese quail can be altered by dietary fatty acid composition and cholesterol intake level and may be related to the severity of aortic plaque deposition in these quail. Obiective for Chapter 4: To examine the independent and/or interactive effects of saturated and unsaturated (i.e. n-6 polyunsaturated lipids)  dietary fat sources and level of cholesterol on plasma lipid concentrations,  endogenous antioxidant status and severity of aortic plaque in atherosclerosis-susceptible Japanese quail. Specific Aims for Chapter 4: i.  Plasma lipid concentrations and presence of aortic plaque will be determined in  atherosclerosis-susceptible Japanese quail fed semi-purified diets differing in dietary fat source (e.g. butter, beef tallow or soybean oil) and cholesterol content (e.g. a high (0.5%, wt/wt) or low (0.05%) level of cholesterol). ii.  Specific tissue antioxidant enzymes and susceptibility of tissues to H -induced 0 2  glutathione depletion and lipid peroxidation in vitro will be examined to determine the role of endogenous antioxidant status in modulating the severity of aortic plaque in atherosclerosis-susceptible Japanese quail fed diets varying in dietary fat source and cholesterol content. iii.  Aortic tissue cholesterol and cholesterol oxide content and associated ultrastructural  changes will be used to characterize the composition of aortic plaque in atherosclerosis-susceptible Japanese quail.  135  Materials and Methods: Animals: Seventy-two, six-week old male atherosclerosis-susceptible Japanese quail (Coturnixjaponica; U.B.C. Quail Genetic Resource Centre, Vancouver, B.C.) treatment groups (n  =  were randomly divided into six dietary  1 2) varying in dietary fat source (i.e. butter, beef tallow and soybean oil) and  cholesterol level (i.e. low, 0.05% (wt/wt) or high, 0.5% (wt/wt) cholesterol). Formulation of semi-synthetic diets: The composition of semi-synthetic diets used in this study differing in both lipid source and cholesterol content is detailed in Table 4.1. A basal diet, containing 3% canola oil (Neptune Food Services, Richmond, B.C.) to provide an adequate supply of essential fatty acids, was formulated with thorough mixing of ingredients prior to the addition of experimental dietary fat sources and sterols. Dietary fat sources consisted of non-salted butter (Dairyworld Foods, Burnaby, B.C.), beef tallow (Cargill Foods, High River, AB)  or soybean oil (Bioforce Canada, Burnaby, B.C.).  No additional  antioxidants were added to diets, with the exception of the vitamin E that was present as a component of the poultry vitamin-premix. Once the basal diet ingredients were thoroughly mixed in an industrialsized stainless steel mixing vat, the powdered diet was pelleted and crumbled in a feed mill (Agriculture Canada Research Station, Agassiz, B.C.). The crumbled basal diet was double-bagged and stored at -15°C until the experimental dietary fat sources could be mixed in. Incorporation of dietary fat sources and sterols into the crumbled basal diet was performed as previously described in Chapter 2. Dietary fats containing sterols at levels reported in Table 4.1 were slowly added to the crumbled basal diet during reblending and mixed in completely using a Hobart mixer with an aluminum bowl over a period of approximately 20-25 minutes. After mixing, individual diets were stored in double, dark plastic bags in a walk-in freezer (-15°C) throughout the experimental study. A sample of each experimental diet was removed for analysis of fatty acid, gross energy and dry matter content as described in Chapter 2. The experimental fat sources were added to the basal diet at a level of 5% to make a final calculated fat content of 8% dietary fat. This level of fat in the diets matches the level of dietary lipid contained in commercial quail feed (Turkey Starter; Otter Co-op, Aldergrove, B.C.). The levels of 136  Table 4.1 Composition of diets fed to atherosclerosissusceptible Japanese quail . 1  Cholesterol level (% by weight):  0.05  0.5  Dietary component (gil OOg) Soy protein meal 2 Ca-free mineral mix 3 4 3 CaCO Poultry vitamin premix 2 5 DL-methionine Choline chloride 2 Cornstarch 6 6 Sucrose 3 Alphacel 2 Monofos Canola oil 6 Dietary fats: Butter, beef tallow, or soybean oil 7 3 Cholesterol Cholic acid 3 1  34.0  34.0  2.0 5.0 0.3 0.4 0.3  2.0 5.0 0.3 0.4 0.3  39.5 2.5 5.0 3.0  39.5 2.5 5.0 3.0  3.0  3.0  5.0 0.05 0.025  5.0 0.50 0.25  Gross energy (kJig) of diets: low cholesterol, butter (15.94), tallow (16.50) and soybean oil (16.56); high cholesterol, butter (16.44), tallow (16.68) and soybean oil (1 6.55). Dry matter content of all diets ranged from 89 91 %. Van Waters & Rogers, Abbotsford, B.C., Canada ICN Biochemicals Inc., Cleveland, OH, USA BDH Chemicals, Toronto, ON, Canada United States Biochemical Co., Cleveland, OH, USA Neptune Food Services, Richmond, B.C., Canada Butter (Dairyworld Foods, Burnaby, B.C.); beef tallow (Cargill Foods, High River, AB); soybean oil (Bioforce Canada, Burnaby, B.C.). -  2  6  137  cholesterol incorporated into diets were 0.05% and 0.5% (wt/wt) for the low cholesterol (on basis of caloric density, 0.0306 mg/kJ) and high cholesterol diets (0.302 mg/kJ), respectively (Table 4.1). As discussed in Chapter 2, the level of 0.05% cholesterol was chosen to equalize all low cholesterol diets for the sterol content naturally present in butter and beef tallow sources. Experimental diets were isonitrogenous and contained a comparable level of energy (e.g. 15.94- 17.00 kJ/g; Table 4.1). Diet gross energy determination: Energy content of all experimental diets was determined using a bomb calorimeter and was corrected for dry weight of diet as described in Chapter 2 (Miller and Payne, 1959). Dietary fatty acid analysis: The fatty acid content of the experimental diets was analyzed by gas chromatography as described in Chapter 2. The results of the fatty acid analyses of diets are presented in Table 4.2. Animal feeding: Quail were housed in heated brooder cages with one treatment group per brooder cage. Feed and distilled deionized water were provided to birds in separate feeding troughs ad ilbitum. Feed was replaced daily to minimize lipid oxidation of diets fed to birds. Experimental jrocedures: After 9 weeks on their respective diets, quail were sacrificed at 09:00 hr.  Quail were  decapitated and trunk blood collected into chilled heparinized tubes and plasma separated by low-speed centrifugation (1000 x g, 5 mm, 4°C). Aliquots of plasma as well as RBCs, heart and liver tissues were collected for analysis of plasma lipids and antioxidant status, respectively, as reported in Chapter 1. In addition, the aortic tree (the brachiocephalic arteries to their bifurcations and the aorta to the iliac branching) was dissected out, opened longitudinally and examined under a 1O-30X dissecting microscope for lesions on the inner wall as described in Chapter 3. Briefly, aortic lesion scores from 0 to 4 were assigned according to Shih eta!. (1983) and Godin eta!. (1994). Scoring was performed by two independent investigators (in a blinded fashion) plaques; 2  =  as follows: 0  6-20 plaques and an affected area less than 50%; 3  area greater than 50%; 4  =  =  =  clean surface; 1  5  =  > 20 plaques with an affected  massive atheromas present. Immediately after scoring was completed,  the aortic tissue was deposited into chilled 50 mM Tris 0.1mM EDTA, pH 7.6 homogenizing buffer 138  Table 4.2 Fatty acid composition of diets fed to atherosclerosis-susceptible Japanese quail.  Diets Butter  Beef tallow  Soybean oil  Area %  Fatty acid: 14:0 16:0 16:1(n-7)  6.5 22.3 1 .0  2.2 18.5 2.0  0.3 9.0 0.2  18:0 18:1(n-9) 1 8:1 (isomers) 1 8:2(n-6) 1 8:3(n-3)  7.4 36.4 1.8 13.7 3.7  10.8 46.7 2.4 13.5 4.0  4.3 47.6 3.4 15.3 2.7  20:0 20:1  0.4 0.8  0.8  Saturates Monounsaturates Polyunsaturates  36.2 40.0 17.4  31.4 51.1 17.5  14.1 52.1 17.9  n-6 n-3  13.7 3.7  13.5 4.0  15.3 2.7  P/Si  0.5  0.6  1 .3  -  polyunsaturated fatty acid/ saturated fatty acid ratio. P/S denotes not detected.  139  prior to homogenization for analysis of antioxidant enzyme activity. The aorta was blotted dry and any adhering tissue was removed before recording the aortic weight. An aliquot (1 .0 mL) of homogenizing buffer was added to the aortic tissue in a test-tube and a homogenate was prepared using a microprobe attachment for a Polytron homogenizer (Brinkmann Instruments, Inc., Westbury, NY) at 50% maximum speed, for 30 seconds (2 X 15 sec). Aortic cytosolic fractions to be used in the enzymatic assays were prepared by centrifugation (12,000 x g, 4°C, 15 mm). The aortic cytosolic fractions were assayed for GSSG-Red, GSH-Px, and SOD activities by the methods outlined in Chapter 1. Additional birds in each treatment group were used to provide aortic specimens for GC-MS analysis of cholesterol oxides, as well as specimens for scanning electron microscopy of aortic lumen cell morphology. Aortic cholesterol oxides: Aortic tissue content of cholesterol and cholesterol oxides was confirmed and quantified by GC MS as described in Chapter 3. Scanning electron microscorv: Aortic specimens were prepared for scanning electron microscopy according to Peng and coworkers (1985). Briefly, samples were immersed in 3% glutaraldehyde buffer, pH 7.6, followed by immersion in an 0.5% 0s0 4 solution, rinsed and dehydrated with ethanol. Samples were then dried by the critical point drying method using CO . Finally, aortic specimens were mounted onto stubs and 2 coated with gold prior to viewing with scanning electron microscope facilities (Electron Microscopy Lab, Dept. of Zoology, U.B.C.). Statistics: All data are expressed as mean ± SEM. One-way analysis of variance (ANOVA; SPSS Inc.) was used to test for differences between experimental treatments. Where differences did exist, the source of the differences at a p multiple range test.  0.05 significance level was identified by the Student-Newman-Keuls  Two-way multiple analysis of variance (MANOVA)  was used to identify any  interactions between dietary fat source and dietary cholesterol level. Linear regression analysis (SPSS) was performed to investigate interactions between plasma lipids and aortic plaque parameters.  140  Results: Fatty acid content of semi-synthetic formulated quail diets: Similar to the dietary fatty acid analysis conducted in Chapter 2, the short-chain saturated fatty acids containing less than 14 carbons were not recorded in the present study (Table 4.2). However, the content of medium-chain myristic acid (C14:O) compared to both beef tallow and soybean oil diets.  was noted to be greater in the butter diets The palmitic acid (C16:O)  and stearic acid  (Cl 8:0) content of both the butter and beef tallow diets was approximately two-fold the content in soybean oil diets.  The beef tallow and soybean oil diets contained a similar amount of oleic acid  (C18:1,n-9), which was greater than that present in the butter diets. The linoleic acid (C18:2,n-6) content of diets containing butter and beef tallow was similar, but contained a lower amount of this fatty acid than soybean oil diets. The linolenic acid (Cl 8:3,n-3) content of all three diets was similar, although the content of this fatty acid in soybean oil diets was slightly lower than the other two diets. The polyunsaturated to saturated fatty acid ratios (P/S) calculated from the fatty acid analysis of the butter and beef tallow diets were 0.5 and 0.6, respectively, while that of the soybean oil diets was 1 .3. The n-6 to n-3 fatty acid ratio (i.e. linoleic to linolenic acid ratio) was 3.7 and 3.4 for butter and beef tallow diets, respectively, and 5.7 for the soybean oil diets (Table 4.2). Animal growth and organ weights: Birds adapted well to the crumbled semi-synthetic formulated diets provided to them. Final body weights of birds were not affected by dietary treatment (range 126 ± 1 g). Similarly, weights of heart tissue were not different between dietary treatment groups (range 0.9 ± 0.1 g). Livers from birds fed high cholesterol diets (range 3.7 ± 0.1 g) weighed significantly (p  0.05) more than liver  tissue from counterparts fed low cholesterol diets (range 2.2 ± 0.1 g). However, liver tissue weights were not influenced by dietary fat source. Plasma lipid profiles: Plasma lipid profiles of quail fed experimental diets are presented in Table 4.3. Plasma free and total cholesterol, as well as triacylglyceride concentrations were significantly (p < 0.05) increased in birds fed the high cholesterol diets (Table 4.3). In contrast, dietary fat source did not have an effect on these same plasma lipid parameters. However, while the difference was not significant, birds fed 141  ‘-a  1.90±0.12  Soybean  2  1  C F  <0.001 N.S.  ±0.9  8.90±0.56  10.9  0.5  56.4±4.7  64.8±5.6  61.2±6.0  <0.001 N.S.  Total cholesterol  5.86±0.36  7.49±0.87  6.89±0.46  0.05  Total cholesterol  Values represent mean ± SEM, n = 1 2. c= cholesterol intake level effect, F = dietary fat source effect by 2-way MANOVA.  2 ANOVA p-value  0.5  8.89±0.77  Free cholesterol  2.45+0.27  Tallow  Plasma lipids:  2.30±0.21  0.05  Free cholesterol  Butter  Dietary Treatment:  Cholesterol level (% by weight):  Plasma lipids: (mmol/L)  Table 4.3 Plasma lipids of atherosclerosis-susceptible Japanese quail fed experimental diets. 1  4.32±0.52  5.76±0.56  5.02±0.65  0.5  <0.001 N.S.  Triacyiglycerides  1.90±0.31  2.17±0.31  2.30±0.57  0.05  Triacylglycerides  the soybean oil diets tended to have slightly lower concentrations of plasma lipids (i.e. total cholesterol and triacyiglycerides) compared to counterparts fed butter or tallow based diets (Table 4.3). Red blood cell and tissue antioxidant status: i. Red blood cell (RBC) antioxidant enzymes: RBCs from Japanese quail did not exhibit detectable CAT activity by the methods used in the present study. RBC activities of the oxygen radical metabolizing enzyme, superoxide dismutase (SOD), were not influenced by dietary cholesterol intake level, or dietary fat source (Table 4.4). However, RBC GSH-Px activity was significantly (p  ..  0.05)  affected by dietary fat source, but was not  influenced by cholesterol intake level (Table 4.4). Birds fed soybean oil diets exhibited greater (p < 0.05) RBC GSH-Px activity than counterparts fed butter or beef tallow diets. ii. Heart antioxidant enzymes: Similar to the results above with RBCs, heart tissue from Japanese quail did not exhibit detectable levels of CAT activity. Neither SOD, nor GSSG-Red activity in heart tissue from quail were different between dietary treatment groups (Table 4.5). Heart GSH-Px activity was significantly (p 0.05)  influenced by dietary fat source, but not by cholesterol intake level (Table 4.5). Activity of  GSH-Px was decreased (p  0.05) in birds fed soybean oil diets compared to those fed butter or beef  tallow diets (Table 4.5). iii. Liver antioxidant enzymes: Liver tissue reactive oxygen species metabolizing antioxidant enzyme activities are presented in Table 4.6a.  Neither CAT, nor SOD activity were influenced by dietary cholesterol intake level or  dietary fat source (Table 4.6a). Similarly, liver tissue activities of GSH-Px and GSSG-Red also were not influenced by dietary treatment in the present study (Table 4.6b). iv. Aortic antioxidant enzymes: Aortic tissue antioxidant enzyme activities are presented in Table 4.7. Activities of GSH-Px, GSSG-Red and SOD in the aortic tissue from atherosclerosis-susceptible Japanese quail were not different between dietary fat source or cholesterol intake level treatment groups in the present study.  143  -  2  1  C F  10.6 ±0.6  N.S. N.S.  GSSG-Red  10.2±0.6  105 ±5  91.4±3.6  92.0±6.1  0.05  N.S. 0.004  GSH-Px  103  ±5  90.9±3.4  86.1 ±3.8  0.5  GSH-Px (nmoles NADPH/min/mg Hb)  Values represent mean .±. SEM, n = 12. GSSG-Red = glutathione reductase; GSH-Px = glutathione peroxidase; SOD = superoxide dismutase. C = cholesterol intake level effect, F = dietary fat source effect by 2-way MANOVA.  ANOVA p-value 3  Antioxidant Enzyme:  -  10.5±0.5  10.4 ±0.5  Tallow  Soybean  10.3±0.5  0.5  9.90±0.65  0.05  GSSG-Red (nmoles NADPH/min/mg Hb)  Butter  Dietary Treatment:  Cholesterol level (% by weight):  : 2 Antioxidant Enzyme  N.S. N.S.  SOD  5.72±0.48  5.49±0.59  5.28±0.57  0.05  0.5  5.52±0.67  6.05±0.82  5.43±0.55  SOD (U/mg Hb)  Table 4.4 Antioxidant enzyme activities in red blood cells of atherosclerosis-susceptible Japanese quail fed experimental diets.’  (31  -a  0.483±0.017  Soybean  2  1  C F  0.441 ±0.007  0.457±0.018  N.S. N.S.  2.22±0.23  2.35±0.21  2.66 ± 0.15  0.05  N.S. 0.024  GSH-Px  1.40±0.13  2.56±0.15  2.28 ± 0.17  0.5  GSH-Px (nmoles NADPH/min/mg tissue wet wt)  Values represent mean ± SEM, n = 1 2. GSSG-Red = glutathione reductase; GSH-Px = glutathione peroxidase; SOD = superoxide dismutase. C = cholesterol intake level effect, F = dietary fat source effect by 2-way MANOVA.  ANOVA p-value 3  0.5  0.447 ± 0.017  GSSG-Red  0.447±0.022  Tallow  Antioxidant Enzyme:  0.457 ± 0.026  0.05  GSSG-Red (nmoles NADPH/min/mg tissue wet wt)  Butter  Dietary Treatment:  Cho’esterol level (% by weight):  Antioxidant Enzyme : 2  1.63±0.09  1.64±0.15  1.35 ± 0.10  0.05  N.S. N.S.  SOD  1.36±0.14  1.41 ±0.16  1.36±0.09  0.5  SOD (U/mg tissue wet wt)  Table 4.5 Antioxidant enzyme activities in heart of atherosclerosis-susceptible Japanese quail fed experimental diets. 1  0)  2  1  C F  8.26 ± 1 .06  6.06 ± 0.31  8.67 ± 1.93  0.05  N.S. N.S.  SOD  7.33 ± 1 .28  5.98 ± 0.76  6.38 ± 0.69  0.5  SOD (U/mg tissue wet wt)  Values represent mean ± SEM, n = 1 2. CAT = catalase, k = first-order rate constant (sec); SOD = superoxide dismutase. C = cholesterol intake level effect, F = dietary fat source effect by 2-way MANOVA.  3 ANOVA p-value N.S. N.S.  6.65 ± 0.67  6.92 ± 0.66  Soybean  CAT  4.84 ± 0.42  8.03 ± 0.43  Tallow  Antioxidant Enzyme:  6.08 ± 0.33  0.5  5.89 ± 1.04  0.05  CAT (k/g tissue wet wt)  Butter  Dietary Treatment:  Cholesterol level (% by weight):  : 2 Antioxidant Enzyme  Table 4.6a Reactive oxygen species metabolizing antioxidant enzyme activities in liver of atherosclerosis-susceptible Japanese quail fed experimental diets. 1  2.74±0.17 2.64±0.08  Tallow  Soybean  2  1  C F  5.33 ±0.38  2.81 ±0.12  N.S. N.S.  GSSG-Red  4.98±0.44  6.67 ± 0.84  0.05  2.88±0.17  2.66 ± 0.11  0.5  N.S. N.S.  GSH-Px  5.20 ±0.42  5.51 ±0.36  5.43 ± 0.33  0.5  GSH-Px (nmoles NADPH/min/mg tissue wet wt)  Values represent mean ± SEM, n = 1 2. GSSG-Red = glutathione reductase; GSH-Px = glutathione peroxidase. C = cholesterol intake level effect, F = dietary fat source effect by 2-way MANOVA.  ANOVA p-value 3  Antioxidant Enzyme:  2.69 ± 0.21  0.05  GSSG-Red (nmoles NADPH/min/mg tissue wet wt)  Butter  Dietary Treatment:  Cholesterol level (% by weight):  : 2 Antioxidant Enzyme  Table 4.6b Glutathione metabolizing antioxidant enzyme activities in liver of atherosclerosis-susceptible 1 Japanese quail fed experimental diets.  -h  0.337±0.015  Soybean  2  1  C F  0.341 ±0.030  0.320±0.013  N.S. N.S.  N.S. N.S.  GSH-Px  0.525±0.054  0.549±0.023  0.476 ± 0.062  0.05  0.497±0.109  0.512±0.027  0.465 ± 0.069  0.5  GSH-Px (nmoles NADPH/min/mg tissue wet wt)  Values represent mean ± SEM, n = 12. GSSG-Red = glutathione reductase; GSH-Px = glutathione peroxidase; SOD = superoxide dismutase. C = cholesterol intake level effect, F = dietary fat source effect by 2-way MANOVA.  3 ANOVA p-value  0.5  0.341 ± 0.020  GSSG-Red  0.389±0.042  Tallow  Antioxidant Enzyme:  0.392 ± 0.040  0.05  Butter  Dietary Treatment:  Cholesterol level (% by weight):  GSSG-Red Antioxidant 2 Enzyme : (nmoles NADPH/min/mg tissue wet wt)  N.S. N.S.  SOD  1.10±0.18  1.29±0.08  1.18 ± 0.1 1  0.05  0.93 ±0.19  1.06 ±0.13  1 .23 ± 0.1 1  0.5  SOD (U/mg tissue wet wt)  Table 4.7 Antioxidant enzyme activities in aorta of atherosclerosis-susceptible Japanese quail fed experimental diets. 1  v. Tissue jlutathione (GSH) content: Levels of glutathione in RBCs (range 4.64 ± 0.22 nmoles GSH/mg RBC), heart tissue (range 1 .88 ± 0.07 nmoles GSH/mg wet wt tissue) and liver tissue (range 3.39 ± 0.59 nmoles GSH/mg wet wt tissue)  in atherosclerosis-susceptible Japanese quail were not affected by dietary fat source or  cholesterol intake level. Red blood cell and tissue susceptibility to forced peroxidation: i. Red blood cell GSH depletion and MDA production: Depletion of GSH in RBCs from atherosclerosis-susceptible Japanese quail was not different between dietary treatment groups (values not reported). The amount of MDA produced in vitro by RBCs incubated with H 0 did not change appreciably with increasing concentrations of peroxidizing 2 agent (data not shown). ii. Heart GSH depletion and TBARs production: The profiles of the in vitro oxidative challenge of heart tissue with increasing concentrations of H 0 are presented in Figure 4.1. As well, the results from the oxidative challenge of heart tissue 2 at a single concentration of H 0 with treatment differences identified are presented in Table 4.8. 2 Depletion of GSH from heart tissue was significantly (p  0.05)  influenced by dietary cholesterol  intake level (Table 4.8, Figure 4.1A, B). Heart tissue from birds fed high cholesterol diets exhibited greater (p < 0.05) depletion of GSH than low cholesterol-fed counterparts (Table 4.8). Dietary fat source did not have an influence on depletion of GSH from quail heart tissue. Susceptibility of heart tissue to TBARs production in vitro did not differ between dietary cholesterol intake level or dietary fat source treatment groups (Table 4.8, Figure 4.1C, D). iii. Liver GSH depletion and TBARs production: The profiles of the in vitro oxidative challenge of liver tissue with increasing concentrations of 0 are shown in Figure 4.2. As well, the results from the oxidative challenge of liver tissue at a 2 H single concentration of H , with treatment differences identified, are presented in Table 4.9. 0 2 Depletion of GSH from liver tissue was significantly (p  0.05) influenced by dietary fat source, but  was not affected by the level of dietary cholesterol intake (Table 4.9, Figure 4.2A, B). GSH depletion was greater (p  0.05) in liver tissue from birds fed soybean oil diets with the low level of 149  0  -  0.00  0.10  0.20  0.30  0  —  -  -  -  C  I  1 3  mM H202 added  2  I  mM H202 added  1.50  4  2.00  5  2,50  N In  0.00  0.10  0.20  0.30  0.40  0.50  0  0.00  D  1  I  0.50  1.50  —  I  3 mM H202 added  2  I  mM H202 ,dded  1.00  4  I  2,00  5  2.50  Figure 4.1 Susceptibility of heart tissue from atherosclerosis-susceptible Japanese quail fed diets varying in dietary fat source and cholesterol -induced depletion of glutathione (GSH) and production of thiobarbituric acid reactive substances (TBARs). (A) heart 0 2 intake level to in vitro H GSH depletion of quail fed low cholesterol diets; (B) heart GSH depletion of quail fed high cholesterol diets; (C) heart TBARs production of quail fed low cholesterol diets; (D) heart TBARs production of quail fed high cholesterol diets. * indicates a significant (p = butter; A = beef tallow; 0 = soybean oil. 0.05) dietary fat source difference.  I-  0  N C., 4)  0.40  0.50  1.00  41  4,  0.50  4,  4,  0.00  g  0  (3  2  1  C F  0.049 N.S.  N.S. N.S.  0.5  0.142 ± 0.020  0.121 ±0.010  0.156 ± 0.021  TBARs (A532)  0.107 ± 0.014  0.142±0.029  0.124 ± 0.013  0.05  TBARs (A532) 1.0 mM H 0 2  Values represent mean ± SEM, n = 12. GSH = glutathione; TBARs = 2-thiobarbituric acid reactive substances. C = cholesterol intake level effect, F = dietary fat source effect by 2-way MANOVA.  ANOVA p-value 2  38.8 ± 7.7  21.6 ± 3.5  Soybean  GSH depletion (%)  30.4±4.5  28.2±5.4  Tallow  29.3 ± 4.9  0.5  24.8 ± 3.7  0.05  Butter  Dietary Treatment:  Cholesterol level (% by weight):  GSH depletion (%) 0.6 mM H 0 2  -induced GSH depletion and TBARs production in atherosclerosis0 2 Table 4.8 Heart homogenate susceptibility to H susceptible Japanese quail fed experimental diets.’  -a 01  5  3  4  10 15  mM H202 added  2  I  5  25  6  0.00  0.60  mM H202 added  20  I-  0  0.00  0.50  1.00  1.50  2.00  0  0  -  -  D  1  5  3  4  15 mM H202 added  10  mM H202 added  2  T  20  4  5  25  Figure 4.2 Susceptibility of liver tissue from atherosclerosis-susceptible Japanese quail fed diets varying in dietary fat source and cholesterol intake -induced depletion of glutathione (GSH) and production of thiobarbituric acid reactive substances (TBARs). (A) liver GSH 0 2 level to in vitro H depletion of quail fed low cholesterol diets; (B) liver GSH depletion of quail fed high cholesterol diets; (C) liver TBARs production of quail fed low cholesterol diets; (D) liver TBARs production of quail fed high cholesterol diets. * indicates a significant (p = butter; A = beef tallow; 0 0.05) dietary fat source difference. soybean oil.  I-  0  1,00  I’,  C  1  C,, lb  0  0  C  S  N  1.50  2.00  20  40  60  N  C  C  80  100  (A)  (21  25.0 ± 4.7  33.3 ± 4.0  Soybean  2  1  C F  N.S. 0.045  0.5  0.260 ± 0.047  0.228 ± 0.024  0.299 ± 0.028  0.001 N.S.  TBARs (A532)  0.723 ± 0.156  0.440 ± 0.008  0.563 ± 0.145  0.05  TBARs (A532) 5.0 mM H 0 2  Values represent mean ± SEM, n = 1 2. GSH = glutathione; TBARs = 2-thiobarbituric acid reactive substances. C = cholesterol intake level effect, F = dietary fat source effect by 2-way MANOVA.  2 ANOVA p-value  32.7 ± 2.4  23.4 ± 2.8  Tallow  GSH depletion (%)  26.7 ± 3.9  0.5  14.5 ± 1.6  0.05  Butter  Dietary Treatment:  Cholesterol level (% by weight):  GSH depletion (%) 0.5 mM H 0 2  -induced GSH depletion and TBARs production in atherosclerosis0 2 Table 4.9 Liver homogenate susceptibility to H susceptible Japanese quail fed experimental diets. 1  cholesterol compared to tissue from counterparts fed butter or tallow diets (Table 4.9, Figure 4.2 A). On the other hand, in vitro production of TBARs from liver tissue was significantly (p  0.05)  influenced by dietary cholesterol intake level, but not by dietary fat source (Table 4.9, Figure 4.2C, D). Liver tissue from birds fed diets containing a high level of cholesterol exhibited reduced (p  0.05)  amounts of TBARs produced when challenged with peroxidizing agent in comparison to tissue from animals fed the low cholesterol diets (Table 4.9, Figure 4.2C, D). Aortic tissue atherosclerotic plaque deposition: The aortic plaque scores and percentage area covered from aortic tissue collected from the experimental atherosclerosis-susceptible Japanese quail are reported in Table 4.10. treatment groups fed high cholesterol diets exhibited significant (p < 0.05)  All dietary  atherosclerotic plaque  deposition in the aortic tree compared to those fed low cholesterol diets (no plaques detected; Table 4.10).  Dietary fat source did not have an effect on the plaque score in the aortae of quail fed high  cholesterol diets. Similar to the results with numerical plaque scores, the percentage of aortic lumen covered by plaque was significantly (p  0.05) influenced by dietary cholesterol intake level, but was  not affected by dietary fat source (Table 4.10). Aortae from birds fed diets containing the high level of cholesterol exhibited significant (p  0.05)  coverage of the lumen surface by plaque material,  compared to tissue from birds fed low cholesterol diets (0% coverage by plaque; Table 4.10). Significant positive correlations were observed between aortic plaque score and plasma total cholesterol concentrations (r  =  0.872, p  plaque and plasma total cholesterol (r  =  =  0.001) as well as between aortic area (%) covered by  0.870, p < 0.001).  Scanning electron micrographs of aortic tissue depicting examples of aortae without plaque (score of zero, with lumen clear of any signs of plaque using a dissecting microscope) and with severe plaque (scores of 4 in vessels, with a high percentage of lumen area covered) are presented in Figures 4.3 and 4.4, respectively. Aortic tissue free from plaque involvement had an undulating, intact surface when scanned by SEM (Figure 4.3).  Ovoid protrusions from the lumen surface likely represented  nuclei and overlying cytoplasm (Figure 4.3; Peng eta!., 1985). Aortae from high cholesterol-fed quail with plaque material visible by dissecting microscope exhibited distinct areas of raised tissue with disruption of the epithelial cells when viewed by SEM (Figure 4.4A, B). 154  C;’ 01  -  2  1  C F  N.D.  N.D.  N.D.  0.05 0.5  <0.001 N.S.  =  79 ±  88±  >  7  4  80± 10  (%)3  Area covered (%)  Area covered  Values represent mean ± SEM, n = 1 2. Plaque score based on scale of 0 (N.D.) = clean surface; 1 = 5 plaques; 2 = 6-20 plaques; 3 4 = massive atheromas seen. Values represent two judges evaluating in a double-blind protocol. Area covered (%) = percent of aortic epithelium covered by plaque, range 0 (N.D.)-100%. C = cholesterol intake level effect, F= dietary fat source effect by 2-way MANOVA.  <0.001 N.S.  3.5 ± 0.2  N.D.  Soybean  ANOVA p-value 4  3.8±0.1  N.D.  Tallow  Plaque score  3.4±0.4  0.5  N.D.  0.05  Butter  Dietary Treatment:  Cholesterol level (% by weight):  Plaque score 2  20 plaques;  Table 4.10 Aortic plaque score and area covered in atherosclerosis-susceptible Japanese quail fed experimental diets. 1  Figure 4.3 A representative scanning electron micrograph of aortic tissue from atherosclerosis susceptible Japanese quail fed a low (0.05%) cholesterol diet. This micrograph depicts the intact, luminal surface of aortic tissue with a plaque score of zero, which was determined using a visual scoring scale.  1 56  A  B  Figure 4.4 Representative scanning electron micrographs of aortic tissue from atherosclerosis susceptible Japanese quail fed a high (0.5%) cholesterol diet. These micrographs depict the atherosclerotic luminal surface of aortic tissue with a plaque score of 4, which was determined using a visual scoring scale. (A) This micrograph depicts the coverage of the aortic lumen by plaque characterized by a focal area of raised tissue; (B) depicts a close-up view of the same plaque area as shown in A, depicting epithelial cell damage in the aortic plaque. 157  Aortic plague cholesterol oxides: The quantitation of cholesterol and cholesterol oxide content of the non-saponifiable extracts of individual aortic tissue samples collected from experimental birds by GC analysis is presented in Table 4.11. Aortic tissue from birds fed high cholesterol diets consistently contained a greater amount of native, unoxidized cholesterol than aortic tissue from counterparts fed the low cholesterol diets (Table 4.11). The presence of cholesterol oxides was detected only in the non-saponifiable extracts of aortic tissue from birds fed the high cholesterol diets. Also, there was individual variability in the profile of cholesterol oxides detectable in aortic tissue between birds fed the different dietary fat sources, as well as within specific dietary fat groups with the same aortic plaque score (Table 4.11). Cholesterol oxides could not be detected in the non-saponifiable extracts from aorta of low cholesterolfed birds (Table 4.11). Three cholesterol oxides (i.e. 5,6a-epoxy-5a-cholesterol, 7f-hydroxycholesterol and 7-ketocholesterol) were present in aortic tissue from birds fed butter, tallow or soybean oil-high cholesterol diets. Additional cholesterol oxides present in the aortae from birds fed the diet containing soybean oil with high cholesterol were: 7a-hydroxycholesterol, 4B-hydroxycholesterol, cholestane-triol, and 25-hydroxycholesterol.  The presence of elevated levels of tissue cholesterol, in addition to  cholesterol oxides in aortae, was observed to coincide with the presence of atherosclerotic plaque in aortae from Japanese quail fed high cholesterol diets (Tables 4.10 and 4.11). The limited number of samples, as well as the individual variability of cholesterol oxide content in aorta from birds within a dietary fat source treatment group, precluded presenting these data using means for each dietary treatment group.  158  CD  01  -a  0.51 0.53  N.D. N.D.  16.52 19.17  4/4/4 3/4/4  0.5% chol.  2.20 2.71  N.D. N.D.  N.D. N.D.  N.D. N.D.  N.D. N.D.  0.44 0.36  N.D. N.D.  N.D. N.D.  N.D. N.D.  0.41 0.65  N.D. N.D.  N.D. N.D.  4fl-OH  N.D. N.D.  7/?-OH  N.D. N.D.  N.D. N.D.  N.D. N.D.  0.53 0.37 N.D. N.D.  N.D. N.D.  N.D. N.D.  N.D. N.D.  N.D. N.D.  0.57 0.38  N.D. N.D.  N.D. N.D.  25-OH  N.D. N.D.  N.D. N.D.  7-keto  N.D. N.D.  triol  0.5% chol.  0.16 N.D. N.D. 0.42 0.61 0.16 N.D. 28.30 4/4/4 0.12 0.25 0.60 0.05 1.06 0.80 0.54 24.82 4/4/4 0.63 0.14 0.36 0.74 0.18 0.08 0.07 23.99 4/4/4 and oxide concentration spectrometry. measure by gas chromatography-mass inctiviaual bird aortic tissue plaque score, cholesterol 2 Cholesterol concentration = mg/g tissue; oxide concentration = mg/g tissue; N.D. = not detected. 7a-OH = 7a-hydroxycholesterol; 5,6aepoxide = 5,6a-epoxy-5a-cholesterol; 7fl-OH = 7fl-hydroxycholesterol; 4l-0H = 41-hydroxycholesterol; triol = cholestane-triol; 7-keto = 7-ketocholesterol; 25-OH = 25-hydroxycholesterol. 5 plaques; 2 = 6-20 plaques; 3 = > 20 plaques; 4 = massive Plaque score is based on scale of 0 (N.D.) = clean surface; 1 = atheromas seen. Values represent 2 judges evaluating in a blinded protocol. x/y/z = individual score for each of three vessels in aortic tree.  0.05% chol.  0/0/0 0/0/0  N.D. 0.26  N.D. N.D.  19.67 19.03  4/4/4 4/4/4  0.5% chol.  Soybean  N.D. N.D.  N.D. N.D.  2.68 2.38  0/0/0 0/0/0  0.05% chol.  Tallow  N.D. N.D.  5,6aepoxide  N.D. N.D.  7a-OH  1.61 2.38  Cholesterol  0/0/0 0/0/0  3 Score  0.05% chol.  Butter  Dietary Treatment  Cholesterol and Oxides 2  Table 4.11 GC quantitation of cholesterol and cholesterol oxide content of aortic tissue from atherosclerosis-susceptible Japanese quail fed 1 experimental diets.  Discussion: The role of dietary fat source in contributing to the development of atherosclerosis is thought to consist of effects on plasma lipid composition, specifically LDL-cholesterol. For example, oleic acid (CT 8:1,n-9), which is present in beef tallow and butter (approx. 44 and 29.5%, respectively), has been found to be just as effective as PUFA in lowering plasma cholesterol concentrations when substituted for saturated fatty acids in the diet (Mattson and Grundy, 1985). A neutral or hypocholesterolemic effect of stearic acid (Cl 8:0), a principal long-chain saturated fatty acid of beef tallow (approx. 30.0%) and cocoa butter (35.0%), has been reported in normocholesterolemic humans (Kris-Etherton et a!., 1993). Plasma cholesterol concentrations in subjects fed the former two types of diets were lower than in those counterparts fed butterfat diets (Kris-Etherton eta!., 1993). Polyunsaturated fatty acids such as linoleic (Cl 8:2,n-6) and linolenic (Cl 8:3,n-3) acids present in significant amounts in soybean oil (approx. 52.6 and 8%, respectively)  have been reported to be hypocholesterolemic when  substituted for saturated fatty acids in the diet (Mott eta!., 1992; Vega eta!., 1982).  Long-term  clinical studies have reported that a reduction in plasma lipid levels is associated with a reduced risk for coronary heart disease in middle-aged hypercholesterolemic men (Lipid Research Clinics Program, 1984). Animal studies provide supporting evidence of the requirement for elevated plasma cholesterol concentrations to induce atherosclerotic lesions in animal feeding trials (Nishina eta!., 1993; Smith and Hilker, 1973). These data, together with epidemiological and clinical reports of an association between increased susceptibility of tissues to lipid oxidation and impaired endogenous antioxidant status in vivo in coronary heart disease patients (Buczynski eta!., 1993; Jayakumari etal., 1 992), suggest a role for lipid peroxidation in the development of atherosclerosis. The present experiment describes the effects of diets containing dietary fat sources varying in degree of saturation (i.e. short versus long-chain saturated fatty acids in butter and beef tallow, respectively) and unsaturation (n-6 polyunsaturated fatty acids in soybean oil)  as well as level of cholesterol intake on the plasma lipid profile, aortic  plaque deposition and associated cholesterol and cholesterol oxide content, as well as the endogenous antioxidant status in atherosclerosis-susceptible Japanese quail.  160  Plasma cholesterol response to dietary fatty acids: The original studies of Keys (1957) and Hegsted (1965) established the “Lipid Hypothesis” that dietary fat sources containing primarily saturated fatty acids were hypercholesterolemic compared to sources of polyunsaturated fatty acids. However, the short-chain saturated fatty acids of chain length from C4:0 to C1O:O have been shown not to have any plasma cholesterol-raising effect in humans fed formula diets containing either butter or a short-chain triacylglyceride preparation isolated from  coconut  oil  (Hashim  et a!.,  1960).  In  studies  with  normocholesterolemic  and  hypercholesterolemic humans consuming self-selected diets containing either butter or margarine, plasma cholesterol levels were not elevated by the butter diet (Flynn et a/., 1991).  In the present  study, atherosclerosis-susceptible Japanese quail fed diets containing butter with a total dietary fat content equivalent to that present in regular commercial bird chow (approx. 10%)  were not  hypercholesterolemic compared to birds fed beef tallow or soybean oil. This result was consistent in birds fed either the low (0.05%) or high (0.5%) level of cholesterol in the diets. Japanese quail fed diets with a higher content of both butter fat and cholesterol (15 and 2%, respectively) for 3 week or 3 month periods also did not exhibit elevated plasma cholesterol concentrations compared to corn or hydrogenated corn oil diets (Toda eta!., 1988). More recently, diets containing 2% cholesterol and 15% dietary fat as medium-chain triacylglycerides (i.e. principal fatty acids were C8:O, 26.1%; C10:0, 20.6%; Cl 2:0, 0.8%) did not increase plasma total cholesterol concentrations, whereas counterparts fed corn oil and palmitic acid diets did exhibit greatly elevated plasma cholesterol levels (Toda and Oku, 1995). The results of the present study parallel the findings reported in Chapter 2, wherein SHR and WKY rats fed semi-synthetic diets containing butter also did not exhibit elevated plasma cholesterol concentrations compared to those fed beef tallow or soybean oil. The long-chain polyunsaturated fatty acids of oilseeds, such as linoleic acid, have been reported to reduce plasma concentrations of total and LDL-cholesterol in patients fed liquid formula diets (Mattson and Grundy, 1985). However, in Chapter 2, SHR and WKY animals fed semi-synthetic diets containing soybean oil diets with either a low or high level of cholesterol did not exhibit reduced plasma cholesterol levels. The plasma cholesterol concentrations of quail fed soybean oil diets herein were not significantly different from counterparts fed diets containing butter or beef tallow. A greater effect 161  of the n-6 polyunsaturated fatty acids in the soybean oil diets on plasma cholesterol levels might have been obtained if a high fat diet (e.g. 16% (wtlwt) or 40% of calories) had been used as previously reported by other workers feeding polyunsaturated fatty acids in diets to rats or primates (Mott et a?., 1992; Ney et a?., 1 991). This approach of greatly exceeding the dietary fat requirement of the quail was not pursued in this study due to the potentially non-physiological effects of feeding high energy atherogenic diets to the atherosclerosis-susceptible quail. Effect of dietary cholesterol on plasma triacylglycerides: A previous animal study demonstrated that cholesterol feeding increases plasma triacylglyceride (TG)  concentrations of rats in a dose-dependent manner (Fungwe et a?., 1992).  These workers  reported that plasma TG concentrations were increased maximally at 0.25% dietary cholesterol supplementation, without further increases with dietary cholesterol levels up to 2%. Lower levels of dietary cholesterol supplementation, from 0.05 to 0.1% cholesterol, did not increase plasma TG levels above that of the basal cholesterol-free diet. Cholesterol feeding was also observed to increase in vitro secretion of VLDL-TG and cholesterol in perfused livers of rats, up to a maximum at a dietary cholesterol level of 0.5% in these same animals (Fungwe eta?., 1992). Subsequent studies examining the effects of dietary cholesterol supplementation on plasma and hepatic TG concentrations in viva and  in vitro indicated that the hypertriacylglyceridemic effect of dietary cholesterol feeding was associated with increased hepatic TG and cholesteryl ester concentrations (Fungwe eta?., 1993, 1994).  The  increased concentrations of hepatic lipids were proposed to be the result not only of the reduced oxidation of fatty acids in the livers of cholesterol-fed rats, but also increased hepatic synthesis of fatty acids and TG (Fungwe et a?., 1993, 1994).  The data from the present study in cholesterol-fed  Japanese quail indicate that feeding a high level of cholesterol resulted in the altered visual appearance of liver tissue, increased liver weights as well as greater plasma TG and cholesterol concentrations. Differences in RBC MDA results between studies: The lack of difference in RBC MDA production with increasing concentrations of H 0 in the 2 present study was in contrast to previous reports of significant MDA production in RBCs from Japanese quail when tertiary-butyl hydroperoxide (t-BHP) was used as the oxidizing agent (Godin et 0 are strong oxidizing agents, the 2 a?., 1994; Godin and Dahlman, 1993). While both t-BHP and H 162  latter was used in these experiments because it is a reactive oxygen species of significance in vivo compared to the synthetic nature of t-BHP. Dietary cholesterol level effects on antioxidant status: The endogenous antioxidant status of diseased aortae, RBCs and platelets from coronary heart disease patients has been reported to be impaired. For example, the activities of specific antioxidant enzymes such as SOD and GSH-Px have been reported to be reduced, while levels of lipid peroxides are increased in tissues from such patients (Buczynski eta!., 1993; Jayakumari eta!., 1992). Animal models of human atherosclerosis have also been demonstrated to exhibit increased levels of lipid oxidation products in diseased aortae in association with decreased activity of specific antioxidant enzymes (Godin at a!., 1994; Wang eta!., 1989). In studies with Japanese quail fed a commercial chow diet supplemented with a high level of cholesterol, Wang and coworkers (1989) reported that aortic tissue from birds fed the atherogenic diet exhibited higher levels of lipid peroxides and reduced activity of GSH-Px. More recently, Godin and coworkers (1994) reported that the activity of GSH-Px and SOD in the plasma was positively correlated with plasma cholesterol and triacylglyceride concentrations, whereas aortic SOD activity was negatively correlated with both plasma cholesterol as well as aortic plaque score in cholesterol-fed (commercial chow supplemented with 1 % cholesterol) atherosclerosis-susceptible Japanese quail. However, other antioxidant enzymes in aortic tissue (i.e. GSH-Px and GSSG-Red)  and RBCs (i.e. GSH-Px, GSSG-Red, and SOD)  were not influenced by  cholesterol feeding in these same animals, suggesting that these endogenous antioxidant components were not involved in determining the susceptibility of these birds to aortic atherosclerotic plaque development (Godin eta!., 1994).  It is noteworthy that very few differences in tissue antioxidant  enzyme activity were observed between atherosclerosis-susceptible and -resistant strains of Japanese quail by these workers. While the activity of aortic tissue GSSG-Red was reduced in atherosclerosis susceptible compared to -resistant quail, activity of this enzyme was not significantly altered in birds with dietary cholesterol-induced aortic plaque (Godin eta!., 1994). On the other hand, while plasma GSH-Px and SOD activities were increased by cholesterol feeding, this response was common to both strains of quail and thus, could not be associated with the deposition of aortic plaque in the susceptible strain. In the present study with atherosclerosis-susceptible Japanese quail fed defined semi-purified 163  diets containing 0.5% cholesterol with 0.25% cholic acid, plasma total cholesterol concentrations were two-fold greater than those previously observed in birds fed chow supplemented with 1 % cholesterol (Godin etaL, 1994). Moreover, the extent of atherosclerotic plaque deposited in aortae of birds herein was also greater than previously observed by Godin and coworkers (1994). Therefore, the finding that there were no effects of dietary cholesterol on the tissue antioxidant enzyme activities examined (i.e. RBC, heart, liver and aorta)  was surprising and suggests that the endogenous antioxidant enzyme  activity of these tissues is relatively insensitive to the effects of hypercholesterolemia in this animal model. However, depletion of GSH from heart tissue in vitro was enhanced in quail fed the high level of dietary cholesterol herein.  This latter result was opposite to the heart GSH depletion results  observed in SHR and WKY animals (i.e. no cholesterol treatment effect) fed a high level of cholesterol in Chapter 2. However, previous studies with wild-type Japanese quail fed commercial chow diets supplemented with cholesterol (1 %) and cholic acid (0.5%) did demonstrate enhanced depletion of heart GSH in vitro (Godin and Dahlman, 1993). Effect of dietary fat source on antioxidant status: The present study represents the first time that the effect of dietary fat source on antioxidant status in the atherosclerosis-susceptible Japanese quail has been investigated. Previous reports have examined the effect of saturated versus polyunsaturated dietary fats on severity of aortic plaque in quail (Toda and Oku, 1995; Toda eta!., 1988; Smith and Hilker, 1973), but no attempt was made to characterize biochemical indices which might be influenced by dietary fatty acid composition. In recent years, concern has been expressed about the effect on lipid peroxidation and antioxidant status in vivo of increased consumption of polyunsaturated fatty acids to reduce the incidence of cardiovascular disease. In fact, several studies have demonstrated an increased requirement for antioxidants when highly polyunsaturated fatty acids are consumed (Skiladóttir et al., 1994; Gonzalez et a!., 1992; De Schrijver eta!., 1992; L’Abbé eta!., 1991).  Previously in Chapter 2, it was demonstrated that a  dietary fat source high in polyunsaturated fatty acids, namely fish oil, could influence various tissue antioxidant enzyme activities in SHR and WKY rats. Glutathione peroxidase is a primary antioxidant enzyme involved in the inactivation and detoxification of lipid peroxides and hydrogen peroxide (Paglia and Valentine, 1967).  It is of interest to note that while GSH-Px activity was increased in RBCs of 164  soybean oil-fed quail, heart tissue activity of this enzyme was reduced in these same animals. It is conceivable that increased incorporation of n-6 fatty acids into RBC membranes could increase the requirement for intracellular GSH-Px activity in these oxygen-carrying cells in soybean oil-fed birds. Aortic plaque development in Japanese quail: The degree of aortic plaque development in atherosclerosis-susceptible Japanese quail observed in Chapter 3 as well as the present study was more severe (scores of 3  -  4 in all three vessels of the  aortic tree) and much less variable than in previously reported studies with cholesterol-fed Japanese quail (Godin etal., 1994; Shih eta!., 1983; Smith and Hilker, 1973). Previous workers using the same strain of atherosclerosis-susceptible Japanese quail (i.e. birds descended from the original line described by Shih et al., 1 983)  as used in Chapter 3 have reported greater variations in plaque scores when  birds were fed commercial chow diets supplemented with cholesterol (1%; Godin et a!., 1994). Similarly, considerable variation in plasma cholesterol and aortic plaque results have been obtained in studies using normal Japanese quail fed high fat (40-53% of calories)  semi-synthetic diets  supplemented with cholesterol (0.5- 1.0% cholesterol, without cholic acid; Smith and Hilker, 1973) or studies with Japanese quail fed a commercial chow diet supplemented with 3.2% cholesterol (Wang et a!., 1989).  Other studies in which the dose-dependent nature of dietary cholesterol-induced  atherosclerosis in susceptible Japanese quail was investigated reported that while there was no incidence of atherosclerotic lesions in aorta of birds fed a basal diet containing 0.06% cholesterol, there was an incremental increase in aortic lesions when the basal diet was supplemented with 0.75% and 1.5% cholesterol (cholesterol:cholic acid 2:1; Radcliffe eta!., 1982). Previously, when regular Japanese quail were fed semi-synthetic diets containing a basal level of 1 .5% corn oil, or 16% beef tallow or corn oil with and without added cholesterol (0.5%), the severity of aortic plaque deposition was observed to vary with dietary fat type, cholesterol level and duration of the feeding period (Smith and Hilker, 1973). Birds fed the dietary fats without cholesterol supplementation did eventually develop aortic plaque after 9 months, although there did not appear to be a dietary fat source treatment effect.  These workers reported that birds fed corn oil diets  supplemented with cholesterol did not exhibit an appreciable increase in aortic plaque deposition, even after 9 months of treatment. In contrast, birds fed beef tallow diets supplemented with cholesterol 165  exhibited increased aortic plaque after 6 and 9 months of feeding (Smith and Hilker, 1973).  It is  noteworthy that another group of birds fed an atherogenic diet containing 20% beef tallow supplemented with 1 % cholesterol achieved aortic scores similar to those observed in Chapter 3 and the present study after 9 months (Smith and Hilker, 1973). Thus, while atherosclerosis can occur spontaneously in Japanese quail, the time and conditions for induction appear to vary between studies due to differences in dietary treatment (i.e. dietary cholesterol intake levels ranging from 0.5 and length of study (i.e. several weeks or months).  -  5%)  The use of an inbred strain of susceptible  Japanese quail can allow greater comparisons between studies varying in dietary treatment effects. In studies with Japanese quail fed diets containing 15% fat comprising corn oil, hydrogenated corn oil, or butterfat with 2% cholesterol, Toda and coworkers (1988) reported that the atherogenicity of butterfat diets was intermediate between corn oil (greatest luminal narrowing) and hydrogenated corn oil (least aortic luminal thickening). However, these workers did not observe any differences in the degree of hypercholesterolemia between these dietary treatment groups. More recently, corn oilor  palmitic  acid-  (15%  fat  plus  2%  cholesterol)  fed  Japanese  quail  exhibited  hypercholesterolemia and severe aortic plaque compared to counterparts fed  marked  medium-chain  triacylglycerides which exhibited only mild increases in plasma cholesterol and aortic thickening (Toda and Oku, 1995).  This latter study clearly showed the relationship between plasma cholesterol  concentrations and aortic plaque development when different dietary lipids are fed to quail. The results of the present study with atherosclerosis-susceptible Japanese quail fed diets containing 8% dietary fat with 0.5% cholesterol and 0.25% cholic acid strongly suggest that the addition of bile acid to diets greatly  increased  dietary  cholesterol  absorption,  hypercholesterolemia achieved in these birds.  and  thereby  enhanced  the  level  of  Thus, the effects of dietary fat source on plasma  cholesterol concentrations were minimal compared to the cholesterol treatment effect in the present study. Aortic tissue cholesterol and cholesterol oxides content: Previously, in Chapter 3, the presence of two cholesterol oxides (i.e. 71-hydroxycholesterol and 7-ketocholesterol) was confirmed in the aortic tissue from atherosclerosis-susceptible Japanese quail fed an atherogenic diet containing beef tallow. The role of oxidized cholesterol species and oxidatively 166  modified LDL in the initiation and progression of atherosclerosis has been substantiated by reports of supporting evidence for the presence of oxidized LDL in the plasma and diseased aortae of animal models (Rosenfeld et a!., 1990; Palinski et a!., 1 990, 1 989) as well as diseased human aortic tissue (Steinbrecher and Lougheed, 1992; Stringer etal., 1989). Also, several species of cholesterol oxide have been confirmed and quantitated in human plasma (Dzeletovic eta!., 1995; Sevanian eta!., 1994; Addis eta!., 1989). The atherogenic potential of cholesterol oxidation products (COPs)  has been  demonstrated by in vitro cell culture (Caboni et al., 1994; Hennig and Boissonneault, 1987; Peng et a!., 1978), intravenous administration to animal models (Peng eta!., 1985)  as well as in animal  feeding studies (Donaldson, 1982). Japanese quail fed oxidized cholesterol in the diet exhibited greater plasma and liver cholesterol concentrations in association with increased severity of atherosclerotic lesions  as  compared  to animals  fed  purified  cholesterol  (Donaldson,  1982).  When  25-  hydroxycholesterol and cholestane-triol were administered intravenously to rabbits, SEM scans of aortic tissue revealed balloon-like protrusions and crater-like defects on the luminal surface, with adhering platelets (Peng et a!., 1985). Transmission EM examination of aortic tissue from these same animals showed vacuolization of cells in combination with edema of tissue indicating endothelial cell injury. Previous studies have indicated a diverse population of COPs species in human plasma (i.e. cholesterol 5,6a- and I-epoxides, the 7-hydroxycholesterol isomers, and 7-ketocholesterol; Dzeletovic etal., 1995; Morin  and  Peng,  1 992)  and  aortic  tissue  (cholest-3,5-diene-7-one,  cholestane-triol,  7-  hydroxycholesterols, cholesterol-S 6a- and f-epoxides, 7-ketocholesterol, 24-hydroxy-, 25-hydroxy,  and 26-hydroxycholesterol; Steinbrecher and Lougheed, 1992; Morin and Peng, 1992). In the present study, atherosclerotic aortae from birds fed butter and beef tallow saturated fat diets contained cholesterol-5,6a-epoxide, 71..-hydroxycholesterol and 7-ketocholesterol, whereas those from birds fed soybean oil contained in addition 7a-hydroxycholesterol, 41.-hydroxycholesterol, cholestane-triol, and 25-hydroxycholesterol.  It is important to note that there was some individual variability in COPs  species present in aortic samples from the same dietary treatment group. While it is noteworthy that only aortic specimens from birds fed n-6 polyunsaturated fatty acids in the soybean oil diet exhibited detectable amounts of the notably cytotoxic 25-hydroxycholesterol and cholestane-triol COPs, the  167  other COPs species have been detected in LDL extracted from diseased human aortic tissue at autopsy (Steinbrecher and Lougheed, 1992). A potential explanation for the diversity of COPs detected in aortic tissue from diseased aortae in the present study, and others, is the fatty acid content of plasma lipoproteins, which would reflect the dietary fatty acid content somewhat (Toda and Oku, 1995; L’Abbé eta!., 1991). In vitro studies examining the susceptibility of LDL to oxidation have reported that LDL with an elevated oleic to linoleic acid ratio are less easily oxidized (Kleinveld etal., 1993). Similarly, Bonanome and coworkers (1992)  reported that LDL from healthy male subjects had greater resistance to in vitro lipid  peroxidation when subjects were fed diets enriched with oleic acid compared to diets high in linoleic acid. When Jialal and coworkers (1991) exposed LDL collected from normolipidemic subjects to in vitro CuSO -catalyzed oxidation, oxidation rates of LDL varied between subjects, with the major COP 4 detected identified as 7-ketocholesterol.  The elevated tissue concentrations of cholesterol in  combination with the detection and quantitation of cholesterol oxides in diseased aortae from atherosclerosis-susceptible Japanese quail confirm the similarities in aortic plaque composition between this animal model and human atherosclerosis.  The SEM micrographs obtained of undiseased and  diseased aortic tissue from experimental quail validates the dissecting microscope scoring protocol for detecting epithelial cell layer damage in these animals.  168  Conclusion: In conclusion, atherosclerosis-susceptible Japanese quail exhibited hypercholesterolemia and hypertriacylglyceridemia when fed experimental diets supplemented with a high level of cholesterol. While these changes in plasma lipid profiles coincided with the deposition of severe aortic plaque in birds fed high cholesterol diets, there were no changes in the tissue antioxidant parameters measured which could be attributed to a cholesterol intake level effect in these same animals. Moreover, dietary fat sources differing in saturated fatty acid composition (i.e. short-chain versus long-chain saturated fatty acids in butter and beef tallow, respectively) and n-6 polyunsaturated fatty acids (i.e. soybean oil) did not result in significant differences in plasma lipid concentrations. Interestingly, RBC and heart GSH-Px activities were different in soybean oil-fed animals. The hypercholesterolemia observed in quail in the present study coincided with not only increased levels of aortic tissue cholesterol, but also the presence of several cholesterol oxide species in the diseased aortic tissue collected from experimental quail. Within each dietary fat group, there was individual tissue variability in the profile and amounts of cholesterol oxides detected, even though plaque scores determined visually were quite similar between birds in a given treatment group. The results of the present study indicate that at the moderate, nutritionally adequate level of dietary fat fed to these quail, neither plasma lipid profile, nor susceptibility to atherosclerosis was affected by dietary fat source in the atherosclerosis-susceptible Japanese quail. While feeding quail a moderate level of fat and a high level of dietary cholesterol with cholic acid resulted in dyslipidemia and severe atherosclerosis, the fact that the endogenous antioxidant status of tissues was not observed to be altered by dietary cholesterol treatment or plaque deposition in these same animals suggests that at the extreme plasma lipid concentrations achieved it was unlikely that tissue antioxidant status was able to modulate the development of atherosclerosis in this animal model. Further studies to investigate the role of dietary fat level and lesser extremes of plasma cholesterol concentrations on aortic plaque development and tissue antioxidant status are indicated by the results reported herein.  169  CHAPTER 5 Influence of increased caloric intake from beef tallow on plasma lipids, antioxidant status and diet-induced atherosclerosis in atherosclerosis-susceptible Japanese quail. Introduction: Evidence derived from both epidemiological (Nordoy and Goodnight, 1990; Lipid Research Clinic Program, 1984)  and animal studies (Toda and Oku, 1995; Nishina eta!., 1993; Smith and Hilker,  1973) have indicated a strong association between plasma cholesterol and atherosclerotic responses with dietary fat or dietary cholesterol intake.  The fatty acyl component of dietary fats, which  comprises fatty acids varying in both the degree of saturation (e.g. saturates, monounsaturates or n-6, n-3 polyunsaturates)  as well as chain length, has been intensely investigated in predicting the  atherogenic response to specific dietary fat sources. A common finding in both animal (Nicolosi et aI., 1990)  and human studies (Shepherd et a!., 1980)  has been the elevated LDL-cholesterol  concentrations observed with dietary treatments rich in saturated fat or cholesterol. Elevated levels of lipid peroxides have also been reported in blood and tissue of individuals with hypercholesterolemia and atherosclerosis, providing supporting evidence of a role for lipid peroxidation in atherosclerosis (Piotrowski eta!., 1990). Although there are many studies that have examined the interaction between dietary fat and cholesterol levels on plasma lipidemia (Lin et a?., 1992), only a few studies have attempted to examine the significance of this interaction on the severity of atherosclerosis (Nishina et a?., 1993). The combination of high fat and cholesterol in diets fed to rabbits has been shown to result in profound changes in aortic lipid peroxidation and antioxidant enzyme activities indicating an important role for endogenous antioxidant systems in preventing tissue lipid peroxidation reactions (Mantha eta!., 1993).  Exogenous antioxidants such as ascorbic acid, a-tocopherol and butylated  hydroxytolulene (BHT) have been shown to reduce plasma cholesterol (Westrope et a?., 1 982) and inhibit lipoprotein oxidation (Morel eta!., 1994), albeit the effectiveness in suppressing atherosclerosis was variable. The Japanese quail has been shown in previous experiments herein (Chapters 3 and 4) and by other investigators (Godin eta!., 1994; Radcliffe eta!., 1982) to develop atherosclerotic plaque when fed diets containing a moderate level of fat with cholesterol. In these studies, a marked increase 170  in plasma total cholesterol concentrations, attributed to feeding high levels of dietary cholesterol, was associated with the development of plaque in these animals. In Chapter 4, atherosclerosis-susceptible Japanese quail fed semi-synthetic diets supplemented with cholesterol and cholic acid developed marked hyperlipidemia and severe aortic plaque. The results of this previous study indicated that the antioxidant status of tissues was not altered by cholesterol feeding or the development of aortic plaque, suggesting that the effect of extreme plasma lipid levels had the greatest effect on susceptibility to the development of aortic plaque in these birds.  Also, previous workers feeding  commercial chow diets supplemented with cholesterol have demonstrated a less severe plasma lipid and aortic plaque response in the Japanese quail (Godin eta!., 1994). Therefore, in the present study, commercial chow diets varying in levels of cholesterol (i.e. 0.05 and 0.5% (wt/wt)) as well as level of saturated fat (i.e. 6 and 12% beef tallow) were used to examine the potential interaction of dietary fat level and cholesterol quantity on plasma lipids, endogenous antioxidant status and susceptibility to atherosclerosis in the atherosclerosis-susceptible Japanese quail.  171  Hypothesis for Chanter 5: Increased total dietary caloric content from beef tallow and dietary cholesterol level have independent or interactive effects on plasma lipids, tissue antioxidant status and severity of aortic plaque in atherosclerosis-susceptible Japanese quail. Obiective for Chapter 5: To examine the potential independent and/or interactive effects of increased caloric intake from saturated fat with dietary cholesterol level on plasma lipid concentrations, endogenous antioxidant status and aortic plaque severity in the atherosclerosis-susceptible Japanese quail. Specific Aims for Chapter 5: i. Plasma total cholesterol and triacylglyceride response and presence of aortic plaque will be determined in atherosclerosis-susceptible Japanese quail fed non-purified, commercial diets differing in dietary fat level (e.g. 6 or 12% beef tallow) and cholesterol content (e.g. a low (0.05%, wt/wt) or high (0.5%)  level of dietary cholesterol). These results with quail fed experimental diets will be  compared to a reference group fed the unsupplemented commercial diet alone. ii. Specific tissue antioxidant enzyme activities and susceptibility of tissues to H -induced 0 2 lipid peroxidation in vitro will be measured in atherosclerosis-susceptible Japanese quail to determine the role of antioxidant status in modulating the severity of aortic plaque when quail are fed diets varying in level of dietary fat and cholesterol. iii. Aortic tissue cholesterol and cholesterol oxide content and associated ultrastructural tissue changes will be used to further characterize the composition of aortic plaque in describing the severity of atherosclerosis in the atherosclerosis-susceptible Japanese quail.  172  Materials and methods: Animals: Sixty, six-week old male atherosclerosis-susceptible Japanese quail (Coturnixjaponica; U.B.C. Quail Genetic Resource Centre, Vancouver, B.C.) were randomly divided into five dietary treatment groups (n  =  12) consisting of a reference group (diet A) fed a commercial Turkey Starter (TS) chow  diet containing 5.4% beef tallow (Otter Co-op, Aldergrove, B.C.)  diet, two groups fed TS  supplemented with 0.6% beef tallow for a total of 6% beef tallow with either a low, 0.05% (wt/wt; diet B) or high, 0.5% (diet C) level of cholesterol, and two groups fed TS supplemented with 6.6% beef tallow for a total of 12% beef tallow with either a low, 0.05% (diet D) or high, 0.5% (diet E) level of cholesterol. Mixing of diets: The composition of the commercial Turkey Starter diets supplemented with beef tallow and cholesterol is summarized in Table 5.1. The basal TS diet was supplemented with beef tallow (Cargill Foods, High River, AB), which was slowly melted over gentle heat (10-15 mm. at 45-50°C) to ensure uniform distribution of cholesterol and cholic acid (2:1 ratio) into the crumbled commercial diet. The additional dietary fat and sterols were thoroughly mixed into diets in an aluminum mixing bowl using a Hobart mixer.  After mixing, individual diets were stored in double, dark plastic bags in a walk-in  freezer (-1 5°C) throughout the experimental study. A sample of each experimental diet was removed for analysis of fatty acid, gross energy and dry matter content as previously described in Chapter 2. Experimental diets were isonitrogenous with diets A 17.21 18.76  -  -  17.97 kJ/g; Table 5.1), and diets D  -  -  C containing a comparable level of energy (e.g.  E containing an greater amount of total energy (e.g.  18.83 kJ/g; Table 5.1) due to the higher amount of dietary fat in these diets.  Dietary fatty acid analysis: The fatty acid content of the basal reference diet and supplemented experimental diets was determined by gas chromatography as previously described in Chapter 3 is summarized in Table 5.2.  173  Table 5.1 Composition and energy content of diets fed to quail. 1  Supplemented Component: (gIlOOg)  A  B  C  D  E  Tallow fat  0.6  0.6  6.6  6.6  Cholesterol  0.05  0.5  0.05  0.5  Cholic Acid  0.025  0.25  0.025  0.25  Crude Lipid (%)  10.0  10.3  10.3  16.1  16.1  Gross Energy (kJ/g)  17.21  17.62  17.97  18.76  18.83  1  Composition of reference diet A (Turkey Starter; TS, Otter Co-op; Aldergrove, B.C.), weight %: Alfalfa meal, 1.5; Corn, 15; Distiller’s grain, 2.5, Fish meal, 5.0; Lime, 4.7; Meat meal, 7.5; Pellet binder, 1.5; Multiphos, 1.0; Salt, 0.2; Soybean meal, 27.4; Tallow, 5.4, Wheat, 2.8; Vitamins and minerals, 0.5; Methionine, 0.025. 2 Diets B-E are diet A with supplemented components.  174  Table 5.2 Fatty acid profile of diets fed to quail. 1 DIETS  Fatty acid (weight %):  A  Saturated Lauric (C12:0) Myristic(C14:0) Palmitic (C16:0) Stearic(C18:0) Arachidic (C20:0) Behenic (C22:0)  0.1 1.5 21.0 9.8 0.3 1.2  Monounsaturated Myristoleic (C14:1) Palmitoleic (C16:1) Oleic (C18:1) Eicosenoic (C20:1)  B  C  D  0.1 1.6 21.0 9.7 0.3 1 .3  0.1 1.6 21.0 9.7 0.3 1.3  0.1 2.2 22.0 10.8 0.2 1.0  0.1 2.2 22.0 10.8 0.2 1.0  0.2 2.9 37.9 0.9  0.2 3.0 37.5 1.4  0.2 3.2 37.5 1.4  0.4 3.3 40.1 0.9  0.4 3.3 40.1 0.9  Polyunsaturated Linoleic (C18:2) Linolenic (Cl 8:3) Arachidonic (C20:4)  20.0 2.3 0.2  19.4 2.4 0.2  19.4 2.4 0.2  15.0 1.8 0.2  15.0 1 .8 0.2  Total Saturates Total Unsaturates P/S n-6/n-3  33.9 64.4 0.66 8.7  33.8 64.1 0.65 8.2  33.8 64.3 0.65 8.2  35.8 61 .7 0.47 8.4  35.6 61.7 0.47 8.4  E  1  Diet A = Turkey Starter (TS); B = TS + 0.05% chol.; C = TS + 0.5% chol.; D = TS + 6% Tallow + 0.05% chol.; E = TS + 6% Tallow + 0.5% chol. P/S = polyunsaturated to saturated fatty acid ratio; n-61n-3 = n-6 to n-3 polyunsaturated fatty acid ratio.  175  Animal feeding: Quail were housed in heated brooder cages with one treatment group per brooder cage. Feed and distilled deionized water were available to birds ad ilbitum.  Diets were replaced daily to minimize  oxidation of dietary lipids in diets fed to birds. Experimental procedures: After 9 weeks on their respective diets, quail were sacrificed at 09:00 hr. Quail were decapitated and trunk blood collected into chilled heparinized tubes and plasma separated as described in Chapter 4. Similarly, aliquots of plasma, RBCs, and heart and liver tissues were collected for analysis of plasma lipids and antioxidant status, respectively, as previously described in Chapter 2. Dissection and scoring of the aortic tree was performed as described in Chapter 4. Similarly, preparation of aortic tissue for antioxidant enzyme analysis was performed as outlined in Chapter 4. Again, additional birds in each dietary treatment group were used to provide aortic specimens for GC-MS analysis of cholesterol oxides and scanning electron microscopy by methods described in Chapter 4. Statistics: All data are expressed as mean ± SEM. One-way analysis of variance (AN OVA; SPSS Inc.) was used to test for differences between experimental treatment groups. Where differences did exist, the source of the differences at a p < 0.05 significance level was identified by the Student-Newman-Keuls multiple range test.  Two-way multiple analysis of variance (MANOVA)  was used to identify any  interactions between level of dietary fat and dietary cholesterol level.  176  Results: Lipid and energy content of experimental diets: The commercial Turkey Starter (TS) diet contained soybean meal, corn and alfalfa meal as primary sources of non-saponifiable lipid and fatty acids derived from vegetable sources, as well as beef tallow, and meat and fish meals as principal sources of lipids derived from animal sources. Supplementation of the TS diet (diet A) with small quantities of beef tallow and cholesterol in diets B and C did not alter the energy content or crude lipid content significantly (Table 5.1). The supplementation of the TS diet with 6.6% tallow (diets D and E)  increased both the total crude lipid and gross energy content,  representative of the amount of tallow added. Fatty acid composition of experimental diets: The fatty acid composition of the experimental diets is presented in Table 5.2. The addition of cholesterol at either supplementation level, 0.05% (wt!wt) in diets B and D or 0.5% in diets C and E, did not have an effect on the dietary fatty acid composition. Beef tallow supplementation of the TS diet resulted in slight increases in the saturated fatty acid (C16:0 and C18:0) content, a relatively larger increase in monounsaturated fatty acid (Cl 8:1 ,n-9) and a decrease in polyunsaturated fatty acid (C18:2,n-6 and C18:3,n-3) content (Table 5.2). These changes in dietary fatty acid composition resulted in a reduction in P/S ratio, but similar n-6/n-3 ratios in diets D and E compared to the reference TS diet (diet A) and diets B and C. Animal growth and organ weights: Final body weights of quail were not affected by dietary fat level or cholesterol intake level treatments (range 1 35 ± 2 g).  Also, weights of heart tissue were not different between dietary  treatment groups (range 1 .0 ± 1 g). On the other hand, livers from birds fed high cholesterol diets (e.g. diets C and E) were significantly (p  .  0.05) heavier (range 3.4 ± 0.3 g) compared to livers  from counterparts fed low cholesterol diets (diets B and D) and the reference TS diet (diet A; range 2.3 ± 0.1 g). Liver tissue weights were not affected by the level of fat in the diet. Plasma lipids of ciuail fed reference and experimental diets: The plasma lipid response of atherosclerosis-susceptible Japanese quail fed the reference TS and experimental diets is shown in Table 5.3. Birds fed the reference TS diet exhibited similar plasma total 177  Table 5.3 Plasma cholesterol and triacylglyceride concentrations in atherosclerosis-susceptible Japanese quail fed experimental diets . 1 2 DIETS ANOVA 3 p-value  A  B  C  D  Total cholesterol (mmol/L)  5.33 ±0.32  7.06 ±0.38  36.8 ±4.9  7.75 ±0.36  43.4 ±3.5  C < 0.001 L N.S. CxL N.S.  Triacylglyceride (mmol/L)  1.89 ±0.19  3.57 ±0.76  7.81 ±1.51  2.73 ±0.38  11.8 ±1.8  C < 0.001 L N.S. CxL 0.05  E  Plasma lipids:  1  Values represent mean ± SEM, (n = 1 2) Diet A = Turkey Starter (TS); B = TS + 0.05% chol.; C = TS + 0.5% chol.; D = TS + Tallow + 0.05% chol.; E = TS + Tallow + 0.5% chol. C = cholesterol treatment effect, L = fat level treatment effect, CxL = cholesterol x fat level treatment interaction by 2-way ANOVA. 2  178  cholesterol levels to counterparts fed TS diets supplemented with 0.05% cholesterol (diets B and D). A significantly (p  0.05) greater concentration of plasma total cholesterol was observed in birds fed  the high cholesterol diets (diets C and E; Table 5.3). The plasma cholesterol concentrations of quail fed diet E were slightly, but not significantly higher than counterparts fed diet C, as shown by the absence of an interaction between dietary cholesterol and fat intake level for plasma total cholesterol. Quail fed the TS reference diet (diet A)  exhibited lower (p < 0.05)  plasma triacylglyceride  concentrations than counterparts fed the tallow and cholesterol supplemented experimental diets (Table 5.3). A significant (p < 0.05)  interaction was observed between dietary fat level and cholesterol  content for plasma triacylglyceride concentrations, as a result of the considerably elevated plasma triacylglyceride concentrations of quail fed diets C and E. RBC and tissue antioxidant status of quail fed reference and experimental diets: i. Red blood cell antioxidant enzymes: The activities of RBC GSH-Px, GSSG-Red and SOD of birds fed reference TS (diet A)  and  experimental diets (diets B to E) were similar, indicating no effect of dietary fat level or cholesterol supplementation level on these RBC antioxidant enzyme activities (Table 5.4). ii. Heart tissue antioxidant enzymes: Heart tissue antioxidant enzyme activities in quail fed the reference TS (diet A) and experimental diets are presented in Table 5.5.  Similar to the results in RBCs, heart tissue from quail fed diets  varying in dietary fat level and cholesterol content did not exhibit any changes in GSH-Px, GSSG-Red and SOD activities (Table 5.5). iii. Liver tissue antioxidant enzymes: The hepatic antioxidant enzyme profiles of quail fed the reference TS (diet A) and experimental diets are presented in Table 5.6. Hepatic CAT activities were greater (p < 0.05) in birds fed the TS reference diet, but were not affected by the level of dietary cholesterol, or the level of dietary fat in the experimental diets (diets B to D; Table 5.6). The presence of the high level of cholesterol in diets C and D significantly (p < 0.05) reduced liver SOD activities compared to counterparts fed a low level of cholesterol by 24 and 26 per cent, respectively. There were no dietary treatment differences in  179  Table 5.4 Antioxidant enzyme activities of red blood cells of atherosclerosis-susceptible Japanese quail fed experimental diets . 1 3 DIETS A  B  C  D  E  ANOVA 4 p-value  Antioxidant Enzyme : 2 Activity SOD (U/mg Hb)  4.52 ±0.49  5.27 ±0.31  5.62 ±0.11  5.42 ±0.24  GSSG-Red (nmoles NADPH/ mm/mg Hb)  10.80 ± 0.35  9.69 ± 0.47  10.20 ±0.30  GSH-Px (nmolesNADPH/ mm/mg Hb)  104 ±5  146 ±9  142 ±5  5.48 ±0.17  N.S. C N.S. L CxL N.S.  8.91  9.42  ± 0.39  ± 0.48  N.S. C L N.S. CxL N.S.  139 ±5  139 ±10  N.S. C N.S. L CxL N.S.  1  Values represent mean ± SEM (n = 12). SOD = superoxide dismutase; GSSG-Red = glutathione reductase; GSH-Px = glutathione peroxidase. Diet A = Turkey Starter (TS); B = TS + 0.05% chol.; C = TS + 0.5% chol, D = TS + Tallow + 0.05% chol.; E = TS + Tallow + 0.5% chol. C = cholesterol treatment effect, L = fat level treatment effect, CxL = cholesterol x fat level treatment interaction by 2-way ANOVA. 2  180  ___________________________________________________  Table 5.5 Antioxidant enzyme activities in heart tissue of atherosclerosis-susceptible Japanese quail fed experimental diets. 1 3 DIETS A  B  C  D  E  ANOVA 4 p-value  C N.S. L N.S. CxL N.S.  Antioxidant Enzyme : 2 Activity SOD (U/mg tissue wet wt)  1.71 ±0.13  1.41 ±0.09  1.31 ±0.17  1.51  1.40  ± 0.07  ± 0.08  GSSG-Red (nmoles NADPH/min/ mg tissue wet wt)  0.48 ±0.02  0.45 ± 0.01  0.47 ±0.01  0.45 ±0.02  0.46 ±0.02  C N.S. L N.S. CxL N.S.  GSH-Px (nmoles NADPH/min/ mg tissue wet wt)  2.59 ±0.19  1 .52 ± 0.29  1.64 ±0.28  1.82 ±0.18  1.89 ±0.21  C N.S. L N.S. CxL N.S.  1  Values represent mean ± SEM (n = 12). SOD = superoxide dismutase; GSSG-Red = glutathione reductase; GSH-Px= glutathione peroxidase. Diet A = Turkey Starter (TS); B = TS + 0.05% chol., C = TS + 0.5% chol.; D = TS + Tallow + 0.05% chol.; E = TS + Tallow + 0.5% chol. C = cholesterol treatment effect, L = fat level treatment effect, CxL = cholesterol x fat level treatment interaction by 2-way ANOVA. 2  181  Table 5.6 Antioxidant enzyme activities in liver tissue of atherosclerosis-susceptible Japanese quail fed experimental diets 1 3 DIETS ANOVA 4 p-value  A  B  C  CAT (kig tissue wet wt)  10.47 ± 1.20  6.22 ± 0.89  5.58 ± 0.39  5.41 ±0.70  5.62 ±0.77  C 0.05 N.S. L CxL N.S.  SOD (U/mg tissue wet wt)  7.24 ±0.69  7.67 ±0.09  5.84 ± 0.32  8.33 ±0.47  6.09 ±0.64  C 0.002 N.S. L CxL N.S.  GSSG-Red (nmoles NADPH/ mm/mg tissue wet wt)  2.60 ±0.14  2.41 ±0.17  2.44 ±0.15  2.66 ± 0.12  2.45 ±0.11  N.S. C N.S. L CxL N.S.  GSH-Px (nmoles NADPH/ mm/mg tissue wet wt)  4.65 ±0.53  3.45 ± 0.21  3.50 ± 0.26  3.50 ±0.26  3.40 ±0.27  N.S. C L N.S. CxL N.S.  D  E  Antioxidant Enzyme Activity : 2  1  Values represent mean ± SEM (n = 12). CAT = catalase, k = first order rate constant (sec; SOD = superoxide dismutase; GSSG-Red = glutathione reductase; GSH-Px = glutathione peroxidase. Diet A = Turkey Starter (TS); B = TS + 0.05% chol.; C = TS + 0.5% chol.; D = TS + Tallow + 0.05% chol.; E = TS + Tallow + 0.5% chol. C = cholesterol treatment effect, L = fat level treatment effect, CxL = cholesterol x fat level treatment interaction by 2-way ANOVA. 2  182  hepatic GSSG-Red and GSH-Px activities of quail ted the reference TS diet (diet A) or the experimental diets (diets B to D; Table 5.6). iv. Aortic tissue antioxidant enzymes: No dietary treatment differences were observed for aortic GSH-Px, GSSG-Red or SOD activities, similar to the results with heart tissue antioxidant enzyme activities (Table 5.7). v. Tissue glutathione (GSH) content: Basal glutathione levels in RBCs, heart and liver homogenates were not significantly altered by the level of cholesterol in the diet, or the level of dietary fat (Table 5.8). RBC and tissue susceptibility to forced peroxidation: i. Red blood cell GSH depletion: Similar to the results of Chapter 4, in the present study depletion of GSH from RBCs was not different between dietary treatment groups of quail fed non-purified commercial TS diets supplemented with beef tallow or cholesterol (data not reported). ii. Heart GSH depletion and TBARs production: The in vitro depletion of GSH in heart tissue was not influenced by the level of cholesterol in the diet (diets C and E) or by the increased caloric contribution of a higher level of fat in the diet (diets D and E; Table 5.9, Figure 5.1A, B). The supplementation of the commercial TS diet (diet A) with added beef tallow (diets D and E) reduced (p 5.9, Figure 5.1C, D).  0.05) the formation of TBARs in heart tissue (Table  Moreover, the combination of the high level of cholesterol with added beef  tallow in diet E further reduced the production of TBARs in heart tissue of quail fed this diet, as demonstrated by the significant interaction observed (Table 5.9, Figure 5.1 D). iii. Liver GSH depletion and TBARS production: Quail fed diets containing high levels of cholesterol (diets C and E) exhibited increased (p< 0.05)  in vitro GSH depletion in hepatic tissue and decreased (p  0.05) formation of TBARs (Table 5.9,  Figure 5.2). However, in contrast to the results obtained with heart tissue, an interaction between level of dietary fat and cholesterol content was not observed for TBARs production in liver tissue.  183  Table 5.7 Antioxidant enzyme activities in aortic tissue of atherosclerosis-susceptible Japanese quail fed experimental diets. 1 3 DIETS A  B  C  D  E  ANOVA 4 p-value  1.31 ±0.32  1.06 ±0.13  1.01 ±0.19  0.96 ±0.12  1.14 ±0.10  N.S. C L N.S. CxL N.S.  0.38 0.04  ±  0.34 0.02  0.30  ± 0.02  ± 0.02  0.32 ±0.01  N.S. C L N.S. CxL N.S.  0.84 ±0.05  0.85 ±0.09  0.85 ±0.10  0.81 ±0.05  0.78 ±0.04  N.S. C L N.S. CxL N.S.  Antioxidant Enzyme Activity : 2  SOD (U/mg tissue wet wt) GSSG-Red (nmoles NADPH/min/ mg tissue wet wt) GSH-Px (nmoles NADPH/min/ mg tissue wet wt)  ±  0.35  1  Values represent mean ± SEM (n = 12). SOD = superoxide dismutase; GSSG-Red = glutathione reductase; GSH-Px = glutathione peroxidase. Diet A = Turkey Starter (TS); B = TS + 0.05% chol.; C = TS + 0.5% chol.; D = TS + Tallow + 0.05% chol.; E = TS + Tallow + 0.5% chol. C = cholesterol treatment effect, L = fat level treatment effect, CxL = cholesterol x fat level treatment interaction by 2-way ANOVA. 2  184  Table 5.8 Basal glutathione levels in heart and liver homogenate and red blood cells of atherosclerosis-susceptible Japanese quail .  2 DIETS A  B  C  D  E  ANOVA 3 p-value  RBC (nmoles GSH/mg RBC)  4.56 ±0.33  4.73 ±0.25  5.08 ±0.13  4.49 ±0.31  4.99 ±0.20  N.S. C N.S. L CxL N.S.  Heart (nmoles GSH/mg tissue wet wt)  1.92 ±0.06  1.99 ±0.07  1.94 ±0.06  2.08 ±0.10  2.06 ±0.06  N.S. C L N.S. CxL N.S.  Liver (nmoles GSH/mg tissue wet wt)  3.21 ±0.26  4.00 ±0.18  3.76 ±0.19  3.50 ±0.22  3.92 ±0.26  N.S. C N.S. L CxL N.S.  Parameter:  1  Values represent mean ± SEM (n = 12). Diet A = Turkey Starter (TS); B = TS + 0.05% chol.; C = TS + 0.5% chol.; D = TS + Tallow + 0.05% chol.; E = TS + Tallow + 0.5% chol. C = cholesterol treatment effect, L = fat level treatment effect, CxL = cholesterol x fat level treatment interaction by 2-way ANOVA. 2  185  Table 5.9 Heart and liver homogenate susceptibility to H -induced GSH depletion and TBARs 0 2 production in atherosclerosis-susceptible Japanese quail fed experimental diets. 1 3 DIETS A  B  C  D  Heart: GSH depletion (%)  26.2 ±5.0  34.1 ±7.0  45.5 ±5.2  34.0 ±2.9  TBARs (A532)  0.12 ±0.02  0.12 ±0.02  0.17 ±0.02  ± 0.01  Liver: GSH depletion (%)  41 .3 ±2.8  32.6 ±5.9  45.4 ± 4.6  E  ANOVA 4 p-value  : 2 Tissue  TBARs (A532)  0.67 0.08  0.31  0.26  ± 0.04  ± 0.02  0.12  33.4 ±5.3 0.09 ± 0.01  32.3 ±3.3  47.5 ±5.2  0.39 ±0.05  0.26 ±0.02  C N.S. L N.S. CxL N.S. C N.S. L 0.016 CxL 0.035  C 0.009 L N.S. CxL N.S. C 0.003 L N.S. CxL N.S.  1  Values represent mean ± SEM (n = 12). GSH = glutathione; TBARs = 2-thiobarbituric acid reactive substances (A532 = absorbance at 532 nm) 2 Heart GSH depletion (%) at 0.6 mM H ; Heart TBARs production at 1 .0 mM H 0 2 ; Liver GSH 0 2 depletion (%) at 0.5 mM H ; Liver TBARs production at 5.0 mM H 0 2 . 0 2 Diet A = Turkey Starter (TS); B = TS + 0.05% chol.; C = TS + 0.5% chol.; D = TS + Tallow + 0.05% chol.; E = TS + Tallow + 0.5% chol. C = cholesterol treatment effect, L = fat level treatment effect, CxL = cholesterol x fat level treatment interaction by 2-way ANOVA.  186  -.3  4  5  0.00  0.00  mM H202 added  0.10  0.10  3  0.20  0.30  0.30 0.20  0.40  0  V  0.00  0.40  2  2.50  0.50  1  2.00  0.50  0  1.50  mM H202 added  1.00  0,60  0.50  C  C  C 0  0.60  0 0.00  20  40  60  0  I  1  *  0,50  1.50  I  3 mM H202 added  2  I  mM H202 added  1.00  4  I  2.00  5  2.50  Figure 5.1 Susceptibility of heart tissue from atherosclerosis-susceptible Japanese quail fed a reference Turkey Starter (TS) diet alone, or TS diets supplemented with varying levels of beef tallow and cholesterol to in vitro H -induced depletion of glutathione (GSH) and production of 0 2 thiobarbituric acid reactive substances (TBARs). (A) heart GSH depletion of quail fed low cholesterol diets; (B) heart GSH depletion of quail fed high cholesterol diets; (C) heart TBARs production of quail fed low cholesterol diets; (D) heart TBARs production of quail fed high cholesterol diets. In A, B, C, and D the reference TS group is included for comparison purposes. * indicates a significant (p 0.05) dietary fat level difference. = TS + 0.6% tallow; A = TS + 6.6% beef tallow: 0 = reference TS group.  I-  0  N C,  4)  0  80  100  -a  co co  3 4  20  5  25  6  S  mM HO2 added  0.00 15  0.00 10  0.50  I—  100  1.60  0.50  1.00  1.60  2,00  mM H202 added  2  2.00  1  40  60  2.50  0  4,  g  2.50  20  40  60  80  80  0  0  D  1  5  /  3  4  /  /  10  15  - - - -  mM H202 added  /  /  -—  mM H202 added  2  - -  T  20  -  5  25  6  Figure 5.2 Susceptibility of liver tissue from atherosclerosis-susceptible Japanese quail fed a reference Turke’ Starter (TS) diet alone, or TS diets supplemented with varying levels of beef tallow and cholesterol to in vitro H -induced depletion of glutathione (GSH) and production of 0 2 thiobarbituric acid reactive substances (TBARs). (A) liver GSH depletion of quail fed low cholesterol diets; (B) liver GSH depletion of quail fed high cholesterol diets; (C) liver TBARs production of quail fed low cholesterol diets; (D) liver TBARs production of quail fed high cholesterol diets. In A, B, C, and D the reference TS group is included for comparison purposes. * indicates a significant (p = TS + 0.6% tallow; A = TS + 6.6% beef tallow; 0 = reference TS group. 0.05) dietary fat level difference.  I-  0  In  0,  4’  4,  100  100  Aortic plague score and percent area covered: The severity of plaque score in aortic tissue from quail determined by visual assessment using a dissecting microscope is summarized in Table 5.10. Birds fed both the TS reference diet (diet A) as well as diets supplemented with a low level of cholesterol (diets B and D) did not exhibit detectable aortic plaque (Table 5.10). On the other hand, inclusion of a high level of cholesterol in diets (diets C and E) resulted in significant (p  0.05) aortic plaque development (Table 5.10). The severity of  aortic plaque in animals fed the latter diets was reduced in quail fed the low fat diet (diet C) compared to counterparts fed the higher level of dietary fat (diet E), as demonstrated by the significant (p 0.05) interaction recorded (Table 5.10). It is noteworthy that the percentage of aortic lumen covered with plaque from birds fed diet E was also significantly (p  0.05) greater than counterparts fed diet  C, as demonstrated by the interaction recorded for this parameter (Table 5.10). Scanning electron micrographs of aortic tissue depicting examples of aortae without plaque (score of zero, with lumen clear of any signs of plaque using a dissecting microscope; Figure 5.3), moderate development of plaque (scores of zero and 2 in vessels; Figure 5.4) and with severe plaque (scores of 4 in vessels, with a high percentage of lumen area covered; Figure 5.5) are presented in Figures 5.3 to 5.5. Similar to the results in Chapter 4, aortic tissue without detectable plaque had an intact, undulating surface when scanned by SEM  (Figure 5.3).  Luminal epithelial cells appeared to be  arranged longitudinally along the lumen wall, with the ovoid raised areas of the lumen surface likely representing nuclei and overlying cytoplasm (Figure 5.3; Peng eta!., 1985).  Aortae which had a  moderate amount of plaque deposition on the lumen surface exhibited slight surface irregularities along with distinct raised areas of cells, possibly representing epithelial cell damage with lipid infiltration and focal areas of cell proliferation (Figure 5.4). Aortae from quail fed diet E containing an elevated level of dietary fat and cholesterol had significant amounts of plaque material visible by dissecting microscope and exhibited distinct areas of raised tissue and clustered areas of enlarged epithelial cells when viewed by SEM (Figure 5.5A, B). Aortic cholesterol and cholesterol oxide content: The aortic tissue from quail fed the reference and experimental diets was further characterized by measuring the cholesterol and cholesterol oxide content of representative aortic tissue specimens 189  Table 5.10 Aortic plaque score and area covered in atherosclerosis-susceptible Japanese quail fed experimental diets. 1 3 DIETS A  B  C  D  E  ANOVA 3 p-value  : 2 Parameter Plaque Score  N.D.  N.D.  0.9 ±0.3  N.D.  Area covered (%)  N.D.  N.D.  16 ±7  N.D.  2.0 ±0.4 44 ±11  C < 0.001 L N.S. CxL 0.02 C < 0.001 N.S. L CxL 0.03  1  Values represent mean ± SEM (n = 12). Plaque score is based on scale of 0 (N.D.) = clean surface; 1 = 5 plaques; 2 = 6-20 plaques; 3 = > 20 plaques; 4 = massive atheromas seen. Values represent 2 judges evaluating in blinded protocol. Values are mean score from three vessels in aortic tree. Area covered (%) = per cent of aortic epithelium covered by plaque, range = 0 (N.D.) to 100% Diet A = Turkey Starter (TS); B = TS + 0.05% chol.; C = TS + 0.5% chol.; D = TS + Tallow + 0.05% chol.; E = TS + Tallow + 0.5% chol. C = cholesterol treatment effect, L = fat level treatment effect, CxL = cholesterol x fat level treatment interaction by 2-way ANOVA. 2  190  Figure 5.3 A representative scanning electron micrograph of aortic tissue from atherosclerosis susceptible Japanese quail fed either a reference Turkey Starter (TS) diet alone; similar results were obtained with aortae from birds fed the TS diets supplemented with a low (0.05%) level of cholesterol (diets B and D). This micrograph depicts the intact, luminal surface of aortic tissue with a plaque score of zero, which was determined using a visual scoring scale. 191  Figure 5.4 A representative scanning electron micrograph of aortic tissue from atherosclerosis susceptible Japanese quail fed a Turkey Starter (TS) diet supplemented with a high (0.5%) level of cholesterol and a minor amount of additional beef tallow (0.6%). This micrograph depicts the moderate coverage of the luminal surface of aortic tissue with a plaque score of 2, which was determined using a visual scoring scale. 192  A  B  Figure 5.5 Representative scanning electron micrographs of aortic tissue from atherosclerosissusceptible Japanese quail fed a Turkey Starter (TS) diet supplemented with a high (0.5%) level of cholesterol and a higher level of beef tallow (6.6%). These micrographs depict the atherosclerotic luminal surface of aorta with a plaque score of 4, which was determined using a visual scoring scale. (A) This micrograph depicts the coverage of the aortic lumen by severe plaque characterized by a focal area of raised tissue; (B) depicts a close-up view of the same plaque area as seen in A, showing enlarged epithelial cells on the surface of the aortic plaque. 193  from birds in each dietary treatment group, with the results presented in Table 5.11. Birds fed the reference TS diet (diet A) and the diets supplemented with the low level of cholesterol (diets B and D)  without any detectable plaque on the luminal surface had similarly low levels of aortic tissue  cholesterol (Table 5.11). In all cases, the combination of the low level of tissue cholesterol and the absence of cholesterol oxides in aortic tissue of quail fed diets A, B and D were associated with an absence of aortic plaque deposition (Table 5.11).  Aortic tissue from quail fed diets containing the  lower level of tallow with high cholesterol (diet C) contained approximately twice the amount of tissue cholesterol compared to counterparts fed diets A and B, as well as detectable levels of a single cholesterol oxide, 5,6a-epoxy-5a-cholesterol (Table 5.11). The individual animal variability in plaque score observed in quail fed diet C was associated with similar variations in both the amount of aortic tissue cholesterol and cholesterol oxide content in these same animals. The absence of detectable cholesterol oxides in aortic tissue from some birds fed diet C corresponded to the relatively lower aortic cholesterol content and plaque scores in these animals (Table 5.1 1). Quail fed diets containing a high level of cholesterol and beef tallow (diet E) also exhibited individual variability in both aortic plaque scores, as well as cholesterol and cholesterol oxide content (Table 5.11). The higher level of tallow in combination with a high level of cholesterol in diet E fed to these quail resulted in the presence of several cholesterol oxides, including 5,6a-epoxy-5a-cholesterol, 71-hydroxycholesterol, cholestane-triol, 7-ketocholesterol and 25-hydroxycholesterol in aortic tissue from these animals (Table 5.1 1). Overall, there was a close association between the severity of plaque scores in aortic tissue with the level of aortic cholesterol and presence of cholesterol oxides in atherosclerosis-susceptible Japanese quail in the present study.  194  -  CD 0,  0/0/0 0/0/0  0/0/0 0/2/0 2/1/2 3/3/4  0/0/0 0/0/0  0/2/1 4/4/4  B B  C C C C  D D  E E 2.63 21.62  2.46 2.31  1.24 1.69 5.60 4.99  0.76 0.63  0.89 0.68  Cholesterol  0.19 0.21  N.D. N.D.  N.D. N.D. 017 1.33  N.D. N.D.  N.D. N.D.  5,6a-epoxide  0.59 0.99  N.D. N.D.  N.D. N.D. N.D. N.D.  N.D. N.D.  N.D. N.D.  7/1-OH  N.D. 0.30  N.D. N.D.  N.D. N.D. N.D. N.D.  N.D. N.D.  N.D. N.D.  triol  0.13 0.08  N.D. N.D.  N.D. N.D. N.D. N.D.  N.D. N.D.  N.D. N.D.  7-keto  N.D. 0.38  N.D. N.D.  N.D. N.D. N.D. N.D.  N.D. N.D.  N.D. N.D.  25-OH  2  Individual bird aortic tissue plaque score, cholesterol and oxide concentration measured by gas chromatography-mass spectrometry. Cholesterol concentration = mg/g tissue; oxide concentration = mg/g tissue; N.D. not detected. 5,6a-epoxide = 5,6a-epoxy-5acholesterol; 7/1-OH = 7/1-hydroxycholesterol; triol = cholestane-triol; 7-keto = 7-ketocholesterol; 25-OH = 25-hydroxycholesterol. Diet A = Turkey Starter (TS); B = TS + 0.05% chol.; C = TS + 0.5% chol.; D = TS + Tallow + 0.05% chol.; E = TS + Tallow + 0.5% chol. Plaque score is based on scale of 0 (N.D.) = clean surface; 1 = 5 plaques; 2 = 6-20 plaques; 3 = > 20 plaques; 4 = massive atheromas seen. Values represent 2 judges evaluating in a blinded protocol. xlylz = individual score for each of three vessels in aortic tree.  0/0/0 0/0/0  4 Score  A A  Dietary Treatment : 3  Cholesterol and Oxides 2  Table 5.11 GC quantitation of cholesterol and cholesterol oxide content of aortic tissue from atherosclerosis-susceptible Japanese quail fed experimental diets. 1  Discussion: Diet composition and fatty acid content: Unlike the protocol in Chapter 4, the basal diet used in the present study consisted of a commercial Turkey Starter (TS)  chow which contained a variety of dietary lipids derived from  vegetable as well as animal sources. additional 0.6% (wt/wt)  The supplementation of the basal TS diet (diet A)  with an  beef tallow and either of the two levels of cholesterol (i.e. 0.05 or 0.5%  cholesterol with 0.025 and 0.25% cholic acid, respectively) resulted in two markedly different levels of dietary cholesterol without altering the total crude lipid content of the diets (diets B and C). As a result, the percent of total calories derived from fat for diets B and C was similar to that of the reference TS diet (diet A; e.g. 22% calories from fat).  Supplementation of the TS diet with an  additional 6.6% (wt/wt) beef tallow (plus cholesterol and cholic acid as for diets B and D) resulted in an energy content of 32% of calories from fat in diets D and E.  For these latter two diets, the  additional saturated fat incorporated into the basal diet resulted in changes to the proportion of specific fatty acids in the diets. Specifically, the proportions of both myristic (increased by 37%) and oleic (increased by 7%) acids were altered in diets D and E, thereby decreasing the dietary lipid P/S ratio (reduced by 27%) in comparison to diets A, B and C. Plasma lipid response to dietary cholesterol levels: Although dietary cholesterol was initially reported to have little effect on plasma cholesterol concentrations (Keys et a!., 1956), it is now recognized to have a role in elevating circulating cholesterol levels (Lipid Research Clinics Program, 1984). However, individual responses to dietary cholesterol can be highly variable, depending on cholesterol metabolism and the level of dietary cholesterol intake (McNamara et a!., 1987).  From the results of the present study and those of  Chapter 4, it appears that the plasma lipid response of atherosclerosis-susceptible Japanese quail fed high cholesterol-cholic acid (0.5% and 0.25%, respectively) diets is analogous to that observed with familial hypercholesterolemia in human subjects (Reynolds, 1989).  These individuals have greatly  elevated plasma cholesterol levels and are at great risk for myocardial infarctions. A dose-dependent increase in plasma cholesterol concentrations has been reported in hamsters (Spady and Dietschy, 1988), guinea pigs (Lin et a!., 1 992) and Japanese quail (Radcliffe et a!., 1 982) fed diets containing 196  different levels of cholesterol. In the present study, the plasma cholesterol concentrations of quail fed high cholesterol diets was clearly elevated after 9 weeks of feeding. Other workers examining the temporal development of hypercholesterolemia in cholesterol-fed quail reported that plasma cholesterol levels were increased 144% after one week on an atherogenic diet (Radcliffe eta!., 1982). In the present study, quail fed diets containing a high level of dietary cholesterol exhibited hypertriacylglyceridemia confirming the results observed previously in Chapter 4. Other workers have also reported a hypertriacylglyceridemic response in Japanese quail fed commercial diets supplemented with 1  -  2% cholesterol (Godin et a!., 1994; Godin and Dahlman, 1993; Radcliffe et a!., 1982).  Fungwe and coworkers (1992, 1993)  reported that rats fed cholesterol in the diet exhibited  hypertriacylglyceridemia in a dose-dependent manner.  These workers were able to associate this  plasma response to dietary cholesterol intake with a suppression in hepatic fatty acid oxidation rate and increased hepatic secretion of VLDL particles (Fungwe eta!., 1993). The hypertriacylglyceridemia observed in cholesterol-fed Japanese quail supports its use as an animal model of human atherosclerosis, since epidemiological studies have reported an association between elevated plasma triacylglycerides and coronary heart disease (Castelli, 1986). Furthermore, the significant interaction noted between dietary cholesterol and fat intake level for plasma triacylglyceride content supports the “Lipid Hypothesis” which states that dietary fat intake level can alter plasma lipid levels and initiate or exacerbate atherogenesis (Roberts, 1992). The markedly greater plasma lipid concentrations and development of aortic plaque observed in quail fed diet E which contained increased levels of both saturated fat and cholesterol supports this hypothesis. Plasma lipid response to dietary fat level: The plasma lipid response of quail fed the two levels of beef tallow with cholesterol do not agree with the results of previous workers who reported a hypocholesterolemic effect of oleic acid (Mattson and Grundy, 1985), a principal fatty acid of beef tallow (approx. 44%).  Quail fed diets containing  0.5% cholesterol with 0.25% cholic acid exhibited similar degrees of hypercholesterolemia regardless of the level of beef tallow in the diet. It is noteworthy, however, that quail fed the high cholesterol diet in combination with a high level of beef tallow did not exhibit a further significant increase in plasma cholesterol levels.  While it is generally regarded that saturated fatty acids contribute to an 197  increase in plasma cholesterol levels, it is conceivable that the increased content of myristic, palmitic and stearic acid in the high tallow diets was not sufficient to result in a further increase in plasma cholesterol concentrations in this group. Also, previous studies in humans fed beef tallow diets have observed a similar neutral effect of dietary stearic acid on plasma cholesterol concentrations (Kris Etherton etal., 1993). Influence of other dietary factors on plasma lipid levels: It is of interest to note that although quail fed high cholesterol diets exhibited hyperlipidemia in both this study and that in Chapter 4, the absolute concentrations of plasma cholesterol obtained in hypercholesterolemic birds was relatively lower herein.  This discrepancy in plasma cholesterol  concentrations between studies may be attributed to the different fibre content of the basal diets used in the two studies. Even though identical levels of dietary cholesterol and cholic acid were used in both studies, the sources of dietary fibre and possibly the amount of soluble and insoluble fibre in the commercial TS (e.g. alfalfa meal, corn, distiller’s grain and soybean meal) was different compared to that of the semi-synthetic diets (e.g. aiphacel) used in Chapter 4. Fibre in the diet has been estimated to have a similar hypocholesterolemic effect in both males and females (Bolton-Smith etal., 1992). According to the results of the present study, the protective effect of dietary fibre in reducing plasma lipid levels suggested by others is likely related to a reduction in the absorption of lipid from the diet. Effect of dietary fat and cholesterol on antioxidant status: The results of the present study indicate a relative insensitivity of antioxidant enzymes in RBCs and heart tissue to elevations in plasma cholesterol and triacyiglycerides in quail fed diets supplemented with cholesterol. It is conceivable that the lack of effect of dietary treatment on GSH concentrations in both RBCs and heart tissue can be attributed to the relatively stable nature of the principal saturated and monounsaturated fatty acids in beef tallow to lipid peroxidation (e.g. palmitic, stearic and oleic acids). In this context, the activity of GSH metabolizing enzymes in RBCs and heart of quail fed a predominantly saturated fat diet would not be altered due to the oxidative stability of diet-derived fatty acids with a low P/S ratio. Support for this explanation is provided by the observed reduction in TBARs production in heart tissue in vitro from birds fed diets containing the high level of beef tallow. Moreover, the additive protective effect of a high cholesterol intake in combination with 198  increased saturated fat for heart TBARs production in birds fed diet E suggests that tissue membrane lipids were stabilized against oxidation by not only an increase in membrane phospholipid saturated fatty acid content, but also the stabilization of membranes by the presence of increased cholesterol. Unlike the minimal changes in antioxidant enzyme activity observed in RBCs and heart, liver tissue CAT and SOD activities were reduced in quail fed the high cholesterol diets. A somewhat surprising observation was the reduction in liver CAT activity in quail fed diets containing even the low level of cholesterol in diet B. The minimal level of cholesterol in this diet resulted in a slight but not significant increase in plasma cholesterol and a significant elevation in plasma triacylglyceride concentrations compared to quail fed TS alone. It is noteworthy that hepatic SOD activity was also reduced in birds fed the high cholesterol diets, regardless of the dietary fat content. A similar result was observed in a previous study conducted in this thesis (Chapter 3), wherein the reduced SOD activity was attributed to the stabilizing effect of cholesterol on tissue membrane lipids against oxidation.  The lack of  differences in hepatic GSSG-Red and GSH-Px activities coincided with similar intracellular GSH levels in livers from experimental quail. Despite the lack of differences in the GSH content and activities of the GSH metabolizing enzymes of liver tissue, the results observed with the in vitro oxidative challenge of liver tissue showed that both GSH depletion and TBARs production were influenced by the level of dietary cholesterol. The greater depletion of GSH in liver tissue from quail fed the high level of dietary cholesterol may be associated with changes in tissue composition due to the increase in liver weight attributable to accumulation of cholesterol and triacylglyceride (Liu eta!., 1995). On the other hand, feeding cholesterol and a high level of beef tallow to quail greatly reduced TBARs production in vitro when liver tissues were challenged with peroxidizing agent. A similar result was observed in previous studies (Chapters 2 and 4)  of this thesis, wherein it was suggested that diets high in cholesterol  resulted not only in enhanced levels of cholesterol in the plasma, but also tissue membranes. Incorporation of greater levels of cholesterol into tissue membranes could conceivably result in a stabilizing effect of membrane phospholipids against oxidation as well as cholesterol acting as a sacrificial antioxidant to spare membrane fatty acids (Smith, 1991). Similar to the results obtained in heart tissue, aortic antioxidant enzyme activities were not significantly altered by the dietary treatments in the present study. These results herein confirm the 199  lack of effect of hypercholesterolemia and aortic plaque deposition on aortic tissue antioxidant status observed in cholesterol-fed atherosclerosis-susceptible Japanese quail in Chapter 4.  While other  workers have reported differences in aortic tissue antioxidant enzyme activities (e.g. SOD; Godin et a?., 1 994) in Japanese quail fed cholesterol, these changes were not unique to the atherosclerosissusceptible strain of these birds and therefore do not appear to be linked to an enhanced susceptibility of aortic tissue to plaque development in these animals. Aortic plaque development: The results of the present study indicate that in the atherosclerosis-susceptible Japanese quail diet-induced risk factors for atherosclerosis, such as hypercholesterolemia and hypertriacylglyceridemia result in the deposition of aortic plaque characterized by increased cholesterol as well as the presence of cholesterol oxides in aortic tissue.  These changes coincided with the severity of aortic plaque  estimated visually using a numerical scale. The supplementation of the TS diet with 0.5% cholesterol resulted in variable degrees of aortic plaque deposition related to the level of fat in the diet. The aortic plaque score of cholesterol-fed birds was greatest in animals fed diets supplemented with both a high level of cholesterol as well as the higher level of saturated fat which indicated a treatment interaction for severity of atherosclerosis in these quail. This observation highlights the fact that while there is little doubt that the appearance of atherosclerotic plaque in birds was associated with the elevation in plasma cholesterol level which was due to the cholesterol-rich diets, the severity of atherosclerosis in these quail was further enhanced by the presence of hypertriacylglyceridemia. It is noteworthy that aortic plaque scores were associated with a significant interaction between dietary cholesterol and fat level which coincided with the results seen with plasma triacylglyceride concentrations but not with plasma total cholesterol concentrations. The absence of atherosclerotic lesions in aorta from birds fed the low cholesterol-cholic acid diets confirms the requirement for a high dietary cholesterol intake and an associated hypercholesterolemia to initiate the deposition of aortic plaque. Supportive evidence for this hypothesis is provided by the low concentrations of aortic tissue cholesterol and the absence of detectable cholesterol oxides in aortic tissue from birds fed low cholesterol diets. Kritchevsky (1970) reported that the extent of atherosclerosis in the rabbit model was greatest when diets were high in cholesterol. 200  Despite the high levels of plasma cholesterol observed in quail fed the atherogenic diets herein, the variable aortic plaque scores and tissue content of cholesterol and cholesterol oxides indicated that individual animal heterogeneity existed in susceptibility to diet-induced atherosclerosis. Other workers have reported that atherogenesis in Japanese quail follows a sequential process that involves tissue disruption and swelling, followed by the appearance of cholesterol-laden foam cells and the formation of plaque characterized by cellular proliferation and narrowing of the lumen (Shih et a!., 1983). Indications of aortic lumen damage accompanied by focal areas of cell proliferation were observed in the scanning electron micrographs from birds with mild to severe plaque, and thereby confirmed the visual scoring method used herein, and in Chapters 3 and 4. Similar to the results in Chapter 4, the profile of cholesterol oxides present in aortic tissue from quail fed the high cholesterol diets varied with the particular treatment as well as with individual birds in a treatment group. The detection of a greater number of cholesterol oxide species in aortic tissue from quail fed the diet containing a high level of cholesterol with a high level of saturated fat, in part, reflects the increased cholesterol content of tissues heavily covered by plaque. Previous workers have reported similar cholesterol oxides recoverable from LDL extracted from human aortic plaque at autopsy (Steinbrecher and Lougheed, 1992).  201  Conclusion: In conclusion, atherosclerosis-susceptible Japanese quail fed commercial Turkey Starter diets supplemented with a high level of cholesterol and a low or high level of saturated fat exhibited hypercholesterolemia and hypertriacylglyceridemia.  The hypertriacylglyceridemia with cholesterol  feeding was further enhanced in quail fed the high level of fat in the diet, which supports the “Lipid Hypothesis” that an increased intake of calories from fat can increase plasma lipid levels and initiate or exacerbate atherogenesis.  This result was further supported by the significant treatment  interactions between dietary cholesterol level and fat intake level observed for both plasma triacylglyceride concentrations as well as aortic plaque score but not plasma total cholesterol concentrations in quail. Moreover, the deposition of atherosclerotic plaque in birds fed high cholesterol and high saturated fat was enhanced compared to those fed the lower level of dietary fat. Despite these effects of diet-induced hypercholesterolemia and hypertriacylglyceridemia on aortic plaque scores, RBC, heart and aortic tissue antioxidant enzyme activities were not altered under these conditions. These results confirm those obtained in Chapter 4 and suggest that antioxidant status does not influence diet-induced susceptibility to the development of atherosclerosis in this animal model under the specific conditions of these studies. Dietary cholesterol level and thereby plasma cholesterol and triacylglyceride levels appear to play a much greater role than specific antioxidant enzymes in the development of aortic plaque in the Japanese quail model which is noted to develop diet-induced atherosclerosis very rapidly.  202  SUMMARY AND GENERAL CONCLUSIONS There is an ample amount of evidence to support the conclusion that diet can play an important role in the pathogenesis of hypertension and hyperlipidemia, two risk factors for the development of cardiovascular disease. Dietary fat composition, proportion of calories in the diet derived from fat, as well as the cholesterol content of the diet are all contributing factors in determining plasma lipid (e.g. total cholesterol and triacylglyceride) concentrations. Numerous studies have shown that dietary fatty acid composition, namely the proportions of saturated, monounsaturated and polyunsaturated (n-6 and n-3) fatty acids, can modulate plasma lipid concentrations. Dietary cholesterol is generally considered to increase plasma cholesterol concentrations when it is consumed at high levels. However, variability in response to dietary cholesterol intake is also known to occur among individuals. While consumption of polyunsaturated fatty acids from vegetable or marine oils is known to reduce plasma cholesterol levels, these fatty acids are also noted to exhibit an enhanced susceptibility to oxidation in food systems as well as in vivo. This characteristic of polyunsaturated fatty acids is of great importance, given the fact that increased levels of lipid peroxides have been demonstrated in the plasma and tissues of hyperlipidemic humans and animal models. Several studies have suggested that in vivo lipid oxidation and alterations in antioxidant status occur both in hypertension as well as atherosclerosis. If diet-induced changes to risk factors for cardiovascular disease, such as hypertension and atherosclerosis, are also demonstrated to have an effect on tissue lipid susceptibility to oxidative challenge and antioxidant enzyme status, this information can potentially be used in developing recommendations to reduce the risk of in vivo lipid oxidation associated with dietary lipids.  More  generally, these investigations will enhance the body of knowledge concerning food safety and processing of foods to maintain the quality of dietary lipids. This thesis reports the results of five studies which were designed to examine the individual and/or interactive effects of dietary fat source and level of cholesterol on plasma lipids and endogenous antioxidant status in two animal models known to exhibit risk factors for the development of cardiovascular disease, the spontaneously hypertensive rat and the atherosclerosis-susceptible Japanese quail.  The spontaneously hypertensive rat (SHR)  was chosen due to its age-dependent  development of hypertension, which can be monitored by systolic blood pressure recordings. The 203  atherosclerosis-susceptible Japanese quail was chosen due to its susceptibility to the development of aortic atherosclerotic plaque in the presence of diet-induced hyperlipidemia in these animals. Chapter 1 compared the plasma lipid concentrations and tissue antioxidant status between hypertensive SHR and their normotensive controls, Wistar Kyoto (WKY) rats fed a non-atherogenic commercial chow diet. This study demonstrated that the genetic susceptibility of the SHR for the development of hypertension coincided with strain differences in plasma cholesterol and triacylglyceride concentrations as well as tissue-specific differences in antioxidant enzyme activities.  A surprising  finding in this study were the relatively lower concentrations of plasma cholesterol and triacylglyceride observed in SHR compared to WKY animals. RBC CAT activity was positively correlated (r p  =  0.026) with SOD activity.  correlated (r=O.709; p  =  =  0.634;  Also, it was noteworthy that RBC SOD activity was positively  0.049) with systolic blood pressure. SHR animals also exhibited greater  concentrations of GSH in RBCs, heart and liver tissues compared to WKY counterparts. Hypertension in SHR coincided with alterations in antioxidant enzyme profiles of RBC and heart, with the latter showing an increased susceptibility to in vitro lipid oxidation. Although hypertension is a recognized factor in the development of human atherosclerosis, hypertensive SHR did not develop atherosclerosis. This study characterized some of the basic metabolic differences in plasma lipids, tissue antioxidant enzyme activities and susceptibility to in vitro oxidative challenge between the hypertensive SHR and normotensive WKY and set the stage for further investigation using this animal model in Chapter 2. The effects of diets containing a low or high level of cholesterol and dietary fat sources differing in proportions of saturated (short-chain versus long-chain saturates) or polyunsaturated (n-6 versus n-3)  fatty acids on plasma lipids, endogenous antioxidant status and the development of  hypertension in SHR and WKY rats were examined in Chapter 2. The semi-synthetic diets used in this experiment were formulated to contain either a low or high level of cholesterol with the fat content of all diets kept constant at a moderate level of 8% (wt/wt), of which 5% was provided by the test fat. This level of dietary fat was chosen because it is considered normal for the rat and meets the nutritional requirements for this animal species.  The relatively lower plasma cholesterol and  triacylglyceride concentrations observed in SHR compared to WKY in Chapter 1 were confirmed in Chapter 2. Animal strain differences in plasma lipid levels were consistent, regardless of the dietary 204  fat source or the level of dietary cholesterol.  Both SHR and WKY animals exhibited reduced body  weight gain and feed efficiency ratios as well as reductions in plasma cholesterol and triacylglyceride concentrations when fed menhaden oil diets. Decreased activities of GSH-Px and SOD in RBCs, heart and liver tissue from animals fed menhaden oil diets coincided with reductions in tissue susceptibility to in vitro H -induced GSH depletion and lipid peroxidation. These results suggest that diet-induced 0 2 alterations to the fatty acid composition of tissue cell membranes may influence the oxidative stability of membrane phospholipids, thereby altering the balance of lipid peroxides in vivo which, in turn, can either be deactivated by antioxidant enzyme activity, or can have inhibitory effects on these same enzyme activities. High cholesterol diets reduced hepatic CAT, SOD and GSH-Px activities and resulted in decreased susceptibility of liver tissue and RBCs to in vitro oxidative challenge. These effects of dietary cholesterol could be associated with incorporation of cholesterol into cell membranes, resulting in increased fatty acid stability against lipid oxidation in vivo. Despite these diet-induced changes to plasma lipids and tissue-specific antioxidant enzyme activities, there was no effect of dietary treatment on systolic blood pressure of SHR or WKY animals. It is conceivable that extending this study in older animals may have resulted in significant changes in the severity of hypertension in the SHR. It can be concluded from this study that the genetic predisposition of the SHR to hypertension and specific strain differences in antioxidant enzyme activity were less sensitive than plasma lipids to dietary fat source and cholesterol content treatment effects. A noteworthy finding of this study was that in vitro H O 2 induced MDA production in RBCs from hypertensive SHR was consistently elevated herein as well as in Chapter 1, suggesting that this observation is a characteristic of RBCs in the SHR. Hypertensive SHR exhibiting diet-induced hypercholesterolemia and hypertriacylglyceridemia did not exhibit signs of atherosclerotic lesions, confirming the resistance of this strain of rat to the development of atherosclerosis. It has been reported that the association between dyslipidemia and atherosclerosis may also involve the presence of increased plasma and tissue levels of lipid oxidation products. Therefore, in Chapter 3, the relative sensitivities of an atherosclerosis-susceptible strain of Japanese quail and a rat model to diet-induced hyperlipidemia and atherosclerosis were compared. Japanese quail exhibited higher concentrations of plasma cholesterol and triacylglycerides than rat counterparts fed similar levels 205  of dietary cholesterol. After a 9-week feeding trial, atherosclerosis was observed only in the quail fed the high cholesterol diet, compared to rat counterparts. The presence of atherosclerotic plaque in aortic tissue of quail was associated with the presence of elevated levels of aortic cholesterol. Moreover, two cholesterol oxides (7R-hydroxycholesterol and 7-ketocholesterol) were identified and quantitated in the non-saponifiable fraction of aortic tissue from atherosclerotic quail. Aortic tissue from rats had relatively lower amounts of tissue cholesterol and did not contain detectable amounts of cholesterol oxides. It was concluded from this study that the atherosclerosis-susceptible Japanese quail develops atherosclerotic plaque in conjunction with elevated levels of plasma cholesterol when these quail are fed an atherogenic diet. The similarity of cholesterol oxides identified in aortic plaque of quail to those noted in diseased human aortae supports the use of the atherosclerosis-susceptible quail as an animal model for human atherosclerosis. Also, the cholesterol and cholesterol oxide content of aortic tissue with plaque agreed with the relative severity of plaque scores determined using a visual scoring method. Once the diet-induced hypercholesterolemia and atherosclerosis were confirmed in the atherosclerosis-susceptible Japanese quail under the conditions employed in Chapter 3, a subsequent study similar in design to Chapter 2 was performed using this strain of Japanese quail. The study in Chapter 4 was designed to examine the effect of different dietary fat sources and cholesterol levels on the plasma lipid profiles and antioxidant status of the atherosclerosis-susceptible Japanese quail. Dietary cholesterol level had a greater effect on plasma cholesterol and triacyiglyceride levels than dietary fat source.  Quail fed high cholesterol diets exhibited severe hypercholesterolemia and  hypertriacylglyceridemia. These elevations in plasma lipids coincided with the presence of extensive atherosclerotic plaque in the aortae of birds fed high cholesterol. However, tissue (e.g. heart, liver and aorta)  and RBC antioxidant parameters were not affected by hyperlipidemia or the presence of  atherosclerotic plaque.  Dietary fat sources differing in fatty acid composition had little effect on  plasma lipid concentrations. However, RBC and heart GSH-Px activities were different in soybean oil fed animals compared to counterparts fed beef tallow or butter diets. Hypercholesterolemia in quail was associated with elevated aortic cholesterol levels as well as the presence of several cholesterol oxides. It was noteworthy that several additional cholesterol oxides (e.g. 5,6a-epoxy-5a-cholesterol, 206  7a-hydroxycholesterol, 41-hydroxycholesterol, cholestane-triol, and 25-hydroxycholesterol)  were  detected in atherosclerotic aortic tissue from quail fed the atherogenic diets. Japanese quail exhibited individual animal variability in the profile and amounts of cholesterol oxides present in atherosclerotic aortic tissue despite the similarities in plaque score determined visually. Scanning electron micrographs of aortic tissue without plaque and tissue with severe plaque also confirmed the presence of focal areas of raised tissue on the luminal surface of atherosclerotic aortae in this study. It was concluded in this study that the lack of an effect of dyslipidemia and severe atherosclerosis on the endogenous antioxidant status of tissues in the atherosclerosis-susceptible Japanese quail suggests that at the extreme plasma lipid concentrations achieved herein it was unlikely that tissue antioxidant status was able to modulate the development of atherosclerosis in this animal model. It remains to be determined if the effect of dietary fat level and less extreme concentrations of plasma cholesterol on aortic plaque development can modify the role of endogenous antioxidant status in the development of atherosclerosis in this animal model. The final study of this thesis examined the role of dietary fat quantity and cholesterol content on the plasma lipid profile, antioxidant status and atherosclerosis in the atherosclerosis-susceptible Japanese quail.  The formulation of diets fed to quail in Chapter 5 was designed to supplement a  commercial Turkey Starter with two levels of cholesterol and beef tallow. Quail fed high cholesterol diets exhibited hypercholesterolemia and hypertriacylglyceridemia regardless of the level of dietary fat. However, an interaction between dietary cholesterol level and fat content was observed for plasma triacylglyceride concentrations in quail fed diets containing high cholesterol in combination with a higher level of tallow. A similar dietary treatment interaction was observed for aortic plaque score in this same group of animals. Similar to the results obtained in Chapter 4, quail fed Turkey Starter diets supplemented with cholesterol and beef tallow exhibited little change in antioxidant enzyme activities of RBCs, heart, liver or aortic tissues. Dietary cholesterol level did, however, significantly reduce the activities of liver CAT and SOD as well as decrease the susceptibility of liver tissue to in vitro H 0 2 induced oxidative challenge. A similar result was observed in heart tissue from animals fed diets high in cholesterol in combination with the higher level of beef tallow. These results may be attributed to the enhanced stability of membrane fatty acids to lipid oxidation due to the increased incorporation of 207  cholesterol into membranes in the first case, and the combination of enhanced membrane cholesterol in addition to increased saturated and monounsaturated fatty acids from dietary beef tallow in the second case. The results of Chapter 5 suggest that in the atherosclerosis-susceptible Japanese quail the development of atherosclerosis is associated with not only hypercholesterolemia but also hypertriacylglyceridemia. Moreover, the latter condition appears to have a potentiating effect on aortic plaque development in quail fed the Turkey Starter based diets used in this study.  It was also  noteworthy in the present study that despite the similar level of dietary cholesterol supplementation as used in Chapter 4, the plasma cholesterol levels of birds were somewhat lower than previously observed in counterparts fed semi-synthetic diets. It was noteworthy that quail fed high cholesterol diets in the present study exhibited greater variability in aortic plaque score than previously observed in Chapter 4. The content of cholesterol and cholesterol oxides detected in atherosclerotic aortic tissue from these same birds paralleled the plaque score assigned to tissues by visual scoring.  Scanning  electron micrographs confirmed the presence of focal areas of raised tissue on the luminal surface of atherosclerotic aortae. In summary, the results of this thesis indicate that the underlying metabolic factors determining the development of hypertension and atherosclerosis are not equivalently linked to the antioxidant status of the animal models employed in this thesis. While some associations between the tissuespecific nature of strain-related differences in antioxidant activity between SHR and WKY animals could be related to the systolic blood pressure recorded in these animals (e.g. RBC SOD activity and systolic blood pressure), the genetic predisposition of the SHR for development of hypertension was not altered by dietary treatment (e.g. cholesterol level and dietary fat source). In contrast, dietary cholesterol and fat level effects on plasma lipids in the atherosclerosis-susceptible Japanese quail were associated with the development and exacerbation, respectively, of aortic plaque in this animal model. These effects of dietary lipids on susceptibility to atherosclerosis were observed to occur in the absence of alterations in RBC or tissue antioxidant status in the Japanese quail. 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Relative retention times of trimethyl-silyl (TMS)-sterol derivatives . 1  Retention time 2 : 3 Sterol 5a-Cholestane  0.721  Cholesta-3,5-dien-7-one  0.988  Cholest-5-en-3B-ol (Cholesterol)  1 .000  Cholest-5-ene-3I-7a-diol (7a-hydroxycholesterol)  1 .009  5,6a-Epoxy-5a-cholestan-31-ol (5, 6a-epoxide)  1 .134  Cholest-5-ene-3B,7B-diol (71,-hydroxycholesterol)  1 .148  Cholest-5-ene-3F,4I-diol (4(-hydroxycholesterol)  1 .164  5a-Cholestane-3f,-5,6B-triol (Cholestane-triol)  1 .297  3I-hydroxychoIest-5-en-7-one (7-ketocholesterol)  1 .320  Cholest-5-ene-3I-25-diol (25-hydroxycholesterol)  1 .378  1  Standard curve constructed with five concentrations of TMS-sterol derivatives with internal standard, 5a-cholestane, with 3 injections per concentration. Response linearity analyzed by plotting the area response ratio of each sterol over the internal standard against the weight ratio of each sterol over the internal standard. 2 Relative retention times expressed in relation to cholesterol. Sterol with common name in parentheses.  229  Table 2. Response linearity of derivatized cholesterol and oxides . 1  Slope  Intercept  2 R,  : 3 Sterol Cholesta-3,5-dien-7-one  1.13  0.001  1.16± 0.10  Cholest-5-en-3l,-ol (Cholesterol)  1 .18  -0.004  1 .00 ± 0.08  Cholest-5-ene-3B-7a-diol (7a-hydroxycholesterol)  0.98  -0.001  0.99 ± 0.07  5,6a-Epoxy-5a-choIestan-3l-ol (5, 6a-epoxid e)  1.01  -0.005  1 .20 ± 0.11  ChoIest-5-ene-31,7B-dioI (7B-hydroxycholesterol)  1 .26  -0.001  1 .03 ± 0.08  Cholest-5-ene-31&4l-dioI (4B-hydroxycholesterol)  0.60  0.001  1.64 ± 0.12  5a-Cholestane-3B-5,61-triol (Cholestane-triol)  1.13  -0.001  0.98 ± 0.09  3R-hydroxycholest-5-en-7-one (7-ketocholesterol)  1.09  -0.001  0.99 ± 0.10  Cholest-5-ene-3I-25-diol (25-hydroxycholesterol)  1 .17  -0.001  0.95 ± 0.79  1  Standard curve constructed with five concentrations of TMS-sterol derivatives with internal standard, 5a-cholestane, with 3 injections per concentration. Response linearity analyzed by plotting the area response ratio of each sterol over the internal standard against the weight ratio of each sterol over the internal standard. 2 Response factor = (VL’IA)(A /W), presented as mean ± SEM, where A = area of sterol standard, 1 = weight of sterol standard and W 1 = area of internal standard, A 1 = weight of internal standard. Sterol with common name in parentheses.  230  

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