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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 INATHEROSCLEROSIS RESISTANT (RAT) AND SENSITIVE (QUAIL) ANIMALS.ByYVONNE VERONICA YUANB.Sc., The University of British Columbia, 1987M.Sc., The University of British Columbia, 1990A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIES(Department of Food Science)We accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIASeptember 1995© Yvonne Veronica Yuan, 1 995In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)_____________________Department of OcXi ScThe University of British ColumbiaVancouver, CanadaDate OCk.L3 Ifc1(DE-6 (2/88)ABSTRACTStudies were performed in two animal models known to be susceptible to (i) age-dependenthypertension and (ii) atherosclerosis, for the purpose of examining the role of specific dietary fatsources and dietary cholesterol intake in modifying hypertension and atherosclerosis, two well definedrisk factors in the development of cardiovascular disease. Despite exhibiting hypertension, thespontaneously hypertensive rat (SHR) had lower (p 0.05) plasma cholesterol and triacyiglycerideconcentrations than normotensive Wistar Kyoto (WKY) controls. SHR and WKY animals exhibitedtissue- and enzyme-specific strain differences in antioxidant status. Systolic blood pressure (SBP) waspositively (r = 0.709, p = 0.049) correlated with RBC superoxide dismutase (SOD) activity andnegatively correlated with liver glutathione reductase (GSSG-Red; r = -0.798, p = 0.018). Dietaryfat source or the proportion of saturated to unsaturated (n-6 or n-3) fatty acids fed at a moderate levelof fat intake (e.g. 22% calories) did not have an effect on plasma lipids, with the exception ofmenhaden oil which significantly reduced both plasma cholesterol and triacylglyceride levels. Therewas no effect of dietary fat source or cholesterol on SBP of SHR or WKY animals. Menhaden oil fedto 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 sameanimals. High levels of dietary cholesterol resulted in reduced (p 0.05) hepatic CAT, SOD and GSHPx activities as well as greater resistance to in vitro lipid peroxidation than in counterparts fed lowcholesterol diets. Despite significant effects of dietary cholesterol and fat source on plasma lipids andantioxidant parameters in hypertensive SHR, changes to SBP were not observed in the present studyinvolving relatively young animals. Similar studies examining the effects of dietary cholesterol intakeand fat source or level on plasma lipids, antioxidant status and aortic plaque were conducted in theatherosclerosis-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 plaquecontaining cholesterol and cholesterol oxides. Aortic atherosclerosis was not associated withalterations 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 anddecreased, respectively, in soybean oil-fed birds. In the final study, an increase in total calories fromsaturated fat (e.g. beef tallow) together with different levels of dietary cholesterol were found to haveinteractive effects on plasma triacylglycerides, and aortic plaque scores of atherosclerosis-susceptibleJapanese quail. These dietary treatments did not have any effect on antioxidant enzymes of heart oraortic tissue. Dietary cholesterol and the higher level of saturated fat did, however, influence hepaticCAT and SOD activities, as well as reducing the susceptibility of liver and heart tissues to in vitro lipidoxidation. It was noteworthy that a significant (p 0.05) interaction was observed between dietarycholesterol and fat level for plasma triacylglycerides but not plasma cholesterol. Elevations in plasmacholesterol and triacylglycerides in atherosclerotic quail paralleled the severity of aortic lesions, asdetermined by plaque score, cholesterol content and presence of cholesterol oxides in aortic tissuefrom these birds. The presence of plasma hypercholesterolemia and hypertriacylglyceridemia in theatherosclerosis-susceptible Japanese quail appeared to play a greater role in determining susceptibilityto the development of aortic plaque than tissue antioxidant status under the experimental conditionsemployed herein. In summary, specific differences in tissue antioxidant enzymes and susceptibility tolipid peroxidation were more evident in the genetically hypertensive SHR rat model, than in diet-inducedatherosclerosis in the atherosclerosis-susceptible Japanese quail.IIITABLE OF CONTENTSPageTITLE PAGEABSTRACTTABLE OF CONTENTS ivLIST OF TABLES viiLIST OF FIGURESLIST OF APPENDICES xiiLIST OF ABBREVIATIONS xiiiACKNOWLEDGEMENTS xviINTRODUCTION 1LITERATURE REVIEW 21. a. Lipid oxidation: reaction mechanism and products 2b. Factors influencing lipid oxidation 4c. Antioxidants 6d. Antioxidants in foods 8e. Lipid autoxidation interactions with other nutrients or cell constituents 112. a. Digestion and absorption of dietary lipids 1 3b. Dietary lipids and dyslipidemias 1 7c. Endogenous antioxidants in vivo 26d. Lipid peroxidation in vivo 30e. Lipid oxidation in heart disease 32f. Oxidized lipid species in plasma 35g. Influence of dietary fatty acids on LDL oxidative stability 35CHAPTER 1:Antioxidant status and plasma lipid levels in spontaneously hypertensive (SHR)and normotensive Wistar Kyoto (WKY) rats 40Introduction 40Hypothesis, Objective and Specific Aims for Chapter 1 43Materials and Methods 44Blood pressure measurement 44Tissue sample preparation 44Tissue antioxidant analysis 45i. Tissue glutathione (GSH; sulfhydryl group) content 45ii. Tissue susceptibility to in vitro forced peroxidation 46iii. Catalase (CAT) activity 47iv. Glutathione peroxidase (GSH-Px) activity 48v. Glutathione reductase (GSSG-Red) activity 48vi. Superoxide dismutase (SOD) activity 49vii. Correction of enzyme activities for blood contamination 50Statistics 50ivnormotensive Wistar Kyoto (WKY) ratsIntroductionHypotheses, Objective and Specific Aims forMaterials and MethodsDiet gross energy determination .Dietary fatty acid analysisStatisticsResultsDiscussionConclusionPage• . . . 51• . . . 63686969717274747677101112CHAPTER 3:Species-related differences in plasma lipids and susceptibility to atherosclerosisbetween atherosclerosis-resistant (rat) and -susceptible (quail) animalsfed diets supplemented with cholesterolIntroductionHypothesis, Objective and Specific Aims for Chapter 3Materials and MethodsAnimals and dietsExperimental proceduresDetermination of aortic plaque scoreAnalysis of cholesterol and cholesterol oxides by gasmass spectrometryResultsDiscussionConclusionchromatography!114114116117117117117119123129132CHAPTER 4:Effect of dietary fat source on aortic plaque, plasma lipids and antioxidant statusof atherosclerosis-susceptible Japanese quailIntroductionHypotheses, Objective and Specific Aims for Chapter 4Materials and MethodsScanning electron microscopyResultsDiscussionConclusionCHAPTER 5:133133135136140141160169Influence of increased caloric intake from beef tallow on plasma lipids, antioxidant statusand diet-induced atherosclerosis in atherosclerosis-susceptible Japanese quailIntroductionHypothesis, Objective and Specific Aims for Chapter 5Materials and MethodsMixing of dietsResultsResultsDiscussionConclusionCHAPTER 2:Effect of saturated and polyunsaturated dietary fat sources on systolic blood pressure,plasma lipids and antioxidant status in spontaneously hypertensive (SHR) andChapter 2170.170• .172• .173.173• . .177VPageDiscussion 196Conclusion 202SUMMARY AND GENERAL CONCLUSIONS 203REFERENCES 210APPENDIX 228viLIST OF TABLESTable Page1.1 Body weight gain and systolic blood pressure of SHR and WKY rats feda standard chow diet 521 .2 Plasma lipids of SHR and WKY rats fed a standard chow diet 531 .3 Antioxidant enzyme activities in red blood cells of SHR and WKY rats feda standard chow diet 541 .4 Antioxidant enzyme activities in heart tissue of SHR and WKY rats feda standard chow diet 551 .5 Antioxidant enzyme activities in liver tissue of SHR and WKY rats feda standard chow diet 571 .6 Tissue glutathione levels of SHR and WKY rats feda standard chow diet 582.1 Composition of diets fed to SHR and WKY rats 732.2 Fatty acid composition of diets fed to SHR and WKY rats 752.3 Body weight gain and systolic blood pressure of SHR and WKY fedexperimental diets 782.4 Plasma lipids of SHR and WKY fed experimental diets 802.5 Antioxidant enzyme activities in red blood cells of SHR and WKY fedexperimental diets 822.6 Antioxidant enzyme activities in heart of SHR and WKY fedexperimental diets 842.7a Reactive oxygen species metabolizing antioxidant enzyme activities in liverof SHR and WKY fed experimental diets 862.7b Glutathione metabolizing antioxidant enzyme activities in liverof SHR and WKY fed experimental diets 872.8 Basal glutathione levels in heart and liver tissue and red blood cellsof SHR and WKY fed experimental diets 892.9 Red blood cell susceptibility toH20-induced GSH depletion and MDA productionin SHR and WKY fed experimental diets 902.10 Heart homogenate susceptibility toH20-induced GSH depletion and TBARs productionin SHR and WKY fed experimental diets 952.11 Liver homogenate susceptibility toH20-induced GSH depletion and TBARs productionin SHR and WKY fed experimental diets 97viiTable Page3.1 Composition of diets fed to Wistar rats and atherosclerosis-susceptibleJapanese quail 1183.2 Plasma lipids of Wistar rats and atherosclerosis-susceptible Japanesequail fed tallow diets containing low and high levels of cholesterol 1 243.3 Aortic plaque score and area covered in Wistar rats and atherosclerosis-susceptibleJapanese quail fed tallow diets containing low and high levels of cholesterol 1 253.4 GC quantitation of cholesterol and cholesterol oxidation products in aortic tissuefrom Wistar rats and atherosclerosis-susceptible Japanese quail fed tallow dietscontaining low and high levels of cholesterol 1 264.1 Composition of diets fed to atherosclerosis-susceptible Japanese quail 1374.2 Fatty acid composition of diets fed to atherosclerosis-susceptibleJapanese quail 1394.3 Plasma lipids of atherosclerosis-susceptible Japanese quailfed experimental diets 1424.4 Antioxidant enzyme activities in red blood cells of atherosclerosis-susceptibleJapanese quail fed experimental diets 1444.5 Antioxidant enzyme activities in heart of atherosclerosis-susceptibleJapanese quail fed experimental diets 1454.6a Reactive oxygen species metabolizing antioxidant enzyme activities in liver ofatherosclerosis-susceptible Japanese quail fed experimental diets 1464.6b Glutathione metabolizing antioxidant enzyme activities in liver of atherosclerosis-susceptible Japanese quail fed experimental diets 1474.7 Antioxidant enzyme activities in aorta of atherosclerosis-susceptibleJapanese quail fed experimental diets 1484.8 Heart homogenate susceptibility toH20-induced GSH depletion and TBARsproduction in atherosclerosis-susceptible Japanese quail fed experimental diets 1514.9 Liver homogenate susceptibility toH20-induced GSH depletion and TBARsproduction in atherosclerosis-susceptible Japanese quail fed experimental diets 1 534.10 Aortic plaque score and area covered in atherosclerosis-susceptibleJapanese quail fed experimental diets 1554.11 GC quantitation of cholesterol and cholesterol oxide content of aortic tissuefrom atherosclerosis-susceptible Japanese quail fed experimental diets 1 595.1 Composition and energy content of diets fed to quail 1745.2 Fatty acid profile of diets fed to quail 175VIIITable Page5.3 Plasma cholesterol and triacyiglyceride concentrations in atherosclerosis-susceptible Japanese quail fed experimental diets 1 785.4 Antioxidant enzyme activities in red blood cells of atherosclerosis-susceptible Japanese quail fed experimental diets 1805.5 Antioxidant enzyme activities in heart tissue of atherosclerosis-susceptible Japanese quail fed experimental diets 1815.6 Antioxidant enzyme activities in liver tissue of atherosclerosis-susceptible Japanese quail fed experimental diets 1 825.7 Antioxidant enzyme activities in aortic tissue of atherosclerosis-susceptible Japanese quail fed experimental diets 1 845.8 Basal glutathione content of heart and liver homogenate and red blood cellsof atherosclerosis-susceptible Japanese quail 1 855.9 Heart and liver homogenate susceptibility toH20-induced GSH depletionand TBARs production in atherosclerosis-susceptible Japanese quailfed experimental diets 1 865.10 Aortic plaque score and area covered in atherosclerosis-susceptibleJapanese quail fed experimental diets 1905.11 GC quantitation of cholesterol and cholesterol oxide content of aortic tissuefrom atherosclerosis-susceptible Japanese quail fed experimental diets 1 95ixLIST OF FIGURESFigure Page1.1 Susceptibility of SHR and WKY red blood cells to in vitroH20-induced oxidative challenge 601 .2 Susceptibility of SHR and WKY heart tissue homogenate to in vitroH0-induced oxidative challenge 611 .3 Susceptibility of SHR and WKY liver tissue homogenate to in vitroH20-induced oxidative challenge 622.1 Susceptibility of red blood cells from SHR and WKY animals feddiets varying in dietary fat source and cholesterol intake level toin vitroH20-induced glutathione (GSH) depletion 912.2 Susceptibility of red blood cells from SHR and WKY animals feddiets varying in dietary fat source and cholesterol intake level toin vitroH20-induced malondialdehyde (MDA) production 932.3 Susceptibility of heart tissue from SHR and WKY animals feddiets varying in dietary fat source and cholesterol intake level toin vitroH20-induced glutathione (GSH) depletion 942.4 Susceptibility of heart tissue from SHR and WKY animals feddiets varying in dietary fat source and cholesterol intake level toin vitroH20-induced thiobarbituric acid reactive substances (TBARS) production 962.5 Susceptibility of liver tissue from SHR and WKY animals feddiets varying in dietary fat source and cholesterol intake level toin vitroH20-induced glutathione (GSH) depletion 992.6 Susceptibility of liver tissue from SHR and WKY animals feddiets varying in dietary fat source and cholesterol intake level toin vitroH20-induced thiobarbituric acid reactive substances (TBARS) production 1003.1 A representative GC-FID chromatogram of derivatized cholesterol oxidestandards with internal standard 1203.2 A representative standard curve depicting the response-linearityof derivatized cholesterol oxide standards using GC-FID 1 223.3 GC-FID chromatogram of atherosclerosis-susceptible Japanese quail aortic tissuederivatized nonsaponifiables 1 273.4 GC-FID chromatogram of Wistar rat aortic tissuederivatized nonsaponifiables 1 284.1 Susceptibility of heart tissue from atherosclerosis-susceptible Japanese quailfed diets varying in dietary fat source and cholesterol intake level toin vitroH20-induced depletion of glutathione (GSH) and productionof thiobarbituric acid reactive substances (TBARs) 1 50xFigure Page4.2 Susceptibility of liver tissue from atherosclerosis-susceptible Japanese quailfed diets varying in dietary fat source and cholesterol intake level toin vitroH20-induced depletion of glutathione (GSH) and productionof thiobarbituric acid reactive substances (TBARs) 1 524.3 A representative scanning electron micrograph of aortic tissue fromatherosclerosis-susceptible Japanese quail fed a low (0.05%) cholesterol diet 1564.4 Representative scanning electron micrographs of aortic tissue fromatherosclerosis-susceptible Japanese quail fed a high (0.5%) cholesterol diet 1575.1 Susceptibility of heart tissue from atherosclerosis-susceptible Japanese quail fed a referenceTurkey Starter (TS) diet alone, or TS diets supplemented with varying levels of beef tallowand cholesterol to in vitroH20-induced depletion of glutathione (GSH) and production ofthiobarbituric acid reactive substances (TBAR5) 1 875.2 Susceptibility of liver tissue from atherosclerosis-susceptible Japanese quail fed a referenceTurkey Starter (TS) diet alone, or TS diets supplemented with varying levels of beef tallowand cholesterol to in vitroH20-induced depletion of glutathione (GSH) and production ofthiobarbituric acid reactive substances (TBAR5) 1 885.3 A representative scanning electron micrograph of aortic tissue from atherosclerosis-susceptible Japanese quail fed the reference Turkey Starter (TS) diet alone 1915.4 A representative scanning electron micrograph of aortic tissue from atherosclerosissusceptible 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%) 1925.5 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%) 193xiLIST OF APPENDICESTable Page1. Relative retention times of TMS-sterol derivatives 2292. Response linearity of derivatized cholesterol and oxides 230xiiLIST OF ABBREVIATIONSa Water activityANOVA Analysis of varianceCAD Coronary artery diseaseCAT CatalaseCHD Coronary heart diseaseCOP Cholesterol oxidation productCVD Cardiovascular diseaseDHA Docosahexaenoic acidDTNB 5,5’-dithiobis-(2-nitrobenzoic acid)EDTA Ethylenediamine tetraacetic acidEPA Eicosapentaenoic acidFER Feed efficiency ratioFFA Free fatty acidGC Gas chromatographyGC-FID Gas chromatography with flame ionization detectorGC-MS Gas chromatography with mass spectrometryGSH Glutathione (reduced form)GSH-Px Selenium-dependent glutathione peroxidaseGSSG Glutathione (oxidized form)GSSG-Red Glutathione reductaseH2O Hydrogen peroxideHb HemoglobinHDL High density lipoproteinhr HourI DL Intermediate density lipoproteinIHD Ischemic heart diseaseLDL Low density lipoproteinXIIIMANOVA Multiple analysis of varianceMDA Malondialdehydemm MinutemLDL Modified low density lipoproteinmM MillimolarmmHg Millimetre of mercurymmol MillimoleMUFA Monounsaturated fatty acidNADPH Nicotinamide adenine dinucleotide phosphate (reduced form)nmole Nanomole102 Singlet oxygen‘OH Hydroxyl radical02’ Superoxide radicalP/S Polyunsaturated to saturated fatty acid ratioPUFA Polyunsaturated fatty acidR’ Lipid free radicalRBC Red blood cellRH Lipid moleculeROOH Lipid hydroperoxideROO’ Lipid peroxy radicalSBP Systolic blood pressuresec SecondSFA Saturated fatty acidSH R Spontaneously hypertensive ratSOD Cu/Zn Superoxide dismutaseTBARs Thiobarbituric acid reactive substancesTCA Trichloroacetic acidTG TriacyiglyceridexivTMS Trimethyl-silylTS Turkey StarterpL MicrolitreVLDL Very low density lipoproteinWKY Wistar Kyoto (rat)wt WeightxvACKNOWLEDGEMENTSI would like to express my gratitude to the many individuals who assisted me during the courseof this study. In particular, I would like to acknowledge the Department of Food Science for the useof the animal and laboratory facilities.I wish to express my sincere gratitude to my thesis advisor, Dr. David D. Kitts, Department ofFood Science, for his dedicated participation in the animal surgeries as well as in the completion of thisthesis. I am also grateful for his neverending support and encouragement throughout the course ofmy 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 forallowing me the use of his laboratory facilities to conduct the biochemical antioxidant enzyme analysesof my experiments. I am also grateful for his untiring editorial assistance in the preparation ofmanuscripts as well as during the preparation of this thesis. His friendship and comraderie outside ofthe 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 JohnVanderstoep of the Department of Food Science and Dr. Jiri Frohlich, Department of Pathology for theirsuggestions 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 wouldlike to thank Ms. Maureen Garnett and Dr. Philip Toleikis, Department of Pharmacology andTherapeutics, 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 helpin 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, Departmentof Chemistry, and Ms. Lina Madilao for allowing me the use of the gas chromatography and massspectrometry facilities as well as their invaluable help with some of trouble-shooting aspects of thisportion of my experiments. Thanks are also given to Dr. Tom Scott, Agriculture Canada ResearchStation, Agassiz, B.C. for his assistance with the pelleting of the formulated semi-synthetic quail dietsused in my experiments.I would like to acknowledge the Dairy Farmers of Canada for their support and funding of thisproject.Finally, I would like to thank my sisters Bernadette (a.k.a. Cha-cha) and Trinie and theirfamilies, and especially my mother, Mrs. Janet M. Yuan, for their good natured support andunderstanding during the course of my studies.xviINTRODUCTIONCardiovascular disease (CVD) is the greatest cause of mortalities in Canada, accounting for39% of all deaths (Heart and Stroke Foundation, 1993). Moreover, 73% of males and 90% of femaleswho die of CVD are 65 years of age and over. Risk factors for the development of CVD andatherosclerosis include: hyperlipidemia, obesity, age, hypertension, diabetes, smoking as well as aheritable genetic component. Dietary fat intake, more specifically, the relative ratio of saturated versusunsaturated fatty acids contained in fat sources, has been a primary focus in the risk for developinghypercholesterolemia 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 ofanimal origin which also contain cholesterol (beef tallow, milk and butter), have been labelled ashypercholesterolemic. Dietary lipids from vegetable and marine sources containing a high proportionof 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 ofatherosclerosis is not necessarily correlated with level of hypercholesterolemia, but rather, can occurat any given level of hypercholesterolemia (Steinberg eta!., 1989). It is noteworthy that levels ofoxidized 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 alsoatherosclerotic 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 arole 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 ofhypertension and atherosclerosis.1LITERATURE REVIEWla. Lipid oxidation: reaction mechanism and products.Lipid oxidation is characterized by a self-catalytic free radical mechanism (autoxidation; Bollandand 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 molecularoxygen are lipid hydroperoxides (ROOH; Farmer eta!., 1942). Hydroperoxides are short-lived, unstablemolecules which undergo further isomerization and decomposition to form a variety of breakdownproducts 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 oxidationreactions, 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 radicalreactions is associated with the production of desirable or undesirable flavours and odours in processedfoods (Ladikos and Lougovois, 1990; Finley and Given, 1 986), as well as reduced availability of certainco-nutrients (Nielsen et a!., 1985a,b; Cuq et a!., 1973). !n vivo, lipid peroxides of dietary orendogenous origin have been implicated in various physiological processes from aging (Halliwell andGutteridge, 1984) to chronic diseases such as atherosclerosis and cancer (Pryor, 1986; Diplock,1986). These effects of ROOH may be mediated, in part, by oxidative damage to membranes insubcellular compartments, resulting in tissue damage.Oxidation of unsaturated fatty acids via interaction of molecular oxygen with the methyleniccarbon (carbon atom neighbour to a double bond or bonds; Farmer eta!., 1942) in a PUFA follows athree-step free radical chain reaction. The process consists of initiation, propagation and terminationstages, as follows (Nawar, 1985; Bateman eta!., 1953; Bolland and Gee, 1946a,b):catalysisInitiation: RH + 02 > R , R00Propagation: R’ +°2 > R00R00 + RH > ROOH + R2Termination: R + R >R + ROO’ > Nonradical productsR00 + R00 >The interaction of unsaturated lipids with molecular oxygen tends to be quite slow for monoenes, withthe rate of reaction increasing rapidly with an increase in unsaturation (Farmer eta!., 1942). Initiationof lipid autoxidation requires catalysis to generate the initial free radical species. Catalysis occursthrough energy provided from radiant heat, transition metal ions or complexes, photochemical reactionswith 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 doublebond (-C=C-) to yield hydroperoxides (ROOH; Labuza, 1971). Once the initial ROOH is formed, theautocatalytic 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 productionof reactive intermediates from reactions such as peroxide decay and chain scission (e.g. at doublebonds; Farmer et 8/., 1942). The production of more stable chemical forms resulting from the decayof hydroperoxides (see page 2) occurs as well at this stage of the reaction. Termination of lipidautoxidation 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 positionwhich 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 andgeometrical 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 indicatorsubstances to determine the extent of lipid oxidation in food systems as well as in vivo. The mostprevalent of these methods is the determination of malondialdehyde, a dialdehyde product of PUFAautoxidation, via the 2-thiobarbituric acid (TBA) reaction using spectrophotometric (Botsoglou et a!.,1994; Buege and Aust, 1978) or chromatographic techniques (Draper eta!., 1993). Determination3of short chain alkanes, namely ethane and pentane in the breath, can also be used as an indicator oflipid oxidation in vivo (Dillard and Tappel, 1989).Lipid oxidation products are not restricted to fatty acids and triacylglycerides, but can alsoinclude sterol oxides present in the lipid system. Various cholesterol oxidation products (COPs) havebeen 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) andtemperature-abused tallow (Park and Addis, 1986a,b). Conversely, the analysis of fresh egg yolk oreggs and freshly prepared freeze-dried egg yolk powder did not indicate the presence of significantlevels 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 assinglet oxygen (102), peroxides, and the hydroxy radical (0H; Smith and Johnson, 1989).Interestingly, the superoxide radical (O2) does not oxidize cholesterol (Smith eta!., 1977). Exposureof cholesterol to lipid autoxidation intermediates during oxidizing conditions, such as extensive heatingof 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 throughthe processing of foods. The commonplace heating of cooking oils and lipid-containing foods as wellas 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 formationof lipid peroxides consumed in the diet. Numerous lipid oxides such as secondary lipid oxidation andco-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 lipidcomposition, 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 andsusceptibility to oxidation, polyunsaturated fatty acids being many times more susceptible than fatty4acids with two or one double bonds (Farmer eta!., 1942). Thus, linolenic acid (C18:3,n-3) containstwo doubly activated methylenic carbons (located between the three double bonds) at positions 11and 14 as sites for initiation of autoxidation, compared to linoleic acid (C18:2,n-6) which has onedoubly activated methylenic carbon at position 11 and oleic acid (C18:1,n-9) with its single doublebond (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 oxygenaddition at the double bond site than are their nonconjugated counterparts (double bonds separatedby 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 thanthe trans isomers, which have a more thermodynamically favoured conformation (Labuza, 1971). Thefact that fatty acid esters, such as the triacyiglycerides, are more stable to autoxidation than free fattyacid molecules is important to the stability of edible oils (Nawar, 1990). Other forms of fatty acidesters, 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 fatglobule membrane (MFGM; Chen and Nawar, 1991), can increase the rate of autoxidation due to theircharacteristic content of polyunsaturated fatty acids.Environmental conditions, such as those present during storage and processing, also have aneffect on the reactivity and stability of lipids to oxidation. The availability of oxygen as a participantin the reaction under normal conditions is not limiting, but at low oxygen pressures, such as undervacuum, the rate increases in proportion to oxygen pressure (Nawar, 1990). Temperature can alsoplay a role in determining oxygen availability. Generally, as the temperature of the lipid autoxidationreaction medium increases, so does the rate of the reaction (Farmer et a!., 1942). However, at hightemperatures, the solubility of oxygen begins to decrease, such that any increase in rate of reactiondue 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, dependingon the water activity (a) of the system (Finley and Given, 1986). Thus, in dehydrated foodscharacterized 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 radicals5and inactivating any metallic catalysts which may be present. Raising the a of a system furtherincreases the mobility of reactants, resulting in an increase in the rate of lipid oxidation (Finley andGiven, 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, andionizing 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 wellas biological tissues in both a free ion or complexed form. Lipid autoxidation reactions are catalysedby these metals through interaction with the fatty acids, hydroperoxides or by activation of molecularoxygen to yield oxygen radicals (Sherwin, 1978; Farmer eta!., 1942). The catalysis of the formationof the highly reactive hydroxyl radical (0H) by iron in the Haber-Weiss cycle (3) plays an importantrole in the initiation of lipid autoxidation in model and biological systems (Graf et a!., 1984):1. Fe +°2 > Fe3 + °22. 202 + 2H > H2O °23. Fe + H20 > 0H + OH- + Fe3Photosensitizing molecules such as hemoglobin or chlorophyll also contain transition metal ions withintheir larger complexes. These trace metal complexes frequently do not occupy all the coordinationsites of the metal ion, thereby allowing the metal to act as a catalyst of lipid autoxidation (Mahoneyand Graf, 1986; Graf eta?., 1984). These reactions are involved in the autoxidation of lipids containedin biological tissues (e.g. meat muscle; Ladikos and Lougovois, 1990; Buckley eta!., 1989) as wellas oils (e.g. soybean oil; Jung eta!., 1991). Exposure of lipids to light energy can have significantsurface effects by catalyzing oxidation of lipids, such as on the exposed surfaces of butter (Luby eta?., 1986a,b).ic. Antioxidants.Control or retardation of the rate of lipid autoxidation can be achieved through the action ofantioxidants. Lipid autoxidation antioxidants include both those naturally occurring in lipid systemssuch as the tocopherols, B-carotene, ascorbic acid and citric acid, as well as synthetic compoundswhich are added to foods to maintain the stability of the lipid component. Synthetic antioxidants6include the phenolic compounds butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),tertiary butyl hydroxyquinone (TBHQ), and propyl gallate. Metal chelating agents such asethylenediamine tetraacetic acid (EDTA) and phytic acid can also act as antioxidants by binding andeffectively removing pro-oxidant metals from the reaction medium (Empson eta!., 1991; Graf andEaton, 1990). Other types of natural, food-derived antioxidant molecules include the phenoliccompounds 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 3-carbon 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 chelatemetals make them ideal antioxidants. Thus, flavonoid compounds exhibit antioxidant activity by actingas free radical acceptors and as chain-breakers. The limited lipid solubility of plant phenols can beimproved through alkylation or esterification to long chain fatty acids or alcohols (Namiki, 1990). Thenatural and synthetic phenolic antioxidants have in common an aromatic carbon ring structure with oneor 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 compoundsenables 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 duringthe propagation stage of lipid autoxidation. The free radical chain inhibitors are able to successfullycompete with the autoxidizable lipid substrates for peroxy radicals, for example, due to the readyabstractability 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, orbe regenerated to its active form by a reducing agent (e.g. ascorbic acid). The regeneration of the7phenolic antioxidants by reducing agents illustrates the “sparing effect” that one antioxidant can haveon another, leading to a synergism, as seen in the example of a-tocopherol and ascorbic acid (KahI andHildebrandt, 1986). An additional example of synergism displayed by the various classes ofantioxidants occurs with the phenolic compounds and metal chelating agents. Chelators act todecrease the concentration of available transition metal ions, effectively removing the catalysts of freeradical production (initiation) and hydroperoxide decomposition (propagation; KahI and Hildebrandt,1986). However, in order to be effective antioxidants, chelates formed must occupy all coordinationsites of a trace metal. Any coordination sites which are left open or that are hydrated can be involvedin oxygen radical production (Graf eta?., 1984). Thus, phytic acid and large chelating organic acidssuch as diethylenetriamine pentaacetic acid have been reported to successfully prevent Fe3 fromacting as a pro-oxidant (Empson et a?., 1991; Graf et a?., 1984). In the case of vegetable oils whichare already rich in naturally occurring tocopherols, the addition of synthetic antioxidants will providelittle added antioxidant protection (KahI and Hildebrandt, 1986).id. Antioxidants in foods.Foods generally contain an endogenous complement of both pro-oxidant as well as antioxidantmolecules. 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 studiesusing model systems, Mahoney and Graf (1986) demonstrated that ascorbic acid exhibited bothantioxidant and pro-oxidant activity for Cu2-mediated 0H production. Ascorbate enhanced 0Hformation when present at low concentrations, by reducing the cuprous ion, similar to the Fe3/F2pathway above. At higher concentrations, ascorbate acted as an antioxidant by scavenging freeradicals. 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 atocopherol can reduce transition metal ions, thereby enhancing the Haber-Weiss cycle (see above, page6) and acting as a pro-oxidant (Mahoney and Graf, 1986).Despite the natural presence of antioxidants, it is frequently necessary to stabilize foods withadded or supplemental antioxidants in order to extend the shelf-life or storage-life of commodities. For8fresh muscle tissue, membrane lipids are generally rich in phospholipid and consequently have a highPUFA content, which is highly susceptible to oxidative attack (Ladikos and Lougovois, 1990; Buckleyet a!., 1989). Different sources of muscle foods can also vary in their sensitivity for development oflipid autoxidation depending upon their relative content of PUFA. Thus, beef is far less susceptible tolipid autoxidation than is chicken meat, which in turn is less sensitive than turkey meat (Ladikos andLougovois, 1990). Living tissues are usually well protected against free radical damage by in vivoenzyme systems and tissue a-tocopherol. However, once meat tissue is damaged, such as by cuttingand slicing, the protection against oxidative stress provided by intact muscle fibres andcompartmentalization of in vivo systems is lost. Differences in stability even exist between intact cutsof meat versus ground tissue which makes lean tissue lipids accessible to contact with heme catalystsof oxidative rancidity. For example, incorporation of a-tocopherol into cellular and subcellularmembranes should influence the stability of meat during storage and processing. Post-mortemapplication of vitamin E, on the other hand, has had only variable success for the inhibition of lipidautoxidation in meat tissue (Buckley eta!., 1989). Supplemental dietary a-tocopherol in the feed ofanimals (swine) for a 10 week period significantly stabilized mitochondrial and microsomal lipids ofpork (Buckley et a!., 1989).Oil seeds and grains are also good sources of antioxidants, including the lipid solubleconstituents (e.g. tocopherols, tocotrienols and B-carotene), trace minerals as cofactors of antioxidantenzymes (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-richfractions of grains contain a variety of compounds with vitamin E activity notably the a, B, y and ótocopherols and tocotrienols. The mineral content of seeds and grains depends somewhat on themineral content of the soil they are grown on. Phenolic acids are especially concentrated in the branfraction 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 toits 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 oxidativedamage can result in decreased yields, rancid off-flavours, discolouration, reduced vitamin and nutrient9content, 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 importantto the oxidative stability of the oil during purification, as well as in the finished product. In fact, theconcentration of tocopherols in finished oils (e.g. soybean oil; 1,100 ppm tocopherol) is oftensufficient to confer considerable shelf-stability (Jung eta!., 1991). However, with processing suchas extensive heating (>70 hours, 135°C), the activity of antioxidants such as a-tocopherol or ascorbylpalmitate is lost. In studies with heated tallow, Park and Addis (1986b) suggested that antioxidantsin heated oils are lost either due to their consumption by excess free radical production, or to aninability to retain functionality at elevated temperatures over a long period of time. This lability ofantioxidants in frying oils no doubt contributes to the presence of lipid oxides in heated frying fats andoils, and consequently in their fried products. Commercially prepared french-fried potatoes from fastfood 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 lipidrancidity due to their mild, delicate flavour, as in fluid milk and butter, as well as the composition ofthe 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 aresurrounded by membrane (milk fat globule membrane, MFGM; Jensen and Clark, 1988). Milk fat isunusual in its composition, in that short and medium-chain fatty acids (C4:0 to C14:0) make upapproximately 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 fattyacid residues (Jensen and Clark, 1988). Adding to the complexity of milk fat are significant amountsof odd-numbered carbon and branched-chain fatty acids. Oxidation of milk fat can be initiated byMFGM components, whose composition reflects its plasma membrane origins, containing neutral lipid,phospholipid and proteins (Keenan eta!., 1988). Thus, oxidative attack of fat globule triacylglyceridesmay be preceded by the oxidation of MFGM phospholipid (Sherwin, 1978). The MFGM contains bothantioxidant and pro-oxidant factors, with some components having a dual character depending onenvironmental conditions. The protein component of the MFGM includes various metalloproteins, some10of 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. Activityof xanthine oxidase may be partially responsible for oxidative attack of milk lipids due to its catalyticproduction of hydrogen peroxide (H20), and superoxide anion (02; Jensen and Clark, 1988). Incontrast, aqueous phase proteins adsorbed onto the surface of the MFGM, such as superoxidedismutases, behave as antioxidants to inhibit milk fat oxidation by dismutating superoxide anions toproduce 02 and H20 (Jensen and Clark, 1988). Acceleration of milk fat lipid peroxidation by theMFGM 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 attributedto antioxidative effects of membrane protein -SH groups, or the presence of Maillard reaction productswhich overcame the pro-oxidative activity of MFGM components. Other dairy products can be seento be sensitive to the photochemical catalysis of lipid autoxidation, as demonstrated by the presenceof 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 todifferent light sources (Luby et a!., 1 986a,b). Also, oxidation of cholesterol in cheese lipid has beenattributed to the catalytic effects of milk trace metals present as protein-metal complexes (Finocchiaroeta!., 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 triacylglyceridefatty acids, but may involve a recently identified conjugated linoleic acid isomer, C18:249’11. Thisconjugated 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, furtherstudies 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 theeffects of lipid autoxidation. Much of the research investigating the reaction rates and products of lipidautoxidation has been performed using model lipid systems, or purified oils high in unsaturated fattyacid content. This work allowed the elucidation of the mechanisms involved in lipid peroxidation;11however, 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 wallcomponents; membrane-bound lipids such as phospholipids). Thus, a variety of co-nutrients existwithin the same environment which can influence both the rate as well as the products of lipid freeradical interactions with oxidizable substrates. Examples of co-nutrients include proteins and aminoacids, carbohydrates, salts as well as lipid- and water-soluble vitamins. The basic side-chains ofprotein amino acid residues may be lost in aldol condensation reactions with carbonyl lipid autoxidationproducts (Nielsen et a!, 1985a,b). For example, casein has been described as having antioxidantactivity due to the fact that sulphur-containing amino acids and proteins are oxidizable by lipidhydroperoxides (McGookin and Augustin, 1991). Polymerization of proteins mediated by lipid peroxidefree radicals has also been described by Roubel and Tappel (1966). Taken together, these reactionsof lipid autoxidation free radicals and hydroperoxide breakdown products with the protein and aminoacid components of food can result in reduced protein functionality and decreased availability ofessential amino acids.The carbohydrate component of foods can have a variety of roles, ranging from catalysis ofhydroperoxide decomposition (Labuza, 1971) to exhibiting antioxidant effects through the formationof 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 acidsor peptides reacted with reducing sugars. The presence of salts such as NaCl in food systems canresult in the acceleration of the oxidation of triacylglycerides, depending on the level of free moisturepresent in the system (Love and Pearson, 1971). The fat soluble vitamins A, 0, E (tocopherols) andK as well as provitamin A (F-carotene) act as antioxidants by being oxidized by lipid hydroperoxidesand their breakdown products (Burton, 1989; Park and Addis, 1 986b). In addition, the water solublevitamins C (ascorbic acid and its lipid soluble form, ascorbyl palmitate) and folate are also susceptibleto oxidative attack (Cort, 1974). These interactions in foods are virtually unavoidable, since they willoccur 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 and12even colours of cooked and cured meats, french-fried potatoes, heated milk, aged cheeses, and theundesirable volatiles associated with oxidative rancidity, but also for alterations to the nutritional valueand 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 offoods 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 oxidationinteractions suggests that the palatability and functionality of lipid-containing foods will render themunusable before significant decreases in nutritional value are reached (Finley and Given, 1 986; Nielseneta?., 1985a).2a. Digestion and absorption of dietary lipids.Digestion and absorption of dietary fat (e.g. triacylglycerides, phospholipids, cholesterol estersand lipid-soluble vitamins) occurs primarily within the lumen of the small intestine (Guyton, 1977). Themajority of dietary lipid is freed from other dietary constituents (i.e. protein and carbohydrate) throughthe action of salivary amylases and gastrointestinal proteases. Minor amounts of fat are digested bylingual and gastric lipases; however, 95-99% of fat digestion occurs in the small intestine (FernandoWarnakulasuriya 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 theaqueous intestinal contents. Gastrointestinal peristaltic movement enhances the breakdown of the fatdroplets, effectively increasing the surface area available for enzymatic hydrolysis. The pancreaticlipases are responsible for the hydrolysis of the triacylglyceride molecules at positions one and threeof the molecule, yielding diacylglycerides (1 ,2-diacylglycerides) as well as monoacylglycerides (2-monoacylglycerides) and free fatty acid molecules. The hydrolysis of triacylglycerides is subject tofeedback inhibition from the accumulation of digestion products. Thus, bile salts play an important rolein the removal of these products via the formation of bile salt micelles. These micelles consist of anonpolar sterol core surrounded by a highly negatively charged polar surface. Monoacyiglycerides andfree fatty acids are miscible within the nonpolar core of the micelles, thus facilitating their removal13from the site of enzymatic hydrolysis. The rate of hydrolysis by pancreatic lipases is somewhatdependent on chain length and degree of saturation. Unsaturated fatty acids are hydrolysed at a fasterrate than are saturated fatty acids (SFA). The bile salt micelles containing hydrolysis products oftriacylglycerides migrate to the intestinal mucosa for absorption of the monoacylglycerides, free fattyacids and glycerol across the epithelial cell brush border membrane. The bile salts are not absorbedthrough the mucosa, but instead act as a type of “ferrying” system and are recycled back into theintestinal contents to form new micelles. The bile salts undergo enterohepatic circulation by beingrecirculated to the liver for release back into the intestinal contents. Also, a certain amount of bile acidsalt is lost in the excreta.The monoacylglycerides and free fatty acids (FFA) from the bile salt micelles become dissolvedin the epithelial cell membrane and immediately diffuse across into the cytosol. Epithelial cell lipasefurther hydrolyses the monoacylglycerides into constituent molecules. When the longer chain fattyacids are absorbed into the mucosal cells, they are re-esterified once more into triacylglyceridemolecules. 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. Thenewly synthesized triacylglycerides (TGs) coalesce into globules along with absorbed cholesterol,phospholipid and newly synthesized phospholipid. These globules, surrounded by a membrane fromthe endoplasmic reticulum, are then extruded into the intercellular spaces as chylomicrons. Shorterchain 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 cellmitochondria for energy.The chylomicrons travel along with the lymph via the lymphatic pump upward to the thoracicduct to be emptied finally into the main circulation. Thus, the chylomicrons are emptied into thevenous blood supply at the juncture of the jugular and subclavian veins. Chylomicrons have a relativelyshort residence time in the plasma, being removed in about one hour, primarily through the capillariesof the adipose tissue. Adipose cells contain high amounts of lipoprotein lipase activity to hydrolysetriacylglycerides to their constituent free fatty acids and glycerol. The FFA diffuse into the adipose14cells where they are re-synthesized into triacylglycerides for storage, until needed elsewhere. Whenthe 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 theblood with the majority of plasma lipids existing as lipoproteins. Lipoproteins are composed ofapproximately 25-33% protein with the remainder as lipid species. The lipoprotein classes consist ofthe TG-rich very low density lipoproteins (VLDL; containing high concentrations of TG with moderateamounts of PL and cholesterol), low density lipoproteins (LDL; consisting of little TG with a highamount of cholesterol) and the high density lipoproteins (HDL; containing approximately 50% proteinwith smaller concentrations of TG and cholesterol). The lipoproteins are synthesized mostly by theliver, with some species also originating from the intestine. They function in the transport of lipidspecies among the tissues, primarily from the liver to other tissues. The protein content of thelipoprotein particles consists of specific classes of apolipoproteins. These apolipoproteins in large partdictate 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 VLDLsynthesized by the liver contain apolipoprotein B-i 00, C and E. Apolipoprotein B-48 is synthesized bythe intestine and is required for chylomicron secretion. On the other hand, apolipoprotein B-100 issynthesized only by the liver and is necessary for VLDL secretion. The primary role of apolipoproteinB-100 is in the recognition of LDL particles by the LDL receptor protein of hepatocytes. Thus, LDLparticles contain only a single apolipoprotein, B-100. HDL lipoprotein particles contain apolipoproteinsA-I, A-Il, A-lV, C and E (depending upon the species of donor). For example, rat HDL contain all fiveof the above apolipoproteins, whereas human HDL lack apolipoproteins E and C (Weisgraber andMahley, 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 byboth the intestine and the liver. Apolipoprotein C consists of a complex of three polypeptides, C-I, C-Iland C-Ill, each with their own activity. Apolipoprotein C-I has activity as another activator of LCAT.15Apolipoprotein C-Il is an activator of lipoprotein lipase (LPL), whereas C-Ill acts as an inhibitor of thisenzyme activity (Norum, 1992; Weisgraber and Mahley, 1983). Finally, apolipoprotein E acts asanother ligand for the LDL receptor protein in hepatocytes.When the chylomicron has been stripped of its triacyiglyceride content through the activity ofLPL, the remaining material (consisting of surface unesterified cholesterol, phospholipids andapolipoproteins) is removed from the circulation by the liver (Norum, 1992). This removal ofchylomicron remnants from the plasma is facilitated by receptors (i.e. apolipoprotein E receptor or LDLreceptor) on the surface of hepatocytes. The formation of the TG-rich VLDL by the liver is dependenton the synthesis or availability of TG in hepatocytes. Thus, VLDL production and secretion is afunction of the balance between the influx of FFA to the liver and their oxidation for energy. The VLDLlipoprotein particles are once more subject to the hydrolysis of their triacylglycerides by LPL of adiposeand 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 LDLparticles from the VLDL remnants in the plasma. This conversion of IDL particles into the cholesterylester-rich LDL lipoproteins is facilitated by the transfer of cholesteryl esters to the precursor VLDL fromcirculating HDL particles. Some LDL cholesteryl ester may originate from direct action of LCAT withoutcholesteryl ester transfer (Frohlich and McLeod, 1987). This process allows the LDL particles to delivertheir cholesterol load to hepatocytes as required (i.e. for the biosynthesis of bile acids). Low densitylipoproteins are taken up by hepatocytes via the LDL receptor pathway which recognizes theapolipoprotein B on the surface of the LDL. This receptor population for uptake of LDL is subject todown-regulation by a saturated fat diet (Woollett eta!., 1992; Fernandez and McNamara, 1991). Adiet high in saturated fatty acids can result in reduced numbers of LDL receptors, resulting inhypercholesterolemia. 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 directlysecreted from the liver or intestine as small sized spheres, or form within the plasma as discoidalparticles developed from VLDL remnants (Norum, 1992; Weisgraber and Mahley, 1983). The newlysecreted HDL particles contain primarily apolipoproteins A-I and A-Il, phospholipids and unesterifiedcholesterol. Once in the plasma, HDL become more spherical in shape due to the catalytic activity of16LCAT 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 bya 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 VLDLin order to reconstitute their cores which have been depleted through the action of LPL. The transferof cholesteryl esters to this lipoprotein fraction is reciprocated by an equimolar transfer of TG to theHDL lipoproteins (Frohlich and McLeod, 1987).2b. Dietary lipids and dyslipidemias.The development of hypercholesterolemia cannot be solely linked to the consumption of dietshigh in animal fat, which have been traditionally labelled as saturated fat diets. A considerable bodyof evidence exists to indicate that not all saturated fatty acids (SFA) have equivalent cholesterolemiceffects (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) fattyacids do not contribute to plasma cholesterol equally. Saturated fatty acids of chain length from C4:Oto C10:0 do not have any plasma cholesterol raising effect, as shown in humans fed formula dietscontaining either butter or a short chain triacylglyceride preparation isolated from coconut oil (Hashimet a!., 1960). Similarly, when Toda and Oku (1995) fed a diet, containing shorter chaintriacylglycerides derived from coconut oil (C8:0, 26%; C 10:0, 20%) with 2% cholesterol, to Japanesequail, plasma total cholesterol and development of aortic lesions were reduced compared to birds fedcorn oil and palmitic acid. This may explain the intermediate effect of diets containing 14% butter faton plasma total cholesterol and TG in rats compared to counterparts fed palm oil, beef tallow, coconutoil 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 produceelevated plasma cholesterol levels in humans and animal models fed diets containing sources of thesefatty acids (Denke and Grundy, 1992; Hayes eta!., 1991). Palmitic acid (C16:0) has intermediateeffects on plasma cholesterol concentrations, being hypercholesterolemic in comparison to diets highin stearic or oleic acids in studies with humans fed liquid formula diets (Bonanome and Grundy, 1988).17However, in studies with baboons fed formula diets containing palmitic acid, plasma total and LDLcholesterol 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 hypercholesterolemiccompared to those fed linolenic acid (Cl 8:2,n-3). Stearic acid (Cl 8:0), a principal saturated fatty acidin animal fats (i.e. beef tallow), was found to reduce human serum cholesterol levels when substitutedfor palmitic acid (C16:0) in formulated liquid diets (Bonanome and Grundy, 1988). The neutralcharacter of stearic acid for plasma lipoprotein composition was further demonstrated in studies withpigs 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). In these studies, there were no differences betweendietary treatment groups for either fasting or postprandial concentrations of cholesterol ortriacylglyceride in total plasma or in the major lipoprotein fractions. Beef tallow not only contains asubstantial 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 dietson cholesterolemia can be attributed to the relative activities of two of the enzyme-mediatedmechanisms of longer chain saturated fatty acid metabolism. The first pathway involves the elongationof palmitic acid to stearic acid via fatty acid elongase activity and in the second, the desaturation ofstearic acid to oleic acid by theA9-desaturase enzyme. Thus, the conversion of dietary fatty acids invivo to less cholesterolemic species may play a significant role in the overall effect of dietary fatcomposition on tissue fatty acid composition and plasma cholesterol concentrations. For example, thehigh ratio of palmitic acid to stearic acid in the plasma TG of humans may be due to the rate-limitingnature of the elongation step in the elongation and desaturation conversion of C16:0 to C18:1 in fattyacid metabolism. While plasma TG levels of stearic acid did not become elevated in studies with a highstearic acid diet, levels of oleic acid did increase in plasma TG as well as cholesteryl esters in humanstudies (Bonanome and Grundy, 1988). Thus, the efficiency of the metabolic conversion of individuallonger chain SFA may influence their effect on plasma lipids.Differences in plasma LDL and HDL concentrations due to saturated fatty acid diets may includeeffects on LDL receptor populations (Fernandez and McNamara, 1991; Fernandez eta!., 1992a,b) orapolipoprotein A-I secretion (Stucchi eta!., 1 991), respectively. In studies with guinea pigs, Fernandez18and McNamara (1991) indicated that animals fed lard-based diets had higher plasma LDL levels thantheir counterparts fed PUFA corn oil. Moreover, the LDL from lard-fed animals had both lower flotationdensities as well as an increased core-to-surface component ratio, the latter parameter being areflection of greater amounts of cholesteryl ester and TG in the LDL core compared to surface proteinand phospholipid components. These alterations in LDL composition coincided with a reduction inapolipoprotein 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 changesto LDL cholesterol concentrations and biochemical composition, as well as changes to theapolipoprotein BIE receptor population resulting in decreased catabolism of LDL cholesterol in theseSFA-fed animals (Fernandez and McNamara, 1991). Similarly, when Woollett and coworkers (1992)fed saturated triacylglyceride diets (hydrogenated coconut oil) to hamsters, LDL receptor activity wasreduced compared to animals fed either a control chow diet or PUFA safflower oil. Animals fed theSEA diet exhibited both increased LDL cholesterol production rates and, consequently, increasedplasma LDL cholesterol levels (Woollett eta!., 1992). Further work investigating the effect of dietarySFA 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 anincreased LDL apolipoprotein B pool size associated with a reduced LDL fractional catabolic rate andan increased flux rate. Catabolism of LDL was related to the apolipoprotein BIE receptor populationas well as radiolabelled plasma LDL disappearance rates (Fernandez eta!., 1992a). Studies with dietscontaining different sources of SEA confirmed that these LDL metabolic parameters were influencedby 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 receptorpopulation 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 determinethe effect of SFA diets on HDL metabolism in cebus monkeys fed coconut oil diets, Stucchi andcoworkers (1991) reported that plasma total, VLDL, LDL and HDL cholesterol levels were increasedcompared to PUFA-fed counterparts. Alteration in the composition of HDL particles from SFA-fedanimals included not only greater amounts of cholesteryl ester and phospholipid, but also decreased19levels of TG and protein. These compositional changes in lipoproteins resulted in an increased corelipid to surface component ratio for HDL from SFA-fed animals. Increased HDL cholesterol was alsoaccompanied by an increase in the apolipoprotein A-I plasma pool due to increased production ofapolipoprotein A-I, as well as a decrease in the apolipoprotein A-I fractional catabolic rate. Elevationsin hepatic tissue levels of apolipoprotein A-I mRNA were also observed in SEA-fed animals (Stucchi eta!., 1991). Thus, dietary SFA can be seen to increase plasma cholesterol concentrations by influencingthe metabolism of more than a single lipoprotein class.Monounsaturated oleic acid (C18:1) is as effective as PUFA at lowering plasma cholesterolwhen substituted for saturates in a liquid formula diet (Mattson and Grundy, 1985). This change inplasma lipid profile was observed to occur without the undesirable concomitant lowering of HDLcholesterol often seen with PUFA diets (Mott eta!., 1992; Vega eta!., 1982). Other studies withwhole food diets fed to humans reported a hypocholesterolemic response with olive oil, but the plasmacholesterol lowering activity of the MUFA olive oil diet was less than that associated with a PUFA cornoil diet (Kris-Etherton et a!., 1993). Plasma cholesterolemic responses to diets high in oleic acid canvary, however, between human studies and those carried out with animal models. Bonanome andGrundy (1988) reported that when humans were fed liquid formula diets high in oleic acid (derivedfrom 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 sourcessuch as lauric and palmitic acids. However, in studies with guinea pigs fed olive oil at either 7.5% or1 5% of diet (wt/wt), plasma total and LDL cholesterol concentrations were increased compared to cornoil-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 ofhepatic apolipoprotein B receptors and a reduced LDL catabolic rate. These workers suggested thatthis 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 documentedin numerous studies in both humans (Kris-Etherton eta!., 1993; Mattson and Grundy, 1985; Vega et20at., 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 dietarysource of PUFA reported that plasma concentrations of total cholesterol were reduced in humans fedliquid formula diets (Hashim eta!., 1960). The hypocholesterolemic effect of diets high in PUFA wasdemonstrated by Vega and coworkers (1982) in humans fed liquid formula diets with 40% of caloriesprovided by either safflower oil or lard. In subjects fed the PUFA diet, plasma total, LDL and HDLcholesterol levels were decreased in comparison to SEA-fed patients (Vega eta!., 1982). Further, theratios 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 observedin 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 from0.37 to 2.1 (Mott et at., 1992). The plasma total, LDL and HDL cholesterol concentrations werereduced by the PUFA diet in non-human primates fed 40% of energy as fat (Mott eta!., 1992). Thesechanges in lipoprotein cholesterol concentrations were associated with decreases in the levels ofapolipoproteins B, A-I and E in PUEA-fed groups. Mattson and Grundy (1985) reported that plasmatotal cholesterol, LDL and HDL cholesterol concentrations were reduced in patients fed high-linoleicacid safflower oil in the diet. The percentage contributions of cholesterol and protein to lipoproteincomposition were not influenced by dietary treatment. Thus, all plasma lipoprotein constituents couldbe seen to be reduced by the PUFA dietary fat in this study (Mattson and Grundy, 1 985). When Neyand coworkers (1991) fed diets containing 16% (wt/wt) corn oil to rats, plasma total, LDL and HDLcholesterol concentrations were reduced compared to counterparts fed SFA diets. However, theplasma concentration of apolipoprotein A-I was greater in corn oil-fed animals. The reduced ratio ofHDL triacylglycerides plus cholesterol to protein in PUFA-fed animals compared to SEA-fed counterpartsindicate 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, studieswith complex whole food diets high in linoleic acid from soybean oil also significantly decreased plasmatotal and LDL cholesterol concentrations (Kris-Etherton eta!., 1993). This study also reported thatapolipoprotein B levels were reduced by the soybean oil diet. However, neither HDL cholesterol nor21apolipoprotein A-I levels were affected by the dietary treatment in this study. Similarly, other workershave 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 (highin linoleic acid), plasma total, VLDL and LDL cholesterol levels were reduced by both of these PUFAdietary lipid sources (McDonald eta?., 1989). On the other hand, HDL cholesterol levels were notaffected by the PUFA dietary treatments in this study.While studies investigating the hypocholesterolemic response to vegetable and oil seed lipidshave 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-loweringagents (Levy and Herzberg, 1995; Ikeda et a?., 1994; Garg et a?., 1988). Consumption of largeamounts of marine oils in the diet has been linked to decreased risk of cardiovascular diseaseassociated with reduced levels of plasma cholesterol, triacylglycerides and decreased plateletaggregating activity (Drevon, 1992; Garg eta?., 1988). The primary fatty acids of interest in themarine oils are the longer chain n-3 PUFA, eicosapentaenoic acid (EPA), C20:5,n-3 anddocosahexaenoic 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 longerchain n-6 and n-3 EFA, respectively (Drevon, 1992). These transformations yield products specific tothe starting precursor since mammalian cells are incapable of transforming n-3 and n-6 fatty acids intoone another. Thus, Cl 8:2,n-6 is biotransformed into y-linolenic acid, Cl 8:3,n-6 via the6-desaturaseenzyme and then elongated to dihomo-y-linolenic acid, C20:3,n-6 which is desaturated by tdesaturase to yield arachidonic acid (AA), C20:4,n-6 which is elongated to C22:4,n-6 before beingacted on by4-desaturase to yield C22:5,n-6. On the other hand, C18:3,n-3 is desaturated bydesaturase to C18:4,n-3, elongated to C20:4,n-3, desaturated to EPA, C20:5,n-3 viaA5-desaturaseand finally elongated to C22:5,n-3 before desaturation to DHA, C22:6,n-3 by4-desaturase (Drevon,1992). From these pathways, it can be seen that the ratio of n-6 to n-3 fatty acids in the diet caninfluence 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 feedingSEA or C18:2,n-6 with marine lipids (i.e. EPA, DHA) can result in alterations in the fatty acid profile22of plasma and tissue TG and phospholipids (SküladOttir eta?., 1994; L’Abbé eta!., 1991; Nalbone eta?., 1989; Garg et al., 1988). In studies with rats fed diets containing beef tallow or safflower oil, bothsupplemented with fish oil, Garg and coworkers (1988) reported that plasma lipoprotein and hepatictriacylglyceride, phospholipid and cholesteryl ester contents of arachidonic acid were reduced in theSFA + fish oil group only. Animals fed safflower + fish oil in the diet had increased levels of Cl 8:2,n-6 and AA in these same plasma and hepatic lipid fractions (Garg eta?., 1988). These alterations in thecomposition of plasma and tissue fatty acid profiles when marine oils are consumed in the diet are dueto 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 and5-desaturaseactivity effectively reduces the biotransformation of C18:2,n-6 to C20:4,n-6 (Drevon, 1992; Garg eta?., 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 A2 which is derived from n-6 precursors, such as AA, has platelet aggregatingactivity and is vasoconstrictive. Conversely, products from EPA, namely thromboxane A3 and PGI3have 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 linkedto 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 ofeffects 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 aninhibitory effect on hepatic TG synthesis, but also by increasing hepatic mitochondrial and peroxisomal1-oxidation of PUFA (Coniglio, 1992; Halminski eta?., 1991). Moreover, Drevon and coworkers haveobserved that postprandial concentrations of free fatty acids were reduced in subjects fed marine oildiets (Rustan eta!., 1993). This situation would, in turn, reduce the availability of fatty acids forhepatic triacylglyceride synthesis. Hypocholesterolemic effects of marine lipids may involve a reduction23in 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 VLDLconcentrations may be due to the reduced levels of plasma free fatty acids causing a reduction in thetransfer 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 theirinfluence on hepatic metabolism of this sterol (Levy and Herzberg, 1995; Smit eta!., 1994). WhenGarg and coworkers (1988) fed rats diets containing 20% (wt/wt) fat provided by either SFA orCl 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 hepaticcholesterol concentrations were reduced by the addition of fish oil to both diets, although the effectwith the PUFA-based diet was greater than that seen with the SFA-based diet. Short-term feedingstudies in rats fed fish oil diets to investigate hepatic bile flow and composition reported reducedplasma cholesterol and TG concentrations; however, liver cholesterol levels were not influenced inthese studies, possibly due to the short time course (14 days) involved (Smit eta!., 1994). Thesestudies in rats with chronically catheterized bile ducts reported that biliary cholesterol secretion wasgreatly 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 extentin fish oil-fed animals. More recently, Levy and Herzberg (1995) reported that in acute bile ductcannulation studies with rats fed fish oil diets not only was the volume of bile secreted increased, butthe total amounts of bile acid, cholesterol and phospholipid secreted were also elevated in animals fedfish oil compared to a corn oil diet. Thus, one of the mechanisms by which dietary fish oil acts toreduce 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, EPAand DHA, on lipid metabolism have yielded some evidence of differential effects between these twolonger chain n-3 PUFA (Ikeda eta!., 1994). In rats fed 10% (wt/wt) fat supplemented with equalproportions of EPA or DHA at 1 % (wt/wt) of the diet, plasma and hepatic cholesterol levels werereduced to a greater extent by DHA than by EPA (Ikeda et a!., 1994). Conversely, plasma24concentrations of TG were decreased to a greater extent in EPA-fed animals than in those fed DHA inthe diet. Reduction of hepatic cholesterol levels may reflect an inhibitory effect of dietary fish oil onthe 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 relateto their effects on lipoprotein composition and size as well as tissue lipoprotein metabolism (Woolletteta!., 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. InPUFA-fed animals, reduced plasma LDL cholesterol concentrations were associated with a reduced LDLcholesterol production rate and an increase in hepatic LDL receptor activity, and thus receptor-dependent LDL transport. Moreover, these workers reported that substituting PUFA for SFA in thediets 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 cholesterolconcentrations 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-to-surface component ratios, reflecting decreased LDL cholesteryl ester and increased phospholipid andprotein content of these LDL. Receptor-mediated binding of LDL to apolipoprotein BIE receptors ofhepatic membranes was two-fold greater in PUFA-fed animals compared with SEA-fed animals. Theseworkers demonstrated that this increased receptor-mediated binding was associated with an increasein the hepatic apolipoprotein B!E receptor number (Fernandez and McNamara, 1991). In further studiesto 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 apolipoproteinB pool size, and thereby a reduced plasma LDL cholesterol concentration in these animals. Theseobservations were accompanied by an increased hepatic receptor-mediated LDL fractional catabolicrate and a reduced LDL cholesterol flux rate (Fernandez et a!., 1992a). Moreover, there was asignificant correlation between hepatic apolipoprotein B/E receptor number and receptor-mediated LDLfractional catabolic rate in vivo. Thus, PUFA diets modulate plasma LDL cholesterol levels in part byaltering lipoprotein composition and receptor-mediated catabolism. Studies have also indicated that25the effect of certain PUFA, e.g. the n-3 fatty acids, on lowering plasma cholesterol is related to theratio of linoleic acid to saturates consumed (Garg eta!., 1989; Garg et a/., 1988). These workersdemonstrated that the beneficial effects of n-3 fatty acids were greater in subjects consuming mainlyanimal fats, as opposed to vegetable oils. Thus, a modest intake of n-3 fatty acids in combination withanimal fats, such as butter, may be of greater benefit than switching to mainly polyunsaturated sourcesof dietary fat (Dairy Bureau of Canada, 1990).2c. Endogenous antioxidants in vivo.The potentially damaging effects of reactive lipid peroxides in vivo are controlled byendogenous antioxidant defenses. These cellular defenses include enzymatic and non-enzymaticcomponents. Enzymes involved in the detoxification of lipid and oxygen radicals include Cu/Znsuperoxide dismutase (SOD; cytoplasm), Mn-SOD (mitochondria), catalase (peroxisomes), the seleniumglutathione peroxidase/reductase redox cycle enzymes (cytoplasm and mitochondria) and finally, thenon-selenium glutathione-S-transferases (cytoplasm). Non-enzymatic cellular antioxidants active in vivoinclude a-tocopherol, B-carotene, ascorbyl palmitate present in membranes as well as the tripeptideglutathione present in the cytoplasm (Reed, 1989). Extracellular antioxidants include plasmacomponents such as uric acid, bilirubin and carnosine (Harris, 1992a; Reed, 1989). In the absence ofadequate endogenous antioxidant defenses, the propagation of free radical reactions can lead to theoxidation of nucleophilic cellular constituents as well as the reaction of secondary lipid autoxidationproducts with nucleophilic macromolecules, such as membrane constituents, enzymes or DNA (Fragaeta!., 1989; Reed, 1989). These events can result in the disruption of cellular membranes and celldeath (cytotoxicity), ultimately leading to tissue damage. The pathological significance of lipidperoxides 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, bothyielding hydrogen peroxide and oxygen as products (Remade eta!., 1992).SOD 2O2 + 2H > H20 + 02H02 + HO2 > H2O + 02The structure of bovine red blood cell Cu/Zn-SOD has been characterized as consisting of two identical151 amino acid residue subunits with two active sites on opposite sides of the enzyme (Harris, 1 992b).26The copper ion which is situated within a deep cleft is easily displaced, whereas zinc is buried insidethe enzyme structure. Studies have shown that zinc can be replaced by other metallic cations suchas cadmium, mercury or cobalt, with only slight decreases in enzyme activity (Harris, 1992b). On theother hand, copper cannot be replaced with another cofactor. Thus, zinc deficiency has little effecton 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 H20. The SOD enzymes are sensitive to theinhibitory effects of H20 concentrations in the reaction environment (Remade et al., 1992). Someinteraction between these two forms of SOD exist, in that the Cu/Zn- and Mn-SOD forms cancompensate for each other in certain deficiency states (Harris, 1992b).Catalase (CAT) is located intracellularly within mitochondria and peroxisomes. This enzymecatalyses two types of reactions. The first involves the decomposition of H20 to water and oxygen,while the second involves the conversion of a hydroperoxide to the corresponding alcohol in thepresence of a hydrogen donor (AH2; Aebi, 1974).CAT 2H0 > 2HO + 02ROOH + AH2 > H20 + ROH + AIn the first reaction, the second molecule of H20 acts as a hydrogen donor for the deactivation of thefirst H2O. Each enzyme molecule contains four ferriprotoporphyrin groups in its structure (MW240,000). The activity of CAT varies between tissues, with the liver and kidney having the greatestamounts, and connective tissue the least. The enzyme is mainly particle-bound within organelles, butis free within red blood cells (Aebi, 1974). Furthermore, species differences have been found to existfor red blood cell (RBC) CAT activity (Godin and Garnett, 1992). For example, while human RBCs arerich 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-glutamylL-cysteinyl-glycine), possess strong antioxidant activity (Reed, 1989). The y-glutamyl linkage rendersthe GSH molecule resistant to proteases. The main role of GSH in vivo is to serve as a primary agent27for deactivating electrophilic free radicals. The antioxidant capacity of thiol groups is a function of thepercentage of the thiol groups in the anion form (Reed, 1989). Thus, while the amino acid cysteinehas a greater number of charged moieties than GSH at pH 7.5, the antioxidant capacity of GSHdominates 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% ofGSH 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 andGSH-S-transferase), cellular GSH also has a protective role to protect protein thiol groups fromoxidation, as an intracellular redox buffer (Moron et a!., 1979). The GSH tripeptide also acts as acysteine reserve when required. The mammalian liver normally has a high concentration of reducedGSH. 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 beenshown to undergo inter-organ circulation via translocation from cells into the plasma (Anderson andMeister, 1980). Metabolism of GSH within the blood is thought to involve the reduction of disulfidebonds of plasma constituents and mobilization of compounds bound by disulfide bridges to plasmaproteins. These products, in turn, are used to synthesize GSSG and low molecular weight derivativesof 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). Cellularlevels 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 indexof in vivo susceptibility to lipid autoxidation, these workers reported increased levels of thiobarbituricacid reactive substances in the intestinal mucosa of experimental animals. In studies varying theprotein content of diets fed to rats, Hum and coworkers (1992) reported an increase in hepatic GSHconcentration and total liver GSH content when dietary casein was increased from zero to twentypercent. A further increase in dietary protein to forty percent did not result in an additional increasein liver GSH content, but did reduce the relative concentration of GSH due to increased liver tissueweight (Hum et a!., 1992). These workers demonstrated a strong correlation between liver GSH28concentration and total GSH content with sulphur amino acid intake. In this study, the plasmaconcentration of free cysteine was a better predictor of liver GSH content than was plasma GSHcontent (Hum et al., 1992). This observation may be due to the sigmoidal relation between plasmaGSH turnover and liver GSH content (Hum eta!., 1992). Moreover, the plasma GSH concentration wasstrongly associated with the absolute turnover rate of plasma GSH. Fasting for 24 to 48 hours canreduce liver GSH in the rat to 50-70% of that in fed counterparts (Reed, 1989). Liver GSH contentcan be altered by variations in diurnal/circadian rhythms (Reed, 1989). This observation may be linkedto the diurnal variation in GSH metabolism (Hum et a!., 1992). Tissue concentrations of GSH aregreater 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 selenium-dependent glutathione peroxidase (GSH-Px) and glutathione reductase (GSSG-Red). These enzymesoccur in the plasma as well as intracellularly (Harris, 1992a; Paglia and Valentine, 1967). Glutathioneperoxidase catalyzes the deactivation of hydrogen peroxide or hydroperoxides using the reducing powerof glutathione (GSH).GSH-Px H20 + 2GSH > 2H0 + GSSGROOH + 2GSH > ROH + H20 + GSSGThese reactions yield oxidized glutathione (GSSG) as one of their products. The GSSG, in turn, isreduced back to its native form via catalysis by GSSG-Red and nicotinamide adenine dinucleotidephosphate (NADPH; Moron eta!., 1979; Paglia and Valentine, 1967).GSSG-Red GSSG + NADPH > 2GSH + NADPGlutathione peroxidase has been considered as a key enzyme of the in vivo antioxidant syste.m underboth normal conditions as well as oxidative stress (Remade eta!., 1992). In studies with culturedfibroblasts and human endothelial cells lower amounts of GSH-Px were required, compared to CAT orSOD, for the same level of antioxidant protection (Remade eta!., 1992). Inhibition of GSH-Px byreactive oxygen species such as 02 and hydroperoxides is possible. Enzyme activity was reduced50% in the presence of 50 pM tertiary butylhydroperoxide (Remade et a!., 1992). The enzyme isdirectly inactivated by the superoxide radical (Turrens, 1991). The activity of GSSG-Red can be29inhibited by the phenylalanine chain of insulin (Long and Carson, 1961), and increased levels of GSSGRed 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 detoxifypotential alkylating agents (Habig eta!., 1974). Glutathione-S-transferases have a high specificity forGSH as reflected by the finding that this substrate is bound with a high affinity to the active site, thusproviding an enzyme-bound glutathione thiolate anion (GS1, which is very effective for conjugateformation (Reed, 1989). These enzymes mediate the conjugation of reactive metabolic intermediatesof xenobiotics with GSH. The reaction of the -SH group of GSH with a xenobiotic to neutralizeelectrophilic sites yielding a more water-soluble product, is catalyzed by GSH-S-transferase (Habig eta!., 1974). The GSH conjugates can be metabolized further by cleavage of the glutamate and glycineresidues, followed by acetylation of the resulting free amino group of the cysteinyl residue to yield amercapturic acid for excretion (Moron eta!., 1979). Further, the glutathione-S-transferases also haveGSH-Px activity (Moron et a!., 1979). Thus, the formation of GSH adducts or other biotransformedproducts 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 modeland food systems are also common to the progression of lipid peroxidation in vivo. Thus, similar tothe situation in food systems, the in vivo concentration of free radicals is proportional to the balancebetween their formation and elimination or deactivation by enzymatic and non-enzymatic scavengers.Free radical production intracellularly can occur through a number of biochemical pathways, includingoxidases (e.g. both xanthine oxidase and glucose oxidase produce°2), flavoprotein dehydrogenases,mitochondrial electron transport proteins, cytochromeP450-dependent enzymes, as well as autoxidationof thiols and hydroquinones (Remade eta!., 1992). The production of reactive oxygen species isfacilitated by the addition of single electrons to molecular oxygen during reduction of°2 to H20 (Harris,1992b). For example, the superoxide radical is formed through the addition of a single electron tomolecular oxygen (Diplock, 1986).02 + le > °230Another pathway for superoxide radical formation is the reaction of molecular oxygen with the ferrousion in the Haber-Weiss cycle (see above, page 6; Graf eta!., 1984). The superoxide radical itself haslimited 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, yieldshydrogen peroxide. When H20 interacts with ferrous ion, the reaction yields the highly reactivehydroxyl radical (OH). The ‘OH radical can then abstract another electron from a neighbouringcompound, such as an unsaturated lipid, to initiate a cascade of lipid autoxidation reactions. Singletoxygen (102), another reactive form of oxygen is produced when°2 absorbs energy, such as inphotochemical reactions involving photons of light. This species of oxygen radical is more selectivein 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°2 and H2O,favouring the generation of the highly reactive hydroxylradical 0H (Zamora eta!., 1991; Leibovitz eta!., 1990; Fraga eta!., 1989). Other sources of lipidautoxidation products occurring in vivo include various exogenous contributions, namely the diet, airpollution and cigarette smoke. These are examples of agents which can exert oxidative stresses onbiological systems. Normally, the compartmentalization of cellular oxidative and antioxidantcomponents 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 disruptionof cell membranes due to the increased polarity of lipid autoxidation products. Thus, membranephospholipid components will undergo degradation due to the polar -OOH group of lipid hydroperoxidesmoving out of the relatively nonpolar environment within the fluid membrane structure. Otherdetrimental effects of oxidative stress in vivo may involve specific biological processes.Malondialdehyde (OHC-CH2-C O), a secondary product of PUFA autoxidation, is capable of crosslinkingproteins, inactivating enzymes and interacting with cellular DNA (Addis, 1986). Fraga and coworkers(1989) demonstrated decreased protein synthesis in rat liver tissue exposed to oxidative stressmediated by halogenated compounds. Furthermore, aging may be the result of cumulative oxidativeevents throughout the lifetime (Simic, 1991; Diplock, 1986). Development of lipofuscin age-pigmentsin older animals is due to the condensation of carbonyl lipid autoxidation products with basic protein31amino acid residues, similar to Maillard reaction products (Pryor, 1986; Reddy etal., 1973). Damageto 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 alsobeen suggested in reference to the elucidation of disease mechanisms (Steinberg et al., 1989). Thelink between age and the development of chronic diseases such as cancer and atherosclerosis isreflected 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 washypothesized to play a major role in the etiology of atherosclerosis (Nordoy and Goodnight, 1990; LipidResearch Clinic Program, 1984). One of the hallmark events in atherogenesis is the appearance oflipid-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 onefactor of many, as there is considerable variation in the expression of atherosclerosis at any given levelof 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 dueto studies performed with cell cultures of foam cell precursors. Monocytes and macrophagesincubated in the presence of high concentrations of native (unoxidized) LDL fail to transform into foamcells (Goldstein et a!., 1979). Moreover, the macrophage LDL receptors are down-regulated in thepresence of elevated levels of LDL (Goldstein et a!., 1979). As well, endogenously inducedhypercholesterolemia was only minimally atherogenic, compared to studies in which commercialcholesterol was fed to subjects resulting in severe atherosclerosis (Addis and Park, 1989). Studies onthe acute response of rabbits to the intravenous administration of various COPs indicated the presenceof 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;32Phase 2. Propagation: accumulation of plaque material;Phase 3. Termination: thrombosis or spasm leading tomyocardial infarction, cardiac muscle damage,or death.From the above mechanism, the progression of atherosclerosis-related events appears to be similar tothose of lipid autoxidation outlined previously (see page 2). Thus, it may be more than coincidentalthat lipid autoxidation is thought to be involved in the progression of this disease process. The primaryevent in the development of atherosclerosis as defined by this pathway is a “response to injury.” Thisis in contrast to the “cause and effect” hypothesis which focuses on the role of dietary fat in diseasedevelopment. 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. Low density lipoproteinscontaining COPs or other lipid oxidation products (LOPs) are referred to as modified LDL (mLDL).Oxidized lipid species have been identified in the plasma lipoproteins and aortic plaque ofatherosclerotic patients (Rosenfeld eta!., 1990; Stringer eta!., 1989). Moreover, several cholesteroloxides have been identified as having cytotoxic, angiotoxic, carcinogenic and mutagenic activity (Smithand Johnson, 1989; Peng eta!., 1992). In contrast, native cholesterol has not been found to beangiotoxic, as have some COPs (Hessler et a!., 1979). Specifically, 25-hydroxycholesterol andcholestane-3I,5a,6f-triol have been found to be particularly toxic to cultured rabbit aortic smoothmuscle cells (Peng eta!., 1978). The COPs may be exerting an effect in vivo through inhibition ofprotein (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 andother leucocytes at the site of injury (Addis, 1990). Circulating monocytes are mobilized to these sitesdue to the bioactivity of mLDLs as strong chemoattractants (Quinn eta!., 1987). The chemotacticactivity of the mLDL has been found to reside in the lipid component, namely lysolecithin (Steinbergeta!., 1989). Once the monocytes have arrived at, and adhered to, the damaged tissue site of thearterial endothelium, they begin to migrate into the arterial intima (the layer of cells below theendothelium). At this time, the monocytes are converted into phagocytic macrophages. Themechanism of this transformation is believed to involve the stimulative activity of the highlyimmunogenic mLDL (Steinberg et a!., 1989). The transformation of macrophages into foam cells33involves the uptake of lipid and cholesteryl esters. Macrophages within the arterial walls caninternalize mLDL, and thus their constituent COPs, by two mechanisms. The first involves theengulfment via phagocytosis of immune complexes of autoantibodies binding mLDL (Steinbrecher andLougheed, 1992). The second route consists of the “Scavenger Pathway” involving an acetyl LDLreceptor 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 LDLreceptors of macrophages. Proliferation of foam cells derived from macrophages and smooth musclecells 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 lipidcontributes to the progressive tissue damage in atherogenesis. Response to tissue injury involvesplatelet 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 therelease of platelet-derived growth factor (Addis, 1990). Finally, extensively raised plaques whichocclude the arterial lumen can be observed. At this stage of the disease, complications can arise, suchas 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 tothe blockage of blood flow during the period of ischemia itself, but rather, the reperfusion phase thatfollows (Bulkley and Morris, 1986). During reperfusion, the tissue undergoes oxidative stress inducedby the sudden restoration of blood circulation and thus, availability of molecular oxygen. It has beensuggested that ischemic injury is a result of the rapid proteolytic conversion of xanthine dehydrogenaseto xanthine oxidase (XOD) at the beginning of the ischemic period (Bulkley and Morris, 1986). Anaccumulation of the enzyme’s substrate, hypoxanthine, via adenine nucleotide degradation also occursat this time. Thus, production of an excess of reactive oxygen species, namely°2’ H20 and ‘OH.can occur upon the restoration of oxygen during reperfusion. These cytotoxic oxygen metabolites canpromote 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 toreperfusion (Bulkley and Morris, 1986). Similarly, the size of myocardial infarcts was shown to bedecreased in canines treated with allopurinol (a specific inhibitor of XOD; Bulkley and Morris, 1986).342f. Oxidized lipid species in plasma.Low levels of lipid peroxides have been reported in human plasma lipoproteins of children, withincreasing 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 absorptionof lipid oxides from the diet (Emanuel eta!., 1991; Steinberg eta!., 1989). Absorption of dietary lipidoxides has been demonstrated in both animal studies (Peng et a!., 1 987; Peng et a!., 1982), as wellas in human trials (Emanuel eta!., 1991). Dietary lipid oxides absorbed across the intestinal mucosaare then incorporated into chylomicrons for transport via the lymph to peripheral tissues as are nativedietary lipids. The profile of post-prandial plasma COPs will reflect those contained in the diet. Thegreater polarity of COPs, relative to sterol esters or triacylglycerides, suggests that they are likely tobe situated within the surface monolayer of plasma lipoproteins. This would allow rapid transfer ofCOPs 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 temporalpattern of absorption for other dietary lipids (Addis, 1990).The in vivo oxidation of lipoprotein lipids, e.g. low-density lipoproteins, requires the initiationof peroxidation of the constituent PUFA. This can be precipitated by the formation of lysolecithin(lecithin from which a fatty acid moiety has been cleaved) by the action of phospholipase A2, and thesubsequent formation of LDL fatty acid peroxides (Steinberg eta!., 1989). Fragments produced fromthe scission of lipid peroxides can then covalently attach to apolipoprotein B, thereby masking the Eamino 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 modelsystems and various food systems, has resulted in queries as to the appropriateness of promoting theconsumption of these lipid sources in modulating the development of atherosclerosis. This is especiallytrue given the concern towards the involvement of lipoprotein oxidized lipid species in the genesis ofatherosclerosis. Enhanced in vivo and in vitro tissue lipid peroxidation has been reported in animalsfed PUFA diets, particularly those containing marine oil PUFA (SküladOttir eta!., 1994; De Schrijver35et a?., 1992; L’Abbé et a!., 1991; Hu et a!., 1989). The majority of these studies evaluated in vivoand tissue levels of thiobarbituric acid reactive substances (TBARs). This assay, although widely usedas an index of lipid peroxidation in animal studies, is regarded as being relatively non-specific in naturesince not only does malondialdehyde (a lipid peroxidation product of PUFA) react with TBA, but otheraldehydes (e.g. 4-hydroxynonenal, hexanal, propanal) will also react with the TBA reagent (Draper eta!., 1993). In an attempt to circumvent this problem, Hu and coworkers (1989) quantitatedconjugated 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 conjugateddienes 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 peroxidationin liver tissue from corn oil-lard-fed rats compared to the fish oil group of animals. These results clearlyreflect the influence of dietary lipid treatment on tissue fatty acid content, and thereby, products oflipid peroxidation. For example, the production of hexanal results only from the breakdown of lipidperoxides 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 fattyacids have multiple nonconjugated double bonds. Further analysis of data from this study revealed asignificant inverse correlation of tissue TBARs, conjugated clienes and total volatiles with the log ofdietary vitamin E content (Hu eta!., 1989). Dietary PUFA and vitamin E contents can influence/n vitroestimates of lipid peroxidation in animal models fed different lipid sources. When L’Abbé andcoworkers (1991) fed menhaden oil to rats over 16 weeks, while total and HDL cholesterol levels werereduced, indices of in vivo lipid peroxidation such as urinary and tissue TBARs were increased andtissue antioxidant enzymes (e.g. superoxide dismutase) were reduced compared to groups fed dietshigh in oleic or linoleic acid. In studies with rats fed beef tallow and fish oil blends, De Schrijver andcoworkers (1992) reported that excretion of urinary malondialdehyde, assayed as TBARs, waselevated when diets containing greater than 1 .8% fish oil were fed to rats. Highly polyunsaturateddietary fatty acids may contribute to oxidative stress in vivo not only due to their relative instabilityto oxidation, but also to effects on endogenous antioxidant enzyme systems. This effect of PUFA invivo is even more evident when systems are challenged with oxidizing agents (Sküladóttir et a!., 1 994)36or cancer inducing agents (Kuratko eta!., 1994). When animals fed fish oil diets were challenged witha free radical generator (methyl ethyl ketone peroxide) in vivo, tissue lipid peroxides and hepatic GSHlevels 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 longerchain PUFA from fish oil-fed rats. Administering an inducer of colon carcinogenesis to rats fed fish oildiets increased liver microsomal levels of TBARs in young animals only (Kuratko eta!., 1994). In thisexperiment, the liver showed susceptibility to microsomal lipid peroxidation which was influenced bydietary lipid source and animal age (Kuratko eta!., 1994).In contrast to the above reports of consistently increased in vivo susceptibility to oxidation fromdietary longer chain PUFA, such as those from marine oils, data relating to the oxidative susceptibilityof 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 andextent of oxidative changes, was inversely correlated with the ratio of oleic to linoleic acid content ofthese particles. Conversely, this ratio was positively correlated with the lag period preceding lipidperoxidation of these LDL particles. Interestingly, despite the fact that these LDL were collected fromvitamin E deficient subjects, they were less susceptible to oxidation than control LDL. The LDL fromvitamin E deficient patients not only contained less vitamin E, but also lower amounts of cholesterylesters 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 tooxidation. When Frankel and coworkers (1994) examined the effect of dietary fish oil supplementationon the oxidative stability of LDL from hypertriacylglyceridemic humans, despite differences in theprofiles of volatiles released from samples from subjects fed fish oil or control diets, the total amountof volatiles was not different. The two groups of LDL samples did not differ in susceptibility tooxidation, although oxidation products were distinct between the two groups of samples (Frankel eta!., 1994).Oxidation of LDL in vivo has been investigated by a variety of methods, includingimmunoassays (Palinski eta!., 1990; Rosenfeld eta!., 1990; Palinski eta!., 1989) and electrophoresis37with chromatographic techniques (Jialal et a!., 1991). Antibodies generated against lipid oxidationproduct-LDL component conjugates (Palinski et al., 1 990) recognize and bind to atherosclerotic lesionsfrom tissue obtained from diseased rabbits (Rosenfeld eta!., 1990; Palinski eta!., 1989). Also, LDLextracted from atherosclerotic lesions was recognized by an antiserum against malondialdehydeconjugated LDL. Moreover, autoantibodies against MDA-LDL conjugates have been observed in serumfrom humans and rabbit animal models (Palinski eta!., 1989). Further evidence of a function for theseautoantibodies against LDL conjugates was reported by Steinbrecher and Lougheed (1992) in studiesinvestigating 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 largeaggregates, 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 asto the extent of oxidation (assessed by degree of apolipoprotein B fragmentation). In association withthe variation in degree of LDL oxidation, uptake by cultured macrophages appeared to be scavengerreceptor-independent (Steinbrecher and Lougheed, 1992). Thus, uptake of mLDL by cells of thearterial intima can also be attributed to phagocytosis of autoantibody-mediated immune complexes ofmLDL. In studies to investigate the susceptibility of LDL to in vitro copper-catalyzed oxidation, Jialaland 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 thisexperiment by in vitro or macrophage modification was 7-ketocholesterol. Preparations of LDL fromdifferent normolipidemic individuals proved to be variable in their susceptibility to induced oxidation.Thus, while a considerable amount of information has been obtained from investigating thesusceptibility of LDL to in vitro transition metal-catalyzed oxidation, several of the parametersmeasured 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 lipidsystems is well recognized, the oxidative stability of PUFA in food systems and in vivo is considerablymore variable due to the presence of pro-oxidants, antioxidant mechanisms and co-nutrients or otherbiomolecules. A large amount of work has been performed over the years to investigate the linkbetween dietary lipid source and plasma lipid profiles involved in the genesis and progression of38cardiovascular disease. In recent years, it has become increasingly clear that while dietary fat sourcemay influence plasma cholesterol concentrations, the latter is not the sole determinant of thedevelopment of atherosclerosis. Rather, in vivo oxidative status as related to the presence of mLDLand oxysterols within plasma and aortic lesions may be more relevant to the risk of development ofheart disease. Thus, the influence of oxidative stress (e.g. from the diet) on endogenous antioxidantstatus (enzymatic and non-enzymatic mechanisms) may play a role in the disease process. Previousworkers have attempted to elucidate the impact of PUFA and cholesterol from dietary lipid sources onindices of in vivo oxidation and susceptibility to atherosclerosis; however, data generated from thesestudies remains somewhat equivocal.The overall purpose of this thesis is to examine the effect of dietary fat sources varying inproportions of saturated (short-chain versus long-chain) and polyunsaturated (n-6 versus n-3) fattyacids and dietary cholesterol level on plasma lipids and antioxidant status in animal models known tobe susceptible to the development of hypertension (spontaneously hypertensive (SHR) rat) andatherosclerosis (atherosclerosis-susceptible Japanese quail).39CHAPTER 1Antioxidant 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 mid1960’s, CVD (ischemic heart disease and stroke) remains the number one cause of mortalities amongCanadian adults (Heart and Stroke Foundation (HSF), 1993). In 1990, approximately 40% of deathsin 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 anindependent 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, actingsynergistically to increase the risk of atherogenesis. For example, the combination of hypertension andhyperlipidemia is known to potentiate the risk for development of CVD in humans (Dzau, 1 990) andexperimental animal models (Yamori eta!., 1976, 1975).The spontaneously hypertensive rat (SHR) developed by Okamoto and Aoki (1963) has beenused as an animal model for human essential hypertension. Efforts to elucidate the underlyingbiochemical mechanisms for the development of hypertension in the SHR have included theexamination of endothelial membrane abnormalities and associated disturbances in ion transport acrosscell membranes (Sagar eta!., 1992; Wu eta!., 1 990). Disturbances in mineral metabolism (i.e. calciumand magnesium) have also been reported to be associated with the development of hypertension inthe SHR (Kitts et a!., 1992; Jones et a!., 1988). While atherosclerosis is difficult to induce throughdietary means in the rat, studies conducted with the SHR have reported the appearance of ring-likeaortic lipid deposits in hyperlipidemic animals fed a highly atherogenic diet (Yamori eta!., 1975). Theselipid deposits within the arteries of hyperlipidemic SHR were observed to occur at branch points of thevessels and were associated with increased endothelial permeability (Yamori et al., 1975).Increasing clinical and epidemiological evidence indicates that the association betweenhypertension and atherosclerosis may be related to the in vivo oxidative status of plasma lipidcomponents and tissues (Gey eta!., 1 991; Stringer et al., 1989). Several studies investigating the role40of oxygen-derived free radicals in CVD have reported enhanced lipid peroxidation and reducedendogenous antioxidant capacity in tissues from patients exhibiting various CVD risk factors (Buczynskieta!., 1993; Jayakumari eta!., 1992; Sagar eta!., 1992; Hunter eta!., 1991). Elevated levels of lipidperoxides have been observed in platelets and red blood cells (RBCs) from patients with coronary heartdisease (CHD; Buczynski eta!., 1993; Jayakumari eta!., 1992). Moreover, these products of lipidoxidation are associated with decreased activities of tissue antioxidant enzymes, such as superoxidedismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) in CHD patients (Buczynski eta!., 1993; Jayakumari eta!., 1992). Also, hypertension has been reported to coincide with increasedproduction of reactive oxygen species (ROS) as well as decreased levels of the antioxidant tripeptideglutathione (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 hypertensionand atherosclerosis.Rodent animal models used in the study of the influence of CVD risk factors such ashypertension and hyperlipidemia on the development of atherosclerosis have included rabbits (Manthaet a!., 1 993), mice (Capel and Dorrell, 1 984) and various strains of rats (Mon eta!., 1 993; Yamori eta!., 1975). Rabbits fed an atherogenic, high cholesterol diet exhibited reduced RBC SOD and GSH-Pxactivities and an increase in RBC CAT activity (Mantha eta!., 1993). On the other hand, activities ofSOD, CAT and GSH-Px were increased in the aorta from high cholesterol-fed rabbits. Some of thesechanges in tissue antioxidant activities in rabbits fed the atherogenic diet were subsequently observedto be reversed with the addition of vitamin E to diets, indicating a protective effect of thisnonenzymatic antioxidant against in vivo oxidative injury (Mantha eta!., 1993). The SHR has beenreported 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 tobe a risk factor associated with atherosclerosis in humans (Dzau, 1990). Furthermore, the heart of theSHR shows greater vulnerability to membrane lipid peroxidation (Toni eta!., 1992).These reports indicate that animal model studies are of value for investigating the interactionof endogenous antioxidant status with CVD risk factors, namely hypertension and dyslipidemia, in thedevelopment of atherosclerosis. However, the majority of the preceding studies were performed with41animals fed highly atherogenic diets without a control group to indicate the antioxidant status of theanimal model on a standard, basal non-atherogenic diet. Thus, further characterization of the in vivoendogenous antioxidant capacity of the SHR on a standard chow diet could potentially contribute tothe elucidation of the role of oxidative status in the development of hypertension, as one risk factorof CVD.42Hypothesis for Chapter 1:Animal models with genotypic differences in CVD risk factors exhibit characteristic differencesin plasma lipid profile and endogenous antioxidant status.Obiective for Chapter 1:To determine whether differences in plasma lipids and endogenous antioxidant status existbetween 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 measuringspecific 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 lipidmetabolism between SHR and WKY rats.43Materials and Methods:Animals:Six-week old, male spontaneously hypertensive rats (SHR) and their normotensivecounterparts, Wistar Kyoto (WKY) rats (Charles River, Montreal, PQ) were fed a standard commercialchow diet (Ralston Purina) for an eight week period. Bomb calorimetry (Miller and Payne, 1959) ofthe chow diet indicated a gross energy content of 16.19 kJIg. All animals were individually housedin 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 animalsbefore blood pressure measurements are performed. Animals had access to distilled deionized waterad ilbitum. Daily feed intake and weekly body weight gain of animals were routinely recordedthroughout the experimental period. Animals were cared for in accordance with the principles of theGuide 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 aftera 1-week training period, to confirm the presence of hypertension in the SHR animals. Measurementswere taken between 1 3:00 and 16:00 hr in conscious rats using an indirect tail-cuff method (HarvardApparatus Ltd., South Natick, MA; Kitts eta!., 1992). Each recorded value represents the mean ofthree 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 anovernight fast, by exsanguination under halothane anaesthesia. Blood was collected into heparinizedtubes for plasma separation at 1000 x g for 5 mm, 4°C. The aortic tree (the brachiocephalic arteriesto their bifurcations and the aorta to the iliac branching) was dissected out, opened longitudinally andexamined under a 10-30X dissecting microscope to confirm the absence of lesions in animals fed abasal non-atherogenic diet. Tissues (heart and liver) were collected into chilled 50 mM Tris 0.1 mM44EDTA, pH 7.6 homogenizing buffer. Aliquots of plasma were analyzed for total cholesterol (Siedel eta!., 1983), triacylglyceride (Ziegenhorn, 1975), and phospholipid (Takayama etal., 1977; BoehringerMannheim, Laval, P0). Red blood cells (RBCs) were washed twice with isotonic (0.9%) saline foruse in biochemical assays. Hemolysates were prepared by diluting RBCs 1:10 with double distilled H20and freeze-thawing 3X in dry-ice/acetone to ensure complete cell disruption. Hemoglobin content ofRBC hemolysates was assayed according to the method of Drabkin and Austin (1935). Heart and liverwere blotted dry, weighed and prepared as 10% homogenates in fresh, chilled homogenizing bufferusing 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 byultracentrifugation at 105,000 x g, 1 5 mm, at 4°C using a Beckman L2-65 ultracentrifuge with an SW4OTi 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 6Bspectrophotometer (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) wasmeasured according to the method of Moron and coworkers (1979) with minor modifications.Aliquots (200 pL) of tissue homogenate diluted 1:2 with cold 0.9% NaCI - 2 mM NaN3 were treatedwith lOOpL ice-cold 25% Trichloroacetic acid (TCA; Sigma, St. Louis, MO) followed by centrifugationat 1 2,000 x g, 4°C, for 5 mm. The supernatant was assayed for acid-soluble sulfhydryl groups at 41 2nm 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 thereaction started with 4OpL cold 3mM DTNBin 0.1 M phosphate buffer, pH 8.0. The absorbance wasread 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 - 2mM NaN3 and centrifuged (12,000 x g, 4°C, 5 mm) followed by a secondwash with 0.9% NaCl - 2 mM NaN3. The pelleted RBCs were then lysed with 50 pL cold doubledistilled H20, followed by the addition of 325 pL cold 5% TCA- 1 mMNa2-ethylenediamine tetraacetic45acid (EDTA), vortexed and centrifuged as above. The cold supernatant, 120 yL, was assayed in 1 .04mL 0.1 M phosphate buffer, pH 8.0 and the reaction started with 40 pL cold 3 mM DTNB. Theabsorbance was read at 41 2 nm after 5 minutes. RBC and tissue GSH contents were calculated fromstandard 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 tissuehomogenates incubated with various concentrations of hydrogen peroxide (H20) prepared in 0.9%NaCI - 2 mM NaN3, followed by determination of acid-soluble sulfhydryl groups as well as 2-thiobarbituric acid reactive substances (TBARs), an indirect measurement of lipid peroxidation.Aliquots (200 pL) of tissue homogenates were incubated with an equal volume of variousconcentrations of H20 (0, 0.05, 0.1, 0.2, 0.3, 0.5 and 1 .0 mM H20 in 0.9% NaCI - 2 mM NaN3) for30 minutes at 37°C. The reaction was terminated by the addition of 100 pL cold 25% TCA andcentrifuged (12,000 x g, 4°C, for 5 mm) to obtain supernatant for use in the determination of acid-soluble sulfhydryl groups as above.Production of TBARs in tissue homogenates following incubation with H20 was determinedaccording to Buege and Aust (1978) with modifications. Aliquots of homogenate (400 pL) wereincubated with an equal volume of various concentrations of H20 (1.0, 1 .5, 2.0, 3.0 and 5.0 mMH20 for heart and 1 .0, 5.0, 10.0, 15.0, 20.0, 30.0 and 40.0 mM H20 for liver, in 0.9% NaCI - 2mM NaN3) for 30 minutes at 37°C. The reaction was terminated by 400pL cold 28% TCA -0.1 MNa-arsenite followed by centrifugation at 1 2,000 x g, 4°C for 5 minutes. An 800 pL aliquot ofsupernatant was mixed with 400pL 0.5% 2-thiobarbituric acid (TBA; Sigma) in 0.025 M NaOH andheated in a boiling water bath for 1 5 minutes. When the tubes were cooled, the absorbance was readat 532 nm.The oxidation of intracellular RBC acid-soluble sulfhydryl group content in response toincreasing concentrations of H20 was measured as follows: aliquots of packed RBCs (50 pL) werepreincubated as 10% suspensions in 0.9% NaCI - 2 mM NaN3 for 5 mm at 37°C. At the end of thepreincubation period, 500 pL of various concentrations of cold H20 (0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5and 1 .0 mM H20 in 0.9% NaCI - 2 mM NaN3) were added to the RBCs, followed by incubation at4637°C for 30 mm. The reaction was terminated by centrifugation (12,000 x g, 4°C, 5 mm) and theRBCs washed with fresh 0.9% NaCI - 2 mM NaN3 and centrifuged again as before. The packed RBCswere lysed with 50 pL double distilled H20 for determination of acid-soluble sulfhydryl groups usingDTNB as described above.Susceptibility of RBCs to in vitro peroxidation was determined by the production ofmalondialdehyde (MDA) and measured according to the method of Stocks and Dormandy (1971) withmodifications as reported by Gilbert and coworkers (1984). Aliquots of packed RBCs (5OpL) werepreincubated as 1 0% suspensions in 0.9% NaCI - 2 mM NaN3 for 5 mm at 37°C. At the end of thepreincubation period, 500pL of various concentrations of cold H20 (1.0, 2.0, 3.0, 4.0, 5.0, 10.0 and20.0 mM H20 in 0.9% NaCI - 2 mM NaN3) were added to the RBCs and incubated at 37°C for 30mm. 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 with500pL 0.5% TBA in 0.025 M NaOH and heated in a boiling water bath for 15 mm. When the tubeswere cooled, absorbances were read at 532 and 453 nm. MDA content of RBCs was calculatedaccording to the method of Gilbert and coworkers (1984). This method corrected the MDAthiobarbituric acid derivative absorbance at 532 nm for interfering absorbance due to the presence ofRBC 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. Enzyme activity was expressed as k/mg protein,where k is the first-order rate constant (sec 1) Cytosolic CAT activity was determined by incubating0.9 mL of tissue supernatant with 1 8 pL 50% diluted ethanol for 30 mm on ice. To this mixture wasthen added lOOpL 10% Triton-X100 in homogenizing buffer before vortexing thoroughly. From thissolution, aliquots of samples (500 pL for heart and 50 pL for liver) were diluted to 10 mL with 50 mMphosphate buffer, pH 7.0 immediately prior to assaying. To perform the assay, 2.0 mL of dilutedsample was added to a 3.0 mL cuvette and the reaction started by adding 1 .0 mL of freshly prepared30 mM H20 in 50 mM phosphate buffer, pH 7.0. After rapid mixing, the rate of decomposition of47H20 was determined from the change of absorbance at 240 nm. The blank for this assay consistedof 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. Samplealiquots (20 pL) were diluted to 10 mL with 50 mM phosphate buffer, pH 7.0 and assayed asdescribed above. Enzyme activity was expressed as kI g hemoglobin (Hb).iv. Glutathione peroxidase (GSH-Px) activity:Tissue GSH-Px (EC.1.11.1.9) activity was assayed according the method of Paglia andValentine (1967) and expressed on the basis of pmoles of nicotinamide adenine dinucleotidephosphate (NADPH) oxidized to NADP per minute per mg protein using the extinction coefficient ofNADPH at 340 nm of 6.22 x 106 pmole’ cm1. Tissue cytosolic GSH-Px was assayed in a 1.5 mLcuvette containing 800 pL 75 mM phosphate buffer, pH 7.0, to which was added 20 pL 60 mMreduced glutathione (GSH), 40 yL of 30 units/mL glutathione reductase (GSSG-Red) in 3 M ammoniumsulphate, 2OpL 0.12 M NaN3, 4OpL 15mM Na2EDTA and 4OpL 3mM NADPH. Aliquots of cytosolicsupernatants of samples diluted 1:2 with double strength Drabkin’s reagent (0.001 6 M KCN - 0.001 2M K3Fe(CN)6 - 0.0238 M NaHCO3) were added to the cuvette (20, 40 and 60 pL) along with doubledistilled H20 to make up a final volume of 1 .1 6 mL. The reaction was started with 40 pL 7.5 mM H20in double distilled H20 and the conversion of NADPH to NADP was monitored by continuous recordingof the absorbance change at 340 nm over 5 mm.Red blood cell GSH-Px activity was determined on RBC hemolysate diluted 1:10 with doubledistilled H20. Diluted RBC hemolysate was then further diluted 1:2 with double strength Drabkin’sreagent and assayed as above, with enzyme activity expressed as nmoles NADPH oxidized to NADPper 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 cytosolicGSSG-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 and4OpL for liver) plus double distilled H20 to make up the volume to 1.16 mL. The reaction was started48with 40 pL 3 mM NADPH and the absorbance at 340 nm was recorded over 5 mm to monitor theconversion of NADPH to NADP.Red blood cell GSSG-Red activity could not be determined due to previously noted limitationsencountered 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 andcoworkers (1975) and expressed as units of SOD per mg protein. One unit of SOD activity is definedas 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 byadding double distilled H2O (0.5 mL for heart and 3.5 mL for liver), 95% ethanol (0.5 mL for heart and1 .0 mL for liver) and chloroform (0.3 mL for heart and 0.6 mL for liver) to tissue supernatant andvortexed vigorously after sitting on ice. Once the supernatants were decolourized, the extractionmixtures were centrifuged at 3,000 x g, 4°C for 5 mm. The resulting supernatant was then furthercentrifuged at 1 2,000 x g, 4°C for 5 mm to remove any sediment. The assay mixture consisted of 1 .0mL 75mM phosphate buffer, pH 7.8, 200pLO.1 M Na2EDTA- 1.5 mg% NaCN, lOOpL 1.5mM NBT(stored in a dark bottle, 4°C) and various aliquots of prepared sample (0, 25, 50, 75, 100, 1 50, 200and 500 pL for both heart and liver) plus double distilled H2O to make up a final volume of 2.95 mL.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. Thelight box consisted of a five-sided wooden box painted inside with white paint with the open end ofthe same dimensions as the light fixture used to illuminate the test tubes. The tubes were vortexedvigorously before each illumination and at the end of the illumination with fluorescent light at aconstant distance and protected from any extraneous room light. The rate of inhibition of NBTreduction by superoxide generated by photoreduction of riboflavin was determined by measuring theabsorbance at 560 nm.Red blood cell SOD activity was determined on 500 pL RBC hemolysate to which was added3.5 mL double distilled H2O, 1.0 mL 95% ethanol and 0.6 mL chloroform for extraction ofcontaminating colour due to the presence of Hb. Once clarified, the extract was centrifuged (3,00049x g, 4°C for 5 mi n) and the supernatant used in the assay as above. Enzyme activity was expressedas 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, itwas necessary to correct tissue cytosolic enzyme activity values for any contribution due to thepresence of contaminating RBCs. For the determination of Hb content, RBC hemolysate and tissuecytosolic fractions were assayed for Hb by the method of Drabkin and Austin (1935). RBC hemolysatewas diluted 1 :10 with double distilled H20 before assaying for Hb. The assay tubes contained 1 .0 mLeach of 2.4 mM KCN and 1 .8 mM K3Fe(CN)6and the reaction was started by the addition of 0.5 mLdiluted RBC hemolysate (plus 0.5 mL double distilled H20) or 1.0 mL tissue supernatant for a finalvolume 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 0.05 significance level. Linearregression analysis (SPSS) was performed to examine associations between tissue antioxidantparameters.50Results:Animal growth and systolic blood pressure:Initial body weights of SHR animals (range 11 6 ± 1 g) were lower (p < 0.05) than for WKYcounterparts (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 greaterfinal body weights for SHR (data not shown). Systolic blood pressure of animals measured at 13weeks of age indicated that SHR were hypertensive compared to WKY animals (Table 1.1). Asexpected, there were no visible signs of atherosclerosis in aorta from SHR or WKY rats when examinedusing a dissecting microscope.Plasma lipid profiles:Both plasma total cholesterol and triacylglyceride concentrations were lower (p < 0.05) inSHR animals compared to WKY counterparts (Table 1.2). However, plasma phospholipidconcentrations were not different between animal strains.Red blood cell and tissue antioxidant enzymes:i. Red blood cell (RBC) antioxidant enzymes:In the RBCs, the activities of CAT and SOD were both greater (p < 0.05) in the SHRcompared 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 waspositively correlated with RBC SOD activity (r = 0.636, p = 0.026) and RBC SOD activity was foundto be positively correlated with systolic blood pressure (r = 0.709, p = 0.049) for SHR and WKYanimals.ii. Heart antioxidant enzymes:Heart tissue from both the SHR and WKY animals had very low levels of CAT activity. Whenthe heart CAT activity was subsequently corrected for the contribution due to presence ofcontaminating RBCs in the cytosol prepared from heart tissue homogenate, CAT activity associatedwith heart cytosol alone was negligible (values not reported). Values for SOD activity in the heart weregreater (p < 0.05) in SHR compared to WKY counterparts (Table 1 .4). Activities of GSSG-Red andGSH-Px in the heart were lower (p 0.05) and unchanged, respectively, in SHR compared to WKY51Table1.1BodyweightgainandsystolicbloodpressureofSHRandWKYratsfedastandardchowdiet1.Parameter:Bodywt.gain2FeedefficiencySystolicblood(g)ratio3pressure(mmHg)Animalstrain4:SHR155±2*0.199±0.003*197±12*WKY97±60.133±0.004132±14Valuesrepresentmean±SEM,n=8.2Bodyweightgainedfrom6to14weeksofage.Feedefficiencyratio=bodyweightgained/feedconsumedfrom6to14weeksofage.SHR=Spontaneouslyhypertensiverat,WKY=WistarKyotorat.*denotesasignificant(p0.05)animalstraindifferencewithinacolumn.aiTable1.2PlasmalipidsofSHRandWKYratsfedastandardchowdiet1.Plasmalipids:(mmol/L)TotalcholesterolTriacylglyceridesPhospholipidsAnimalstrain2:SHR1.46±0.03*0.335±0.032*1.17±0.06WKY1.75±0.080.640±0.0311.28±0.06Valuesrepresentmean±SEM,n=8.2SHR=Spontaneouslyhypertensiverat,WKYWistarKyotorat.*denotesasignificant(p0.05)animalstraindifferencewithinacolumn.01 0Table1.3AntioxidantenzymeactivitiesinredbloodcellsofSHRandWKYratsfedastandardchowdiet.’AntioxidantEnzyme2:CATGSH-PxSOD(k/mgHb)(nmolesNADPH/min/mgHb)(U/mgHb)Animalstrain3:SHR0.061±0.002*60.0±1.65.64±0.34*WKY0.052±0.00252.0±4.04.20±0.231Valuesrepresentmean±SEM,n=8.2CAT=catalase,k=first-orderrateconstant(sec1);GSH-Px=glutathioneperoxidase;SOD=superoxidedismutase.SHR=Spontaneouslyhypertensiverat,WKY=WistarKyotorat.*denotesasignificant(p0.05)animalstraindifferencewithinacolumn.C,,Table1.4Antioxidantenzymeactivitiesinheart tissueofSHRandWKYratsfedastandardchowdiet.1Antioxidantenzyme2:GSSG-RedGSH-PxSOD(nmolesNADPH/(nmolesNADPH/(U/mgprotein)mm/mgprotein)mm/mgprotein)Animalstrain3:SHR19.6±0.6*114±828.1±0.9*WKY30.5±0.2110±520.8±1.11Valuesrepresentmean±SEMIn=8.2GSSG-Red=glutathionereductase;GSH-Px=glutathioneperoxidase;SOD=superoxidedismutase.SHR=Spontaneouslyhypertensiverat,WKY=WistarKyotorat.*denotesasignificant(p0.05)animalstraindifferencewithinacolumn.01animals (Table 1 .4). Heart SOD activity was positively correlated with systolic blood pressure in SHRand 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 tissuewere greater (p 0.05) in the SHR compared to WKY animals (Table 1 .5). Liver GSH-Px and GSSGRed activities were found to be negatively correlated (r = -0.682, p = 0.015) for SHR and WKYanimals. Liver CAT and GSSG-Red activities were found to be positively (r = 0.781, p = 0.022) andnegatively (r = -0.798, p = 0.018) correlated, respectively, with systolic blood pressure in SHR andWKY 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 levelsof acid-soluble sulfhydryl groups as GSH were consistently greater (p < 0.05) in tissue collected fromSHR animals when compared to WKY counterparts (Table 1.6). Heart GSH was found to be negativelycorrelated with heart GSSG-Red activity (r = -0.801, p = 0.002). Liver GSH was negativelycorrelated with liver GSH-Px activity (r = -0.708, p = 0.010). Also, liver GSH was positivelycorrelated with systolic blood pressure in SHR and WKY animals (r = 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 andliver homogenates) and RBCs were exposed to varying concentrations of hydrogen peroxide (H20)in vitro and the depletion of GSH recorded. In addition, heart and liver TBARs and red blood cell MDAproduction was also evaluated as an indicator of susceptibility to in vitro lipid peroxidation. Theprofiles 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 WKYanimals at concentrations of 0.4 and 0.5 mM H2O (Figure 1.1A). However, at the higherconcentrations of H20 used to evaluate the production of MDA as an indicator of lipid peroxidation56Table1.5AntioxidantenzymeactivitiesinlivertissueofSHRandWKYratsfedastandardchowdiet1.Antioxidantenzyme2:CATGSSG-RedGSH-PxSOD.(k/mgprotein)(nmolesNADPH/(nmolesNADPH/(U/mgprotein)mm/mgprotein)mm/mgprotein)Animalstrain3:SHR0.426±0.040*69.3±6.0*118±11*278±19WKY0.318±0.04093.9±8.3202±24236±241Valuesrepresentmean±SEM,n8.2CAT=catalase,k=first-orderrateconstant(sec’);GSSG-Red=glutathionereductase;GSH-Px=glutathioneperoxidase;SOD=superoxidedismutase.SHR=Spontaneouslyhypertensiverat,WKY=WistarKyotorat.*denotesasignificant(p0.05)animalstraindifferencewithinacolumn.C,,Table1.6TissueglutathionelevelsinSHRandWKYratsfedastandardchowdiet1.TissueGSH2:RedbloodcellsHeartLiver(nmoles/mgRBC)(nmoles/mgtissue)(nmoles/mgtissue)Animalstrain3:SHR1.47±0.04*1.98±0.02*5.36±0.12*WKY1.28±0.031.76±0.093.94±0.311Valuesaremean±SEM,n8.2GSH=glutathioneSHRSpontaneouslyhypertensiverat,WKY=WistarKyotorat.*denotesasignificant(p0.05)animalstraindifferencewithinacolumn.01in RBCs, the hypertensive rats exhibited greater (p 0.05) MDA production at the addedH2Oconcentrations of 3-5mM (Figure 1.1B).ii. Tissue susceptibility to in vitro oxidative challenge:Depletion of GSH in heart tissue homogenate was not different between animal strains (Figure1.2A). On the other hand, SHR animals had greater (ps. 0.05) levels of induced TBARs productioncompared to WKY counterparts at added H20 concentrations of 1 .5 - 3.0 mM (Figure 1 .2B). Livertissue homogenate from SHR animals had a greater (p.0.05) susceptibility to GSH depletion atlevels of added H20 of 1 .0 and 5.0 mM compared to WKY animals (Figure 1 .3A). However,production of TBARs in liver tissue homogenates exposed to varying concentrations of H20 was notdifferent between SHR and WKY animal strains (Figure 1.38).590 0, 4, 0 SFigure1 .1SusceptibilityofSHRandWKYredbloodcellstoinvitroH20-iriducedoxidativechallenge.(A)Depletionofglutathione(GSH);(B)Productionofmalondialdehyde(MDA).*indicatesasignificant(p0.05)animalstraindifference.=SHR;A=WKY.C 0 4, 4,100 80 60 40 20 00.000.100.200.300.400.500.60mMH202added700600500400300200100 0 0.005.0010.0015.0020.0025.00mMH202added0) 01000.50*C 0 4- 4, 0. 4,.mMH202addedmMH202addedFigure1.2SusceptibilityofSHRandWKYhearttissuehomogenatetoinvitroH20-inducedoxidativechallenge.(A)Depletionofglutathione(GSH);(B)Productionofthiobarbituricacidreactivesubstances(TBARs).*indicatesasignificant(p0.05)animalstraindifference.=SHR;A=WKY.B80 60 40 20 0C’,LO0.380.250.130.000.000.250.500.751.001.250.001.002.003.004.000) -C 0 4- 0 0. C VFigure1.3SusceptibilityofSHRandWKYlivertissuehomogenatetoinvitroH20-inducedoxidativechallenge.(A)Depletionofglutathione(GSH);(B)Productionofthiobarbituricacidreactivesubstances(TBARs).*indicatesasignificant(p0.05)animalstraindifference.A=SHR;A=WKY.100 80 60 40 20 00.001.002.003.004.005.006.00mMH202added(.4 to2.001.501.000.500.000.0010.0020.0030.0040.0050.00mMH202added0) F’.)Discussion:Clinical and epidemiological studies investigating the interaction of CVD risk factors such ashypertension, smoking and dyslipidemia are suggestive of a role for in vivo lipid peroxidation in theinitiation of atherosclerosis (Buczynski et al., 1993; Jayakumari et a!., 1992; Sagar et a!., 1992; Geyeta)., 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 tomacromolecules such as oxidized LDL (Wu eta!., 1990). The SHR exhibits several characteristics thatsuggest the involvement of reactive oxygen species (ROS) and lipid peroxidation in the developmentof 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) andred 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 astandard 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 weeksof age is consistent with previous findings in both our laboratory (Kitts et a!., 1992) and others(Janero and Burghardt, 1988; Jones et a!., 1988). Plasma lipids (i.e. total cholesterol andtriacylglycerides) were lower in chow-fed SHR animals compared to WKY counterparts. Mon andcoworkers (1993) reported a similar result with SHR and WKY fed extremely high levels of saturateddietary fat (25% suet) and cholesterol (5% cholesterol, 2% cholate) in the diet. Elevated plasmatriacylglycerides 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 andWKY rats in the present study likely represent differences in lipid metabolism between these two inbredstrains of rat. It is generally recognized that the initiation of atherosclerosis as a result of cholesteroldeposition 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 isconsistent with the relative resistance of this animal species to atherosclerosis.63RBC and tissue antioxidant status:Previous workers have reported several differences in the endogenous antioxidant statusbetween 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 andSOD activities in RBCs compared to WKY animals, while the activity of the glutathione metabolizingenzyme, GSH-Px, was not different between animal strains. The increased activity of CAT in SHRRBCs may indicate up-regulation of this enzyme’s activity, possibly to compensate for the increasedproduction of H20generated from SOD activity. The positive correlation of the activities of these twoenzymes in the RBCs would be consistent with such a possibility. Increased RBC SOD activitysuggests a greater requirement for antioxidant enzyme protection against the production of ROS andin vivo lipid peroxidation in SHR animals.The antioxidant capacity of the SHR myocardium is of special interest, given the previousreports of hypertrophy of this tissue in the SHR (Kashli et a!., 1977). While heart tissue activities ofCAT were below the level of detection, SOD activity was greater in SHR than WKY. This resultparalleled the situation in SHR RBCs above. This finding may indicate a generalized compensatoryantioxidant enzyme response to enhanced levels of ROS production in SHR tissues. Previous workershave reported greater heart GSH-Px activity in SHR at 6 weeks of age and lower activity in SHR at 1 6weeks of age (Ito eta!., 1992). The results of the present study in 14 week old animals are likelyintermediate in the temporal pattern of GSH-Px activity. While the activity of heart tissue GSH-Px wasnot 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 activityis 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 andRBCs. Liver tissue activities of CAT were greater in SHR compared to WKY, while the activity of SODwas not different. Both liver GSH-Px and GSSG-Red activities were reduced in SHR compared to WKYanimals, with GSH-Px activity negatively correlated with liver tissue GSH content, suggesting a64protective effect of the greater levels of nonenzymatic antioxidant GSH content against tissue lipidperoxidation 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 depletiondid not differ between animal strains, but, production of TBARs was greater in SHR animal heart tissuecompared to WKY. Similar findings of enhanced susceptibility of SHR myocardium to lipid peroxidationhave been noted by others using a variety of in vitro oxidation systems, such as Fe2/ascorbate (Itoeta!., 1992), xanthine oxidase (superoxide radical production; Janero and Burghardt, 1989) or in vivoadministration of doxorubicin, a cardiotoxic drug (Toni eta!., 1992). In all these cases, formation oflipid peroxidation products (i.e. TBARs or MDA) was enhanced in SHR heart tissue or isolatedmembrane preparations. The antioxidant enzyme analyses in the present study indicate that thegreater susceptibility of the SHR myocardium to oxidative damage may be due to the presence ofhigher levels of H20 resulting from the greater SOD activity in the SHR heart, in the presence ofvanishingly low levels of CAT activity.When RBCs were subjected to similar in vitro forced peroxidation conditions, depletion of GSHwas lower in SHR animals, while production of MDA, at higher levels of pro-oxidant was enhanced inSHR RBCs. These seemingly contradictory results can be explained on the basis that at the lowerconcentrations of H20 used in the GSH depletion assay, the elevated levels of GSH in the RBCs of theSHR were sufficient to inactivate the peroxidizing agent. The greater SOD activity in RBCs from SHRanimals (as an in vivo source of H20), could contribute to the greater production of MDA in RBCs fromSHR animals.While both the heart and RBCs from SHR demonstrate a greater susceptibility of these tissuesto lipid peroxidation, in vitro oxidative challenge of liver tissue was quite different from these twotissues. Depletion of liver tissue GSH was greater in SHR animals than WKY, despite the greater levelof tissue GSH in SHR animals. On the other hand, production of TBARs from liver tissue was notdifferent between these two animal strains. The lower activities of the GSH metabolizing enzymes inSHR liver tissue may be responsible for the enhanced depletion of GSH observed in the SHR. Thereduced activity of GSH-Px for inactivating peroxides, in association with reduced regeneration of GSH65reducing equivalents by GSSG-Red enzyme activity, would be consistent with the enhanced GSHdepletion observed in the liver tissue of SHR animals. In contrast, the higher concentrations of H20used in the TBARs assay not only exhausted the supply of GSH in the liver tissue, but may also haveinactivated antioxidant enzymes by binding to enzyme protein moieties resulting in similar productionof TBARs in liver tissue from SHR and WKY.The endogenous antioxidant status has been similarly investigated in clinical andepidemiological studies with subjects exhibiting various risk factors of CVD, including hypertension andatherosclerosis. Hypertensive patients have been reported to have reduced levels of ascorbic acid aswell as SOD activity in diseased aortae compared to normal tissue from normotensive controls, andit has been suggested that the reduced antioxidant capacity of aortic tissue in hypertensive subjectsmay 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, indicatingpossible 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 PMNchemiluminescence (a measure of ROS production) was increased in hypertensive patients (Sagar eta!., 1992). These workers also showed that levels of GSH and SOD activity in PMN were bothinversely correlated with the degree of hypertension in these patients. Conversely, in the presentstudy, 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 inthe SHR.More recently, Buczynski and coworkers (1993) reported that platelets from patients withcoronary artery disease (CAD) exhibited increased aggregability (which may predispose to atheromaformation) in association with increased levels of cellular TBARs, while platelet activities of theantioxidant 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 inpatients with CAD, and the extent of elevation of plasma lipid peroxides appeared to increase withincreasing severity of CAD. Although RBC activities of CAT and GSH-Px in these patients with CADwere normal, SOD activity was decreased (Jayakumari eta!., 1992). Those patients with CAD who66were also diabetic exhibited the greatest levels of plasma lipid peroxides compared to smokers andhypertensive patients. Taken together, these findings suggest that subjects which have several riskfactors for CVD and exhibit atherosclerotic vascular changes have a reduced tissue antioxidantcapacity and thereby, increased levels of tissue lipid peroxides. In the present study, while the SHRanimal model exhibited hypertension, there were no visible signs of atherosclerosis. This finding isconsistent 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 secondaryhyperinsulinemia compared to its WKY counterpart (Hulman et a!., 1993; Finch et a!., 1990). Instudies 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 withinsufficient plasma insulin levels exhibited elevated GSSG-Red activity in red blood cells (Long andCarson, 1961). Hypertensive SHR animals exhibited reduced levels of GSSG-Red activity in hearttissue in the present study, which may be related to their relative insulin resistance andhyperinsulinemia (Hulman eta!., 1993).67Conclusion:In summary, hypertension in the SHR fed a normal (commercial rat chow) diet was associatedwith alterations in antioxidant profiles of RBC, liver and heart tissues, the latter also showing anincreased susceptibility to in vitro lipid peroxidation. The plasma cholesterol and triacylglycerideconcentrations of the SHR were lower in comparison to WKY counterparts. Underlying metabolicfactors that may directly or indirectly regulate in vivo antioxidant status and lipid metabolism appearto reflect the genotype of the hypertensive SHR animal model, compared to its normotensive WKYcounterpart. Further experiments will explore the influence of dietary lipid source and cholesterolcontent on the antioxidant status and lipidemia in hypertensive SHR and normotensive WKY rats.68CHAPTER 2Effect of saturated and polyunsaturated dietary fat sources on systolic blood pressure,plasma lipids and antioxidant status in spontaneously hypertensive (SHR) andnormotensive 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 inplasma cholesterol is associated with a 2% decrease in the incidence of IHD in middle aged men (LipidResearch Clinic Program, 1 984). Therefore, much research has investigated the role of dietary fatintake 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 thedevelopment of hypercholesterolemia (Keys eta!., 1957). Diets high in monounsaturated fatty acidsand marine oil long-chain PUFA have been found to be relatively hypocholesterolemic andhypotriacylglyceridemic, respectively (Bairati eta?., 1992; Mattson and Grundy, 1985). As well, therole of dietary cholesterol intake and its interaction with dietary fat source in determining plasma lipidlevels 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 influenceblood pressure in the treatment of cardiovascular disease (H SF, 1993). The influence of dietary fattyacid composition on blood pressure consists primarily of effects on the biosynthesis of vasoactiveagents following the elongation and desaturation of essential fatty acids. Thus, consumption of marineoils 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 theoxidative load of plasma lipid constituents and membrane lipids in vivo. Studies have indicated notonly that lipid oxidation cannot be completely inhibited by the addition of antioxidants to fish oil diets69(Gonzalez et a!., 1992), but also that tissue levels of lipid oxidation products are enhanced in animalsfed 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 intoplasma 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 isthought to play a significant role in the development of atherosclerosis. The susceptibility ofmembrane lipids and plasma lipoproteins to lipid oxidation will depend on in vivo antioxidant status asinfluenced 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 beenused in a variety of metabolic studies as an animal model of human essential hypertension. Previouslyin Chapter 1, the SHR was demonstrated to differ from the normotensive WKY rat in the activity ofseveral tissue antioxidant enzymes, as well as in the susceptibility of tissues to in vitro oxidativechallenge. Moreover, plasma lipid profiles of the chow-fed SHR were observed to differ from thoseof 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 providefurther insight into the effects of dietary fatty acid composition on cholesterolemia, hypertension andsusceptibility of tissues to lipid oxidation.70Hypotheses for Chapter 2:i. Genetic predisposition for a risk factor of atherosclerosis, namely hypertension, can bemodulated by diet.ii. Dietary fat source and level of cholesterol may have independent, or possibly interactiveeffects on hyperlipidemia and associated changes in endogenous antioxidant status in hypertensiveanimals.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 endogenousantioxidant 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 andlevels of cholesterol intake on plasma lipid profiles.ii. The relative effect of atherogenic diets on specific tissue antioxidant enzymes andsusceptibility of tissues toH20-induced glutathione depletion and lipid peroxidation in vitro will bedetermined in SHR and WKY rats.71Materials and Methods:Animals:Sixty-four, six week old, male spontaneously hypertensive rats (SHR) and sixty-fournormotensive Wistar Kyoto (WKY) rats (Charles River, Montreal, PQ) were randomly divided into eightdietary treatment groups differing in dietary fat source (i.e. butter, beef tallow, soybean oil, andmenhaden 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 andcholesterol content and are detailed in Table 2.1. Each dietary fat source was obtained in sufficientquantities of single batches from respective suppliers to ensure consistency of fatty acid compositionin 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 thoroughmixing of ingredients prior to the addition of experimental dietary fat sources and sterols. Dietary fatsources 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 thevitamin E that was present as a component of the vitamin mixture. Both the butter and tallow fatswere liquefied using a short time - mild heat (10-15 mm at 45-50°C) treatment to facilitate the uniformdistribution of cholesterol and cholic acid (2:1 ratio, to ensure maximal absorption of dietarycholesterol) into diets. No heat treatment was necessary for the soybean or menhaden oils. Sterolswere added slowly to the liquefied experimental fat sources and thoroughly mixed to ensure uniformincorporation. Dietary fats containing sterols at levels reported in Table 2.1 were slowly added to thebasal diet during reblending and mixed in completely using a Hobart mixer with an aluminum bowl overa period of approximately 20-25 minutes. After mixing, individual diets were stored in double, darkplastic bags in a walk-in freezer (-15°C) throughout the experimental study. A sample of eachexperimental diet was removed for analysis of fatty acid, gross energy and dry matter content.72Table 2.1 Composition of diets fed to SHR and WKY rats1.Cholesterol level(% by weight) 0.05 0.5Dietarycomponent (gil OOg)Casein2 25.0 25.0Ca-free mineral mix2 3.5 3.5CaCO3 2.0 2.0Vitamin mix2 3.0 3.0DL-methionine4 0.3 0.3Choline chloride5 0.2 0.2Cornstarch6 47.0 47.0Sucrose 3.0 3.0Alphacel2 5.0 5.0Monofos5 3.0 3.0Canola oil6 3.0 3.0Dietary fats:Butter, beef tallow,soybean or menhaden oil7 5.0 5.0Cholesterol2 0.05 0.50Cholic acid2 0.025 0.251 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 mattercontent of all diets ranged from 89 - 93%.2 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.6 Neptune Food Services, Richmond, B.C., Canada.Butter (Dairyworld Foods, Burnaby, B.C.); beef tallow (Cargill Foods, HighRiver, AB); soybean oil (Bioforce Canada, Burnaby, B.C.); menhaden oil(Zapata Haynie, Reedville, VA).73Experimental fats were added to the basal diet at a level of 5% to make a final calculated fatcontent of 8% dietary fat. This level of fat in the diets meets the nutritional recommendations for thelaboratory rat (eg. 5-10%) as outlined by the National Academy of Sciences (1978) and the CanadianCouncil on Animal Care (Vol.2, 1984). The amount of butter fat added to experimental diets wascalculated 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 inbutter (approx. 0.2%; Jensen and Clark, 1988) and beef tallow (approx. 0.1 %) sources. Analysis ofthe diets for cholesterol confirmed the expected uniform content of sterol in both individual lowcholesterol and high cholesterol diets. Experimental diets were isonitrogenous and contained acomparable 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 wascorrected for dry weight of diet (Miller and Payne, 1959). The energy value represents the amountof 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 BF3(Nwokolo et a!., 1988) and analyzed for component fatty acids using a Varian Model 3700 gaschromatograph 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 (Yuanand Kitts, 1 994; Kitts et a!., 1 992) was also used in this experiment. The meal feeding scheduleensures a similar postprandial time period in animals before blood pressure measurements areperformed. All diets were replaced daily to minimize autoxidation of dietary lipids during the 6 hourfeeding period.74Table 2.2 Fatty acid composition of diets fed to SHR and WKY rats.DietsFatty acid Butter Beef Soybean Menhadentallow oil oil14:016:016:1(n-7)18:018:1(n-9)1 8:1 (isomers)1 8:2(n-6)1 8:3(n-3)20:020:120:4(n-6)20:5(n-3)22:022: 5(n-3)22:6(n-3)SaturatesMonounsaturatesPolyunsaturatesn-6n-3P/Si8.425.21.29.034.61.78.63.142.737.511.78.63.10.32.921.02.512.947.82.37.52.736.852.710.27.52.70.3Area %0.39.10.24.347.63.415.32.70.40.814.152.117.915.32.71 .34.613.46.73.233.113.34.70.41.60.57.30.91.25.522.541.432.813.818.71.51 P/s = polyunsaturated fatty- denotes not detected.acid! saturated fatty acid ratio.75Blood pressure measurements:Systolic blood pressure recordings were taken using a tail-cuff sphyngomanometer (HarvardApparatus) between 15:00 - 17:00 hr in SHR and WKY rats fed semi-synthetic diets, as reported inChapter 1.Experimental procedures:After 8 weeks on the experimental diets, non-fasted animals were sacrificed by exsanguinationunder halothane anaesthesia at 1 3:00 hr. Animals were sacrificed in a non-fasted state to minimizechanges to antioxidant status due to possible stresses of fasting as well as any uncontrolled nutritionaleffect on antioxidant levels attributed to diet withdrawal. Blood and tissues (i.e. heart and liver) werecollected and prepared for plasma lipid analysis and antioxidant status, respectively, as previouslydescribed in Chapter 1.Analytical methods:Plasma lipids were quantified using the procedures given in Chapter 1. Similarly, all enzymaticand nonenzymatic antioxidant measurements made in RBC, heart and liver tissues were performedaccording to the methods described in Chapter 1.Statistics:All data are expressed as mean ± standard error of the mean (SEM). One-way analysis ofvariance (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 wasidentified 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 anddietary fat source. In order to separate treatment differences due to animal strain, dietary fat sourceor 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 cholesterollevel (0.05% versus 0.5%) or one-way ANOVA for dietary fat source (butter, tallow, soybean oil andmenhaden oil) differences. Linear regression analysis (SPSS) was performed to examine associationsbetween tissue antioxidant parameters.76Results: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 containapproximately 15% of total fatty acids as chain lengths of C:4 to C:1 2 (Kitts and Jones, 1995; Jensenand Clark, 1988). The weight percentage of myristic (C14:O) acid was greater in butter diets than indiets containing beef tallow, soybean or menhaden oils. Butter and beef tallow diets contained similaramounts of palmitic (C16:0) and stearic (C18:O) acids, which were greater than those found insoybean and menhaden oil diets. Beef tallow and menhaden oil diets contained very similar amountsof oleic (C18:1,n-9) acid, which were greater than those found in butter and soybean oil diets; theselatter two diets also contained very similar amounts of Cl 8:1. Soybean and menhaden oil dietscontained similar quantities of linoleic (Cl 8:2,n-6) acid which, in turn, were greater than in butter andbeef tallow diets. The content of linolenic (C18:3,n-3) acid was greater in menhaden oil dietscompared to butter, beef tallow and soybean oil diets. The menhaden oil diets were unique in theircontent of eicosapentaenoic (C20:5,n-3; EPA) and docosahexaenoic (C22:6,n-3; DHA) acids. On thebasis 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 andmenhaden oil diets was 1 .5. The ratio of n-6 to n-3 fatty acids was 2.8 for butter and beef tallowdiets, 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 aresummarized 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 wasnot influenced by animal strain or dietary cholesterol level. However, body weight gain was influencedby dietary fat source (p < 0.05), as animals fed menhaden oil diets gained less weight than otherdietary fat groups (Table 2.3). The feed efficiency ratio (FER) of SHR and WKY animals wasinfluenced (p < 0.05) by both animal strain and dietary fat source as demonstrated by the interactionrecorded. SHR animals had lower (p < 0.05) FER values than WKY counterparts. Animals fed77Table2.3BodyweightgainandsystolicbloodpressureofSHRandWKYfedexperimentaldiets.1BodywtgainFERSystolicblood(9)pressure2(mmHg)SHRWKYSHRWKYSHRWKYDietaryTreatment:Butter0.05%chol.168±5157±100.204±.0080.224±.008197±10147±70.5%chol.172±7154±90.198±.0080.220±.006174±4131±5Tallow0.05%chol.166±3154±70.190±.0070.220±.008187±10125±60.5%chol.167±8164±70.198±.0080.236±.008186±8140±6Soybean 0.05%chol.154±4163±100.167±.0080.228±.013180±6128±70.5%chol.156±7167±70.176±.0100.228±.009187±8134±8Menhaden 0.05%chol.131±5126±80.157±.0040.175±.012194±6133±100.5%chol.133±9137±110.162±.0090.190±.011190±6140±7BodywtgainFERSystolicbloodpressureANOVAp-value3AN.S.<0.001<0.001CN.S.N.S.N.S.F<0.001<0.001N.S.MANOVAp-value3AxFN.S.0.023N.S.Valuesrepresentmean±SEM,n=8.SHR=Spontaneouslyhypertensiverat,WKY=WistarKyotorat;FER=Feedefficiencyratio=feedintake(g)/bodyweightgained(g)during8weekfeedingstudy.Systolicbloodpressuremeasuredinanimalsat13weeksofage.A=animalstraineffect,C=cholesterolintakeleveleffect,F=dietaryfatsourceeffectby3-wayMANOVA;treatmentinteractionsby3-wayMANOVA.2 303menhaden oil diets exhibited reduced (p 0.05) FER values compared to other dietary fat groups andthis 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 lowcholesterol diets (range 12.1 ± 0.1 versus 10.1 j 0.1 g) and high cholesterol diets (19.0± 0.1versus 1 5.4 ± 0.1 g). Dietary cholesterol level, but not fat source, had a significant (p < 0.05) effecton liver weight. Heart weights of SHR (range 0.9 ± 0.1 g) and WKY (range 1.0 ± 0.1 g) were notsignificantly 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 werehypertensive (p < 0.05) compared to WKY counterparts. Systolic blood pressure was not affectedby 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 totalcholesterol concentrations were significantly (p < 0.05) affected by animal strain, dietary cholesterolintake level as well as dietary fat source (Table 2.4). SHR animals had lower (p 0.05) plasma freeand total cholesterol levels compared to WKY counterparts (Table 2.4). Also, animals fed dietscontaining 0.5% cholesterol exhibited increased (p 0.05) levels of plasma free and total cholesterol,compared to counterparts fed diets containing 0.05% cholesterol, as expected. Animals fed menhadenoil diets had reduced (p 0.05) levels of plasma free cholesterol compared to other dietary fatgroups, 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 fatsource. Also, plasma total cholesterol levels of WKY animals fed 0.05% cholesterol diets were notaffected by dietary fat source. However, in WKY rats fed 0.5% cholesterol diets, plasma totalcholesterol was greatest in tallow-fed animals and lowest in the menhaden oil-fed group. Theseobservations demonstrate the interaction recorded between animal strain, dietary fat source andcholesterol level for plasma total cholesterol concentrations. Plasma triacylglyceride levels weresignificantly (p 0.05) influenced by dietary cholesterol intake level and dietary fat source (Table79Table2.4PlasmalipidsofSHRandWKYfedexperimentaldiets.’Plasmalipids:FreecholesterolTotalcholesterolTriacyiglycerides(mmol/L)SHRWKYSHRWKYSHRWKYDietaryTreatment:Butter0.05%chol.0.76±0.060.5%chol.1.09±0.120.05%chol.0.5%chol.Soybean 0.05%chol.0.5%chol.Menhaden 0.05%chol.0.5%chol.Plasmalipids0.80±0.071.05±0.110.68±0.060.98±0.140.40±0.030.56±0.041.40±0.192.35±0.381.43+2.76+1.30+1.95+0.70+0.94+Freecholesterol0.200.440.130.310.040.111.71±0.051.98±0.051.84±0.072.01±0.111.75±0.091.89±0.091.68±0.051.92±0.132.82±0.10.4.90±0.202.98+6.04+2.96+4.14+3.04+3.68+Totalcholesterol0.090.340.090.250.080.341.33±0.071.29±0.090.96±0.060.78±0.111.38+1.03+1.11+1.07+0.46+0.43+0.120.080.100.110.050.061.27+0.88+1.50+0.96+0.52+0.41+Triacylglycerides0.150.080.090.060.050.05ANOVAp-value2A C F MANOVAp-value2AxCAxFCxFAxCxF<0.001<0.001<0.0010.0070.023N.S.N.S.<0.001<0.001<0.001<0.001<0.001<0.001<0.001N.S.<0.001<0.0010.034N.S.0.022N.S.1Valuesrepresentmean±SEM,n=8.SHR=Spontaneouslyhypertensiverat,WKY=WistarKyotorat.2A=animalstraineffect,C=cholesterolintakeleveleffect,F=dietaryfatsourceeffectby3-wayMANOVA;treatmentinteractionsby3-wayMANOVA.TallowC2.4). However, animal strain did not influence plasma triacylglyceride levels. Animals fed dietscontaining 0.5% cholesterol exhibited reduced (p 0.05) levels of plasma triacylglycerides comparedto those fed 0.05% cholesterol diets (Table 2.4). Also, animals fed menhaden oil diets had reducedplasma 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 semi-synthetic diets containing 0.05% cholesterol were elevated in comparison to values reported in chow-fed animals in Chapter 1. Moreover, both SHR and WKY animals fed low cholesterol semi-syntheticdiets containing butter, beef tallow, and soybean oil as principal fat sources, exhibited relatively greaterplasma triacylglyceride levels than chow-fed animals. The exceptions to this observation were boththe SHR and WKY animals fed the menhaden oil diets. Both animal strains exhibited uniformly reducedplasma 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 WKYcounterparts when treatment groups were pooled for dietary fat source and cholesterol level (Table2.5). 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). Soybeanoil-fed animals had higher (p.0.05) RBC SOD activity than menhaden oil-fed counterparts whentreatment groups were pooled for animal strain and dietary cholesterol level. Animals fed dietscontaining 0.5% cholesterol exhibited higher (p< 0.05) activities of RBC SOD when treatments werepooled for animal strain and dietary fat source. Activity of RBC glutathione peroxidase (GSH-Px) wassignificantly (p 0.05) affected by animal strain and dietary fat source but was not influenced bydietary cholesterol intake level (Table 2.5). SHR animals exhibited greater (p<0.05) activities of RBCGSH-Px than WKY animals when treatment groups were pooled for dietary fat source and cholesterollevel. Animals fed menhaden oil diets exhibited lower (p 0.05) RBC GSH-Px activity than otherdietary fat groups (Table 2.5). Other dietary fat source treatment effects on RBC GSH-Px activity81Table2.5AntioxidantenzymeactivitiesinredbloodcellsofSHRandWKYfedexperimentaldiets.1AntioxidantEnzyme2:CATGSH-PxSOD(k/mgHb)(nmolesNADPH/min/mgHb)(U/mgHb)SHRWKYSHRWKYSHRWKYDietaryTreatment:Butter0.05%chol.0.058±.0040.051±.00562.9±4.956.6±2.16.94±0.487.91±0.550.5%chol.0.064±.0050.052±.00457.9±1.561.1±5.16.67±0.447.33±0.29Tallow0.05%chol.0.055±.0030.054±.00566.1±1.169.2±5.86.00±0.337.15±0.270.5%chol.0.062±.0030.048±.00164.9±3.258.5±4.07.43±0.867.65±0.39Soybean 0.05%chol.0.064±.0020.048±.00165.6±4.756.6±2.46.47±0.247.66±0.380.5%chol.0.066±.0030.050±.00264.8±2.355.4+2.56.80±0.178.47±0.26Menhaden 0.05%chol.0.065±.0030.054±.00256.0±3.749.2±3.86.13±0.446.34±0.300.5%chol.0.066±.0040.056±.00458.0±3.346.3±2.76.85±0.426.75±0.26AntioxidantEnzymeCATGSH-PxSODANOVAp-value3A<0.0010.002<0.001CN.S.N.S.0.044FN.S.<0.0010.0291Valuesrepresentmean±SEM,n=8.SHR=Spontaneouslyhypertensiverat,WKY=WistarKyotorat.2CAT=catalase,k=first-orderrateconstant(sec1);GSH-Px=glutathioneperoxidase;SOD=superoxidedismutase.A=animalstraineffect,C=cholesterolintakeleveleffect,F=dietaryfatsourceeffectby3-wayMANOVA.included differences between animals fed butter and tallow diets, as well as differences betweenanimals 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 levelsof heart tissue CAT activity (values not reported) after correction for enzyme activity attributed to thepresence of contaminating RBCs in the heart tissue homogenate preparation. Heart SOD activity wassignificantly (p 0.05) influenced by dietary fat source but not by animal strain or dietary cholesterolintake level (Table 2.6). Animals fed butter and soybean oil diets exhibited greater (p 0.05) heartSOD activities than those fed menhaden oil diets when treatment groups were pooled for animal strainand dietary cholesterol intake level (Table 2.6). The activities of heart GSH-Px and glutathionereductase (GSSG-Red) were both significantly (p 0.05) influenced by animal strain and dietary fatsource. Dietary cholesterol intake did not affect either heart GSH-Px or GSSG-Red activities. HeartGSH-Px activity was lower (p 0.05) in SHR animals compared to WKY animals when treatmentgroups were pooled for dietary fat source and cholesterol level. Animals fed menhaden oil dietsexhibited 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 whentreatment groups were pooled for dietary fat source and cholesterol level (Table 2.6). Also, butter-and tallow-fed animals had greater (p < 0.05) activities of heart GSSG-Red than those fed menhadenoil 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 dietarycholesterol intake level but was not affected by dietary fat source (Table 2.7a). When treatmentgroups 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. Animals fed diets containing 0.5%cholesterol exhibited reduced (p 0.05) liver CAT activities when treatment groups were pooled foranimal strain and dietary fat source. Dietary fat source and cholesterol intake level both had asignificant (p 0.05) influence on liver SOD activities (Table 2.7a). However, liver SOD activitieswere not different between the two animal strains. Animals fed menhaden oil diets were observed to83DietaryTreatment:Butter0.05%chol.0.5%chol.TallowTable2.6AntioxidantenzymeactivitiesinheartofSHRandWKYfedexperimentaldiets.1AntioxidantEnzyme2:GSSG-RedGSH-PxSOD(nmolesNADPH/min/mgprotein)(nmolesNADPH/min/mgprotein)(U/mgprotein)SHRWKYSHRWKYSHRWKYSoybeanMenhaden20.1±0.619.2±0.9110±7.5120±6.547.9±4.152.0±5.119.2±1.020.0±1.7114±8.0110±5.851.7±7.249.4±2.90.05%chol.20.2±0.618.6±1.695.8±7.4112±8.147.6±4.747.5±4.10.5%chol.20.5±0.616.9±0.8108±11.7114±8.245.9±2.845.2±1.50.05%chol.18.5±1.617.2±0.797.9±11.5114±4.947.5±2.548.4±2.60.5%chol.18.1±1.117.2±0.4108±14.0119±7.248.1±2.956.9±5.30.05%chol.17.2±1.015.5±1.869.0±6.274.3±2.238.8±2.944.7±3.60.5%chol.17.3±2.115.0±1.681.6±16.791.7±18.840.8±4.144.1±2.5AntioxidantEnzymeGSSG-RedGSH-PxSODANOVAp-value3A0.0140.024N.S.CN.S.N.S.N.S.F0.001<0.0010.0141Valuesrepresentmean.±.SEM,n=8.SHR=Spontaneouslyhypertensiverat,WKY=WistarKyotorat.2GSSG-Red=glutathionereductase;GSH-Px=glutathioneperoxidase;SOD=superoxidedismutase.A=animalstraineffect,C=cholesterolintakeleveleffect,F=dietaryfatsourceeffectby3-wayMANOVA.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. LiverGSH-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) liverGSH-Px activities compared to WKY counterparts when treatment groups were pooled for dietary fatsource and cholesterol intake level. Also, liver GSH-Px activities were reduced (p < 0.05) in animalsfed 0.5% dietary cholesterol when treatment groups were pooled for animal strain and dietary fatsource. WKY animals fed soybean oil diets had greater (p < 0.05) liver GSH-Px activities thanmenhaden oil-fed counterparts (Table 2.7b). Activity of liver GSSG-Red was greater (p 0.05) inSHR 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 oiland 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 semi-synthetic diets in the present study showed similarities as well as some differences, which resultedin 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 straindifferences in RBC CAT activity were obtained in animals fed either rat chow in Chapter 1, or semi-synthetic diets in the present study. RBC GSH-Px and SOD activities were relatively higher in chow-fed WKY than SHR in Chapter 1, but these trends were reversed in animals fed the semi-synthetic, lowcholesterol diets in the present study. Activity of heart GSSG-Red was greater in SHR than WKYanimals fed the semi-synthetic diets which was opposite to the results obtained Chapter 1 with chowfed animals. Animals fed semi-synthetic diets in the present study, did not exhibit strain differencesfor 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-fedSHR and WKY animals, was lower in SHR fed semi-synthetic diets in the present study. Differencesbetween animal strains for liver CAT, SOD and GSH-Px activities in SHR and WKY animals fed semi85Table27aReactiveoxygenspeciesmetabolizingantioxidantenzymeactivitiesinliverofSHRandWKYfedexperimentaldiets.1AntioxidantEnzyme2:CATSOD(k/mgprotein)(U/mgprotein)SHRWKYSHRWKYDietaryTreatment:Butter0.05%chol..359±.004.283±.02799.2±5.496.8±4.50.5%chol..367±.019.225±.03585.2±4.488.3±3.8Tallow0.05%chol..375±.018.320±.03888.7±3.999.6±4.60.5%chol..332±.041.279±.02291.7±7.096.0±10.7Soybean 0.05%chol..300±.011.348±.02692.8±1 1.0102±5.60.5%chol..366±.031.248±.01795.7±1.193.9±1.0Menhaden 0.05%chol..356±.023.308±.01187.0±4.779.6±3.00.5%chol..332±.017.300±.01373.2+4.078.1±4.2AntioxidantEnzymeCATSODANOVAp-value3A<0.001N.S.C0.0450.04FN.S.<0.001Valuesrepresentmean±SEMIn=8.2CAT=catalase,k=first-orderrateconstant(sec);SOD=superoxidedismutase.A=animalstraineffect,C=cholesterolintakeleveleffect,F=dietaryfatsourceeffectby3-wayMANOVA.Table2.7bGlutathionemetabolizingantioxidantenzymeactivitiesinliverofSHRandWKYfedexperimentaldiets.1AntioxidantEnzyme2:GSSG-RedGSH-Px(nmolesNADPH/min/mgprotein)(nmolesNADPH/min/mgprotein)SHRWKYSHRWKYDietaryTreatment:Butter0.05%chol.61.3±2.652.9±1.6131±18271±250.5%chol.75.9±0.658.4±3.2135±3195±32Tallow0.05%chol.60.2±0.951.0±1.7148±4281±120.5%chol.76.3±3.258.6±3.6156±3282±8Soybean 0.05%chol.61.0±3.356.7±1.5170±9357±140.5%chol.89.2±2.060.6±1.2192±1234±41Menhaden 0.05%chol.72.8±4.358.0±5.0201±12187±110.5%chol.84.1±6.072.8±8.5174±16194±14AntioxidantEnzymeGSSG-RedGSH-PxANOVAp-value3A<0.001<0.001C<0.0010.022F0.0490.001MANOVAp-value3AxCN.S.0.014AxFN.S.<0.001CxFN.S.N.S.AxCxFN.S..005Valuesrepresentmean±SEM,n=8.SHR=Spontaneouslyhypertensiverat,WKY=WistarKyotorat.2GSSG-Red=glutathionereductase;GSH-Px=glutathioneperoxidase.A=animalstraineffect,C=cholesterolintakeleveleffect,F=dietaryfatsourceeffect;treatmentinteractionsby3-wayMANOVA.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 andopposite to the results obtained for chow-fed animals in Chapter 1. Despite these alterations in thespecific 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. Heartand liver tissue concentrations of GSH were greater (p 0.05) in SHR animals compared to WKYcounterparts (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 toother dietary fat groups when treatment groups were pooled for animal strain and dietary cholesterolintake level (Table 2.8). RBC GSH concentrations were significantly (p < 0.05) reduced in animalsfed menhaden oil diets compared to counterparts fed the other dietary fats when treatment groupswere pooled for animal strain and dietary cholesterol intake level (Table 2.8). Animal strain and dietarycholesterol 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 wellas heart and liver homogenates to increasing concentrations of hydrogen peroxide (H20) in vitro, andmonitoring the depletion of GSH. Heart and liver TBARs and red blood cell MDA production wereevaluated as further indicators of in vitro lipid peroxidation. The profiles of the in vitro forcedperoxidation of RBCs, heart and liver tissues are presented in Figures 2.1 to 2.6. As well, the resultsfrom the oxidative challenge of RBCs, heart and liver at a single concentration of H20,with treatmentdifferences 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 animalscompared to other dietary fat groups (Table 2.9, Figure 2.1A, B, C, D). However, neither animal strainnor dietary cholesterol level influenced RBC GSH depletion. Production of MDA in RBCs was influenced88Table2.8BasalglutathionelevelsinheartandlivertissueandredbloodcellsofSHRandWKYfedexperimentaldiets.1HeartRBCLiver(nmolesGSHImgtissuewetwt.)(nmolesGSH/mgRBC)(nmolesGSH/mgtissuewetwt.)SHRWKYSHRWKYSHRWKYDietaryTreatment:Butter0.05%chol.1.76±.0321.57±.0811.06±.0371.07±.0225.76±.4045.06±1250.5%chol.1.82±.0681.51±.0731.04+.0621.13±.0645.36±.2855.35+.188Tallow0.05%chol.1.80±.0711.60±.0661.07+.0331.09±0625.10±.1444.98+.3940.5%chol.1.74±.1071.63±.0531.16±.0431.16±.0526.19±.0164.72±.205Soybean 0.05%chol.1.96±.0941.66±.0251.19±.0521.20±.0315.70±.c625.70±.2800.5%chol.1.86±.0541.72±.0521.17±.0381.17±.0346.03±.0144.53±.121Menhaden 0.05%chol.1.72±.0761.54±.0781.16±.180.960±.0415.68±.3485.24±.1930.5%chol.1.70±.1371.59±.0601.06±.036.991±.0534.93±.4384.43±.172HeartRBCLiverANOVAp-value2A<0.001N.S.0.014CN.S.N.S.N.S.F0.0490.025N.S.1Valuesrepresentmean±SEM,n=8.SHR=Spontaneouslyhypertensiverat,WKY=WistarKyotorat.GSH=glutathione.2A=animalstraineffect,C=cholesterolintakeleveleffect,F=dietaryfatsourceeffectby3-wayMANOVA.CDTable2.9RedbloodcellsusceptibilitytoH20-inducedGSHdepletionandMDAproductioninSHRandWKYfedexperimentaldiets.1GSHdepletion(%)MDA(nmoles/gRBC)0.5mMH2025.0mMH202SHRWKYSHRWKYDietaryTreatment:Butter0.05%chol.54.9±3.8868.4±3.39183±11.7186±6.490.5%chol.48.0±15.070.3±5.09180±29.9148±10.3Tallow0.05%chol.64.8±4.3875.2±7.45220±5.25204±8.400.5%chol.65.8±5.4463.9±5.82176±16.8104±11.1Soybean 0.05%chol.54.6±4.8262.2±1.25222±7.00184±12.00.5%chol.55.3±1.3360.1±1.74171±3.82127±13.7Menhaden 0.05%chol.50.0±4.2553.4±4.76136±33.1181±31.40.5%chol.48.2±2.1649.8±1.79107±20.4122±21.4GSHdepletion(%)MDA(nmolesfgRBC)ANOVAp-value2AN.S.0.001CN.S.<0.001F<0.0010.003MANOVAp-value2AxF1Valuesrepresentmean±SEM,n=8.SHR=Spontaneouslyhypertensiverat,WKY=WistarKyotorat.GSH=glutathione;MDA=malondialdehyde.2A=animalstraineffect,C=cholesterolintakeleveleffect,F=dietaryfatsourceeffectby3-wayMANOVA;treatmentinteractionsby3-wayMANOVA.N.S.0.0260100g C‘CmMH202addedmMH202addedmMH202addedFigure2.1SusceptibilityofredbloodcellsfromSHRandWKYanimalsfeddietsvaryingindietaryfatsourceandcholesterolintakeleveltoinvitroH20-inducedglutathione(GSH)depletion.(A)SHR,lowcholesteroldiets;(B)SHR,highcholesteroldiets;(C)WKY,lowcholesteroldiets;(0)WKY,highcholesteroldiets.*indicatesasignificant(p..0.05)dietaryfatsourcedifference.=butter;A=beeftallow;0=soybeanoil;•=menhadenoil.g VmMH202added1000.000,250.500.751.001.25V Cg V0.000.250.500.751.001.251.25CD(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 whentreatment groups were pooled for dietary fat source and cholesterol level. Animals fed menhaden oildiets 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 (Table2.9).ii. Heart GSH derletion and TBARs production:Heart tissue GSH depletion was significantly (p 0.05) influenced by animal strain and dietaryfat source at the lower concentrations of added H20 (0.05 and 0.1 mM H20; Figure 2.3A, B). SHRanimals exhibited lower (p 0.05) depletion of heart tissue GSH than WKY counterparts at 0.05 and0.1 mM added H20 (Table 2.10; Figure 2.3A, B, C, D). Also, SHR animals fed soybean oil andmenhaden oil diets exhibited reduced (p.0.05) depletion of heart GSH at 0.1 mM added H20,compared to WKY counterparts (Table 2.10; Figure 2.3A, B, C, D). However, these treatmentdifferences were not maintained at the higher concentrations of added peroxidizing agent (Figure 2.3A,B, C, D). Production of lipid peroxidation products (TBARs) in heart tissue was significantly (p0.05) influenced by dietary fat source (Table 2.10; Figure 2.4A, B). However, animal strain anddietary 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 TBARsof animals fed menhaden oil diets were significantly (p < 0.05) lower than for other dietary fat groupsat 1-3mM of added H20 (Table 2.10). However, when the animal strains were examined individually,the dietary fat source effect was significant only in SHR animals (Figure 2.4A, B, C, D). Thisobservation confirms the significant (p 0.05) interaction recorded between animal strain and dietaryfat 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 animalstrain, 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 were92600600400I 2 C) I 2mMH202addedFigure2.2SusceptibilityofredbloodcellsfromSHRandWKYanimalsfeddietsvaryingindietaryfatsourceandcholesterolintakeleveltoinvitroH20-inducedmalondialdehyde(MDA)production.(A)SHR,lowcholesteroldiets;(B)SHR,highcholesteroldiets;(C)WKY,lowcholesteroldiets;(D)WKY,highcholesteroldiets.*indicatesasignificant(p.0.05)dietaryfatsourcedifference.=butter;A=beeftallow;0=soybeanoil;•=menhadenoil.I 2600500400300200100 03002000610152025mMH202added6005000510162025400mMH202added300200100100 0600500400300200100 006101520250010152025mMH202addedCD C,)100100C C 0 C 0.0 0.000.25mMff202addedmMH202added0,000.250.500.751.001.25mMH202addedmMff202addedFigure2.3SusceptibilityofhearttissuefromSHRandWKYanimalsfeddietsvaryingindietaryfatsourceandcholesterolintakeleveltoinvitroH202-inducedglutathione(GSH)depletion.(A)SHR,lowcholesteroldiets;(B)SHR,highcholesteroldiets;(C)WKY,lowcholesteroldiets;(D)WKY,highcholesteroldiets.*indicatesasignificant(p0.05)dietaryfatsourcedifference.=butter;A=beeftallow;0=soybeanoil;•=menhadenoil.80 60 40 200.500.761.001.501.251.50C C 0. C100 0.000.250.500.751.00100 80 60 40 20 00.000.250.500.761,001.251.601.251.50CDTable2.10HearthomogenatesusceptibilitytoH20-inducedGSHdepletionandTBARsproductioninSHRandWKYfedexperimentaldiets.’ GSHdepletion(%)TBARs(A532)0.1mMH202.0mMH20SHRWKYSHRWKYDietaryTreatment:Butter0.05%chol.46.7±5.2950.0±6.840.187±.0090.156±.0180.5%chol.52.2±3.4156.8±5.860.200±.0240.172±.017Tallow0.05%chol.43.9±3.5857.8±5.080.174±.0110.150±.0130.5%chol.48.7±4.2653.2±4.090.231±.0210.160±.008Soybean 0.05%chol.40.5±3.0052.2±3.730.161±.0070.179±.0190.5%chol.36.4±3.6340.4±3.740.182±.0370.184±.021Menhaden 0.05%chol.35.8±2.8245.9±3.250.109±.0190.166±.0240.5%chol.38.9±2.7446.4±1.520.088±.0190.132±.031GSHdepletion(%)TBARs(A532)ANOVAp-value2A<0.001N.S.CN.S.N.S.F<0.001<0.001MANOVAp-value2AxFN.S.<0.0011Valuesrepresentmean±SEM,n=8.SHR=Spontaneouslyhypertensiverat,WKY=WistarKyotorat.GSH=glutathione;TBARs=2-thiobarbituricacidreactivesubstances.2A=animalstraineffect,C=cholesterolintakeleveleffect,F=dietaryfatsourceeffectby3-wayMANOVA;treatmentinteractionsby3-wayMANOVA.Ca C,’N S 0) I. N ImMH202addedN In I-mMH202addedFigure2.4SusceptibilityofhearttissuefromSHRandWKYanimalsfeddietsvaryingindietaryfatsourceandcholesterolintakeleveltoinvitroH20-inducedthiobarbituricacidreactivesubstances(TBARS)production.(A)SHR,lowcholesteroldiets;(B)SHR,highcholesteroldiets;(C)WKY,lowcholesteroldiets;(D)WKY,highcholesteroldiets.*indicatesasignificant(p0.05)dietaryfatsourcedifference.=butter;A=beeftallow;0=soybeanoil;•=menhadenoil.AmM11202added00.500.400.300.200.100.000.500.400.300.200.100.000.500.400.300.200.100.000.500,400.300.200.100.00CIIIIN 0 In S0123456mM11202addedD •TIIII0156012345234CD 0)Table2.11LiverhomogenatesusceptibilitytoH202-inducedGSHdepletionandTBARsproductioninSHRandWKYfedexperimentaldiets.1GSHdepletion(%)TBARs(A532)0.5mMH20240.0mMH202SHRWKYSHRWKYDietaryTreatment:Butter0.05%chol.32.8±1.039.9±2.80.531±.0090.545±.0100.5%chol.27.2±3.041.6±5.10.490±.0040.289+.019Tallow0.05%chol.33.0±2.131.3±5.60.666±.1620.776±.0580.5%chol.37.4±8.964.2±0.40.436±.0260.214±.027Soybean 0.05%chol.36.0±5.839.2±4.50.654±.0460.525±.0380.5%chol.48.2±0.860.5±4.60.536±.0410.303±.052Menhaden 0.05%chol.25.5±2.232.1±2.00.240±.0700.296±.0560.5%chol.34.0±3.546.3±2.40.154±.0270.156±.032GSHdepletion(%)TBARs(A532)ANOVAp-value2A0.0110.014C<0.001<0.001F0.041<0.001MANOVAp-value2AxCN.S.0.005CxFN.S.0.0061Valuesrepresentmean±SEM,n=8.SHR=Spontaneouslyhypertensiverat,WKY=WistarKyotorat.GSH=glutathione;TBARs=2-thiobarbituricacidreactivesubstances.2A=animalstraineffect,C=cholesterolintakeleveleffect,F=dietaryfatsourceeffectby3-wayMANOVA;treatmentinteractionsby3-wayMANOVA.pooled for dietary fat source and cholesterol intake level (Table 2.11). Animals fed diets containing0.5% 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 treatmentgroups 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) influencedby dietary fat source and cholesterol intake level (Table 2.11; Figure 2.6A, B, C, D). Animals fedbutter, tallow and soybean oil diets exhibited greater (p < 0.05) levels of TBARs when liver tissuewas incubated with 1-40 mM added H20, compared to those fed menhaden oil diets (Table 2.11;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 thediet (Table 2.11). There tended to be a relatively greater influence of dietary cholesterol intake levelon liver TBARs production in WKY animals compared to their SHR counterparts as demonstrated bythe interaction (p 0.05) recorded between these two variables (Table 2.11).98100g CmMH202addedmMH202addedC CmMH202addedmMH202addedFigure2.5SusceptibilityoflivertissuefromSHRandWKYanimalsfeddietsvaryingindietaryfatsourceandcholesterolintakeleveltoinvitroH20-inducedglutathione(GSH)depletion.(A)SHR,lowcholesteroldiets;(B)SHR,highcholesteroldiets;(C)WKY,lowcholesteroldiets;(D)WKY,highcholesteroldiets.*indicatesasignificant(p0.05)dietaryfatsourcedifference.=butter;Abeeftallow;0=soybeanoil;•=menhadenoil.01234561000123456C C0123450123456CD CD1.001.00N C., C, I-0102030405001020304050mMH202addedmMH202addedI’, C,mMH202addedmMH202addedFigure2.6SusceptibilityoflivertissuefromSHRandWKYanimalsfeddietsvaryingindietaryfatsourceandcholesterolintakeleveltoinvitroH20-inducedthiobarbituricacidreactivesubstances(TBARS)production.(A)SHR,lowcholesteroldiets;(B)SHR,highcholesteroldiets;(C)WKY,lowcholesteroldiets;(D)WKY,highcholesteroldiets.*indicatesasignificant(p0.05)dietaryfatsourcedifference.=butter;A=beeftallow;0=soybeanoil;•=menhadenoil.In C, I-N 4.,In 0, I-0.800.600.400.200.000.800.600.400.200.001.000.800.600.400.200,001.000.800.600.400.200.000102030406001020304050- 0 0Discussion:The role of dietary fat as a source of saturated, monounsaturated, or polyunsaturated n-6 andlong-chain n-3 fatty acids in determining plasma lipid concentrations has been the subject of extensivestudy. It is now well recognized that while medium-chain saturated fatty acids (i.e. myristic and Iauricacids) 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; Bonanomeand 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 contrastto 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 chainpolyunsaturated fatty acids (e.g. eicospentaenoic (EPA) and docosahexaenoic acids (DHA)) are ableto reduce plasma cholesterol levels as well as having beneficial effects on hypertension and bloodclotting 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 fromvarious essential fatty acids, including thromboxanes involved in platelet aggregation andprostaglandins involved in vasodilatation (Drevon, 1992). However, the increased incorporation ofpolyunsaturated fatty acids from marine or vegetable oil dietary fat sources into plasma lipoprotein andcell 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 antioxidantmolecules, 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 dietscontaining dietary fat sources varying in degree of saturation or unsaturation (n-6 or n-3polyunsaturated fatty acids) as well as the level of cholesterol intake on the plasma lipid profile andantioxidant status of SHR and WKY rats.101Feed efficiency and plasma lipids:The consistently reduced FER in SHR fed semi-synthetic diets is similar to previous studies fromthis 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) wouldlikely result in reduced energy accretion and feed efficiency similar to that previously reported indiabetic animal models (Wohaieb and Godin, 1987a,b).A considerable body of evidence indicates that dietary fatty acid composition can influence thepartitioning 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 nowwell recognized that polyunsaturated fatty acids are oxidized more rapidly for energy than long-chainsaturated 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 anddegree of unsaturation (Leyton et a?., 1987). The short- and medium-chain fatty acids whichpredominate in butterfat (C:4to C:14, approx. 25% of total fatty acids; Jensen and Clark, 1988) aremuch more rapidly oxidized than palmitic or stearic acids and hence are neither incorporated intoplasma 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, theunsaturated 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 effectof experimental diets due to variations in substrate utilization for energy or deposition in body storesand thereby energy balance in studies using different dietary fat sources (Takeuchi eta?., 1995; Suand Jones, 1993; Shimomura eta?., 1990). While Su and Jones (1993) reported that whole bodyenergy gains differed between animals fed high-fat vegetable, animal or marine oil based diets, neitherbody weight gained nor energy expenditure differed between treatment groups. Similarly, in thepresent study, while the oxidation of the individual dietary fatty acids would differ between animalsfed the butter, tallow and soybean oil diets, the substrate utilization and energy accretion from thesediets might not be different, as demonstrated by the similar body weight gain, FER and plasma total102cholesterol and triacylglyceride concentrations between these three dietary fat sources. The decreasein body weight gained and FER of animals fed menhaden oil diets in the present study is consistentwith previous reports of reduced energy gain and energy efficiency in animals fed fish oil diets (Su andJones, 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 sourcesin studies from Jones and coworkers (Jones eta!., 1995; Su and Jones, 1 993) likely reflects the highfat content (22%) of the diets fed to animals in these studies, in contrast to the nutritionally adequatelevel of dietary fat (8%) for laboratory rats used in the present study. In other studies when rats werefed long-chain PUFAs characteristic of fish oil (i.e. EPA, DHA) in the diet, lipid utilization for energy wasdecreased (Rustan eta!., 1993). Rustan and coworkers (1993) demonstrated that reduced oxidationof long-chain n-3 fatty acids for energy was associated with reduced plasma concentrations oftriacylglyceride, phospholipids and free fatty acids. Taken together, these studies indicate that througheffects on substrate utilization for oxidation (i.e. energy) or deposition, dietary fatty acid compositioncan influence energy accretion and animal growth characteristics.The lower concentrations of plasma lipids observed in SHR animals compared to WKY animalsfed formulated semi-synthetic diets in the present study are consistent with the results from Chapter1 in chow-fed animals and those of others (Mon eta!., 1993; McGregor eta!., 1981). These strain-related differences in relative plasma lipid concentrations persisted in the present study, regardless ofthe dietary fat source or cholesterol level fed to the animals. Differences in cholesterol metabolismbetween SHR and WKY animals have been demonstrated in vitro using radiolabelled acetate precursorincorporation into cholesterol as an index of hepatic cholesterol synthesis (Iritani et al., 1977). Theseworkers reported a reduced incorporation of acetate into cholesterol in liver tissue slices from SHRanimals compared to their WKY counterparts. The relative metabolic differences underlying plasmalipid levels between SHR and WKY did not appear to be altered by feeding the formulated, semisynthetic diets in the present study as compared to the complex, commercial chow diet fed to animalsin Chapter 1. However, it is interesting to note that animals fed semi-synthetic diets containing thelow (0.05%) level of cholesterol in the present study exhibited higher plasma lipid levels as well asdifferences in tissue antioxidant status compared to those fed chow diets in Chapter 1. These103differences further substantiate the argument that the SHR and WKY animals fed chow diets in Chapter1 should only be used as reference groups for the qualitative comparison of results, rather than as truedietary treatment controls in the present study. For the purposes of the present study with SHR andWKY animals fed semi-synthetic diets containing a low or high level of cholesterol, the groups fed0.05% cholesterol in the diet are considered as controls for those fed the high (0.5%) level of dietarycholesterol.While the hypotriacylglyceridemic effect of dietary fish oil is well recognized, the effect onplasma cholesterol concentrations can be variable (Ikeda eta!., 1 994; Bairati eta!., 1992; De Schrijvereta!., 1992; Coniglio, 1992; L’Abbé eta!., 1991; Garg eta!., 1988). Mechanisms whereby dietaryfish oil exerts a plasma lipid-lowering effect include the inhibition of the synthesis and secretion oftriacylglyceride-rich VLDL particles as well as effects on fatty acid oxidation and esterification (Rustaneta!., 1993; Coniglio, 1992; Halminski eta!., 1991). The n-3 PUFA of dietary fish oil influencedtriacylglyceride metabolism via decreased mobilization of fatty acids from adipose tissue as substratesfor triacylglyceride synthesis, as well as inhibitory effects on enzymes involved in fatty acidesterification (Rustan eta!., 1993; Halminski eta!., 1991). Moreover, increased hepatic mitochondrialand 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 oftriacylglycerides for incorporation into chylomicron and VLDL particles, hence lowering postprandiallevels of plasma triacylglycerides in animals fed fish oil compared to other saturated or n-6 PUFAdietary fat sources as observed in the present study.Previous workers who have reported reduced plasma cholesterol levels when feeding fish oildiets 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 lowerlevels of fat were fed to animals, as in the present experiment, plasma total cholesterol levels were notreduced by dietary fish oil (McGregor eta!., 1981). The plasma cholesterol lowering effects of dietaryfish 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 HMGCoA reductase activity (Ikeda etal., 1994), as well as effects on bile acid flow and composition (Levy104and Herzberg, 1995; Smit et a/., 1994). In studies to compare the effects of individual fish oil n-3fatty acids on lipid metabolism, Ikeda and coworkers (1994) reported that plasma cholesterolconcentrations 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 VLDLcholesteryl ester (Rustan et a!., 1988). Dietary fish oil has been demonstrated to enhance bilesecretion (Levy and Herzberg, 1995) as well as the amount of cholesterol in bile secreted by rats (Smitet a!., 1994; De Schrijver et a!., 1992). It is plausible that fish oil n-3 fatty acids influence plasmacholesterol levels through effects on hepatic cholesterol handling as well as plasma lipoproteincomposition.Systolic blood pressure:The hypertension observed in SHR animals at 1 3 weeks of age is consistent with the resultsfrom Chapter 1 as well as previous reports from this laboratory (Kitts et a!., 1992), but differs fromother workers who reported a hypotensive effect of dietary fish oil and butterfat compared to corn oilin older (26 weeks of age) SHRs (Karanja eta!., 1989). However, these workers did not begin toobserve a significant hypotensive effect of 1 8% menhaden oil or butterfat diets until animals were 1 6weeks of age (Karanja eta!., 1989). Thus, the results of thepresent study in 14-week old animals arelikely intermediate in the temporal lowering of blood pressure by dietary fish oil, as well as reflectingthe lower fat content of the diets used herein. In studies with stroke-prone SHR fed diets containing20% milk fat (butterfat), the incidence of cerebrovascular disease was lowered in the absence of areduction in blood pressure (Ikeda eta!., 1987). The content of short-chain saturated fatty acids andthe relatively high level of palmitoleic acid (C16:1) unique to butterfat was suggested to have a rolein 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 dietfor 1 6 weeks was not reduced compared to animals fed a highly saturated fat diet. These workersalso demonstrated that SHR animals had greater platelet aggregation activity than their WKYcounterparts. Saturated fat diets elicited a similar platelet response as did hypertension in animals andthis effect was not significantly reduced by the PUFA diet in the SHR (McGregor eta!., 1981).105Animal 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 WKYanimals. Studies with diabetic rats and food-deprived rats have reported alterations in tissueantioxidant 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 enzyme-specific changes in antioxidant status (Xia et a!., 1995). It is important to recognize that despite anincrease or decrease in the activity of an individual antioxidant enzyme, the susceptibility of a tissueto oxidative stress may not be altered. Tissue antioxidant status usually reflects a balance betweenenzymatic 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 effecton animal strain differences for the glutathione metabolizing enzymes than those involved inmetabolizing reactive oxygen species. These diet-induced effects on enzyme activity were alsoobserved to be organ-specific. Tissue antioxidant status is known to be sensitive to changes innutrition 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 theantioxidant enzyme activities of animals fed semi-synthetic diets in the present study to those fed acommercial chow in Chapter 1, it is evident that various antioxidant enzyme activities can also bealtered by subtle changes in dietary composition, including the source of dietary fibre or the presenceof sterols, such as cholesterol. Evidence indicates that both soluble and insoluble non-starchpolysaccharides reduce plasma lipids to an extent that is related to the viscosity of the fibre in thegastrointestinal tract (Abbey et a!., 1993). These effects of dietary fibre on plasma lipid dispositionmay have had an indirect effect on tissue antioxidant enzyme activities as well, since there wereconsiderable differences in fibre sources between the chow and semi-synthetic diets used in Chapter1 and the present study, respectively. Another important factor to consider was the absolute amountof lipid included in the two types of diet. The chow diet used in Chapter 1 contained tallow as theprincipal fat in addition to lipids contributed by wheat and soybean meals included in the complex diet,106resulting in approximately 5% total crude lipid. In contrast, the semi-synthetic diets used hereincontained 8% dietary lipid, derived from the experimental fats (5%) and canola oil (3%), present toprovide adequate essential fatty acids. In addition, the low cholesterol semi-synthetic diets contained0.05% 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, butcould also be a factor in determining antioxidant status. It is known that the functional state ofspecific enzymes (e.g. glucose-6-phosphate dehydrogenase; G6PDH) can be subject to modulation bychanges in dietary lipid composition (Mohan eta!., 1991). Also, tissue antioxidant enzyme activity andsusceptibility 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 influencemembrane fluidity and stability (Kirby eta!., 1980) and therefore could also represent a source ofdifferences between animals fed the two types of diet. Despite the similar gross energy content ofthe chow and semi-synthetic diets, the different fibre and lipid sources, as well as the presence of asmall amount of cholesterol in the low cholesterol diet, should not be overlooked in attempting toexplain 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 dietsvarying in n-6 and n-3 PUFA composition (Kuratko eta!., 1994; SkiladOttir eta!., 1994; De Schrijvereta!., 1992; Gonzalez eta!., 1992; L’Abbé eta!., 1991; Mohan eta!., 1991; Huetal., 1989; Nalboneeta!., 1989). Studies with rats fed diets varying in n-6 and n-3 PUFA content from corn and olive oilshave reported that RBC membrane fluidity and stability can be greatly altered by dietary fatty acidcomposition (Periago eta!., 1990). Moreover, various in vivo biochemical parameters involving plasmamembrane fluidity can be altered by membrane fatty acid composition, such as insulin receptor bindingand uptake (Storlien eta!., 1987) and cation flux (Schedl eta!., 1989). In the present study, RBCSOD and GSH-Px activities were both decreased in animals fed menhaden oil diets, while RBC CATactivity was not influenced by dietary fat source. A positive correlation between RBC CAT and SODactivities was previously observed in chow-fed SHR and WKY in Chapter 1, suggesting a compensatorymechanism for elevated SOD activity. L’Abbé and coworkers (1991) reported an inverse correlation107between the ratio of tissue SOD/GSH-Px activities with urinary TBARs. These workers suggested thatthe balance in activity between these two enzymes is a factor in tissue susceptibility to lipidperoxidation. Moreover, despite the decrease in RBC SOD and GSH-Px activities in animals fedmenhaden oil in the present study, both the in vitroH20-induced production of MDA as well as GSHdepletion in RBCs were actually decreased. The reduced susceptibility of RBCs from menhaden oil-fedanimals to induced oxidative stress suggests that dietary n-3 PUFA affected RBC membrane fluidityand stability (Periago eta!., 1990). Increased incorporation of n-3 PUFA into RBC membranes canresult in changes to the membrane cholesterol/phospholipid ratio to stabilize membrane fluidity (Periagoeta!., 1990). Similarly, animals fed the high cholesterol diets exhibited reduced susceptibility to H20-induced MDA production in RBCs, suggesting that increased membrane stability due to cholesterolincorporation (Kirby eta!., 1980) reduced oxidation of membrane n-3 PUFA. The greater RBC GSH-Pxactivity observed in tallow-fed rats compared to those fed butter or soybean oil diets suggests that thestability of RBC membrane fatty acids to lipid oxidation can be altered by the ratio of C18:2,n-6 toC18:3,n-3 in the dietary fat. The competition for6-desaturase activity by linoleic and linolenic acidscould be responsible for an improved linolenate desaturation in the presence of reduced quantities oflinoleic acid, such as may be the case for both butter and tallow diets compared to soybean oil dietsused in the present study. This effect of dietary fatty acid composition could conceivably result in theincreased incorporation of linolenic acid and its metabolites into RBC membranes. These n-3polyunsaturated fatty acids are known to exhibit enhanced susceptibility to lipid peroxidation, therebypossibly contributing to an increased requirement for intracellular RBC GSH-Px activity to inactivatelipid peroxides formed in vivo.As a muscular tissue primarily involved in oxygen exchange, the heart uses circulating free fattyacids as an energy source as opposed to lipoprotein sources of lipid. This may explain the relativeinsensitivity 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 ortriacylglyceride concentrations in the aforementioned studies, myocardial phospholipid fatty acids werefound to reflect dietary fatty acid composition. In the present study, activities of heart SOD, GSH-Pxand GSSG-Red were reduced in SHR and WKY animals fed menhaden oil. L’Abbé and coworkers108(1991) also reported that heart SOD activity was reduced in rats fed diets containing 20% menhadenoil. Other workers have reported that rats restricted to 60% of normal energy intake exhibited reducedheart SOD activity (Xia eta!., 1995). The fact that animals fed menhaden oil diets exhibited reducedbody weight gain and FER values could suggest that diet-induced changes in fatty acid substrateavailability 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-fedanimals did not result in enhanced susceptibility to H2O-induced lipid oxidation. The decreasedsusceptibility of heart tissue from animals fed menhaden oil diets to in vitro GSH depletion and TBARsformation agrees with the results in RBCs above. Therefore, despite the decreased activities of theantioxidant enzymes measured in heart tissue from animals fed menhaden oil diets, susceptibility tolipid oxidation appeared to be lower in heart tissue from these animals.Dietary fatty acid composition conceivably influences hepatic membrane lipid composition viatriacylglyceride synthesis and secretion. Several reports have demonstrated an influence of dietary fatsource on hepatic membrane fatty acid profiles (Jones et a!., 1 995; De Schrijver et a!., 1992) andsusceptibility to lipid peroxidation (L’Abbé eta!., 1991; Hu eta!., 1989). It is well recognized that therelatively high content of n-3 PUFA provided by marine oil diets can result in the replacement ofarachidonic 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 membranefluidity, but also enzymatic activity, namely mixed function oxidases (Saito et a!., 1 990) andcyclooxygenases 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 mayconceivably reduce the activity of cyclooxygenase and lipooxygenase enzymes for which arachidonicacid is a substrate, thereby reducing the load of reactive oxygen species (ROS) produced from thesebiosynthetic pathways. Decreased production of ROS in vivo would result in a reduced necessity fortissue antioxidant enzyme activity. This hypothesis is supported by the fact that liver GSH-Px and SODactivities 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.109An alternative explanation for the decreased hepatic GSH-Px activity in menhaden oil-fed animals isthe 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 fed18% 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 theoxidative 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 tissueand 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 reactionof lipid peroxidation products with membrane proteins have been identified in tissues from animals fedfish oil diets (Nalbone eta!., 1989; Reddy eta!., 1973). The increased peroxisomal li-oxidation oflong-chain n-3 PUFA from dietary marine oils suggests a potential increase in the production of H20in vivo (Mohan et a!., 1991). Thus, reactive oxygen species as well as products of lipid peroxidationmay be present in vivo, necessitating their detoxification by CAT and GSH-Px. However, excessamounts 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 elevatedconcentrations of H20 and lipid hydroperoxides (ROOH), respectively (Remade et a!., 1992). Thus,the reduced activities of SOD and GSH-Px in tissues from animals fed diets containing menhaden oilmay 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 inthe 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, thereduced susceptibility toH20-induced GSH depletion and TBARs production of liver tissue from SHRand WKY fed menhaden oil may be related to the lower body weight gain and FER of these animalsand possibly to a relative reduction in available energy stores associated with the marine oil diets (Xiaeta!., 1995; Su and Jones, 1993).While neither heart antioxidant enzyme activities nor susceptibility toH20-induced oxidativestress were affected by the level of dietary cholesterol, animals fed the high cholesterol diets exhibited110reduced hepatic CAT, SOD and GSH-Px activities, and increased hepatic GSSG-Red activity. It hasbeen hypothesized by Smith (1991) that cholesterol may itself act as an antioxidant molecule bystabilizing cell membranes and preventing peroxidation of membrane fatty acids. The presence ofincreased membrane cholesterol due to dietary cholesterol supplementation could reduce therequirement for antioxidant enzyme activity due to increased membrane lipid stability associated withthe replacement of phospholipid polyunsaturated fatty acids with cholesterol in tissue membranes. Thereduced susceptibility to H2O-induced TBARs production of liver tissue from animals fed highcholesterol diets supports this concept of increased membrane stability due to cholesterolincorporation. Feeding rats cholesterol results in a fatty liver as evidenced by the greater liver weightsand abnormal macroscopic appearance as noted in the present study. The increased susceptibility toH2O-induced GSH depletion of liver from animals fed high cholesterol diets, therefore, suggests thatthe fatty liver observed in these animals may be regarded as an additional factor contributing toincreased GSH depletion.Other considerations:It has been shown that the development of insulin resistance in rats fed high fat diets (Barnardet 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, whichhas 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 consideredthe possibility of the effect of a relative selenium (Se) deficiency on antioxidant enzyme activity inthese animals (L’Abbé eta!., 1991; Nalbone eta!., 1989). Feeding animals either 20% menhaden oilor 12.5% salmon oil in the diet did not result in a functional Se deficiency as demonstrated by thetissue activities of GSH-Px in these studies (L’Abbé eta!., 1991; Nalbone eta!., 1989). Similarly, inthe present study, while GSH-Px activities were reduced in RBCs and heart of SHR and WKY animalsfed menhaden oil diets, the variable effect on hepatic GSH-Px activities in SHR compared to WKYanimals indicates that functional Se deficiency did not occur in menhaden oil-fed animals in the presentstudy.111Conclusion:In conclusion, hypertensive SHR exhibited reduced plasma lipids and FER values in combinationwith tissue-specific and enzyme-specific alterations in antioxidant status compared to normotensiveWKY animals. The differences in heart and liver tissue GSH levels and GSH depletion paralleled theactivity of GSSG-Red in these tissues and may indicate an up-regulation of this enzyme activity tomaintain 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 acompensatory 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 theprevious results with chow-fed animals in Chapter 1, suggesting that this may be a useful marker ofRBCs 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 potentialdifferences in fatty acid substrate availability in animals fed these diets. The reduced plasma lipidconcentrations in animals fed menhaden oil diets are consistent with the effects of long-chain n-3PUFA on lipoprotein metabolism and fatty acid peroxisomal B-oxidation. Both the decreased tissueGSH-Px and SOD activities in RBCs, heart and liver tissue from animals fed menhaden oil diets, inaddition to the reduction in tissue susceptibility to in vitro H20-induced GSH depletion and lipidperoxidation in these same animals, may be related to the influence of dietary fatty acids on membranefatty acid composition. Diet-induced alterations to cell membrane fatty acid stability to lipid oxidationmay be due to the replacement of n-6 (i.e. arachidonic acid) by n-3 fatty acids (eicosapentaenoic anddocosahexaenoic acids) in membrane phospholipids.Diets containing a high level of cholesterol increased plasma cholesterol levels as expected, aswell as inducing pathophysiological changes to the liver, as reflected by liver size and weight. Theseeffects of feeding high cholesterol diets to both SHR and WKY animals were associated with areduction in the activity of hepatic CAT, SOD and GSH-Px, possibly indicating a reduced requirementfor antioxidant enzyme activity due to increased membrane fatty acid stability to lipid oxidationresulting from the potential incorporation of cholesterol into cell membranes. This result was further112substantiated by the reduced susceptibility to in vitroH20-induced lipid peroxidation of liver tissue,and also RBCs from animals fed high cholesterol diets. However, the increased susceptibility to GSHdepletion of liver tissue from these same animals is evidence for a pathophysiological effect of the highcholesterol diets on liver tissue. In summary, dietary fat source and dietary cholesterol content hada greater influence on animal strain differences in plasma lipid levels as demonstrated by theexperimental treatment interactions observed than on animal strain differences in tissue antioxidantstatus parameters. The one exception to this was the activity of liver GSH-Px, suggesting that thisliver antioxidant enzyme was particularly sensitive to tissue compositional changes induced by dietarytreatment interactions. Finally, it was noteworthy that semi-synthetic diets fed to the SHR and WKYanimals in the present study resulted in alterations in antioxidant status parameters when comparedto the chow diet in Chapter 1, demonstrating the subtle effects of non-purified dietary components onin vivo antioxidant status.113CHAPTER 3Species-related differences in plasma lipids and susceptibility to atherosclerosisbetween atherosclerosis-resistant (rat) and -susceptible (quail) animalsfed diets supplemented with cholesterol.Introduction:Numerous animal models have been utilized to study the effects of dietary fat source andcholesterol intake level on atherosclerosis. Through the use of genetic selection for specific traits ofinterest, models for elevated plasma lipids (Watanabe Heritable Hyperlipidemic (WHHL) rabbit; Bilheimereta!., 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 animalmodels 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 abilityof 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 WHHLrabbit as a model for human familial hyperlipidemia (Kita et a!., 1981). Often, the interaction ofexperimental treatment effects with the lipid and lipoprotein metabolism of animals does not mimic thatof 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 versusthat of humans (Chapman, 1 980), differences in lipoprotein composition (Terpstra et a!., 1982; Millsand 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 hasbeen successful in accelerating the rate of atherogenesis, in comparison to the natural, but lengthydevelopment of the disease in these animals (Masuda and Ross, 1990a). Studies in non-humanprimates have indicated that the cellular events and tissue morphological changes which occur duringthe progression of atherosclerosis are similar whether animals are fed diets inducing modest levels ofhypercholesterolemia 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 suggested114the 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 componentsof arterial plaque composition in humans (Steinbrecher and Lougheed, 1992) and various animalspecies (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 nutritionalbiochemistry studies. When high levels of cholesterol are fed to rats, changes in plasma lipid profilesare 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 inatherosclerosis research (Stewart-Phillips et a?., 1992; Shih eta?., 1983). Japanese quail (Coturnixjaponica) made hyperlipidemic by dietary cholesterol supplementation develop atherosclerotic lesionsin the aorta in a dose-dependent manner (Radcliffe eta?., 1982). Moreover, studies feeding oxidizeddietary cholesterol to Japanese quail resulted in greater severity of aortic lesions than was observedwith purified cholesterol (Donaldson, 1982).115Hypothesis for Chapter 3:Differences in the susceptibility of animal models to diet-induced hyperlipidemia contribute tospecies-specific variations in the development of atherosclerosis.Obiective for Chapter 3:To link the relative differences in susceptibility to diet-induced atherosclerosis in theatherosclerosis-susceptible Japanese quail and -resistant rat to species-related variations inhyperlipidemia and aortic sterol content.Specific Aims for Chapter 3:i. A comparison between species for susceptibility to diet-induced hyperlipidemia will be madeby feeding diets varying in cholesterol content with a constant level of fat for a 9 week period to theWistar rat (a species showing a high degree of resistance to the development of atherosclerosis) andthe atherosclerosis-susceptible Japanese quail (known to develop aortic atherosclerotic plaques whenfed cholesterol-rich diets).ii. Gas chromatography combined with mass spectrometry (GC-MS) will be used to identifyand quantitate the cholesterol and cholesterol oxide content in aortic tissue samples.116Materials and Methods:Animals and diets:Sixteen male Wistar rats (5 weeks of age; Charles River, Montreal, PQ) and 1 6 male Japanesequail (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 wereselected for susceptibility to cholesterol-induced atherosclerosis (Shih et a!., 1983). Animals (8 pergroup for rats and quail) were fed semi-synthetic diets (Table 3.1) varying in protein source (caseinand soy protein for rat and quail diets, respectively) and containing 3% canola oil (to provide essentialfatty acids) and 5% beef tallow. Quail diets were mixed, pelleted and crumbled at the AgricultureCanada 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 experimentalperiod. Animals had access to distilled deionized water at all times. Animals were separately housedwith a 14-hr light/i 0-hr dark cycle and ambient temperature. Birds were housed in brooder cages ina separate room with lighting and temperature controls.Experimental procedures:At the end of the experimental feeding period blood was collected into heparinized tubes fromrats by exsanguination under halothane anaesthesia (4% halothane at a flow rate of 4L/ mm forinduction, and 2.5% at a flow rate of 2 L/min for maintenance), while quail were decapitated and trunkblood 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 inChapter 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 andthe aorta to the iliac branching) was dissected out, opened longitudinally and examined under a iO-30Xdissecting microscope for lesions on the inner wall. Aortic lesion scores from 0 to 4 were assignedaccording to Shih et a!. (1983) and Godin eta!. (1994). Scoring was performed by two independentinvestigators (in a blinded fashion) as follows: 0 = clean surface; 1 = < 5 plaques; 2 = 6-20 plaques117Table 3.1. Composition of diets fed to Wistar rats and atherosclerosis-susceptibleJapanese quail.Rat diet Quail dietCholesterol level(% by weight) 0.05 0.5 0.05 0.5Dietarycomponent (gil OOg)Casein’ 25.0 25.0 - -Soy protein meal2 - - 34.0 34.0Ca-free mineral mix1 3.5 3.5 2.0 2.0CaCO3 2.0 2.0 5.0 5.0Vitamin mix1 3.0 3.0 0.3 0.3DL-methionine4 0.3 0.3 0.4 0.4Choline chloride2 0.2 0.2 0.3 0.3Cornstarch5 47.0 47.0 39.5 39.5Sucrose 3.0 3.0 2.5 2.5Alphacel’ 5.0 5.0 5.0 5.0Monofos2 3.0 3.0 3.0 3.0Canola oil5 3.0 3.0 3.0 3.0Beef tallow6 5.0 5.0 5.0 5.0Cholesterol’ 0.05 0.50 0.05 0.50Cholic acid1 0.025 0.25 0.025 0.251 ICN Biochemicals Inc., Cleveland, OH, USA2 Van Waters & Rogers, Abbotsford, B.C., CanadaBDH Chemicals, Toronto, ON, CanadaUnited States Biochemical Co., Cleveland, OH, USANeptune Food Services, Richmond, B.C., Canada6 Cargill Foods, High River, AB, Canada118and 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) withconfirmation 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 internalstandard (5a-cholestane, lOOpg) was added to samples before lipid extraction. The extracted lipidswere evaporated to dryness under a stream of N2 and subjected to a cold saponification with 1 N KOH(in methanol) overnight at room temperature (25°C). The saponified samples were extracted withdiethyl ether (3X) and washed with 0.5 N KOH (1X) and distilled deionized water (2X).Nonsaponifiables were dried using anhydrous Na2SO4 before reducing the sample volume with a N2stream for transfer to Reacti-Vials (Pierce Chemical Co., Rockford, IL). Samples were dried undervacuum to remove traces of moisture, before solubilization in 200 p1 dry pyridine. A 100 p1 aliquot ofsample was derivatized with 50 p1 Sylon BTZ (Supelco, Inc., Oakville, ON) and the reaction allowedto 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 (4l-hydroxycholesterol), cholest-5-ene- 3R, 7a-diol (7a-hydroxycholesterol), cholest-5-ene-3I&71-diol (7I-hydroxycholesterol), 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-ene-7-one (7-ketocholesterol); Steraloids, Inc., Wilton, N.H., USA) and samples were analyzed using a DB1 column (15 m X 0.25 mm i.d., 0.1 p film thickness; J & W Scientific, Inc., Folsom, CA, USA) on aCarlo Erba gas chromatograph (Carlo Erba Strumentazione, Italy) equipped with a flame ionizationdetector (GC-FID). A representative chromatogram of derivatized cholesterol oxide standards is shownin Figure 3.1. Carrier gas used was He with N2 as the make-up gas. Injector and detectortemperatures were 250°C and 280°C, respectively, oven temperature was programmed from 1 80°Cto 250°C at 3C° per minute, final temperature was held for 1 5 minutes. During GC-FID analysis,119Is5L4JiLFigure 3.1 A representative GC-FID chromatogram of derivatized cholesterol oxide standards withinternal standard (IS) = 5a-cholestane. 1 = cholesta-3,5-dien-7-one; 2 = cholesterol; 3 = 7a-hydroxycholesterol; 4 = 5,6a-epoxy-5a-cholesterol; 5 = 7f-hydroxycholesterol; 6 = 4B-hydroxycholesterol; 7 = cholestane-triol; 8 = 7-ketocholesterol; 9 = 25-hydroxycholesterol.120chromatograms were stored and analyzed using a Hewlett-Packard HP 3393A integrator (HewlettPackard 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. Mass spectra were scannedbetween mass range m/e = 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 derivatizedsterol. Response linearity of GC-FID to trimethyl silyl (TMS) ether sterols was confirmed byconstructing a calibration curve of the response to mixtures of varying amounts of sterols with aconstant amount of internal standard (IS; 5a-cholestane). Mixtures of standards were prepared suchthat concentrations of sterols ranged from 10 nglpL to 1 .Opg/pL with the ratio of IS:standard rangingfrom 1:1 to 100:1. Response linearity was analyzed by plotting the area response ratio of each sterolto IS versus the weight ratio of the sterol to IS (Figure 3.2). Refer to Appendix Tables 1 and 2 foradditional information regarding relative retention times and response linearity of derivatized sterolsdetermined by GC-FID.Statistics:All data are expressed as the mean ± SEM. Statistical analysis was performed using Student’st-test at a significance level of 0.05.121Calibration curve for standard #5:7 B — hydroxycholesterolFigure 3.2 A representative standard curve depicting the response linearity of derivatized cholesteroloxide standards using GC-FID. Standard curve is that of 7-hydroxychoIesterol, r = 0.995.0.4.’004.’II1.251.000.750.500.250.000.00 0.25 0.50 0.75 1.00Weight ratio, Std.IIS1.25122Results:Plasma lipid profiles of rat and quail:Plasma lipid profiles of Wistar rats and atherosclerosis-susceptible Japanese quail aresummarized 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, withquail exhibiting a much greater increase (ca. lOX) in plasma total cholesterol between low and highcholesterol-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-susceptibleJapanese quail (Table 3.3). The severe hyperlipidemia of high cholesterol-fed quail was associatedwith severe atherosclerotic plaque (reflected in the high plaque score and high percentage of lumenarea covered). No visible atherogenesis was observed by dissecting microscope in aortae from quailfed low dietary cholesterol or from rats fed either diet. Aortic tissue cholesterol and cholesteroloxidation product content (COPs; Table 3.4) coincided with the plaque scores obtained from visualmicroscopic evaluation of the vessel lumen walls. Aortic tissue cholesterol content was greater in quailthan in rats regardless of dietary cholesterol content. Feeding high levels of cholesterol to animalsresulted in an increase in aortic cholesterol content in quail only. Rat aortic tissue cholesterol contentwas not altered by cholesterol feeding. Combined gas chromatography and mass spectrometry (GCMS) 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 notdetected in the nonsaponifiable extracts from aortae of low cholesterol-fed quail (Figure 3.3a) or fromeither dietary groups of rats (Fig. 3.4a,b).123Table3.2.PlasmalipidsofWistarratsandatherosclerosis-susceptibleJapanesequailfedtallowdietscontaininglowandhighlevelsofcholesterol.1Plasmalipids2:FreecholesterolTotalcholesterolTriacyiglycerides(mmol/L)Cholesterollevel(%byweight):0.050.50.050.50.050.5Animalspecies:Quail1.55±0.099.08±0.17*597±0.2553.1±2.5*1.47±0.124.12±O.36Rat1.82±0.083.55±0.21*3.06±0.126.22±0.46*1.40±0.200.99±0.07*1Valuesrepresentmean±SEMIn=8.2*denotesasignificant(p0.05)differencebetweencholesterollevelsacrossarow.Table3.3.AorticplaquescoreandareacoveredinWistarratsandatherosclerosis-susceptibleJapanesequailfedtallowdietscontaininglowandhighlevelsofcholesterol.1Plaquescore2Areacovered(%)3Cholesterollevel(%byweight):0.050.50.050.5Animalspecies:QuailN.D.3.7±0.2N.D.61±10RatN.D.N.D.N.D.N.D.1Valuesrepresentmean±SEM,n=8.2Plaquescorebasedonscaleof0(N.D.)=cleansurface;1=5plaques;2=6-20plaques;3=>20plaques;4=massiveatheromasobserved.Valuesrepresenttwojudgesevaluatinginablindedprotocol.Areacovered(%)=percentofaorticepitheliumcoveredbyplaque,range0(N.D.)-100%.- c,1Table3.4.GCquantitationofcholesterolandcholesteroloxidationproductsinaortictissuefromWistarratsandatherosclerosis-susceptibleJapanesequailfedtallowdietscontaininglowandhighlevelsofcholesterol.1Sterol2:Cholesterol7R-hydroxycholesterol7-ketocholesterol(mglg)Cholesterollevel(%byweight):0.050.50.050.50.050.5Animalspecies:Quail3.19±0.9111.48±2.13*N.D.0.24±0.03N.D.0.27±0.04Rat0.93±0.040.91±0.49N.D.N.D.N.D.N.D.1Valuesrepresentmean±SEM,n=8.2Valuesexpressedonbasisoftissuewetweight.N.D.=nonedetected.*denotesasignificant(p0.05)differencebetweencholesterollevelsacrossarow.r%) C)A15 20 25time (mm.)Figure 3.3 GC-Fl 0 chromatogram of atherosclerosis-susceptible Japanese quail aortic tissue dervatizednonsaponifiables. (A) 0.05% cholesterol diet, (B) 0.5% cholesterol diet. 1 = internal standard, 5a-cholestane; 2 = cholesterol; 3 = 7B-hydroxycholesterol; 4 = 7-ketocholesterol.12B3-j30127A30time (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.128Discussion:While both the quail and the rat are HDL predominant animals, their individual responses tofeeding 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 greaterthan their rat counterparts at both the low and high levels of dietary cholesterol. This speciesdifference has been attributed to the presence of the large, absorptive TG-rich portomicrons (largeVLDL) 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 toanimals, with the quail showing dramatic increases in both plasma total cholesterol and TGconcentrations. 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 cholesterolfeeding (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 thoseobserved in the present study only after 9 months on these experimental diets. The large increase inplasma total (free and esterified) cholesterol concentration observed in cholesterol-fed quail reflectsboth the increase in plasma free cholesterol levels as well as the increase in cholesteryl ester levels (anincrease 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 plasmatotal and esterified cholesterol were increased, but no change was observed in the relative proportionof esterified cholesterol (ca. 70%). However, these workers did observe a dose-dependent elevationin arterial tissue cholesterol with a greater percentage in the esterified form when birds were fed highcholesterol diets (Radcliffe eta!., 1982). In the present study, greater levels of aortic cholesterol wereobserved 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 wasdemonstrated a9ain in the present study by the absence of atherosclerotic plaque and the considerably129lower aortic cholesterol content in the normotensive Wistar rat fed a hypercholesterolemic diet. Inmarked contrast, several avian species (chicken, pigeon and quail) do develop atherosclerotic plaquein 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 cholesterolsupplementation (0.75%, 1 .5%) than those used in the present study, have also reported thedevelopment of macroscopic atherosclerotic lesions in the aorta of Japanese quail. One of the riskfactors 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, suchas the resistant pigeon and quail strains (Godin eta!., 1994; Chapman, 1980).Modified (oxidized) lipid species have been identified in the plasma lipoproteins and aorticplaque of atherosclerotic humans (Steinbrecher and Lougheed, 1992; Stringer eta!., 1 989) and animalmodels (Rosenfeld eta!., 1990; Palinski eta!., 1989). Furthermore, the presence of COPs in circulatinglipoprotein has been demonstrated in healthy humans (Dzeletovic et a!., 1995) and monkeys (Pengeta!., 1982). The atherogenic potential of COPs has been demonstrated by in vitro cell culture (Pengeta!., 1985, 1978) as well as in animal feeding studies (Donaldson, 1982). In a study whereinJapanese quail were fed either purified cholesterol or oxidized cholesterol, those birds fed the latterdiets exhibited greater plasma and liver cholesterol concentrations in association with increasedseverity of atherosclerotic lesions as compared to animals fed purified cholesterol (Donaldson, 1982).Differences in aortic lesion severity were also observed between animals fed purified cholesterolcompared to USP cholesterol diets. The latter diet resulted in greater lesion severity than purifiedcholesterol, but less so than oxidized cholesterol feeding. The supplementation of oxidized cholesteroldiets with antioxidants (synthetic phenolics or a-tocopherol) reduced severity of aortic lesions, but didnot completely prevent the development of atherosclerosis in those animals (Donaldson, 1982).While it is well known that atherosclerotic plaque contains large amounts of cholesterol, tissueculture 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 anonsaturable fashion (Steinberg eta!., 1989). Several COPs have been identified as having cytotoxic,130angiotoxic, 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 culturedrabbit aortic smooth muscle cells (Peng eta!., 1978). Thus, potentially angiotoxic cholesterol oxidesmay be present in the lower density lipoprotein fractions of animals which develop atheroscleroticlesions. 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 thepresence of these same COPs in the LDL extracted from human atherosclerotic plaques. The presenceof 7l.-hydroxychoIesterol in tissues may be due to the autoxidation of cholesterol, whereas its 7a-hydroxycholesterol isomer is known to be a product of in viva oxidation in bile acid synthesis (Smithand Johnson, 1989). Other workers have been able to confirm the identity of several COPs (cholest3,5-diene-7-one, cholestane-triol, 7-hydroxycholesterols, 7-ketocholesterol, 24-hydroxy-, 25-hydroxy-and 26-hydroxycholesterol) in human aortic specimens (Morin and Peng, 1992). Detectable amountsof 7-ketocholesterol have also been reported in aortic tissue from rat, cat, bovine, horse and baboonsamples (Morin and Peng, 1992). The present study represents the first time that COPs have beenmeasured in aortic plaque material collected from atherosclerosis-susceptible Japanese quail.In the present study, precautions were taken to minimize exposure of sample lipid extracts toexcess oxygen. One indicator of breakdown of cholesterol oxides during sample preparation is thepresence of cholest-3,5-diene-7-one due to the instability of 7-ketocholesterol through thermaldehydration (Park and Addis, 1985). None of the experimental samples had detectable amounts of thiscompound 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. TheseCOPs are identical to those reported from aortic plaques from both humans as well as other animalmodels, thereby further validating the use of the atherosclerosis-susceptible Japanese quail for researchin the area of experimental atherogenesis.131Conclusion:While both the rat and the Japanese quail are HDL predominant species, atherosclerosis wasinducible only in the latter animal model. This result was associated with species differences in plasmalipid profiles as observed with the greater response to cholesterol feeding in the quail. Aortic tissuesterol content was also shown to vary between animal models. Quail aortae exhibited greatercholesterol contents than corresponding tissues from similarly treated rats, which could be associatedwith differences in plasma cholesterol concentrations between species. Moreover, the presence of thecholesterol oxides (7B-hydroxycholesterol and 7-ketocholesterol) in aortic plaque from atherosclerosissusceptible Japanese quail similar to those observed in human aortic plaque were identified using GCMS. These findings further substantiate the use of the atherosclerosis-susceptible Japanese quail inexperimental atherosclerosis research.132CHAPTER 4Effect of dietary fat source on aortic plaque, plasma lipids and antioxidant statusof atherosclerosis-susceptible Japanese quail.Introduction:Studies conducted with tissues collected from patients with coronary heart disease suggestan association between increased susceptibility to lipid peroxidation and reduced levels of specificantioxidant enzymes in the plasma and red blood cells (Jayakumari eta!., 1 992), platelets (Buczynskieta!., 1993) and diseased aortic tissue (Hunter eta!., 1991). Further evidence of a role for lipidoxidation and in vivo antioxidant status in atherosclerosis is the presence of oxidized LDL in aorticplaque 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 tostudy 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 whichdevelops when diets containing a high level of cholesterol are fed to these birds indicates that the cellpopulations involved in the initiation and propagation phases of plaque development are very similarbetween the quail and human atherosclerosis (Shih, 1983; Shih eta!., 1983; McCormick eta!., 1982).The characterization of atherosclerosis-susceptible and -resistant strains of Japanese quail hascontributed 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 atherosclerosisin Japanese quail, Godin and coworkers (1994) reported that differences between atherosclerosis-susceptible 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 toexhibit a much greater increase in plasma lipid concentrations in response to feeding a high level ofcholesterol in the diet than the rat, the latter species generally being considered to be resistant to thedevelopment of atherosclerosis (Bishop, 1980). The greatly increased plasma lipid concentrations133observed in these quail coincided with the appearance of severe atherosclerotic plaque in the aortaeof 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 temporaldevelopment of aortic plaque in relation to plasma cholesterol and triacylglyceride concentrations inthe 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 dietaryfat sources on plasma lipid concentrations and the severity of aortic plaque development in theJapanese quail in light of the association between dietary fatty acid composition and hyperlipidemiaas 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 sourcesand cholesterol intake levels on plasma lipid response, aortic plaque deposition as well as aorticcholesterol and cholesterol oxide composition in atherosclerosis-susceptible Japanese quail.Furthermore, the effect of dietary fat source on endogenous antioxidant status was studied todetermine the potential association between severity of aortic plaque deposition and the antioxidantstatus of selected tissues of these birds.134Hypotheses for Chapter 4:i. Dietary fat sources differing in fatty acid composition (i.e. saturated versus unsaturatedfatty acids) and cholesterol intake level have independent or interactive effects on plasma lipidresponse and aortic plaque development in atherosclerosis-susceptible Japanese quail.ii. The endogenous antioxidant status of atherosclerosis-susceptible Japanese quail can bealtered by dietary fatty acid composition and cholesterol intake level and may be related to the severityof 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-6polyunsaturated lipids) dietary fat sources and level of cholesterol on plasma lipid concentrations,endogenous antioxidant status and severity of aortic plaque in atherosclerosis-susceptible Japanesequail.Specific Aims for Chapter 4:i. Plasma lipid concentrations and presence of aortic plaque will be determined inatherosclerosis-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 H20-inducedglutathione depletion and lipid peroxidation in vitro will be examined to determine the role ofendogenous antioxidant status in modulating the severity of aortic plaque in atherosclerosis-susceptibleJapanese quail fed diets varying in dietary fat source and cholesterol content.iii. Aortic tissue cholesterol and cholesterol oxide content and associated ultrastructuralchanges will be used to characterize the composition of aortic plaque in atherosclerosis-susceptibleJapanese quail.135Materials and Methods:Animals:Seventy-two, six-week old male atherosclerosis-susceptible Japanese quail (Coturnixjaponica;U.B.C. Quail Genetic Resource Centre, Vancouver, B.C.) were randomly divided into six dietarytreatment groups (n = 1 2) varying in dietary fat source (i.e. butter, beef tallow and soybean oil) andcholesterol 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 andcholesterol content is detailed in Table 4.1. A basal diet, containing 3% canola oil (Neptune FoodServices, Richmond, B.C.) to provide an adequate supply of essential fatty acids, was formulated withthorough 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 additionalantioxidants were added to diets, with the exception of the vitamin E that was present as a componentof the poultry vitamin-premix. Once the basal diet ingredients were thoroughly mixed in an industrial-sized stainless steel mixing vat, the powdered diet was pelleted and crumbled in a feed mill (AgricultureCanada 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 sourcesand sterols into the crumbled basal diet was performed as previously described in Chapter 2. Dietaryfats containing sterols at levels reported in Table 4.1 were slowly added to the crumbled basal dietduring reblending and mixed in completely using a Hobart mixer with an aluminum bowl over a periodof approximately 20-25 minutes. After mixing, individual diets were stored in double, dark plastic bagsin a walk-in freezer (-15°C) throughout the experimental study. A sample of each experimental dietwas removed for analysis of fatty acid, gross energy and dry matter content as described in Chapter2.The experimental fat sources were added to the basal diet at a level of 5% to make a finalcalculated fat content of 8% dietary fat. This level of fat in the diets matches the level of dietary lipidcontained in commercial quail feed (Turkey Starter; Otter Co-op, Aldergrove, B.C.). The levels of136Table 4.1 Composition of diets fed to atherosclerosis-susceptible Japanese quail1.Cholesterol level(% by weight): 0.05 0.5Dietarycomponent (gil OOg)Soy protein meal2 34.0 34.0Ca-free mineral mix3 2.0 2.0CaCO34 5.0 5.0Poultry vitamin premix2 0.3 0.3DL-methionine5 0.4 0.4Choline chloride2 0.3 0.3Cornstarch6 39.5 39.5Sucrose 2.5 2.5Alphacel3 5.0 5.0Monofos2 3.0 3.0Canola oil6 3.0 3.0Dietary fats:Butter, beef tallow, orsoybean oil7 5.0 5.0Cholesterol3 0.05 0.50Cholic acid3 0.025 0.251 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) andsoybean oil (1 6.55). Dry matter content of all diets ranged from 89 - 91 %.2 Van Waters & Rogers, Abbotsford, B.C., CanadaICN Biochemicals Inc., Cleveland, OH, USABDH Chemicals, Toronto, ON, CanadaUnited States Biochemical Co., Cleveland, OH, USA6 Neptune Food Services, Richmond, B.C., CanadaButter (Dairyworld Foods, Burnaby, B.C.); beef tallow (Cargill Foods, HighRiver, AB); soybean oil (Bioforce Canada, Burnaby, B.C.).137cholesterol incorporated into diets were 0.05% and 0.5% (wt/wt) for the low cholesterol (on basisof 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 cholesteroldiets for the sterol content naturally present in butter and beef tallow sources. Experimental diets wereisonitrogenous 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 wascorrected 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 asdescribed 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. Feedand distilled deionized water were provided to birds in separate feeding troughs ad ilbitum. Feed wasreplaced 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 weredecapitated and trunk blood collected into chilled heparinized tubes and plasma separated by low-speedcentrifugation (1000 x g, 5 mm, 4°C). Aliquots of plasma as well as RBCs, heart and liver tissues werecollected 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 iliacbranching) was dissected out, opened longitudinally and examined under a 1O-30X dissectingmicroscope for lesions on the inner wall as described in Chapter 3. Briefly, aortic lesion scores from0 to 4 were assigned according to Shih eta!. (1983) and Godin eta!. (1994). Scoring was performedby two independent investigators (in a blinded fashion) as follows: 0 = clean surface; 1 = 5plaques; 2 = 6-20 plaques and an affected area less than 50%; 3 = > 20 plaques with an affectedarea greater than 50%; 4 = 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 buffer138Table 4.2 Fatty acid composition of diets fed to atherosclerosis-susceptibleJapanese quail.DietsButter Beef Soybeantallow oilFatty acid: Area %14:016:016:1(n-7)18:018:1(n-9)1 8:1 (isomers)1 8:2(n-6)1 8:3(n-3)20:020:1SaturatesMonounsaturatesPolyunsaturatesn-6n-3P/SiP/S polyunsaturated fatty- denotes not detected.acid/ saturated fatty acid ratio.6.522.31 .07.436.41.813.73.70.836.240.017.413.73.70.52.218.52.010.846.72.413.54.031.451.117.513.54.00.60.39.00.24.347.63.415.32.70.40.814.152.117.915.32.71 .3139prior to homogenization for analysis of antioxidant enzyme activity. The aorta was blotted dry and anyadhering tissue was removed before recording the aortic weight. An aliquot (1 .0 mL) of homogenizingbuffer was added to the aortic tissue in a test-tube and a homogenate was prepared using a micro-probe 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 enzymaticassays were prepared by centrifugation (12,000 x g, 4°C, 15 mm). The aortic cytosolic fractions wereassayed for GSSG-Red, GSH-Px, and SOD activities by the methods outlined in Chapter 1. Additionalbirds in each treatment group were used to provide aortic specimens for GC-MS analysis of cholesteroloxides, 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 GCMS as described in Chapter 3.Scanning electron microscorv:Aortic specimens were prepared for scanning electron microscopy according to Peng andcoworkers (1985). Briefly, samples were immersed in 3% glutaraldehyde buffer, pH 7.6, followed byimmersion in an 0.5% 0s04 solution, rinsed and dehydrated with ethanol. Samples were then driedby the critical point drying method using CO2. Finally, aortic specimens were mounted onto stubs andcoated with gold prior to viewing with scanning electron microscope facilities (Electron MicroscopyLab, 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, thesource of the differences at a p 0.05 significance level was identified by the Student-Newman-Keulsmultiple range test. Two-way multiple analysis of variance (MANOVA) was used to identify anyinteractions between dietary fat source and dietary cholesterol level. Linear regression analysis (SPSS)was performed to investigate interactions between plasma lipids and aortic plaque parameters.140Results:Fatty acid content of semi-synthetic formulated quail diets:Similar to the dietary fatty acid analysis conducted in Chapter 2, the short-chain saturated fattyacids 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) was noted to be greater in the butter dietscompared to both beef tallow and soybean oil 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 insoybean 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 thisfatty 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 thebutter and beef tallow diets were 0.5 and 0.6, respectively, while that of the soybean oil diets was1 .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 andbeef 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. Finalbody weights of birds were not affected by dietary treatment (range 126 ± 1 g). Similarly, weightsof heart tissue were not different between dietary treatment groups (range 0.9 ± 0.1 g). Livers frombirds fed high cholesterol diets (range 3.7 ± 0.1 g) weighed significantly (p 0.05) more than livertissue from counterparts fed low cholesterol diets (range 2.2 ± 0.1 g). However, liver tissue weightswere 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 andtotal cholesterol, as well as triacylglyceride concentrations were significantly (p < 0.05) increased inbirds fed the high cholesterol diets (Table 4.3). In contrast, dietary fat source did not have an effecton these same plasma lipid parameters. However, while the difference was not significant, birds fed141Table4.3Plasmalipidsofatherosclerosis-susceptibleJapanesequailfedexperimentaldiets.1Cholesterollevel(%byweight):DietaryTreatment:Plasmalipids:FreecholesterolTotalcholesterolTriacylglycerides(mmol/L)0.050.50.050.50.050.5Butter2.30±0.218.89±0.776.89±0.4661.2±6.02.30±0.575.02±0.65Tallow2.45+0.2710.9±0.97.49±0.8764.8±5.62.17±0.315.76±0.56Soybean1.90±0.128.90±0.565.86±0.3656.4±4.71.90±0.314.32±0.52Plasmalipids:FreecholesterolTotalcholesterolTriacyiglyceridesANOVAp-value2C<0.001<0.001<0.001FN.S.N.S.N.S.1Valuesrepresentmean±SEM,n=12.2c=cholesterolintakeleveleffect,F=dietaryfatsourceeffectby2-wayMANOVA.‘-athe soybean oil diets tended to have slightly lower concentrations of plasma lipids (i.e. total cholesteroland 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 thepresent 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 notinfluenced 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 exhibitdetectable levels of CAT activity. Neither SOD, nor GSSG-Red activity in heart tissue from quail weredifferent between dietary treatment groups (Table 4.5). Heart GSH-Px activity was significantly (p0.05) influenced by dietary fat source, but not by cholesterol intake level (Table 4.5). Activity ofGSH-Px was decreased (p 0.05) in birds fed soybean oil diets compared to those fed butter or beeftallow diets (Table 4.5).iii. Liver antioxidant enzymes:Liver tissue reactive oxygen species metabolizing antioxidant enzyme activities are presentedin Table 4.6a. Neither CAT, nor SOD activity were influenced by dietary cholesterol intake level ordietary fat source (Table 4.6a). Similarly, liver tissue activities of GSH-Px and GSSG-Red also werenot 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 notdifferent between dietary fat source or cholesterol intake level treatment groups in the present study.143Table4.4Antioxidantenzymeactivitiesinredbloodcellsofatherosclerosis-susceptibleJapanesequailfedexperimentaldiets.’AntioxidantEnzyme2:GSSG-RedGSH-PxSOD(nmolesNADPH/min/mgHb)(nmolesNADPH/min/mgHb)(U/mgHb)Cholesterollevel(%byweight):0.050.50.050.50.050.5DietaryTreatment:Butter9.90±0.6510.3±0.592.0±6.186.1±3.85.28±0.575.43±0.55Tallow10.4±0.510.5±0.591.4±3.690.9±3.45.49±0.596.05±0.82Soybean-10.6±0.610.2±0.6105±5103±55.72±0.485.52±0.67AntioxidantEnzyme:GSSG-RedGSH-PxSODANOVAp-value3CN.S.N.S.N.S.FN.S.0.004N.S.1Valuesrepresentmean.±.SEM,n=12.2GSSG-Red=glutathionereductase;GSH-Px=glutathioneperoxidase;SOD=superoxidedismutase.C=cholesterolintakeleveleffect,F=dietaryfatsourceeffectby2-wayMANOVA.-Table4.5Antioxidantenzymeactivitiesinheartofatherosclerosis-susceptibleJapanesequailfedexperimentaldiets.1AntioxidantEnzyme2:GSSG-RedGSH-PxSOD(nmolesNADPH/min/mgtissuewetwt)(nmolesNADPH/min/mgtissuewetwt)(U/mgtissuewetwt)Cho’esterollevel(%byweight):0.050.50.050.50.050.5DietaryTreatment:Butter0.457±0.0260.447±0.0172.66±0.152.28±0.171.35±0.101.36±0.09Tallow0.447±0.0220.457±0.0182.35±0.212.56±0.151.64±0.151.41±0.16Soybean0.483±0.0170.441±0.0072.22±0.231.40±0.131.63±0.091.36±0.14AntioxidantEnzyme:GSSG-RedGSH-PxSODANOVAp-value3CN.S.N.S.N.S.FN.S.0.024N.S.1Valuesrepresentmean±SEM,n=12.2GSSG-Red=glutathionereductase;GSH-Px=glutathioneperoxidase;SOD=superoxidedismutase.C=cholesterolintakeleveleffect,F=dietaryfatsourceeffectby2-wayMANOVA.-a (31Table4.6aReactiveoxygenspeciesmetabolizingantioxidantenzymeactivitiesinliverofatherosclerosis-susceptibleJapanesequailfedexperimentaldiets.1AntioxidantEnzyme2:CATSOD(k/gtissuewetwt)(U/mgtissuewetwt)Cholesterollevel(%byweight):0.050.50.050.5DietaryTreatment:Butter5.89±1.046.08±0.338.67±1.936.38±0.69Tallow8.03±0.434.84±0.426.06±0.315.98±0.76Soybean6.92±0.666.65±0.678.26±1.067.33±1.28AntioxidantEnzyme:CATSODANOVAp-value3CN.S.N.S.FN.S.N.S.1Valuesrepresentmean±SEM,n=12.2CAT=catalase,k=first-orderrateconstant(sec);SOD=superoxidedismutase.C=cholesterolintakeleveleffect,F=dietaryfatsourceeffectby2-wayMANOVA.0)Table4.6bGlutathionemetabolizingantioxidantenzymeactivitiesinliverofatherosclerosis-susceptibleJapanesequailfedexperimentaldiets.1AntioxidantEnzyme2:GSSG-RedGSH-Px(nmolesNADPH/min/mgtissuewetwt)(nmolesNADPH/min/mgtissuewetwt)Cholesterollevel(%byweight):0.050.50.050.5DietaryTreatment:Butter2.69±0.212.66±0.116.67±0.845.43±0.33Tallow2.74±0.172.88±0.174.98±0.445.51±0.36Soybean2.64±0.082.81±0.125.33±0.385.20±0.42AntioxidantEnzyme:GSSG-RedGSH-PxANOVAp-value3CN.S.N.S.FN.S.N.S.1Valuesrepresentmean±SEM,n=12.2GSSG-Red=glutathionereductase;GSH-Px=glutathioneperoxidase.C=cholesterolintakeleveleffect,F=dietaryfatsourceeffectby2-wayMANOVA.Table4.7Antioxidantenzymeactivitiesinaortaofatherosclerosis-susceptibleJapanesequailfedexperimentaldiets.1AntioxidantEnzyme:GSSG-RedGSH-PxSODANOVAp-value3CN.S.N.S.N.S.FN.S.N.S.N.S.1Valuesrepresentmean±SEM,n=12.2GSSG-Red=glutathionereductase;GSH-Px=glutathioneperoxidase;SOD=superoxidedismutase.C=cholesterolintakeleveleffect,F=dietaryfatsourceeffectby2-wayMANOVA.AntioxidantEnzyme2:GSSG-RedGSH-PxSOD(nmolesNADPH/min/mgtissuewetwt)(nmolesNADPH/min/mgtissuewetwt)(U/mgtissuewetwt)0.050.50.050.50.050.5Cholesterollevel(%byweight):DietaryTreatment:Butter0.392±0.0400.341±0.0200.476±0.0620.465±0.0691.18±0.111.23±0.11Tallow0.389±0.0420.320±0.0130.549±0.0230.512±0.0271.29±0.081.06±0.13Soybean0.337±0.0150.341±0.0300.525±0.0540.497±0.1091.10±0.180.93±0.19-hv. Tissue jlutathione (GSH) content:Levels of glutathione in RBCs (range 4.64 ± 0.22 nmoles GSH/mg RBC), heart tissue (range1 .88 ± 0.07 nmoles GSH/mg wet wt tissue) and liver tissue (range 3.39 ± 0.59 nmoles GSH/mg wetwt tissue) in atherosclerosis-susceptible Japanese quail were not affected by dietary fat source orcholesterol 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 differentbetween dietary treatment groups (values not reported). The amount of MDA produced in vitro byRBCs incubated with H20 did not change appreciably with increasing concentrations of peroxidizingagent (data not shown).ii. Heart GSH depletion and TBARs production:The profiles of the in vitro oxidative challenge of heart tissue with increasing concentrationsof H20 are presented in Figure 4.1. As well, the results from the oxidative challenge of heart tissueat a single concentration of H20 with treatment differences identified are presented in Table 4.8.Depletion of GSH from heart tissue was significantly (p 0.05) influenced by dietary cholesterolintake level (Table 4.8, Figure 4.1A, B). Heart tissue from birds fed high cholesterol diets exhibitedgreater (p < 0.05) depletion of GSH than low cholesterol-fed counterparts (Table 4.8). Dietary fatsource did not have an influence on depletion of GSH from quail heart tissue. Susceptibility of hearttissue to TBARs production in vitro did not differ between dietary cholesterol intake level or dietary fatsource 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 ofH20 are shown in Figure 4.2. As well, the results from the oxidative challenge of liver tissue at asingle concentration of H20, with treatment differences identified, are presented in Table 4.9.Depletion of GSH from liver tissue was significantly (p 0.05) influenced by dietary fat source, butwas not affected by the level of dietary cholesterol intake (Table 4.9, Figure 4.2A, B). GSH depletionwas greater (p 0.05) in liver tissue from birds fed soybean oil diets with the low level of149N C.,4) 0 I-Figure4.1Susceptibilityofhearttissuefromatherosclerosis-susceptibleJapanesequailfeddietsvaryingindietaryfatsourceandcholesterolintakeleveltoinvitroH20-induceddepletionofglutathione(GSH)andproductionofthiobarbituricacidreactivesubstances(TBARs).(A)heartGSHdepletionofquailfedlowcholesteroldiets;(B)heartGSHdepletionofquailfedhighcholesteroldiets;(C)heartTBARsproductionofquailfedlowcholesteroldiets;(D)heartTBARsproductionofquailfedhighcholesteroldiets.*indicatesasignificant(p0.05)dietaryfatsourcedifference.=butter;A=beeftallow;0=soybeanoil.0 4, 4,0.000.501.001.502.002,50mMH202addedCg 4, 41N In0.000.501.001.502,002.50mMH202,dded0.500.40-0.300.20-0.10-0.00—D0.500.400.300.200.100.00II01234mMH202added501II—II2345mMH202added- 0Table4.8HearthomogenatesusceptibilitytoH202-inducedGSHdepletionandTBARsproductioninatherosclerosis-susceptibleJapanesequailfedexperimentaldiets.’GSHdepletion(%)TBARs(A532)0.6mMH2021.0mMH202Cholesterollevel(%byweight):0.050.50.050.5DietaryTreatment:Butter24.8±3.729.3±4.90.124±0.0130.156±0.021Tallow28.2±5.430.4±4.50.142±0.0290.121±0.010Soybean21.6±3.538.8±7.70.107±0.0140.142±0.020GSHdepletion(%)TBARs(A532)ANOVAp-value2C0.049N.S.FN.S.N.S.1Valuesrepresentmean±SEM,n=12.GSH=glutathione;TBARs=2-thiobarbituricacidreactivesubstances.2C=cholesterolintakeleveleffect,F=dietaryfatsourceeffectby2-wayMANOVA.(3100-a 01C C2.001.501,000.600.00S C2.001.50-1.000.50-0.00Figure4.2Susceptibilityoflivertissuefromatherosclerosis-susceptibleJapanesequailfeddietsvaryingindietaryfatsourceandcholesterolintakeleveltoinvitroH20-induceddepletionofglutathione(GSH)andproductionofthiobarbituricacidreactivesubstances(TBARs).(A)liverGSHdepletionofquailfedlowcholesteroldiets;(B)liverGSHdepletionofquailfedhighcholesteroldiets;(C)liverTBARsproductionofquailfedlowcholesteroldiets;(D)liverTBARsproductionofquailfedhighcholesteroldiets.*indicatesasignificant(p0.05)dietaryfatsourcedifference.=butter;A=beeftallow;0soybeanoil.80 60 40 2001234560mMH202addedC12345mMH202addedN C,, lb 0 I-N I’, 0 I-DT4_______________________I05101520mMH202added250510152025mMH202addedTable4.9LiverhomogenatesusceptibilitytoH202-inducedGSHdepletionandTBARsproductioninatherosclerosis-susceptibleJapanesequailfedexperimentaldiets.1GSHdepletion(%)TBARs(A532)0.5mMH2025.0mMH202Cholesterollevel(%byweight):0.050.50.050.5DietaryTreatment:Butter14.5±1.626.7±3.90.563±0.1450.299±0.028Tallow23.4±2.832.7±2.40.440±0.0080.228±0.024Soybean33.3±4.025.0±4.70.723±0.1560.260±0.047GSHdepletion(%)TBARs(A532)ANOVAp-value2CN.S.0.001F0.045N.S.1Valuesrepresentmean±SEM,n=12.GSH=glutathione;TBARs=2-thiobarbituricacidreactivesubstances.2C=cholesterolintakeleveleffect,F=dietaryfatsourceeffectby2-wayMANOVA.(21 (A)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 fromanimals 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 theexperimental atherosclerosis-susceptible Japanese quail are reported in Table 4.10. All dietarytreatment groups fed high cholesterol diets exhibited significant (p < 0.05) atherosclerotic plaquedeposition in the aortic tree compared to those fed low cholesterol diets (no plaques detected; Table4.10). Dietary fat source did not have an effect on the plaque score in the aortae of quail fed highcholesterol diets. Similar to the results with numerical plaque scores, the percentage of aortic lumencovered by plaque was significantly (p 0.05) influenced by dietary cholesterol intake level, but wasnot affected by dietary fat source (Table 4.10). Aortae from birds fed diets containing the high levelof 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 totalcholesterol concentrations (r = 0.872, p = 0.001) as well as between aortic area (%) covered byplaque and plasma total cholesterol (r = 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 severeplaque (scores of 4 in vessels, with a high percentage of lumen area covered) are presented in Figures4.3 and 4.4, respectively. Aortic tissue free from plaque involvement had an undulating, intact surfacewhen scanned by SEM (Figure 4.3). Ovoid protrusions from the lumen surface likely representednuclei and overlying cytoplasm (Figure 4.3; Peng eta!., 1985). Aortae from high cholesterol-fed quailwith plaque material visible by dissecting microscope exhibited distinct areas of raised tissue withdisruption of the epithelial cells when viewed by SEM (Figure 4.4A, B).154Table4.10Aorticplaquescoreandareacoveredinatherosclerosis-susceptibleJapanesequailfedexperimentaldiets.1Plaquescore2Areacovered(%)3Cholesterollevel(%byweight):0.050.50.050.5DietaryTreatment:ButterN.D.3.4±0.4N.D.80±10TallowN.D.3.8±0.1N.D.88±4SoybeanN.D.3.5±0.2N.D.79±7PlaquescoreAreacovered(%)ANOVAp-value4C<0.001<0.001FN.S.N.S.1Valuesrepresentmean±SEM,n=12.2Plaquescorebasedonscaleof0(N.D.)=cleansurface;1=5plaques;2=6-20plaques;3=>20plaques;4=massiveatheromasseen.Valuesrepresenttwojudgesevaluatinginadouble-blindprotocol.Areacovered(%)=percentofaorticepitheliumcoveredbyplaque,range0(N.D.)-100%.C=cholesterolintakeleveleffect,F=dietaryfatsourceeffectby2-wayMANOVA.- C;’01Figure 4.3 A representative scanning electron micrograph of aortic tissue from atherosclerosissusceptible 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 visualscoring scale.1 56ABFigure 4.4 Representative scanning electron micrographs of aortic tissue from atherosclerosissusceptible Japanese quail fed a high (0.5%) cholesterol diet. These micrographs depict theatherosclerotic luminal surface of aortic tissue with a plaque score of 4, which was determined usinga visual scoring scale. (A) This micrograph depicts the coverage of the aortic lumen by plaquecharacterized by a focal area of raised tissue; (B) depicts a close-up view of the same plaque area asshown in A, depicting epithelial cell damage in the aortic plaque.157Aortic plague cholesterol oxides:The quantitation of cholesterol and cholesterol oxide content of the non-saponifiable extractsof individual aortic tissue samples collected from experimental birds by GC analysis is presented inTable 4.11. Aortic tissue from birds fed high cholesterol diets consistently contained a greater amountof 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 extractsof aortic tissue from birds fed the high cholesterol diets. Also, there was individual variability in theprofile of cholesterol oxides detectable in aortic tissue between birds fed the different dietary fatsources, 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 cholesterol-fed birds (Table 4.11). Three cholesterol oxides (i.e. 5,6a-epoxy-5a-cholesterol, 7f-hydroxycholesteroland 7-ketocholesterol) were present in aortic tissue from birds fed butter, tallow or soybean oil-highcholesterol diets. Additional cholesterol oxides present in the aortae from birds fed the diet containingsoybean oil with high cholesterol were: 7a-hydroxycholesterol, 4B-hydroxycholesterol, cholestane-triol,and 25-hydroxycholesterol. The presence of elevated levels of tissue cholesterol, in addition tocholesterol oxides in aortae, was observed to coincide with the presence of atherosclerotic plaque inaortae from Japanese quail fed high cholesterol diets (Tables 4.10 and 4.11). The limited number ofsamples, as well as the individual variability of cholesterol oxide content in aorta from birds within adietary fat source treatment group, precluded presenting these data using means for each dietarytreatment group.158Table4.11GCquantitationofcholesterolandcholesteroloxidecontentofaortictissuefromatherosclerosis-susceptibleJapanesequailfedexperimentaldiets.1-a 01 CDCholesterolandOxides2DietaryScore3Cholesterol7a-OH5,6a-7/?-OH4fl-OHtriol7-keto25-OHTreatmentepoxideButter0.05%chol.0/0/01.61N.D.N.D.N.D.N.D.N.D.N.D.N.D.0/0/02.38N.D.N.D.N.D.N.D.N.D.N.D.N.D.0.5%chol.4/4/416.52N.D.0.510.41N.D.N.D.0.57N.D.3/4/419.17N.D.0.530.65N.D.N.D.0.38N.D.Tallow0.05%chol.0/0/02.68N.D.N.D.N.D.N.D.N.D.N.D.N.D.0/0/02.38N.D.N.D.N.D.N.D.N.D.N.D.N.D.0.5%chol.4/4/419.67N.D.N.D.0.44N.D.N.D.0.53N.D.4/4/419.03N.D.0.260.36N.D.N.D.0.37N.D.Soybean0.05%chol.0/0/02.20N.D.N.D.N.D.N.D.N.D.N.D.N.D.0/0/02.71N.D.N.D.N.D.N.D.N.D.N.D.N.D.0.5%chol.4/4/428.30N.D.0.160.610.16N.D.0.42N.D.4/4/424.820.121.060.600.050.250.800.544/4/423.990.180.140.630.080.070.360.74inctiviaualbirdaortictissueplaquescore,cholesterolandoxideconcentrationmeasurebygaschromatography-massspectrometry.2Cholesterolconcentration=mg/gtissue;oxideconcentration=mg/gtissue;N.D.=notdetected.7a-OH=7a-hydroxycholesterol;5,6a-epoxide=5,6a-epoxy-5a-cholesterol;7fl-OH=7fl-hydroxycholesterol;4l-0H=41-hydroxycholesterol;triol=cholestane-triol;7-keto=7-ketocholesterol;25-OH=25-hydroxycholesterol.Plaquescoreisbasedonscaleof0(N.D.)=cleansurface;1=5plaques;2=6-20plaques;3=>20plaques;4=massiveatheromasseen.Valuesrepresent2judgesevaluatinginablindedprotocol.x/y/z=individualscoreforeachofthreevesselsinaortictree.Discussion:The role of dietary fat source in contributing to the development of atherosclerosis is thoughtto 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 beenfound to be just as effective as PUFA in lowering plasma cholesterol concentrations when substitutedfor saturated fatty acids in the diet (Mattson and Grundy, 1985). A neutral or hypocholesterolemiceffect 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 lowerthan in those counterparts fed butterfat diets (Kris-Etherton eta!., 1993). Polyunsaturated fatty acidssuch as linoleic (Cl 8:2,n-6) and linolenic (Cl 8:3,n-3) acids present in significant amounts in soybeanoil (approx. 52.6 and 8%, respectively) have been reported to be hypocholesterolemic whensubstituted for saturated fatty acids in the diet (Mott eta!., 1992; Vega eta!., 1982). Long-termclinical studies have reported that a reduction in plasma lipid levels is associated with a reduced riskfor coronary heart disease in middle-aged hypercholesterolemic men (Lipid Research Clinics Program,1984). Animal studies provide supporting evidence of the requirement for elevated plasma cholesterolconcentrations to induce atherosclerotic lesions in animal feeding trials (Nishina eta!., 1993; Smith andHilker, 1973). These data, together with epidemiological and clinical reports of an association betweenincreased susceptibility of tissues to lipid oxidation and impaired endogenous antioxidant status in vivoin coronary heart disease patients (Buczynski eta!., 1993; Jayakumari etal., 1 992), suggest a role forlipid peroxidation in the development of atherosclerosis. The present experiment describes the effectsof diets containing dietary fat sources varying in degree of saturation (i.e. short versus long-chainsaturated fatty acids in butter and beef tallow, respectively) and unsaturation (n-6 polyunsaturatedfatty acids in soybean oil) as well as level of cholesterol intake on the plasma lipid profile, aorticplaque deposition and associated cholesterol and cholesterol oxide content, as well as the endogenousantioxidant status in atherosclerosis-susceptible Japanese quail.160Plasma 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 comparedto sources of polyunsaturated fatty acids. However, the short-chain saturated fatty acids of chainlength from C4:0 to C1O:O have been shown not to have any plasma cholesterol-raising effect inhumans fed formula diets containing either butter or a short-chain triacylglyceride preparation isolatedfrom coconut oil (Hashim et a!., 1960). In studies with normocholesterolemic andhypercholesterolemic 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 presentstudy, atherosclerosis-susceptible Japanese quail fed diets containing butter with a total dietary fatcontent equivalent to that present in regular commercial bird chow (approx. 10%) were nothypercholesterolemic compared to birds fed beef tallow or soybean oil. This result was consistent inbirds fed either the low (0.05%) or high (0.5%) level of cholesterol in the diets. Japanese quail feddiets with a higher content of both butter fat and cholesterol (15 and 2%, respectively) for 3 weekor 3 month periods also did not exhibit elevated plasma cholesterol concentrations compared to cornor hydrogenated corn oil diets (Toda eta!., 1988). More recently, diets containing 2% cholesterol and15% 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 counterpartsfed 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 andWKY rats fed semi-synthetic diets containing butter also did not exhibit elevated plasma cholesterolconcentrations compared to those fed beef tallow or soybean oil.The long-chain polyunsaturated fatty acids of oilseeds, such as linoleic acid, have been reportedto 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 dietscontaining soybean oil diets with either a low or high level of cholesterol did not exhibit reduced plasmacholesterol levels. The plasma cholesterol concentrations of quail fed soybean oil diets herein werenot significantly different from counterparts fed diets containing butter or beef tallow. A greater effect161of the n-6 polyunsaturated fatty acids in the soybean oil diets on plasma cholesterol levels might havebeen obtained if a high fat diet (e.g. 16% (wtlwt) or 40% of calories) had been used as previouslyreported 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 quailwas not pursued in this study due to the potentially non-physiological effects of feeding high energyatherogenic 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 workersreported that plasma TG concentrations were increased maximally at 0.25% dietary cholesterolsupplementation, without further increases with dietary cholesterol levels up to 2%. Lower levels ofdietary cholesterol supplementation, from 0.05 to 0.1% cholesterol, did not increase plasma TG levelsabove that of the basal cholesterol-free diet. Cholesterol feeding was also observed to increase in vitrosecretion of VLDL-TG and cholesterol in perfused livers of rats, up to a maximum at a dietarycholesterol level of 0.5% in these same animals (Fungwe eta?., 1992). Subsequent studies examiningthe effects of dietary cholesterol supplementation on plasma and hepatic TG concentrations in viva andin vitro indicated that the hypertriacylglyceridemic effect of dietary cholesterol feeding was associatedwith increased hepatic TG and cholesteryl ester concentrations (Fungwe eta?., 1993, 1994). Theincreased concentrations of hepatic lipids were proposed to be the result not only of the reducedoxidation of fatty acids in the livers of cholesterol-fed rats, but also increased hepatic synthesis of fattyacids and TG (Fungwe et a?., 1993, 1994). The data from the present study in cholesterol-fedJapanese quail indicate that feeding a high level of cholesterol resulted in the altered visual appearanceof 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 H20 in thepresent study was in contrast to previous reports of significant MDA production in RBCs fromJapanese quail when tertiary-butyl hydroperoxide (t-BHP) was used as the oxidizing agent (Godin eta?., 1994; Godin and Dahlman, 1993). While both t-BHP and H20 are strong oxidizing agents, the162latter was used in these experiments because it is a reactive oxygen species of significance in vivocompared 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 heartdisease patients has been reported to be impaired. For example, the activities of specific antioxidantenzymes such as SOD and GSH-Px have been reported to be reduced, while levels of lipid peroxidesare increased in tissues from such patients (Buczynski eta!., 1993; Jayakumari eta!., 1992). Animalmodels of human atherosclerosis have also been demonstrated to exhibit increased levels of lipidoxidation products in diseased aortae in association with decreased activity of specific antioxidantenzymes (Godin at a!., 1994; Wang eta!., 1989). In studies with Japanese quail fed a commercialchow diet supplemented with a high level of cholesterol, Wang and coworkers (1989) reported thataortic tissue from birds fed the atherogenic diet exhibited higher levels of lipid peroxides and reducedactivity of GSH-Px. More recently, Godin and coworkers (1994) reported that the activity of GSH-Pxand SOD in the plasma was positively correlated with plasma cholesterol and triacylglycerideconcentrations, whereas aortic SOD activity was negatively correlated with both plasma cholesterolas 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 bycholesterol feeding in these same animals, suggesting that these endogenous antioxidant componentswere not involved in determining the susceptibility of these birds to aortic atherosclerotic plaquedevelopment (Godin eta!., 1994). It is noteworthy that very few differences in tissue antioxidantenzyme activity were observed between atherosclerosis-susceptible and -resistant strains of Japanesequail by these workers. While the activity of aortic tissue GSSG-Red was reduced in atherosclerosissusceptible compared to -resistant quail, activity of this enzyme was not significantly altered in birdswith dietary cholesterol-induced aortic plaque (Godin eta!., 1994). On the other hand, while plasmaGSH-Px and SOD activities were increased by cholesterol feeding, this response was common to bothstrains of quail and thus, could not be associated with the deposition of aortic plaque in the susceptiblestrain. In the present study with atherosclerosis-susceptible Japanese quail fed defined semi-purified163diets containing 0.5% cholesterol with 0.25% cholic acid, plasma total cholesterol concentrations weretwo-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 hereinwas also greater than previously observed by Godin and coworkers (1994). Therefore, the finding thatthere 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 enzymeactivity of these tissues is relatively insensitive to the effects of hypercholesterolemia in this animalmodel. However, depletion of GSH from heart tissue in vitro was enhanced in quail fed the high levelof dietary cholesterol herein. This latter result was opposite to the heart GSH depletion resultsobserved in SHR and WKY animals (i.e. no cholesterol treatment effect) fed a high level of cholesterolin Chapter 2. However, previous studies with wild-type Japanese quail fed commercial chow dietssupplemented with cholesterol (1 %) and cholic acid (0.5%) did demonstrate enhanced depletion ofheart 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 antioxidantstatus in the atherosclerosis-susceptible Japanese quail has been investigated. Previous reports haveexamined the effect of saturated versus polyunsaturated dietary fats on severity of aortic plaque inquail (Toda and Oku, 1995; Toda eta!., 1988; Smith and Hilker, 1973), but no attempt was made tocharacterize biochemical indices which might be influenced by dietary fatty acid composition. In recentyears, concern has been expressed about the effect on lipid peroxidation and antioxidant status in vivoof increased consumption of polyunsaturated fatty acids to reduce the incidence of cardiovasculardisease. In fact, several studies have demonstrated an increased requirement for antioxidants whenhighly polyunsaturated fatty acids are consumed (Skiladóttir et al., 1994; Gonzalez et a!., 1992; DeSchrijver eta!., 1992; L’Abbé eta!., 1991). Previously in Chapter 2, it was demonstrated that adietary fat source high in polyunsaturated fatty acids, namely fish oil, could influence various tissueantioxidant enzyme activities in SHR and WKY rats. Glutathione peroxidase is a primary antioxidantenzyme involved in the inactivation and detoxification of lipid peroxides and hydrogen peroxide (Pagliaand Valentine, 1967). It is of interest to note that while GSH-Px activity was increased in RBCs of164soybean oil-fed quail, heart tissue activity of this enzyme was reduced in these same animals. It isconceivable that increased incorporation of n-6 fatty acids into RBC membranes could increase therequirement 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 observedin Chapter 3 as well as the present study was more severe (scores of 3 - 4 in all three vessels of theaortic tree) and much less variable than in previously reported studies with cholesterol-fed Japanesequail (Godin etal., 1994; Shih eta!., 1983; Smith and Hilker, 1973). Previous workers using the samestrain of atherosclerosis-susceptible Japanese quail (i.e. birds descended from the original line describedby Shih et al., 1 983) as used in Chapter 3 have reported greater variations in plaque scores whenbirds 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 instudies using normal Japanese quail fed high fat (40-53% of calories) semi-synthetic dietssupplemented 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 (Wanget a!., 1989). Other studies in which the dose-dependent nature of dietary cholesterol-inducedatherosclerosis in susceptible Japanese quail was investigated reported that while there was noincidence 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 levelof 1 .5% corn oil, or 16% beef tallow or corn oil with and without added cholesterol (0.5%), theseverity of aortic plaque deposition was observed to vary with dietary fat type, cholesterol level andduration of the feeding period (Smith and Hilker, 1973). Birds fed the dietary fats without cholesterolsupplementation did eventually develop aortic plaque after 9 months, although there did not appearto be a dietary fat source treatment effect. These workers reported that birds fed corn oil dietssupplemented with cholesterol did not exhibit an appreciable increase in aortic plaque deposition, evenafter 9 months of treatment. In contrast, birds fed beef tallow diets supplemented with cholesterol165exhibited increased aortic plaque after 6 and 9 months of feeding (Smith and Hilker, 1973). It isnoteworthy that another group of birds fed an atherogenic diet containing 20% beef tallowsupplemented with 1 % cholesterol achieved aortic scores similar to those observed in Chapter 3 andthe present study after 9 months (Smith and Hilker, 1973). Thus, while atherosclerosis can occurspontaneously in Japanese quail, the time and conditions for induction appear to vary between studiesdue to differences in dietary treatment (i.e. dietary cholesterol intake levels ranging from 0.5- 5%)and length of study (i.e. several weeks or months). The use of an inbred strain of susceptibleJapanese 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, hydrogenatedcorn oil, or butterfat with 2% cholesterol, Toda and coworkers (1988) reported that the atherogenicityof butterfat diets was intermediate between corn oil (greatest luminal narrowing) and hydrogenatedcorn oil (least aortic luminal thickening). However, these workers did not observe any differences inthe degree of hypercholesterolemia between these dietary treatment groups. More recently, corn oil-or palmitic acid- (15% fat plus 2% cholesterol) fed Japanese quail exhibited markedhypercholesterolemia and severe aortic plaque compared to counterparts fed medium-chaintriacylglycerides which exhibited only mild increases in plasma cholesterol and aortic thickening (Todaand Oku, 1995). This latter study clearly showed the relationship between plasma cholesterolconcentrations and aortic plaque development when different dietary lipids are fed to quail. The resultsof the present study with atherosclerosis-susceptible Japanese quail fed diets containing 8% dietaryfat with 0.5% cholesterol and 0.25% cholic acid strongly suggest that the addition of bile acid to dietsgreatly increased dietary cholesterol absorption, and thereby enhanced the level ofhypercholesterolemia achieved in these birds. Thus, the effects of dietary fat source on plasmacholesterol concentrations were minimal compared to the cholesterol treatment effect in the presentstudy.Aortic tissue cholesterol and cholesterol oxides content:Previously, in Chapter 3, the presence of two cholesterol oxides (i.e. 71-hydroxycholesterol and7-ketocholesterol) was confirmed in the aortic tissue from atherosclerosis-susceptible Japanese quailfed an atherogenic diet containing beef tallow. The role of oxidized cholesterol species and oxidatively166modified LDL in the initiation and progression of atherosclerosis has been substantiated by reports ofsupporting evidence for the presence of oxidized LDL in the plasma and diseased aortae of animalmodels (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 oxidehave 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 beendemonstrated by in vitro cell culture (Caboni et al., 1994; Hennig and Boissonneault, 1987; Peng eta!., 1978), intravenous administration to animal models (Peng eta!., 1985) as well as in animalfeeding studies (Donaldson, 1982). Japanese quail fed oxidized cholesterol in the diet exhibited greaterplasma and liver cholesterol concentrations in association with increased severity of atheroscleroticlesions as compared to animals fed purified cholesterol (Donaldson, 1982). When 25-hydroxycholesterol and cholestane-triol were administered intravenously to rabbits, SEM scans of aortictissue revealed balloon-like protrusions and crater-like defects on the luminal surface, with adheringplatelets (Peng et a!., 1985). Transmission EM examination of aortic tissue from these same animalsshowed 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. cholesterol5,6a- 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 presentstudy, atherosclerotic aortae from birds fed butter and beef tallow saturated fat diets containedcholesterol-5,6a-epoxide, 71..-hydroxycholesterol and 7-ketocholesterol, whereas those from birds fedsoybean oil contained in addition 7a-hydroxycholesterol, 41.-hydroxycholesterol, cholestane-triol, and25-hydroxycholesterol. It is important to note that there was some individual variability in COPsspecies present in aortic samples from the same dietary treatment group. While it is noteworthy thatonly aortic specimens from birds fed n-6 polyunsaturated fatty acids in the soybean oil diet exhibiteddetectable amounts of the notably cytotoxic 25-hydroxycholesterol and cholestane-triol COPs, the167other 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 aortaein the present study, and others, is the fatty acid content of plasma lipoproteins, which would reflectthe dietary fatty acid content somewhat (Toda and Oku, 1995; L’Abbé eta!., 1991). In vitro studiesexamining the susceptibility of LDL to oxidation have reported that LDL with an elevated oleic tolinoleic 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 lipidperoxidation when subjects were fed diets enriched with oleic acid compared to diets high in linoleicacid. When Jialal and coworkers (1991) exposed LDL collected from normolipidemic subjects to invitro CuSO4-catalyzed oxidation, oxidation rates of LDL varied between subjects, with the major COPdetected identified as 7-ketocholesterol. The elevated tissue concentrations of cholesterol incombination with the detection and quantitation of cholesterol oxides in diseased aortae fromatherosclerosis-susceptible Japanese quail confirm the similarities in aortic plaque composition betweenthis animal model and human atherosclerosis. The SEM micrographs obtained of undiseased anddiseased aortic tissue from experimental quail validates the dissecting microscope scoring protocol fordetecting epithelial cell layer damage in these animals.168Conclusion:In conclusion, atherosclerosis-susceptible Japanese quail exhibited hypercholesterolemia andhypertriacylglyceridemia 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 inbirds fed high cholesterol diets, there were no changes in the tissue antioxidant parameters measuredwhich could be attributed to a cholesterol intake level effect in these same animals. Moreover, dietaryfat sources differing in saturated fatty acid composition (i.e. short-chain versus long-chain saturatedfatty acids in butter and beef tallow, respectively) and n-6 polyunsaturated fatty acids (i.e. soybeanoil) did not result in significant differences in plasma lipid concentrations. Interestingly, RBC and heartGSH-Px activities were different in soybean oil-fed animals. The hypercholesterolemia observed in quailin the present study coincided with not only increased levels of aortic tissue cholesterol, but also thepresence of several cholesterol oxide species in the diseased aortic tissue collected from experimentalquail. Within each dietary fat group, there was individual tissue variability in the profile and amountsof cholesterol oxides detected, even though plaque scores determined visually were quite similarbetween birds in a given treatment group.The results of the present study indicate that at the moderate, nutritionally adequate level ofdietary fat fed to these quail, neither plasma lipid profile, nor susceptibility to atherosclerosis wasaffected by dietary fat source in the atherosclerosis-susceptible Japanese quail. While feeding quaila moderate level of fat and a high level of dietary cholesterol with cholic acid resulted in dyslipidemiaand severe atherosclerosis, the fact that the endogenous antioxidant status of tissues was notobserved to be altered by dietary cholesterol treatment or plaque deposition in these same animalssuggests that at the extreme plasma lipid concentrations achieved it was unlikely that tissueantioxidant 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 cholesterolconcentrations on aortic plaque development and tissue antioxidant status are indicated by the resultsreported herein.169CHAPTER 5Influence of increased caloric intake from beef tallow on plasma lipids, antioxidant statusand diet-induced atherosclerosis in atherosclerosis-susceptible Japanese quail.Introduction:Evidence derived from both epidemiological (Nordoy and Goodnight, 1990; Lipid Research ClinicProgram, 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 responseswith dietary fat or dietary cholesterol intake. The fatty acyl component of dietary fats, whichcomprises 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 theatherogenic 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-cholesterolconcentrations observed with dietary treatments rich in saturated fat or cholesterol. Elevated levelsof lipid peroxides have also been reported in blood and tissue of individuals with hypercholesterolemiaand 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 betweendietary fat and cholesterol levels on plasma lipidemia (Lin et a?., 1992), only a few studies haveattempted to examine the significance of this interaction on the severity of atherosclerosis (Nishina eta?., 1993). The combination of high fat and cholesterol in diets fed to rabbits has been shown to resultin profound changes in aortic lipid peroxidation and antioxidant enzyme activities indicating animportant role for endogenous antioxidant systems in preventing tissue lipid peroxidation reactions(Mantha eta!., 1993). Exogenous antioxidants such as ascorbic acid, a-tocopherol and butylatedhydroxytolulene (BHT) have been shown to reduce plasma cholesterol (Westrope et a?., 1 982) andinhibit lipoprotein oxidation (Morel eta!., 1994), albeit the effectiveness in suppressing atherosclerosiswas variable.The Japanese quail has been shown in previous experiments herein (Chapters 3 and 4) andby other investigators (Godin eta!., 1994; Radcliffe eta!., 1982) to develop atherosclerotic plaquewhen fed diets containing a moderate level of fat with cholesterol. In these studies, a marked increase170in plasma total cholesterol concentrations, attributed to feeding high levels of dietary cholesterol, wasassociated with the development of plaque in these animals. In Chapter 4, atherosclerosis-susceptibleJapanese quail fed semi-synthetic diets supplemented with cholesterol and cholic acid developedmarked hyperlipidemia and severe aortic plaque. The results of this previous study indicated that theantioxidant status of tissues was not altered by cholesterol feeding or the development of aorticplaque, suggesting that the effect of extreme plasma lipid levels had the greatest effect onsusceptibility to the development of aortic plaque in these birds. Also, previous workers feedingcommercial chow diets supplemented with cholesterol have demonstrated a less severe plasma lipidand 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 levelof saturated fat (i.e. 6 and 12% beef tallow) were used to examine the potential interaction of dietaryfat level and cholesterol quantity on plasma lipids, endogenous antioxidant status and susceptibilityto atherosclerosis in the atherosclerosis-susceptible Japanese quail.171Hypothesis for Chanter 5:Increased total dietary caloric content from beef tallow and dietary cholesterol level haveindependent or interactive effects on plasma lipids, tissue antioxidant status and severity of aorticplaque in atherosclerosis-susceptible Japanese quail.Obiective for Chapter 5:To examine the potential independent and/or interactive effects of increased caloric intake fromsaturated fat with dietary cholesterol level on plasma lipid concentrations, endogenous antioxidantstatus 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 willbe determined in atherosclerosis-susceptible Japanese quail fed non-purified, commercial diets differingin 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 becompared to a reference group fed the unsupplemented commercial diet alone.ii. Specific tissue antioxidant enzyme activities and susceptibility of tissues toH20-inducedlipid peroxidation in vitro will be measured in atherosclerosis-susceptible Japanese quail to determinethe role of antioxidant status in modulating the severity of aortic plaque when quail are fed dietsvarying in level of dietary fat and cholesterol.iii. Aortic tissue cholesterol and cholesterol oxide content and associated ultrastructural tissuechanges will be used to further characterize the composition of aortic plaque in describing the severityof atherosclerosis in the atherosclerosis-susceptible Japanese quail.172Materials 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 treatmentgroups (n = 12) consisting of a reference group (diet A) fed a commercial Turkey Starter (TS) chowdiet containing 5.4% beef tallow (Otter Co-op, Aldergrove, B.C.) diet, two groups fed TSsupplemented 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 andcholesterol is summarized in Table 5.1. The basal TS diet was supplemented with beef tallow (CargillFoods, High River, AB), which was slowly melted over gentle heat (10-15 mm. at 45-50°C) to ensureuniform distribution of cholesterol and cholic acid (2:1 ratio) into the crumbled commercial diet. Theadditional dietary fat and sterols were thoroughly mixed into diets in an aluminum mixing bowl usinga Hobart mixer. After mixing, individual diets were stored in double, dark plastic bags in a walk-infreezer (-1 5°C) throughout the experimental study. A sample of each experimental diet was removedfor analysis of fatty acid, gross energy and dry matter content as previously described in Chapter 2.Experimental diets were isonitrogenous with diets A - C containing a comparable level of energy (e.g.17.21 - 17.97 kJ/g; Table 5.1), and diets D - E containing an greater amount of total energy (e.g.18.76 - 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 wasdetermined by gas chromatography as previously described in Chapter 3 is summarized in Table 5.2.173Table 5.1 Composition and energy content of diets fed to quail.1Supplemented Component: (gIlOOg) A B C D ETallow fat 0.6 0.6 6.6 6.6Cholesterol 0.05 0.5 0.05 0.5Cholic Acid 0.025 0.25 0.025 0.25Crude Lipid (%) 10.0 10.3 10.3 16.1 16.1Gross Energy (kJ/g) 17.21 17.62 17.97 18.76 18.831 Composition of reference diet A (Turkey Starter; TS, Otter Co-op; Aldergrove, B.C.), weight %: Alfalfameal, 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.174Table 5.2 Fatty acid profile of diets fed to quail.1PolyunsaturatedLinoleic (C18:2)Linolenic (Cl 8:3)Arachidonic (C20:4)20.02.30.219.4 19.4 15.02.4 2.4 1.80.2 0.2 0.215.01 .80.21 Diet A = Turkey Starter (TS); B = TS + 0.05% chol.; C =D = TS + 6% Tallow + 0.05% chol.; E = TS + 6% TallowP/S = polyunsaturated to saturated fatty acid ratio; n-61n-3 =TS + 0.5% chol.;+ 0.5% chol.n-6 to n-3 polyunsaturated fatty acid ratio.DIETSA B C D EFatty acid (weight %):SaturatedLauric (C12:0) 0.1 0.1 0.1 0.1 0.1Myristic(C14:0) 1.5 1.6 1.6 2.2 2.2Palmitic (C16:0) 21.0 21.0 21.0 22.0 22.0Stearic(C18:0) 9.8 9.7 9.7 10.8 10.8Arachidic (C20:0) 0.3 0.3 0.3 0.2 0.2Behenic (C22:0) 1.2 1 .3 1.3 1.0 1.0MonounsaturatedMyristoleic (C14:1) 0.2 0.2 0.2 0.4 0.4Palmitoleic (C16:1) 2.9 3.0 3.2 3.3 3.3Oleic (C18:1) 37.9 37.5 37.5 40.1 40.1Eicosenoic (C20:1) 0.9 1.4 1.4 0.9 0.9Total Saturates 33.9 33.8 33.8 35.8 35.6Total Unsaturates 64.4 64.1 64.3 61 .7 61.7P/S 0.66 0.65 0.65 0.47 0.47n-6/n-3 8.7 8.2 8.2 8.4 8.4175Animal feeding:Quail were housed in heated brooder cages with one treatment group per brooder cage. Feed anddistilled deionized water were available to birds ad ilbitum. Diets were replaced daily to minimizeoxidation 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 decapitatedand trunk blood collected into chilled heparinized tubes and plasma separated as described in Chapter4. Similarly, aliquots of plasma, RBCs, and heart and liver tissues were collected for analysis of plasmalipids and antioxidant status, respectively, as previously described in Chapter 2. Dissection and scoringof the aortic tree was performed as described in Chapter 4. Similarly, preparation of aortic tissue forantioxidant enzyme analysis was performed as outlined in Chapter 4. Again, additional birds in eachdietary treatment group were used to provide aortic specimens for GC-MS analysis of cholesteroloxides 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.) wasused to test for differences between experimental treatment groups. Where differences did exist, thesource of the differences at a p < 0.05 significance level was identified by the Student-Newman-Keulsmultiple range test. Two-way multiple analysis of variance (MANOVA) was used to identify anyinteractions between level of dietary fat and dietary cholesterol level.176Results:Lipid and energy content of experimental diets:The commercial Turkey Starter (TS) diet contained soybean meal, corn and alfalfa meal as primarysources 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. Supplementationof the TS diet (diet A) with small quantities of beef tallow and cholesterol in diets B and C did not alterthe energy content or crude lipid content significantly (Table 5.1). The supplementation of the TS dietwith 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 ofcholesterol at either supplementation level, 0.05% (wt!wt) in diets B and D or 0.5% in diets C andE, did not have an effect on the dietary fatty acid composition. Beef tallow supplementation of theTS diet resulted in slight increases in the saturated fatty acid (C16:0 and C18:0) content, a relativelylarger 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 compositionresulted in a reduction in P/S ratio, but similar n-6/n-3 ratios in diets D and E compared to the referenceTS 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 leveltreatments (range 1 35 ± 2 g). Also, weights of heart tissue were not different between dietarytreatment 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 liversfrom counterparts fed low cholesterol diets (diets B and D) and the reference TS diet (diet A; range2.3 ± 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 andexperimental diets is shown in Table 5.3. Birds fed the reference TS diet exhibited similar plasma total177Table 5.3 Plasma cholesterol and triacylglyceride concentrations in atherosclerosis-susceptibleJapanese quail fed experimental diets1.DIETS2A B C D E ANOVAp-value3Plasma lipids:Total cholesterol 5.33 7.06 36.8 7.75 43.4 C < 0.001(mmol/L) ±0.32 ±0.38 ±4.9 ±0.36 ±3.5 L N.S.CxL N.S.Triacylglyceride 1.89 3.57 7.81 2.73 11.8 C < 0.001(mmol/L) ±0.19 ±0.76 ±1.51 ±0.38 ±1.8 L N.S.CxL 0.051 Values represent mean ± SEM, (n = 1 2)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 leveltreatment interaction by 2-way ANOVA.178cholesterol 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 fedthe high cholesterol diets (diets C and E; Table 5.3). The plasma cholesterol concentrations of quailfed diet E were slightly, but not significantly higher than counterparts fed diet C, as shown by theabsence 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 triacylglycerideconcentrations than counterparts fed the tallow and cholesterol supplemented experimental diets (Table5.3). A significant (p < 0.05) interaction was observed between dietary fat level and cholesterolcontent for plasma triacylglyceride concentrations, as a result of the considerably elevated plasmatriacylglyceride 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) andexperimental diets (diets B to E) were similar, indicating no effect of dietary fat level or cholesterolsupplementation 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 experimentaldiets are presented in Table 5.5. Similar to the results in RBCs, heart tissue from quail fed dietsvarying in dietary fat level and cholesterol content did not exhibit any changes in GSH-Px, GSSG-Redand SOD activities (Table 5.5).iii. Liver tissue antioxidant enzymes:The hepatic antioxidant enzyme profiles of quail fed the reference TS (diet A) and experimentaldiets are presented in Table 5.6. Hepatic CAT activities were greater (p < 0.05) in birds fed the TSreference diet, but were not affected by the level of dietary cholesterol, or the level of dietary fat inthe experimental diets (diets B to D; Table 5.6). The presence of the high level of cholesterol in dietsC and D significantly (p < 0.05) reduced liver SOD activities compared to counterparts fed a low levelof cholesterol by 24 and 26 per cent, respectively. There were no dietary treatment differences in179Table 5.4 Antioxidant enzyme activities of red blood cells of atherosclerosis-susceptible Japanesequail fed experimental diets1.DIETS3A B C D E ANOVAp-value4Antioxidant EnzymeActivity2:SOD 4.52 5.27 5.62 5.42 5.48 C N.S.(U/mg Hb) ±0.49 ±0.31 ±0.11 ±0.24 ±0.17 L N.S.CxL N.S.GSSG-Red 10.80 9.69 10.20 8.91 9.42 C N.S.(nmoles NADPH/ ± 0.35 ± 0.47 ±0.30 ± 0.39 ± 0.48 L N.S.mm/mg Hb) CxL N.S.GSH-Px 104 146 142 139 139(nmolesNADPH/ ±5 ±9 ±5 ±5 ±10mm/mg Hb)1 Values represent mean ± SEM (n = 12).2 SOD = superoxide dismutase; GSSG-Red = glutathione reductase; GSH-Px = glutathioneperoxidase.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 leveltreatment interaction by 2-way ANOVA.C N.S.L N.S.CxL N.S.180Table 5.5 Antioxidant enzyme activities in heart tissue of atherosclerosis-susceptible Japanesequail fed experimental diets.1DIETS3ANOVA___________________________________________________p-value4Antioxidant EnzymeActivity2:SOD 1.51 1.40 C N.S.(U/mg tissue ± 0.07 ± 0.08 L N.S.wet wt) CxL N.S.0.46 C N.S.±0.02 L N.S.CxL N.S.1.89 C N.S.±0.21 L N.S.CxL N.S.1 Values represent mean ± SEM (n = 12).2 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 leveltreatment interaction by 2-way ANOVA.A B C D E1.71±0.130.48±0.022.59±0.191.31±0.170.47±0.01GSSG-Red(nmoles NADPH/min/mg tissue wet wt)GSH-Px(nmoles NADPH/min/mg tissue wet wt)1.41±0.090.45± 0.011 .52± 0.290.45±0.021.64 1.82±0.28 ±0.18181Table 5.6 Antioxidant enzyme activities in liver tissue of atherosclerosis-susceptible Japanese quailfed experimental diets1DIETS3AntioxidantEnzyme Activity2:A B C D E ANOVAp-value4CAT(kig tissuewet wt)SOD(U/mg tissuewet wt)GSSG-Red(nmoles NADPH/mm/mg tissue wetwt)GSH-Px(nmoles NADPH/mm/mg tissue wetwt)5.41 5.62 C 0.05±0.70 ±0.77 L N.S.CxL N.S.6.09 C 0.002±0.64 L N.S.CxL N.S.2.45 C N.S.±0.11 L N.S.CxL N.S.3.40 C N.S.±0.27 L N.S.CxL N.S.SOD = superoxide dismutase; GSSG-Red =10.47± 1.207.24±0.692.60±0.144.65±0.536.22± 0.897.67±0.092.41±0.173.45± 0.215.58± 0.395.84± 0.322.44±0.153.50± 0.268.33±0.472.66± 0.123.50±0.261 Values represent mean ± SEM (n = 12).2 CAT = catalase, k = first order rate constant (sec;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 leveltreatment interaction by 2-way ANOVA.182hepatic GSSG-Red and GSH-Px activities of quail ted the reference TS diet (diet A) or the experimentaldiets (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 thelevel 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 notdifferent between dietary treatment groups of quail fed non-purified commercial TS diets supplementedwith 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 thediet (diets C and E) or by the increased caloric contribution of a higher level of fat in the diet (dietsD and E; Table 5.9, Figure 5.1A, B). The supplementation of the commercial TS diet (diet A) withadded beef tallow (diets D and E) reduced (p 0.05) the formation of TBARs in heart tissue (Table5.9, Figure 5.1C, D). Moreover, the combination of the high level of cholesterol with added beeftallow in diet E further reduced the production of TBARs in heart tissue of quail fed this diet, asdemonstrated 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 betweenlevel of dietary fat and cholesterol content was not observed for TBARs production in liver tissue.183Table 5.7 Antioxidant enzyme activities in aortic tissue of atherosclerosis-susceptible Japanesequail fed experimental diets.1DIETS3Antioxidant EnzymeActivity2:A B C D E ANOVAp-value4SOD(U/mg tissuewet wt)1.31 1.06±0.32 ±0.131.01 0.96±0.19 ±0.121.14 C N.S.±0.10 L N.S.CxL N.S.GSSG-Red(nmoles NADPH/min/mg tissue wet wt)GSH-Px(nmoles NADPH/min/mg tissue wet wt)0.32 C N.S.±0.01 L N.S.CxL N.S.0.78 C N.S.±0.04 L N.S.CxL N.S.1 Values represent mean2 SOD = superoxide dismutase;peroxidase.Diet A = Turkey Starter (TS); B =0.05% chol.; E = TS + Tallow + 0.5% chol.C = cholesterol treatment effect, L = fat level treatment effect, CxL = cholesterol x fat leveltreatment interaction by 2-way ANOVA.0.38 0.35 0.34 0.30± 0.04 ± 0.02 ± 0.02 ± 0.020.84 0.85 0.85 0.81±0.05 ±0.09 ±0.10 ±0.05± SEM (n = 12).GSSG-Red = glutathione reductase; GSH-Px = glutathioneTS + 0.05% chol.; C = TS + 0.5% chol.; D = TS + Tallow +184Table 5.8 Basal glutathione levels in heart and liver homogenate and red blood cells ofatherosclerosis-susceptible Japanese quail .DIETS2A B C D E ANOVAp-value3Parameter:RBC(nmoles GSH/mgRBC)Heart(nmoles GSH/mgtissue wet wt)Liver(nmoles GSH/mgtissue wet wt)4.99 C N.S.±0.20 L N.S.CxL N.S.2.06 C N.S.±0.06 L N.S.CxL N.S.3.92 C N.S.±0.26 L N.S.CxL N.S.1 Values represent mean ± SEM (n = 12).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 leveltreatment interaction by 2-way ANOVA.4.56 4.73 5.08 4.49±0.33 ±0.25 ±0.13 ±0.311.92 1.99 1.94 2.08±0.06 ±0.07 ±0.06 ±0.103.21 4.00 3.76 3.50±0.26 ±0.18 ±0.19 ±0.22185Table 5.9 Heart and liver homogenate susceptibility toH20-induced GSH depletion and TBARsproduction in atherosclerosis-susceptible Japanese quail fed experimental diets.1DIETS31 Values represent mean ± SEM (n = 12). GSH = glutathione; TBARs =substances (A532 = absorbance at 532 nm)2-thiobarbituric acid reactive2 Heart GSH depletion (%) at 0.6 mM H20; Heart TBARs productiondepletion (%) at 0.5 mM H20; Liver TBARs production at 5.0 mM H20.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 leveltreatment interaction by 2-way ANOVA.at 1 .0 mM H20; Liver GSHA B C D E26.2±5.00.12±0.0234.1±7.00.12±0.02Tissue2:Heart:GSHdepletion (%)TBARs(A532)Liver:GSHdepletion (%)TBARs(A532)45.5±5.20.17±0.0245.4± 4.60.26± 0.0234.0±2.90.12± 0.0132.3±3.30.39±0.05ANOVAp-value4C N.S.L N.S.CxL N.S.C N.S.L 0.016CxL 0.035C 0.009L N.S.CxL N.S.C 0.003L N.S.CxL N.S.41 .3 32.6±2.8 ±5.933.4±5.30.09± 0.0147.5±5.20.26±0.020.670.080.31± 0.041861000 4)N C, 0 I-C 0 C CFigure5.1Susceptibilityofhearttissuefromatherosclerosis-susceptibleJapanesequail fedareferenceTurkeyStarter(TS)dietalone,orTSdietssupplementedwithvaryinglevelsofbeeftallowandcholesteroltoinvitroH20-induceddepletionofglutathione(GSH)andproductionofthiobarbituricacidreactivesubstances(TBARs).(A)heartGSHdepletionofquailfedlowcholesteroldiets;(B)heartGSHdepletionofquailfedhighcholesteroldiets;(C)heartTBARsproductionofquail fedlowcholesteroldiets;(D) heartTBARsproductionofquailfedhighcholesteroldiets.InA,B,C,andDthereferenceTSgroupisincludedforcomparisonpurposes.*indicatesasignificant(p0.05)dietaryfatleveldifference.=TS+0.6%tallow;A=TS+6.6%beeftallow:0=referenceTSgroup.80 60 40 20 00.000.501.001.502.002.50mMH202added0.600.500.400.300.200.100.000.000,501.001.502.00mMH202added2.5000,600.500.400.30V0.200.100.00*012345mMH202addedIIII01234mMH202added5-.31001004, 4’mMHO2addedmMH202addedFigure5.2Susceptibilityoflivertissuefromatherosclerosis-susceptibleJapanesequail fedareferenceTurke’Starter(TS)dietalone,orTSdietssupplementedwithvaryinglevelsofbeeftallowandcholesteroltoinvitroH20-induceddepletionofglutathione(GSH)andproductionofthiobarbituricacidreactivesubstances(TBARs).(A)liverGSHdepletionofquailfedlowcholesteroldiets;(B)liverGSHdepletionof quailfedhighcholesteroldiets;(C)liverTBARsproductionofquailfedlowcholesteroldiets;(D) liverTBARsproductionofquailfedhighcholesteroldiets.InA,B,C,andDthereferenceTSgroupisincludedforcomparisonpurposes.*indicatesasignificant(p0.05)dietaryfatleveldifference.=TS+0.6%tallow;A=TS+6.6%beeftallow;0=referenceTSgroup.80 6080406020g 4,400123456mMH202added0123456mMH202added0, In 0 I-2.502.001.601.000.500.002.50D2,00-—1.60/S//100//TI—-------0.500.00101520250510152025-a co coAortic plague score and percent area covered:The severity of plaque score in aortic tissue from quail determined by visual assessment using adissecting microscope is summarized in Table 5.10. Birds fed both the TS reference diet (diet A) aswell as diets supplemented with a low level of cholesterol (diets B and D) did not exhibit detectableaortic plaque (Table 5.10). On the other hand, inclusion of a high level of cholesterol in diets (dietsC and E) resulted in significant (p 0.05) aortic plaque development (Table 5.10). The severity ofaortic plaque in animals fed the latter diets was reduced in quail fed the low fat diet (diet C) comparedto counterparts fed the higher level of dietary fat (diet E), as demonstrated by the significant (p0.05) interaction recorded (Table 5.10). It is noteworthy that the percentage of aortic lumen coveredwith plaque from birds fed diet E was also significantly (p 0.05) greater than counterparts fed dietC, as demonstrated by the interaction recorded for this parameter (Table 5.10).Scanning electron micrographs of aortic tissue depicting examples of aortae without plaque (scoreof zero, with lumen clear of any signs of plaque using a dissecting microscope; Figure 5.3), moderatedevelopment of plaque (scores of zero and 2 in vessels; Figure 5.4) and with severe plaque (scoresof 4 in vessels, with a high percentage of lumen area covered; Figure 5.5) are presented in Figures5.3 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 bearranged longitudinally along the lumen wall, with the ovoid raised areas of the lumen surface likelyrepresenting nuclei and overlying cytoplasm (Figure 5.3; Peng eta!., 1985). Aortae which had amoderate amount of plaque deposition on the lumen surface exhibited slight surface irregularities alongwith distinct raised areas of cells, possibly representing epithelial cell damage with lipid infiltration andfocal areas of cell proliferation (Figure 5.4). Aortae from quail fed diet E containing an elevated levelof dietary fat and cholesterol had significant amounts of plaque material visible by dissectingmicroscope and exhibited distinct areas of raised tissue and clustered areas of enlarged epithelial cellswhen 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 characterizedby measuring the cholesterol and cholesterol oxide content of representative aortic tissue specimens189Table 5.10 Aortic plaque score and area covered in atherosclerosis-susceptible Japanese quail fedexperimental diets.1DIETS3A B C D E ANOVAp-value3Parameter2:Plaque Score N.D. N.D. 0.9 N.D. 2.0 C < 0.001±0.3 ±0.4 L N.S.CxL 0.02Area covered N.D. N.D. 16 N.D. 44 C < 0.001(%) ±7 ±111 Values represent mean ± SEM (n = 12).2 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 blindedprotocol. 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 leveltreatment interaction by 2-way ANOVA.L N.S.CxL 0.03190Figure 5.3 A representative scanning electron micrograph of aortic tissue from atherosclerosissusceptible Japanese quail fed either a reference Turkey Starter (TS) diet alone; similar results wereobtained 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 scoreof zero, which was determined using a visual scoring scale.191Figure 5.4 A representative scanning electron micrograph of aortic tissue from atherosclerosissusceptible Japanese quail fed a Turkey Starter (TS) diet supplemented with a high (0.5%) level ofcholesterol and a minor amount of additional beef tallow (0.6%). This micrograph depicts themoderate coverage of the luminal surface of aortic tissue with a plaque score of 2, which wasdetermined using a visual scoring scale.192ABFigure 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 ofcholesterol and a higher level of beef tallow (6.6%). These micrographs depict the atheroscleroticluminal 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 focalarea of raised tissue; (B) depicts a close-up view of the same plaque area as seen in A, showingenlarged epithelial cells on the surface of the aortic plaque.193from birds in each dietary treatment group, with the results presented in Table 5.11. Birds fed thereference TS diet (diet A) and the diets supplemented with the low level of cholesterol (diets B andD) without any detectable plaque on the luminal surface had similarly low levels of aortic tissuecholesterol (Table 5.11). In all cases, the combination of the low level of tissue cholesterol and theabsence of cholesterol oxides in aortic tissue of quail fed diets A, B and D were associated with anabsence of aortic plaque deposition (Table 5.11). Aortic tissue from quail fed diets containing thelower level of tallow with high cholesterol (diet C) contained approximately twice the amount of tissuecholesterol compared to counterparts fed diets A and B, as well as detectable levels of a singlecholesterol oxide, 5,6a-epoxy-5a-cholesterol (Table 5.11). The individual animal variability in plaquescore observed in quail fed diet C was associated with similar variations in both the amount of aortictissue cholesterol and cholesterol oxide content in these same animals. The absence of detectablecholesterol oxides in aortic tissue from some birds fed diet C corresponded to the relatively lower aorticcholesterol content and plaque scores in these animals (Table 5.1 1). Quail fed diets containing a highlevel of cholesterol and beef tallow (diet E) also exhibited individual variability in both aortic plaquescores, as well as cholesterol and cholesterol oxide content (Table 5.11). The higher level of tallowin combination with a high level of cholesterol in diet E fed to these quail resulted in the presence ofseveral 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 ofaortic cholesterol and presence of cholesterol oxides in atherosclerosis-susceptible Japanese quail inthe present study.194Table5.11GCquantitationofcholesterolandcholesteroloxidecontentofaortictissuefromatherosclerosis-susceptibleJapanesequailfedexperimentaldiets.1CholesterolandOxides2DietaryScore4Cholesterol5,6a-epoxide7/1-OHtriol7-keto25-OHTreatment3:A0/0/00.89N.D.N.D.N.D.N.D.N.D.A0/0/00.68N.D.N.D.N.D.N.D.N.D.B0/0/00.76N.D.N.D.N.D.N.D.N.D.B0/0/00.63N.D.N.D.N.D.N.D.N.D.C0/0/01.24N.D.N.D.N.D.N.D.N.D.C0/2/01.69N.D.N.D.N.D.N.D.N.D.C2/1/25.60017N.D.N.D.N.D.N.D.C3/3/44.991.33N.D.N.D.N.D.N.D.D0/0/02.46N.D.N.D.N.D.N.D.N.D.D0/0/02.31N.D.N.D.N.D.N.D.N.D.E0/2/12.630.190.59N.D.0.13N.D.E4/4/421.620.210.990.300.080.38Individualbirdaortictissueplaquescore,cholesterolandoxideconcentrationmeasuredbygaschromatography-massspectrometry.2Cholesterolconcentration=mg/gtissue;oxideconcentration=mg/gtissue;N.D.notdetected.5,6a-epoxide=5,6a-epoxy-5a-cholesterol;7/1-OH=7/1-hydroxycholesterol;triol=cholestane-triol;7-keto=7-ketocholesterol;25-OH=25-hydroxycholesterol.Diet A=TurkeyStarter(TS);B=TS+0.05%chol.;C=TS+0.5%chol.;D=TS+Tallow+0.05%chol.;E=TS+Tallow+0.5%chol.Plaquescoreisbasedonscaleof0(N.D.)=cleansurface;1=5plaques;2=6-20plaques;3=>20plaques;4=massiveatheromasseen.Valuesrepresent2judgesevaluatinginablindedprotocol.xlylz=individualscoreforeachofthreevesselsinaortictree.- CD 0,Discussion:Diet composition and fatty acid content:Unlike the protocol in Chapter 4, the basal diet used in the present study consisted of acommercial Turkey Starter (TS) chow which contained a variety of dietary lipids derived fromvegetable as well as animal sources. The supplementation of the basal TS diet (diet A) with anadditional 0.6% (wt/wt) 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 levelsof dietary cholesterol without altering the total crude lipid content of the diets (diets B and C). As aresult, the percent of total calories derived from fat for diets B and C was similar to that of thereference TS diet (diet A; e.g. 22% calories from fat). Supplementation of the TS diet with anadditional 6.6% (wt/wt) beef tallow (plus cholesterol and cholic acid as for diets B and D) resultedin an energy content of 32% of calories from fat in diets D and E. For these latter two diets, theadditional saturated fat incorporated into the basal diet resulted in changes to the proportion of specificfatty 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 cholesterolconcentrations (Keys et a!., 1956), it is now recognized to have a role in elevating circulatingcholesterol levels (Lipid Research Clinics Program, 1984). However, individual responses to dietarycholesterol can be highly variable, depending on cholesterol metabolism and the level of dietarycholesterol intake (McNamara et a!., 1987). From the results of the present study and those ofChapter 4, it appears that the plasma lipid response of atherosclerosis-susceptible Japanese quail fedhigh cholesterol-cholic acid (0.5% and 0.25%, respectively) diets is analogous to that observed withfamilial hypercholesterolemia in human subjects (Reynolds, 1989). These individuals have greatlyelevated plasma cholesterol levels and are at great risk for myocardial infarctions. A dose-dependentincrease 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 containing196different levels of cholesterol. In the present study, the plasma cholesterol concentrations of quail fedhigh cholesterol diets was clearly elevated after 9 weeks of feeding. Other workers examining thetemporal development of hypercholesterolemia in cholesterol-fed quail reported that plasma cholesterollevels 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 exhibitedhypertriacylglyceridemia confirming the results observed previously in Chapter 4. Other workers havealso reported a hypertriacylglyceridemic response in Japanese quail fed commercial diets supplementedwith 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 exhibitedhypertriacylglyceridemia in a dose-dependent manner. These workers were able to associate thisplasma response to dietary cholesterol intake with a suppression in hepatic fatty acid oxidation rateand increased hepatic secretion of VLDL particles (Fungwe eta!., 1993). The hypertriacylglyceridemiaobserved in cholesterol-fed Japanese quail supports its use as an animal model of humanatherosclerosis, since epidemiological studies have reported an association between elevated plasmatriacylglycerides and coronary heart disease (Castelli, 1986). Furthermore, the significant interactionnoted 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 initiateor exacerbate atherogenesis (Roberts, 1992). The markedly greater plasma lipid concentrations anddevelopment of aortic plaque observed in quail fed diet E which contained increased levels of bothsaturated 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 agreewith the results of previous workers who reported a hypocholesterolemic effect of oleic acid (Mattsonand Grundy, 1985), a principal fatty acid of beef tallow (approx. 44%). Quail fed diets containing0.5% cholesterol with 0.25% cholic acid exhibited similar degrees of hypercholesterolemia regardlessof the level of beef tallow in the diet. It is noteworthy, however, that quail fed the high cholesteroldiet in combination with a high level of beef tallow did not exhibit a further significant increase inplasma cholesterol levels. While it is generally regarded that saturated fatty acids contribute to an197increase in plasma cholesterol levels, it is conceivable that the increased content of myristic, palmiticand stearic acid in the high tallow diets was not sufficient to result in a further increase in plasmacholesterol concentrations in this group. Also, previous studies in humans fed beef tallow diets haveobserved a similar neutral effect of dietary stearic acid on plasma cholesterol concentrations (KrisEtherton 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 inboth this study and that in Chapter 4, the absolute concentrations of plasma cholesterol obtained inhypercholesterolemic birds was relatively lower herein. This discrepancy in plasma cholesterolconcentrations between studies may be attributed to the different fibre content of the basal diets usedin the two studies. Even though identical levels of dietary cholesterol and cholic acid were used inboth studies, the sources of dietary fibre and possibly the amount of soluble and insoluble fibre in thecommercial TS (e.g. alfalfa meal, corn, distiller’s grain and soybean meal) was different compared tothat of the semi-synthetic diets (e.g. aiphacel) used in Chapter 4. Fibre in the diet has been estimatedto 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 plasmalipid 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 RBCsand heart tissue to elevations in plasma cholesterol and triacyiglycerides in quail fed dietssupplemented with cholesterol. It is conceivable that the lack of effect of dietary treatment on GSHconcentrations in both RBCs and heart tissue can be attributed to the relatively stable nature of theprincipal 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 heartof quail fed a predominantly saturated fat diet would not be altered due to the oxidative stability ofdiet-derived fatty acids with a low P/S ratio. Support for this explanation is provided by the observedreduction in TBARs production in heart tissue in vitro from birds fed diets containing the high level ofbeef tallow. Moreover, the additive protective effect of a high cholesterol intake in combination with198increased saturated fat for heart TBARs production in birds fed diet E suggests that tissue membranelipids were stabilized against oxidation by not only an increase in membrane phospholipid saturatedfatty 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 tissueCAT and SOD activities were reduced in quail fed the high cholesterol diets. A somewhat surprisingobservation was the reduction in liver CAT activity in quail fed diets containing even the low level ofcholesterol in diet B. The minimal level of cholesterol in this diet resulted in a slight but not significantincrease in plasma cholesterol and a significant elevation in plasma triacylglyceride concentrationscompared to quail fed TS alone. It is noteworthy that hepatic SOD activity was also reduced in birdsfed the high cholesterol diets, regardless of the dietary fat content. A similar result was observed ina previous study conducted in this thesis (Chapter 3), wherein the reduced SOD activity was attributedto the stabilizing effect of cholesterol on tissue membrane lipids against oxidation. The lack ofdifferences in hepatic GSSG-Red and GSH-Px activities coincided with similar intracellular GSH levelsin livers from experimental quail. Despite the lack of differences in the GSH content and activities ofthe GSH metabolizing enzymes of liver tissue, the results observed with the in vitro oxidative challengeof liver tissue showed that both GSH depletion and TBARs production were influenced by the level ofdietary cholesterol. The greater depletion of GSH in liver tissue from quail fed the high level of dietarycholesterol may be associated with changes in tissue composition due to the increase in liver weightattributable 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 vitrowhen liver tissues were challenged with peroxidizing agent. A similar result was observed in previousstudies (Chapters 2 and 4) of this thesis, wherein it was suggested that diets high in cholesterolresulted 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 astabilizing effect of membrane phospholipids against oxidation as well as cholesterol acting as asacrificial antioxidant to spare membrane fatty acids (Smith, 1991).Similar to the results obtained in heart tissue, aortic antioxidant enzyme activities were notsignificantly altered by the dietary treatments in the present study. These results herein confirm the199lack of effect of hypercholesterolemia and aortic plaque deposition on aortic tissue antioxidant statusobserved in cholesterol-fed atherosclerosis-susceptible Japanese quail in Chapter 4. While otherworkers have reported differences in aortic tissue antioxidant enzyme activities (e.g. SOD; Godin eta?., 1 994) in Japanese quail fed cholesterol, these changes were not unique to the atherosclerosis-susceptible strain of these birds and therefore do not appear to be linked to an enhanced susceptibilityof aortic tissue to plaque development in these animals.Aortic plaque development:The results of the present study indicate that in the atherosclerosis-susceptible Japanese quaildiet-induced risk factors for atherosclerosis, such as hypercholesterolemia and hypertriacylglyceridemiaresult in the deposition of aortic plaque characterized by increased cholesterol as well as the presenceof cholesterol oxides in aortic tissue. These changes coincided with the severity of aortic plaqueestimated visually using a numerical scale. The supplementation of the TS diet with 0.5% cholesterolresulted in variable degrees of aortic plaque deposition related to the level of fat in the diet. The aorticplaque score of cholesterol-fed birds was greatest in animals fed diets supplemented with both a highlevel of cholesterol as well as the higher level of saturated fat which indicated a treatment interactionfor severity of atherosclerosis in these quail. This observation highlights the fact that while there islittle doubt that the appearance of atherosclerotic plaque in birds was associated with the elevationin plasma cholesterol level which was due to the cholesterol-rich diets, the severity of atherosclerosisin these quail was further enhanced by the presence of hypertriacylglyceridemia. It is noteworthy thataortic plaque scores were associated with a significant interaction between dietary cholesterol and fatlevel which coincided with the results seen with plasma triacylglyceride concentrations but not withplasma total cholesterol concentrations. The absence of atherosclerotic lesions in aorta from birds fedthe low cholesterol-cholic acid diets confirms the requirement for a high dietary cholesterol intake andan associated hypercholesterolemia to initiate the deposition of aortic plaque. Supportive evidence forthis hypothesis is provided by the low concentrations of aortic tissue cholesterol and the absence ofdetectable 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 incholesterol.200Despite 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 thatindividual animal heterogeneity existed in susceptibility to diet-induced atherosclerosis. Other workershave reported that atherogenesis in Japanese quail follows a sequential process that involves tissuedisruption and swelling, followed by the appearance of cholesterol-laden foam cells and the formationof 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 inthe scanning electron micrographs from birds with mild to severe plaque, and thereby confirmed thevisual 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 fromquail fed the high cholesterol diets varied with the particular treatment as well as with individual birdsin a treatment group. The detection of a greater number of cholesterol oxide species in aortic tissuefrom 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 havereported similar cholesterol oxides recoverable from LDL extracted from human aortic plaque at autopsy(Steinbrecher and Lougheed, 1992).201Conclusion:In conclusion, atherosclerosis-susceptible Japanese quail fed commercial Turkey Starter dietssupplemented with a high level of cholesterol and a low or high level of saturated fat exhibitedhypercholesterolemia and hypertriacylglyceridemia. The hypertriacylglyceridemia with cholesterolfeeding was further enhanced in quail fed the high level of fat in the diet, which supports the “LipidHypothesis” that an increased intake of calories from fat can increase plasma lipid levels and initiateor exacerbate atherogenesis. This result was further supported by the significant treatmentinteractions between dietary cholesterol level and fat intake level observed for both plasmatriacylglyceride concentrations as well as aortic plaque score but not plasma total cholesterolconcentrations in quail. Moreover, the deposition of atherosclerotic plaque in birds fed high cholesteroland 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 aorticplaque scores, RBC, heart and aortic tissue antioxidant enzyme activities were not altered under theseconditions. These results confirm those obtained in Chapter 4 and suggest that antioxidant status doesnot influence diet-induced susceptibility to the development of atherosclerosis in this animal modelunder the specific conditions of these studies. Dietary cholesterol level and thereby plasma cholesteroland triacylglyceride levels appear to play a much greater role than specific antioxidant enzymes in thedevelopment of aortic plaque in the Japanese quail model which is noted to develop diet-inducedatherosclerosis very rapidly.202SUMMARY AND GENERAL CONCLUSIONSThere is an ample amount of evidence to support the conclusion that diet can play an importantrole in the pathogenesis of hypertension and hyperlipidemia, two risk factors for the development ofcardiovascular disease. Dietary fat composition, proportion of calories in the diet derived from fat, aswell 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 fattyacid composition, namely the proportions of saturated, monounsaturated and polyunsaturated (n-6 andn-3) fatty acids, can modulate plasma lipid concentrations. Dietary cholesterol is generally consideredto increase plasma cholesterol concentrations when it is consumed at high levels. However, variabilityin response to dietary cholesterol intake is also known to occur among individuals. While consumptionof polyunsaturated fatty acids from vegetable or marine oils is known to reduce plasma cholesterollevels, these fatty acids are also noted to exhibit an enhanced susceptibility to oxidation in foodsystems 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 andtissues of hyperlipidemic humans and animal models. Several studies have suggested that in vivo lipidoxidation 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 andatherosclerosis, are also demonstrated to have an effect on tissue lipid susceptibility to oxidativechallenge and antioxidant enzyme status, this information can potentially be used in developingrecommendations to reduce the risk of in vivo lipid oxidation associated with dietary lipids. Moregenerally, these investigations will enhance the body of knowledge concerning food safety andprocessing of foods to maintain the quality of dietary lipids.This thesis reports the results of five studies which were designed to examine the individualand/or interactive effects of dietary fat source and level of cholesterol on plasma lipids and endogenousantioxidant status in two animal models known to exhibit risk factors for the development ofcardiovascular disease, the spontaneously hypertensive rat and the atherosclerosis-susceptibleJapanese quail. The spontaneously hypertensive rat (SHR) was chosen due to its age-dependentdevelopment of hypertension, which can be monitored by systolic blood pressure recordings. The203atherosclerosis-susceptible Japanese quail was chosen due to its susceptibility to the development ofaortic atherosclerotic plaque in the presence of diet-induced hyperlipidemia in these animals.Chapter 1 compared the plasma lipid concentrations and tissue antioxidant status betweenhypertensive SHR and their normotensive controls, Wistar Kyoto (WKY) rats fed a non-atherogeniccommercial chow diet. This study demonstrated that the genetic susceptibility of the SHR for thedevelopment of hypertension coincided with strain differences in plasma cholesterol and triacylglycerideconcentrations as well as tissue-specific differences in antioxidant enzyme activities. A surprisingfinding in this study were the relatively lower concentrations of plasma cholesterol and triacylglycerideobserved in SHR compared to WKY animals. RBC CAT activity was positively correlated (r = 0.634;p = 0.026) with SOD activity. Also, it was noteworthy that RBC SOD activity was positivelycorrelated (r=O.709; p = 0.049) with systolic blood pressure. SHR animals also exhibited greaterconcentrations of GSH in RBCs, heart and liver tissues compared to WKY counterparts. Hypertensionin SHR coincided with alterations in antioxidant enzyme profiles of RBC and heart, with the lattershowing an increased susceptibility to in vitro lipid oxidation. Although hypertension is a recognizedfactor 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 antioxidantenzyme activities and susceptibility to in vitro oxidative challenge between the hypertensive SHR andnormotensive 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 sourcesdiffering in proportions of saturated (short-chain versus long-chain saturates) or polyunsaturated (n-6versus n-3) fatty acids on plasma lipids, endogenous antioxidant status and the development ofhypertension in SHR and WKY rats were examined in Chapter 2. The semi-synthetic diets used in thisexperiment were formulated to contain either a low or high level of cholesterol with the fat contentof all diets kept constant at a moderate level of 8% (wt/wt), of which 5% was provided by the testfat. This level of dietary fat was chosen because it is considered normal for the rat and meets thenutritional requirements for this animal species. The relatively lower plasma cholesterol andtriacylglyceride concentrations observed in SHR compared to WKY in Chapter 1 were confirmed inChapter 2. Animal strain differences in plasma lipid levels were consistent, regardless of the dietary204fat source or the level of dietary cholesterol. Both SHR and WKY animals exhibited reduced bodyweight gain and feed efficiency ratios as well as reductions in plasma cholesterol and triacylglycerideconcentrations when fed menhaden oil diets. Decreased activities of GSH-Px and SOD in RBCs, heartand liver tissue from animals fed menhaden oil diets coincided with reductions in tissue susceptibilityto in vitroH20-induced GSH depletion and lipid peroxidation. These results suggest that diet-inducedalterations to the fatty acid composition of tissue cell membranes may influence the oxidative stabilityof membrane phospholipids, thereby altering the balance of lipid peroxides in vivo which, in turn, caneither be deactivated by antioxidant enzyme activity, or can have inhibitory effects on these sameenzyme activities. High cholesterol diets reduced hepatic CAT, SOD and GSH-Px activities and resultedin decreased susceptibility of liver tissue and RBCs to in vitro oxidative challenge. These effects ofdietary cholesterol could be associated with incorporation of cholesterol into cell membranes, resultingin increased fatty acid stability against lipid oxidation in vivo. Despite these diet-induced changes toplasma lipids and tissue-specific antioxidant enzyme activities, there was no effect of dietary treatmenton systolic blood pressure of SHR or WKY animals. It is conceivable that extending this study in olderanimals may have resulted in significant changes in the severity of hypertension in the SHR. It can beconcluded from this study that the genetic predisposition of the SHR to hypertension and specific straindifferences in antioxidant enzyme activity were less sensitive than plasma lipids to dietary fat sourceand cholesterol content treatment effects. A noteworthy finding of this study was that in vitro H2O-induced MDA production in RBCs from hypertensive SHR was consistently elevated herein as well asin Chapter 1, suggesting that this observation is a characteristic of RBCs in the SHR. HypertensiveSHR exhibiting diet-induced hypercholesterolemia and hypertriacylglyceridemia did not exhibit signs ofatherosclerotic lesions, confirming the resistance of this strain of rat to the development ofatherosclerosis.It has been reported that the association between dyslipidemia and atherosclerosis may alsoinvolve the presence of increased plasma and tissue levels of lipid oxidation products. Therefore, inChapter 3, the relative sensitivities of an atherosclerosis-susceptible strain of Japanese quail and a ratmodel to diet-induced hyperlipidemia and atherosclerosis were compared. Japanese quail exhibitedhigher concentrations of plasma cholesterol and triacylglycerides than rat counterparts fed similar levels205of dietary cholesterol. After a 9-week feeding trial, atherosclerosis was observed only in the quail fedthe high cholesterol diet, compared to rat counterparts. The presence of atherosclerotic plaque inaortic 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 andquantitated in the non-saponifiable fraction of aortic tissue from atherosclerotic quail. Aortic tissuefrom rats had relatively lower amounts of tissue cholesterol and did not contain detectable amountsof cholesterol oxides. It was concluded from this study that the atherosclerosis-susceptible Japanesequail develops atherosclerotic plaque in conjunction with elevated levels of plasma cholesterol whenthese quail are fed an atherogenic diet. The similarity of cholesterol oxides identified in aortic plaqueof quail to those noted in diseased human aortae supports the use of the atherosclerosis-susceptiblequail as an animal model for human atherosclerosis. Also, the cholesterol and cholesterol oxide contentof aortic tissue with plaque agreed with the relative severity of plaque scores determined using a visualscoring method.Once the diet-induced hypercholesterolemia and atherosclerosis were confirmed in theatherosclerosis-susceptible Japanese quail under the conditions employed in Chapter 3, a subsequentstudy similar in design to Chapter 2 was performed using this strain of Japanese quail. The study inChapter 4 was designed to examine the effect of different dietary fat sources and cholesterol levelson 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 thandietary fat source. Quail fed high cholesterol diets exhibited severe hypercholesterolemia andhypertriacylglyceridemia. These elevations in plasma lipids coincided with the presence of extensiveatherosclerotic plaque in the aortae of birds fed high cholesterol. However, tissue (e.g. heart, liver andaorta) and RBC antioxidant parameters were not affected by hyperlipidemia or the presence ofatherosclerotic plaque. Dietary fat sources differing in fatty acid composition had little effect onplasma lipid concentrations. However, RBC and heart GSH-Px activities were different in soybean oilfed animals compared to counterparts fed beef tallow or butter diets. Hypercholesterolemia in quailwas associated with elevated aortic cholesterol levels as well as the presence of several cholesteroloxides. It was noteworthy that several additional cholesterol oxides (e.g. 5,6a-epoxy-5a-cholesterol,2067a-hydroxycholesterol, 41-hydroxycholesterol, cholestane-triol, and 25-hydroxycholesterol) weredetected in atherosclerotic aortic tissue from quail fed the atherogenic diets. Japanese quail exhibitedindividual animal variability in the profile and amounts of cholesterol oxides present in atheroscleroticaortic tissue despite the similarities in plaque score determined visually. Scanning electron micrographsof aortic tissue without plaque and tissue with severe plaque also confirmed the presence of focalareas of raised tissue on the luminal surface of atherosclerotic aortae in this study. It was concludedin this study that the lack of an effect of dyslipidemia and severe atherosclerosis on the endogenousantioxidant status of tissues in the atherosclerosis-susceptible Japanese quail suggests that at theextreme plasma lipid concentrations achieved herein it was unlikely that tissue antioxidant status wasable to modulate the development of atherosclerosis in this animal model. It remains to be determinedif the effect of dietary fat level and less extreme concentrations of plasma cholesterol on aortic plaquedevelopment can modify the role of endogenous antioxidant status in the development ofatherosclerosis in this animal model.The final study of this thesis examined the role of dietary fat quantity and cholesterol contenton the plasma lipid profile, antioxidant status and atherosclerosis in the atherosclerosis-susceptibleJapanese quail. The formulation of diets fed to quail in Chapter 5 was designed to supplement acommercial Turkey Starter with two levels of cholesterol and beef tallow. Quail fed high cholesteroldiets exhibited hypercholesterolemia and hypertriacylglyceridemia regardless of the level of dietary fat.However, an interaction between dietary cholesterol level and fat content was observed for plasmatriacylglyceride concentrations in quail fed diets containing high cholesterol in combination with ahigher level of tallow. A similar dietary treatment interaction was observed for aortic plaque score inthis same group of animals. Similar to the results obtained in Chapter 4, quail fed Turkey Starter dietssupplemented with cholesterol and beef tallow exhibited little change in antioxidant enzyme activitiesof RBCs, heart, liver or aortic tissues. Dietary cholesterol level did, however, significantly reduce theactivities of liver CAT and SOD as well as decrease the susceptibility of liver tissue to in vitro H20-induced oxidative challenge. A similar result was observed in heart tissue from animals fed diets highin cholesterol in combination with the higher level of beef tallow. These results may be attributed tothe enhanced stability of membrane fatty acids to lipid oxidation due to the increased incorporation of207cholesterol into membranes in the first case, and the combination of enhanced membrane cholesterolin addition to increased saturated and monounsaturated fatty acids from dietary beef tallow in thesecond case. The results of Chapter 5 suggest that in the atherosclerosis-susceptible Japanese quailthe development of atherosclerosis is associated with not only hypercholesterolemia but alsohypertriacylglyceridemia. Moreover, the latter condition appears to have a potentiating effect on aorticplaque development in quail fed the Turkey Starter based diets used in this study. It was alsonoteworthy in the present study that despite the similar level of dietary cholesterol supplementationas used in Chapter 4, the plasma cholesterol levels of birds were somewhat lower than previouslyobserved in counterparts fed semi-synthetic diets. It was noteworthy that quail fed high cholesteroldiets in the present study exhibited greater variability in aortic plaque score than previously observedin Chapter 4. The content of cholesterol and cholesterol oxides detected in atherosclerotic aortic tissuefrom these same birds paralleled the plaque score assigned to tissues by visual scoring. Scanningelectron micrographs confirmed the presence of focal areas of raised tissue on the luminal surface ofatherosclerotic aortae.In summary, the results of this thesis indicate that the underlying metabolic factors determiningthe development of hypertension and atherosclerosis are not equivalently linked to the antioxidantstatus of the animal models employed in this thesis. While some associations between the tissue-specific nature of strain-related differences in antioxidant activity between SHR and WKY animals couldbe related to the systolic blood pressure recorded in these animals (e.g. RBC SOD activity and systolicblood pressure), the genetic predisposition of the SHR for development of hypertension was not alteredby dietary treatment (e.g. cholesterol level and dietary fat source). In contrast, dietary cholesterol andfat level effects on plasma lipids in the atherosclerosis-susceptible Japanese quail were associated withthe development and exacerbation, respectively, of aortic plaque in this animal model. These effectsof dietary lipids on susceptibility to atherosclerosis were observed to occur in the absence of alterationsin RBC or tissue antioxidant status in the Japanese quail. It is possible that the rapid development ofaortic tissue lesions in the atherosclerosis-susceptible Japanese quail occur prior to the manifestationof significant biochemical changes associated with the gradual, cumulative nature of oxidative damagein vivo. Future directions suggested by the results herein include examining the temporal association208between the development and progression of (i) hypertension in the SHR and (ii) the development ofatherosclerosis in the Japanese quail in relation to diet-induced alterations to antioxidant status. Thesestudies would conceivably further characterize the association between antioxidant status and dietrelated alterations to these specific risk factors for cardiovascular disease.209REFERENCESAbbey, M., Triantafilidis, C. & Topping, D.L. (1993). Dietary non-starch polysaccharides interact withcholesterol and fish oil in their effects on plasma lipids and hepatic lipoprotein receptor activity in rats.J. Nutr., 123, 900-908.Addis, P.B. (1986). 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Improved method for enzymatic determination of serum triglycerides. Clin.Chem., 21, 1627-1629227APPENDIX228Table 1. Relative retention times of trimethyl-silyl (TMS)-sterol derivatives1.Retention time2Sterol3:5a-Cholestane 0.721Cholesta-3,5-dien-7-one 0.988Cholest-5-en-3B-ol 1 .000(Cholesterol)Cholest-5-ene-3I-7a-diol 1 .009(7a-hydroxycholesterol)5,6a-Epoxy-5a-cholestan-31-ol 1 .134(5, 6a-epoxide)Cholest-5-ene-3B,7B-diol 1 .148(71,-hydroxycholesterol)Cholest-5-ene-3F,4I-diol 1 .164(4(-hydroxycholesterol)5a-Cholestane-3f,-5,6B-triol 1 .297(Cholestane-triol)3I-hydroxychoIest-5-en-7-one 1 .320(7-ketocholesterol)Cholest-5-ene-3I-25-diol 1 .378(25-hydroxycholesterol)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 arearesponse ratio of each sterol over the internal standard against the weight ratio of each sterol over theinternal standard.2 Relative retention times expressed in relation to cholesterol.Sterol with common name in parentheses.229Table 2. Response linearity of derivatized cholesterol and oxides1.Slope Intercept R,2Sterol3:Cholesta-3,5-dien-7-one 1.13 0.001 1.16± 0.10Cholest-5-en-3l,-ol 1 .18 -0.004 1 .00 ± 0.08(Cholesterol)Cholest-5-ene-3B-7a-diol 0.98 -0.001 0.99 ± 0.07(7a-hydroxycholesterol)5,6a-Epoxy-5a-choIestan-3l-ol 1.01 -0.005 1 .20 ± 0.11(5, 6a-epoxid e)ChoIest-5-ene-31,7B-dioI 1 .26 -0.001 1 .03 ± 0.08(7B-hydroxycholesterol)Cholest-5-ene-31&4l-dioI 0.60 0.001 1.64 ± 0.12(4B-hydroxycholesterol)5a-Cholestane-3B-5,61-triol 1.13 -0.001 0.98 ± 0.09(Cholestane-triol)3R-hydroxycholest-5-en-7-one 1.09 -0.001 0.99 ± 0.10(7-ketocholesterol)Cholest-5-ene-3I-25-diol 1 .17 -0.001 0.95 ± 0.79(25-hydroxycholesterol)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 arearesponse ratio of each sterol over the internal standard against the weight ratio of each sterol over theinternal standard.2 Response factor = (VL’IA)(A1/W , presented as mean ± SEM, where A = area of sterol standard,A1 = area of internal standard, = weight of sterol standard and W1 = weight of internal standard.Sterol with common name in parentheses.230

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