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An investigation of L-carnitine treatment in the hyperlipidemic rabbit James, Leighton Rolston 1986

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AN INVESTIGATION OF L-CARNITINE TREATMENT IN THE HYPERLIPIDEMIC RABBIT BY LEIGHTON ROLSTON JAMES B.Sc.(HON), Simon Fr a s e r U n i v e r s i t y , 1983 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department o f Pathology) We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA October 1986 ® Leighton R o l s t o n James, 1986 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree a t the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department or by h i s o r her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date #M i E - 6 ( 3 / 8 1 ) ABSTRACT Cardiovascular disease, specifically coronary heart disease, remains the leading cause of morbidity and mortality among the adult population i n North America and Western Europe. Hyperlipidemia ranks as one of the most important risk factors for cardiovascular disease. Thus, the need for effective therapy in the management and treatment of hyperlipidemia remains high. At present, dietary manipulations and/or drug therapy are the methods of choice i n the management and treatment of hyperlipidemia. Though most of the currently used hypolipidemic drugs are very effective i n reducing plasma lipi d s , many of these have unpleasant side-effects. The search for effective hypolipidemic agents, with relatively few side-effects, continues. substance which has been reported to possess lipid-lowering properties. It i s required for the optimum oxidation of long-chain fatty acids. In addition, i t functions as a buffer for coenzyme A pools within the c e l l . The present study was designed to examine the hypolipidemic effect of 4 weeks of L-carnitine treatment (170 mg/kg b.w/day) i n New Zealand White rabbits fed a high-fat diet. In particular, the effect of L-carnitine treatment on plasma concentrations of cholesterol, triglycerides, VLDL and 3 125 HDL-cholesterol were examined. In addition, H-glycerol and I-VLDL turnover studies were conducted in order to examine the effect of treatment on VLDL kinetics. In rabbits fed the high-fat diet, plasma total cholesterol, and triglycerides, cholesterol, apoprotein B and total protein associated with the VLDL particle increased significantly. There were no significant changes i n HDL-cholesterol and plasma triglycerides. One such compound presently under consideration i s L-carnitine ( hydroxy-*V -trimethylaminobutyrate). This acid i s a naturally occurring i i The fractional catabolic rate for VLDL-triglycerides and VLDL-apoprotein B were significantly reduced in the hyperlipidemic state. In addition, the transport rate for these two components of the VLDL particle were moderately elevated. With hyperlipidemia, plasma concentrations of free carnitine, acetylcarnitine, acylcarnitine and total carnitine were increased. Although carnitine levels were increased, the relative percentage of acetyl and acyl carnitine esters within the plasma pool were unchanged. Liver and skeletal muscle long-chain acylcarnitines were also significantly increased. On the other hand, the l i v e r concentrations of free carnitine, short-chain acylcarnitine and total carnitine were significantly reduced. L-carnitine treatment of the hyperlipidemic rabbit produced significant reductions i n plasma concentrations of total cholesterol, triglycerides, VLDL-triglycerides, VLDL-cholesterol and VLDL total protein. It had no effect on plasma HDL-cholesterol. Liver and skeletal muscle carnitine levels i n the hyperlipidemic carnitine-treated animals were normalized. Although treatment significantly elevated a l l plasma carnitine fractions well above those seen i n the hyperlipidemic untreated animals, the percentage of acetyl- and acylcarnitines remained unchanged. The fractional catabolic rate of VLDL-triglycerides returned to control values with L-carnitine treatment. Treatment had no effect on VLDL-apoprotein B kinetics. On the basis of these results, i t was concluded that the reduction i n plasma triglycerides i n the hyperlipidemic rabbit following L-carnitine treatment was due to an increase i n the catabolism of VLDL-triglycerides. TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES v i i LIST OF FIGURES v i i i LIST OF PLATES i x ACKNOWLEDGEMENTS x ABBREVIATIONS x i 1 INTRODUCTION 1.1 The Met a b o l i c Role o f L - c a r n i t i n e 1 1.2 T r i g l y c e r i d e s y n t h e s i s versus ft - o x i d a t i o n 3 1.3 T r i g l y c e r i d e Transport: The metabolism of chylomicrons and very low d e n s i t y l i p o p r o t e i n s . 5 1.4 C h o l e s t e r o l metabolism 7 a) C h o l e s t e r o l Synthesis 7 b) C h o l e s t e r o l Catabolism 8 1.5 C h o l e s t e r o l Transport a) Metabolism of low d e n s i t y l i p o p r o t e i n s (LDL) 8 b) HDL Metabolism 9 1.6 R a t i o n a l e 10 1.7 S p e c i f i c Aims 13 2 MATERIALS AND METHODS 2.1 Study Design 15 2.2 Blood C o l l e c t i o n 16 2.3 P r e p a r a t i o n o f High f a t D i e t 17 2.4 Determination o f Plasma Parameters 2.4.1 Free, A c y l , and T o t a l C a r n i t i n e 17 2.4.2 A c e t y l c a r n i t i n e 18 iv 2.4.3 D-fi -hydroxybutyrate 19 2.4.4 Total Cholesterol 20 2.4.5 Triglycerides 20 2.4.6 HDL-cholesterol 21 2.4.7 Glucose 21 2.5 Histologic Studies 21 2.6 Determination of Tissue Carnitines 22 2.7 Lipoprotein Electrophoresis 23 2.8 Lipoprotein Turnover studies 24 2.8.1 VLDL isolation 24 2.8.2 Determination of VLDL-TG, VLDL-cholesterol and VLDL-ApoB 25 2.8.3 Radioiodination of VLDL 25 2.8.4 Characterization of iodination lipoproteins 26 3 2.8.5 Preparation of injectable H-glycerol 27 2.8.6 Turnover Study Protocol 28 2.8.7 Isolation of VLDL from Post-Injection Plasma 29 2.8.8 Estimation of VLDL-ApoB Specific Activity 29 2.8.9 Estimation of VLDL-TG Specific Activity 30 2.9 KINETIC ANALYSIS 2.9.1 VLDL-Triglycerides 31 2.9.2 VLDL-ApoB 32 2.10 STATISTICAL ANALYSIS 35 3. RESULTS 3.1 Food Consumption and Body Weight 36 3.2 Plasma Lipid parameters a) Cholesterol and Triglycerides 36 b) Plasma VLDL parametrs and HDL-cholesterol 41 V 3.3 D- ^  -Hydroxybutyrate 44 3.4 Plasma glucoose 44 3.5 Lipoprotein Electrophoresis 44 3.6 Kinet i c Analysis a) VLDL-TG metabolism 46 b) VLDL-ApoB metabolism 50 3.7 Plasma Carnitines 55 3.8 Tissue Carnitines 59 3.9 Histology a) Gross Vis u a l Changes 62 b) Histologic Findings 62 4 DISCUSSION 70 5 CONCLUSIONS 85 6 APPENDIX Appendix A: Calculation of the FCR f o r Monoexponential Decay ... 87 Appendix B: Analysis of blood l e v e l data i n a Two-Pool System: Calculation of curve parameters 90 Appendix C: Kinet i c Definitions 92 Appendix D: An investigation of L-carnitine treatment i n Hypertriglyceridemia: Results of studies i n Yucatan minipigs 94 LIST of REFERENCES 103 vi LIST OF TABLES NUMBER TITLE PAGE I Daily food consumptions and i n i t i a l and f i n a l body weight f o r the groups of rabbits involved i n the study 37 I I Plasma l i p i d s and c a r n i t i n e values for rabbits maintained on regular rabbit chow (group N) for 8 weeks. 40 I I I Plasma HDL-cholesterol and VLDL parameters i n untreated hyperlipidemic rabbits (Group H) 42 IV Plasma HDL-cholesterol and VLDL parameters i n hyper-lipidemic rabbits treated with L-carnitine (Group C) 43 V Fractional catabolic rates (FCRs) and transport rates of VLDL-TG metabolism i n groups N and C 51 VI Fractional catabolic rates and transport rates of VLDL-ApoB i n normals and hyperlipidemic rabbits before and a f t e r L-carnitine treatment. 56 VII Changes i n plasma carnitines levels i n rabbits fed a high-fat d i e t (Group H) 57 VIII Plasma c a r n i t i n e levels i n hyperlipidemic rabbits (Group C) before and a f t e r L-carnitine treatment 58 IX Muscle ca r n i t i n e levels i n normal rabbits, hyper-lipidemic rabbits and hyperlipidemic rabbits treated with L-carnitine f o r 1 month 60 X L i v e r c a r n i t i n e levels i n normal rabbits, hyperlipidemic rabbits and hyperlipidemic rabbits treated with L-c a r n i t i n e f o r 1 month 61 XI Plasma ele c t r o l y t e s and glucose i n adult male Yucatan miniature pigs 99 XII Plasma l i p i d values i n adult male Yucatan miniature pigs 100 XIII Serum levels of glutamateoxalate transaminase, creatine kinase, albumin and t o t a l protein i n adult male Yucatan miniature pigs 101 XIV Plasma free, a c y l , a c e t y l , and t o t a l c arnitine i n adult male yucatan miniature pigs and i n men 102 v i i LIST OF FIGURES NUMBER TITLE PAGE 1. The general two-pool model of Gurpide et a l . 33 2. Plasma cholesterol and t r i g l y c e r i d e s levels i n hyper-lipidemic rabbits 38 3. Plasma cholesterol and t r i g l y c e r i d e s levels i n hyper-lipidemic rabbits treated with L-carnitine 39 4. Lipoprotein electrophoresis of human plasma and baseline plasma from NZW rabbit 45 5. Lipoprotein electrophoresis of human plasma and NZW rabbits' plasma 45 6. Lipoprotein electrophoresis of NZW rabbit plasma 45 7. Representative VLDL-TG s p e c i f i c a c t i v i t y - t i m e curve f o r normal rabbits 47 8. Representative VLDL-TG s p e c i f i c a c t i v i t y time curve f o r hyperlipedemic rabbit p r i o r to L-carnitine treatment 48 9. Representative s p e c i f i c a c t i v i t y - t i m e curve f o r VLDL-TG turnover i n hyperlipedemic rabbits a f t e r 1 month on L-carn i t i n e 49 125 10. Representative curve f o r the decay of 12I-VLDL-ApoB following the i n j e c t i o n of autologous I-VLDL i n normal rabbits 52 125 11. Representative curve f o r the decay of I-VLDL-ApoB i n hyperlipidmic rabbits 53 125 12. Representative decay curve f o r I-VLDL-ApoB i n hyperlipidemic rabbits treated with L-carnitine f o r one month 54 13 Typical VLDL-TG tracer curve obtained following the i n j e c t i o n of H-glycerol into normal rabbits 88 14 Typical specif i C j ^ a c t i v i t y - t i m e curve f o r the disappearance of I-VLDL-ApoB from the plasma of normal rabbits 91 v i i i LIST OF PLATES NUMBER TITLE PAGE 1. Light micrographs of a section of the l i v e r from a group N rabbit 63 2. O i l red 0 stained section of the l i v e r from a group N rabbit 64 3. Hematoxylin-eosin stained section of the l i v e r from a hyperlipedemic rabbit (group H) 66 4. O i l red 0 stained section of the l i v e r from a hyperlipidemic rabbit showing extensive f a t t y i n f i l t r a t i o n 67 5. Light micrograph of a l i v e r section from a hyperlipidemic rabbit showing neutral l i p i d droplets 68 6. O i l red 0 stained section of l i v e r from a c a r n i t i n e -treated hyperlipidemic rabbit 69 i x ACKNOWLEDGEMENTS I would l i k e to thank a l l those individuals who contributed to the successful completion of t h i s document. In p a r t i c u l a r , I would l i k e to express my gratitude to the members of my supervisory committee f o r t h e i r valuable contributions. I am indebted to my supervisor, Dr. David Seccombe, f o r h i s invaluable support, and contributions during the course of t h i s study and preparation of t h i s thes i s . I wish to express special thanks to Mr. Chuck Yeung of IPC Systems (Vancouver) f o r allowing me the use of hi s microcomputer, and to Drs. Ed Jones (Department of Pathology, Shaughnessy Hospital) and David Foster (Department of Biomedical engineering, University of Washington) for t h e i r expertise. F i n a l l y , I would l i k e to express my deepest appreciation to my wife, Joan Bernard, f o r of f e r i n g support and encouragement during the d i f f i c u l t periods of t h i s project. During the course of t h i s study, I obtained f i n a n c i a l assistance from the B r i t i s h Columbia Heart Foundation. The project was founded by a grant from the B r i t i s h Columbia Health Care and Research foundation. x ABBREVIATIONS oC y ANT CoA Acetyl-CoA Acyl-CoA TG VLDL VLDL-TG VLDL-ApoB IDL HDL HDL-Chol. LDL LDL-cholesterol ACAT HMG-COA HMG-CoA Reductase FCR umole uM S.D N.S. Alpha Beta Gamma Adenine nucleotide translocase Coenzyme A Acetyl-coenzyme A Acyl-coenzyme A Triglycerides Very low density lipoprotein Very low density lipoprotein t r i g l y c e r i d e s Very low density lipoprotein apoprotein B. Intermediate density lipoprotein High density lipoprotein High density lipoprotein cholesterol Low density lipoprotein Low density lipoprotein cholesterol Acyl CoA: cholesterol acyltransferase 3-Hydroxy-3-methylglutaryl CoA 3-Hydroxy-3-methylglutaryl CoA Reductase Fractional catabolic rate micromoles micromoles per l i t e r or micromolar Standard deviation Not s i g n i f i c a n t x i LCAT L e c i t h i n : cholesterol acyltransferase NZW rabbits New Zealand White rabbits w/w weight/weight v/v volume/volume xii 1. INTRODUCTION Over the past two decades, i t has been wel l established that a p o s i t i v e c o r r e l a t i o n e x i s t s between abnormal levels of plasma l i p i d s and coronary heart disease (1-3). Diet, exercise and drug therapy are t y p i c a l l y used i n the management of hyperlipidemic patients. Some of the more commonly used medications such as c l o f i b r a t e and n i c o t i n i c acid have several w e l l known side-effects (4,5). For t h i s reason they are frequently withheld and introduced only a f t e r other means of l i p i d control have f a i l e d . E f f o r t s are underway to i d e n t i f y alternate hypolipidemic agents which are e s s e n t i a l l y free of these side-effects. L-carnitine ( y3 -hydroxyl- If -trimethylaminobutyrate) i s one such agent receiving consideration. This water-soluble acid which i s synthesized i n the body and i s a normal constituent of the d i e t , i s essential f o r optimal oxidation of long-chain f a t t y acids (C > 8) i n the mitochondria (6). Studies which have examined the ef f e c t of L-carnitine treatment on serum l i p i d s have, to date, u t i l i z e d i n d i r e c t approaches with inconclusive r e s u l t s . In order to circumvent some of the weaknesses of these e a r l i e r protocols, a study was designed to examine the e f f e c t of L-carnitine treatment on lipoprotein k i n e t i c s i n an animal model. 1.1 THE METABOLIC ROLE OF L-CARNITINE In l i v i n g systems, acyl groups undergo an ATP-dependent ac t i v a t i o n process which re s u l t s i n the production of acyl-coenzyme A (7). P r i o r to gaining entry to the s i t e of ^ - o x i d a t i o n i n the matrix of the mitochondrion, these acyl-coenzyme A species must f i r s t undergo a reversible trans-e s t e r i f i c a t i o n reaction with L-carnitine, y i e l d i n g acylcarnitine. This 1 reaction i s catalyzed by carnitine acyltransferase. I t i s only a f t e r t h i s e s t e r i f i c a t i o n reaction that long-chain f a t t y acids can pass through the inner mitochondrial membrane. This membrane i s normally impermeable to a l l coenzyme A moieties, both free and e s t e r i f i e d . Once formed, acylcarnitines are transported across the inner mitochondrial membrane by means of a car n i t i n e : acylcarnitine translocase system. On the inner aspect of the mitochondrial membrane, they are tr a n s e s t e r i f i e d to form acyl-CoAs which then undergo oxidation (8-11). Thus f a r , three d i s t i n c t c arnitine acyltransferases have been i d e n t i f i e d i n mitochondria. Carnitine palmitoyltransferase i s exclusively a mitochondrial enzyme found i n l i v e r , heart, kidney and sk e l e t a l muscle. I t has peak a c t i v i t y with f a t t y acids of carbon chain length of C-^- C±Q' Beyond C^g the enzyme exhibits decreasing a c t i v i t y with f a t t y acids of increasing chain length (14-17). Carnitine acetyltransferase and octanoyltransferase have been shown to be present i n mitochondria, peroxisomes and microsomes (18). Acetyltransferase i s s p e c i f i c f o r C 0- CR with peak a c t i v i t y at C_- C 0, while the octanoyl-/ b Z J transferase a c t i v i t y overlaps the a c t i v i t i e s of the palmitoyl- and a c e t y l -transferase (17,19). Carnitine palmitoyltransferase and carnitine acetyltransferase may possess two separate a c t i v i t i e s (12,13); one located on the outer portion of the inner mitochondrial membrane and the other on the inner surface of that membrane. A s i m i l i a r finding has not been reported f o r the octanoyltransferase. In addition to i t s r o l e i n optimizing f a t t y acid oxidation, L-carnitine plays a central r o l e i n regulating the a c t i v i t y of adenyl nucleotide translocase (ANT) (20). This enzyme which catalyzes the exchange of ADP and ATP across the inner mitochondrial membrane d i r e c t l y influences the energy charge of the c e l l (21). Accumulation of acyl-CoA's of chain length longer than eight carbons on either side of the inner mitochondrial membrane i n h i b i t s 2 ANT (22-24). This r e s u l t s i n a s i g n i f i c a n t decrease i n the energy charge of the c e l l . L-carnitine and i t s associated enzyme systems are capable of modulating the acyl-CoA/CoASH r a t i o and i t i s through t h i s mechanism that they are believed to influence the a c t i v i t y of ANT (20). L-carnitine and carnitine acetyltransferase are capable of modulating the acetylCoA/CoASH r a t i o i n various tissues. In t h i s r o l e , c a r n i t i n e i s believed to "buffer" mitochondrial acetylCoA by promoting the s h i f t of acetyl groups from the mitochondria to the cytosol (25-28). Since c e l l u l a r c a r n i t i n e concentrations are normally higher than those of CoASH (12,29), i t i s conceivable that the extramitochondrial acetylcarnitine/carnitine r a t i o w i l l buffer fluctuations i n mitochondrial acetylCoA. I t has been suggested that L-carnitine may serve a metabolic r o l e i n the oxidation of excess extramitochondrial acetylCoA (30,31). Peroxisomal ^ -oxidation i s incomplete ( i . e . only chain-shortening of very long-chain f a t t y acids such as u r i c i c acid occur i n these organelles). This r e s u l t s i n the production of short-chain acyl-CoAs which must be transferred out of the peroxisome to the mitochondria (32,33). L-carnitine i s probably involved i n shu t t l i n g these ^ - o x i d a t i o n chain-shortened products. Peroxisomal ca r n i t i n e acetyltransferase and octanoyltansferase are believed to function i n t h i s transfer proccess (34) . 1.2 TRIGLYCERIDE SYNTHESIS versus -OXIDATION: Once formed, acyl-CoAs can be used either f o r the synthesis of t r i g l y c e r i d e s and complex l i p i d s or they can be degraded v i a -oxidation. Acyl-CoAs which are transported into the mitochondrial matrix are degraded to acetyl-CoA which i n turn may be either shunted into the t r i c a r b o x y l i c acid cycle (TCA) or used f o r ketone body production. Lopes-Cardozo et. a l . (35) 3 have shown that due to the high a f f i n i t y of the TCA enzyme c i t r a t e synthase for acetyl-CoA, the use of the l a t t e r substrate i n the TCA cycle takes precedence over i t s use for ketone body production. At times of high rates of P -oxidation, c i t r a t e synthase becomes saturated thereby s h i f t i n g the bias of the acetylCoA pool i n favour of ketone body production. The p a r t i t i o n i n g of f a t t y acyl-CoA between ^ - o x i d a t i o n and t r i g l y c e r i d e synthesis seems to be of major importance i n the regulation of yfl-oxidation and t r i g l y c e r i d e synthesis. As such, a reciprocal relationship has been established between the rate of ketogenesis and the rate of t r i g l y c e r i d e synthesis. For instance, i t has been demonstrated that i f acylcarnitine formation i s s p e c i f i c a l l y i n h i b i t e d , the rate of g l y c e r o l i p i d synthesis increases and can reach maximal rates even i n l i v e r s from starved animals. (36,37). This indicates that the rate of f a t t y acid oxidation may influence the rate of t r i g l y c e r i d e and very low density lipoprotein (VLDL) synthesis by the l i v e r . I t i s evident that the net d i r e c t i o n of movement of f a t t y acyl-CoA i s controlled by a number of inter r e l a t e d factors. Included among these are: a) The n u t r i t i o n a l and endocrine state of the organism Fasting (36), diabetes (37) and hyperthyroidism (38) lead to increased oxidation and decreased e s t e r i f i c a t i o n of free f a t t y acids by perfused l i v e r and cultured hepatocytes. b) Fatty acid binding protein Fatty acids which are bound to t h i s protein are p r e f e r e n t i a l l y activated and e s t e r i f i e d i n the smooth endoplasmic reticulum as opposed to undergoing t r a n s e s t e r i f i c a t i o n to L-carnitine and subsequent oxidation i n mitochondria (39). 4 c) The a v a i l a b i l i t y of competing oxidizable substrates The most important competing oxidizable substrates are pyruvate and extra-mitochondrial NADH. In order fo r y6-oxidation to occur, upon entry i n t o the mitochondria, acyl-CoA's must compete with other available substrates (e.g pyruvate) f o r available NAD+ and the electron transport chain. Thus, at r e l a t i v e l y high concentrations of pyruvate, f a t t y acid oxidation i s i n h i b i t e d i n the perfused heart whereas glucose oxidation i s accelerated (40). Cytosolic substrates, such as lactate and ethanol which produce NADH when oxidized, i n h i b i t f a t t y acid oxidation. This i n h i b i t i o n occurs because the cyto s o l i c and mitochondrial NAD/NADH pools are linked by the malate aspartate shuttle (41). d) Malonyl-CoA Malonyl-CoA, the end-product of the committed step i n f a t t y acid synthesis, i s capable of modulating the a c t i v i t i e s of outer ca r n i t i n e palymitoyltransferase (CPT 1) and car n i t i n e acetyltransferase of l i v e r mitochondria (41-43). High levels of malonyl-CoA (carbohydrate feeding) leads to suppression of these two enzymes and enhancement of t r i g l y c e r i d e synthesis; whereas low levels of malonyl-CoA (uncontrolled diabetes, f a t t y meal) has the opposite e f f e c t (44,45). Additional studies indicate that t h i s r o l e of malonyl-CoA may be more complex than was i n i t i a l l y thought. 1.3 TRIGLYCERIDE TRANSPORT The metabolism of chylomicrons and very low density lipoproteins (VLDL) Triglycerides synthesized i n the l i v e r or i n the intestines are transported by two major classes of lipoproteins, namely very low density 5 lipoproteins (VLDL) and chylomicrons. Chylomicrons, the largest of the lipoproteins, are formed i n the i n t e s t i n a l mucosa and serve mainly to transport and de l i v e r t r i g l y c e r i d e s , cholesterol and phospholipids of dietary o r i g i n to other tissues. As they move from the lymph to the venous plasma, chylomicrons acquire apoproteins C and E and lose t r i g l y c e r i d e s and phospholipids v i a the hydrolytic action of the enzyme, lipoprotein lipase, which i s attached to the c a p i l l a r y endothelium. The released free f a t t y acids, glycerol and lysophospholipids are used for energy production and membrane synthesis. The net r e s u l t i s that chylomicrons are converted to smaller ch o l e s t e r o l - r i c h p a r t i c l e s , chylomicron remnants, which are taken up by hepatocytes (46-48). VLDL are t r i g l y c e r i d e - r i c h lipoproteins produced primarily by the l i v e r . In addition to t r i g l y c e r i d e s , cholesterol, cholesterol esters and phospholipids, VLDL contains apoproteins B, C and E. The major function of VLDL i s to transport t r i g l y c e r i d e s from the l i v e r to other tissues. VLDL-tr i g l y c e r i d e s may be derived from chylomicron remnants or from newly synthesized hepatic stores. As they move through the c i r c u l a t i o n , VLDL acquire apoprotein C and lose apoprotein E; apoprotein B i s retained. In addition, t r i g l y c e r i d e s and phospholipids are l o s t through the action of lipoprotein lipase. Overall, VLDL i s transformed into smaller p a r t i c l e s c a l l e d intermediate density lipoproteins (IDL) which i n human beings are further catabolized to low density lipoproteins (LDL) (49). Disturbances i n the metabolism of chylomicrons and VLDL may lead to the development of hypertriglyceridemia (i.e f a s t i n g plasma t r i g l y c e r i d e levels th greater than the 95 percentile f o r age and sex) (50). Primary hyper-triglyceridemia may r e s u l t from one or more of the following: i ) overproduction of t r i g l y c e r i d e s , i i ) overproduction of apoprotein B and i i i ) decreased clearance of t r i g l y c e r i d e - r i c h p a r t i c l e s (VLDL and chylomicrons) 6 These defects are usually inherited as autosomal recessive t r a i t s (4). 1.4 CHOLESTEROL METABOLISM A d i r e c t involvement of L-carnitine i n cholesterol metabolism has not yet been established. However, the re s u l t s of a few studies i n hyperlipidemic patients suggest that L-carnitine may a f f e c t plasma cholesterol concentration (51,52). The average North American adult consumes about 500mg of cholesterol/day of which 40 - 60% i s absorbed. Cholesterol i s absorbed i n the free form (53) and subsequently i s e s t e r i f i e d i n the enterocyte by the action of acyl CoA: cholesterol acyltransferase (ACAT). Once e s t e r i f i e d , cholesterol i s incorporated together with t r i g l y c e r i d e s into chylomicrons for transport i n the blood (53,54). a) Cholesterol Synthesis In mammalian c e l l s , endogenously synthesized cholesterol i s derived from a c y t o s o l i c acetyl-CoA pool. The acetate within t h i s pool i s derived from many reactions including the catabolism of long-chain f a t t y acids. The i n i t i a l reaction i n cholesterol synthesis i s the condensation of two units of acetyl-CoA to form acetoacetyl-CoA. This reaction i s catalyzed by a thiolase. One molecule of acetyl-CoA i s combined with acetoacetyl-CoA i n the presence of water to form 3-hydroxy-3-methlglutaryl-CoA (HMG-CoA). This reaction i s catalyzed by HMG-CoA synthase. The next reaction, which involves the reduction of HMG-CoA to mevalonate i n the presence of NADH , i s the rate-l i m i t i n g reaction f o r cholesterol synthesis. This reaction i s catalyzed by HMG-CoA reductase. Because of i t s central r o l e i n cholesterol biosynthesis, 7 HMG-CoA reductase i s very t i g h t l y regulated. I t s a c t i v i t y appears to be modulated by such metabolites as mevalonate and oxysterols (derived from cholesterol). The l a t e r reactions i n the synthetic pathway involve a series of activations (by ATP) followed by condensations leading to the production of squalene. Subsequently squalene i s oxidized, then reduced twice forming cholesterol (see ref 55). In adults, about lOOOmg of cholesterol i s synthesized v i a t h i s route on a d a i l y basis (53,45). About one half of t h i s i s made i n the l i v e r . b) Cholesterol catabolism In normal mammals, cholesterol i s catabolized exclusively by the l i v e r . Greater than 50% of d a i l y synthesized and ingested cholesterol i s metabolized to b i l e acids. The remainder i s used for such purposes as steroid hormone synthesis and c e l l membrane assembly. B i l e acids, together with some cholesterol, are secreted into b i l e which passes into the gut. Some of the secreted cholesterol and b i l e are reabsorbed while the remainder i s passed i n the feces. Although some cholesterol i s l o s t v i a the sk i n , the feces i s by far the major route for excretion of cholesterol and i t s catabolic products (53-55). 1.5 CHOLESTEROL TRANSPORT. a) Metabolism of Low Density Lipoproteins (LDL) In human beings, low density lipoproteins (LDL) are derived largely from the catabolism of VLDL. They are p a r t i c u l a r l y r i c h i n cholesterol 8 (approximately 50% by weight) which i s present mainly as cholesterol esters. They also contain smaller quantities of t r i g l y c e r i d e s (approximately 10%). The major apoprotein i s apoprotein B which constitutes more than 95% of the protein mass within LDL p a r t i c l e s (49). LDL i s the major cholesterol carrying lipoprotein i n humans and under normal conditions i s the most important source of cholesterol f o r most c e l l s (56). LDL i s removed from the plasma by either hepatocytes or peripheral c e l l s . Removal of LDL may be receptor-mediated or receptor-independent (57-59). The receptor-mediated pathway appears to be responsible f o r as much as 80% of LDL uptake i n humans. The remainder i s taken up v i a receptor-independent pathways (57). The c e l l surface receptor involved i n the uptake of LDL i s known as ApoB-100/Apo E (or LDL) receptor and interacts with either ApoB- or ApoE-containing lipoproteins (58,59). Reduction i n the a c t i v i t y of the B-100/E receptor r e s u l t s i n decreased clearance of LDL. This ultimately leads to the development of hyper-cholesterolemia (57). This mechanism appears to be the most common underlying cause of hypercholesterolemia. However, i t has recently been shown that overproduction of LDL by d i r e c t secretion (i.e by routes independent of VLDL catabolsim) may also contribute to hypercholesterolemia (60,61). b) HDL Metabolism The immediate precursors of plasma HDL are 'nascent' HDL p a r t i c l e s . These 'nascent' p a r t i c l e s may be produced from the intravascular catabolism of VLDL and chylomicrons and by hepatocytes and enterocytes. (62-64). The major function of t h i s class of lipoproteins i s to transport excess cholesterol from peripheral c e l l s back to the l i v e r (reverse cholesterol transport). In t h i s process the nascent HDL p a r t i c l e i s eventually trans-formed to a mature HDL (HDL- and HDL_) p a r t i c l e which i s removed from the 9 c i r c u l a t i o n by ApoE and non ApoE receptors (65,66). The d e t a i l s of t h i s conversion and the mechanisms of 'reverse cholesterol transport' are s t i l l not c l e a r l y understood. Low levels of plasma HDL (hypoalphalipoproteinemia) are usually (but not always) associated with increased r i s k of atherosclerosis. In the majority of cases, t h i s condition i s inherited and both HDL2 and HDL ^ a r e greatly reduced i n plasma (49). Hyperalphalipoproteinemia (increased levels of HDL) can be inherited. Individuals who are affected by t h i s condition usually have increased longevity. 1.6. RATIONALE There i s l i t t l e doubt that there e x i s t s a d i s t i n c t relationship between disorders of lipoprotein metabolism and cardiovascular disease. Numerous studies have established a p o s i t i v e relationship between hypercholesterolemia and atherosclerosis (67-69). Therefore, the management and treatment of hypercholesterolemia remains an important goal of many c l i n i c s throughout North America and Western Europe. Despite e a r l i e r b e l i e f s , there i s some evidence suggesting that hypertriglyceridemia may, as w e l l , act as an independent r i s k factor i n the development of coronary heart disease (70,71). Using multivariate s t a t i s t i c a l analysis, Carlson et. a l . (70) were able to demonstrate that f a s t i n g plasma t r i g l y c e r i d e s acted as an independent r i s k factor i n myocardial infractions (MI) . Brunzell et. a l . (71) found that below the age of 60, hypertriglyceridemia (with or without hypercholesteremia) was the most common l i p i d disorder i n survivors of Mis. A few other studies (72,73,79,80) seem to support these observations. In addition to being at increased r i s k of 10 developing coronary heart disease, hypertriglyceridemics often have painful eruptive xanthomatosis and episodic abdominal pain with or without pancreatitis (74). Currently, treatment of the hyperlipidemic i n d i v i d u a l involves the use of a combination of d i e t , exercise and drug therapy. Due to the unpleasant side-effects (e.g abdominal pain, nausea, skin rashes, l i v e r dysfunction) the commonly used hypolipidemic drugs such as c l o f i b r a t e and n i c o t i n i c acid, are usually withheld u n t i l a l l other means of l i p i d control are exhausted. Consequently, alternate hypolipidemic drugs have been sought. On account of the r o l e of L-carnitine i n f a t t y acid metabolism, i t i s possible that exogenously administered L-carnitine may be able to s h i f t the metabolic bias of acyl-CoAs away from e s t e r i f i c a t i o n and synthesis of t r i g l y c e r i d e s towards acylcarnitine formation and ^ - o x i d a t i o n . Such a s h i f t w i l l lead to a decrease i n the synthesis of t r i g l y c e r i d e and VLDL and an increase i n the rate of ft -oxidation. Since c i t r a t e synthase i s saturated at r e l a t i v e l y high rates of ^ 2-oxidation, exogenous L-carnitine could t h e o r e t i c a l l y produce an increase i n the production of ketone bodies ( & -hydroxybutyrate and acetoacetate). Studies have indicated that L-carnitine lowers serum cholesterol (51,52,75), t r i g l y c e r i d e s (51,76-78) and free f a t t y acids (76,81) and increases ^-hydroxybutyrate (81,82) and high density lipoprotein-cholesterol (76,78,83). However, i n other studies (78,82,84) L-carnitine has been found to have no s i g n i f i c a n t e f f e c t on either plasma t r i g l y c e r i d e s or (5-hydroxybutyrate. The current study was prompted by an e a r l i e r investigation (85) into the e f f e c t of L-carnitine treatment on plasma l i p i d s i n a 32 yr o l d hyper-t r i g lyceridemic patient. The r e s u l t s of that study indicated that L-carnitine may have lipid-lowering properties. However, because of possible v a r i a t i o n 11 due to d i e t , i t could not be concluded that the observed changes were so l e l y due to L-carnitine. Therefore, a study was designed to examine the e f f e c t of L-carnitine 3 treatment on serum lipoprotein k i n e t i c s . In p a r t i c u l a r , [ H]-glycerol and 125 [ I]-VLDL turnover studies were conducted i n hyperlipidemic and normo-lipidemic rabbits i n order to study the e f f e c t of L-carnitine on VLDL-t r i g l y c e r i d e and VLDL-apoprotein B metabolism. Yucatan miniature pigs were o r i g i n a l l y selected as the model for carrying out these studies but due to recurring problems (see Appendix D ), New Zealand white (NZW) rabbits were eventually chosen to complete the study. The rabbit was chosen because i t has plasma lipoprotein classes comparable to those of man (86-88), i s inexpensive and easy to handle and maintain (89,90). Moreover, rabbits respond rather r a p i d l y to changes i n dietary cholesterol and fats (89) and provide adequate blood and tissue samples for molecular and biochemical studies. The New Zealand White rabbits were chosen primarily because they are hyperresponders with respect to hyperlipidemia and atherosclerosis (89,91). One of the major drawbacks of the rabbit model i s that atherogenic diets cause extreme hypercholesteremia and l i p i d storage i n many body organs. In addition, under the usual experimental conditions applied i n the laboratory, the rabbit develops atherosclerotic lesions unlike those found i n humans (90). However, i t i s now known that by c a r e f u l l y selecting the type of f a t ( e.g peanut or coconut o i l versus o l i v e o i l or butter) and the concentration of cholesterol (eg. 0.1 to 0.5% versus 1-3%) i n the d i e t that the degree of hypercholesteremia and the type of atherosclerotic lesion which develops may be modulated (92). In order to moderate the degree of hypercholesterolemia, the cholesterol content of the d i e t was set at 0.5% (weight/weight). Corn o i l (5%) was added 12 to serve as a vehicle f o r the cholesterol (93). Turnover studies were selected because, unlike the measurement of plasma free f a t t y acids, /5 -hydroxybutyrate and t r i g l y c e r i d e s which give i n d i r e c t information about VLDL metabolism, they give a d i r e c t assessment of the ef f e c t of treatment on the metabolism of VLDL. The k i n e t i c s of VLDL metabolism was selected because i n the cholesterol/fat fed rabbits VLDL i s the lipoprotein which i s primarily affected (94-96). I t has been suggested (97) that L-carnitine may a f f e c t cholesterol synthesis by af f e c t i n g c e l l u l a r acetyl-CoA l e v e l s . Dietary cholesterol appears to be the prime source of plasma cholesterol i n the hyperlipidemic rabbit. Rabbit l i v e r HMG-CoA reductase a c t i v i t y , which i s normally very low i s further reduced i n the hypercholesterolemic state (142). Thus, i t could be argued that L-carnitine would be un l i k e l y to have any e f f e c t on cholesterol levels i n t h i s model. On account of the role of L-carnitine i n l i p i d metabolism, i t was decided that studying the e f f e c t of L-carnitine on the ki n e t i c s of VLDL-TG metabolism (rather than the k i n e t i c s of VLDL-cholesterol metabolism) would p o t e n t i a l l y provide more information. The k i n e t i c s of VLDL-ApoB metabolism were studied because unlike the other apoproteins of the VLDL p a r t i c l e , apoprotein B remains with the VLDL p a r t i c l e throughout the catabolism to IDL and subsequently LDL (98,99). Therefore, following the apoB was l i k e l y to provide more valuable information about VLDL catabolism than would be obtained by studying the other VLDL-apoproteins. 1.7 SPECIFIC AIMS. a) The induction of hyperlipidemia i n New Zealand White (NZW) rabbits. 13 b) The determination of the plasma levels of free, a c y l , acetyl and t o t a l L-apoprotein B, t o t a l VLDL protein, and HDL-cholesterol i n normolipidemic and hyperlipidemic rabbits. c) The determination of l i v e r and muscle free c a r n i t i n e , short-chain acyl c a r n i t i n e , long-chain acyl carnitine and t o t a l c a r n i t i n e i n normolipidemic, untreated hyperlipidemic and carnitine-treated hyperlipidemic NZW rabbits. d) The determination of the k i n e t i c parameters of VLDL-triglycerides and VLDL-apoprotein B i n normolipidemic and hyperlipidemic NZW rabbits before and af t e r L-carnitine treatment. e) The examination of the l i v e r histology i n normolipidemic rabbits, untreated hyperlipidemic rabbits and hyperlipidemic rabbits treated with L-carnitine. c a r n i t i n e , VLDL-triglcerides, VLDL-cholesterol, VLDL-14 2. MATERIALS and METHODS 2.1 STUDY DESIGN Twelve male NZW rabbits (2.5 - 3.0 kg) were divided i n t o three groups as follows: GROUP C The rabbits i n t h i s group were placed on a high-fat d i e t containing 0.5% cholesterol and 5.0% corn o i l (w/w) for a one month period. At the end of 125 3 th i s period, [ I]-VLDL and [ H]-glycerol turnover studies were performed according to the protocol outlined elsewhere i n t h i s text (see section 2.8.6). The animals were then given L-carnitine f o r one month while being maintained on the same d i e t . L-carnitine (a g i f t from Sigma-Tau, Italy) was given i n the drinking water r e s u l t i n g i n a f i n a l dose of 170 mg/kg. During the treatment period, the d a i l y water intake of each animal was recorded for the purpose of ca l c u l a t i n g the d a i l y c a r n i t i n e intake. Daily water intake was calculated as the difference between the quantity of water given and the sum of that wasted (collected i n 12.7 x 10.3 x 4.8 cm pans placed i n the cages) and that remaining at time of feeding on the following day. At the end of t h i s period the turnover studies were repeated. In t h i s way, each animal served as i t s own control. I t i s important to note that each animal was injected with i t s own VLDL so as to minimize the formation of antibodies i n the period between the f i r s t and second turnover study. GROUP H The rabbits i n t h i s group were fed the same d i e t as the group C rabbits. This group received no L-carnitine and remained on the high-fat d i e t for the f u l l duration of the study (two months). No turnover studies were done i n t h i s group. However, weekly blood specimens were taken. The data from t h i s group were useful as control values f o r the Group C animals. 15 GROUP N: This group consisted of age and weight matched rabbits which were fed Purina rabbit chow (Ralston Purina Canada Inc., Mississauga, Ontario). 125 3 After two months, VLDL was i s o l a t e d , radioiodinated and I-VLDL and H-glycerol turnover studies were carri e d out. The rabbits were housed i n ind i v i d u a l cages and were allowed free access to food and water, except during turnover studies. The d a i l y food consumption of each animal i n each group was recorded throughout the course of the study. At the end of the study period, each animal was administered (1.0 ml) Euthanyl Forte (sodium pentobarbitol, 540 mg/ml; MTC Pharmacueticals, Mississuaga, Ontario, CAN.) v i a a marginal ear vein. Immediately a f t e r death, l i v e r and muscle specimens were collected and appropriately processed f o r h i s t o l o g i c a l and biochemical analyses. Tissue was f l a s h frozen and stored at -70°C u n t i l analyzed. Specimens for h i s t o l o g i c a l analyses were f i x e d i n either 10% neutral buffered formalin or i n Bouin's solution. 2.2 BLOOD COLLECTION Fasted blood (16-18 hours) was routinely collected from either the marginal ear vein or central ear artery into tubes containing EDTA (1.0 mg/ml) as an anti-coagulant. During c o l l e c t i o n , each animal was held i n a re s t r a i n i n g box (Lab Products Inc. Mayweed, NJ). Plasma was obtained following centrifugation at 500 x g f o r f i f t e e n minutes at 4°C. Each sample was divided into two portions, one of which was stored at -20°C and the other at 4°C. The -20°C samples were used for the determination of plasma carnitines (free, a c y l - , a c e t y l - , and t o t a l carnitines) and -hydroxybutyrate. The 4°C samples were used f o r the determination of plasma cholesterol, t r i g l y c e r i d e s and HDL-cholesterol and f o r the i s o l a t i o n of VLDL. 16 2.3 PREPARATION OF HIGH-FAT DIET (5% Corn O i l / 0.5% Cholesterol) A Blakeslee food mixer supplied by A l l e n - Bradley (Gault, Ontario.) was used for preparing the high f a t d i e t . Cholesterol was obtained from the Sigma Chemical Company (St. Louis, MO. USA). To prepare the d i e t , Purina rabbit chow was mixed with heated corn o i l (60°C) f o r approximately three minutes. Cholesterol was then added and the mix was blended for an additional three minutes to ensure that the p e l l e t s were uniformly coated with cholesterol. 2.4 DETERMINATION OF PLASMA PARAMETERS 2.4.1 Free, Acyl-, and Total L-carnitine Plasma free, a c y l - , and t o t a l L-carnitine were determined by a radiometric method o r i g i n a l l y described by Parvin and Pande (100) and la t e r modified by Seccombe et. a l . (101). B a s i c a l l y , the method involves i n i t i a l deproteinization of 0.1 ml of serum or plasma with 0.4 mis of 0.087 moles/L zinc sulfate (ZnSO^) followed by neutralization with 0.4 mis of 0.083M barium hydroxide (Ba(OH) 2). The supernatant (0.1 ml) obtained a f t e r centrifugation at 1500 x g for 10 minutes at 4°C, was used for the determination of free L-carnitine according to the following reaction: 14 14 L-carnitine + [1- C]AcetylCoA » [1- C]Acetylcarnitine + CoASH This reaction i s catalyzed by car n i t i n e acetyltransferase (EC 2.3.1.7). The 14 labelled acetylcarnitine formed i s separated from the unreacted [1- C] acetylCoA by adsorption of the l a t e r to activated charcoal i n the presence of ethanol and phosphoric acid. Stoichiometric conversion of L-carnitine to acetylcarnitine i s assured by the inclus i o n of N-ethylmaleimide (NEM) which 17 binds CoASH s h i f t i n g the equilibrium to the r i g h t . In order to determine t o t a l L-carnitine, the sequence of addition of zinc sulfate and barium hydroxide was reversed. Plasma was i n i t i a l l y incubated with barium hydroxide at 60°C for one hour p r i o r to the addition of the zinc sulfate. The r e s u l t i n g supernatant was used for the determination of t o t a l L-carnitne as described above for free c a r n i t i n e . Acylcarnitine was calculated as the difference between t o t a l and free c a r n i t i n e . 14 A l l of the chemicals, except [1- C]AcetylCoA, were purchased from the 14 Sigma Chemical Company. [1- C]AcetylCoA, with a s p e c i f i c a c t i v i t y of 55 mCi/mmol, was obtained from the Amersham Corporation ( Arlington Heights, IL, USA ). 2.4.2 Acetylcarnitine Deproteinized plasma (ZnSO^ / Ba(OH)2) was used for the determination of acetylcarnitine by a radiometric method o r i g i n a l l y described by Pande and Caramancion (102). In t h i s assay, acetylcarnitine i s determined by coupling carnitine acetyltransferase (CAT) to c i t r a t e synthase, CS, (EC 4.1.3.7) and measuring the amount of c i t r a t e formed according to the following reactions: CAT CoASH + Acetyl-L-carnitine '** L-carnitine + AcetylCoA AcetylCoA + [U-]Oxaloacetate ^ [ U - C ] C i t r a t e + [U- 1 4C]Oxaloacetate. Excess oxaloacetate i s converted to aspartate and subsequently removed by adsorption to an anion exchange r e s i n (AG 50WX8, BIO-RAD Laboratories (CANADA) 14 Inc., Mississuaga, Ontario). The remaining r a d i o a c t i v i t y ([U- C]Citrate) i s determined by l i q u i d s c i n t i l l a t i o n counting. Since oxaloacetate i s r e l a t i v e l y 18 unstable, i t i s freshly prepared from [U- C]Aspartate i n a reaction catalysed by glutamate-oxalatetransaminase, GOT, (EC 2.6.1.1). Excess oxaloacetate i s removed as aspartate by the reverse reaction which i s catalysed by the same enzyme. A l l of the chemicals used and the detailed procedures followed were as described i n the o r i g i n a l method (102). 2.4.3 D-/3 -Hydroxybutyrate D-p -hydroxybutyrate was determined by an enzymatic fluorometric method (103) as modified by Seccombe et a l . (101). The assay was carri e d out on ZnS0 4 / Ba(OH) 2 deproteinized plasma. A l l chemicals were supplied by the Sigma Chemical Company. Fluorometric measurements were made using an Aminco Bowman fluorometer (American Instruments Company Inc., S i l v e r Spring, MD). -hydroxybutyrate i s oxidized by D-^ -hydroxybutyrate dehydrogenase (EC 1.1.1.30) i n the presence of NAD+. The amount of D-jS - hydroxybutyrate o r i g i n a l l y present i s estimated by fluorometrically measuring the quantity of NADH formed. A b r i e f summary of the assay procedure follows. To 0.2 ml of deproteinized plasma, 0.1 ml of Barnstead deionized water and 0.15 ml of an assay mix (containing 75 umoles of carbonate-bicarbonate buffer, pH 9.4, 3.0 umoles NAD+ and 0.1 un i t of D-^ -hydroxybutyrate dehydrogenase) was added. The reaction was allowed to proceed f o r 75 - 90 minutes at room temperature. At the end of t h i s period, the reaction was terminated by the addition of 1.55 mis of 0.1M NaOH / l.OmM EDTA to each tube. Fluorescence was determined at an ex c i t a t i o n wavelength of 360nm and emission wavelength of 460nm. 19 2.4.4 Total Cholesterol Total cholesterol was determined on an Abbott Bichromatic Analyzer (Abbott, Toronto, Ontario) using k i t s obtained from Boehringer Mannheim Diagnostica (Montreal, Canada). The underlying p r i n c i p l e i n t h i s method i s the coupling of cholesterol oxidation by cholesterol oxidase to the formation of a colorimetric compound (104,105). Hydrogen peroxide, formed during the oxidation of cholesterol reacts with 4-aminophenazone and phenol i n the presence of peroxidase to form a coloured compound, 4-(p-benzoquinone-monoimino)-phenazone. The i n t e n s i t y of the colour developed i s proportional to the amount of cholesterol o r i g i n a l l y present and i s monitored as the increase i n absorbance at 565nm. I t i s important to note that i n order to determine t o t a l cholesterol, any cholesterol esters present must f i r s t be hydrolyzed p r i o r to analysis (104,105). 2.4.5 Triglycerides Plasma t r i g l y c e r i d e s were measured on an Abbott Bichromatic Analyzer using k i t s obtained from Boerhinger Mannheim Diagnostica. Triglycerides are hydrolyzed by a b a c t e r i a l lipase y i e l d i n g glycerol and free f a t t y acids. Glycerol i s converted to dihydroxyacetone by coupling to glycerol kinase, GK (E.C. 2.7.1.30) and glycerol phosphate oxidase, GPO, (E.C. 1.1.1.8). Hydrogen peroxide formed i n the GPO-catalyzed reaction participates i n the formation of a colorimetric compound (106) s i m i l i a r to that described f o r the determination of t o t a l cholesterol. The i n t e n s i t y of t h i s product i s proportional to the glycerol concentration. 20 2.4.6 HDL-Cholesterol Plasma HDL-cholesterol was measured by i n i t i a l heparin-manganese p r e c i p i t a t i o n of plasma (107,108) followed by the measurement of cholesterol. A b r i e f outline of the procedure i s as follows: HDL p r e c i p i t a t i n g reagent was prepared by mixing 2.0 mis of 2.0M manganous chloride / 0.15M sodium chloride with 1.0 ml Heparin (10,000 U) and 1.0 ml of 0.15M sodium chloride. This reagent (0.05 ml) was added to plasma (0.50 ml) while vortexing and the the mixture was allowed to stand at 4°C f o r 20 minutes. The supernatant obtained a f t e r centrifugation at 2500 x g for 30 minutes at 4°C was used f o r the determination of cholesterol as outlined above. 2.4.7 Glucose Plasma glucose was determined by the glucose oxidase method (109) on an ASTRA-8 machine (Beckman Instruments Inc., Palo A l t o , CA). The method employs the enzymatic reaction of ^ -D-glucose with oxygen and measures oxygen consumption according to the follwing reaction: glucose oxidase ^-D-glucose + 0 2 ^ gluconic acid + H 20 2 The observed rate of oxygen depletion i s d i r e c t l y proportional to the glucose concentration i n the sample. 2.5 HISTOLOGIC STUDIES Liver was i n i t i a l l y f i x e d i n either 10% neutral buffered formalin or i n 21 Bouin's solution, then dehydrated i n increasing concentrations of ethanol. The specimen was cleared with xylene, then embedded i n parafilm wax, i n preparation for sectioning. Sections (4.0 micrometers) were prepared and stained with haematoxylin and eosin as described (110). Other sections were stained f o r neutral l i p i d s by the O i l Red O method (111). These studies were conducted i n collaboration with Dr. Jones, Department of Pathology, Shaughnessy Hospital, Vancouver, B.C. 2.6 DETERMINATION OF TISSUE CARNITINES Free and short-chain acylcarnitine levels were determined from perchloric acid extracts of 100 - 125mg of muscle and l i v e r (112,113). Perchloric acid (6%) was used f o r deproteinization of the tissue a f t e r disruption by a polytron homogenizer (Brinkmann Instruments, Rexdale, Ontario, CANADA). The homogenate (0.05 ml) was saved for the estimation of non-collagenous protein (NCP) and the remainder was centrifuged at 1700 x g f o r f i f t e e n minutes at 4°C. After noting the volume of the supernatant, an aliquot was used for the determination of free carnitine following neutralization with 2M potassium carbonate / 0.5M triethanolamine solution. In t h i s procedure, long-chain acylcarnitines are pelleted with the deproteinized material. Therefore, the p e l l e t was saved f o r the determination of these esters. The t o t a l c a r n i t i n e content of the acid-soluble extract ( i . e . free c a r n i t i n e plus short-chain acylcarnitines) was measured following incubation of the perchloric acid extract (0.3 ml) with 0.2 mis of 2.0M potassium hydroxide (KOH) at 75°C for 2.0 hours. Subsequently, the excess KOH was neutralized with 6% perchloric acid (0.15 mis). The difference between t h i s t o t a l c a r n i t i n e value and free c a r n i t i n e was taken as a measure of the short-22 chain acylcarnitine concentration of the extract. KOH (2.0 M,0.2 ml) was added to the p e l l e t and the mixture incubated at 75°C for 2.0 hours. The digest was neutralised with s u f f i c i e n t 6% perchloric acid (0.4 to 0.6 mis) to ensure the complete p r e c i p i t a t i o n of a l l s o l u b i l i z e d protein. The neutralized digest was centrifuged at 1700 x g f o r 30 minutes at 4°C. The volume of the supernatant was noted. An aliquot (0.3 mis) of t h i s supernatant was used f o r the determination of c a r n i t i n e . This value was taken as an index of the quantity of long-chain acylcarnitine esters o r i g i n a l l y present i n the p e l l e t . In a l l three instances, c a r n i t i n e was assayed as described f o r the determination of free c a r n i t i n e above. Tissue non-collagenous protein (NCP) was determined according to Lowry e t . a l . (114). Tissue carnitine concentrations were routinely expressed as umoles/g NCP. Total tissue carnitine was taken as being equal to the sum of the free c a r n i t i n e s , short-chain acylcarnitines and long-chain acylcarnitines. 2.7 LIPOPROTEIN ELECTROPHORESIS Agarose gel electrophoresis was carried out as described (115), using plasma from 16-18 hour fasted animals. A l l the materials required f o r the electrophoresis were supplied by Corning Medical (Palo A l t o , CA, USA). The procedure may be summarised as follows: Aliquots (0.01 mL) of plasma were transferred to wells on the agarose gel using a Hamilton Syringe and the gel was placed i n the upper chamber of the electrophoresis c e l l . The lower, divided chamber, of the c e l l was f i l l e d with 0.05 moles/L b a r b i t a l buffer, pH 8.6 and electrophoresis was c a r r i e d out for 35 mins. The agarose f i l m was allowed to dry, then stained with 12 mis of Fat Red 7B s t a i n (0.1875 mg/ml of methanol) for 5 minutes. The agarose f i l m was 23 destained by r i n s i n g with 50% methanol. 2.8 LIPOPROTEIN TURNOVER STUDIES S t e r i l e conditions were employed throughout. A l l s t e r i l e disposable items were purchased from Becton Dickinson (Mississauga, Ontario, Canada). S t e r i l e saline and water were supplied by Travenol Canada Inc. (Mississauga, Ontario). Gel f i l t r a t i o n columns (1.0 x 20 cm) were obtained from BIO-RAD. 2.8.1 VLDL Iso l a t i o n Animals being fed regular rabbit chow were fasted f o r 18 hours p r i o r to c o l l e c t i o n of blood f o r VLDL i s o l a t i o n . On the other hand, those rabbits maintained on the high f a t d i e t were routinely fasted f o r 24 hours so as to minimize the contamination of VLDL preparation with chylomicrons. VLDL was isol a t e d by f l o a t a t i o n as described (116). For normal rabbits at least 15 - 20 mis of plasma was required i n order to obtain s u f f i c i e n t VLDL to carry out a turnover study. In the case of hyperlipidemic rabbits 5 - 1 0 mis of plasma was usually s u f f i c i e n t . The i s o l a t i o n procedure may be summarized as follows. S t e r i l e s a l i n e was underlaid with plasma i n 16 x 76mm Quick-Seal centrifuge tubes (Beckman Instruments Inc.). The tubes were sealed using gas-s t e r i l i z e d metal tube-sealing caps and centrifuged at 112,000 x g for 18 hours at 15°C i n a Beckman L8-70M ultracentifuge. VLDL was recovered from the top of the tubes using s t e r i l e , pyrogen-free syringes and needles. The lipoproteins were resuspended i n s t e r i l e saline and the centrifugation repeated as above. The washed VLDL was used for radioiodination as w i l l be described shortly. Portions of the preparation were saved f o r the determination of plasma VLDL-TG, VLDL-cholesterol and VLDL-apoB. I t should be 24 noted that VLDL not being used for turnover studies was isolat e d under non -s t e r i l e conditions. 2.8.2 Determination of VLDL-TG/ VLDL-Cholesterol and VLDL-ApoB The t r i g l y c e r i d e and cholesterol content of the lipoprotein preparation was measured as described e a r l i e r f o r plasma t r i g l y c e r i d e and cholesterol measurement. VLDL-ApoB was determined as described by Egusa et. a l . (117). The lipoprotein solution (0.5 mis) was added to 0.5 mis of 100% isopropanol i n 12 x 75 mm heavy-walled conical polystyrene centrifuge tubes. The tubes were mixed f o r about one minute and incubated at room temperature overnight. Precipitated ApoB was pelleted by centrifugation at 1000 x g f o r 30 minutes. A modified Lowry protein assay (118) was used to estimate the protein concentration of the supernatant and the i n i t i a l lipoprotein preparation. The difference i n protein concentration between the former and the l a t e r was taken as an estimate of the ApoB concentration. Plasma VLDL-TG, VLDL-Cholesterol, VLDL-apoB was calculated by mu l t i -plying the values obtained f o r the lipoprotein preparation by the appropriate d i l u t i o n factor. 2.8.3 Radioiodination of VLDL VLDL was iodinated by the iodine monochloride method of McFarlane (119) as modified by Fidge and Poulis (120). The solution (2.0 mis) of VLDL 125 containing 1.0 to 2.0 mg of protein was mixed with 1.0 mCi Na I (Amersham, USA) i n a 12 x 75 mm s t e r i l e tube containing 0.0033M IC1 ( s u f f i c i e n t to give an iodine / protein r a t i o of 10:1) and about of 0.05 mis of 0.4M glycine buffer, pH 10.0 (prepared with s t e r i l e water). The iodinated VLDL was passed 25 through a s t e r i l e column (1.0 x 20 cm) of Sephadex G50 (medium) equilibrated with 0.4M glycine buffer, pH 10.0. The eluent (1.0 mis) was collected into 125 s t e r i l e 13 x 100 mm tubes and those fractions containing the I-VLDL (cloudy) were pooled and passed through a second Sephadex G50 column equilibrated with s t e r i l e injectable s a l i n e i n order remove free iodine. I f upon characterization of the iodinated lipoprotein (see below) more than 5% 125 free iodine was found to be associated with i t , the I-VLDL was passed through a t h i r d column of Sephadex G50 equilibrated with s t e r i l e s a l i n e . The iodinated VLDL was s t e r i l i z e d by the addition of gentamycin sulfate (100 ug/ml) followed by passage through a s t e r i l e 0.22um low-binding m i l l i p o r e f i l t e r ( M illipore Corporation, Bedford, MA.) into s t e r i l e tubes. 2.8.4 Characterization of Iodinated Lipoproteins A d i l u t e d (1:100) preparation of the iodinated lipoprotein was used for the purpose of characterization as previously described (117,120-122). A l l procedures were done i n duplicate f o r each lipoprotein preparation and a l l steps were ca r r i e d out i n 12 x 75 mm gamma tubes (Fischer S c i e n t i f i c , Vancouver B.C.). 125 a) Percentage of I_ bound to Apollpoprotein B The d i l u t e d lipoprotein solution (0.1 mis) was mixed with 0.2 mis of unlabelled low density lipoprotein, LDL (to serve as a c a r r i e r f o r ApoB) and 0.3 mis of 100% isopropanol (117). The mixture was mixed and l e f t to stand at room temperature f o r 10 minutes. The precipitated ApoB was pelleted by centrifugation at 1700 x g fo r 30 minutes and the supernatant was transferred to another tube. The r a d i o a c t i v i t y i n the p e l l e t and supernatant were determined using a 4 channel automatic gamma counter (LKB - WALLAC, QY 20101 26 Turku 10, Finland). The counter was calibrated with a Co standard supplied by LKB-Wallac. The e f f i c i e n c y of each channel was used to calculate the disintegrations per minute (dpm). b) Percentage of I_ bound to Lipids The d i l u t e d lipoprotein solution (0.01 mis) was mixed with 0.01 mis of LDL and 0.2 mis of methanol. Following the addition of 0.3 mis of chloroform, the contents of the tubes were mixed, 1.0 ml of d i e t h y l ether was added and the tubes were l e f t at -20°C f o r 10 minutes. The tubes were centrifuged at 1700 x g f o r 30 minutes and the p e l l e t and supernatant separated. The r a d i o a c t i v i i t y i n the supernatant and the p e l l e t was determined as previously 125 described. The t o t a l r a d i o a c t i v i t y i n the supernatant represented I bound to l i p i d s (120-122). 125 125 c) Percentage I associated with I-VLDL: 125 The d i l u t e d I-VLDL solution (O.lmls) was mixed with 0.3 mis of 5% bovine serum albumin i n saline and 0.3 mis of 10% t r i c h l o r o a c e t i c acid i n sa l i n e . The contents of the tube were mixed and then centrifuged at 1700 x g for 10 minutes. The supernatant and p e l l e t were separated and the r a d i o a c t i v i t y determined. The r a d i o a c t i v i t y i n the supernatant was associated with free iodine (120-122). 3 2.8.5 Preparation of Injectable H-Glycerol 3 [2- H]glycerol i n absolute ethanol, with a s p e c i f i c a c t i v i t y of 1.0 mCi/mmol was purchased from the Amersham Corporation. The radiochemical purity 3 of the H-glycerol was checked by t h i n layer chromatography i n two systems: a) n-butanol saturated with water and b) chloroform / acetone / 5N ammonia 27 10:80:10 (v/v/v). The purity was found to be approximately 98 % using both systems. 3 H-glycerol was prepared to give a f i n a l concentration of 40-50 uCi / ml. Each animal received with 1.5 to 2.0 mis. The ethanol was evaporated under a stream of nitrogen and then redissolved i n 2.0 mis of s t e r i l e injectable s a l i n e . The f i n a l solution was s t e r i l i z e d by passing i t through a 0.22um m i l l i p o r e f i l t e r . Aliquots (0.1 mis) of a 1:100 di l u t e d preparation of the s t e r i l i z e d glycerol solution was mixed with 5.0 mis of aqueous s c i n t i l l a n t , ACS (Amersham Corporation), and the r a d i o a c t i v i t y was measured i n an LKB 1217 Rackbeta s c i n t i l l a t i o n counter (LKB - WALLAC, Finland). 2.8.6 Turnover Study Protocol Two days before i n j e c t i o n and up to two weeks a f t e r , each animal was given potassium iodide i n the drinking water (average d a i l y consumption of 125 approximately 200 mg) so as to block thyroid uptake of any free I-iodine 125 which may be present i n the I-VLDL preparation. Turnover studies were routinely carried out i n two rabbits on the same day, and thus the times of i n j e c t i o n were staggered so as to avoid overlapping of the time of blood c o l l e c t i o n f o r each animal. Each animal was fasted f o r 12 hours p r i o r to the i n j e c t i o n of the radionuclides. On the day of the turnover study, a blood sample was taken f o r the determination of baseline parameters. 125 3 At time t = 0, 1 5 - 2 5 uCi of I-VLDL and 70 - 100 uCi of H-glycerol were injected i n t o the marginal ear vein of each rabbit. Each i n j e c t i o n was followed by saline (3.0 mis) At specified i n t e r v a l s , 3 - 5 mis of blood was withdrawn from the marginal vein or central artery of the other ear into tubes containing EDTA. The rabbits were allowed free access to water throughout the study and remained fa s t i n g u n t i l a f t e r the 12 hour sample. The rabbits were 28 fasted f o r an additional twelve hours, a f t e r which the 24 hour sample was taken. This procedure served to l i m i t the contribution of i n t e s t i n a l l y derived p a r t i c l e s (containing ApoB and TG) to the VLDL pool. 2.8.7 Iso l a t i o n of VLDL from Post - Injection Plasma VLDL was obtained from 1.5 to 3.0 mis of plasma as described above. The volume of the VLDL preparation was recorded and 0.025 mis was used to determine the r a d i o a c t i v i t y . The remainder of the VLDL solution was used for the determination of the s p e c i f i c a c t i v i t i e s of VLDL-TG and VLDL-apoB. 2.8.8 Estimation of VLDL-ApoB Spe c i f i c A c t i v i t y ApoB was precipitated from VLDL using isopropanol as described (117). Lipoprotein solution containing 20 - 100 ug of protein was adjusted to 0.5 mis with s a l i n e , then added drop by drop to 100% isopropanol while mixing. The mixture was incubated at room temperature for about 12 hours. ApoB was pelleted by centrifugation (1700 x g / 30 minutes / 4°C). The supernatant was aspirated. The p e l l e t was washed twice with 1.0 ml of isopropanol-water (1:1), once with 1.5 mis of isopropanol and once with 1.5 mis of Barnstead deionized water. The ApoB was r e s o l u b i l i z e d by incubation with 0.3 - 0.6 mis of 1.0M sodium hydroxide (depending on the quantity of protein present) f o r 24 hours. An aliquot of the ApoB solution was used to measure the r a d i o a c t i v i t y . Plasma VLDL-ApoB s p e c i f i c a c t i v i t y was estimated from knowledge of the t o t a l amount of r a d i o a c t i v i t y used, the amount of r a d i o a c t i v i t y recovered and the d i l u t i o n of the VLDL solution r e l a t i v e to plasma. The s p e c i f i c a c t i v i t y was expressed i n dpm / ml (of plasma). 29 2.8.9 Estimation of VLDL-TG Spe c i f i c A c t i v i t y Triglycerides were extracted from VLDL by a modification of the method of Folch et. a l . (123). The lipoprotein solution (0.5 mis) was mixed with 10 mis of chloroform -methanol 2:1 (v/v) i n stoppered pyrex tubes and mixed. Sodium chloride (0.05 M, 1.5 mis) was added to the tubes, the contents mixed and centrifuged at 550 x g f o r 10 minutes at 4°C. The aqueous phase was aspirated and 2.5 mis of chloroform-methanol-0.05M sodium chloride (3:47:50 (v/v/v)) was added to the organic phase. The tubes were agitated, centrifuged and the aqueous phase aspirated. To separate phospholipids from t r i g l y c e r i d e s , approximately 0.7 g of a z e o l i t e mixture (copper sulfate / Lloyds reagent / calcium hydroxide / z e o l i t e 1:2:2:20 (w/w/w/w)) was added to each tube. The tubes were allowed to stand at room temperature for 10 minutes and were occasionally mixed during t h i s period. The particulate matter was pelleted by centrifugation at 1000 x g f o r 25 minutes at 4°C. The supernatant was f i l t e r e d and evaporated under a stream of a i r . The extracted t r i g l y c e r i d e s were r e s o l u b i l i z e d i n 0.25 mis of isopropanol. Both t r i t i a t e d and iodinated t r i g l y c e r i d e s are found i n the extract. 3 However, only H-triglycerides r e f l e c t endogenous synthesis. Since the pulse-125 3 height spectrum of I completely overlaps that of H (124) , the 3 r a d i o a c t i v i t y associated with H-TG was determined i n a Beckman LS 9000 3 s c i n t i l l a t i o n counter with windows manually adjusted to discriminate H from 1 ?R I (153). The isopropanol solution (0.05 - 0.1 ml) was added to 10 ml of ACS and 1.0 ml of de-ionized water and the r a d i o a c t i v i t y determined. From knowledge of the volume of lipoprotein solution extracted, the t o t a l 3 r a d i o a c t i v i t y recovered ( i . e . that due to H-TG), the extraction e f f i c i e n c y and d i l u t i o n of the VLDL preparation r e l a t i v e to plasma, the s p e c i f i c a c t i v i t y 30 of plasma VLDL-TG was calculated. The e f f i c i e n c y of extraction was estimated by two methods. In the f i r s t method, the t r i g l y c e r i d e concentration i n the VLDL preparation was measured so as to allow estimation of the t o t a l mass of t r i g l y c e r i d e s (mg) which was being extracted. The amount of t r i g l y c e r i d e recovered was determined from the t r i g l y c e r i d e concentration of the isopropanol solution. The difference between the recovered amount and that which was extracted was taken to represent losses. 14 In the second method, a known quantity of glycerol t r i [ l - C]oleate, with a s p e c i f i c a c t i v i t y of 56 mCi/mmol (Amersham Corporation) was mixed with 0.5 mis of unlabelled rabbit VLDL and t r i g l y c e r i d e s extracted. An aliquot of the extract was used for measuring the r a d i o a c t i v i t y . In both cases, the ef f i c i e n c y of extraction was greater than 95%. 2.9 KINETIC ANALYSIS 2.9.1 VLDL-Triglycerides The f r a c t i o n a l catabolic rate (FCR) for the removal of VLDL-TG from plasma was estimated as o r i g i n a l l y described by Farquhar et. a l . (125). In t h i s method, the slope of the log-linear phase of the decline i n s p e c i f i c a c t i v i t y of VLDL-TG af t e r the peak of the curve was used to estimate the h a l f -l i f e b v extrapolation back to zero. The FCR was then calculated from the relationship FCR = In 2 / t ^ ( s e e appendix A). Plasma VLDL-TG transport rate (production rate under steady-state conditions) was determined by multiplying the mass of VLDL-TG i n the plasma compartment by the f r a c t i o n a l catabolic rate (FCR). Plasma VLDL-TG mass was calculated by multiplying the plasma VLDL-TG concentration by the estimated 31 plasma volume. The plasma volume was taken to be equivalent to the i n i t i a l 125 volume of d i s t r i b u t i o n of I-VLDL. The l a t t e r was calculated from the 125 d i l u t i o n of the injected I-VLDL by extrapolation to time t = 0 (126). For normals (group N), the steady-state mass could not always be calculated by measuring VLDL-TG at each experimental time point because of the low levels of t r i g l y c e r i d e s and the limited amount of specimen. However, p r i o r to the turnover studies, good agreement was found to e x i s t between the VLDL-TG concentration at t = 0 and the VLDL concentration throughout the study up to and including that at 12 hours. This was done by following the v a r i a t i o n i n VLDL-TG concentration i n one normal rabbit over a period of 12 hours. The VLDL-TG concentration at t=0 was used to calculate VLDL-TG steady state mass. Steady-state mass was obtained by multiplying the VLDL-TG concentration 125 at t = 0 by the i n i t i a l volume of d i s t r i b u t i o n of the injected I-VLDL. 2.9.2 VLDL-ApoB Analysis of the k i n e t i c s of VLDL-apoB metabolism was carr i e d out by application of the two pool model of Gurpide et. a l . (127) (see figure 1.) Pool A includes the plasma compartment and possibly the extravascular space, while the r e s t of the body i s represented by pool B. Tracer i s considered to be administered into pool A and a l l subsequent sampling i s also considered to be from that pool. At present, the physical constraints on pools A and B have not yet been c l e a r l y defined. The use of t h i s model ( f i g . 1) i s relevant to the metabolism of VLDL-apoB because i ) i t allows independent entry and e x i t of metabolites from both pools and i i ) the primary pool into which tracer i s injected (pool A) i s not necessarily confined to the intravascular space. Both of these conditions have been found to be applicable to VLDL-apoB metabolism. The relevance of t h i s model to VLDL-apoB metabolism i n man and pigs has recently been discussed 32 F i g . 1: The general two-pool model o r i g i n a l l y described by Gurpide et. a l . (127) which was used i n the analysis of VLDL-ApoB k i n e t i c s . 33 (128,129). According to t h i s two pool model, the decay of VLDL-apoB s p e c i f i c a c t i v i t y , SA(t) i s biexponential and i s represented by the function (127) where SA(t) = cAe - " t + C B e ~ ' 5 t SA(t) i s VLDL-apoB s p e c i f i c a c t i v i t y determined at various times, t , a f t e r administration of the tracer, e i s the natural logarithm base. 0( and p are rate constants which are determined from the h a l f - l i v e s of t h e i r respective exponential on the basis of the relationship k = 0.693 / t y 2 f o r monoexponential decay (appendix A). C, and C D are constants estimated from the points at which the respective exponentials intercept the y-axis. The curve parameters o( , ^ , C A and C B may be manually estimated as indicated i n appendix B. These values may then be used to estimate the k i n e t i c parameters of VLDL-ApoB metabolism. The f r a c t i o n a l catabolic rate f o r the removal of VLDL-ApoB from pool A (excluding material that may recycle between A and B) i s given by the following equation (127): FCRA = «CA + /3CB  CA + CB The production rate of VLDL-apoB which represents synthesis into pool A, but excludes apoB that may recycle between A and B i s given by the product of the FCR and the steady-state mass of VLDL-ApoB. In the present study, curve parameters were calculated on an IPC microcomputer (IPC Systems, Vancouver, B.C. CAN.) using a "curve stripping" 34 program which incorporated a nonlinear least square analysis method f o r f i t t i n g the exponentials (129). The estimation of the FCR was also incorporated i n t o the program. 2.10 STATISTICAL ANALYSIS A l l s t a t i s t i c a l analyses were carr i e d out using the ABSTAT s t a t i s t i c a l program (Anderson-Bell Company, USA). The program was run on an IPC XT portable microcomputer (I n t e l l i g e n t Series) supplied by IPC Systems. For the comparison of differences between the mean values i n two d i f f e r e n t groups, the student t t e s t f o r unpaired data was used. Within the same group of rabbits, differences between means were compared using the student t t e s t f o r paired data. Differences were considered s i g n i f i c a n t at P < 0.05, where P represents the p r o b a b i l i t y f o r two-tailed te s t s . 35 3. RESULTS 3.1 FOOD CONSUMPTION and BODY WEIGHT The average d a i l y food consumption and mean body weights at the commencement and conclusion of the study are presented i n Table I. Overall, Group N had the highest d a i l y food intake. However, the differences i n food consumption between the groups were not s t a t i s t i c a l l y s i g n i f i c a n t . The body weights at the end of the study period were s i m i l i a r f o r a l l three groups. 3.2 PLASMA LIPID PARAMETERS a) Cholesterol and Triglycerides The v a r i a t i o n of plasma cholesterol and t r i g l y c e r i d e s i n rabbits fed a high-fat d i e t f o r eight weeks (Group H) i s shown i n f i g . 2. The v a r i a t i o n of plasma cholesterol and t r i g l y c e r i d e s i n rabbits (group C) which were exposed to the same d i e t as group H, except that they were administered L-carnitine f o r the f i n a l 4 weeks, i s presented i n f i g . 3. I t can be re a d i l y seen that baseline values (i.e those at the s t a r t of the study) f o r both cholesterol and t r i g l y c e r i d e s i n groups H and C were si m i l a r to those f o r group N (Table I I ) . The elevations i n l i p i d s seen over the f i r s t 4 weeks of exposure to the d i e t i n groups H and C were s i m i l i a r . L i p i d levels continued to r i s e i n group H over the f i n a l four weeks of the study. In both groups, the change i n cholesterol was more s t r i k i n g . In the case of group H, cholesterol increased from a baseline mean value of 50 mg/dl to more than 2000 mg/dl af t e r eight weeks on the d i e t . This change was s i g n i f i c a n t (p < 0.005). Despite the fa c t that the mean value of plasma 36 Table I Daily food consumption and i n i t i a l and f i n a l body weight f o r the groups of rabbits involved i n the study, n = 4 for a l l groups. Food Consumption (g/day) I n i t i a l Body F i n a l Body Weight (Kg) Weight (Kg) Group N (Regular Chow) 227 + 36 2.6 + 0.3 3.9 + 0.5 Group H (Corn o i l & Cholesterol) 187 + 17 2.9 + 0.3 3.9 + 0.3 Group C (Corn o i l , Cholesterol & carnitine) 176 + 33 2.6 + 0.3 4.1 + 0.3 Values represent the mean S.D. 37 3 0 0 0 T F i g . 2: Plasma cholesterol (x x) and t r i g l y c e r i d e s (o o) levels i n hyperlipidemic rabbits (group H). n = 4. 1 " P < 0.005 and * P < 0.001 versus time, t = 0. 38 2000 1800--1600 1400--CP 1200 O pr 1 0 0 0 y 8 0 0 o o 600 + 4Q0--200--0 - ' I I I I I H I I I I I I H I I I I H 1 I I I I I I I II I I I I I I I I I I I I I I I I I I I I H 0 2 4 S. -1 1111111111111111111111111111 T I M E ( w e e k s ) F i g . 3: Plasma cholesterol ( x — x ) and t r i g l y c e r i d e s (o o) concentrations i n hyperlipidemic rabbits (group C). The rabbits were given L-carnitine for one month while being maintained on a high-fat d i e t . t P < 0 . 005 vs. time, t = 0. * P < 0.025 and # P < 0.05 vs. 4 weeks. Daily L-carnitine started. The d a i l y intake was 170 +_ 40 mg/kg. 39 Table I I Plasma l i p i d s and car n i t i n e values f o r rabbits maintained on regular rabbit chow (Group N) f o r 8 weeks, n = 4. Parameter Concentration Cholesterol (mg/dL) 55.0 _+ 18.2 Triglycerides (mg/dL) 40.0 +_ 11.4 HDL - Choi. (mg/dL) 36.7 + 11.0 VLDL - Choi. (mg/dL) 1.43 +_ 0.68 VLDL - TG. (mg/dL) 6.20 + 3.0 VLDL protein (mg/dL) 1.43 + 0.70 VLDL - ApoB (mg/dL) 0.62 +_ 0.29 Free c a r n i t i n e (umoles/L) 21.0 _+ 6.0 Acylcarnitines (umoles/L) 13.0 +_ 3.0 Total c a r n i t i n e (umoles/L) 34.0 +_ 9.0 Acylcarnitine 0.38 +_ 0.03 Total c a r n i t i n e A l l values represents the mean + S.D. 40 t r i g l y c e r i d e s doubled over the eight week period, t h i s change was not s i g n i f i c a n t . In group C, a f t e r 4 weeks on the high-fat d i e t , plasma cholesterol had increased s i g n i f i c a n t l y (from 50 mg/dl to 1700 mg/dl, P < .005). The increase i n t r i g l y c e r i d e s was not s i g n i f i c a n t . After 4 weeks on L-carnitine, plasma cholesterol and t r i g l y c e r i d e s i n group C decreased s i g n i f i c a n t l y (see f i g . 3.) Plasma t r i g l y c e r i d e s decreased by 50% while cholesterol decreased by 35%. (b) Plasma VLDL parameters and HDL-cholesterol The values f o r plasma VLDL parameters and HDL-cholesterol f o r groups H and C are presented i n Tables I I I and IV. Comparison of the data i n Tables I I I and IV with that i n Table I I indicates that baseline values f o r groups H and C were s i m i l a r to those f o r group N. I t i s apparent that over the 8 week period, there was a progressive increase i n a l l parameters i n group H. Except f o r HDL-cholesterol, s i g n i f i c a n t increases occurred i n a l l parameters. Among the various parameters, VLDL-cholesterol showed the largest change, increasing almost 200 f o l d from an i n i t i a l value of 3.6 + 2.5 mg/dl to 1328 + 472 mg/dl. VLDL-TG showed the smallest change, increasing from 9.1 +_ 3.0 mg/dl to 82.0 +_ 45.1 mg/dl (see table I I I ) . Over the f i r s t 4 weeks, the effects of the d i e t on l i p i d parameters i n * group C were not s i g n i f i c a n t l y d i f f e r e n t from those i n group H (Table IV). VLDL-cholesterol showed the largest change, while the change i n VLDL-TG was the smallest among those VLDL parameters which were s i g n i f i c a n t l y altered by the d i e t . HDL-cholesterol was not affected. After 4 weeks on L-carnitine, plasma VLDL-cholesterol, VLDL-TG and VLDL protein i n group C decreased s i g n i f i c a n t l y (Table IV). The concentration of 41 Table I I I Plasma HDL-Cholesterol and VLDL parameters i n hyperlipidemic rabbits (Group H). n = 4 Parameter Baseline 4 weeks 8 weeks (mg/dl) HDL-Chol. 31.4 + 1.4 32.3 + 6.1 38.8 + 10.3 VLDL-TG 9.1 + 3.0 42.8 + 33.8 82.0 +_ 45.1 *N.S *p < .05 VLDL-Chol. 3.6 + 2.5 697 + 152 1328 + 472 *p < .005 VLDL-ApoB 0.79 +_ 0.21 24.2 _+ 9.9 29.8 _+ 16.6 *p < .02 VLDL protein 1.8 + 0.4 55.5 _+ 16.4 83.5 _+ 35.0 * p < .01 A l l values represent the mean +_ S.D. 4 weeks, 8 weeks versus baseline. 42 Table IV Plasma HDL-Cholesterol and VLDL parameters i n hyperlipidemic rabbits treated with L-carnitine (Group C). n = 4 Parameter Baseline 4 weeks 8 weeks (mg/dl) HDL-Chol. 22.5 + 6.1 34.3 + 10.8 33.0 + 8.5 VLDL-TG 10.3 +_ 3.2 36.4 + 5.4 16.75 + 6.3 *P < .01 *P < .05 VLDL-Chol. 1.90 + 0.26 661.5 + 40.8 308.0 + 77.1 * P < .005 *P < .02 VLDL-ApoB 1.89 + 1.80 26.6 + 11.4 19.2 + 13.3 P < .025 *N.S. VLDL protein 2.16 +_ 0.50 54.9 +_ 19.5 32.0 +_ 19.0 * P < .001 *P < .02 Values represent the mean +_ S.D. L-carnitine treatment was started at 4 weeks. The d a i l y consumption was 170 +_ 40 mg/kg b.w. * 4 weeks versus baseline. 8 weeks versus 4 weeks. 43 plasma VLDL-TG decreased by 50%, while VLDL-cholesterol declined by 37%. The VLDL protein concentration decreased by 40% while VLDL-apoB decreased by about 35% with the treatment. However, the change i n VLDL-apoB was not s i g n i f i c a n t . HDL-cholesterol was unchanged following 1 month of L-carnitine treatment. I t i s worth noting that the concentration of plasma VLDL-cholesterol and plasma cholesterol d i d not decrease by the same quantity; plasma VLDL-cholesterol decreased by about 350 mg/dl while plasma cholesterol decreased by 600mg/dl. The findings f o r plasma VLDL-TG and plasma t r i g l y c e r i d e s were s i m i l i a r , with VLDL-TG decreasing by 20 mg/dl while plasma t r i g l y c e r i d e s decreased by 40 mg/dl. 3.3 D- f$ -HYDROXYBUTYRATE Fasting levels of D-y# -Hydroxybutyrate tended to be higher i n the untreated rabbits (Group H) than i n the treated group (Group C). However, these differences were not s i g n i f i c a n t . One month of L-carnitine therapy had no s i g n i f i c a n t e f f e c t on plasma levels of D--hydroxybutyrate. 3.4 PLASMA GLUCOSE Fasting plasma glucose levels were measured at the beginning and end of the study. There was no s i g n i f i c a n t difference between f a s t i n g glucose levels i n the three groups of rabbits. 3.5 LIPOPROTEIN ELECTROPHORESIS Electrophoresis of plasma from a l l three groups p r i o r to introducing the high f a t d i e t indicated that rabbit lipoproteins have s l i g h t l y greater mobility than those of human beings (Fig. 4). There was a s t r i k i n g reduction i n the in t e n s i t y of the ©(-band and a 44 Alpha Pre-beta-Beta l\m m M mm Origin I -4 F i q . 4 : L i p o p r o t e i n e l e c t r o p h o r e s i s o f h u m a n p l a s m a a n d b a s e l i n e p l a s m a f r o m NZW r a b b i t s . L a n e 1 : h u m a n p l a s m a ; l a n e s 2 t o 6 : p l a s m a f r o m 1 8 h o u r - f a s t e d r a b b i t s . Alpha Pre-beta m Beta . Origin I I 3 ( _ , 4 8 F i g . 5 : L i p o p r o t e i n e l e c t r o p h o r e s i s o f h u m a n a n d NZW r a b b i t s ' p l a s m a . L a n e s 1 & 5 : h u m a n ; l a n e s 2 , 3 ; 6 & 7 : NZW r a b b i t s ' p l a s m a 2 w e e k s a f t e r s t a r t i n g o n a h i g h f a t d i e t ( 0 . 5 % c h o l e s t e r o l / 5 % c o r n o i l ) ; l a n e s 4 & 8 : p l a s m a f r o m NZW r a b b i t s c o n s u m i n g r e g u l a r r a b b i t c h o w . A l l r a b b i t s w e r e f a s t e d f o r 1 8 h o u r . T h e h u m a n s u b j e c t w a s f a s t e d f o r 1 2 - 1 4 h o u r s . Alpha Pre-beta Beta Origin | | W K w w F i g . 6 : L i p o p r o t e i n e l e c t r o p h o r e s i s o f NZW r a b b i t s ' p l a s m a . L a n e s 1 & 2 : 1 m o n t h o n h i g h - f a t d i e t ; l a n e s 3 & 4 : 2 w e e k s o n l - c a r n i t i n e ; l a n e s 5 & 6 : 1 m o n t h o n l - c a r n i t i n e . A l l r a b b i t s w e r e f a s t e d f o r 1 8 h o u r s p r i o r t o t h e c o l l e c t i o n o f b l o o d . 4 3 concomitant increase i n the i n t e n s i t y and s i z e of the 8 and pre-P bands was a l l that was evident i n the electrophoretogram. TheoC-band appeared to be completely absent (Fig. 6, lanes 1 & 2). As L-carnitine treatment progressed the in t e n s i t y and s i z e of the broad beta / pre-beta band decreased. The 0C-band remained absent a f t e r 1 month of L-carnitine ( f i g . 6). 3.6 KINETIC ANALYSIS a) VLDL-TG metabolism A representative VLDL-TG s p e c i f i c a c t i v i t y - t i m e curve obtained following 3 the i n j e c t i o n of H-glycerol i n normal rabbits (Group N) i s presented i n Figure 7. This curve may be divided into 3 phases: i) an i n i t i a l rapid r i s e , found to peak at 1 - 3 hours i i ) an early, f a s t decay phase l a s t i n g 2 - 6 hours i i i ) a slow decay phase l a s t i n g f o r the rest of the study A representative curve f o r VLDL-TG turnover i n group C p r i o r to L-ca r n i t i n e therapy i s shown i n Figure 8. The shape of the VLDL-TG s p e c i f i c a c t i v i t y - t i m e curve f o r group C rabbits on a high-fat d i e t p r i o r to L-car n i t i n e treatment was noticeably d i f f e r e n t from that f o r chow-fed animals (group N). In f a c t , i n group C, the curve obtained f o r VLDL-TG s p e c i f i c a c t i v i t y over 12 - 30 hours could not be c l e a r l y resolved i n t o the rapid and slow decline phases. 3 A representative curve f o r VLDL-TG metabolism following H-glycerol i n j e c t i o n i n t o hyperlipidemic rabbits a f t e r L-carnitine treatment i s shown i n Figure 9. Comparison of t h i s curve with that i n Figure 7 indicates that with (Fig. 5). In f a c t , a f t e r 1 month, a broad band 46 F i g . 7: Representative VLDL-TG s p e c i f i c a c t i v i t y - t i m e curve obtained following the i n j e c t i o n of H-glycerol into a rabbit consuming regular lab. chow. The h a l f - l i f e (t- ,») was obtained by v i s u a l inspection of the linear portion of the decay curve. The FCR was estimated from the relationship FCR = In 2 / t ^ 2 (see appendix A). 47 10000 T F i g . 8: Representative VLDL-TG s p e c i f i c activity-time curve f o r hyperlipidemic rabbit (group C) p r i o r to L-carnitine treatment. 48 1 'iiiiiiiiiiiiiiiiiiiiiiiiimiiiiiiiiiiiiii minium min i in IIIIHIIIIIIHIIHHIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIHIIIIIHIIIIIHIIIIIIIIIIIIIIIIIIIIIIHUII IIIIIIIIIIIIIIIIIIIIIIIIIIIIIMIIIIIH O 3 6 9 12 15 18 21 T i m e ( H r s ) F i g . 9: Representative s p e c i f i c activity-time curve f o r VLDL-TG turnover i n group C af t e r 1 month of L-carnitine. The d a i l y consumption of L-carnitine was 170 + 40 mg/kg. 49 treatment, the shape of the s p e c i f i c a c t i v i t y - t i m e curves i n Group C approached the shape of those i n Group N. FCR was calculated from the apparent log-linear phase of the decline i n s p e c i f i c a c t i v i t y of VLDL-TG as indicated i n the previous chapter (see section 2.9.1). The FCR values f o r groups C and N are presented i n Table V along with the VLDL-TG transport rates f o r the two groups. The FCR for VLDL-TG i n the hyperlipidemic rabbits was found to be 0.069 +_ 0.024 hr 1. This value was s i g n i f i c a n t l y lower (P < .001) than the value of .308 _+ .035 hr. 1 found f o r the FCR i n group N. Following 1 month of L-carnitine therapy, the mean value of the FCR for VLDL-TG i n group C increased by 3.5 times to 0.239 +_ 0.033 hr. 1. The post-treatment FCR was s i g n i f i c a n t l y higher (P < 0.01) than the pre-treatment value and was not s i g n i f i c a n t l y d i f f e r e n t from the FCR for VLDL-TG metabolism i n normal rabbits. The transport of VLDL-TG i n group C increased from 5.23 +_ 2.97 mg.hr. 1 p r i o r to treatment to 7.47 +_ 2.60 mg.hr. 1 following L-carnitine therapy. However, t h i s increase f a i l e d to a t t a i n s t a t i s t i c a l significance. The transport rate f o r VLDL-TG i n group N (3.26 _+ 2.29 mg/hr) tended to be lower than that i n group C both p r i o r and subsequent to L-carnitine therapy. However, these differences were not s i g n i f i c a n t . b) VLDL-ApoB Metabolism Representative decay curves f o r VLDL-ApoB decay following i n j e c t i o n of 125 autologous I-VLDL into normals (group N) and hyperlipidemic rabbits p r i o r to L-carnitine (group C) are shown i n Figures 10 and 11. As i s demonstrated, i n both cases the decay curve may be resolved into two exponentials by the method of curve s t r i p p i n g (see Appendix B and ref 127). 50 Table V Fractional catabolic rate (FCR) and transport rate f o r VLDL-TG metabolism i n Groups N and C. Group N (n=3) Group C (n=4) Pre-treatment Post-treatment FCR (hr 1) 0.308 + 0.035 0.069 + 0.024 0.239 + 0.033 P < 0.001 # P < 0.01 Transport Rate (mg/hr) 3.26 + 2.29 5.23 + 2.97 * N.S. 7.47 _+ 2.60 #N.S. A l l values represent mean +_ S.D. * Group C (Pre-treatment) versus Group N. Post-treatment versus Pre-treatment. 51 IO 6 8 10 T I M E ( h r s ) F i g . 10: Representative ?curve for the decay of I-VLDL-apoB following the in j e c t i o n of autologous I-VLDL into normal rabbits. The FCR was calculated from the curve parameters er, /3 , Cft and Cfi (refer to section 2.9.2 and appendix B f o r furthur explanation). 52 1000001 T CL. Q -< o UJ CL c B = 100000 100000 10000 M l i n t i n i M)lllllinMMItnM^MMtHIIHIHIIIIIIIIMIttHIIMIIIIIIIIIIIIIIIIIIIIIIIIIIIIItl)IM)llltlllllllllllllll 0 2 4 6 8 10 T I M E ( h r s ) 125, F i g . 11: Representative curve for the decay of I-VLDL-ApoB i n hyper-lipidemic rabbits (group C). 53 1O00OO1 T 54 Examination of the shape of both curves c l e a r l y indicates that i n the case of hyperlipidemic rabbits the decay of VLDL-ApoB s p e c i f i c a c t i v i t y i s r e l a t i v e l y sluggish i n comparison with that i n the normal rabbits. This i s obvious from the flatness of the curve i n f i g . 11. In f a c t , a f t e r 12 hours, the s p e c i f i c a c t i v i t y of VLDL-ApoB i n the hyperlipidemic rabbits i s 25-30% of i t s value at t = 0, whereas i n the normal rabbits i t i s only about 8%. F l a t VLDL-ApoB decay curves were obtained for a l l the rabbits i n group C p r i o r to L-carnitine therapy. The shape of the VLDL-ApoB curves i n group C rabbits was not markedly affected by L-carnitine therapy. (Fig. 11 and 12). FCR values f o r the removal of VLDL-ApoB from pool A (plasma and possibly the intravascular space) of the two pool model are presented i n Table VI. The FCR f o r the hyperlipidemic rabbits i s not affected following L-carnitine therapy, (see Table VI). The FCR of VLDL-ApoB i n group C was s i g n i f i c a n t l y lower (P < .02) than that i n group N. The FCR i n group C was unchanged by L-c a r n i t i n e treatment. The production rate f o r VLDL-ApoB i s presented i n Table VI. The VLDL-ApoB production rate was not s i g n i f i c a n t l y d i f f e r e n t from control values. L-carnitine treatment di d not s i g n i f i c a n t l y a l t e r t h i s rate. 3.7 PLASMA CARNITINES The r e s u l t s f o r plasma carnitine levels i n groups H and C are presented i n Tables VII and VIII respectively. Plasma car n i t i n e baseline values were not s i g n i f i c a n t l y d i f f e r e n t between the three groups. Over the f i r s t 4 weeks of the study plasma free , a c y l , acetyl and t o t a l c a r n i t i n e levels i n group H increased s i g n i f i c a n t l y over baseline values (Table V I I ) . Acyl c a r n i t i n e and acetylcarnitine increased by approximately 200%. For the duration of the study, plasma free, acetyl,acyl and t o t a l c arnitine levels i n group H remained 55 Table VI Fractional catabolic rate (FCR) and transport rate of VLDL-ApoB i n normals (Group N) and and hyperlipidemic rabbits (Group C) before and af t e r L-carnitine treatment. Group N (n = 3) Pre-treatment Post-treatment Group C (n = 4) FCR (hr 1) 1.23 + 0.42 0.040 + 0.015 0.042 + 0.019 *P < 0.02 *P < 0.02 Transport 1.43 + 0.79 1.74 + 0.87 1.26 + 0.50 Rate * Group C (Pre-treatment) versus Group N. Post-treatment versus Pre-treatment. (mg/hr) N.S "N.S Values represent mean +_ S.D. * 56 Table VII Changes i n plasma Carnitine levels i n rabbits fed a high-fat d i e t (Group H) n = 4. Carnitine Baseline 2 weeks 4 weeks 6 weeks 8 weeks (uM) Free 17.8 + 2.4 25.4 + 5.9 28.9 + 3.6 27.8 + 5.6 32.2 + 4.2 *P < .005 *P < .005 Acyl 5.5 + 1.5 16.2 + 3.5 16.0 + 5.3 18.0 + 7.8 15.4 + 5.2 *P < .02 *P < .05 Acetyl 4.2 + 1.2 13.3 + 3.5 13.3 + 6.2 17.2 + 6.4 13.2 + 4.4 *P < .05 *P < .05 Total 23.5 + 2.3 41.6 + 8.2 44.8 + 2.9 45.8 _+ 4.5 47.5 _+ 8.0 *P < .005 *P < .02 Acetyl 0.76 + 0.11 0.81 + 0.07 0.81 + 0.12 0.95 + 0.05 0.85 + 0.03 Acyl *N.S. *N.S. Acyl 0.24 + 0.07 0.39 + 0.06 0.35 +_ 0.09 0.39 + 0.15 0.32 + 0.06 Total * * N.S. N.S. Values represents mean +_ S.D. * 4 weeks, 8 weeks versus baseline. 57 Table VIII Plasma carnitine levels i n hyperlipidemic rabbits (Group C) before and af t e r L-carnitine treatment, n = 4 Carnitine Baseline 2 weeks 4 weeks 6 weeks 8 weeks (uM) Free 15.3 + 5.8 20.4 + 4.6 29.6 + 3.5 98.3 + 37.2 109.8 + 23.7 *P < .01 P < .005 Acyl 3.6 + 2.4 4.4 + 1.8 11.1 + 3.5 58.6 + 11.8 33.8 + 11.2 *P < .001 P < .05 Acetyl 3.2 + 2.0 4.0 _+ 1.8 8.6 _+ 2.2 53.7 + 14.3 26.9 +• 13.0 *P < .001 P < .05 Total 18.9 + 3.7 24.8 + 3.8 40.7 + 1.7 156.9 + 40.3 143.6 + 29.5 *P < .005 P < .005 Acetyl 0.89 + 0.02 0.90 + 0.09 0.77 + 0.08 0.90 + 0.07 0.79 + 0.19 *N.S N.S. Acyl Acyl 0.22 +_ 0.18 0.18 + 0.09 0.27 + 0.09 0.37 + 0.10 0.24 + 0.06 Total * N.S N.S. Values represents mean _+ S.D. # L-carnitine started. The o v e r a l l d a i l y consumption of car n i t i n e was 170 +_ 40 mg/kg. * 4 weeks versus baseline. 8 weeks versus 4 weeks (See methods f o r experimental d e t a i l s ) . 58 unchanged. The r a t i o of acetylcarnitine to acylcarnitine and acylcarnitine to t o t a l c a r n i t i n e i n the group H fluctuated throughout the course of the study but d i d not change s i g n i f i c a n t l y . Comparison of the data i n Table VII with those i n Table VIII indicates that over the i n i t i a l 4 weeks of the study, plasma carnitines underwent si m i l a r changes i n both groups C and H. L-carnitine treatment produced large increases i n plasma free c a r n i t i n e , acetylcarnitine, acylcarnitine and t o t a l L-carnitine i n group C. A l l four parameters increased during the f i r s t 2 weeks of L-carnitine administration (see Table V I I I ) . Although t o t a l c a r n i t i n e increased, the r e l a t i v e percentage of c a r n i t i n e c i r c u l a t i n g i n ester form (acetyl- and acyl-) d i d not change s i g n i f i c a n t l y . 3.8 TISSUE CARNITINES Although there was no s i g n i f i c a n t difference i n muscle t o t a l c a r n i t i n e levels between the three groups (see Table IX), long-chain acylcarnitine levels were s i g n i f i c a n t l y higher i n group H animals than i n groups N and C. The l i v e r s of the hyperlipidemic rabbits (group H) had s i g n i f i c a n t l y lower levels of free, short-chain acyl and t o t a l c a r n i t i n e than both groups C and N (Table X). In contrast, the levels of l i v e r long-chain acylcarnitine within t h i s group were s i g n i f i c a n t l y higher than the levels found i n groups C and N. This i s s i m i l a r to the s i t u a t i o n found for muscle car n i t i n e levels and may indicate an accumulation of long chain esters i n l i v e r and muscle secondary to the development of the hyperlipidemia. I t i s of interest that L-c a r n i t i n e treatment of the hyperlipidemic rabbit was able to normalize the levels of long-chain acylcarnitine within both tissues. 59 Table IX Muscle c a r n i t i n e levels i n normal rabbits (Group N), hyperlipidemic rabbits (Group H) and hyperlipidemic rabbits treated with L-carnitine f o r 1 month (Group C). n = 4 f o r each group. Group Free car n i t i n e Short - chain Long - chain Total c a r n i t i n e acylcarnitine acylcarnitine (umoles/g NCP) (umoles/g NCP) (umoles/g NCP) (umoles/g NCP) Group N 5.32 + 1.80 7.32 + 3.02 0.31 + 0.11 13.00 + 4.82 Group H 6.34 + 1.01 6.19 + 1.18 0.52 + 0.06 13.10 + 1.96 Group C 7.63 + 2.53 10.48 + 3.73 0.34 + 0.09 18.44 + 5.60 A l l values represent the mean + S.D. P < 0.05 Group H versus Groups N,C. 6 0 Table X Liver carnitines i n normal rabbits (Group N), hyperlipidemic rabbits (Group H) and hyperlipidemic rabbits treated with L - car n i t i n e f o r 1 month (Group C). n = 4 f o r each group. Group Free c a r n i t i n e Short - chain Long - chain Total c a r n i t i n e acylcarnitine acylcarnitine (umoles/g NCP) (umoles/g NCP) (umoles/g NCP) (umoles/g NCP) Group N 3.82 + 1.01 2.04 + 0.68 0.13 + 0.05 6.00 + 0.40 Group H 2.25 + 0.11 0.82 + 0.36 0.40 + 0.06 3.47 + 0.29 * * P < .05 P < .05 WP < .001 VP < .001 Group C 4.51 + 0.74 2.26 + 0.06 0.11 + 0.04 6.90 + 1.10 A l l values represent the mean _+ S.D. * Group H versus Groups N and C. Group H versus Groups N and C. 61 3.9 HISTOLOGY a) Gross Visual Changes The l i v e r s of the rabbits on the high f a t d i e t (Group H) and those on the high f a t d i e t treated with carnitine (Group C) appeared pale yellowish brown as compared to the redish brown colour of the group N rabbits. In addition, the l i v e r s of the group H animals exhibited decreased s t r u c t u r a l i n t e g r i t y and tended to f a l l apart quite e a s i l y . b) H i s t o l o g i c a l Findings Haematoxylin and Eosin (H&E) section of l i v e r from group N rabbits indicated normal l i v e r histology. At low power (x80) d i s t i n c t l i v e r sinusoids and portal space (i.e that region containing the b i l e ductule, terminal hepatic venule and hepatic a r t e r i o l e ) were evident. At higher magnification (x500) hepatocytes had c e n t r a l l y placed nuclei with rather prominent n u c l e o l i . Cytoplasm displayed a combination of granularity, especially i n the perinuclear area, and vacuolation i n other regions. Sinusoidal and/or Kupffer c e l l s were quite evident as indicated by the elongated nuc l e i and sparse cytoplasm (plate 1). O i l Red 0 s t a i n revealed the presence of l i p i d deposits i n group N l i v e r sections. These droplets were more abundant around the portal space (plate 2). In accordance with the gross appearance, H&E sections revealed the presence of s t r i k i n g f a t t y changes i n the l i v e r of group H animals. At low power, the hepatocytes appeared swollen and vacuolated. The vessels of the portal space were normal except f o r the occasional h i s t i o c y t e . The pre-terminal venules were quite noticeable. However, the l i v e r sinusoids, and i n many cases, the central vein were not as obvious. At higher magnification 62 Plate 1: Light micrograph of a section of l i v e r from a group N rabbit. S = l i v e r sinusoids. The arrow points to nuclei of either endothelial or Kupffer c e l l s . Hematoxylin-eosin, x500 63 Plate 2: O i l red 0 stained section of the l i v e r from a group N rabbit. Neutral l i p i d s (dark red spots) appear to be more abundant i n the region surrounding the portal space (P). C = central vein (terminal hepatic venule). x80 64 (x500), most hepatocytes contained several vacuoles presumably representing l i p i d droplets. In some cases, the n u c l e i were either indented or displaced from t h e i r central location (plate 3). Sinusoidal and/or Kupffer c e l l s were evident. However, i t could not be ascertained whether or not t h e i r cytoplasm contained any vacuoles. At low power, O i l Red 0 stained l i v e r sections from group H animals, revealed the presence of neutral l i p i d droplets throughout the l i v e r (plate 4). At higher magnification (x500) i t was obvious that the l i p i d droplets were located within the hepatocytes (plate 5). No e x t r a c e l l u l a r f a t deposits were evident. Haematoxylin and eosin sections of l i v e r from the rabbits treated with L-ca r n i t i n e (group C) revealed the presence of f a t t y changes. These changes appeared to be s i m i l i a r to those seen i n group H. O i l Red 0 stained l i v e r sections from a l l animals i n group C revealed general sparing of the areas immediately surrounding the portal space (i.e the periportal region). The greatest quantities of l i p i d deposits were located around the terminal hepatic venule (central vein). This region represents microcirculatory zone 3 i n the hepatic acinus of Rappaport's model (130) (plate 6). 65 Plate 3: Hematoxylin-eosin stained section of the l i v e r from a hyperlipidemic rabbit (group H). The swollen appearance of the hepatocytes i s quite evident. L = neutral l i p i d droplets. x500 66 Plate 4: O i l red 0 stained section of the l i v e r from a hyperlipidemic rabbit (group H) showing extensive f a t t y i n f i l t r a t i o n (red spots). P = portal space. C = central vein. x80 Plate 5: Light micrograph of a l i v e r section from a hyperlipidemic rabbit (group H). I t can be seen that there are neutral l i p i d droplets (NL) within the hepatocytes. O i l Red 0, x500. 68 Plate 6: O i l red O stained section of the l i v e r from a carnitine-treated hyperlipidemic rabbit (group C). The rabbit was treated with L-carnitine for 1 month. Sparing of the periportal region (PR) i s quite obvious. C = terminal hepatic venule. x80 69 4. DISCUSSION. Each rabbit i n the study had a f a s t i n g plasma glucose determined i n order to r u l e out the existence of diabetes, a condition i n which a secondary hyperlipidemia frequently develops. The f a s t i n g glucose values obtained for a l l animals agreed we l l with reported values i n normal rabbits (131). The weight gain among the three groups of animals was not s i g n i f i c a n t l y d i f f e r e n t from each other, i n spite of the f a c t that group N rabbits tended to consume more food than the other groups. The higher food consumption by Group N may have been due to the lower energy content of the chow with respect to the high-fat d i e t . I n i t i a l l y , attempts were made to induce hypertriglyceridemia i n rabbits i n order to determine whether or not exogenous L-carnitine would have any e f f e c t on VLDL-triglyceride metabolism. In an e f f o r t to achieve t h i s goal, rabbits i n groups C and H were fed a d i e t supplemented with 5% corn o i l and 0.5% cholesterol (W/W). The l e v e l of exogenous cholesterol was set at 0.5% i n order to moderate the extreme hypercholesterolemia which usually develops i n rabbits fed d i e t s supplemented with 1-3% cholesterol (132). In spite of t h i s , levels of plasma cholesterol increased dramatically i n contrast to t r i g l y c e r i d e l e v e l s . At the end of the f i r s t month of feeding, groups C and H had mean plasma cholesterol levels greater than 1000 mg/dl, while t r i g l y c e r i d e levels were less than 100 mg/dl. These findings are i n agreement with those of other investigators (132-134). Rabbits fed a cholesterol d i e t with or without o i l , r apidly develop a severe hypercholesterolemia. The hypertriglyceridemia which develops i s not as severe. I t i s believed that the absence of an appropriate homeostatic mechanism f o r e f f e c t i v e l y regulating whole body cholesterol metabolism together with a very e f f i c i e n t absorption system f o r cholesterol renders the 7 0 rabbit very susceptible to the developemnt of hypercholestermia (135). The a l t e r a t i o n i n plasma t r i g l y c e r i d e s which accompanies the hypercholesterolemia i s thought to be a secondary phenomenon. High plasma cholesterol levels are believed to i n h i b i t lipoprotein lipase which i s involved i n the hydrolysis of VLDL-trglycerides (136,137). This i n h i b i t i o n i s due to the presence of oxysterols which are derivatives of cholesterol (55). Decreased hydrolysis of VLDL-TG eventually leads to increased plasma t r i g l y c e r i d e concentration. Over the second month of the study, group H was continued on the high f a t d i e t . As was expected, plasma cholesterol and t r i g l y c e r i d e s continued to increase, reaching values of 2010 _+ 617 mg/dl and 129 _+ 60 mg/dl respectively. The large standard deviation r e f l e c t s the wide v a r i a t i o n i n response to the d i e t within the group. Rabbits i n group C were treated i n a manner s i m i l i a r to that f o r group H except that they received L-carnitine i n t h e i r water (daily consumption 170 _+ 40 mg/kg). The concentration of plasma cholesterol and t r i g l y c e r i d e s decreased by 35% and 50% respectively i n group C following L-carnitine treatment. These changes were s t a t i s t i c a l l y s i g n i f i c a n t . These res u l t s support e a r l i e r reports (51,52,76,78) which suggest that L-carnitine may lower both cholesterol and t r i g l y c e r i d e s by d i v e r t i n g acyl groups away from e s t e r i f i c a t i o n reactions towards ^ -oxidation i n the mitochondria. Some workers have suggested that t h i s increased f l u x of acyl groups through the ^ -oxidation system i s p a r a l l e l e d by s i g n i f i c a n t increases i n plasma ^-hydroxybutyrate levels (77,81,82,138). This suggestion i s based upon the assumption that ^ -hydroxybutyrate i s a r e l i a b l e indicator of ^ - o x i d a t i o n . In a recent report by Brady e t . a l (139), obese Zucker rats receiving subcutaneous doses of L-carnitine, had s i g n i f i c a n t reductions i n plasma t r i g l y c e r i d e s . This reduction was ascribed to decreased hepatic t r i g l y c e r i d e synthesis and/or secretion. L-carnitine was found to have no e f f e c t on 71 mitochondrial state 3 oxidation. In the current study, L-carnitine had no e f f e c t on plasma ^-hydroxy-butyrate l e v e l s . Plasma ^-hydroxybutyrate turns over very ra p i d l y ( t ^ ^ = ^ minutes (140)), therefore i t would l i k e l y be very d i f f i c u l t to detect increased production by following plasma ^-hydroxybutyrate chronically. Many of the studies which reported increased plasma ^-hydroxybutyrate following L-c a r n i t i n e treatment were of acute design and would therefore be more l i k e l y to a l t e r plasma ^-hydroxybutyrate l e v e l s . Although ^-hydroxybutyrate levels were unchanged i n the current study, i t remains that the e f f e c t of L-carnitine on plasma t r i g l y c e r i d e may be through increased diversion of acyl groups away from e s t e r i f i c a t i o n towards oxidation. The p o s s i b i l i t y that L-carnitine affected synthesis and/or degradation of t r i g l y c e r i d e s w i l l be discussed l a t e r i n connection with the results of the k i n e t i c studies. The mechanisms underlying the decrease i n plasma cholesterol are unknown. Over the past f i v e years, several reports have appeared i n d i c a t i n g that c a r n i t i n e lowers plasma cholesterol i n d i f f e r e n t hyperlipidemic states (52,52,75,79). However, no satisfactory explanation concerning the mechanism of t h i s action has ever been put forward. In the hypercholesterolemic rabbit, the major source of plasma cholesterol i s VLDL and, to a lesser extent LDL secreted by the l i v e r (94-96,141,154,). In addition, plasma t o t a l cholesterol can be affected by cholesterol released i n t o plasma by non-hepatic c e l l s (i.e by reverse cholesterol transport) (55). Exogenous cholesterol i s primarily responsible f o r the increase i n plasma cholesterol (135) and the i n h i b i t i o n of HMG-CoA reductase i n the cholesterol-fed rabbit (142). I t therefore seems un l i k e l y that L-carnitine exerts i t s e f f e c t on plasma cholesterol by modulating the r a t i o of c e l l u l a r acylCoA /CoASH and thereby cholesterol synthesis. Secretion of VLDL- and LDL-cholesterol and the rate of reverse cholesterol transport were not investigated i n t h i s study. 72 Reverse cholesterol transport seems to be maintained primarily by LCAT (143) i n that i n h i b i t i o n of t h i s enzyme leads to a reduction i n reverse cholesterol transport. Whether or not l - c a r n i t i n e has an i n h i b i t o r y e f f e c t on LCAT i s unknown. The p o s s i b i l i t y e x i s t that c a r n i t i n e may decrease plasma cholesterol by decreasing the rate of synthesis and/or secretion of VLDL and LDL-cholesterol by the l i v e r i n the cholesterol fed rabbit. For such mechanisms to be e f f e c t i v e , L-carnitine would have to influence one or more of a number of c e l l u l a r processes involved i n cholesterol homeostasis. Such mechanisms could include (55): a) uptake of lipoproteins v i a receptor-mediated pathways b) net uptake of free cholesterol from lipoproteins by l i p i d transfer. c) cholesterol synthesis d) e f f l u x of cholesterol from c e l l s v i a HDL. e) e s t e r i f i c a t i o n of cholesterol by acyl CoA: cholesterol a c y l -transf erase (ACAT). f) hydrolysis of cholesterol esters, by neutral cholesterol esterase. In the cholesterol-fed rabbit, the uptake of chylomicron remnants by the l i v e r i s normal, while the uptake of VLDL remants i s severely depressed (144,145). This decreased uptake of VLDL remnants promotes further increases i n plasma cholesterol. The k i n e t i c data (to be discussed later) indicate that the catabolism of VLDL was increased. One possible mechanism f o r t h i s increased catabolism could be through increased uptake of of VLDL remnants. Binding studies were not done, and therefore i t i s d i f f i c u l t to draw conclusions with respect to the e f f e c t of L-carnitine on VLDL remnants removal 73 by the l i v e r . Increased uptake of LDL-cholesterol by peripheral c e l l s could also r e s u l t i n decreased plasma cholesterol l e v e l s . However, i t i s not known whether L-c a r n i t i n e has any e f f e c t on the apoB-100/E receptor a c t i v i t y . A decrease i n the quantity of i n t r a c e l l u l a r cholesterol available f o r export could t h e o r e t i c a l l y lead to a reduction i n plasma cholesterol l e v e l s . Such a decrease would occur i f the i n t r a c e l l u l a r a c t i v i t y of ACAT increased and that of cholesterol esterase decreased. However, i t i s not known whether L-carnitine affects either of these two enzymes. F i n a l l y , i t i s possible that c a r n i t i n e may a f f e c t plasma cholesterol i n d i r e c t l y by a f f e c t i n g the synthesis of b i l e acids. Since, greater than 50% of t o t a l body cholesterol i s metabolised to b i l e acid, then any factor a f f e c t i n g b i l e acid metabolism i s very l i k e l y to eventually a f f e c t plasma cholesterol l e v e l s . The r a t e - l i m i t i n g step i n b i l e acid synthesis i s the hydroxylation of cholesterol to 7QC -hydroxycholesterol. This reaction i s catalyzed by 70( -hydroxylase (146). The mechanisms and regulation of subsequent reactions i n the synthesis pathway are largely unknown. For example, l i t t l e i s known about the mechanism and regulation of the ^ -oxidation reaction and side-chain cleavage which produces c h o l i c acid from 3<X ,7 0C ,12 0 ( -trihydroxy- 5/3-cholestanoic acid. I t i s possible that c a r n i t i n e may act to regulate the rate of ^ - o x i d a t i o n and side-chain cleavage. In the conversion of 3QC ,7 OC ,120(-trihydroxy-5^ -cholestanoic acid to c h o l i c acid, propionyl CoA i s released. Thus, i n conditions i n which hepatocyte cholesterol levels are high, i t i s expected that hepatocyte propionly-CoA levels may r i s e secondary to increased b i l e acid synthesis. Propionyl-CoA may be r a t e - l i m i t i n g f o r the production of c h o l i c acid. Removal of propionate may accelerate t h i s reaction. Orally administered L-carnitine i s known to increase the excretion of propionate as 74 propionyl-carnitine i n patients with propionic acidemia (147). Thus, i t i s conceivable that when the i n f l u x of cholesterol into the l i v e r i s high (cholesterol feeding) and the levels of propiony1-CoA are increased, that L-carn i t i n e may decrease levels of propionly CoA thereby enhancing the a v a i l a b i l i t y of free coenzyme A and increasing the rate of b i l e acid synthesis. The net e f f e c t would be to decrease the a v a i l a b i l i t y of cholesterol. Further studies are required to assess the effects of propionyl-CoA on hepatocyte 7 0< -hydroxylase and to determine the effects of L-carnitine treatment on 7 o( -hydroxylase a c t i v i t y and b i l e acid secretion. Plasma c a r n i t i n e levels were monitored i n order to examine the relationship between plasma car n i t i n e and plasma l i p i d s . During the i n i t i a l 4 weeks on the d i e t , plasma free, a c y l - , a c e t y l - and t o t a l L-carnitine increased s i g n i f i c a n t l y above baseline. These values remained s i g n i f i c a n t l y above baseline throughout the study. In t h i s regard, i t i s interesting to note that with prolonged starvation (72 hours), a condition i n which there i s increased f a t mobilization, increases i n serum levels of free, a c y l - and t o t a l c a r n i t i n e were observed i n rats (101). In the same study, f a t feeding (long- and medium-chain trig l y c e r i d e s ) was found to produce an increase i n serum a c y l -and t o t a l c a r n i t i n e and and a decrease i n free c a r n i t i n e . Because of L-carnitine's r o l e i n f a t t y acid metabolism, the increase i n plasma free f a t t y acid levels which usually occurs with f a t feeding could lead to an increased demand for L-carnitine. In such si t u a t i o n s , an increase i n ace t y l - , a c y l - and t o t a l c a r n i t i n e would be expected. The increases i n plasma carn i t i n e tend to support t h i s argument and may represent increased synthesis and/or release of car n i t i n e from other tissues. However, the r a t i o s of acetylcarnitine / acylcarnitine and acylcarnitine / t o t a l c a r n i t i n e were unaffected by the d i e t . This raises the p o s s i b i l i t y that the increase i n 75 plasma t o t a l L-carnitine may have resulted from increased movement of L-carnitine from tissue (muscle or l i v e r ) to plasma. Carnitine treatment produced large increases i n plasma t o t a l c a r n i t i n e , with most of the increase being due primarily to an increase i n free carnitine and secondarily to an increase i n acetylcarnitine. However, the r a t i o s of acetyl/acyl and a c y l / t o t a l were unaffected. These observations may simply r e f l e c t d i r e c t interaction of the administered L-carnitine with the acyl-CoA pools. Total muscle carnitine levels between the three groups were not s t a t i s t i c a l l y d i f f e r e n t . Nonetheless, i t i s interesting to note that muscle long-chain acyl c a r n i t i n e levels i n the fat-fed group were higher than i n the L-carnitine-treated and chow-fed groups. Elevated levels of long-chain acyl carnitines have been previously reported i n diabetic r a t myocardium (149), i n adipose tissue of chronic uremic patients undergoing hemodialysis (84), i n the l i v e r of f a s t i n g rats (150) and i n the l i v e r , adipose tissue and heart of corn o i l - f e d rats (150). Tissue levels of long-chain acylcarnitine are believed to be a r e l i a b l e index of activated f a t t y acids (i.e Acyl CoA's) i n that tissue content of long-chain acylcarnitines varies inversely with the rate of f a t t y acid synthesis and d i r e c t l y with /^-oxidation (150). Thus, i n conditions i n which P-oxidation i s favoured (e.g f a t feeding, f a s t i n g , diabetes), the l e v e l of acylcarnitine increases. Lopaschuk et. a l . (149) have suggested that the increase i n long-chain acyl-CoA levels that occur i n f a t feeding re s u l t s i n the i n h i b i t i o n c a r n i t i n e : acylcarnitine translocase. An i n h i b i t i o n of t h i s enzyme would eventually lead to a build-up of long-chain acylcarnitines within the tissues. In the present study, the increased levels of muscle long-chain acyl 76 carnitines i n the f a t - f e d animals may have been related to increased levels of long-chain acyl-CoA r e s u l t i n g from increased dietary intake of free f a t t y acids. The transfer of the acyl groups to c a r n i t i n e combined with an i n h i b i t i o n of the c a r n i t i n e : acylcarnitine translocase system may have contributed to the build-up of long-chain acylcarnitine. In the treated group, L-carnitine prevented the build-up of long-chain acylcarnitine i n muscle. A s i m i l a r finding has been reported f o r long-chain acylcarnitines i n the myocardium of diabetic rats administered L-carnitine (149). Carnitine i s believed to reverse the i n h i b i t i o n of the c a r n i t i n e : acylcarnitine translocase system through the increased formation of long-chain ac y l c a r n i t i n e from acyl-CoA allowing f o r the regular transport of long-chain acylcarnitine i n t o the mitochondria and a return to normalcy (149). Additional studies are needed to elucidate the mechanisms underlying the accumulation of long-chain acylcarnitines i n muscle with f a t feeding and i t s reversal following L-carnitine treatment Liver levels of free, short-chain acylcarnitine and t o t a l c a r n i t i n e decreased with f a t feeding r e l a t i v e to levels found i n the chow-fed controls. Carnitine reversed t h i s decrease. Long-chain acylcarnitine levels were increased r e l a t i v e to the other two groups. This increase i n long-chain acyl c a r n i t i n e may have been due p a r t l y to increased formation of long-chain acylcarnitines and p a r t l y to i n h i b i t i o n of the c a r n i t i n e : acylcarnitine translocase by long-chain acyl-CoAs. The decrease i n l i v e r free, short-chain a c y l - and t o t a l c a r n i t i n e and increase i n plasma free, a c e t y l - , a c y l - and t o t a l c a r n i t i n e i n the fat-fed animals may be related to i n h i b i t o n of ^ -oxidation and increased movement of free and short-chain acylcarnitines from l i v e r to plasma. Depression of -oxidation (e.g glucose loading) leads to decreased formation of a c e t y l -77 c a r n i t i n e (150). Short-chain f a t t y acids are able to move f r e e l y across the inner mitochondrial membrane (6). Since a short-chain acylcarnitine transferase i s present i n the endoplasmic recticulum (18) then, the levels of these esters i n plasma may be independent of the ca r n i t i n e : acylcarnitne translocase system. Moreover, the l i v e r c a r n i t i n e pool (unlike that of muscle) i s i n rapid equilibrium with the plasma pool (151) and l i v e r perfusion studies have established that free and acetylcarnitine are rapidly released by the l i v e r i n t o the blood (12). Once i n the plasma, free and short-chain acylcarnitines may be excreted i n urine or u t i l i s e d by peripheral tissues (12). In t h i s regard i t i s interesting to note that high levels of L-car n i t i n e and acetylcarnitine have been found i n the brain where they may play a c r u c i a l r o l e i n the formation of acetylcholine and i n the regulation of "Y -aminobutyrate levels i n cerebro-spinal f l u i d (152). The continued movement of acetylcarnitine from l i v e r to plasma together with the decreased formation of short-chain esters i n the l i v e r (due to the i n h i b i t i o n of j2 -oxidation) could eventually lead to depletion of l i v e r L-carnitine stores. The f a c t that the treated group had normal l i v e r c a r n i t i n e levels may simply r e f l e c t a replenishment of l i v e r c a r n i t i n e stores. In the hypercholesterolemic rabbit, a large percentage of plasma cholesterol and t r i g l y c e r i d e s are carried primarily by VLDL p a r t i c l e s having beta mobility (154). LDL carries a smaller percentage of the cholesterol i n t h i s perturbed state (96). With t h i s i n mind, various VLDL parameters were examined to determine how they would be affected by L-carnitine treatment. A l l measured plasma VLDL parameters (VLDL-TG, VLDL-ApoB, VLDL-cholesterol and VLDL protein) increased over the f i r s t 4 weeks of exposure to the high f a t d i e t . VLDL-cholesterol had the largest change, increasing by more than 200 f o l d . VLDL-TG increased by 4 f o l d . In the second 4 weeks of the experiment, 78 plasma VLDL-cholesterol and VLDL-triglyceride doubled. This was re f l e c t e d i n a doubling of plasma t o t a l cholesterol and t r i g l y c e r i d e s . Plasma VLDL-cholesterol decreased by 50% i n the L-carnitine-treated group. This decrease was p a r a l l e l e d by a 40% decrease i n plasma cholesterol. A s i m i l a r pattern was noted f o r plasma VLDL-triglycerides and t o t a l t r i g l y c e r i d e s . These res u l t s indicate that i n the hypercholesteremic rabbit changes i n plasma cholesterol and t r i g l y c e r i d e s are not s o l e l y due to changes i n VLDL composition. The VLDL-ApoB and t o t a l protein levels which were elevated by the f a t d i e t , decreased following L-carnitine treatment. The decrease i n VLDL t o t a l protein achieved significance whereas the reduction i n apoB d i d not. These data indicate that other VLDL associated proteins, i n addition to apoB, decreased. A number of studies (91,155) have reported increased plasma levels of HDL-cholesterol i n the hypercholesterolemic rabbits, while others have found HDL-cholesterol to be unaltered (94,96) or reduced (156). In the present study, neither the high-fat d i e t nor L-carnitine treatment affected plasma HDL-cholesterol. Agarose gel electrophoresis of plasma revealed that the high-fat d i e t produced increased width and int e n s i t y of the beta and pre-beta bands and a decrease i n the si z e and in t e n s i t y of the 0(-band. Following L-carnitine treatment, the width and int e n s i t y of t h i s broad beta/pre-beta band decreased, but the 0<-band was not affected. I t i s now known that feeding rabbits a d i e t supplemented with cholesterol and f a t leads to the appearance of VLDL withyS-mobility i.e ^ -VLDL (96,154). This a l t e r a t i o n i n mobility from pre-beta to beta i s due to changes i n the si z e and composition of the VLDL p a r t i c l e s (96). In the present study, the composition of the VLDL p a r t i c l e was markedly altered by the the high d i e t . The appearance of a 79 "broad" beta band may be explained p a r t l y i n terms of the changes i n the l i p i d composition of these VLDL p a r t i c l e s . The reason for the decline i n int e n s i t y of the 0<-band (implying reduced HDL) i s not c l e a r . The finding of decreased i n t e n s i t y of the 0<-band seem to contradict the observation that HDL-cholesterol remained unchanged during the course of the current study. Other investigators (96,156) have reported decreased HDL staining upon polyacrylamide gel electrophoresis of hypercholesterolemic plasma. The observation may be a r t i f a c t u a l i n that ultrace n t r i f u g a t i o n and electrophoresis of the "HDL" f r a c t i o n showed the presence of an 0<-band. In addition, normal levels of HDL were found on chemical analysis. In rabbits fed the high f a t d i e t , the f r a c t i o n a l catabolic rate (FCR) of VLDL-TG was s i g n i f i c a n t l y reduced r e l a t i v e to the chow-fed rabbits. The transport rate of VLDL-TG out of the plasma compartment (which represents production rate under steady-state conditions) was not s i g n i f i c a n t l y affected by the d i e t . The decrease i n the FCR of VLDL-TG would be expected given the in h i b i t o r y e f f e c t of increasing plasma cholesterol levels on lipoprotein lipase (136,137). I n h i b i t i o n of lipoprotein lipase would decrease l i p o l y s i s , and lead to a reduction i n the catabolic rate of VLDL-TG. The lack of e f f e c t of the d i e t on VLDL-TG transport rate can be accounted for on the basis of the increased VLDL-TG pool s i z e found i n the hyperlipidemic rabbits. This r e s u l t s i n transport rates (transport rate = FCR x pool size) which are close to, or higher than, those i n chow fed rabbits. L-carnitine treatment increased the FCR of VLDL-TG i n hyperlipidemic rabbits, i n d i c a t i n g that VLDL-TG was being cleared at a faster rate from the plasma. This increase resulted i n a mean FCR which was not s t a t i s t i c a l l y d i f f e r e n t from that f o r the chow-fed animals. This increase i n FCR may have 80 been due to increased lipoprotein lipase a c t i v i t y following L-carnitine treatment. L-carnitine may have d i r e c t l y modulated the a c t i v i t y of lipoprotein lipase or lipoprotein lipase a c t i v i t y may have increased subsequent to the decrease i n plasma cholesterol. Although the resu l t s of a few studies (78,157) suggest that L-carnitine therapy increases heparin releasable lipase a c t i v i t y (i.e lipoprotein lipase a c t i v i t y ) , other studies have reported f i n d i n g no e f f e c t (139,148). A previous study i n rabbits has demonstrated that that lipoprotein lipase i s i n h i b i t e d i n the presence of high levels of plasma cholesterol (136). I t thus seems appropriate to suggest that, i n the present study, lipoprotein lipase a c t i v i t y may have increased secondarily to the decrease i n plasma cholesterol l e v e l s . The transport rate of VLDL-TG was not s i g n i f i c a n t l y altered following L-carn i t i n e treatment. Since transport rate i s equal to the production rate under steady-state conditions, these re s u l t s indicate that L-carnitine had no ef f e c t on VLDL-TG synthesis. Brady et. a l . (139) reported that subcutaneous doses of L-carnitine (administered f o r 2 to 3 months) were found to produce a s i g n i f i c a n t reduction i n plasma t r i g l y c e r i d e levels and hepatic t r i g l y c e r i d e secretion i n female obese zucker r a t s , while o r a l doses were i n e f f e c t i v e . The differences between the resu l t s of that study and the resu l t s of the present study may be a function of differences i n the methodology used f o r assessing hepatic t r i g l y c e r i d e secretion, species differences and/or d i f f e r e n t route of admininistration of L-carnitine. The f r a c t i o n a l catabolic rate of VLDL-ApoB was decreased i n the hyperlipidemic rabbits r e l a t i v e to the chow-fed rabbits. Similar r e s u l t s have been reported by others (158,159). This decrease i n FCR i s thought to r e f l e c t a decreased catabolism of the 'abnormal1 VLDL which i s secreted by the rabbit l i v e r i n the hypercholesteremic state. On the other hand, the transport rate of VLDL-ApoB was moderately elevated i n the hyperlipedemic rabbit compared to 81 normals despite the reduced FCR. As i n the case of VLDL-TG, t h i s i s most l i k e l y due to large pool sizes (determined from steady state masses of VLDL-ApoB) i n the hyperlipidemic rabbits (160). The FCR f o r VLDL-ApoB was unaffected by L-carnitine treatment. Although there tended to be a decrease i n the VLDL production rate with L-carnitine treatment, t h i s decrease f a i l e d to reach significance. Since FCR was unaffected, then t h i s moderate decrease i n the production rate can be attributed to a decrease i n the VLDL-apoB pool s i z e . I t should be pointed out that the transport rate f o r VLDL-ApoB was s i m i l a r i n the normals and hyperlipidemic rabbits. This finding i s contrary to previous reports (158-160) and may be a r e s u l t of abnormally high f r a c t i o n a l catabolic rates calculated f o r the normal rabbits. The r e s u l t s of the k i n e t i c studies indicate that f a t feeding reduces the FCRs of VLDL-ApoB and VLDL-TG, whereas the production of these two moieties are unaffected. L-carnitine treatment normalised the FCR of VLDL-TG, but had no e f f e c t on the FCR of VLDL-ApoB. The transport of both moieties was unaffected. This seemingly d i f f e r e n t i a l e f f e c t of L-carnitine on VLDL-TG and VLDL-apoB metabolism i s not unexpected i n that, at least i n humans, i t has been shown that the metabolism of VLDL-ApoB i s disassociated from that of VLDL-TG and that changes i n the rates of secretion or catabolism of one may not be accompanied by changes i n the other (176). Fat-feeding lead to the development of a hepatic steatosis (fatty l i v e r ) . The f a t was uniformly d i s t r i b u t e d throughout the l i v e r . L-carnitine treatment did not reverse the steatosis, but i t d i d reduce the density of f a t deposits i n the periportal area. These findings are s i m i l i a r to those found i n an e a r l i e r study by Seccombe et. a l (85). In that study, 16 weeks of L-carnitne 82 therapy d i d not reverse the hepatic steatosis, but there was a reduction i n l i p i d deposits within the l i v e r . The f a t t y l i v e r s developed i n the fat-fed rabbits may have resulted from increased production of VLDL by the l i v e r . Kroon et a l (161) have recently reported that (2 -VLDL i n plasma of the fat-fed rabbits was of hepatic rather than of i n t e s t i n a l , o r i g i n . The ^5-VLDL that i s produced i s cho l e s t e r o l - r i c h and i s poorly catabolized by lipoprotein lipase. In addition i t appears to be poorly endocytosed by hepatocytes once released (144). Thus the accumulation of l i p i d s i n the l i v e r of fat-fed rabbits could have resulted mainly from over production of ^ -VLDL rather than from uptake of previously secreted lipoproteins. An increase i n VLDL-triglyceride synthesis could have also contributed to the f a t t y l i v e r since f a t feeding has been shown to lead to increased l i v e r t r i g l y c e r i d e levels i n rats (148). Interesting, exogenous L-carnitine was found to prevent t h i s increase. The hepatocytes i n the periportal region (microcirculatory zone 1 of the hepatic acinus) are at higher oxygen tension and are metabolically more active (130). I t i s possible that these c e l l s may be capable of secreting f!> -VLDL at a higher rate. They therefore do not accumulate these p a r t i c l e s except i n situations where the i n f l u x of cholesterol i s so high that i t accumulates within the c e l l at a faster rate than i t can be exported. The sparing of the peripo r t a l area i n l i v e r sections from treated rabbits could therefore be the resul t s of higher VLDL secretion rates. A l t e r n a t i v e l y , the observed sparing of the periportal area could be a r e f l e c t i o n of a metabolic gradient of d i s t r i b u t i o n of administered L-carnitine i n the l i v e r , since i t i s known that zone 1 hepatocytes receive blood richer i n nutrients. Because of the higher oxygen tension, the mitochondria within t h i s zone may be able to respond to L-carnitine treatment with enhanced &-83 oxidation. 84 5. CONCLUSIONS The following conclusions can be drawn from the r e s u l t s of t h i s study: 1) In rabbits fed the high-fat d i e t , plasma t o t a l cholesterol, and t r i g l y c e r i d e s , cholesterol, apoprotein B and t o t a l protein associated with the VLDL p a r t i c l e increased s i g n i f i c a n t l y . There were no s i g n i f i c a n t changes i n HDL-cholesterol and plasma t r i g l y c e r i d e s . 2) The f r a c t i o n a l catabolic rate f o r VLDL-triglycerides and VLDL-apoprotein B was s i g n i f i c a n t l y reduced i n the hyperlipidemic state. 3) Plasma concentrations of free c a r n i t i n e , acetylcarnitine, acylcarnitine and t o t a l c a r n i t i n e increased with the high-fat d i e t . Although plasma c a r n i t i n e levels were increased, the r e l a t i v e percentage of a c e t y l - and acylcarnitine within the plasma pool were unchanged. 4) The high-fat d i e t s i g n i f i c a n t l y increased l i v e r and s k e l e t a l muscle long-chain acylcarnitines. On the other hand, the d i e t caused l i v e r free c a r n i t i n e , short-chain acylcarnitine and t o t a l c a r n i t i n e to be s i g n i f i c a n t l y reduced. 5) L-carnitine treatment of the hyperlipidemic rabbit produced s i g n i f i c a n t reductions i n plasma concentrations of t o t a l cholesterol, t r i g l y c e r i d e s , VLDL-t r i g l y c e r i d e s , VLDL-cholesterol and VLDL t o t a l protein. I t had no e f f e c t on plasma HDL-cholesterol. 6) Liver and s k e l e t a l muscle car n i t i n e levels i n the hyperlipidemic c a r n i t i n e -85 treated animals were normalized following treatment. Although treatment s i g n i f i c a n t l y elevated a l l plasma ca r n i t i n e fractions well above those seen i n the hyperlipidemic untreated animals, the r e l a t i v e percentage of acetyl and acyl esters within the plasma pool remained unchanged. 7) The f r a c t i o n a l catabolic rate of VLDL-triglycerides returned to control values with L-carnitine treatment. Treatment had no e f f e c t on VLDL-apoprotein B k i n e t i c s . 8) On the basis of these r e s u l t s , i t was concluded that the reduction i n plasma t r i g l y c e r i d e s i n the hyperlipidemic rabbit following L-carnitine treatment was due to an increase i n the catabolism of VLDL-triglycerides. 86 APPENDIX A Calculation of the Fractional Catabolic Rate (FCR) for monoexponentia1 decay: Application to the determination of FCR f o r VLDL-TG A t y p i c a l VLDL-TG tracer curve obtained following the i n j e c t i o n of H-glycerol i n t o normal rabbits i s presented i n f i g . 13. The curve may be resolved into 3 phases. An i n i t i a l rapid r i s e , an early f a s t decay followed by a slow decay phase. In humans, the decay of VLDL-TG s p e c i f i c a c t i v i t y i s s i m i l a r to that shown for the rabbits (125,162). Moreover, i t has been shown that t h i s decay curve may be approximated t o " f i i a t of a monoexponential curve since the t a i l of the curve i s not considered to contribute s i g n i f i c a n t l y to the magnitude of the o v e r a l l catabolic rate of VLDL-TG (125,162). In t h i s instance, only the rapid decline phase i s used i n the estimation of FCR. The h a l f - l i f e ( t ^ ^ ) °^ the decay of t h i s i n i t i a l phase was v i s u a l l y estimated and from the value of ^1/2 ^ e f r a c t i o n a l catabolic rate was estimated (125). The mathematical derivation of the relationship between a n d t h e rcR ^ s a s follows. Let A = Quantity of r a d i o a c t i v i t y i n i t i a l l y injected. The decay, dA/dt, i s proportional to A or dA/dt = -kA Separating the variables gives dA/A = -kdt and by integration we get In A = - Kt + C. (1) where C i s a constant and In i s the natural logarithm. At t = 0, A = A Q and therefore In A Q = C 87 O 2 A- 6 8 10 T I M E ( h r s ) F i g . 13: Typical VLDL-TG tracer curve obtained following the i n j e c t i o n of H-glycerol into normal rabbits. The curve may be resolved into three phases: an i n i t i a l rapid r i s e , an early f a s t decay followed by a slow decay phase. 88 Thus equation (1) becomes In A/AQ = -kt or In AQ/A = k t (2) At time t " t 2 / 2 ' a = A Q / 2 and upon substitution into equation (2), we w i l l obtain In 2 = k . t 1 / 2 (3) In equation (3), k represents the f r a c t i o n a l catabolic rate (FCR) f o r monoexponential decay (125). As a r e s u l t the equation may be rewritten as FCR = In 2 / t 1 / 2 (4) t ^ 2 can be found d i r e c t l y from the s p e c i f i c a c t i v i t y versus time curve by v i s u a l inspection and the FCR may then be estimated using equation (4). Alt e r n a t i v e l y , a p l o t of In A versus time w i l l have a slope of k (see equation 2). This method of estimating the FCR may occasionally underestimate the FCR value because of the error involved i n f i t t i n g the curve by eye and because the t a i l of the VLDL-TG curve i s now known to contribute to varying degrees to the FCR i n d i f f e r e n t disease conditions (162,163,164). In f a c t , more detailed methods, namely multicompartmental analysis (164), are now a v i l a b l e for the estimation of the FCR of VLDL-TG catabolism, but the use of these methods are outside the scope of t h i s study and w i l l not be discussed. At t h i s stage, i t should be pointed out that the shape of the VLDL-TG decay curve may vary from indi v i d u a l to indiv i d u a l (animal to animal). In some cases, as i n hypercholesterolemic rabbits, two d i s t i n c t decay phases may not be evident. However, regardless of the shape of the curve, the ca l c u l a t i o n of FCR remains the same. 89 APPENDIX B Analysis of Blood Level data i n a Two-Pool system: Calculation of curve  parameters. 125 The disappearance of I-VLDL-ApoB i n plasma generally conforms to a biexponential function. As a r e s u l t , the two pool model of Gurpide et. a l (127) i s usually used to describe the metabolism of VLDL-ApoB. These authors developed a series of mathematical formulae for estimating the rate constants (including FCR's), production rates and pool sizes i n two-pool systems. A l l of these formulae are functions of ce r t a i n parameters of the decay curve. Therefore, the curve analysis method w i l l be b r i e f l y summarised i n t h i s section. 125 A t y p i c a l curve f o r the decay of I-VLDL-ApoB s p e c i f i c a c t i v i t y 125 following the i n j e c t i o n of I-VLDL into chow-fed rabbit i s presented i n f i g . 14. As shown, the disappearance curve can be resolved into two exponential functions with rate constants OC and respectively. The f i r s t exponential ( o< * -line) i s obtained by "curve-peeling" (curve stripping) (127). The value of the second exponential ( ^ - l i n e ) at a given time i s subtracted from the corresponding value of the i n i t i a l phase of the curve. A p l o t of the new values versus time w i l l y i e l d a l i n e with slope equal to - OC and a y-intercept equal to C A. The slope of the second exponential has a value of -y3 and y-axis intercept of Cg. 0 < , ^ , C^ and Cg may then be used to estimate the value of the FCR as described i n the text. * see Appendix C for d e f i n i t i o n 90 1ooooo 210000 Q _ Q 5 -< O G: o Q J Q _ O O 1000 + 100 4 6 TIME ( h r s ) 125, F i g . 14: Typical s p e c i f i c a c t i v i t y - t i m e curve f o r the disappearance of I -VLDL-apoB f^pm the plasma of normal rabbits following the i n j e c t i o n of autologous I-VLDL. 91 APPENDIX C KINETIC DEFINITIONS i) Compartment a) An anatomical, physiological, chemical or physical subdivision of a system, throughout which the r a t i o of concentration of tracer to tracee i s uniform at any given time (165). b) A subdivision of a model used for the analysis of experimental observation (165). i i ) Pool The t o t a l amount of substance i n a system or subsystem (165). i i i ) Steady-state The condition i n which the amount of tracee (or of tracer) i s constant over the duration of the experiment (165). (iv) Fractional Catabolic Rate (FCR) The f r a c t i o n a l catabolic rate f o r a p a r t i c l e i n compartment I represents the p r o b a b i l i t y per u n i t time f o r a p a r t i c l e to leave that compartment without ever returning (166). An equivalent d e f i n i t i o n i s the f r a c t i o n of the amount of material present that i r r e v e r s i b l y leaves a given compartment without ever returning. (v) Transport Rate (165-167) The amount of material crossing a boundary per u n i t time or more precisely the rate at which material leaves a compartment i r r e v e r s i b l y . I t i s given by 92 the product of the steady-state mass (M^ ) and the f r a c t i o n a l catabolic rate I t i s also sometimes known as the i r r e v e r s i b l e disposal rate (IDR), metabolic rate (MR) or catabolic rate, CR. In steady state, any i r r e v e r s i b l e loss must be replaced, thus, t h i s quantity also represents the rate of entry of new (nonrecycled) material into that compartment and i s referred to as the production rate. (vi) Curve-peeling (Curve stripping) A process of resolving a curve into the sum of i t s indi v i d u a l exponentials by sequentially extracting the indi v i d u a l exponentials (166). 93 Appendix D An Investigation of L-carnitine Treatment i n Hypertriglyceridemia: Results of  studies i n Yucatan Minipigs O r i g i n a l l y the current study was designed to examine the e f f e c t of L-car n i t i n e treatment on hypertriglyceridemia i n the Yucatan miniature pig. The pig was selected as the model of choice because i t has been shown to be an excellent animal model for the study of many human disorders including those of the blood, cardiovascular system and those of l i p i d metabolism (90,168). Yucatan miniature pigs were chosen on the basis of the r e l a t i v e l y small s i z e of the adult and because of t h e i r r e l a t i v e d o c i l i t y (169). On account of the d i f f i c u l t i e s experienced i n working with t h i s animal model, and the low rate of success of the various procedures, i t was decided to search f o r an alternative model. In t h i s section of the text , the progress of the work with the Yucatan minipigs, the d i f f i c u l t i e s experienced and the data co l l e c t e d w i l l be b r i e f l y summarised. Animals Five adult male Yucatan Miniature pigs (2.5 - 4 years old) were o r i g i n a l l y obtained from the Swine Research Center at Colorado State University. One animal succumbed to hypothermia while another had to be k i l l e d because of systemic i n f e c t i o n which developed following blood vessel catheterization. Autopsy revealed that a l o c a l i n f e c t i o n had developed around the catheter and tracked into the c i r c u l a t i o n (sinus tracking). 94 Handling of Animals A Panepinto S l i n g (169) obtained from the Colorado Swine Research Center was used f o r re s t r a i n i n g the pigs during a l l routine procedures. Implantation of Venous Catheters I n i t i a l l y a 60 cm s i l a s t i c l i n e (Medical Grade; int e r n a l diameter, ID, of 0.04 i n . , outer diameter, OD, of 0.085 in.) was implanted into the r o s t r a l vein of the auricular h e l i x of each pig. The l i n e was advanced 20 cm into the vessel such that the t i p was located i n the maxillary vein. The li n e s were supplied by the Dow Corning Corporation, Midland, Michigan, USA. S i l a s t i c tubing was found to be unsuitable f o r long term sequential blood sampling because the l i n e s occasionally collapsed during the withdrawal of blood and some l i n e s ruptured 2 weeks to one month following implantation into the pigs. I t was therefore decided to t r y Tygon Microbore tubing (ID .04 i n . , OD .07 i n . , w a l l .015 in) which was previously found to be much more durable and more suitable f o r long term implantation i n t o these pigs (personal communication: Dr. Jim T e r r i s , Armed Forces University of the Health Sciences, Bethesda, Maryland.). Due to frequent blockage of the Microbore l i n e s , a larger tygon tubing (ID 0.0625 i n , OD 0.125 in) was f i n a l l y selected f o r future catheterizations. The tygon tubings were purchased from Norton P l a s t i c s , Cleveland, Ohio, USA. To reduce the incidence of sinus tracking, a velor cuff was glued around a l l l i n e s and positioned approximately 1.0 to 2.0 inches below the skin when the l i n e was implanted (170). The li n e s were gas - s t e r i l i z e d and a l l l i nes were implanted i n a s t e r i l e environment while the animals were under general anesthesia. On account of the small s i z e of the r o s t r a l vein of the ear and the s i z e of the l i n e which were f i n a l l y selected, the li n e s were implanted 95 into the external jugular vein and tunnelled subcutaneously to the interscapular region of the neck. Each animal was allowed to recover f o r one week before proceeding with the study. However, i n spite of a l l the precautions taken, including d a i l y cleansing of the wounds with antiseptic solution, l o c a l i z e d i n f e c t i o n remained a troublesome problem. Maintenance of Venous Lines I n i t i a l l y , the l i n e s were flushed d a i l y with 4% sodium c i t r a t e solution. Heparin was not used because i t i s known to release the enzyme lipoprotein lipase which hydrolyzes VLDL-triglycerides (171) and thus would inte r f e r e with the study. I t was found that despite d a i l y flushing, l i n e patency could not be e f f e c t i v e l y maintained due to leaching of the c i t r a t e . To overcome t h i s problem, i t was decided to switch to an 8% solution of sodium c i t r a t e i n 50% dextrose (172). The 'flushing solution' was prepared by the Shaughnessy, Children's and Grace Pharmacy, Vancouver, B.C. Since t h i s solution i s viscous leaching of the c i t r a t e was minimized. The l i n e s were flushed as follows: a) the 8.0% sodium c i t r a t e / 50 % dextrose solution was removed. b) the l i n e s were flushed with s t e r i l e s a l i n e . c) the l i n e s were then f i l l e d with a known volume of the 8.0% sodium c i t r a t e / 50 % dextrose. This volume was calculated on the basis of the length and in t e r n a l diameter of the implanted l i n e . In many cases blood could not be withdrawn from the l i n e s , but i t was always possible to i n j e c t solution with l i t t l e pressure. This indicated that the t i p of the catheter (cut at an acute angle to f a c i l i t a t e implantation) rested against the w a l l of the vessel creating a v a l v e - l i k e action whenever negative pressure was applied. To minimize future occurrence of t h i s problems 96 the proximal t i p of the li n e s were cut bl u n t l y p r i o r to implantation. In f a c t , t h i s l a t t e r action seemed to increase the l i n e patency period from a couple of weeks to a couple of months. Diet. For normotriglyceridemic studies, the pigs were maintained on adult swine r a t i o n (14% protein, 70.7% carbohydrates (mainly starch), 3.5% f a t s , 3.5% vitamin-mineral mix and 8% fiber) supplied by Buckerfield's, Abbortsford, B.C. For hypertriglyceridemic studies, the pigs were maintained on an i s o c a l o r i c , isonitrogenous d i e t containing 40% sucrose (14% protein, 1.8% f a t , 81.3% carbohydrates (50% sucrose),1.2% f i b r e s , 3.5 vitamin-mineral mix). The d i e t was prepared from crushed corn, p u r i f i e d casein (Biochemical Corporation, Bethesda, MD) sucrose c r y s t a l s and the University of B r i t i s h Columbia (U.B.C) vitamin-mineral premix A (Animal Sciences Dept. U.B.C). Methodologies The various methods employed for the measurement of plasma l i p i d s and car n i t i n e are detailed i n the Materials and Methods section of t h i s text. Serum elec t r o l y t e s and serum levels of l i v e r enzymes were measured on an Ektachem 700 machine using k i t s supplied by the manufacturer (Eastman Kodak Co. Rochester, N.Y. USA). Preliminary Data Plasma levels of elec t r o l y t e s and fasting glucose (Table XI), l i p i d s (Table XII) and l i v e r enzymes (Table XIII) were well within the normal range of values f o r adult pigs (173). However, fa s t i n g levels of cholesterol (58.0 -97 69.0 mg/dl), t r i g l y c e r i d e s (12.0 - 32.0 mg/dl) and HDL-cholesterol (35.0 -43.0 mg/dl) were lower than i n humans (Cholesterol = 150 - 270 mg/dl, t r i g l y c e r i d e s = 50 - 260 mg/dl and HDL-cholesterol = 35.0 - 70.0 mg/dl respectively). Lipoprotein electrophoresis (LPE) indicated that the pigs' VLDL, LDL and HDL had the same mobility as those i n humans. However i n a l l cases there was a s l i g h t decrease i n the LDL band. Fasting serum t o t a l L-carnitine and the r a t i o s of acetylcarnitine /acylcarnitine and a c y l c a r n i t i n e / t o t a l c a r n i t i n e were found to be lower i n Yucatan Miniature pigs than i n Humans (Table XIV). I n i t i a l l y the pigs were maintained on the low f a t , high carbohydrate hyperlipidemic d i e t f o r two months. However, there appeared to be no e f f e c t on plasma l i p i d s (data not shown). The pigs were then switched to a high f a t d i e t (containing 20% safflower o i l ) because i t was previously reported that t h i s d i e t produced hyperlipidemia i n domestic pigs (174,175). The pigs were kept on t h i s d i e t f o r 6 weeks during which time plasma l i p i d s were monitored. Plasma l i p i d s were also unaffected by t h i s d i e t . On the basis of these findings, i t s was inferred that the Yucatan miniature p i g was either very r e s i s t a n t to the development of dietary -induced hypertriglyceridemia using the methods employed i n t h i s study or the feeding period was too short. The model was abandoned. 98 Table XI Plasma el e c t r o l y t e s and glucose i n adult male Yucatan miniature pigs. The pigs were fasted f o r 24 hours p r i o r to blood c o l l e c t i o n . n = 3. Parameter Plasma concentration Potassium (mM) 4.1 - 4.4 Sodium (mM) 150 - 152 Chloride (mM) 99 - 101 Calcium (mM) 7.4 - 8.3 Phosphorus (mg/dL) 5.8 Anion gap 26.1 - 33.4 Glucose (mg/dL) 59 - 65 99 Table XII Plasma l i p i d values i n adult male Yucatan miniature pigs. Animals were fasted for 24 hours p r i o r to blood c o l l e c t i o n , n = 3. Parameter Plasma value Triglycerides (mg/dL) 12.0 - 32.0 Cholesterol (mg/dL) 58.0 - 69.0 Free f a t t y acid (uEq/L) 202 - 372 HDL-cholesterol (mg/dL) 35.0 - 43.0 100 Table XIII Serum levels of glutamate - oxalate transaminase (GOT), creatine kinase (CK), albumin, and t o t a l protein i n adult male Yucatan miniature pigs. The animals were fasted f o r 24 hours p r i o r to blood c o l l e c t i o n . Parameter Concentration GOT (IU/L) 34.0 - 48.0 CK (IU/L) 189 - 554 Albumin (g/dL) 4.0 - 4.4 Protein (g/dL) 7.3 - 7.7 101 Table XIV Plasma free L-carnitine (free), acylcarnitine (acyl), acetylcarnitine (acetyl) and t o t a l L-carnitine (total) i n adult male Yucatan miniature pigs (n = 3) and i n normal male human subjects. The pigs were fasted f o r 24 hours while the human subjects were fasted f o r 12 hours. 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