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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 F r 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 in THE FACULTY OF GRADUATE STUDIES (Department o f P a t h o l o g y )  We a c c e p t t h i s t h e s i s a s c o n f o r m i n g to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA O c t o b e r 1986 ® L e i g h t o n R o l s t o n James, 1986  iE-6  In p r e s e n t i n g requirements  this thesis f o r an  of  British  it  freely available  agree t h a t for  that  Library  s h a l l make  for reference  and  study.  I  f o r extensive copying of  h i s or  be  her  g r a n t e d by  shall  not  be  of  The U n i v e r s i t y o f B r i t i s h 1956 Main Mall V a n c o u v e r , Canada V6T 1Y3  Date  (3/81)  #M  of  further this  Columbia  thesis  head o f  this  my  It is thesis  a l l o w e d w i t h o u t my  permission.  Department  the  representatives.  copying or p u b l i c a t i o n  f i n a n c i a l gain  University  the  s c h o l a r l y p u r p o s e s may  understood  the  the  I agree that  permission by  f u l f i l m e n t of  advanced degree a t  Columbia,  department or for  in partial  written  ABSTRACT  Cardiovascular disease, leading  s p e c i f i c a l l y coronary heart disease, remains the  cause of morbidity and mortality among the adult population i n  America and Western Europe.  North  Hyperlipidemia ranks as one of the most important  r i s k f a c t o r s f o r cardiovascular disease.  Thus, the need f o r e f f e c t i v e therapy  i n the management and treatment of hyperlipidemia remains high. At present, choice  d i e t a r y manipulations and/or drug therapy are the methods  i n the management and treatment of hyperlipidemia.  currently lipids,  used  hypolipidemic  drugs are very e f f e c t i v e  many of these have unpleasant s i d e - e f f e c t s .  of  Though most of the in  reducing  plasma  The search f o r e f f e c t i v e  hypolipidemic agents, with r e l a t i v e l y few s i d e - e f f e c t s , continues. One  such  compound  presently under consideration i s  hydroxy-*V -trimethylaminobutyrate).  This  acid  is  a  L-carnitine (  n a t u r a l l y occurring  substance which has been reported to possess lipid-lowering properties. required f o r the optimum oxidation of long-chain f a t t y acids.  It i s  In addition, i t  functions as a buffer f o r coenzyme A pools within the c e l l . The weeks  present study was designed to examine the hypolipidemic e f f e c t of  of  rabbits treatment  L - c a r n i t i n e treatment fed  a  high-fat  diet.  (170 mg/kg b.w/day)  in  In p a r t i c u l a r ,  effect  the  on plasma concentrations of c h o l e s t e r o l ,  studies  were  Zealand of  White  L-carnitine  triglycerides,  3 HDL-cholesterol were examined. In addition,  New  4  VLDL  and  125  H-glycerol and  I-VLDL turnover  conducted i n order to examine the e f f e c t of treatment  on  VLDL  kinetics. In  rabbits  fed  the  high-fat  diet,  plasma  total  cholesterol,  and  t r i g l y c e r i d e s , c h o l e s t e r o l , 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 .  HDL-cholesterol  There were no s i g n i f i c a n t changes  and plasma t r i g l y c e r i d e s . i i  in  The  f r a c t i o n a l catabolic rate f o r VLDL-triglycerides  B were s i g n i f i c a n t l y reduced i n the hyperlipidemic transport  state.  and VLDL-apoprotein In  addition,  rate f o r these two components of the VLDL p a r t i c l e were  the  moderately  elevated. With  hyperlipidemia,  acetylcarnitine,  plasma  concentrations  of  l e v e l s were increased,  carnitine  esters within the plasma pool were unchanged.  other  the  liver  Although  the r e l a t i v e percentage of acetyl and  long-chain a c y l c a r n i t i n e s hand,  carnitine,  a c y l c a r n i t i n e and t o t a l c a r n i t i n e were increased.  carnitine  muscle  free  L i v e r and  skeletal  were also s i g n i f i c a n t l y increased.  concentrations  of  free  carnitine,  acyl  On  the  short-chain  a c y l c a r n i t i n e and t o t a l c a r n i t i n e were s i g n i f i c a n t l y reduced. L-carnitine  treatment of the hyperlipidemic  rabbit produced  significant  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 , VLDLtriglycerides,  VLDL-cholesterol  and VLDL t o t a l protein.  I t had no e f f e c t on  plasma HDL-cholesterol. Liver  and  skeletal  muscle  carnitine  levels  in  the  hyperlipidemic  carnitine-treated animals were normalized.  Although treatment s i g n i f i c a n t l y  elevated  well  a l l plasma  hyperlipidemic  carnitine  fractions  above  those  untreated animals, the percentage of a c e t y l - and  seen  in  the  acylcarnitines  remained unchanged. The  f r a c t i o n a l catabolic rate of  values with L-carnitine treatment.  VLDL-triglycerides  returned to control  Treatment had no e f f e c t on VLDL-apoprotein  B kinetics. On plasma  the basis of these r e s u l t s , triglycerides  treatment was  in  the  i t was  concluded that the  hyperlipidemic  rabbit  due to an increase i n the catabolism of  following  reduction  in  L-carnitine  VLDL-triglycerides.  TABLE OF CONTENTS  ABSTRACT  i  L I S T OF TABLES  v i i  L I S T OF FIGURES  viii  L I S T OF PLATES  ix  ACKNOWLEDGEMENTS  x  ABBREVIATIONS 1  x i  INTRODUCTION 1.1  The M e t a b o l i c R o l e 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 v e r s u s 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 m e t a b o l i s m o f c h y l o m i c r o n s  and v e r y l o w d e n s i t y l i p o p r o t e i n s . 1.4  5  C h o l e s t e r o l metabolism  7  a)  Cholesterol Synthesis  7  b)  Cholesterol Catabolism  8  1.5  2  i  Cholesterol Transport a)  M e t a b o l i s m o f low d e n s i t y l i p o p r o t e i n s (LDL)  8  b)  HDL M e t a b o l i s m  9  1.6  Rationale  10  1.7  S p e c i f i c Aims  13  MATERIALS AND METHODS 2.1  Study Design  15  2.2  Blood C o l l e c t i o n  16  2.3  Preparation o f High f a t D i e t  17  2.4  D e t e r m i n a t i o n 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  Acetylcarnitine  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  H i s t o l o g i c 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 VLDL-ApoB  and 25  2.8.3  Radioiodination of VLDL  25  2.8.4  Characterization of i o d i n a t i o n lipoproteins  26  3  2.9  2.8.5  Preparation of i n j e c t a b l e  2.8.6  Turnover Study Protocol  28  2.8.7  I s o l a t i o n of VLDL from Post-Injection Plasma  29  2.8.8  Estimation of VLDL-ApoB S p e c i f i c A c t i v i t y  29  2.8.9  Estimation of VLDL-TG S p e c i f i c A c t i v i t y  30  27  KINETIC ANALYSIS 2.9.1  VLDL-Triglycerides  31  2.9.2  VLDL-ApoB  32  2.10 3.  H-glycerol  STATISTICAL ANALYSIS  35  RESULTS 3.1  Food Consumption and Body Weight  3.2  Plasma L i p i d parameters  36  a) Cholesterol and T r i g l y c e r i d e s  36  b)  41  Plasma VLDL parametrs and HDL-cholesterol V  3.3  D- ^ -Hydroxybutyrate  44  3.4  Plasma glucoose  44  3.5  Lipoprotein Electrophoresis  44  3.6  Kinetic Analysis a)  VLDL-TG metabolism  46  b)  VLDL-ApoB metabolism  50  3.7  Plasma C a r n i t i n e s  55  3.8  Tissue C a r n i t i n e s  59  3.9  Histology a)  Gross V i s u a l Changes  62  b)  H i s t o l o g i c Findings  62  4  DISCUSSION  70  5  CONCLUSIONS  85  6  APPENDIX Appendix A: C a l c u l a t i o n o f the FCR f o r Monoexponential Decay ... Appendix B:  87  A n a l y s i s of blood l e v e l data i n a Two-Pool System: C a l c u l a t i o n o f curve parameters  90  Appendix C:  Kinetic Definitions  92  Appendix D:  An i n v e s t i g a t i o n o f L - c a r n i t i n e treatment i n Hypertriglyceridemia: Results of studies i n Yucatan minipigs  94  LIST o f REFERENCES  103  vi  LIST OF TABLES  NUMBER  I  TITLE  PAGE  D a i l y food consumptions and i n i t i a l and f i n a l body weight f o r the groups of r a b b i t s involved i n the study  37  II  Plasma lipids and c a r n i t i n e values for rabbits maintained on regular r a b b i t chow (group N) f o r 8 weeks.  40  III  Plasma HDL-cholesterol and VLDL parameters i n hyperlipidemic r a b b i t s (Group H)  42  untreated  IV  Plasma HDL-cholesterol and VLDL parameters i n hyperl i p i d e m i c r a b b i t s t r e a t e d w i t h L - c a r n i t i n e (Group C)  43  V  F r a c t i o n a l c a t a b o l i c rates (FCRs) and transport rates of VLDL-TG metabolism i n groups N and C  51  VI  F r a c t i o n a l c a t a b o l i c rates and transport rates of VLDLApoB i n normals and hyperlipidemic r a b b i t s before and a f t e r L - c a r n i t i n e treatment.  56  VII  Changes i n plasma c a r n i t i n e s l e v e l s i n r a b b i t s h i g h - f a t d i e t (Group H)  57  VIII  Plasma c a r n i t i n e l e v e l s i n hyperlipidemic r a b b i t s (Group C) before and a f t e r L - c a r n i t i n e treatment  58  Muscle c a r n i t i n e l e v e l s i n normal l i p i d e m i c r a b b i t s and hyperlipidemic w i t h L - c a r n i t i n e f o r 1 month  60  IX  X  rabbits, rabbits  fed  a  hypertreated  L i v e r c a r n i t i n e l e v e l s i n normal r a b b i t s , hyperlipidemic r a b b i t s and hyperlipidemic r a b b i t s treated w i t h Lc a r n i t i n e f o r 1 month  61  XI  Plasma e l e c t r o l y t e s and glucose i n a d u l t miniature pigs  99  XII  Plasma l i p i d values i n a d u l t male Yucatan miniature pigs  100  XIII  Serum l e v e l s of glutamateoxalate transaminase, c r e a t i n e kinase, albumin and t o t a l p r o t e i n i n a d u l t male Yucatan miniature pigs  101  Plasma f r e e , a c y l , a c e t y l , and t o t a l c a r n i t i n e i n a d u l t male yucatan miniature pigs and i n men  102  XIV  vii  male  Yucatan  LIST OF FIGURES  NUMBER  TITLE  PAGE  1.  The general two-pool model o f Gurpide e t a l .  33  2.  Plasma c h o l e s t e r o l lipidemic rabbits  38  3.  Plasma c h o l e s t e r o l and t r i g l y c e r i d e s l e v e l s lipidemic rabbits treated with L-carnitine  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 human plasma and b a s e l i n e plasma from NZW r a b b i t  45  5.  Lipoprotein electrophoresis r a b b i t s ' plasma  45  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 plasma  45  7.  Representative normal r a b b i t s  47  8. 9.  10.  11. 12.  13 14  and t r i g l y c e r i d e s l e v e l s i n hyper-  o f human  i n hyper-  plasma  and  NZW  39  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  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 r a b b i t p r i o r t o L - c a r n i t i n e treatment  48  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 r a b b i t s a f t e r 1 month on Lcarnitine  49  125 Representative curve f o r the decay o f I-VLDL-ApoB f o l l o w i n g the i n j e c t i o n o f autologous I-VLDL i n normal r a b b i t s 125 Representative curve f o r the decay of I-VLDL-ApoB i n hyperlipidmic r a b b i t s 125 Representative decay curve f o r I-VLDL-ApoB in hyperlipidemic r a b b i t s t r e a t e d w i t h L - c a r n i t i n e f o r one month T y p i c a l VLDL-TG t r a c e r curve obtained f o l l o w i n g the i n j e c t i o n o f H-glycerol i n t o normal r a b b i t s 12  Typical specif i C j ^ a c t i v i t y - t i m e curve for the disappearance o f I-VLDL-ApoB from the plasma o f normal r a b b i t s  viii  52 53  54 88  91  LIST OF PLATES  NUMBER 1.  TITLE  PAGE  L i g h t micrographs of a s e c t i o n of the l i v e r from a group N rabbit  63  2.  O i l red rabbit  s e c t i o n of the l i v e r from a group  N  64  3.  Hematoxylin-eosin stained s e c t i o n of the l i v e r from hyperlipedemic r a b b i t (group H)  a  4.  5. 6.  0 stained  O i l red 0 stained s e c t i o n of hyperlipidemic rabbit showing infiltration  the l i v e r from a extensive fatty  Light micrograph of a liver section from hyperlipidemic r a b b i t showing n e u t r a l l i p i d d r o p l e t s O i l red 0 stained s e c t i o n o f l i v e r from t r e a t e d hyperlipidemic r a b b i t  ix  66  a  67  a  carnitine-  68 69  ACKNOWLEDGEMENTS  I  would  successful express  like  t o thank a l l those i n d i v i d u a l s who c o n t r i b u t e d t o the  completion  my  o f t h i s document.  In particular,  I would  g r a t i t u d e t o the members o f my supervisory committee  like to f o r their  valuable c o n t r i b u t i o n s . I am indebted t o my supervisor, support,  Dr. David Seccombe,  f o r h i s invaluable  and c o n t r i b u t i o n s during the course o f t h i s study and preparation o f  this thesis. I  wish  t o express s p e c i a l thanks t o Mr.  Chuck Yeung  o f IPC Systems  (Vancouver) f o r a l l o w i n g me the use o f h i s microcomputer, and t o Drs. Ed Jones (Department o f Pathology,  Shaughnessy Hospital) and David Foster  (Department  of Biomedical engineering, U n i v e r s i t y o f Washington) f o r t h e i r e x p e r t i s e . F i n a l l y , I would l i k e t o express my deepest a p p r e c i a t i o n t o my w i f e , Joan Bernard,  f o r o f 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 p r o j e c t . During the course o f t h i s study, I obtained f i n a n c i a l a s s i s t a n c e from the British  Columbia Heart Foundation.  The p r o j e c t was founded by a grant  the B r i t i s h Columbia Health Care and Research foundation.  x  from  ABBREVIATIONS  oC  Alpha Beta  y  Gamma  ANT  Adenine nucleotide translocase  CoA  Coenzyme A  Acetyl-CoA  Acetyl-coenzyme A  Acyl-CoA  Acyl-coenzyme A  TG  Triglycerides  VLDL  Very low density  VLDL-TG  Very low density l i p o p r o t e i n t r i g l y c e r i d e s  VLDL-ApoB  Very low density l i p o p r o t e i n apoprotein B.  IDL  Intermediate density  HDL  High density  HDL-Chol.  High density l i p o p r o t e i n c h o l e s t e r o l  LDL  Low density  LDL-cholesterol  Low density l i p o p r o t e i n c h o l e s t e r o l  ACAT  Acyl CoA: c h o l e s t e r o l  HMG-COA  3-Hydroxy-3-methylglutaryl CoA  HMG-CoA Reductase  3-Hydroxy-3-methylglutaryl CoA Reductase  FCR  Fractional catabolic  umole  micromoles  uM  micromoles per l i t e r o r micromolar  S.D  Standard d e v i a t i o n  N.S.  Not s i g n i f i c a n t  xi  lipoprotein  lipoprotein  lipoprotein  lipoprotein  acyltransferase  rate  LCAT  Lecithin: cholesterol  NZW r a b b i t s  New Zealand White r a b b i t s  w/w  weight/weight  v/v  volume/volume  xii  acyltransferase  1. INTRODUCTION  Over  the past two decades,  correlation heart the  i t has been w e l l e s t a b l i s h e d t h a t a p o s i t i v e  e x i s t s between abnormal l e v e l s o f plasma  disease  (1-3).  Diet,  such  side-effects introduced  Some of the more commonly used  as c l o f i b r a t e and n i c o t i n i c a c i d have  (4,5).  and coronary  e x e r c i s e and drug therapy a r e t y p i c a l l y used i n  management o f hyperlipidemic p a t i e n t s .  medications  lipids  For t h i s  reason  they  several  a r e frequently  well  known  withheld  only a f t e r other means of l i p i d c o n t r o l have f a i l e d .  and  E f f o r t s are  underway t o i d e n t i f y a l t e r n a t e hypolipidemic agents which a r e e s s e n t i a l l y f r e e of these s i d e - e f f e c t s . ( y3 -hydroxyl- If -trimethylaminobutyrate)  L-carnitine  receiving consideration. body  and i s a  i s one such  This water-soluble a c i d which i s synthesized i n the  normal c o n s t i t u e n t o f the d i e t ,  i s essential  o x i d a t i o n o f long-chain f a t t y acids (C > 8) i n the mitochondria which  agent  f o r optimal (6).  Studies  have examined the e f f e c t o f L - c a r n i t i n e 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 w i t h i n c o n c l u s i v e r e s u l t s . I n order t o  circumvent designed  some  o f the weaknesses of these e a r l i e r p r o t o c o l s ,  t o examine  the e f f e c t of L - c a r n i t i n e  treatment  a  study  was  on l i p o p r o t e i n  k i n e t i c s i n an animal model.  1.1  THE METABOLIC ROLE OF L-CARNITINE  In process  living which  systems,  acyl  groups undergo  an ATP-dependent a c t i v a t i o n  r e s u l t s i n the production o f acyl-coenzyme A  (7). P r i o r t o  gaining entry t o the s i t e o f ^ - o x i d a t i o n i n the matrix of the mitochondrion, these  acyl-coenzyme  esterification  A  reaction  species with  must f i r s t  L-carnitine,  1  undergo  a  reversible  yielding acylcarnitine.  transThis  reaction  i s c a t a l y z e d by c a r n i t i n e a c y l t r a n s f e r a s e .  I t i s only  after  this  e s t e r i f i c a t i o n r e a c t i o n t h a t long-chain f a t t y acids can pass through the inner mitochondrial membrane. This membrane i s normally impermeable t o a l l coenzyme A  moieties,  both  transported  and e s t e r i f i e d .  Once formed,  a c y l c a r n i t i n e s are  across the inner mitochondrial membrane by means o f a c a r n i t i n e :  acylcarnitine  translocase system.  membrane, they oxidation  free  On the inner aspect o f the mitochondrial  a r e t r a n s e s t e r i f i e d t o form acyl-CoAs which then undergo  (8-11).  Thus f a r , three d i s t i n c t c a r n i t i n e a c y l t r a n s f e r a s e s have been i d e n t i f i e d i n mitochondria. C a r n i t i n e p a l m i t o y l t r a n s f e r a s e i s e x c l u s i v e l y a mitochondrial enzyme found i n l i v e r , heart, kidney and s k 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 o f carbon chain length o f C-^ ±Q'  Beyond  - C  C^g the enzyme  e x h i b i t s decreasing a c t i v i t y w i t h f a t t y acids o f i n c r e a s i n g chain length (1417). C a r n i t i n e a c e t y l t r a n s f e r a s e and octanoyltransferase have been shown t o be present i n mitochondria,  peroxisomes and microsomes (18).  is  w i t h peak a c t i v i t y a t C_- C ,  specific  transferase transferase  for C - C 0  R  /  b  activity  0  Z  Acetyltransferase  while  J  overlaps the a c t i v i t i e s of the p a l m i t o y l - and a c e t y l -  (17,19).  Carnitine  palmitoyltransferase  a c e t y l t r a n s f e r a s e may possess two separate a c t i v i t i e s (12,13); the  outer  the octanoyl-  and  carnitine  one located on  p o r t i o n o f the inner mitochondrial membrane and the other  inner surface o f t h a t membrane.  on the  A s i m i l i a r f i n d i n g has not been reported f o r  the octanoyltransferase. In a d d i t i o n t o i t s r o l e i n o p t i m i z i n g f a t t y a c i d plays  a  central  role  i n regulating  the a c t i v i t y  oxidation, L-carnitine o f adenyl  translocase  (ANT) (20).  ATP  the inner mitochondrial membrane d i r e c t l y influences  across  charge  o f the c e l l (21).  nucleotide  This enzyme which c a t a l y z e s the exchange o f ADP and the energy  Accumulation of acyl-CoA's of chain length  longer  than e i g h t carbons on e i t h e r s i d e of the inner mitochondrial membrane i n h i b i t s 2  ANT (22-24). the  cell.  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 L-carnitine  and i t s associated enzyme systems  are capable  of of  modulating the acyl-CoA/CoASH r a t i o and i t i s through t h i s mechanism t h a t they are b e l i e v e d t o i n f l u e n c e the a c t i v i t y o f ANT (20). L - c a r n i t i n e and c a r n i t i n e a c e t y l t r a n s f e r a s e are capable o f modulating the acetylCoA/CoASH r a t i o i n various t i s s u e s . to  In t h i s role, carnitine i s believed  "buffer" mitochondrial acetylCoA by promoting the s h i f t o f a c e t y l  from  the mitochondria  concentrations conceivable  t o the c y t o s o l  are normally  higher  (25-28). than those  Since  cellular  o f CoASH  groups  carnitine  (12,29),  i tis  t h a t the extramitochondrial a c e t y l c a r n i t i n e / c a r n i t i n e r a t i o  will  b u f f e r f l u c t u a t i o n s i n mitochondrial acetylCoA. I t has been suggested t h a t L - c a r n i t i n e may serve a metabolic r o l e i n the oxidation  o f excess  oxidation  i s incomplete ( i . e .  acids  extramitochondrial acetylCoA (30,31).  Peroxisomal ^ -  only chain-shortening o f very long-chain f a t t y  such as u r i c i c a c i d occur i n these o r g a n e l l e s ) .  This r e s u l t s  production  o f short-chain acyl-CoAs which must be t r a n s f e r r e d  peroxisome  t o the mitochondria (32,33).  out o f the  L - c a r n i t i n e i s probably i n v o l v e d i n  s h u t t l i n g these ^ - o x i d a t i o n chain-shortened products. acetyltransferase  i n the  Peroxisomal  carnitine  and octanoyltansferase are b e l i e v e d t o f u n c t i o n  i n this  t r a n s f e r proccess (34) .  1.2  TRIGLYCERIDE SYNTHESIS versus  Once  formed,  triglycerides Acyl-CoAs acetyl-CoA  acyl-CoAs  -OXIDATION:  can be  used  either  and complex l i p i d s o r they can be  f o r the synthesis o f  degraded  via  -oxidation.  which are transported i n t o the mitochondrial matrix are degraded t o which  i n t u r n may be e i t h e r  shunted i n t o the t r i c a r b o x y l i c  c y c l e (TCA) o r used f o r ketone body production.  3  Lopes-Cardozo e t .  acid  a l . (35)  have for  shown that due t o the high a f f i n i t y o f the TCA enzyme acetyl-CoA,  citrate  the use o f the l a t t e r substrate i n the TCA  precedence over i t s use f o r ketone body production.  synthase  cycle  takes  A t times o f high rates o f  P - o x i d a t i o n , c i t r a t e synthase becomes saturated thereby s h i f t i n g the b i a s  of  the acetylCoA pool i n favour o f ketone body production. The  p a r t i t i o n i n g o f 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 t o be o f major importance i n the r e g u l a t i o n of yfl-oxidation and triglyceride  synthesis.  As  established  between  the r a t e  synthesis.  For instance,  such,  a  reciprocal  r e l a t i o n s h i p has been  o f ketogenesis and the r a t e o f t r i g l y c e r i d e  i t has been demonstrated t h a t  formation  i s specifically  increases  and can reach maximal rates even i n l i v e r s from  (36,37).  This i n d i c a t e s that the r a t e o f f a t t y a c i d o x i d a t i o n may  the  i n h i b i t e d , the r a t e  i f acylcarnitine  of g l y c e r o l i p i d starved  synthesis animals. influence  r a t e o f t r i g l y c e r i d e and very low density l i p o p r o t e i n (VLDL) synthesis by  the l i v e r . It  i s evident that the net d i r e c t i o n of movement o f f a t t y  c o n t r o l l e d by a number o f i n t e r r e l a t e d f a c t o r s .  acyl-CoA i s  Included among these a r e :  a) The n u t r i t i o n a l and endocrine s t a t e o f the organism Fasting  (36), diabetes  oxidation  (37) and hyperthyroidism  (38) lead  to  increased  and decreased e s t e r i f i c a t i o n o f f r e e f a t t y acids by perfused  liver  and c u l t u r e d hepatocytes. b) F a t t y a c i d binding p r o t e i n Fatty  acids which are bound t o t h i s p r o t e i n are p r e f e r e n t i a l l y a c t i v a t e d  esterified  i n the smooth endoplasmic reticulum  transesterification  as opposed  and  t o undergoing  t o L - c a r n i t i n e and subsequent o x i d a t i o n i n mitochondria  (39).  4  c) The a v a i l a b i l i t y o f competing o x i d i z a b l e substrates The  most  important competing o x i d i z a b l e substrates are pyruvate  mitochondrial  NADH.  I n order f o r y6-oxidation t o occur,  mitochondria, acyl-CoA's pyruvate)  must compete w i t h other  f o r a v a i l a b l e NAD  +  and e x t r a -  upon entry i n t o the  available  substrates (e.g  and the e l e c t r o n t r a n s p o r t chain.  r e l a t i v e l y h i g h concentrations o f pyruvate,  Thus, a t  fatty acid oxidation i s inhibited  i n the perfused heart whereas glucose o x i d a t i o n i s accelerated (40). C y t o s o l i c substrates, such as l a c t a t e and ethanol which produce NADH when oxidized,  inhibit  fatty acid oxidation.  This i n h i b i t i o n occurs because the  c y t o s o l i c and mitochondrial NAD/NADH pools are l i n k e d by the malate  aspartate  s h u t t l e (41). d)  Malonyl-CoA  Malonyl-CoA, the end-product o f the committed step i n f a t t y a c i d s y n t h e s i s , i s capable o f modulating the a c t i v i t i e s o f outer c a r n i t i n e p a l y m i t o y l t r a n s f e r a s e (CPT  1) and c a r n i t i n e a c e t y l t r a n s f e r a s e o f l i v e r mitochondria (41-43).  High  l e v e l s o f malonyl-CoA (carbohydrate feeding) leads t o suppression o f these two enzymes  and enhancement o f t r i g l y c e r i d e s y n t h e s i s ;  malonyl-CoA (44,45).  (uncontrolled diabetes,  fatty  whereas low l e v e l s o f  meal) has the opposite  A d d i t i o n a l studies i n d i c a t e t h a t t h i s r o l e o f malonyl-CoA  effect may be  more complex than was i n i t i a l l y thought.  1.3 TRIGLYCERIDE TRANSPORT The metabolism o f chylomicrons and very low d e n s i t y l i p o p r o t e i n s (VLDL)  Triglycerides transported  synthesized  i n the l i v e r  o r i n the i n t e s t i n e s are  by two major c l a s s e s o f l i p o p r o t e i n s ,  5  namely very  low d e n s i t y  l i p o p r o t e i n s (VLDL) and chylomicrons. Chylomicrons, intestinal  the l a r g e s t  o f the l i p o p r o t e i n s ,  mucosa and serve mainly t o t r a n s p o r t and d e l i v e r  cholesterol  E  the  i n the  triglycerides,  and phospholipids o f d i e t a r y o r i g i n t o other t i s s u e s .  move from the lymph t o the venous plasma, and  are formed  As  they  chylomicrons acquire apoproteins  C  and lose t r i g l y c e r i d e s and phospholipids v i a the h y d r o l y t i c a c t i o n o f enzyme,  lipoprotein  lipase,  which  i s attached  t o the c a p i l l a r y  endothelium. The released f r e e f a t t y a c i d s , g l y c e r o l and lysophospholipids are used  f o r energy production and membrane s y n t h e s i s .  The net r e s u l t  chylomicrons a r e converted t o smaller c h o l e s t e r o l - r i c h p a r t i c l e s , remnants, which are taken up by hepatocytes VLDL In  phospholipids,  (46-48).  to triglycerides,  cholesterol,  VLDL contains apoproteins B,  cholesterol  C and E.  triglycerides synthesized  be  hepatic  derived  stores.  from As  chylomicron  triglycerides  lipoprotein  lipase.  intermediate  remnants  they move through  apoprotein C and lose apoprotein E;  addition,  called  may  density  VLDL  development  tissues.  VLDL-  o r from  newly VLDL  apoprotein B i s r e t a i n e d .  i s transformed i n t o  In  the a c t i o n o f  smaller  l i p o p r o t e i n s (IDL) which i n human  f u r t h e r c a t a b o l i z e d t o low d e n s i t y l i p o p r o t e i n s (LDL) Disturbances  and  the c i r c u l a t i o n ,  and phospholipids are l o s t through  Overall,  esters  The major f u n c t i o n o f  i s t o t r a n s p o r t t r i g l y c e r i d e s from the l i v e r t o other  acquire  chylomicron  a r e t r i g l y c e r i d e - r i c h l i p o p r o t e i n s produced p r i m a r i l y by the l i v e r .  addition  VLDL  i s that  particles  beings  are  (49).  i n the metabolism o f chylomicrons and VLDL may lead t o the  o f h y p e r t r i g l y c e r i d e m i a ( i . e f a s t i n g plasma  triglyceride  levels  th greater  than  triglyceridemia overproduction decreased  the 95 may  p e r c e n t i l e f o r age and sex) result  from  of triglycerides,  clearance  one o r more  of  (50). the  Primary  hyper-  following:  i)  i i ) overproduction o f apoprotein B and i i i )  o f 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 6  chylomicrons)  These defects are u s u a l l y i n h e r i t e d as autosomal recessive t r a i t s (4).  1.4 CHOLESTEROL METABOLISM  A been  d i r e c t involvement o f L - c a r n i t i n e i n c h o l e s t e r o l metabolism has not y e t established.  patients  However,  the r e s u l t s o f a few studies i n  suggest t h a t L - c a r n i t i n e may a f f e c t plasma c h o l e s t e r o l  hyperlipidemic concentration  (51,52). The average North American a d u l t consumes about 500mg of which 40 - 60% i s absorbed. and  o f cholesterol/day  Cholesterol i s absorbed i n the f r e e form (53)  subsequently i s e s t e r i f i e d i n the enterocyte by the a c t i o n o f a c y l CoA:  cholesterol  acyltransferase  incorporated  together  (ACAT).  Once e s t e r i f i e d ,  cholesterol  is  w i t h t r i g l y c e r i d e s i n t o chylomicrons f o r transport i n  the blood (53,54).  a)  C h o l e s t e r o l Synthesis  In mammalian c e l l s , a  endogenously synthesized c h o l e s t e r o l i s derived from  c y t o s o l i c acetyl-CoA pool.  many  The acetate w i t h i n t h i s pool i s derived  from  reactions i n c l u d i n g the catabolism o f long-chain f a t t y a c i d s . The  i n i t i a l r e a c t i o n i n c h o l e s t e r o l synthesis i s the condensation o f two  u n i t s o f acetyl-CoA t o form acetoacetyl-CoA. thiolase. presence  This r e a c t i o n i s catalyzed by a  One molecule o f acetyl-CoA i s combined w i t h acetoacetyl-CoA i n the o f water t o form  3-hydroxy-3-methlglutaryl-CoA  r e a c t i o n i s catalyzed by HMG-CoA synthase.  (HMG-CoA). This  The next r e a c t i o n , which involves  the reduction o f HMG-CoA t o mevalonate i n the presence o f NADH , i s the r a t e limiting HMG-CoA  r e a c t i o n f o r c h o l e s t e r o l synthesis. reductase.  This r e a c t i o n i s catalyzed  by  Because o f i t s c e n t r a l r o l e i n c h o l e s t e r o l b i o s y n t h e s i s ,  7  HMG-CoA reductase modulated  i s very t i g h t l y regulated.  by  such  cholesterol).  The  appears  metabolites as mevalonate and oxysterols  t o be  (derived  from  l a t e r reactions i n the synthetic pathway involve a s e r i e s  of a c t i v a t i o n s (by ATP) squalene.  Its activity  followed by condensations leading t o the production of  Subsequently  squalene  i s oxidized,  then reduced  twice  forming  c h o l e s t e r o l (see r e f 55). In a d u l t s , about lOOOmg of c h o l e s t e r o l i s synthesized v i a t h i s route on a d a i l y basis  b)  (53,45).  Cholesterol  About one h a l f o f t h i s i s made i n the l i v e r .  catabolism  In normal mammals, c h o l e s t e r o l i s c a t a b o l i z e d e x c l u s i v e l y by the l i v e r . Greater to  than 50% o f d a i l y synthesized and ingested c h o l e s t e r o l i s metabolized  b i l e acids.  synthesis  The remainder i s used f o r such purposes as s t e r o i d  and c e l l  membrane assembly.  Bile  acids,  together  c h o l e s t e r o l , a r e secreted i n t o b i l e which passes i n t o the gut.  hormone  with  some  Some o f the  secreted c h o l e s t e r o l and b i l e are reabsorbed w h i l e the remainder i s passed i n the feces. far  Although some c h o l e s t e r o l i s l o s t v i a the s k i n ,  the feces  the major route f o r excretion o f c h o l e s t e r o l and i t s c a t a b o l i c  i s by  products  (53-55).  1.5 CHOLESTEROL TRANSPORT. a) Metabolism o f Low Density Lipoproteins (LDL)  In human beings, the  catabolism  low density l i p o p r o t e i n s (LDL) are derived l a r g e l y from  o f VLDL.  They  are p a r t i c u l a r l y  8  rich  in  cholesterol  (approximately  50% by weight) which i s present mainly as c h o l e s t e r o l  esters.  They a l s o c o n t a i n smaller q u a n t i t i e s of t r i g l y c e r i d e s (approximately 10%). The major  apoprotein  i s apoprotein B which c o n s t i t u t e s more than  95%  of  the  p r o t e i n mass w i t h i n LDL p a r t i c l e s (49). LDL i s the major c h o l e s t e r o l c a r r y i n g l i p o p r o t e i n i n humans and under normal c o n d i t i o n s i s the most important source of c h o l e s t e r o l f o r most c e l l s (56). LDL i s removed from the plasma by e i t h e r hepatocytes  or p e r i p h e r a l c e l l s .  receptor-independent  (57-59).  Removal of LDL may be receptor-mediated or 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 i n v o l v e d in  the  uptake  of  LDL  i s known as ApoB-100/Apo E  (or LDL)  receptor  and  i n t e r a c t s w i t h e i t h e r ApoB- or ApoE-containing l i p o p r o t e i n s (58,59). Reduction clearance  i n the a c t i v i t y of the B-100/E receptor r e s u l t s  of  LDL.  cholesterolemia  This  (57).  ultimately  This  leads t o  mechanism  the  appears  in  development to  be  the  decreased of  hyper-  most common  underlying cause of hypercholesterolemia. However, i t has r e c e n t l y been shown that  overproduction of LDL by d i r e c t s e c r e t i o n ( i . e by routes independent  of  VLDL catabolsim) may a l s o c o n t r i b u t e t o 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 i n t r a v a s c u l a r catabolism of  VLDL  and chylomicrons and by hepatocytes and enterocytes. (62-64). The  major f u n c t i o n of t h i s c l a s s of l i p o p r o t e i n s i s t o t r a n s p o r t excess  cholesterol  from  transport).  In  formed  to  a  peripheral  c e l l s back t o the  liver  (reverse  cholesterol  t h i s process the nascent HDL p a r t i c l e i s e v e n t u a l l y  mature HDL  (HDL- and HDL_)  9  p a r t i c l e which i s removed  trans-  from  the  circulation  by  ApoE  and non ApoE receptors (65,66).  The  details  of  this  conversion and the mechanisms of 'reverse c h o l e s t e r o l t r a n s p o r t ' are s t i l l not c l e a r l y understood. Low l e v e l s of plasma HDL  (hypoalphalipoproteinemia)  are u s u a l l y (but not  always) associated w i t h increased r i s k of a t h e r o s c l e r o s i s . cases,  this  condition i s  i n h e r i t e d and both  In the majority of  HDL and HDL  ^  2  greatly  a r e  reduced i n plasma (49). Hyperalphalipoproteinemia Individuals  who  are  (increased  a f f e c t e d by  this  l e v e l s of HDL)  can  be  inherited.  c o n d i t i o n u s u a l l y have  increased  longevity.  1.6. RATIONALE There disorders  i s l i t t l e doubt t h a t there e x i s t s a d i s t i n c t r e l a t i o n s h i p of  lipoprotein  metabolism and  c a r d i o v a s c u l a r disease.  studies have e s t a b l i s h e d a p o s i t i v e r e l a t i o n s h i p between and  a t h e r o s c l e r o s i s (67-69).  hypercholesterolemia  remains  Therefore, an  Numerous  hypercholesterolemia  the management and  important goal of many  between  treatment  clinics  of  throughout  North America and Western Europe. Despite  earlier  beliefs,  h y p e r t r i g l y c e r i d e m i a may,  there  as w e l l ,  is  Carlson e t .  triglycerides (MI) .  al.  hypertriglyceridemia  et.  these  the  (70) were able t o demonstrate t h a t f a s t i n g plasma  al.  (71)  found  that  myocardial  below  the  infractions  age  of  60,  (with or without hypercholesteremia) was the most common  l i p i d d i s o r d e r i n s u r v i v o r s of Mis. support  that  Using m u l t i v a r i a t e s t a t i s t i c a l  acted as an independent r i s k f a c t o r i n  Brunzell  suggesting  a c t as an independent r i s k f a c t o r i n  development of coronary heart disease (70,71). analysis,  some evidence  observations.  In  A few other s t u d i e s (72,73,79,80) seem t o a d d i t i o n t o being  10  at  increased  risk  of  developing eruptive  coronary heart disease, xanthomatosis  pancreatitis  and  h y p e r t r i g l y c e r i d e m i c s o f t e n have  episodic  abdominal  usually  (e.g abdominal p a i n , used hypolipidemic withheld  until  nausea,  that  metabolic  s k i n rashes,  the  drugs such as c l o f i b r a t e and n i c o t i n i c a c i d ,  are  a l l other means of  liver  lipid  control  bias  lead  of  to  theoretically  acyl-CoAs  away from  esterification  exogenous  have  of  Such a s h i f t VLDL  and  L-carnitine  an at  could  bodies ( & -  acetoacetate). indicated  triglycerides  However, no  the  synthesis  increase i n the production of ketone  that  L-carnitine  (51,76-78)  and  free  lowers  serum  fatty  acids  increases ^-hydroxybutyrate (81,82) and high density  to  i t is  Since c i t r a t e synthase i s saturated  ^2-oxidation,  of  produce an  have  (76,78,83).  and  a c y l c a r n i t i n e formation and ^ - o x i d a t i o n .  rates  hydroxybutyrate and  (51,52,75),  metabolism,  a decrease i n the synthesis of t r i g l y c e r i d e and  high  Studies  exhausted.  drugs have been sought.  increase i n the r a t e of ft - o x i d a t i o n . relatively  are  exogenously administered L - c a r n i t i n e may be able to s h i f t  t r i g l y c e r i d e s towards will  Due t o the unpleasant s i d e -  account of the r o l e of L - c a r n i t i n e i n f a t t y a c i d  possible  without  dysfunction)  Consequently, a l t e r n a t e hypolipidemic On  or  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.  commonly  with  (74).  C u r r e n t l y , treatment of the hyperlipidemic  effects  pain  painful  cholesterol (76,81)  and  lipoprotein-cholesterol  i n other studies (78,82,84) L - c a r n i t i n e has been found  significant effect  on  either  plasma  t r i g l y c e r i d e s or  (5-  hydroxybutyrate. The effect  current study was prompted by an e a r l i e r i n v e s t i g a t i o n (85) i n t o the of  L-carnitine  t r i g lyceridemic p a t i e n t . may  treatment  on plasma l i p i d s i n a  32  yr  old  hyper-  The r e s u l t s of that study i n d i c a t e d t h a t L - c a r n i t i n e  have l i p i d - l o w e r i n g p r o p e r t i e s .  11  However,  because of p o s s i b l e v a r i a t i o n  due t o d i e t ,  i t could not be concluded that the observed changes were s o l e l y  due t o L - c a r n i t i n e . Therefore,  a  study  was designed t o examine the e f f e c t  of  L-carnitine  3  treatment  on serum l i p o p r o t e i n 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  lipidemic  turnover  rabbits  in  studies  were conducted i n  order  t o study the e f f e c t  t r i g l y c e r i d e and VLDL-apoprotein B metabolism. originally  selected  recurring  hyperlipidemic of  and  L-carnitine  on  (see Appendix D ),  New  VLDL-  Yucatan miniature pigs  as the model f o r c a r r y i n g out these studies but  problems  normo-  Zealand white (NZW)  were  due  rabbits  to were  eventually chosen t o complete the study. The  rabbit  comparable maintain  was  t o those (89,90).  dietary  chosen of man  (86-88),  Moreover,  cholesterol  and  because  chosen  hyperlipidemia One cause  primarily  lipoprotein  (89) and provide adequate  because  they are  and a t h e r o s c l e r o s i s  The New  classes  t o handle  respond rather r a p i d l y t o  samples f o r molecular and biochemical s t u d i e s . were  plasma  i s inexpensive and easy  rabbits  fats  i t has  blood  changes and  and in  tissue  Zealand White r a b b i t s  hyperresponders  with  respect  to  (89,91).  of the major drawbacks of the r a b b i t model i s t h a t atherogenic d i e t s  extreme hypercholesteremia and l i p i d storage i n many body  addition,  under  the usual experimental conditions applied i n the  organs.  In  laboratory,  the r a b b i t develops a t h e r o s c l e r o t i c l e s i o n s u n l i k e those found i n humans (90). However, peanut  i t i s now known t h a t by c a r e f u l l y s e l e c t i n g the type of f a t  or  cholesterol  coconut o i l versus o l i v e o i l or butter) and the (eg.  hypercholesteremia  0.1  to  0.5% versus 1-3%)  (  e.g  concentration  of  i n the d i e t that the  degree  and the type of a t h e r o s c l e r o t i c l e s i o n which develops  of may  be modulated (92). In order t o moderate the degree of hypercholesterolemia, content of the d i e t was  set a t 0.5%  (weight/weight). 12  the c h o l e s t e r o l  Corn o i l (5%) was added  t o serve as a v e h i c l e f o r the c h o l e s t e r o l (93). Turnover studies were selected because, free  fatty  a c i d s , /5 -hydroxybutyrate  u n l i k e the measurement o f plasma  and t r i g l y c e r i d e s which  give  indirect  information about VLDL metabolism, they give a d i r e c t assessment of the e f f e c t of treatment on the metabolism o f VLDL. selected  because  The k i n e t i c s o f VLDL metabolism  was  i n the c h o l e s t e r o l / f a t f e d r a b b i t s VLDL i s the l i p o p r o t e i n  which i s p r i m a r i l y a f f e c t e d (94-96). It  has been  synthesis appears  by  suggested (97) t h a t  affecting  cellular  L-carnitine  acetyl-CoA  may  levels.  affect  cholesterol  Dietary  cholesterol  t o be the prime source of plasma c h o l e s t e r o l i n the  rabbit.  Rabbit l i v e r HMG-CoA reductase a c t i v i t y ,  i s f u r t h e r reduced  hyperlipidemic  which i s normally very low  i n the hypercholesterolemic s t a t e (142).  Thus,  i t could  be argued that L - c a r n i t i n e would be u n l i k e l y t o have any e f f e c t on c h o l e s t e r o l levels  i n this  metabolism, kinetics  model.  i t was  On  account of the r o l e  of L-carnitine  i n lipid  decided that studying the e f f e c t o f L - c a r n i t i n e  on the  o f 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 other  kinetics  o f VLDL-ApoB metabolism were studied because  apoproteins of the VLDL p a r t i c l e ,  particle  throughout  Therefore, about  VLDL  the catabolism  apoprotein B remains w i t h  t o IDL and subsequently  than would be obtained by studying  the other  SPECIFIC AIMS.  a) The i n d u c t i o n of hyperlipidemia  i n New Zealand White (NZW) r a b b i t s .  13  (98,99).  information  apoproteins.  1.7  the  the VLDL  LDL  f o l l o w i n g the apoB was l i k e l y t o provide more valuable catabolism  unlike  VLDL-  b) The determination o f the plasma l e v e l s o f f r e e , VLDL-triglcerides,  carnitine, apoprotein B,  t o t a l VLDL p r o t e i n ,  hyperlipidemic  rabbits.  c)  The  long-chain  untreated hyperlipidemic  a c e t y l and t o t a l L-  VLDL-cholesterol,  VLDL-  and HDL-cholesterol i n normolipidemic and  determination o f l i v e r and muscle f r e e  carnitine,  acyl,  carnitine,  short-chain  acyl  a c y l c a r n i t i n e and t o t a l c a r n i t i n e i n normolipidemic, and c a r n i t i n e - t r e a t e d hyperlipidemic  NZW r a b b i t s .  d) The determination o f the k i n e t i c parameters of VLDL-triglycerides apoprotein B i n normolipidemic and hyperlipidemic  and VLDL-  NZW r a b b i t s before and a f t e r  L - c a r n i t i n e treatment. e) The examination o f the l i v e r h i s t o l o g y i n normolipidemic r a b b i t s , untreated hyperlipidemic  r a b b i t s and hyperlipidemic  14  r a b b i t s treated with L - c a r n i t i n e .  2. MATERIALS and METHODS  2.1 STUDY DESIGN Twelve follows: GROUP C  male NZW r a b b i t s (2.5 - 3.0 kg) were d i v i d e d i n t o three groups as  The r a b b i t s i n t h i s group were placed on a h i g h - f a t d i e t  containing  0.5% c h o l e s t e r o l and 5.0% corn o i l (w/w) f o r a one month period. A t the end o f 125 this  period,  3  [  I]-VLDL  and [ H]-glycerol turnover studies were  performed  according t o the protocol o u t l i n e d elsewhere i n t h i s t e x t (see s e c t i o n 2.8.6). The  animals  were  then given L - c a r n i t i n e  maintained on the same d i e t .  f o r one month w h i l e  L - c a r n i t i n e (a g i f t from Sigma-Tau,  being  I t a l y ) was  given i n the d r i n k i n g water r e s u l t i n g i n a f i n a l dose o f 170 mg/kg. During the treatment purpose  period,  o f c a 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.  calculated of and  the d a i l y water intake o f each animal was recorded f o r the D a i l y water  intake was  as the d i f f e r e n c e between the quantity o f water given and the  t h a t wasted ( c o l l e c t e d i n 12.7 x 10.3 x 4.8 cm pans placed i n that  this  remaining a t time o f feeding on the f o l l o w i n g day.  period  served  the turnover studies were repeated.  as i t s own c o n t r o l .  injected  In this  I t i s important t o note t h a t  sum  the cages)  A t the end o f  way, each each  animal  animal was  w i t h i t s own VLDL so as t o minimize the formation o f antibodies i n  the period between the f i r s t and second turnover study. GROUP H rabbits. for  The r a b b i t s i n t h i s group were fed the same d i e t as the group  C  This group received no L - c a r n i t i n e and remained on the h i g h - f a t d i e t  the f u l l duration o f the study (two months).  done i n t h i s group.  No turnover  studies  were  However, weekly blood specimens were taken. The data from  t h i s group were u s e f u l as c o n t r o l values f o r the Group C animals.  15  GROUP N: This group c o n s i s t e d of age and weight matched r a b b i t s which were fed Purina  rabbit  chow (Ralston Purina  Canada  Inc.,  Mississauga, O n t a r i o ) . 125  After  two  months, VLDL was i s o l a t e d ,  r a d i o i o d i n a t e d and  3 I-VLDL and  H-  g l y c e r o l turnover s t u d i e s were c a r r i e d out. The r a b b i t s were housed i n i n d i v i d u a l cages and were allowed f r e e t o food and water,  except during turnover s t u d i e s . The d a i l y food consumption  of each animal i n each group At  the  Euthanyl  Mississuaga, liver  and  was recorded throughout the course of the study.  end of the study p e r i o d ,  Forte  access  (sodium  each animal was administered (1.0  pentobarbitol,  Ontario, CAN.)  540  mg/ml;  MTC  ml)  Pharmacueticals,  v i a a marginal ear v e i n . Immediately a f t e r death,  muscle specimens were c o l l e c t e d and  a p p r o p r i a t e l y processed  for  h i s t o l o g i c a l and biochemical analyses. Tissue was f l a s h f r o z e n and stored a t 70°C u n t i l analyzed.  Specimens f o r h i s t o l o g i c a l analyses were f i x e d i n e i t h e r  10% n e u t r a l b u f f e r e d f o r m a l i n or i n Bouin's s o l u t i o n .  2.2 BLOOD COLLECTION Fasted  blood  (16-18  hours)  was r o u t i n e l y  collected  from  e i t h e r the  marginal ear v e i n or c e n t r a l ear a r t e r y i n t o tubes c o n t a i n i n g EDTA (1.0 mg/ml) as an anti-coagulant. During c o l l e c t i o n , each animal was h e l d i n a r e s t r a i n i n g box  (Lab  Products  Inc.  Mayweed,  NJ).  Plasma  c e n t r i f u g a t i o n a t 500 x g f o r f i f t e e n minutes a t 4°C.  was  obtained  following  Each sample was d i v i d e d  i n t o two p o r t i o n s , one of which was stored a t -20°C and the other a t 4°C. -20°C acyl-,  samples  were used f o r the determination of plasma  a c e t y l - , and t o t a l c a r n i t i n e s ) and  16  (free,  -hydroxybutyrate. The 4°C samples  were used f o r the determination of plasma c h o l e s t e r o l , c h o l e s t e r o l and f o r the i s o l a t i o n of VLDL.  carnitines  The  t r i g l y c e r i d e s and  HDL-  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, used f o r preparing the high f a t d i e t .  Ontario.) was  Cholesterol was obtained from the Sigma  Chemical Company (St. L o u i s , MO. USA). To prepare the d i e t , Purina r a b b i t chow was  mixed  with  Cholesterol  was  heated  corn o i l (60°C)  f o r approximately  three  minutes.  then added and the mix was blended f o r an a d d i t i o n a l  three  minutes t o ensure t h a t the p e l l e t s were uniformly coated w i t h c h o l e s t e r o l .  2.4  DETERMINATION OF PLASMA PARAMETERS  2.4.1  Free, A c y l - , and T o t a l L - c a r n i t i n e  Plasma radiometric  free,  acyl-,  determined  by a  method  o r i g i n a l l y described by P a r v i n and Pande (100) and  later  modified by Seccombe e t . deproteinization zinc  and t o t a l  a l . (101).  L-carnitine  were  B a s i c a l l y , the method involves  initial  o f 0.1 ml of serum o r plasma w i t h 0.4 mis o f 0.087  moles/L  s u l f a t e (ZnSO^) followed by n e u t r a l i z a t i o n w i t h 0.4 mis o f 0.083M barium  hydroxide (Ba(OH) ). The supernatant (0.1 ml) obtained a f t e r c e n t r i f u g a t i o n a t 2  1500  x g f o r 10 minutes a t 4°C, was used f o r the determination o f f r e e  L-  c a r n i t i n e according t o the f o l l o w i n g r e a c t i o n : 14 L - c a r n i t i n e + [1- C]AcetylCoA  »  14 [1- C ] A c e t y l c a r n i t i n e + CoASH  This  r e a c t i o n i s catalyzed by c a r n i t i n e a c e t y l t r a n s f e r a s e  (EC 2.3.1.7).  The 14  labelled  acetylcarnitine  formed  i s separated from  the unreacted  [1- C]  acetylCoA by adsorption o f the l a t e r t o a c t i v a t e d charcoal i n the presence of ethanol  and phosphoric  acetylcarnitine  acid.  Stoichiometric conversion of L - c a r n i t i n e  i s assured by the i n c l u s i o n of N-ethylmaleimide 17  to  (NEM) which  binds CoASH s h i f t i n g the e q u i l i b r i u m t o the r i g h t . In order t o determine t o t a l L - c a r n i t i n e , the sequence o f a d d i t i o n o f z i n c s u l f a t e and barium hydroxide was reversed. Plasma was i n i t i a l l y incubated w i t h barium  hydroxide  a t 60°C f o r one hour p r i o r t o the a d d i t i o n of the z i n c  sulfate.  The r e s u l t i n g supernatant was used f o r the determination of t o t a l L-  carnitne  as described above f o r f r e e c a r n i t i n e .  A c y l c a r n i t i n e was c a l c u l a t e d  as the d i f f e r e n c e between t o t a l and f r e e c a r n i t i n e . A l l o f the chemicals, Sigma  Chemical  Company.  14 except [1- C]AcetylCoA, 14 [1- C]AcetylCoA,  were purchased from the  with a specific a c t i v i t y  of 55  mCi/mmol, was obtained from the Amersham Corporation ( A r l i n g t o n Heights, I L , USA ). 2.4.2  Acetylcarnitine  Deproteinized plasma (ZnSO^ / Ba(OH) ) was used f o r the determination of 2  acetylcarnitine  by  Caramancion (102). carnitine  a  radiometric method o r i g i n a l l y described by  I n t h i s assay,  Pande and  a c e t y l c a r n i t i n e i s determined by coupling  a c e t y l t r a n s f e r a s e (CAT) t o c i t r a t e synthase,  CS,  (EC 4.1.3.7) and  measuring the amount o f c i t r a t e formed according t o the f o l l o w i n g r e a c t i o n s : CAT CoASH + A c e t y l - L - c a r n i t i n e  AcetylCoA + [ U - ] O x a l o a c e t a t e  '** L - c a r n i t i n e + AcetylCoA  ^  [U-C]Citrate + [U- C]Oxaloacetate. 14  Excess  oxaloacetate  i s converted t o aspartate  and subsequently  removed by  adsorption t o an anion exchange r e s i n (AG 50WX8, BIO-RAD Laboratories (CANADA) 14 Inc.,  Mississuaga, O n t a r i o ) . The remaining r a d i o a c t i v i t y ([U- C ] C i t r a t e ) i s  determined by l i q u i d s c i n t i l l a t i o n counting. 18  Since oxaloacetate i s r e l a t i v e l y  unstable, i t i s f r e s h l y prepared from [U- C]Aspartate i n a r e a c t i o n c a t a l y s e d by glutamate-oxalatetransaminase, removed  GOT,  (EC 2.6.1.1).  Excess oxaloacetate i s  as aspartate by the reverse r e a c t i o n which i s c a t a l y s e d by the same  enzyme. All  o f the chemicals used and the d e t a i l e d procedures f o l l o w e d 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 f l u o r o m e t r i c  (103) as modified by Seccombe e t a l . /  Ba(OH)  2  d e p r o t e i n i z e d plasma.  Chemical Company.  method  (101). The assay was c a r r i e d out on ZnS0  4  A l l chemicals were s u p p l i e d by the Sigma  Fluorometric measurements were made u s i n g an Aminco Bowman  fluorometer (American Instruments Company Inc., S i l v e r Spring, MD). -hydroxybutyrate (EC  1.1.1.30) i n the presence o f NAD . +  originally NADH formed. To and  i s o x i d i z e d by D-^ -hydroxybutyrate dehydrogenase The amount  o f D-jS - hydroxybutyrate  present i s estimated by f l u o r o m e t r i c a l l y measuring the q u a n t i t y o f A b r i e f summary o f the assay procedure f o l l o w s .  0.2 ml o f d e p r o t e i n i z e d plasma,  0.1 ml o f Barnstead d e i o n i z e d  water  0.15 ml o f an assay mix (containing 75 umoles o f carbonate-bicarbonate  buffer,  pH  9.4, 3.0 umoles NAD  dehydrogenase) minutes  and 0.1 u n i t  +  o f D-^ -hydroxybutyrate  was added. The r e a c t i o n was allowed t o proceed f o r 75 - 90  a t room temperature.  A t the end o f t h i s p e r i o d , the r e a c t i o n was  terminated by the a d d i t i o n o f 1.55 mis o f 0.1M NaOH / l.OmM EDTA t o each tube. Fluorescence was determined a t an e x c i t a t i o n wavelength o f 360nm and emission wavelength o f 460nm.  19  2.4.4  Total  Total (Abbott,  Cholesterol  cholesterol Toronto,  was  Ontario)  determined using  on an  Abbott  k i t s obtained  Bichromatic  Analyzer  Boehringer  Mannheim  from  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  oxidation  of  cholesterol  by c h o l e s t e r o l oxidase t o the formation of a c o l o r i m e t r i c  (104,105).  Hydrogen  peroxide,  formed  during the o x i d a t i o n  of  compound  cholesterol  reacts w i t h 4-aminophenazone and phenol i n the presence of peroxidase t o a coloured compound, 4-(p-benzoquinone-monoimino)-phenazone. the  The i n t e n s i t y of  colour developed i s proportional t o the amount of c h o l e s t e r o l  present  and  important  is  to  cholesterol  monitored  note  that  esters  as the increase i n absorbance in  present  order must  to first  determine be  total  hydrolyzed  form  at  originally  565nm.  It  cholesterol, prior  is any  to  analysis  Bichromatic  Analyzer  (104,105).  2.4.5  Triglycerides  Plasma using  t r i g l y c e r i d e s were measured on an  k i t s obtained from Boerhinger Mannheim Diagnostica.  hydrolyzed  by  a  Triglycerides  b a c t e r i a l l i p a s e y i e l d i n g g l y c e r o l and  G l y c e r o l i s converted t o dihydroxyacetone (E.C.  Abbott  free  fatty  acids.  by coupling t o g l y c e r o l kinase,  2.7.1.30) and g l y c e r o l phosphate oxidase, GPO,  are  GK  (E.C. 1.1.1.8). Hydrogen  peroxide formed i n the GPO-catalyzed r e a c t i o n p a r t i c i p a t e s i n the formation of a  colorimetric  determination  of  compound total  (106)  similiar  cholesterol.  proportional t o the g l y c e r o l  The  concentration.  20  to  that  intensity  described of  this  for  the  product  is  2.4.6  HDL-Cholesterol  Plasma  HDL-cholesterol was  measured  by  initial  heparin-manganese  p r e c i p i t a t i o n of plasma (107,108) followed by the measurement of c h o l e s t e r o l . A b r i e f o u t l i n e of the procedure i s as f o l l o w s : HDL  precipitating  c h l o r i d e / 0.15M  reagent was prepared by mixing 2.0 mis of  sodium c h l o r i d e  0.15M  sodium c h l o r i d e .  while  v o r t e x i n g and  minutes. minutes  The at  manganous  w i t h 1.0 ml Heparin (10,000 U) and 1.0 ml of  This reagent (0.05 ml) was added t o plasma (0.50 the  supernatant 4°C  2.0M  the mixture was allowed t o stand obtained  at  after centrifugation at  was used f o r the determination of  4°C  for  ml) 20  2500 x g f o r 30  cholesterol  as  outlined  above.  2.4.7  Glucose  Plasma  glucose was determined by the glucose oxidase method (109) on  ASTRA-8 machine (Beckman Instruments Inc., Palo A l t o , CA). the  enzymatic  reaction  of  ^ -D-glucose w i t h  oxygen  and  an  The method employs measures  oxygen  consumption according t o the f o l l w i n g r e a c t i o n : glucose oxidase ^-D-glucose  +  0  ^  2  gluconic acid  +  H 0 2  2  The observed r a t e of oxygen d e p l e t i o n i s d i r e c t l y p r o p o r t i o n a l t o 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 e i t h e r 10% n e u t r a l buffered f o r m a l i n or  21  in  Bouin's s o l u t i o n , specimen for  then dehydrated i n i n c r e a s i n g concentrations  o f ethanol. The  was c l e a r e d w i t h xylene, then embedded i n p a r a f i l m wax, i n preparation  sectioning.  haematoxylin  Sections  (4.0 micrometers)  and eosin as described  (110).  were prepared and stained  Other sections were stained f o r  n e u t r a l l i p i d s by the O i l Red O method (111). c o l l a b o r a t i o n w i t h Dr.  Jones,  with  These studies were conducted i n  Department of Pathology, Shaughnessy H o s p i t a l ,  Vancouver, B.C.  2.6 DETERMINATION OF TISSUE CARNITINES  Free and short-chain a c y l c a r n i t i n e l e v e l s were determined from p e r c h l o r i c acid  e x t r a c t s o f 100 - 125mg of muscle and l i v e r (112,113).  (6%)  was  used  f o r d e p r o t e i n i z a t i o n o f the t i s s u e  polytron homogenizer (Brinkmann Instruments, homogenate (NCP) 4°C.  after  Rexdale,  Perchloric disruption  and the remainder was centrifuged a t 1700 x g f o r f i f t e e n  determination carbonate  /  solution.  protein  minutes a t  an a l i q u o t was used f o r the  of free carnitine following neutralization with 0.5M triethanolamine  by a  Ontario, CANADA). The  (0.05 ml) was saved f o r the estimation o f non-collagenous  A f t e r noting the volume o f the supernatant,  acid  In this  2M  procedure,  a c y l c a r n i t i n e s are p e l l e t e d w i t h the deproteinized m a t e r i a l .  potassium long-chain  Therefore, the  p e l l e t was saved f o r the determination o f these e s t e r s . The carnitine of  total  carnitine  acid  e x t r a c t (0.3 ml) w i t h 0.2 mis  (KOH) a t 75°C f o r 2.0 hours.  Subsequently,  n e u t r a l i z e d w i t h 6% p e r c h l o r i c a c i d (0.15 mis). total  extract  plus short-chain a c y l c a r n i t i n e s ) was measured f o l l o w i n g  the p e r c h l o r i c  hydroxide  content of the a c i d - s o l u b l e  o f 2.0M the excess  (i.e.  free  incubation potassium KOH  The d i f f e r e n c e between  was this  c a r n i t i n e value and f r e e c a r n i t i n e was taken as a measure of the short-  22  chain a c y l c a r n i t i n e concentration of the e x t r a c t . KOH  (2.0 M,0.2  ml) was added t o the p e l l e t and the mixture incubated  75°C f o r 2.0 hours. acid  The d i g e s t was n e u t r a l i s e d w i t h s u f f i c i e n t 6%  perchloric  (0.4 t o 0.6 mis) t o 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. 4°C.  The n e u t r a l i z e d d i g e s t was c e n t r i f u g e d a t 1700 x g f o r 30 minutes a t  The volume of the supernatant was noted.  An a l i q u o t (0.3 mis) of  supernatant was used f o r the determination of c a r n i t i n e . as  at  an  index  this  This value was  of the q u a n t i t y of long-chain a c y l c a r n i t i n e  esters  taken  originally  present i n the p e l l e t . In  a l l three  determination was  instances,  c a r n i t i n e was assayed as  of f r e e c a r n i t i n e above.  determined  concentrations  according  were  to  described  for  Tissue non-collagenous p r o t e i n  Lowry  r o u t i n e l y expressed  et.al. as  (114). umoles/g  c a r n i t i n e was taken as being equal t o the sum of the f r e e  Tissue NCP.  the (NCP)  carnitine  Total  tissue  carnitines,  short-  chain a c y l c a r n i t i n e s and long-chain a c y l c a r n i t i n e s .  2.7 LIPOPROTEIN ELECTROPHORESIS Agarose plasma  from  gel  e l e c t r o p h o r e s i s was c a r r i e d out as described  16-18 hour f a s t e d animals.  (115),  using  A l l the materials required f o r  e l e c t r o p h o r e s i s were supplied by Corning Medical (Palo  Alto,  CA,  USA).  the The  procedure may be summarised as f o l l o w s : Aliquots using  (0.01  mL)  of  gel the  The lower, d i v i d e d chamber, of the c e l l was f i l l e d w i t h  moles/L b a r b i t a l b u f f e r ,  35 mins. Red  agarose  a Hamilton Syringe and the g e l was placed i n the upper chamber  electrophoresis c e l l . 0.05  of plasma were t r a n s f e r r e d t o w e l l s on the  pH 8.6 and e l e c t r o p h o r e s i s was c a r r i e d out f o r  The agarose f i l m was allowed t o dry, then s t a i n e d w i t h 12 mis of Fat  7B s t a i n (0.1875 mg/ml of methanol) f o r 5 minutes.  23  The agarose f i l m  was  destained by r i n s i n g w i t h  50% methanol.  2.8 LIPOPROTEIN TURNOVER STUDIES S t e r i l e c o n d i t i o n s were employed throughout. A l l s t e r i l e disposable items were purchased from Becton Dickinson (Mississauga,  Ontario, Canada).  Sterile  s a l i n e and water were supplied by Travenol Canada Inc. (Mississauga, O n t a r i o ) . Gel  filtration  columns  (1.0 x 20 cm) were obtained  from  BIO-RAD.  2.8.1 VLDL I s o l a t i o n Animals  being fed regular r a b b i t chow were f a s t e d f o r 18 hours p r i o r  collection  of  blood f o r VLDL i s o l a t i o n .  On the other  maintained  on the high f a t d i e t were r o u t i n e l y f a s t e d f o r 24 hours so  minimize the contamination of VLDL preparation w i t h  hand,  those  to  rabbits as  to  chylomicrons.  VLDL was i s o l a t e d by f l o a t a t i o n as described (116). For normal r a b b i t s a t l e a s t 15 - 20 mis of plasma was required i n order t o o b t a i n s u f f i c i e n t VLDL t o c a r r y out a turnover study.  In the case of hyperlipidemic r a b b i t s 5 - 1 0  mis  of plasma was u s u a l l y 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. Sterile  s a l i n e was  underlaid  w i t h plasma i n  c e n t r i f u g e tubes (Beckman Instruments I n c . ) .  16  x  76mm  Quick-Seal  The tubes were sealed using gas-  s t e r i l i z e d metal tube-sealing caps and c e n t r i f u g e d a t 112,000 x g f o r 18 hours a t 15°C of  the  i n a Beckman L8-70M u l t r a c e n t i f u g e . tubes  lipoproteins repeated described  using  were  sterile,  resuspended  as above.  VLDL was recovered from the  pyrogen-free in  sterile  syringes  s a l i n e and  and  needles.  determination of plasma  Portions  of  the  preparation  The  the c e n t r i f u g a t i o n  The washed VLDL was used f o r r a d i o i o d i n a t i o n as  shortly.  top  were  saved  will for  be the  VLDL-TG, VLDL-cholesterol and VLDL-apoB. I t should be  24  noted  t h a t VLDL not being used f o r turnover studies was i s o l a t e d under non -  s t e r i l e conditions.  2.8.2 Determination o f VLDL-TG/ VLDL-Cholesterol The was  and VLDL-ApoB  t r i g l y c e r i d e and c h o l e s t e r o l content of the l i p o p r o t e i n  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  preparation  and c h o l e s t e r o l  measurement. VLDL-ApoB was  determined  as described by Egusa  e t . a l . (117).  The  l i p o p r o t e i n s o l u t i o n (0.5 mis) was added t o 0.5 mis o f 100% isopropanol i n 12 x  75  mm heavy-walled  mixed  f o r about  c o n i c a l polystyrene c e n t r i f u g e tubes.  one minute and incubated a t room  The  temperature  tubes were overnight.  P r e c i p i t a t e d ApoB was p e l l e t e d by c e n t r i f u g a t i o n a t 1000 x g f o r 30 minutes. A modified  Lowry  protein  assay  (118) was  used  t o estimate  the  protein  concentration o f the supernatant and the i n i t i a l l i p o p r o t e i n preparation.  The  d i f f e r e n c e i n p r o t e i n concentration between the former and the l a t e r was taken as an estimate o f the ApoB concentration. Plasma  VLDL-TG, VLDL-Cholesterol,  VLDL-apoB was c a l c u l a t e d by m u l t i -  p l y i n g the values obtained f o r the l i p o p r o t e i n preparation by the  appropriate  dilution factor. 2.8.3 R a d i o i o d i n a t i o n o f VLDL VLDL was i o d i n a t e d by the i o d i n e monochloride method o f McFarlane as  modified  by  Fidge  and P o u l i s (120).  The s o l u t i o n  (119)  (2.0 mis) o f VLDL 125  containing USA) an  1.0 t o 2.0 mg o f p r o t e i n was mixed w i t h 1.0 mCi Na  I  (Amersham,  i n a 12 x 75 mm s t e r i l e tube c o n t a i n i n g 0.0033M IC1 ( s u f f i c i e n t t o give iodine  buffer,  / p r o t e i n r a t i o of 10:1) and about  pH 10.0 (prepared w i t h s t e r i l e water). 25  o f 0.05 mis o f 0.4M  glycine  The i o d i n a t e d VLDL was passed  through with  a s t e r i l e column (1.0 x 20 cm) o f Sephadex G50 (medium)  0.4M g l y c i n e b u f f e r ,  pH 10.0.  equilibrated  The eluent (1.0 mis) was c o l l e c t e d  into  125 sterile  13 x  (cloudy)  100 mm tubes and those  were  equilibrated  pooled  fractions  w i t h s t e r i l e i n j e c t a b l e s a l i n e i n order remove f r e e  iodine. I f  free  iodine  second  I-VLDL  G50 column  characterization  a  the  Sephadex  upon  and passed through  containing  o f the iodinated l i p o p r o t e i n (see below) more than 5% 125  was found t o be associated w i t h i t , the  I-VLDL was passed  through a t h i r d column o f Sephadex G50 e q u i l i b r a t e d w i t h s t e r i l e s a l i n e . iodinated ug/ml)  VLDL was s t e r i l i z e d by the a d d i t i o n o f gentamycin  followed  by passage through a s t e r i l e 0.22um  The  s u l f a t e (100  low-binding  millipore  f i l t e r ( M i l l i p o r e Corporation, Bedford, MA.) i n t o s t e r i l e tubes. 2.8.4  Characterization A  the  o f Iodinated  Lipoproteins  d i l u t e d (1:100) preparation o f the iodinated l i p o p r o t e i n  purpose o f c h a r a c t e r i z a t i o n as previously  procedures steps  were  were  described  was used f o r  (117,120-122).  done i n d u p l i c a t e f o r each l i p o p r o t e i n preparation  carried  out i n 12 x 75 mm  gamma  tubes  (Fischer  All  and a l l  Scientific,  Vancouver B.C.). 125 a) Percentage o f The  diluted  unlabelled  I_ bound t o Apollpoprotein  B  l i p o p r o t e i n s o l u t i o n (0.1 mis) was mixed w i t h  low density l i p o p r o t e i n ,  0.2 mis o f  LDL (to serve as a c a r r i e r f o r ApoB) and  0.3 mis o f 100% isopropanol (117).  The mixture was mixed and l e f t t o stand a t  room  The p r e c i p i t a t e d  temperature  centrifugation to  another  f o r 10 minutes.  ApoB was p e l l e t e d  by  a t 1700 x g f o r 30 minutes and the supernatant was t r a n s f e r r e d  tube.  The r a d i o a c t i v i t y  i n the  pellet  and supernatant  were  determined using a 4 channel automatic gamma counter (LKB - WALLAC, QY 20101  26  Turku 10, by  Finland).  LKB-Wallac.  The counter was c a l i b r a t e d w i t h a  The  efficiency  Co standard supplied  of each channel was used t o  calculate  the  d i s i n t e g r a t i o n s per minute (dpm).  b) Percentage of The  I_ bound t o L i p i d s  d i l u t e d l i p o p r o t e i n s o l u t i o n (0.01 mis) was mixed w i t h 0.01  LDL and 0.2 mis of methanol.  contents of the tubes were mixed,  the  tubes were l e f t a t -20°C f o r 10 minutes. f o r 30  radioactiviity  of  Following the a d d i t i o n of 0.3 mis of chloroform,  the  1700 x g  mis  minutes and  the  1.0 ml of d i e t h y l ether was added  pellet  The tubes were and  supernatant  and  centrifuged  at  separated.  The  i n the supernatant and the p e l l e t was determined as p r e v i o u s l y 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 w i t h I-VLDL: 125 The  diluted  bovine  serum  saline.  The  for  10  I-VLDL  s o l u t i o n (O.lmls) was mixed w i t h 0.3 mis  albumin i n s a l i n e and 0.3 mis of 10%  trichloroacetic  of  5%  acid  in  contents of the tube were mixed and then c e n t r i f u g e d a t 1700 x g  minutes.  The  r a d i o a c t i v i t y determined.  supernatant  and  pellet  were  separated  and  the  The r a d i o a c t i v i t y i n the supernatant was associated  w i t h f r e e i o d i n e (120-122). 3 2.8.5 P r e p a r a t i o n of I n j e c t a b l e  H-Glycerol  3 [2- H ] g l y c e r o l i n absolute e t h a n o l , w i t h 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 p u r i t y 3 of the  H-glycerol was checked by t h i n layer chromatography  n-butanol  saturated w i t h water  i n two systems: a)  and b) chloroform / acetone 27  /  5N  ammonia  10:80:10  (v/v/v).  The p u r i t y was found t o be approximately 98 % using  both  systems. 3  H-glycerol was prepared t o g i v e a f i n a l concentration o f 40-50 u C i / ml. Each animal received w i t h 1.5 t o 2.0 mis. The  ethanol  redissolved sterilized  was evaporated  under  a  stream  of n i t r o g e n and  i n 2.0 mis o f s t e r i l e i n j e c t a b l e s a l i n e .  by passing i t through a 0.22um m i l l i p o r e f i l t e r .  mis) o f a 1:100 d i l u t e d preparation o f the s t e r i l i z e d mixed the  The f i n a l s o l u t i o n  with  5.0 mis o f aqueous s c i n t i l l a n t ,  then was  A l i q u o t s (0.1  glycerol solution  was  ACS (Amersham Corporation), and  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, F i n l a n d ) . 2.8.6 Turnover Study P r o t o c o l Two given  days  before i n j e c t i o n and up t o two weeks a f t e r ,  potassium  approximately  i o d i d e i n the d r i n k i n g water (average d a i l y  animal  was  consumption o f  200 mg) so as t o block t h y r o i d uptake o f any f r e e 125  which may be present i n the Turnover  each  125 I-iodine  I-VLDL preparation.  s t u d i e s were r o u t i n e l y c a r r i e d out i n two r a b b i t s on the same  day, and thus the times o f i n j e c t i o n were staggered so as t o avoid overlapping of the time o f blood c o l l e c t i o n f o r each animal. Each animal was f a s t e d f o r 12 hours p r i o r t o the i n j e c t i o n o f the r a d i o n u c l i d e s .  On the day of the turnover  study, a blood sample was taken f o r the determination o f b a s e l i n e parameters. 125 3 At time t = 0, 1 5 - 2 5 u C i o f I-VLDL and 70 - 100 u C i o f H-glycerol were injected  into  the marginal ear v e i n o f each  rabbit.  followed  by s a l i n e (3.0 mis) A t s p e c i f i e d i n t e r v a l s ,  Each  injection  was  3 - 5 mis of blood was  withdrawn from the marginal v e i n o r c e n t r a l a r t e r y o f the other ear i n t o tubes containing EDTA.  The r a b b i t s were allowed f r e e access t o water throughout the  study and remained f a s t i n g u n t i l a f t e r the 12 hour sample.  28  The r a b b i t s  were  fasted  f o r an a d d i t i o n a l twelve hours,  taken.  This  procedure  derived p a r t i c l e s  a f t e r which the 24 hour  served t o l i m i t the  contribution of  sample  was  intestinally  (containing ApoB and TG) t o the VLDL pool.  2.8.7 I s o l a t i o n o f VLDL from Post - I n j e c t i o n Plasma VLDL was obtained from 1.5 t o 3.0 mis o f plasma as described above. volume  o f the VLDL  determine  preparation was recorded and 0.025  the r a d i o a c t i v i t y .  mis was  The  used t o  The remainder of the VLDL s o l u t i o n was used f o r  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 S p e c i f i c A c t i v i t y ApoB was Lipoprotein  p r e c i p i t a t e d from VLDL using isopropanol as described  s o l u t i o n containing  (117).  20 - 100 ug o f p r o t e i n was adjusted t o 0.5  mis w i t h s a l i n e , then added drop by drop t o 100% isopropanol w h i l e mixing. The mixture  was  incubated  a t room temperature f o r  about  12 hours.  ApoB  was  p e l l e t e d by c e n t r i f u g a t i o n (1700 x g / 30 minutes / 4°C). The supernatant was a s p i r a t e d . The p e l l e t was washed twice w i t h 1.0 ml o f isopropanol-water once  (1:1),  w i t h 1.5 mis o f isopropanol and once w i t h 1.5 mis o f Barnstead deionized  water.  The ApoB was r e s o l u b i l i z e d by incubation w i t h 0.3 - 0.6 mis o f  sodium  hydroxide  1.0M  (depending on the q u a n t i t y o f p r o t e i n present) f o r 24 hours.  An a l i q u o t o f the ApoB s o l u t i o n was used t o measure the r a d i o a c t i v i t y .  Plasma  VLDL-ApoB  amount  s p e c i f i c a c t i v i t y was estimated from knowledge o f the t o t a l  of r a d i o a c t i v i t y used,  the amount o f 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 s o l u t i o n r e l a t i v e t o plasma. i n dpm / ml (of plasma).  29  The s p e c i f i c a c t i v i t y was  expressed  2.8.9  Estimation of VLDL-TG S p e c i f i c A c t i v i t y T r i g l y c e r i d e s were extracted from VLDL by a m o d i f i c a t i o n of the method of  Folch e t . a l . (123). The l i p o p r o t e i n s o l u t i o n (0.5 mis) was mixed w i t h 10 mis of chloroform methanol M,  2:1  (v/v) i n stoppered pyrex tubes and mixed.  Sodium c h l o r i d e  -  (0.05  1.5 mis) was added t o the tubes, the contents mixed and c e n t r i f u g e d a t 550  x  g  f o r 10 minutes a t 4°C.  chloroform-methanol-0.05M organic  phase.  aspirated. of  a  The aqueous phase was a s p i r a t e d and 2.5  of  sodium c h l o r i d e (3:47:50 (v/v/v)) was added t o  The tubes were a g i t a t e d ,  c e n t r i f u g e d and the  aqueous  the phase  To separate phospholipids from t r i g l y c e r i d e s , approximately 0.7  g  z e o l i t e mixture (copper s u l f a t e / Lloyds reagent / calcium hydroxide  /  z e o l i t e 1:2:2:20 (w/w/w/w)) was added t o each tube. stand  mis  The tubes were allowed t o  a t room temperature f o r 10 minutes and were o c c a s i o n a l l y  t h i s period.  The p a r t i c u l a t e matter was  g f o r 25 minutes a t 4°C. stream  mixed  during  p e l l e t e d by c e n t r i f u g a t i o n a t 1000  x  The supernatant was f i l t e r e d and evaporated under  a  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 i o d i n a t e d t r i g l y c e r i d e s are found i n the e x t r a c t . 3 However, only H - t r i g l y c e r i d e s r e f l e c t endogenous synthesis. Since the p u l s e 125 3 height spectrum of I completely overlaps t h a t of H (124) , the 3 r a d i o a c t i v i t y associated w i t h H-TG was determined i n a Beckman LS 9000 3 scintillation 1  counter w i t h windows manually adjusted to d i s c r i m i n a t e  H  from  ?R  I (153). The isopropanol s o l u t i o n (0.05 - 0.1 ml) was added t o 10 ml of and  1.0  knowledge  ml of  radioactivity  of de-ionized water the  volume  of  and  the  radioactivity  determined.  l i p o p r o t e i n s o l u t i o n extracted, 3  recovered ( i . e . t h a t due to  H-TG),  the  ACS From  total  the e x t r a c t i o n 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 t o plasma, the s p e c i f i c a c t i v i t y 30  of plasma VLDL-TG  was c a l c u l a t e d .  The e f f i c i e n c y o f e x t r a c t i o n was estimated by two methods.  In the  first  method, the t r i g l y c e r i d e concentration i n the VLDL preparation was measured so as t o a l l o w 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 o f 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 s o l u t i o n . The d i f f e r e n c e between the  recovered  amount and t h a t which was extracted was  taken  to  represent  losses. 14 In the second method, a known quantity o f g l y c e r o l t r i [ l a  C]oleate, w i t h  s p e c i f i c a c t i v i t y o f 56 mCi/mmol (Amersham Corporation) was mixed w i t h  mis  o f u n l a b e l l e d r a b b i t VLDL and t r i g l y c e r i d e s e x t r a c t e d .  extract  was  used  f o r measuring  the r a d i o a c t i v i t y .  0.5  An a l i q u o t of the  I n both  cases,  the  e f f i c i e n c y o f e x t r a c t i o n was greater than 95%.  2.9 KINETIC ANALYSIS 2.9.1 VLDL-Triglycerides The  fractional  c a t a b o l i c r a t e (FCR) f o r the removal  plasma was estimated as o r i g i n a l l y described by Farquhar this  method,  o f VLDL-TG  e t . a l . (125).  the slope o f the l o g - l i n e a r phase o f the d e c l i n e  from In  i n specific  a c t i v i t y o f VLDL-TG a f t e r the peak of the curve was used t o estimate the h a l f life  e x t r a p o l a t i o n back t o zero.  b v  the r e l a t i o n s h i p FCR = In 2 / t ^ ( Plasma conditions)  VLDL-TG was  transport  s e e  rate  The FCR was then c a l c u l a t e d from  appendix A ) . (production  by  under  steady-state  determined by m u l t i p l y i n g the mass of VLDL-TG i n the plasma  compartment by the f r a c t i o n a l c a t a b o l i c r a t e (FCR). calculated  rate  Plasma VLDL-TG mass  m u l t i p l y i n g the plasma VLDL-TG concentration by the  31  was  estimated  plasma  volume.  volume  of  The plasma volume was taken t o be equivalent t o  the  initial  125  dilution normals  distribution  of 125  of the i n j e c t e d (group N),  I-VLDL.  The l a t t e r was  calculated  from  the  I-VLDL by e x t r a p o l a t i o n t o time t = 0 (126).  For  the steady-state mass could not always be  calculated  by  measuring VLDL-TG a t each experimental time p o i n t because of the low l e v e l s of triglycerides turnover  and  the  s t u d i e s , good  concentration  However,  agreement was found t o e x i s t  prior  between  12 hours.  VLDL-TG  concentration  VLDL-TG  concentration  the VLDL-TG  This was done by f o l l o w i n g the v a r i a t i o n  i n one normal r a b b i t over a p e r i o d of a t t=0  12  was used t o c a l c u l a t e VLDL-TG  hours. steady  application  of  the k i n e t i c s of VLDL-apoB metabolism was  o f the two pool model o f Gurpide e t .  The state  I-VLDL.  carried  out by  a l . (127) (see f i g u r e  A includes the plasma compartment and p o s s i b l y the extravascular  w h i l e the r e s t of the body i s represented by pool B. be  in  VLDL-ApoB Analysis  Pool  to  Steady-state mass was obtained by m u l t i p l y i n g 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 i n j e c t e d 2.9.2  t o the  a t t = 0 and the VLDL concentration throughout the study up  and i n c l u d i n g t h a t a t  mass.  l i m i t e d amount of specimen.  1.)  space,  Tracer i s considered  to  administered i n t o pool A and a l l subsequent sampling i s a l s o considered t o  be from t h a t p o o l .  A t present, the p h y s i c a l c o n s t r a i n t s on pools A and B have  not y e t been c l e a r l y defined. The use of t h i s model ( f i g . 1) i s r e l e v a n t t o 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 i n t o which t r a c e r i s i n j e c t e d  (pool  A)  i s not  n e c e s s a r i l y confined t o the i n t r a v a s c u l a r space. Both of these c o n d i t i o n s have been model  found t o be a p p l i c a b l e t o VLDL-apoB metabolism. t o VLDL-apoB  The relevance  metabolism i n man and p i g s has r e c e n t l y been 32  of  this  discussed  F i g . 1: The general two-pool model o r i g i n a l l y described by Gurpide e t . a l . (127) which was used i n the a n a l y s i s of VLDL-ApoB k i n e t i c s .  33  (128,129). According  to  this  two pool model,  the decay  of  VLDL-apoB  specific  a c t i v i t y , SA(t) i s b i e x p o n e n t i a l and i s represented by the f u n c t i o n (127)  where  SA(t) =  c  SA(t)  i s VLDL-apoB  e A  -"  t  +  C  B ~' e  5 t  specific  activity  determined  at  various  times, t , a f t e r a d m i n i s t r a t i o n of the t r a c e r , e i s the n a t u r a l logarithm base. 0( and p  are r a t e constants which are determined from the  half-lives  of t h e i r r e s p e c t i v e exponential on the b a s i s  r e l a t i o n s h i p k = 0.693 / t y  f o r monoexponential decay  2  of  the  (appendix  A). C, and C  D  are constants estimated from the p o i n t s a t which  the r e s p e c t i v e exponentials i n t e r c e p t the y - a x i s .  The curve parameters o( , ^ in  appendix  B.  These  , C  values  A  and C  may be manually estimated as i n d i c a t e d  B  may then be  used  to  estimate  the  kinetic  parameters of VLDL-ApoB metabolism. The  fractional  (excluding  material  c a t a b o l i c r a t e f o r the removal of VLDL-ApoB from pool that  may  r e c y c l e between A and  B)  i s given  by  A the  f o l l o w i n g equation (127): FCR  A  =  «C  A  C  /3C  +  A  +  C  B  B  The production r a t e of VLDL-apoB which represents synthesis i n t o pool but  A,  excludes apoB t h a t may r e c y c l e between A and B i s given by the product of  the FCR and the steady-state mass of In  the  present  study,  VLDL-ApoB.  curve  parameters were  microcomputer (IPC Systems, Vancouver, 34  B.C.  CAN.)  calculated  on  an  IPC  using a "curve s t r i p p i n g "  program  which incorporated  a nonlinear l e a s t  f i t t i n g the exponentials (129).  square a n a l y s i s method f o r  The estimation o f the FCR was a l s o incorporated  i n t o the program.  2.10 STATISTICAL ANALYSIS All program portable  statistical (Anderson-Bell  analyses were c a r r i e d out using the ABSTAT  statistical  Company, USA). The program was run on an  IPC  XT  microcomputer ( I n t e l l i g e n t Series) supplied by IPC Systems. For the  comparison o f d i f f e r e n c e s 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 o f r a b b i t s ,  differences data.  between means were compared using the student t t e s t  f o r paired  Differences were considered s i g n i f i c a n t a t P < 0.05, where P represents  the p r o b a b i l i t y f o r t w o - t a i l e d t e s t s .  35  3. RESULTS  3.1  FOOD CONSUMPTION and BODY WEIGHT  The  average  daily  food  consumption  and mean body  weights  commencement and conclusion o f the study are presented i n Table Group N had the h i g h e s t d a i l y food i n t a k e . consumption  between the groups  However,  a t the  I . Overall,  the d i f f e r e n c e s i n food  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 a t the end o f the study period were s i m i l i a r f o r a l l three groups.  3.2 a)  PLASMA LIPID PARAMETERS C h o l e s t e r o l and T r i g l y c e r i d e s The  variation  o f plasma c h o l e s t e r o l and t r i g l y c e r i d e s i n r a b b i t s f e d a  h i g h - f a t d i e t f o r e i g h t weeks (Group H) i s shown i n f i g . plasma to  2. The v a r i a t i o n o f  c h o l e s t e r o l and t r i g l y c e r i d e s i n r a b b i t s (group C) which were  exposed  the same d i e t as group H, except t h a t they were administered L - c a r n i t i n e  f o r the f i n a l 4 weeks, i s presented i n f i g . 3. It the  can be r e a d i l y seen t h a t b a s e l i n e values ( i . e those a t the s t a r t o f  study)  f o r both c h o l e s t e r o l and t r i g l y c e r i d e s i n groups H  and C  were  s i m i l a r t o those f o r group N (Table I I ) . The  e l e v a t i o n s i n l i p i d s seen over the f i r s t 4 weeks o f exposure t o the  d i e t i n groups H and C were s i m i l i a r . H  over  L i p i d l e v e l s continued t o r i s e i n group  the f i n a l four weeks o f the study.  cholesterol  I n both groups,  the change i n  was more s t r i k i n g .  In the case o f group H, c h o l e s t e r o l increased from a b a s e l i n e mean value of 50 mg/dl t o more than 2000 mg/dl a f t e r e i g h t 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 f a c t t h a t the mean value o f plasma  36  Table  I  D a i l y food consumption and i n i t i a l and f i n a l body weight f o r the groups o f r a b b i t s involved i n the study, n = 4 f o r a l l groups.  Food Consumption (g/day)  I n i t i a l Body Weight (Kg)  F i n a l Body 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  3000 T  F i g . 2: Plasma c h o l e s t e r o l (x x) and t r i g l y c e r i d e s (o hyperlipidemic r a b b i t s (group H). n = 4. 1 " P < 0.005  and * P < 0.001 versus time, t = 0.  38  o)  levels i n  2000  1800-1600  1400-CP  1200  O  pr  y  oo  1000  800  600 +  4Q0-200-0-'  S. -1 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 1111111111111111111111111111 I I I I I I I II I III I I H  0  2  4  TIME  (weeks)  F i g . 3: Plasma c h o l e s t e r o l ( x — x ) and t r i g l y c e r i d e s (o o) concentrations i n hyperlipidemic r a b b i t s (group C ) . The r a b b i t s were given L - c a r n i t i n e f o r one month while being maintained on a h i g h - f a t d i e t .  t  P < 0 . 005 v s . time, t = 0.  * P < 0.025 and P < 0.05 v s . 4 weeks. #  D a i l y L - c a r n i t i n e s t a r t e d . The d a i l y intake was 170 +_ 40 mg/kg.  39  Table I I  Plasma l i p i d s and c a r n i t i n e values f o r r a b b i t s maintained on r e g u l a r chow (Group N) f o r 8 weeks, n = 4.  Parameter Cholesterol  Concentration (mg/dL)  55.0 _+ 18.2  T r i g l y c e r i d e s (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 p r o t e i n  (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  13.0 +_ 3.0  (umoles/L)  Total c a r n i t i n e (umoles/L)  34.0 +_ 9.0  Acylcarnitine Total c a r n i t i n e  0.38 +_ 0.03  A l l values represents the mean + S.D.  40  rabbit  triglycerides  doubled  over  the  e i g h t week  period,  this  change  was  not  plasma c h o l e s t e r o l  had  significant. In group C,  a f t e r 4 weeks on the h i g h - f a t d i e t ,  increased s i g n i f i c a n t l y (from 50 mg/dl t o 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 .  A f t e r 4 weeks on L - c a r n i t i n e ,  plasma  c h o l e s t e r o l 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 . Plasma t r i g l y c e r i d e s decreased by 50%  (b)  while cholesterol  3.)  decreased by 35%.  Plasma VLDL parameters and HDL-cholesterol The  values  f o r plasma VLDL parameters and HDL-cholesterol f o r groups  and  C are presented i n Tables I I I and IV.  III  and IV w i t h t h a t i n Table I I i n d i c a t e s t h a t b a s e l i n e values f o r groups  H  Comparison o f the data i n Tables H  and C were s i m i l a r t o those f o r group N. It  i s apparent  increase  i n a l l parameters  significant  increases  parameters, fold  t h a t over the 8 week p e r i o d , i n group  occurred  H.  there was  Except  i n a l l parameters.  progressive  f o r HDL-cholesterol, Among  the  various  VLDL-cholesterol showed the l a r g e s t change, i n c r e a s i n g almost 200  from an i n i t i a l value of 3.6 + 2.5 mg/dl t o 1328 +  showed  a  the  smallest change,  472  mg/dl.  VLDL-TG  i n c r e a s i n g from 9.1 +_ 3.0 mg/dl t o 82.0 +_  45.1  mg/dl (see t a b l e I I I ) . Over  the f i r s t 4 weeks,  the e f f e c t s 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  VLDL-cholesterol showed the l a r g e s t change, the the  (Table I V ) .  w h i l e the change i n VLDL-TG  smallest among those VLDL parameters which were s i g n i f i c a n t l y a l t e r e d diet.  was by  HDL-cholesterol was not a f f e c t e d .  A f t e r 4 weeks on L - c a r n i t i n e , 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 I V ) .  41  The concentration of  Table I I I Plasma HDL-Cholesterol and VLDL parameters i n h y p e r l i p i d e m i c r a b b i t s H). n = 4  Parameter (mg/dl)  Baseline  4 weeks  8 weeks  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  697 + 152  1328 + 472  VLDL-Chol.  3.6 + 2.5  *p < .005 VLDL-ApoB  0.79 +_ 0.21  24.2 _+ 9.9  29.8 _+ 16.6  *p < .02 VLDL p r o t e i n  1.8 + 0.4  55.5 _+ 16.4 *  A l l values represent the mean +_ S.D. 4 weeks, 8 weeks versus b a s e l i n e .  42  p < .01  83.5 _+ 35.0  (Group  Table IV Plasma HDL-Cholesterol and VLDL parameters i n hyperlipidemic w i t h L - c a r n i t i n e (Group C ) . n = 4  rabbits  treated  Parameter (mg/dl)  Baseline  4 weeks  8 weeks  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  661.5 + 40.8  308.0 + 77.1  VLDL-Chol.  1.90 + 0.26  *  VLDL-ApoB  1.89 + 1.80  P < .005  26.6 + 11.4 P < .025  VLDL p r o t e i n  2.16 +_ 0.50  54.9 +_ 19.5  *  P < .001  *P < .02  19.2 + 13.3 *N.S. 32.0 +_ 19.0 *P < .02  Values represent the mean +_ S.D. L - c a r n i t i n e treatment was s t a r t e d a t 4 weeks. The d a i l y consumption was 170 +_ 40 mg/kg b.w.  * 4 weeks versus b a s e l i n e . 8 weeks versus 4 weeks.  43  plasma VLDL-TG decreased by 50%,  w h i l e VLDL-cholesterol d e c l i n e d by 37%.  The  VLDL p r o t e i n concentration decreased by 40% w h i l e VLDL-apoB decreased by about 35% w i t h 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 f o l l o w i n g 1 month of L - c a r n i t i n e is  treatment. I t  worth noting t h a t the concentration of plasma VLDL-cholesterol and  cholesterol decreased  d i d not decrease by the same quantity;  plasma  VLDL-cholesterol  by about 350 mg/dl w h i l e plasma c h o l e s t e r o l decreased by  The f i n d i n g s f o r plasma VLDL-TG and plasma t r i g l y c e r i d e s were VLDL-TG  decreasing  by  plasma  600mg/dl.  similiar,  with  20 mg/dl w h i l e 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  untreated  levels  of  D-y# -Hydroxybutyrate  tended t o be  r a b b i t s (Group H) than i n the t r e a t e d group  these d i f f e r e n c e s were not  significant.  (Group  higher  i n the  C).  However,  One month of L - c a r n i t i n e therapy had  no s i g n i f i c a n t e f f e c t on plasma l e v e l s of D - - h y d r o x y b u t y r a t e . 3.4  PLASMA GLUCOSE Fasting  plasma glucose l e v e l s were measured a t the beginning and end  of  the study. There was no s i g n i f i c a n t d i f f e r e n c e between f a s t i n g glucose l e v e l s i n the three groups of r a b b i t s .  3.5 LIPOPROTEIN ELECTROPHORESIS E l e c t r o p h o r e s i s of plasma high  f a t diet  indicated  from a l l three groups p r i o r t o i n t r o d u c i n g the  that rabbit  lipoproteins  have  slightly  greater  m o b i l i t y than those of human beings (Fig. 4 ) . There  was  a  s t r i k i n g reduction i n the i n t e n s i t y of the ©(-band  44  and  a  Alpha Pre-beta-  l\m  Beta  m M mm  I -4  Origin  Fiq. 4: Lipoprotein NZW r a b b i t s . Lane 1:  electrophoresis human p l a s m a ;  o f human 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 l a n e s 2 t o 6: p l a s m a f r o m 18 h o u r - f a s t e d  rabbits.  m  Alpha Pre-beta Beta  II  .  Origin  8  3 _,4 (  Fig.  5: Lipoprotein  & 5:  human;  high  f a t diet  rabbits  lanes  electrophoresis  (0.5% c h o l e s t e r o l / 5 %  consuming  The human s u b j e c t  of  h u m a n a n d NZW r a b b i t s '  2 , 3 ; 6 & 7 : NZW r a b b i t s ' regular  rabbit  was f a s t e d  corn  chow.  f o r 12 -  o i l ) ; A l l  14  plasma  2 weeks  lanes  rabbits  4 & 8: were  plasma.  after  plasma  fasted  Lanes  starting from  f o r 18  on  1 a  NZW hour.  hours.  Alpha Pre-beta  | | WK w w  Beta Origin  Fig.  6:  month  on high-fat  month  on  collection  Lipoprotein  electrophoresis  diet;  l-carnitine. of  of  NZW r a b b i t s '  lanes  3 & 4:  2 weeks  A l l  rabbits  were  blood.  43  plasma.  on l-carnitine;  fasted  f o r  18 h o u r s  Lanes lanes prior  1 & 2:  1  5 & 6: to  1  the  concomitant (Fig. 5 ) .  increase i n the  intensity  and s i z e of the 8 and  pre-P  bands  In f a c t , a f t e r 1 month, a broad band  was a l l t h a t was evident i n the electrophoretogram. TheoC-band appeared t o be completely absent ( F i g . 6, lanes 1 & 2 ) . As  L - c a r n i t i n e treatment progressed the i n t e n s i t y and s i z e of the  broad  The 0C-band remained absent a f t e r 1 month of  beta / pre-beta band decreased. 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 3 the  injection  Figure 7.  of  H-glycerol i n normal r a b b i t s (Group N)  following  i s presented  in  This curve may be d i v i d e d i n t o 3 phases:  i ) an i n i t i a l r a p i d r i s e , found t o peak a t 1 - 3 hours i i ) an e a r l y , 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 r e s t of the study A  representative  carnitine  therapy  activity-time carnitine (group  N).  activity  curve  f o r VLDL-TG turnover i n group  i s shown i n Figure 8.  curve  treatment  f o r group  The shape of the  C r a b b i t s on a h i g h - f a t  C  prior  VLDL-TG  diet  to  specific  prior  to  was n o t i c e a b l y d i f f e r e n t from t h a t f o r chow-fed  In f a c t ,  i n group C,  the curve obtained  f o r VLDL-TG  over 12 - 30 hours could not be c l e a r l y resolved i n t o  L-  L-  animals specific  the  rapid  and slow d e c l i n e phases. 3 A  representative  following  H-glycerol  i n j e c t i o n i n t o hyperlipidemic r a b b i t s a f t e r L - c a r n i t i n e treatment  i s shown i n  Figure 9.  curve  f o r VLDL-TG  metabolism  Comparison of t h i s curve w i t h t h a t i n Figure 7 i n d i c a t e s t h a t w i t h  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 f o l l o w i n g the i n j e c t i o n o f H-glycerol i n t o a r a b b i t consuming regular l a b . chow. The h a l f - l i f e (t- ,») was obtained by v i s u a l inspection o f the l i n e a r p o r t i o n o f the decay curve. The FCR was estimated from the r e l a t i o n s h i p FCR = In 2 / t ^ (see appendix A ) .  47  2  10000  T  F i g . 8: 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 h y p e r l i p i d e m i c r a b b i t (group C) p r i o r t o L - c a r n i t i n e treatment.  48  1  'iiiiiiiiiiiiiiiiiiiiiiiiimiiiiiiiiiiiiii minium  O  3  6  min i in  IIIIHIIIIIIHIIHHIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIHIIIIIHIIIIIHIIIIIIIIIIIIIIIIIIIIIIHUII  9  12 Time  15  18  IIIIIIIIIIIIIIIIIIIIIIIIIIIIIMIIIIIH  21  (Hrs)  F i g . 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 group C a f t e r 1 month o f L - c a r n i t i n e . The d a i l y consumption o f L - c a r n i t i n e was 170 + 40 mg/kg.  49  treatment,  the shape  o f the s p e c i f i c  activity-time  curves  i n Group  C  approached the shape o f those i n Group N. FCR was c a l c u l a t e d from the apparent l o g - l i n e a r phase o f the d e c l i n e i n s p e c i f i c a c t i v i t y o f VLDL-TG as i n d i c a t e d i n the previous chapter (see s e c t i o n 2.9.1). the  The FCR values f o r groups C and N are presented i n Table V along w i t h  VLDL-TG t r a n s p o r t rates f o r the two groups.  hyperlipidemic  rabbits  The FCR f o r VLDL-TG i n the  was found t o be 0.069 +_ 0.024 h r . 1  This value  s i g n i f i c a n t l y lower (P < .001) than the value o f .308 _+ .035 h r .  1  was  found f o r  the FCR i n group N. Following  1 month o f L - c a r n i t i n e therapy,  the mean value o f the FCR f o r  VLDL-TG i n group C increased by 3.5 times t o 0.239 +_ 0.033 h r . .  The  1  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 f o r VLDL-TG  metabolism  in  normal r a b b i t s . The t r a n s p o r t o f VLDL-TG i n group C prior  t o treatment  increased from 5.23 +_ 2.97  t o 7.47 +_ 2.60 mg.hr.  1  following  L-carnitine  mg.hr.  1  therapy.  However, t h i s increase f a i l e d t o a t t a i n s t a t i s t i c a l s i g n i f i c a n c e . The be  t r a n s p o r t r a t e f o r VLDL-TG i n group N (3.26 _+ 2.29 mg/hr) tended  lower  than  that  i n group C both p r i o r  and subsequent  to  to L-carnitine  therapy. However, these d i f f e r e n c e s 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 f o l l o w i n g  injection  of  125 autologous  I-VLDL i n t o normals (group N) and hyperlipidemic r a b b i t s p r i o r  to L - c a r n i t i n e (group C) are shown i n Figures 10 and 11. in  both  method  As i s demonstrated,  cases the decay curve may be r e s o l v e d i n t o two exponentials by the o f curve s t r i p p i n g (see Appendix B and r e f 127). 50  Table  V  F r a c t i o n a l c a t a b o l i c r a t e (FCR) and transport r a t e f o r VLDL-TG metabolism i n Groups N and C.  Group N (n=3)  FCR (hr ) 1  Group C (n=4)  0.308 + 0.035  Pre-treatment  Post-treatment  0.069 + 0.024  0.239 + 0.033  P < 0.001 Transport Rate (mg/hr)  3.26 + 2.29  5.23 + 2.97 * N.S.  A l l values represent mean +_ S.D. * Group C (Pre-treatment) versus Group N. Post-treatment versus Pre-treatment.  51  # P < 0.01 7.47 _+ 2.60 #  N.S.  IO  6  TIME  8  10  (hrs)  F i g . 10: Representative curve f o r the decay o f I-VLDL-apoB f o l l o w i n g the i n j e c t i o n o f autologous I-VLDL i n t o normal r a b b i t s . The FCR was c a l c u l a t e d from the curve parameters er, /3 , C and C (refer t o s e c t i o n 2.9.2 and appendix B f o r f u r t h u r explanation). ?  ft  52  fi  1000001  T  c  CL.  B  =  100000  Q  100000  -<  o UJ CL  10000  MlintiniM)lllllinMMItnM^MMtHIIHIHIIIIIIIIMIttHIIMIIIIIIIIIIIIIIIIIIIIIIIIIIIIItl)IM)llltlllllllllllllll  0  2  4  6  8  10  TIME ( h r s )  F i g . 11: Representative curve l i p i d e m i c r a b b i t s (group C ) .  f o r the decay o f  53  125, I-VLDL-ApoB  i n hyper-  1O00OO1  T  54  Examination case  of  of  the shape of both curves c l e a r l y i n d i c a t e s t h a t  hyperlipidemic  relatively  the  r a b b i t s the decay of VLDL-ApoB s p e c i f i c a c t i v i t y  s l u g g i s h i n comparison w i t h t h a t i n the normal r a b b i t s .  obvious from the f l a t n e s s of the curve i n f i g .  11.  is  This  is  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 i t s value a t t = 0,  in  r a b b i t s i s 25-30%  whereas i n the normal r a b b i t s i t i s only about 8%.  of Flat  VLDL-ApoB decay curves were obtained f o r a l l the r a b b i t s i n group C p r i o r L - c a r n i t i n e therapy.  The shape of the VLDL-ApoB curves i n group C r a b b i t s  not markedly a f f e c t e d by FCR  L - c a r n i t i n e therapy.  (Fig. 11 and  intravascular  The  FCR f o r the hyperlipidemic  therapy,  (see  space)  Table V I ) .  lower (P < .02)  of the two pool model are presented i n r a b b i t s i s not a f f e c t e d f o l l o w i n g  The FCR of VLDL-ApoB i n group C was  than t h a t i n group N.  was  12).  values f o r the removal of VLDL-ApoB from pool A (plasma and  the  to  possibly  Table  VI.  L-carnitine significantly  The FCR i n group C was unchanged by  L-  c a r n i t i n e treatment. The production r a t e 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-  c a r n i t i n e treatment d i d not s i g n i f i c a n t l y a l t e r t h i s r a t e .  3.7  PLASMA CARNITINES The  r e s u l t s f o r plasma c a r n i t i n e l e v e l s i n groups H and C  are presented  i n Tables VII and V I I I r e s p e c t i v e l y . Plasma c a r n i t i n e b a s e l i n e values were not significantly the  d i f f e r e n t between the three groups.  study plasma f r e e ,  acyl,  Over the f i r s t 4 weeks  a c e t y l and t o t a l c a r n i t i n e l e v e l s i n group  increased s i g n i f i c a n t l y over baseline values (Table V I I ) . acetylcarnitine  increased  by approximately 200%.  of H  A c y l c a r n i t i n e and  For the duration  of  the  study, plasma f r e e , 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 l e v e l s i n group H remained  55  Table VI F r a c t i o n a l c a t a b o l i c r a t e (FCR) and transport r a t e o f VLDL-ApoB i n normals (Group N) and and hyperlipidemic r a b b i t s (Group C) before and a f t e r Lc a r n i t i n e treatment.  Group N (n = 3)  Group C (n = 4) Pre-treatment  FCR (hr ) 1  Transport Rate (mg/hr)  1.23 + 0.42  0.040  1.43 + 0.79  Values represent mean +_ S.D. * Group C (Pre-treatment) versus Group N. Post-treatment versus Pre-treatment.  56  + 0.015  Post-treatment  0.042  + 0.019  *P < 0.02  *P < 0.02  1.74 + 0.87  1.26 + 0.50  *N.S  "N.S  Table V I I Changes i n plasma C a r n i t i n e l e v e l s i n r a b b i t s f e d a h i g h - f a t d i e t (Group H) n = 4. Carnitine (uM)  Baseline  2 weeks  4 weeks  6 weeks  8 weeks  Free  17.8 + 2.4  25.4 + 5.9  28.9 + 3.6  27.8 + 5.6  32.2 + 4.2  *P < .005 Acyl  5.5 + 1.5  16.2 + 3.5  16.0 + 5.3  *P < .005 18.0 + 7.8  *P < .02 Acetyl  4.2 + 1.2  13.3 + 3.5  13.3 + 6.2  *P < .05 17.2 + 6.4  *P < .05 Total  23.5 + 2.3  41.6 + 8.2  44.8 + 2.9  0.76 + 0.11  Acyl Total  0.24 + 0.07  0.81 + 0.07  0.81 + 0.12  45.8 _+ 4.5  0.35 +_ 0.09 * N.S.  Values represents mean +_ S.D. * 4 weeks, 8 weeks versus b a s e l i n e .  57  47.5 _+ 8.0 *P < .02  0.95 + 0.05  *N.S. 0.39 + 0.06  13.2 + 4.4 *P < .05  *P < .005 Acetyl Acyl  15.4 + 5.2  0.85 + 0.03 *N.S.  0.39 + 0.15  0.32 + 0.06 * N.S.  Table V I I I Plasma c a r n i t i n e l e v e l s i n hyperlipidemic r a b b i t s (Group C) before and a f t e r L - c a r n i t i n e treatment, n = 4  Carnitine (uM)  Baseline  2 weeks  4 weeks  6 weeks  8 weeks  Free  15.3 + 5.8  20.4 + 4.6  29.6 + 3.5  98.3 + 37.2  109.8 + 23.7  *P Acyl  3.6 + 2.4  4.4 + 1.8  <  .01  11.1 + 3.5  P < .005 58.6 + 11.8  *P < .001 Acetyl  3.2 + 2.0  4.0 _+ 1.8  8.6 _+ 2.2  P < .05 53.7 + 14.3  *P < .001 Total  18.9 + 3.7  24.8 + 3.8  40.7 + 1.7  0.89 + 0.02  Acyl Total  0.22 +_ 0.18  0.90 + 0.09  0.77 + 0.08  156.9 + 40.3  0.27 + 0.09 * N.S  143.6 + 29.5 P < .005  0.90 + 0.07  *N.S 0.18 + 0.09  26.9 +• 13.0 P < .05  *P < .005 Acetyl Acyl  33.8 + 11.2  0.79 + 0.19 N.S.  0.37 + 0.10  0.24 + 0.06 N.S.  Values represents mean _+ S.D. # L - c a r n i t i n e s t a r t e d . The o v e r a l l d a i l y consumption o f c a r n i t i n e was 170 +_ 40 mg/kg. * 4 weeks versus b a s e l i n e . 8 weeks versus 4 weeks (See methods f o r experimental d e t a i l s ) .  58  unchanged. total  The r a t i o of a c e t y l c a r n i t i n e t o a c y l c a r n i t i n e and a c y l c a r n i t i n e t o  c a r n i t i n e i n the group H f l u c t u a t e d 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 that  over  similar  of the data i n Table VII w i t h those i n Table  the  i n i t i a l 4 weeks of the  changes i n both groups C and H.  increases  i n plasma f r e e c a r n i t i n e ,  L-carnitine  in  group C.  the  plasma  indicates  carnitines  underwent  L - c a r n i t i n e treatment produced large  a c e t y l c a r n i t i n e , a c y l c a r n i t i n e and t o t a l  A l l four parameters increased during the  weeks of L - c a r n i t i n e administration increased,  study,  VIII  relative  first  2  (see Table V I I I ) . Although t o t a l c a r n i t i n 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  ( a c e t y l - 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  levels  there was  between  the  no s i g n i f i c a n t d i f f e r e n c e i n muscle  three groups (see Table  IX),  total  long-chain  carnitine  acylcarnitine  l e v e l s 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 The  l i v e r s of the hyperlipidemic r a b b i t s (group  lower l e v e l s of f r e e , and  N (Table X).  H)  had  C.  significantly  short-chain a c y l and t o t a l c a r n i t i n e than both groups C  In contrast,  the l e v e l s of l i v e r long-chain a c y l c a r n i t i n e  w i t h i n t h i s group were s i g n i f i c a n t l y higher than the l e v e l s found i n groups and N. may  C  This i s s i m i l a r t o the s i t u a t i o n found f o r muscle c a r n i t i n e l e v e l s and  indicate  an  accumulation  of  long chain esters  secondary t o the development of the hyperlipidemia. carnitine  treatment  in  and  muscle  I t i s of i n t e r e s t t h a t L-  of the hyperlipidemic r a b b i t was  able t o  l e v e l s of long-chain a c y l c a r n i t i n e w i t h i n both t i s s u e s .  59  liver  normalize  the  Table IX Muscle c a r n i t i n e l e v e l s i n normal r a b b i t s (Group N), hyperlipidemic r a b b i t s (Group H) and hyperlipidemic r a b b i t s treated w i t h L - c a r n i t i n e f o r 1 month (Group C ) . n = 4 f o r each group.  Long - chain acylcarnitine (umoles/g NCP)  Total carnitine  (umoles/g NCP)  Short - c h a i n acylcarnitine (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  Group  Free c a r n i t i n e  A l l values represent the mean + S.D. P < 0.05 Group H versus Groups N,C.  60  (umoles/g NCP)  Table X L i v e r c a r n i t i n e s i n normal r a b b i t s (Group N), hyperlipidemic r a b b i t s (Group H) and hyperlipidemic r a b b i t s t r e a t e d w i t h L - c a r 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 (umoles/g NCP)  Short - chain acylcarnitine (umoles/g NCP)  Long - chain acylcarnitine (umoles/g NCP)  Total carnitine (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 Group C  4.51 + 0.74  * P < .05 2.26 + 0.06  A l l values represent the mean _+ S.D.  *  Group H versus Groups N and C. Group H versus Groups N and C.  61  W  P < .001 0.11 + 0.04  V  P < .001  6.90 + 1.10  3.9 HISTOLOGY a)  Gross V i s u a l Changes The l i v e r s of the r a b b i t s on the high f a t d i e t (Group H) and those on the  high f a t d i e t t r e a t e d w i t h c a r n i t i n e (Group C)  appeared pale y e l l o w i s h  as compared t o the r e d i s h brown colour o f the group N r a b b i t s .  brown  In addition,  the l i v e r s o f the group H animals e x h i b i t e d decreased s t r u c t u r a l i n t e g r i t y and tended t o f a l l apart q u i t e 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) s e c t i o n of l i v e r from  i n d i c a t e d normal l i v e r h i s t o l o g y . and  portal  space  group  N  rabbits  A t low power (x80) d i s t i n c t l i v e r sinusoids  ( i . e t h a t region containing  the b i l e  hepatic venule and hepatic a r t e r i o l e ) were evident.  ductule,  A t higher  terminal  magnification  (x500) hepatocytes had c e n t r a l l y placed n u c l e i w i t h rather prominent n u c l e o l i . Cytoplasm  displayed  a  combination  of granularity,  perinuclear area, and vacuolation i n other regions. cells  were  quite  especially  in  the  S i n u s o i d a l and/or Kupffer  evident as i n d i c a t e d by the elongated  nuclei  and  sparse  cytoplasm (plate 1 ) . O i l Red 0 s t a i n revealed the presence o f l i p i d deposits i n group N sections.  These  d r o p l e t s were more abundant around the p o r t a l space  liver (plate  2). In presence  accordance  with  the gross appearance,  H&E  sections  o f 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.  power,  the hepatocytes appeared swollen and vacuolated.  portal  space  were  normal except f o r the occasional  terminal venules were q u i t e n o t i c e a b l e . many  revealed  cases,  A t low  The v e s s e l s o f the  histiocyte.  The  pre-  However, the l i v e r s i n u s o i d s , and i n  the c e n t r a l v e i n were not as obvious.  62  the  A t higher  magnification  P l a t e 1: L i g h t micrograph of a s e c t i o n of l i v e r from a group N r a b b i t . S = l i v e r sinusoids. The arrow p o i n t s t o n u c l e i of e i t h e r e n d o t h e l i a l or Kupffer c e l l s . Hematoxylin-eosin, x500  63  P l a t e 2: O i l red 0 stained s e c t i o n of the l i v e r from a group N r a b b i t . Neutral l i p i d s (dark red spots) appear t o be more abundant i n the region surrounding the p o r t a l space (P). C = c e n t r a l v e i n (terminal hepatic venule). x80  64  (x500),  most hepatocytes contained several vacuoles presumably  l i p i d droplets. from  In some cases,  the n u c l e i were e i t h e r indented or d i s p l a c e d  t h e i r c e n t r a l l o c a t i o n (plate 3).  evident.  representing  S i n u s o i d a l and/or Kupffer c e l l s  However, i t could not be ascertained whether or not t h e i r  were  cytoplasm  contained any vacuoles. At  low  power,  O i l Red 0 stained l i v e r sections from group  revealed  the  4).  higher magnification (x500) i t was obvious t h a t the  At  H  animals,  presence of n e u t r a l l i p i d d r o p l e t s throughout the l i v e r  were located w i t h i n the hepatocytes  (plate 5).  lipid  (plate droplets  No e x t r a c e l l u l a r f a t deposits  were evident. Haematoxylin and e o s i n sections of l i v e r from the r a b b i t s t r e a t e d w i t h Lcarnitine  (group  appeared  t o be  Oil  Red  C) revealed the presence of f a t t y  changes.  These  changes  s i m i l i a r t o those seen i n group H. 0 s t a i n e d l i v e r sections from a l l animals i n group  C  revealed  general sparing of the areas immediately surrounding the p o r t a l space ( i . e the periportal around  the  region).  The greatest q u a n t i t i e s of l i p i d deposits were  terminal hepatic venule  microcirculatory  zone  3  (central v e i n ) .  i n the hepatic acinus of  (plate 6).  65  This region Rappaport's  located  represents model  (130)  P l a t e 3: Hematoxylin-eosin stained s e c t i o n of the l i v e r from a hyperlipidemic r a b b i t (group H). The swollen appearance of the hepatocytes i s q u i t e evident. L = n e u t r a l l i p i d d r o p l e t s . x500  66  P l a t e 4: O i l red 0 stained s e c t i o n 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 = p o r t a l space. C = c e n t r a l v e i n . x80  P l a t e 5: L i g h t micrograph of a l i v e r s e c t i o n from a hyperlipidemic (group H). I t can be seen that there are n e u t r a l l i p i d droplets (NL) the hepatocytes. O i l Red 0, x500.  68  rabbit within  P l a t e 6: O i l red O stained s e c t i o n of the l i v e r from a c a r n i t i n e - t r e a t e d hyperlipidemic r a b b i t (group C). The r a b b i t was treated with L-carnitine for 1 month. Sparing of the p e r i p o r t a l region (PR) i s q u i t e obvious. C = terminal hepatic venule. x80  69  4. DISCUSSION.  Each r a b b i t i n the study had a f a s t i n g plasma glucose determined i n order to  rule  out  the existence of diabetes,  hyperlipidemia  frequently develops.  a condition i n  which  a  secondary  The f a s t i n g glucose values obtained  for  a l l animals agreed w e l l w i t h reported values i n normal r a b b i t s (131). The  weight gain among the three groups of animals was not  significantly  d i f f e r e n t from each other, i n s p i t e of the f a c t that group N r a b b i t s tended to consume more food than the other groups. N  The higher food consumption by Group  may have been due t o the lower energy content of the chow w i t h  respect  to  the h i g h - f a t d i e t . Initially, in  order  effect  were made to induce h y p e r t r i g l y c e r i d e m i a  t o determine whether or not exogenous L - c a r n i t i n e  on V L D L - t r i g l y c e r i d e metabolism.  rabbits 0.5%  attempts  in  would  goal, and  set a t 0.5%  in  t o moderate the extreme hypercholesterolemia which u s u a l l y develops  in  In s p i t e of t h i s , contrast  while  any  groups C and H were fed a d i e t supplemented w i t h 5% corn o i l  r a b b i t s fed d i e t s supplemented w i t h 1-3%  groups  have  In an e f f o r t t o achieve t h i s  c h o l e s t e r o l (W/W). The l e v e l of exogenous c h o l e s t e r o l was  order  i n rabbits  c h o l e s t e r o l (132).  l e v e l s of plasma c h o l e s t e r o l increased d r a m a t i c a l l y i n  t o 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 C  and H had mean plasma c h o l e s t e r o l l e v e l s greater than  t r i g l y c e r i d e l e v e l s were l e s s than 100 mg/dl.  of  feeding,  1000  mg/dl,  These f i n d i n g s are  in  agreement w i t h those of other i n v e s t i g a t o r s (132-134). Rabbits severe as  hypercholesterolemia.  severe.  mechanism together  fed a c h o l e s t e r o l d i e t w i t h or without o i l ,  It for  rapidly  develop  a  The h y p e r t r i g l y c e r i d e m i a which develops i s not  i s b e l i e v e d that the absence of an  e f f e c t i v e l y regulating  whole  body  appropriate  homeostatic  cholesterol  metabolism  w i t h a very e f f i c i e n t absorption system f o r c h o l e s t e r o l renders  70  the  rabbit  very  susceptible t o the developemnt of hypercholestermia  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 is  thought t o be a secondary phenomenon.  (135).  The  hypercholesterolemia  High plasma c h o l e s t e r o l l e v e l s are  b e l i e v e d t o i n h i b i t l i p o p r o t e i n l i p a s e which i s involved i n the h y d r o l y s i s  of  VLDL-trglycerides  of  (136,137).  This  inhibition i s  o x y s t e r o l s which are d e r i v a t i v e s of c h o l e s t e r o l (55).  due t o  the  presence  Decreased h y d r o l y s i s of  VLDL-TG eventually leads t o 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 diet.  As  was expected,  plasma c h o l e s t e r o l 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 r e s p e c t i v e l y . The  large  standard d e v i a t i o n r e f l e c t s the wide v a r i a t i o n i n response t o  diet within  the  the group.  Rabbits i n group C were treated i n a manner s i m i l i a r t o that f o r group except  H  t h a t they received L - c a r n i t i n e i n t h e i r water ( d a i l y consumption 170 _+  40 mg/kg). The concentration of plasma c h o l e s t e r o l and t r i g l y c e r i d e s decreased by 35% and 50% r e s p e c t i v e l y i n group C f o l l o w i n g L - c a r n i t i n e treatment. 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 r e s u l t s support e a r l i e r reports  (51,52,76,78)  which suggest that L - c a r n i t i n e may  triglycerides  by  diverting  towards ^ - o x i d a t i o n i n the this  increased  paralleled  flux  by  of  This  mitochondria.  a c y l groups  suggestion  in  is  a  triglycerides. synthesis  doses  of  L-carnitine,  the  ^ -oxidation  based upon the assumption  secretion.  obese Zucker  rats  had s i g n i f i c a n t reductions  L-carnitine  71  system  is  levels  that  ^-  ^-oxidation.  This reduction was ascribed t o decreased hepatic  and/or  and  e s t e r i f i c a t i o n reactions  plasma ^-hydroxybutyrate  recent report by Brady e t . a l (139),  subcutaneous  cholesterol  Some workers have suggested t h a t  through  hydroxybutyrate i s a r e l i a b l e i n d i c a t o r of In  lower both  a c y l groups away from  s i g n i f i c a n t increases  (77,81,82,138).  These  was found to have  receiving in  plasma  triglyceride no  effect  on  mitochondrial In  the current study,  butyrate minutes  state 3 oxidation.  levels. (140)),  L - c a r n i t i n e had no e f f e c t on plasma ^ - h y d r o x y -  Plasma ^-hydroxybutyrate turns over very r a p i d l y ( t ^ ^ therefore  i t would  l i k e l y be  very  difficult  to  =  ^  detect  increased production by f o l l o w i n g plasma ^-hydroxybutyrate c h r o n i c a l l y . Many of the studies which reported increased plasma ^ - h y d r o x y b u t y r a t e f o l l o w i n g Lc 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 alter  plasma ^-hydroxybutyrate l e v e l s . Although  ^-hydroxybutyrate  levels  were unchanged i n the current study, i t remains t h a t the e f f e c t of L - c a r n i t i n e on  plasma t r i g l y c e r i d e may be through increased d i v e r s i o n of a c y l groups away  from  e s t e r i f i c a t i o n towards o x i d a t i o n .  The  possibility  that  L-carnitine  a f f e c t e d 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 w i t h the r e s u l t s of the k i n e t i c s t u d i e s . The mechanisms underlying the decrease i n plasma c h o l e s t e r o l are unknown. Over  the  past  carnitine  f i v e years,  lowers  (52,52,75,79).  plasma  several reports have  cholesterol  in  major  different  indicating  hyperlipidemic  that states  However, no s a t i s f a c t o r y explanation concerning the mechanism  of t h i s a c t i o n has ever been put forward. the  appeared  In the hypercholesterolemic r a b b i t ,  source of plasma c h o l e s t e r o l i s VLDL and,  secreted by the l i v e r (94-96,141,154,).  t o a l e s s e r extent  LDL  In a d d i t i o n , plasma t o t a l c h o l e s t e r o l  can be a f f e c t e d by c h o l e s t e r o l released i n t o plasma by non-hepatic c e l l s by  reverse  c h o l e s t e r o l transport)  (55).  Exogenous c h o l e s t e r o l i s  (i.e  primarily  responsible f o r the increase i n plasma c h o l e s t e r o l (135) and the i n h i b i t i o n of HMG-CoA reductase i n the unlikely  that  L-carnitine  modulating  the  ratio  synthesis.  Secretion  of of  c h o l e s t e r o l - f e d r a b b i t (142). exerts  i t s effect  on  plasma  c e l l u l a r acylCoA /CoASH and VLDL- and LDL-cholesterol  It  seems  cholesterol  thereby  and the r a t e  c h o l e s t e r o l transport were not i n v e s t i g a t e d i n t h i s study.  72  therefore  by  cholesterol of  reverse  Reverse (143)  in  cholesterol  that  transport seems t o be maintained p r i m a r i l y by  i n h i b i t i o n of t h i s enzyme leads t o  c h o l e s t e r o l transport.  a  reduction  in  LCAT  reverse  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 t h a t c a r n i t i n e may decrease plasma c h o l e s t e r o l decreasing by  the  the r a t e of synthesis and/or s e c r e t i o n of VLDL and LDL-cholesterol  liver  effective,  i n the c h o l e s t e r o l fed r a b b i t .  L-carnitine  would  For  such  mechanisms  have t o influence one or more of a  c e l l u l a r processes involved i n c h o l e s t e r o l homeostasis. include  by  to  be  number  of  Such mechanisms could  (55): a) uptake of l i p o p r o t e i n s v i a receptor-mediated pathways b)  net  uptake  of  free cholesterol  from  lipoproteins  by  lipid  cholesterol  acyl-  transfer. c) c h o l e s t e r o l  synthesis  d) e f f l u x of c h o l e s t e r o l from c e l l s v i a e)  esterification  HDL.  of c h o l e s t e r o l by a c y l  CoA:  t r a n s ferase (ACAT). f)  hydrolysis  of  cholesterol  esters,  by  neutral  cholesterol  esterase. In the c h o l e s t e r o l - f e d r a b b i t , liver  is  normal,  (144,145). in the  while  the uptake of chylomicron remnants by  the uptake of VLDL remants  is  the  severely  depressed  This decreased uptake of VLDL remnants promotes f u r t h e r  increases  plasma c h o l e s t e r o l . The k i n e t i c data (to be discussed l a t e r ) i n d i c a t e that catabolism  increased Binding  of  VLDL was  increased.  One p o s s i b l e  mechanism  catabolism could be through increased uptake of of studies  were  not  done,  and  therefore i t i s  VLDL  difficult  for  this  remnants. to  draw  conclusions w i t h respect t o the e f f e c t of L - c a r n i t i n e on VLDL remnants removal  73  by the  liver.  Increased uptake of LDL-cholesterol by p e r i p h e r a l c e l l s could a l s o r e s u l t in  decreased plasma c h o l e s t e r o l l e v e l s .  carnitine A  Such  i t i s not known whether L-  has any e f f e c t on the apoB-100/E receptor a c t i v i t y .  decrease  export  However,  i n the quantity of i n t r a c e l l u l a r c h o l e s t e r o l  could t h e o r e t i c a l l y lead t o a reduction i n plasma c h o l e s t e r o l 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  and t h a t of c h o l e s t e r o l esterase decreased. L-carnitine  However,  for  levels.  ACAT  increased  i t i s not known whether  a f f e c t s e i t h e r of these two enzymes.  Finally,  i t is  p o s s i b l e that c a r n i t i n e may  affect  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 a c i d s . of  available  total  body  cholesterol  affecting  bile  a c i d metabolism i s very l i k e l y t o  plasma  cholesterol  Since, greater than 50%  i s metabolised t o b i l e  acid,  then  eventually  any  factor  affect  plasma  cholesterol levels. The  r a t e - l i m i t i n g step i n b i l e a c i d synthesis i s the  c h o l e s t e r o l t o 7QC -hydroxycholesterol. hydroxylase  (146).  This r e a c t i o n i s catalyzed by 70(  The mechanisms and r e g u l a t i o n of subsequent reactions  the synthesis pathway are l a r g e l y unknown. the  mechanism  cleavage  which  cholestanoic  and  r e g u l a t i o n of the  produces  acid.  hydroxylation  ^ -oxidation reaction ,7 0C ,12 0(  I t i s p o s s i b l e t h a t c a r n i t i n e may  acid to cholic acid,  and  of 3QC ,7 OC  propionyl CoA  conditions i n which hepatocyte c h o l e s t e r o l l e v e l s are  expected  t h a t hepatocyte propionly-CoA l e v e l s may  acid.  administered  side-chain  5/3-  -trihydroxy-  Thus, i n  cholic  in  act t o regulate the r a t e  of ^ - o x i d a t i o n and side-chain cleavage. In the conversion  b i l e a c i d synthesis.  -  For example, l i t t l e i s known about  c h o l i c a c i d from 3<X  t r i h y d r o x y - 5 ^ -cholestanoic  of  is high,  r i s e secondary t o  ,120(-  released. i t is increased  Propionyl-CoA may be r a t e - l i m i t i n g f o r the production of  Removal  of propionate may  accelerate  this  reaction.  Orally  L - c a r n i t i n e i s known to increase the e x c r e t i o n of propionate  74  as  propionyl-carnitine conceivable  that  (cholesterol carnitine  i n p a t i e n t s w i t h propionic acidemia (147). when  the i n f l u x o f c h o l e s t e r o l i n t o  Thus, i t i s  the l i v e r  i s high  feeding) and the l e v e l s of propiony1-CoA are increased,  may  availability  decrease of free  synthesis.  The  cholesterol.  levels  o f propionly  coenzyme  net e f f e c t  A  CoA  and increasing  would  be  thereby  enhancing  the  of b i l e  acid  the r a t e  t o decrease  that L-  the a v a i l a b i l i t y  of  Further studies are required t o assess the e f f e c t s o f p r o p i o n y l -  CoA on hepatocyte 7 0< -hydroxylase and t o determine the e f f e c t s of L - c a r n i t i n e treatment on 7 o( -hydroxylase a c t i v i t y and b i l e a c i d  Plasma  carnitine  levels  were  monitored  secretion.  i n order  r e l a t i o n s h i p between plasma c a r n i t i n e and plasma l i p i d s .  t o examine  the  During the i n i t i a l 4  weeks on the d i e t , plasma f r e e , a c y l - , a c e t y l - and t o t a l L - c a r n i t i n e increased significantly  above  baseline.  b a s e l i n e throughout the study. w i t h prolonged s t a r v a t i o n  These values  remained  s i g n i f i c a n t l y above  I n t h i s regard, i t i s i n t e r e s t i n g t o note t h a t  (72 hours),  a c o n d i t i o n i n which there i s increased  f a t m o b i l i z a t i o n , increases i n serum l e v e l s o f f r e e , a c y l - and t o t a l c a r n i t i n e were  observed  medium-chain  i n r a t s (101).  I n the same study,  f a t feeding  (long- and  t r i g l y c e r i d e s ) was found t o produce an increase i n serum  acyl-  and t o t a l c a r n i t i n e and and a decrease i n f r e e c a r n i t i n e . Because o f L - c a r n i t i n e ' s r o l e  i n f a t t y a c i d metabolism,  the increase i n  plasma f r e e f a t t y a c i d l e v e l s which u s u a l l y occurs w i t h f a t feeding could lead to  an increased demand f o r L - c a r n i t i n e .  I n such s i t u a t i o n s ,  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 would be expected. carnitine and/or  The increases i n plasma  tend t o support t h i s argument and may represent increased synthesis  release  acetylcarnitine unaffected  an increase i n  by  o f c a r n i t i n e from other /  acylcarnitine  the d i e t .  tissues.  However,  and a c y l c a r n i t i n e /  total  the r a t i o s of carnitine  were  This r a i s e s the p o s s i b i l i t y t h a t the increase i n  75  plasma  total  L-carnitine  may have r e s u l t e d from increased movement of  L-  c a r n i t i n e from t i s s u e (muscle or l i v e r ) t o plasma. Carnitine  treatment produced large increases i n plasma t o t a l  carnitine,  w i t h most of the increase being due p r i m a r i l y t o an increase i n f r e e c a r n i t i n e and  secondarily  acetyl/acyl reflect  and  t o an increase i n a c e t y l c a r n i t i n e . a c y l / t o t a l were unaffected.  However,  the r a t i o s  These observations  may  d i r e c t i n t e r a c t i o n of the administered L - c a r n i t i n e w i t h the  of  simply acyl-CoA  pools.  Total  muscle  carnitine  statistically different. long-chain  levels  between  Nonetheless,  the  three  groups  were  not  i t i s i n t e r e s t i n g t o note t h a t muscle  a c y l c a r n i t i n e l e v e l s i n the f a t - f e d group were higher than i n the  L-carnitine-treated  and chow-fed groups.  Elevated l e v e l s of long-chain  acyl  c a r n i t i n e s have been previously reported i n d i a b e t i c r a t myocardium (149),  in  adipose t i s s u e of chronic uremic patients undergoing hemodialysis (84), i n the l i v e r of f a s t i n g r a t s (150)  and i n the l i v e r , adipose t i s s u e and heart of corn  o i l - f e d r a t s (150). Tissue index  of  l e v e l s of long-chain a c y l c a r n i t i n e are believed t o be a activated  long-chain  f a t t y acids ( i . e Acyl CoA's) i n that t i s s u e  acylcarnitines  varies  inversely  synthesis and d i r e c t l y w i t h /^-oxidation P-oxidation  i s favoured (e.g  of a c y l c a r n i t i n e increases. increase the  fat  w i t h the  (150).  feeding,  Thus,  fasting,  Lopaschuk e t . a l .  (149)  rate  of  enzyme would eventually  a c y l c a r n i t i n e translocase.  content fatty  i n conditions diabetes),  of acid  i n which  the  level  have suggested that  i n long-chain acyl-CoA l e v e l s t h a t occur i n f a t feeding  inhibition carnitine:  reliable  results  the in  An i n h i b i t i o n of t h i s  lead t o a build-up of long-chain a c y l c a r n i t i n e s w i t h i n  the t i s s u e s . In  the  present study,  the increased l e v e l s of muscle  76  long-chain  acyl  carnitines of  i n the f a t - f e d animals  long-chain acyl-CoA  acids.  The  transfer the  may  have been r e l a t e d t o increased  r e s u l t i n g from increased d i e t a r y intake of f r e e f a t t y of  the  carnitine:  a c y l groups t o  carnitine  combined  acylcarnitine  translocase  system  inhibition  of  contributed  t o the build-up of long-chain a c y l c a r n i t i n e .  In the treated group, acylcarnitine acylcarnitines (149).  is  with may  an have  the build-up of  long-chain  A s i m i l a r f i n d i n g has been reported f o r  long-chain  the myocardium of d i a b e t i c r a t s administered  Carnitine  acylcarnitine  L - c a r n i t i n e prevented  i n muscle. in  levels  believed t o reverse the i n h i b i t i o n of  the  carnitine: of  long-  chain a c y l c a r n i t i n e from acyl-CoA allowing f o r the regular transport of  long-  chain  translocase  L-carnitine  acylcarnitine  Additional  into  studies  are  system through the increased formation  the mitochondria and a r e t u r n t o needed t o e l u c i d a t e the  normalcy  mechanisms  accumulation of long-chain a c y l c a r n i t i n e s i n muscle  (149).  underlying  the  w i t h f a t feeding and  its  r e v e r s a l f o l l o w i n g L - c a r n i t i n e treatment  Liver  total  carnitine  decreased w i t h f a t feeding r e l a t i v e t o l e v e l s found i n the chow-fed  controls.  Carnitine  levels  of  reversed  free,  this  short-chain a c y l c a r n i t i n e and  decrease.  Long-chain  increased r e l a t i v e t o the other two groups. carnitine  may  acylcarnitines  have and  to i n h i b i t i o n  levels  were  This increase i n long-chain a c y l  been due p a r t l y t o increased partly  acylcarnitine  of  the  formation  of  carnitine:  long-chain  acylcarnitine  translocase by long-chain acyl-CoAs. The increase animals  decrease  i n l i v e r free,  i n plasma f r e e , may be  acetyl-,  r e l a t e d to  short-chain a c y l - and t o t a l c a r n i t i n e a c y l - and t o t a l c a r n i t i n e i n the  i n h i b i t o n of ^ - o x i d a t i o n and increased  of f r e e and short-chain a c y l c a r n i t i n e s from l i v e r t o plasma. oxidation  (e.g  glucose  loading) leads t o  77  decreased  fat-fed movement  Depression of  formation  of  and  -  acetyl-  carnitine inner  (150).  Short-chain f a t t y acids are able t o move f r e e l y across the  mitochondrial  membrane  (6).  Since  a  short-chain  acylcarnitine  transferase i s present i n the endoplasmic recticulum (18) then, these  esters  translocase  the l e v e l s of  i n plasma may be independent of the c a r n i t i n e : a c y l c a r n i t n e  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 r a p i d e q u i l i b r i u m w i t h the plasma pool (151) and l i v e r perfusion studies have e s t a b l i s h e d t h a t f r e e and a c e t y l c a r n i t i n e a r e r a p i d l y released by the  liver  into  acylcarnitines (12).  the blood  may  In this  (12).  Once i n the plasma,  be excreted i n u r i n e o r u t i l i s e d  free by  and  short-chain  peripheral  tissues  regard i t i s i n t e r e s t i n g t o note t h a t high l e v e l s o f L-  c a r n i t i n e and a c e t y l c a r n i t i n e have been found i n the b r a i n where they may play a  c r u c i a l r o l e i n the formation o f a c e t y l c h o l i n e and i n the r e g u l a t i o n of "Y -  aminobutyrate l e v e l s i n cerebro-spinal f l u i d (152). acetylcarnitine  The continued movement of  from l i v e r t o plasma together w i t h the decreased formation o f  short-chain e s t e r s i n the l i v e r (due t o the i n h i b i t i o n of j2 -oxidation)  could  e v e n t u a l l y lead t o d e p l e t i o n o f l i v e r L - c a r n i t i n e s t o r e s . The simply  In  fact reflect  t h a t the t r e a t e d group had normal l i v e r c a r n i t i n e  may  a replenishment of l i v e r c a r n i t i n e s t o r e s .  the hypercholesterolemic  cholesterol  levels  rabbit,  a  large  percentage  o f plasma  and t r i g l y c e r i d e s are c a r r i e d p r i m a r i l y by VLDL p a r t i c l e s having  beta m o b i l i t y (154).  LDL c a r r i e s a smaller percentage o f the c h o l e s t e r o l i n  t h i s perturbed s t a t e (96).  With t h i s i n mind,  various VLDL parameters were  examined t o determine how they would be a f f e c t e d by L - c a r n i t i n e 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 o f exposure t o the high f a t diet. fold.  VLDL-cholesterol  had the l a r g e s t change,  VLDL-TG increased by 4 f o l d . 78  i n c r e a s i n g by more than  200  I n the second 4 weeks o f the experiment,  plasma VLDL-cholesterol and V L D L - t r i g l y c e r i d e doubled. This was r e f l e c t e d i n a doubling  o f plasma  total  cholesterol  and t r i g l y c e r i d e s .  Plasma  c h o l e s t e r o l decreased by 50% i n the L - c a r n i t i n e - t r e a t e d group. was p a r a l l e l e d by a 40% decrease i n plasma c h o l e s t e r o l . noted  This  decrease  A s i m i l a r p a t t e r n was  f o r plasma V L D L - t r i g l y c e r i d e s and t o t a l t r i g l y c e r i d e s .  indicate  VLDL-  These  t h a t i n the hypercholesteremic r a b b i t changes i n plasma  results  cholesterol  and t r i g l y c e r i d e s are not s o l e l y due t o changes i n VLDL composition. The diet,  VLDL-ApoB  and t o t a l p r o t e i n l e v e l s which were elevated by  decreased f o l l o w i n g L - c a r n i t i n e treatment.  protein data  the f a t  The decrease i n VLDL t o t a l  achieved s i g n i f i c a n c e whereas the r e d u c t i o n i n apoB d i d not. These  indicate  that  other VLDL associated p r o t e i n s , i n a d d i t i o n t o apoB,  decreased. A  number  o f s t u d i e s (91,155) have reported increased  plasma l e v e l s  HDL-cholesterol i n the hypercholesterolemic r a b b i t s ,  w h i l e others have  HDL-cholesterol  t o be  (156).  study,  the h i g h - f a t d i e t nor L - c a r n i t i n e treatment  neither  unaltered (94,96) o r reduced  I n the  of  found present  a f f e c t e d plasma  HDL-cholesterol.  Agarose  g e l e l e c t r o p h o r e s i s of plasma revealed  t h a t the h i g h - f a t  produced  increased width and i n t e n s i t y o f the beta and pre-beta bands  decrease  i n the s i z e and i n t e n s i t y o f the 0(-band.  treatment, decreased, rabbits of  the width  intensity  of t h i s  but the 0<-band was not a f f e c t e d .  and a  Following L - c a r n i t i n e  broad  beta/pre-beta  I t i s now known t h a t  band feeding  a d i e t supplemented w i t h c h o l e s t e r o l and f a t leads t o the appearance  VLDL withyS-mobility  from  pre-beta  VLDL  particles  particle  and  diet  was  i . e ^-VLDL (96,154).  This a l t e r a t i o n i n m o b i l i t y  t o beta i s due t o changes i n the s i z e and composition (96). markedly  I n the present study,  the composition  a l t e r e d by the the high d i e t .  79  o f the  o f the VLDL  The appearance  of a  "broad" beta band may be explained p a r t l y i n terms o f the changes i n the l i p i d composition o f these VLDL p a r t i c l e s . The reason f o r the d e c l i n e i n i n t e n s i t y o f the 0<-band (implying reduced HDL) i s not c l e a r . The f i n d i n g of decreased i n t e n s i t y of the 0<-band seem t o c o n t r a d i c t the observation t h a t HDL-cholesterol remained unchanged during course  o f the current study.  decreased  HDL  staining  hypercholesterolemic ultracentrifugation presence  upon  plasma.  polyacrylamide  The  gel  observation may be  and e l e c t r o p h o r e s i s  0<-band.  o f an  Other i n v e s t i g a t o r s (96,156)  o f the "HDL"  have  reported  electrophoresis artifactual fraction  the  of  i n that  showed the  I n a d d i t i o n , normal l e v e l s o f HDL were  found  on  chemical a n a l y s i s .  In r a b b i t s f e d the high f a t d i e t , VLDL-TG  was  transport  significantly  rate  the f r a c t i o n a l c a t a b o l i c r a t e (FCR) of  reduced r e l a t i v e t o the chow-fed  o f VLDL-TG out o f the plasma  production r a t e under steady-state conditions) by  the d i e t .  inhibitory  compartment  r a b b i t s . The  (which  represents  was not s i g n i f i c a n t l y a f f e c t e d  The decrease i n the FCR o f VLDL-TG would be expected given the  e f f e c t o f i n c r e a s i n g plasma c h o l e s t e r o l  l i p a s e (136,137).  levels  on l i p o p r o t e i n  I n h i b i t i o n of l i p o p r o t e i n l i p a s e would decrease  and lead t o a reduction i n the c a t a b o l i c r a t e o f VLDL-TG.  lipolysis,  The lack o f e f f e c t  of the d i e t on VLDL-TG t r a n s p o r t r a t e can be accounted f o r on the b a s i s o f the increased VLDL-TG pool s i z e found i n the hyperlipidemic r a b b i t s . This r e s u l t s in  t r a n s p o r t r a t e s (transport r a t e = FCR x pool size) which are c l o s e t o , o r  higher than, those i n chow f e d r a b b i t s . L-carnitine  treatment  rabbits,  indicating  plasma.  This  different  increased the FCR of VLDL-TG  i n hyperlipidemic  t h a t VLDL-TG was being c l e a r e d a t a f a s t e r r a t e from the  increase  r e s u l t e d i n a mean FCR which was  from t h a t f o r the chow-fed animals.  80  not  statistically  This increase i n FCR may  have  been  due  t o increased l i p o p r o t e i n  treatment.  L-carnitine  lipoprotein  lipase  subsequent few  have  have  activity  directly  or lipoprotein  lipase  (78,157)  suggest that  following  modulated  activity  t o the decrease i n plasma c h o l e s t e r o l .  studies  releasable  may  lipase  L-carnitine  the a c t i v i t y  may  have  f i n d i n g no e f f e c t (139,148).  of  increased  Although the r e s u l t s of a therapy  increases  lipase a c t i v i t y (i.e lipoprotein lipase a c t i v i t y ) ,  reported  L-carnitine  other  heparin studies  A previous study i n r a b b i t s  has  demonstrated t h a t t h a t l i p o p r o t e i n l i p a s e i s i n h i b i t e d i n the presence o f high l e v e l s o f plasma c h o l e s t e r o l in  the present  secondarily The  study,  (136). I t thus seems appropriate t o suggest t h a t ,  lipoprotein  activity  may  have  increased  t o the decrease i n plasma c h o l e s t e r o l l e v e l s .  transport r a t e o f VLDL-TG was not s i g n i f i c a n t l y a l t e r e d f o l l o w i n g L-  carnitine  treatment.  Since transport r a t e i s equal t o the production  under steady-state c o n d i t i o n s , e f f e c t on VLDL-TG synthesis. doses  lipase  of L-carnitine  significant  these r e s u l t s i n d i c a t e t h a t L - c a r n i t i n e had no Brady e t .  a l . (139) reported t h a t subcutaneous  (administered f o r 2 t o 3 months) were found t o produce a  reduction i n plasma t r i g l y c e r i d e l e v e l s and hepatic  triglyceride  s e c r e t i o n i n female obese zucker r a t s , w h i l e o r a l doses were i n e f f e c t i v e . differences study  rate  The  between the r e s u l t s o f that study and the r e s u l t s o f the present  may be a f u n c t i o n of d i f f e r e n c e s i n the methodology used f o r  hepatic t r i g l y c e r i d e s e c r e t i o n ,  assessing  species d i f f e r e n c e s and/or d i f f e r e n t route o f  admininistration of L-carnitine. The  fractional  catabolic  rate  o f VLDL-ApoB was  hyperlipidemic r a b b i t s r e l a t i v e t o the chow-fed r a b b i t s . been reported by others (158,159). a  decreased  in  the  S i m i l a r r e s u l t s have  This decrease i n FCR i s thought t o r e f l e c t  decreased catabolism o f the 'abnormal VLDL which i s secreted by the r a b b i t 1  l i v e r i n the hypercholesteremic s t a t e .  On the other hand, the transport  rate  of VLDL-ApoB was moderately elevated i n the hyperlipedemic r a b b i t compared t o  81  normals  despite  likely  the reduced FCR.  As i n the case o f VLDL-TG,  t h i s i s most  due t o large pool s i z e s (determined from steady s t a t e masses o f VLDL-  ApoB) i n the hyperlipidemic r a b b i t s (160). The FCR f o r VLDL-ApoB was unaffected by L - c a r n i t i n e treatment. there  tended  t o be a decrease i n the VLDL production r a t e  treatment,  this  decrease  unaffected,  then  this  failed  moderate  t o reach decrease  that  the t r a n s p o r t  rate  hyperlipidemic  rabbits.  160)  be  and may  with L-carnitine  significance.  Since  i n the production  a t t r i b u t e d t o a decrease i n the VLDL-apoB pool s i z e . f o r VLDL-ApoB was  Although  rate  FCR  was  can be  I t should be pointed out  similar  i n the normals and  This f i n d i n g i s contrary t o previous reports (158-  a r e s u l t of abnormally  high  fractional  c a t a b o l i c rates  c a l c u l a t e d f o r the normal r a b b i t s . The FCRs  r e s u l t s of the k i n e t i c studies i n d i c a t e t h a t f a t feeding reduces the  o f VLDL-ApoB and VLDL-TG, whereas the production of these two  are unaffected. no  effect  on  unaffected. VLDL-apoB been  L - c a r n i t i n e treatment normalised the FCR o f VLDL-TG, but had the FCR o f VLDL-ApoB.  The transport  o f both  moieties  was  This seemingly d i f f e r e n t i a l e f f e c t o f L - c a r n i t i n e on VLDL-TG and metabolism i s not unexpected i n t h a t ,  shown  VLDL-TG  moieties  that  and t h a t  a t l e a s t i n humans,  the metabolism o f VLDL-ApoB i s d i s a s s o c i a t e d from  i t has that of  changes i n the rates o f s e c r e t i o n o r catabolism of one  not be accompanied by changes i n the other  may  (176).  Fat-feeding lead t o the development o f a hepatic s t e a t o s i s ( f a t t y 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 . did in  not reverse the s t e a t o s i s , the p e r i p o r t a l area.  L - c a r n i t i n e treatment  but i t d i d reduce the d e n s i t y o f f a t deposits  These f i n d i n g s are s i m i l i a r t o those found  e a r l i e r study by Seccombe e t .  a l (85).  82  i n an  I n t h a t study, 16 weeks o f L-carnitne  therapy  d i d not reverse the hepatic s t e a t o s i s , but there was a reduction  in  l i p i d deposits w i t h i n the l i v e r . The  f a t t y l i v e r s developed i n the f a t - f e d r a b b i t s may have r e s u l t e d from  increased production o f VLDL by the l i v e r .  Kroon e t a l (161) have  t h a t (2 -VLDL i n plasma o f the f a t - f e d r a b b i t s was o f hepatic  reported  than o f i n t e s t i n a l ,  origin.  rather  The ^5-VLDL t h a t i s produced i s c h o l e s t e r o l - r i c h  and i s poorly c a t a b o l i z e d by l i p o p r o t e i n l i p a s e . poorly  recently  I n a d d i t i o n i t appears t o be  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 f a t - f e d r a b b i t s could have r e s u l t e d mainly from over production  o f ^ -VLDL  rather  than  from  uptake  of previously  secreted  lipoproteins. An increase i n V L D L - t r i g l y c e r i d e synthesis could have a l s o contributed t o the  f a t t y l i v e r since f a t  triglyceride  feeding has been shown t o lead t o increased  l e v e l s i n r a t s (148).  found t o prevent t h i s  liver  I n t e r e s t i n g , exogenous L - c a r n i t i n e  was  increase.  The hepatocytes i n the p e r i p o r t a l region (microcirculatory zone 1 of the hepatic acinus) are a t higher oxygen tension and are m e t a b o l i c a l l y more a c t i v e (130). a  I t i s p o s s i b l e t h a t these c e l l s may be capable of s e c r e t i n g f!> -VLDL a t  higher  situations  rate.  They  where  therefore do not accumulate these p a r t i c l e s except i n  the i n f l u x o f c h o l e s t e r o l i s so high  w i t h i n the c e l l a t a f a s t e r r a t e than i t can be exported. periportal  that  i t accumulates  The sparing o f the  area i n l i v e r sections from t r e a t e d r a b b i t s could therefore be the  r e s u l t s o f higher VLDL s e c r e t i o n r a t e s . Alternatively,  the observed sparing of the p e r i p o r t a l area could  be  a  r e f l e c t i o n of a metabolic gradient o f d i s t r i b u t i o n of administered L - c a r n i t i n e in  the l i v e r ,  i n nutrients. this  zone  since i t i s known t h a t zone 1 hepatocytes r e c e i v e blood r i c h e r Because o f the higher oxygen tension,  the mitochondria  may be able t o respond t o L - c a r n i t i n e treatment w i t h  83  within  enhanced  &-  oxidation.  84  5. CONCLUSIONS  The f o l l o w i n g conclusions can be drawn from the r e s u l t s of t h i s study:  1)  In  rabbits  f e d the h i g h - f a t  diet,  plasma  total  cholesterol,  and  t r i g l y c e r i d e s , c h o l e s t e r o l , apoprotein B and t o t a l p r o t e i n associated w i t h 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 HDL-cholesterol  2)  in  and plasma t r i g l y c e r i d e s .  The f r a c t i o n a l c a t a b o l i c r a t e f o r V L D L - t r i g l y c e r i d e s 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 f r e e c a r n i t i n e , a c e t y l c a r n i t i n e , a c y l c a r n i t i n e and total levels  c a r n i t i n e increased w i t h the h i g h - f a t d i e t . were increased,  Although plasma c a r n i t i n e  the r e l a t i v e percentage of a c e t y l - and  acylcarnitine  w i t h i n the plasma pool were unchanged.  4)  The h i g h - f a t 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 a c y l c a r n i t i n e s . On the other hand, the d i e t caused l i v e r f r e e c a r n i t i n e , short-chain a c y l c a r n i t i n e and t o t a l c a r n i t i n e t o be s i g n i f i c a n t l y reduced.  5)  L-carnitine  treatment of the hyperlipidemic r a b b i t  produced  significant  reductions i n plasma concentrations of t o t a l c h o l e s t e r o l , t r i g l y c e r i d e s , VLDLtriglycerides,  VLDL-cholesterol  and VLDL t o t a l p r o t e i n .  I t had no e f f e c t on  plasma HDL-cholesterol.  6) L i v e r and s k e l e t a l muscle c a r n i t i n e l e v e l s 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 c a r n i t i n e f r a c t i o n s w e l l above those seen i n the  hyperlipidemic untreated animals,  the r e l a t i v e percentage of a c e t y l  and  a c y l e s t e r s w i t h i n the plasma pool remained unchanged.  7)  The  f r a c t i o n a l c a t a b o l i c r a t e of  values w i t h L - c a r n i t i n e treatment.  V L D L - t r i g l y c e r i d e s returned t o  control  Treatment had no e f f e c t on VLDL-apoprotein  B kinetics.  8)  On  plasma  the b a s i s of these r e s u l t s , i t was concluded t h a t triglycerides i n  the  hyperlipidemic  rabbit  the  reduction  following  L-carnitine  treatment was due t o an increase i n the catabolism of V L D L - t r i g l y c e r i d e s .  86  in  APPENDIX A  Calculation  o f the F r a c t i o n a l Catabolic Rate (FCR) f o r monoexponentia1 decay:  A p p l i c a t i o n t o the determination o f FCR  A  f o r VLDL-TG  t y p i c a l VLDL-TG t r a c e r curve obtained f o l l o w i n g the i n j e c t i o n o f  glycerol  into  normal  r a b b i t s i s presented i n f i g . 13.  resolved i n t o 3 phases.  An i n i t i a l r a p i d r i s e ,  H-  The curve may  be  an e a r l y 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  shown f o r the r a b b i t s (125,162).  to  that  Moreover, i t has been shown t h a t t h i s decay  curve may be approximated t o " f i i a t o f a monoexponential curve since the t a i l o f the  curve i s not considered t o contribute  s i g n i f i c a n t l y t o the magnitude  the o v e r a l l c a t a b o l i c r a t e o f VLDL-TG (125,162).  In t h i s instance,  r a p i d d e c l i n e phase i s used i n the estimation o f FCR. the ^1/2  of  only the  The h a l f - l i f e ( t ^ ^ ) °^  decay o f 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 ^  e  f  r a c  t i o n a l c a t a b o l i c r a t e was estimated  d e r i v a t i o n o f the r e l a t i o n s h i p between  a n d  t h e  (125). rcR  ^  s a s  The mathematical follows.  Let A = Quantity o f r a d i o a c t i v i t y i n i t i a l l y i n j e c t e d . The decay, dA/dt, i s proportional t o A o r dA/dt  = -kA  Separating the v a r i a b l e s gives dA/A = -kdt and by i n t e g r a t i o n we get In A = - Kt + C.  (1)  where C i s a constant and In i s the n a t u r a l At t = 0, In A  A = A Q  Q  and therefore  = C  87  logarithm.  O  2  6  A-  TIME  8  10  (hrs)  F i g . 13: T y p i c a l VLDL-TG t r a c e r curve obtained f o l l o w i n g the i n j e c t i o n of Hg l y c e r o l i n t o normal r a b b i t s . The curve may be resolved i n t o three phases: an i n i t i a l r a p i d r i s e , an e a r l y f a s t decay followed by a slow decay phase.  88  Thus equation (1) becomes In A/A  Q  =  - k t or  At time " 2 / 2 ' t  t  a  =  A  In A /A Q  Q/2  =  kt  (2)  and upon s u b s t i t u t i o n i n t o equation (2), we  w i l l obtain In 2 = k . t In  (3)  1 / 2  equation (3), k represents the f r a c t i o n a l c a t a b o l i c r a t e  (FCR) f o r  monoexponential decay (125). As a r e s u l t the equation may be r e w r i t t e n as FCR = In 2 / t  t^  1  /  (4)  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  2  curve  by v i s u a l i n s p e c t i o n and the FCR may then be estimated using equation (4). Alternatively,  a  plot  o f In A versus time w i l l have a slope of k (see  equation 2 ) . This value the to  method o f estimating the FCR may o c c a s i o n a l l y underestimate the FCR  because o f the e r r o r involved i n f i t t i n g the curve by eye and because t a i l of the VLDL-TG  the FCR  i n d i f f e r e n t disease c o n d i t i o n s  d e t a i l e d methods, for  curve i s now known t o c o n t r i b u t e t o v a r y i n g  namely multicompartmental  (162,163,164).  a n a l y s i s (164),  the e s t i m a t i o n o f the FCR o f VLDL-TG catabolism,  methods  degrees  In f a c t ,  are now a v i l a b l e  but the use o f  a r e outside the scope o f t h i s study and w i l l not be  more  discussed.  these At  t h i s stage, i t should be pointed out t h a t the shape o f the VLDL-TG decay curve may vary from i n d i v i d u a l t o i n d i v i d u a l (animal t o animal).  I n some cases, as  i n hypercholesterolemic r a b b i t s , two d i s t i n c t decay phases may not be evident. However,  regardless o f the shape o f the curve, the c a l c u l a t i o n o f FCR remains  the same.  89  APPENDIX B  Analysis  o f Blood  L e v e l data i n a Two-Pool  system:  Calculation  of  curve  I-VLDL-ApoB i n plasma g e n e r a l l y conforms  to a  parameters.  125 The  disappearance  of  biexponential f u n c t i o n .  As a r e s u l t ,  the two pool model of Gurpide  (127) i s u s u a l l y used t o describe the metabolism of VLDL-ApoB. developed  these  These authors  a s e r i e s o f mathematical formulae f o r estimating the r a t e constants  (including FCR's), of  et. a l  production r a t e s and pool s i z e s i n two-pool systems. A l l  formulae are functions o f c e r t a i n parameters of the decay  Therefore,  the curve  a n a l y s i s method w i l l be b r i e f l y summarised  curve. i n this  section. 125 A  typical  curve  f o r the decay 125  f o l l o w i n g the i n j e c t i o n o f 14.  As shown,  of  I-VLDL-ApoB  specific  activity  I-VLDL i n t o chow-fed r a b b i t i s presented i n f i g .  the disappearance curve can be resolved i n t o two  functions w i t h r a t e constants OC and  respectively.  exponential  The f i r s t exponential ( o<  * - l i n e ) i s obtained by "curve-peeling" (curve s t r i p p i n g ) the  second  exponential  corresponding values  versus  value time  will A  from the  A p l o t of the new  y i e l d a l i n e w i t h slope equal t o - OC and  a  y-  The slope of the second exponential has a value o f -y3  y - a x i s i n t e r c e p t o f Cg. 0 < , ^ ,  C^ and Cg may then be used t o estimate the  value o f the FCR as described i n the t e x t .  *  The value of  i s subtracted  o f the i n i t i a l phase of the curve.  i n t e r c e p t equal t o C . and  ( ^ - l i n e ) a t a given time  (127).  see Appendix C f o r d e f i n i t i o n  90  1ooooo  210000 Q_ Q  5 -< O  G:  o  QJ Q_ OO  1000  100  +  4  6  TIME  (hrs)  125, F i g . 14: T y p i c a l 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 o f IVLDL-apoB f^pm the plasma of normal r a b b i t s f o l l o w i n g the i n j e c t i o n o f autologous I-VLDL.  91  APPENDIX C KINETIC DEFINITIONS  i)  Compartment a)  An anatomical,  system,  throughout  physiological,  which  chemical or p h y s i c a l s u b d i v i s i o n of  the r a t i o of concentration of t r a c e r t o tracee  a is  uniform a t any given time (165). b)  A  s u b d i v i s i o n of a model used f o r  the  a n a l y s i s of  experimental  observation (165).  ii)  Pool The t o t a l amount of substance i n a system or subsystem (165).  iii)  Steady-state The  c o n d i t i o n i n which the amount of tracee (or of t r a c e r )  over the d u r a t i o n of the experiment  (iv)  F r a c t i o n a l C a t a b o l i c Rate The  is  constant  (165).  (FCR)  f r a c t i o n a l c a t a b o l i c r a t e 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 t o leave t h a t compartment without ever r e t u r n i n g (166). of  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  m a t e r i a l present t h a t i r r e v e r s i b l y leaves a given compartment without ever  returning.  (v) The  Transport  Rate (165-167)  amount of m a t e r i a l c r o s s i n g a boundary per u n i t time or more  the r a t e a t which m a t e r i a l leaves a compartment i r r e v e r s i b l y .  92  precisely  I t i s given by  the  product  of the steady-state  mass (M^) and the f r a c t i o n a l c a t a b o l i c  I t i s a l s o sometimes known as the i r r e v e r s i b l e d i s p o s a l r a t e (IDR),  rate  metabolic  r a t e (MR) o r c a t a b o l i c r a t e , CR. In quantity  steady s t a t e ,  any i r r e v e r s i b l e l o s s must  be  replaced,  thus,  this  a l s o represents the r a t e of entry of new (nonrecycled) material i n t o  t h a t compartment and i s r e f e r r e d t o as the production r a t e .  (vi) Curve-peeling (Curve s t r i p p i n g ) A  process  of  resolving  a  curve  into  the sum  of  its  individual  exponentials by s e q u e n t i a l l y e x t r a c t i n g the i n d i v i d u a l exponentials (166).  93  Appendix D  An I n v e s t i g a t i o n o f L - c a r n i t i n e Treatment i n Hypertriglyceridemia: R e s u l t s of studies i n Yucatan M i n i p i g s  Originally  the c u r r e n t study was designed t o examine the e f f e c t  o f L-  c a r n i t i n e treatment on h y p e r t r i g l y c e r i d e m i a i n the Yucatan miniature p i g . The pig  was  s e l e c t e d as the model o f choice because i t has been shown t o be an  e x c e l l e n t animal model of  the blood,  Yucatan  f o r the study o f many human d i s o r d e r s i n c l u d i n g  cardiovascular system and those o f l i p i d metabolism  those  (90,168).  miniature p i g s were chosen on the b a s i s of the r e l a t i v e l y small  size  of the a d u l t and because o f t h e i r r e l a t i v e d o c i l i t y (169). On model,  account o f the d i f f i c u l t i e s experienced i n working w i t h t h i s and the low r a t e of success of the v a r i o u s procedures,  t o search f o r an a l t e r n a t i v e model. of  animal  i t was decided  I n t h i s s e c t i o n o f the t e x t , the progress  the work w i t h the Yucatan minipigs,  the d i f f i c u l t i e s experienced and the  data c o l l e c t e d w i l l be b r i e f l y summarised.  Animals Five originally  adult  male  obtained  Yucatan  from  Miniature pigs (2.5 - 4 years  the Swine  Research  Center  old) were  a t Colorado  State  U n i v e r s i t y . One animal succumbed t o hypothermia w h i l e another had t o be k i l l e d because  o f systemic  infection  which  developed  following  blood  c a t h e t e r i z a t i o n . Autopsy revealed t h a t a l o c a l i n f e c t i o n had developed the c a t h e t e r and tracked i n t o the c i r c u l a t i o n (sinus t r a c k i n g ) .  94  vessel around  Handling o f Animals A  Panepinto S l i n g (169) obtained from the Colorado Swine Research Center  was used f o r r e s t r a i n i n g the pigs during a l l r o u t i n e procedures.  Implantation o f Venous Catheters Initially ID,  a  o f 0.04 i n . ,  rostral  60 cm  silastic  outer diameter,  l i n e (Medical Grade; OD,  internal  diameter,  o f 0.085 in.) was implanted i n t o  v e i n o f the a u r i c u l a r h e l i x o f each p i g .  the  The l i n e was advanced 20 cm  i n t o the v e s s e l such t h a t the t i p was located i n the m a x i l l a r y v e i n . The l i n e s were supplied by the Dow Corning Corporation, Midland, Michigan, USA. S i l a s t i c tubing was found t o be unsuitable f o r long term sequential blood sampling  because  the l i n e s o c c a s i o n a l l y c o l l a p s e d during the withdrawal  of  blood and some l i n e s ruptured 2 weeks t o one month f o l l o w i n g implantation i n t o the p i g s .  I t was therefore decided t o t r y Tygon Microbore tubing (ID .04 i n . ,  OD .07 i n . , w a l l .015 in) which was p r e v i o u s l y found t o be much more and  more  s u i t a b l e f o r long term implantation  into  these  pigs  durable (personal  communication: Dr. J i m T e r r i s , Armed Forces U n i v e r s i t y of the Health Sciences, Bethesda,  Maryland.).  Due  larger  tygon  future  catheterizations.  t o frequent blockage of the Microbore  tubing (ID 0.0625 i n , OD 0.125 in) was The  tygon  tubings  were  finally  lines,  a  selected f o r  purchased  from  Norton  P l a s t i c s , Cleveland, Ohio, USA. To reduce the incidence o f sinus t r a c k i n g , a v e l o r c u f f was glued around all  l i n e s and p o s i t i o n e d approximately 1.0 t o 2.0 inches below the s k i n when  the  l i n e was implanted  were  The l i n e s were gas - s t e r i l i z e d and a l l  implanted i n a s t e r i l e environment w h i l e the animals were under  anesthesia. the  (170).  On  lines general  account of the small s i z e of the r o s t r a l v e i n of the ear and  s i z e o f the l i n e which were f i n a l l y s e l e c t e d , 95  the l i n e s  were  implanted  into  the  external  interscapular week  before  precautions  jugular  vein  region of the neck. proceeding taken,  with  and  tunnelled  subcutaneously  to  the  Each animal was allowed t o recover f o r one  the  study.  However,  in  spite  of  i n c l u d i n g d a i l y c l e a n s i n g of the wounds w i t h  all  the  antiseptic  s o l u t i o n , 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 w i t h 4% sodium c i t r a t e s o l u t i o n . Heparin  was  not used because i t i s known t o release the  lipase  which  w i t h the study.  enzyme l i p o p r o t e i n  hydrolyzes VLDL-triglycerides (171) and thus I t was found t h a t despite d a i l y f l u s h i n g ,  would  interfere  l i n e patency could  not be e f f e c t i v e l y maintained due t o leaching of the c i t r a t e . To overcome t h i s problem,  i t was  decided t o switch t o an 8% s o l u t i o n of sodium c i t r a t e i n 50%  dextrose  (172).  The  ' f l u s h i n g s o l u t i o n ' was  Children's and Grace Pharmacy, Vancouver, B.C.  prepared by the  Shaughnessy,  Since t h i s s o l u t i o n i s viscous  leaching of the c i t r a t e was minimized. The l i n e s were flushed as f o l l o w s : a) the 8.0%  sodium c i t r a t e / 50 % dextrose s o l u t i o n was removed.  b) the l i n e s were flushed w i t h 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 w i t h a known volume of the 8.0% 50  %  dextrose.  sodium c i t r a t e /  This volume was c a l c u l a t e d on the b a s i s of the  length  and  i n t e r n a l diameter of the implanted l i n e . In always the  many cases blood could not be withdrawn from the l i n e s , p o s s i b l e t o i n j e c t s o l u t i o n w i t h l i t t l e pressure.  t i p of the catheter (cut a t an acute angle to  rested  but  was  This i n d i c a t e d t h a t  facilitate  implantation)  against the w a l l of the v e s s e l c r e a t i n g a v a l v e - l i k e a c t i o n  negative pressure was a p p l i e d .  it  whenever  To minimize future occurrence of t h i s problems  96  the proximal t i p o f the l i n e s were c u t b l u n t l y p r i o r t o implantation. In f a c t , t h i s l a t t e r a c t i o n seemed t o increase the l i n e patency p e r i o d from a couple o f weeks t o a couple of months.  Diet. For normotriglyceridemic s t u d i e s , the pigs were maintained on a d u l t swine ration  (14% p r o t e i n ,  70.7% carbohydrates  (mainly s t a r c h ) , 3.5%  fats,  3.5%  vitamin-mineral mix and 8% f i b e r ) supplied by B u c k e r f i e l d ' s , Abbortsford, B.C. For  hypertriglyceridemic studies,  isonitrogenous carbohydrates was  prepared  Bethesda,  diet  the pigs were maintained on an i s o c a l o r i c ,  containing 40% sucrose (14% p r o t e i n ,  (50% sucrose),1.2% f i b r e s , from crushed corn,  1.8%  f a t , 81.3%  3.5 vitamin-mineral mix).  p u r i f i e d casein  (Biochemical  The  diet  Corporation,  MD) sucrose c r y s t a l s and the U n i v e r s i t y o f B r i t i s h Columbia (U.B.C)  vitamin-mineral premix A (Animal Sciences Dept. U.B.C).  Methodologies The carnitine Serum  various  are d e t a i l e d i n the M a t e r i a l s and Methods s e c t i o n  electrolytes  Ektachem  methods employed f o r the measurement of plasma  700  and  serum l e v e l s o f l i v e r enzymes were  of  lipids this  and text.  measured on  machine using k i t s supplied by the manufacturer (Eastman  an  Kodak  Co. Rochester, N.Y. USA).  P r e l i m i n a r y Data Plasma (Table  levels  o f e l e c t r o l y t e s and f a s t i n g glucose  (Table  XI), lipids  XII) and l i v e r enzymes (Table XIII) were w e l l w i t h i n the normal  range  of values f o r a d u l t pigs (173). However, f a s t i n g l e v e l s o f c h o l e s t e r o l (58.0 -  97  69.0  mg/dl),  43.0  mg/dl)  triglycerides  triglycerides were =  lower 50  (12.0 - 32.0 mg/dl) and HDL-cholesterol  than i n humans  - 260  mg/dl and  (Cholesterol  =  HDL-cholesterol =  (35.0 -  150  - 270  mg/dl,  35.0  - 70.0  mg/dl  respectively). 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 (LPE) i n d i c a t e d t h a t the p i g s ' VLDL,  LDL and  HDL had the same m o b i l i t y 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 /acylcarnitine  serum and  total  L - c a r n i t i n e and  the r a t i o s  of a c e t y l c a r n i t i n e  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 t o be  lower  in  Yucatan Miniature pigs than i n Humans (Table XIV). Initially  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 t o be no e f f e c t on plasma  lipids  (data not shown).  The pigs were then switched t o a high f a t  diet  (containing 20% safflower o i l )  because i t was p r e v i o u s l y reported  this  d i e t produced hyperlipidemia i n domestic pigs (174,175).  The p i g s  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  that were  monitored.  Plasma l i p i d s were a l s o unaffected by t h i s d i e t . On miniature  the b a s i s p i g was  o f these f i n d i n g s , i t s was  inferred  that  the Yucatan  e i t h e r very r e s i s t a n t t o the development o f d i e t a r y  induced h y p e r t r i g l y c e r i d e m i a using the methods employed i n t h i s study feeding p e r i o d was too short. The model was abandoned.  98  -  o r the  Table XI Plasma e l e c t r o l y t e s and glucose i n a d u l t male Yucatan miniature pigs were f a s t e d f o r 24 hours p r i o r t o 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  Calcium  (mM)  7.4 - 8.3  - 101  Phosphorus (mg/dL)  5.8  Anion gap  26.1 - 33.4  Glucose  (mg/dL)  59 - 65  99  pigs.  The  Table X I I Plasma l i p i d values i n a d u l t male Yucatan miniature p i g s . f o r 24 hours p r i o r t o 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 a c i d  (uEq/L)  202 - 372  HDL-cholesterol  (mg/dL)  35.0 - 43.0  100  Animals were f a s t e d  Table X I I I  Serum l e v e l s of glutamate - oxalate transaminase (GOT), c r e a t i n e kinase (CK), albumin, and t o t a l p r o t e i n i n a d u l t male Yucatan miniature p i g s . The animals were f a s t e d f o r 24 hours p r i o r t o blood c o l l e c t i o n . Concentration  Parameter 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 f r e e L - c a r n i t i n e ( f r e e ) , a c y l c a r n i t i n e ( a c y l ) , a c e t y l c a r n i t i n e (acetyl) and t o t a l L - c a r n i t i n e (total) i n a d u l t male Yucatan miniature pigs (n = 3) and i n normal male human subjects. The pigs were f a s t e d f o r 24 hours w h i l e the human subjects were f a s t e d f o r 12 hours. 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