Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The carnitine and carnitine ester content of rat bile and human duodenal fluid Hamilton, Jennifer Jane 1986

You don't seem to have a PDF reader installed, try download the pdf

Item Metadata

Download

Media
[if-you-see-this-DO-NOT-CLICK]
UBC_1986_A6_7 H35.pdf [ 4.74MB ]
Metadata
JSON: 1.0096708.json
JSON-LD: 1.0096708+ld.json
RDF/XML (Pretty): 1.0096708.xml
RDF/JSON: 1.0096708+rdf.json
Turtle: 1.0096708+rdf-turtle.txt
N-Triples: 1.0096708+rdf-ntriples.txt
Original Record: 1.0096708 +original-record.json
Full Text
1.0096708.txt
Citation
1.0096708.ris

Full Text

THE CARNITINE AND CARNITINE ESTER CONTENT OF RAT B I L E AND HUMAN DUODENAL FLUID By JENNIFER JANE HAMILTON B.Sc,  The U n i v e r s i t y  o f G u e l p h , 1983  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in THE FACULTY OF GRADUATE Division  o f Human  We a c c e p t t h i s t h e s i s to the r e q u i r e d  STUDIES  Nutrition  as c o n f o r m i n g standard  THE UNIVERSITY OF BRITISH COLUMBIA August ©J' C o p y r i g h t : J e n n i f e r  1986 J a n e H a m i l t o n , 1986  i i ABSTRACT Carnitine long-chain transport  i s necessary  fatty fatty  mitochondrial  acids. acids  f o r the b e t a - o x i d a t i o n of  Carnitine  across  membrane.  esters  the otherwise  Although  tissues,  i n many  of  i n the gastrointestinal  completely  investigated.  absorption  have  sources  study  investigated  t h e amounts  bile  were  bile  was s t u d i e d .  bile-rich Bile  content  measured.  Then,  fluid  was c o l l e c t e d  was from  and c a r n i t i n e  a radiochemical  samples with  were  male  contribution  and c a r n i t i n e  esters  of carnitine content  o f human  eleven  rats.  anaesthetized  Bile  flow  was  and i t s e s t e r s assay.  quantitated  measured  were  Specific  after  first  determined types of  extracting  The o r i g i n o f  carnitine  i n t h e b i l e was i n v e s t i g a t e d  i n d i r e c t l y by  measuring  the carnitine  f o r 72 h o u r s  tetradecylglycidic palmitoyltransferase the  amounts  content  or o r a l l y acid,  and t y p e s  I.  in rat  i n the  and m e t h a n o l .  fasted  of  investigated.  carnitine  chloroform  a r e unknown.  of t h e i n t e s t i n a l lumen.  the o r i g i n  gravimetrically  esters  have n o t been carnitine  F i n a l l y , the carnitine  duodenal  roles  b u t t h e t r e a t m e n t and  of c a r n i t i n e  adult  carnitine  of  the possible  (pentobarbital)  using  and p o s s i b l e  tract  The k i n e t i c s  inner  has been  i n the i n t e s t i n a l tract  to the carnitine  First,  the content  determined  of c a r n i t i n e  This bile  been  impermeable  carnitine  investigated carnitine  a r e produced to  of b i l e from  administered  an i n h i b i t o r o f These  treatments  of c a r n i t i n e  esters  rats  who  the  were  with carnitine differently found  affect  i n the l i v e r  i i i and  extrahepatically.  the  serum and l i v e r  if  The b i l e c a r n i t i n e carnitine  s i m i l a r i t i e s existed.  bile  was f u r t h e r  intravenously  studied  aspiration  The o r i g i n  and d e t e r m i n i n g  with  suspected  using  carnitine  esters  I t appears  directly  from  that  t h e newly  carnitine  pool  and a l s o  carnitine  from  serum.  liver  before  entering  carnitine  esters  metabolic  state  was  also  found  intubation  the bile.  at the time  common a s s u m p t i o n  if  i s the sole  that  This  might  the bile  or stored  hepatic  and amounts o f to reflect the  of b i l e formation. fluid.  with  carnitine  disputes  f o rcarnitine carnitine  general  The  was s i m i l a r t o  i s present i n  the theory  excretion. depletion  i n the b i l e i s not reabsorbed  occur  Carnitine  i n r a t bile challenges the  dietary  I t also  that  hepatic  e s t e r i f i e d i n the  appear  esters  both  uptake of  The t y p e s  carnitine  only  route  i tsuggests  carnitine  enters  thus,  of carnitine  intestinal tract.  addition,  and l o n g - c h a i n  i n human b i l e - r i c h d u o d e n a l  i n rat bile.  urine  after  The l a t t e r i s f i r s t  that  the  carnitine  i n the bile,  discovery  from t e n  i n r a t b i l e r e l a t i v e to other  following  of long-chain  The  i n the bile.  injection.  synthesized  percentage found  i nthe  ^C]carnitine  was c o l l e c t e d  of t o t a l carnitine  were found  tissues.  of carnitine  t o see  c h o l e l i t h i a s i s by d u o d e n a l  nasogastric  quantities  treatments  i t s recovery  fluid  pancreozymin-cholecystokinin Large  these  by a d m i n i s t e r i n g  Human b i l e - r i c h d u o d e n a l patients  after  was c o m p a r e d t o  malabsorption  that In  could  result  i n the intestine. syndromes.  i v  T A B L E OF  CONTENTS  Abstract  i  i  List  of Tables  i x  List  of Figures  x i  Acknowledgements List  x i i  of Abbreviations  xiii  Chapter  1  P U R P O S E AND  ORGANIZATION  Chapter  2  L I T E R A T U R E REVIEW  1 2  2.1 D i s c o v e r y  of C a r n i t i n e  2  2.2 C h e m i s t r y  of C a r n i t i n e  2  2.3 F u n c t i o n 2.4 S y n t h e s i s 2.5 P r e s e n c e 2.6 C a r n i t i n e 2.6.1  of Carnitine of C a r n i t i n e  6  and D i s t r i b u t i o n o f C a r n i t i n e and t h e I n t e s t i n e  Carnitine  2.6.2 O r a l 2.6.3  4  8  i n Food  Administration  8 of Carnitine  Carnitine i n the Contents Gastrointestinal Tract.  2.6.6  Carnitine Intestine  Acyltransferases  Carnitine  Degradation  9  2.7 C a r n i t i n e  10  i n the 11  i nthe  Intestine 2.6.7 C a r n i t i n e  8  of the  2.6.4 T r a n s p o r t K i n e t i c s o f C a r n i t i n e i n the I n t e s t i n a l Mucosa 2.6.5  6  12 Malabsorption  Metabolism  During  12  Fasting  2.8 E f f e c t s o f T e t r a d e c y l g l y c i d i c Carnitine Metabolism  Acid  13 on 14  V  Chapter  3  3.1  EXPERIMENTAL The C a r n i t i n e of  D E S I G N AND R A T I O N A L E  and C a r n i t i n e  Ester  Content  Rat B i l e  3.2 T h e O r i g i n  19 of Carnitine  3.2.1 T h e E f f e c t s o f the Content o f 3.2.2 T h e E f f e c t s o f Acid Treatment Content of Rat  i n Rat B i l e  20  a 72 H o u r F a s t o n Rat B i l e Tetradecylglycidic on t h e C a r n i t i n e Bile  3.2.3 T h e R e c o v e r y o f Intravenously Injected [ C]Carnitine i n Rat B i l e . . . . 3.3  Chapter 4.1  The C a r n i t i n e C o n t e n t Duodenal F l u i d 4  of Bile-Rich  4.2 H o u s i n g  23  of Animals  Duodenal  21  23  24  4.3 R a t B i l e C o l l e c t i o n 4.4 Human B i l e - R i c h  21  22  and I s o t o p e s  and Care  20  Human  METHODS AND M A T E R I A L S Chemicals  18  24 Fluid Collection....  4.5 D e t e r m i n a t i o n o f L - C a r n i t i n e  26  i n Serum and  Bile  26  4.5.1  P r i n c i p l e of the Carnitine  4.5.2  Procedure  4.6 A c e t y l c a r n i t i n e  Used Analysis  Assay  26 28 30  4.6.1  P r i n c i p l e o f Method  30  4.6.2  Procedure  31  4.7 C a r n i t i n e  Ester  Used Separation  33  4.7.1  P r i n c i p l e o f Method  33  4.7.2  V e r i f i c a t i o n o f Method  33  4.7.3 P r o c e d u r e U s e d 4.8 F a s t i n g P r o c e d u r e s  36 36  vi  4.9  Tetradecylglycidic and A d m i n i s t r a t i o n  4.10 Chapter  Analysis  5  THE  6.1  37 37  CARNITINE  AND  RAT  CARNITINE  ESTER  BILE  39  Results  5.2 Chapter  Preparation  of Data  CONTENT OF 5.1  Acid  39  5.1.1  Bile  Flow.  39  5.1.2  Carnitine  Content  5.1.3  Carnitine  Esters  5.1.4  Serum C a r n i t i n e  of B i l e  39  i n Bile  39 44  Discussion 6  THE  44 ORIGIN  The E f f e c t s Carnitine 6.1.1  OF  CARNITINE  I N RAT  o f a 72 H o u r F a s t  on t h e  Content  BILE  of Rat B i l e  51  Results 6.1.1.1  51 Bile  Flow  and Weight L o s s  51  6.1.1.2 C a r n i t i n e  Content  51  6.1.1.3  Esters  Carnitine  of the B i l e . . . in Bile  6.1.1.4 S e r u m C a r n i t i n e  6.2  Results 6.2.1.1  55 55  6.1.2 D i s c u s s i o n The E f f e c t s o f T e t r a d e c y l g l y c i d i c Acid T r e a t m e n t on t h e C a r n i t i n e C o n t e n t o f R a t Bile 6.2.1  51  55  60 60  Bile  Flow  and W e i g h t  6.2.1.2 C a r n i t i n e  Content  6.2.1.3  Esters  Carnitine  6.2.1.4 Serum C a r n i t i n e 6.2.2 D i s c u s s i o n  Loss  of the B i l e . . . in Bile  60 60 65 65 65  vii  6.3  The R e c o v e r y  of Intravenously  [ 14c]Carnitine Chapter  7  Injected 69  i n Rat B i l e  THE C A R N I T I N E CONTENT OF HUMAN 72  B I L E - R I C H DUODENAL F L U I D 7.1  Results  72  7.2  Discussion  72  Chapter  8  CHANGES  I N THE C A R N I T I N E CONTENT OF  RAT B I L E 8.1  The E f f e c t  8.3  The E f f e c t Carnitine  8.4  9 .1  of Saline  of P e n t o b a r b i t a l Content  on t h e  9  80 82 82  Summary 9.1.1  Carnitine  9.1.2  Origin  9.1.3  The T r a n s p o r t  9.1.4  Possible  Suggestions  of Carnitine  Functions  82  i n the Bile into  Bile...  83  of Carnitine i n 83  Bile forAdditional  84  Work  of Carnitine  and 84  Esters of Carnitine i n  85  Absorption  The M a l a b s o r p t i o n Carnitine  82  of the B i l e  of C a r n i t i n e  The I n v o l v e m e n t Fat  9.2.3  Content  The A b s o r p t i o n its  9.2.2  79  79  of the B i l e  CONCLUSIONS  9.2.1  78  on t h e  Summary  the 9.2  Infusion  C a r n i t i n e Content of the B i l e The E f f e c t o f I n t e r r u p t i o n o f t h e E n t e r o h e p a t i c C i r c u l a t i o n on t h e C a r n i t i n e Content of the B i l e  8.2  Chapter  78  DURING C O L L E C T I O N  of  Biliary 86  viii  9.2.4  The E f f e c t s o f H e p a t i c on B i l i a r y C a r n i t i n e  9.2.5  Biliary Studies  9.2.6  9.3  Carnitine  Dysfunction  i n Body  86 Pool 87  Carnitine Carnitine  Absorption Deficiency  i n Systemic Syndromes  Implications  88  References Appendix  89  1  The R e c o v e r y  of Intravenously  [ C]Carnitine  Injected  i n Rat B i l e  1 4  Al.l  98  Methods  98  Al.1.1  Purity  of Isotope  98  Al.l.2  Surgical  Procedure  98  Al.l.3  Analysis  of Samples  98  A1.2 R e s u l t s Appendix 2 T h e E f f e c t o f S a l i n e I n f u s i o n on B i l e C o l l e c t i o n and t h e C a r n i t i n e C o n t e n t of Rat B i l e Appendix  Appendix Appendix  3  4 5  The E f f e c t o f P e n t o b a r b i t a l on t h e C a r n i t i n e Content of Rat B i l e  106  P o t e n t i a l Mechanisms f o r the T r a n s p o r t Carnitine  Micellar  A5.2  The E f f e c t s Carnitine Other 6  103  105  A5.1  A5.3  99  The E f f e c t o f I n t e r r u p t i o n o f t h e E n t e r o h e p a t i c C i r c u l a t i o n on t h e C a r n i t i n e Content of Rat B i l e  of  Appendix  87  Pull  into  Rat B i l e  107  Theory  of B i l e  Content  107 Acid  Infusion  of the B i l e  Observations C a r n i t i n e C o n t e n t o f Serum o f Cystic Fibrosis Patients  on t h e 108 109  I l l  L I S T OF The  TABLES  E f f e c t s o f F a s t i n g on t h e R e l a t i v e Amounts o f C a r n i t i n e and C a r n i t i n e E s t e r s i n R a t Liver  The Amounts o f C a r n i t i n e a n d Carnitine Esters i n the Bile of Fed R a t s The  Concentration of A c e t y l c a r n i t i n e i n R a t B i l e A f t e r 15 a n d 1 2 0 Minutes of B i l e C o l l e c t i o n  The C o n c e n t r a t i o n s o f C a r n i t i n e a n d C a r n i t i n e E s t e r s i n R a t Serum After Bile Collection The C o n c e n t r a t i o n 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 i n t h e B i l e of Fed and F a s t e d R a t s The Amounts o f F r e e , A c y l Carnitine i n the Bile Fasted Rats The  and T o t a l o f Fed and  C o n c e n t r a t i o n s o f C a r n i t i n e and i t s E s t e r s i n t h e Serum o f F e d , F a s t e d a n d TDGA T r e a t e d R a t s  The C o n c e n t r a t i o n s o f F r e e , A c y l a n d Total Carnitine i n the B i l e of F a s t e d and TDGA-Treated R a t s The Amounts o f F r e e , A c y l Carnitine i n the Bile and T D G A - T r e a t e d R a t s  and T o t a l of Fasted  The C o n c e n t r a t i o n s o f C a r n i t i n e a n d C a r n i t i n e E s t e r s i n Human Duodenal F l u i d After S t i m u l a t i o n o f B i l e Flow The  Relative Quantities of Carnitine and C a r n i t i n e E s t e r s i n B i l e R i c h Human D u o d e n a l F l u i d  The  S p e c i f i c A c t i v i t y of Carnitine i n t h e Serum and B i l e o f R a t s Two H o u r s A f t e r t h e I n t r a v e n o u s I n j e c t i o n o f [ -"-^C ] C a r n i t i n e  X  Table  13.  The  C o n c e n t r a t i o n s o f F r e e and C a r n i t i n e i n t h e Serum of Cystic Fibrosis Patients  Total I l l  xi  L I S T OF  FIGURES  Fig.  1.  The  Structure  of C a r n i t i n e  Fig.  2.  The  Structure Acid  of  Fig.  Fig.  Fig.  Fig.  Fig.  Fig.  Fig.  Fig.  Fig.  3.  4.  5.  6.  7.  8.  9.  10.  11.  Tetradecylglycidic 16  Thin-layer Chromatographic V e r i f i c a t i o n of the S e p a r a t i o n of Long-chain Carnitine Esters from Other Types of C a r n i t i n e by L i p i d E x t r a c t i o n Bile  The  The  Bile  The  Bile  The  The  3  Flow During B i l e from Fed R a t s  35  Collection 40  C o n c e n t r a t i o n 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 i n B i l e from F e d R a t s C o l l e c t e d a t 15 M i n u t e Intervals  41  Amounts o f F r e e , A c y l and T o t a l Carnitine in Bile Collected f r o m F e d R a t s a t 15 M i n u t e Intervals  42  Flow During B i l e C o l l e c t i o n f r o m F e d a n d 72 H o u r F a s t e d Rats  52  Amounts o f C a r n i t i n e and C a r n i t i n e E s t e r s i n the B i l e of Fed and F a s t e d R a t s I m m e d i a t e l y A f t e r the I n i t i a t i o n of B i l e Collection  56  Flow Rates During B i l e C o l l e c t i o n f r o m 72 H o u r F a s t e d and T e t r a d e c y l g l y c i d i c Acid Treated Rats  61  Amounts o f C a r n i t i n e and C a r n i t i n e E s t e r s i n the B i l e of F a s t e d and T e t r a d e c y l g l y c i d i c A c i d T r e a t e d R a t s 15 a n d 105 Minutes A f t e r the I n i t i a t i o n of Bile Collection  66  Appearance Injected [ u l of B i l e at V a r i o u s Hours A f t e r  of I n t r a v e n o u s l y C ] C a r n i t i n e i n 50 of 6 Rats C o l l e c t e d I n t e r v a l s up t o 2 Injection  1 4  100  ACKNOWLEDGEMENTS  The  author  helped  -Dr.  would  with this  like  t o thank  the following  P e t e r Hahn f o r h i s s u p e r v i s i o n  t h e i r time  -Leighton  who  project:  - c o m m i t t e e members, D r . S h e i l a for  people  Innis  and g r e a t  friendship.  and Dr J o s e p h  Leichter  and guidance.  James and D r . D a v i d  Seccombe f o r t h e i r  kind  assistance.  -Marta  Taller, Livia  Nada Hahn f o r t h e i r  S r u b i s k i , Wendy C a n n o n , V i c k y L o u i e a n d technical  a d v i c e , and ever  stimulating  conversation.  -the  Cystic  F i b r o s i s Foundation  o f Canada f o r f i n a n c i a l  support.  -both  h e r h u s b a n d , Drew M e i k l e , a n d h e r p a r e n t s  support.  f o r moral  xiii L I S T OF  ABBREVIATIONS  BW  body  weight  °C  degrees  c-  centi-  Ci  Curie  CAT  carnitine  CoA  coenzyme  CPT  carnitine  palmitoyltransferase  CV  coefficient  of  DPM  disintegrations  EDTA  ethylenediaminetetraacetate  g  gram  GOT  glutamate-oxaloacetate transaminase  h  hour  HEPES  N-2-hydroxyethylpiperazine-N'-2-  Celsius  acetyltransferase A  variation  ethanesulphonic  per  minute  acid  IP  intraperitoneally  IV  intravenously  1  liter  m-  milli-  M  molar  min  minute  mol  mole  n-  nano-  NEM  N-ethylmaleimide  SE  standard  TDGA  tetradecylglycidic  error acid  xiv TLC  thin  layer  rpm  rotations  u-  micro-  UBC  University  v  volume  chromatography per  minute  of B r i t i s h  Columbia  CHAPTER 1  P u r p o s e and  Organization  1  The  metabolism  many o r g a n s . intestine, of  this  of c a r n i t i n e  The p r e s e n c e  and r o l e  duodenal  in rat bile fluid.  T h i s might  i n the i n t e s t i n e  metabolic  role  experimental described. Chapter  includes  a discussion  investigations. are  summarized  further are also  studies  In Chapter  Nine  presented.  a r e r e p o r t e d and Chapter  of other  Eight  related  the experimental  and c o n c l u s i o n s a r e made. and t h e p o s s i b l e  experiment a r e  work a r e d e s c r i b e d i n  S i x and S e v e n .  of the r e s u l t s  study i s  Three, the  The e x p e r i m e n t a l r e s u l t s Five,  to t h i s  f o r each  i n this  a potential  organ.  relevant  In Chapter  The methods used  i n Chapters  i n human  an endogenous s o u r c e o f  i n this  d e s i g n and r a t i o n a l e  Four.  discussed  Two.  The o b j e c t i v e  and h e l p t o e l u c i d a t e  A review of the l i t e r a t u r e  i n the  t h e c o n t e n t and o r i g i n o f  reveal  for carnitine  i n Chapter  ignored.  and measure i t s p r e s e n c e  carnitine  presented  of c a r n i t i n e  however, has been l a r g e l y  work was t o i n v e s t i g a t e  carnitine  has been w i d e l y s t u d i e d i n  results  Suggestions f o r  implications  of t h i s  work  CHAPTER 2  Literature  Review  2 2.1  Discovery In  1905  Carnitine  c a r n i t i n e was  laboratories (Gulewitsch carnitine  of  as  a  and was  independently  component  Krimberg;  determined  of  Liebig's  Kutscher). in  1927  discovered meat  The  by  extract  structure  (Tomita  two  and  of  S e n d j u ) and  in  1  1952  i t was  Tenebrio  identified  molitor  (Carter  carnitine  in lipid  Fraenkel;  Fritz).  2.2  Chemistry The  of  of  short-chain  (2-6  long-chain  (14  et  a l . , 1952). was  carnitine,  the  isomer. hydroxyl  carbons),  1955  mealworm, the  (Friedman  or  1.  role  of  is  the  and  L-carnitine  acids of  are  (8-12  more c a r b o n s ) c a r n i t i n e  short  and  d i s t r i b u t e d i n aqueous  carnitine  esters  medium-chain  migrate  to  the  bound  carnitine  medium-chain  uniformly  form  carbons)  esters.  carnitine  solutions air/water  to  Free  esters  but  and  are  long-chain  interphase  (Pande,  . Yalkowsky  esters  with  and  Zografi  b e t w e e n 8 and  micelles.  The  ( 1 9 7 0 ) showed 16  critical  decreases with  increasing  showed also  the  gamma-trimethylamino-  group  and  form  for By  known  Fatty  carnitine  1981)  factor  i s shown i n F i g u r e  active to  growth  Carnitine  3-hydroxybutyrate,  enzymically  a  metabolism  structure  biologically  as  carbons micellar  molecular  that  an  uneven  occurs  at  concentrations  are  the  carnitine  amphiphiles  and  concentration  weight.  d i s t r i b u t i o n of below  that  Pande  (1981)  palmitoylcarnitine critical  micellar  3  +  CH  3  CH - N-CH -CH-CH -COO3  2  CH  2  OH  3  FIGURE 1 The  structure  of  carnitine.  4  concentration. distributed  Long-chain  carnitine esters  i n s o l u t i o n s of methanol  a r e evenly-  and c h l o r o f o r m  (Pande,  1981) .  2.3 F u n c t i o n The  principal  long-chain membrane occurs  of C a r n i t i n e  fatty  function  acids  of c a r n i t i n e i s to transport  across  to the mitochondrial  (Fritz,  acyl-CoA  1968).  formed  mitochondrial formation  the inner matrix  where  I t i s necessary  of f a t t y  beta-oxidation  because  i n the c y t o s o l cannot  membrane w h i c h  mitochondrial  cross  i s impermeable  fatty  the inner t o CoA.  acylcarnitine i s catalyzed  by c a r n i t i n e  palmitoyltransferase  I (CPT I , E.C.2.3.1.21) w h i c h i s  located  side  on t h e o u t e r  membrane. membrane and  i s facilitated  Parvin,  ester of  short  i s catalyzed  of the c a r n i t i n e ester  across  by a c y l c a r n i t i n e t r a n s l o c a s e  by CPT I I w h i c h  i s located  C a r n i t i n e can then  return  Carnitine  i n the beta-oxidation  i s also  and medium-chain to acyl-CoA  n o t needed  Shorter-chain  shorten  (Pande  to the outer  this  very  the  on t h e i n s i d e  o f t h e membrane and r e p e a t  activated  of  mitochondrial  1976) and t h e d i s s o c i a t i o n o f t h e c a r n i t i n e  t h e membrane.  side  is  The t r a n s p o r t  of the inner  The  fatty within  f o r membrane fatty  long-chain these  involved  fatty  acids fatty acids  acids.  Usually  the mitochondria  transport may  process. of  these are and c a r n i t i n e  (Aas and Bremer,  1968).  a r i s e as a r e s u l t o f o x i d a t i o n  acids.  Peroxisomes  independently  partially  of c a r n i t i n e  5 (Osmundsen and N e a t , bound  1981).  esters  f o r complete  Peroxisomal  octanoyl-  maintenance (Pearson  mitochondrial (Bremer,  may  Rebouche,  1967).  for this  process  may  be i n v o l v e d i n  also  acyl-CoA/CoA  A c e t y l c a r n i t i n e i s formed  freeing  play  fatty  a role  CoA w i t h i n t h e acid  oxidation  excreted  acids  that  accumulate  1984).  undergo  high  colleagues  carnitine  to accept  (Ohtani  a r e formed  i s again  of these  used  ratio  (Engel  deficiency the organic  leave  et a l . , 1982).  within  that  high  the mitochondria  to buffer  conditions,  then  metabolic  acid  the c e l l  T h e same s i t u a t i o n o c c u r s  (1984) have h y p o t h e s i z e d  valproyl-CoA  of the  carnitine  The c a r n i t i n e e s t e r s  therapy  errors of  acyl-CoA/CoA  An a p p a r e n t  or  intramitochondr,ial  at the point  an a b n o r m a l l y  i n the urine.  acid  i n certain disease  A s a r e s u l t o f some i n b o r n  as t h e c a r n i t i n e i s used  t h e CoA.  both  et a l . ,  c a r n i t i n e a c e t y l - and  mitochondrial  f o r further  also  organic  causing  valproic  In  and Tubbs,  shortening  results  are  of the inner  states.  metabolism,  defect  (Bieber  as  1983).  toxicity  from  are required  carnitine esters  matrix  Carnitine  and  oxidation  c a r n i t i n e and a c e t y l - C o A  chain  are then  1983).  Shorter-chain  ratio  esters  to the mitochondria  and m i t o c h o n d r i a l  transferases  (Bremer,  from  The s h o r t e n e d  t o c a r n i t i n e and t r a n s f e r r e d  carnitine  the  1979).  and during  T h u r s t o n and l e v e l s of and  the acyl-CoA/CoA  that ratio.  carnitine administration  allows  6  the  e x c r e t i o n of the accumulated  and  alleviates  2.4  Synthesis In  free  first  the eventual  and  2.5  Presence  (Bohmer,  Muscle  and  carnitine  mammals.  two  pool.  kidney  turnover  of the t o t a l  this  liver, i s limited  to  and  in  Engel,  and L i n d s t e d t , 1976). and  and L i n d s t e d t , 1976) and  compartment  three  carnitine  1983; Rebouche  (Rebouche and E n g e l ,  carnitine  rate  body  i s i n the serum, l i v e r  t u r n i n g over  The  to produce  i n the  model  compartment  as a s e p a r a t e  (Rebouche and E n g e l ,  triple been  of c a r n i t i n e  includes a small  carnitine  and  model pool  in serum  a large stable defines the  having  1983).  The  kidneys.  1983) have  to e x p l a i n the d i s t r i b u t i o n  of r a p i d l y  muscle and  The  process  of C a r n i t i n e  (Cederblad  (Cederblad models  Kim,  I n humans,  but i n r a t s ,  (Rebouche and E n g e l ,  hypothesized  pool  95-98%  98% i n r a t s  compartment  i n the  and  1974).  contains  double  ( P a i k and  (E.C.1.14.11.1)  1979),  and D i s t r i b u t i o n  dogs  remaining Both  steps  synthesis occurs  brain (Englard,  liver  and  hydroxylase  i n carnitine  the  1984)  trimethyllysine  ( L i n d s t e d t and L i n d s t e d t , 1 9 6 5 ) .  step  kidney  man  form  lysine  h y d r o x y l a t i o n o f g a m m a - b u t y r o b e t a i n e by  gamma b u t y r o b e t a i n e carnitine  mitochondria  protein-bound  There are s e v e r a l a d d i t i o n a l  before  the  of C a r n i t i n e  the s y n t h e s i s of c a r n i t i n e ,  1975).  from  t h e symptoms o f c a r n i t i n e d e f i c i e n c y .  methionine  final  acid  a very  Both models  liver high have  7  been m a t h e m a t i c a l l y radiolabelled calculated of  by  other  found  Yue  (1975).  tracer  No  and  using  excretion  i n the  Fritz  Methods f o r  the  assessment  serum c a r n i t i n e  concentrations  the  Carnitine  deficiency the  subclinical  carnitine  The  i n d e x of  esterified and al.,  1978)  Rebouche,  i s very  metabolic  additional individual.  depletion rare  were  also  excretion,  The  information  and  For  metabolic  specific about  fat  are  (Mitchell,  The  and  low  only  1978b).  a l t h o u g h not  always  importance  of  known.  serum c a r n i t i n e instance, of  (Brass  (Seccombe  aciduria  (Engel  esters  metabolic  is a  highly  fasting  content  carnitine  the  are  Linstedt,  levels  is indicative dietary  and  i s not of  status  between m u s c l e  distinct.  state.  some t y p e s of  1984).  amount  Mcintosh  carnitine  (Cederblad  esterification  1978), a h i g h or  of  deficiencies  serum c a r n i t i n e  Hoppel,  results  breakdown or  serum c a r n i t i n e  symptoms a r e  d e g r e e of  than the  B r o o k s and  correlation  s e v e r e muscle  recognized,  useful  i s no  B o t h m u s c l e and c a s e of  the  i s known.  There  in  greater  carnitine  from  i n both c a s e s ,  Similar  (1962) and  controversial.  1976).  was  urine.  o t h e r means o f  than u r i n a r y ,  data obtained  e x p e r i m e n t s but  carnitine  carnitine  found  tested  state  and  present of  et  the  give  8  2.6  C a r n i t i n e i n the I n t e s t i n e  2.6.1  C a r n i t i n e i n Food The  carnitine  laboratories; 1978a). present  2.6.2  content  the r e s u l t s  has been s t u d i e d by many  have been r e v i e w e d  C a r n i t i n e i s most abundant  (Mitchell,  i n meats b u t i s a l s o  i n plants.  O r a l A d m i n i s t r a t i o n of C a r n i t i n e The  Karpati  absorption  of c a r n i t i n e  and c o l l e a g u e s  (1975).  carnitine  to a carnitine  carnitine  concentration  observations  after  associated  with  urine carnitine  and  Rebouche, 1 9 8 4 ) .  students.  levels  absorption  oral  then,  similar  authors.  carnitine,  t h e symptoms  d e f i c i e n c y disappeared, increased  (Gilbert,  and serum  1985; E n g e l  using  twelve  apparently  studied  healthy  Two grams o f L - c a r n i t i n e were a d m i n i s t e r e d  and t o t a l  subsequent  Since  I n 1983, Bach and c o l l e a g u e s  s i x of the students;  Free  with  oral  p a t i e n t , t h e serum  increased.  carnitine  d e m o n s t r a t e d by  Upon a d m i n i s t e r i n g  deficient  treatment  and  carnitine  was f i r s t  have been made by many o t h e r  Generally,  to  of food  the remainder  serum c a r n i t i n e  24 h o u r s .  received a  were m o n i t o r e d  Carnitine levels  peaked  placebo.  during the after  3.5  h o u r s and had n o t r e t u r n e d  to baseline levels  the  i n v e s t i g a t o r s have r e p o r t e d  24 hour p e r i o d .  serum c a r n i t i n e after 1978).  an o r a l  Other  levels  dose  are s t i l l  elevated  a t t h e end o f that  i n humans 4 h o u r s  (Seccombe e t a l . , 1984; F r o h l i c h  Similar observations  orally  have been made i n r a t s .  et a l . ,  9  G u d j o n s s o n and c o l l e a g u e s intraluminal in  (1984) o b s e r v e d  dose o f l a b e l l e d  carnitine,  after  labelled  carnitine  an  t h e amount o f l a b e l  t h e serum had n o t y e t peaked 8 h o u r s l a t e r .  G r o s s and H e n d e r s o n (1984) f o u n d of  that  Similarly,  35% o f an i n t r a l u m i n a l  dose  i n t h e serum 7 h o u r s a f t e r  administration.  2.6.3  Carnitine  i n the Contents  of the  Gastrointestinal  Tract Sachan and Ruark rats  to determine the content  intestinal only and  (1985) s t u d i e d  free  lumen and t i s s u e . carnitine  medium-chain  intestinal  lumen.  tissue,  esters  esters  may  that  the d i e t s  were f o u n d this  these  have been r e l e a s e d  and  i n the  contained  esters,  short  i n the  fraction  short  adult  carnitine  carnitine  formed due t o i n t r a l u m i n a l  produced  decreased. medium-chain  from i n t e s t i n a l  e s t e r i f i c a t i o n or  by i n t e s t i n a l m i c r o o r g a n i s m s .  Investigations rats  Although  With f a s t i n g ,  The a u t h o r s h y p o t h e s i z e d carnitine  o f endogenous  and l o n g - c h a i n  carnitine  f e d and f a s t e d  of the i n t e s t i n a l contents of  by Hahn and c o w o r k e r s (1985) f o u n d  carnitine  e s t e r i f i c a t i o n increased  the  gastrointestinal  was  due  tract.  to p r e f e r e n t i a l  I t was  absorption  that  the degree of  throughout  the l e n g t h of  hypothesized of f r e e  suckling  that  carnitine.  this  10 2.6.4  Kinetics  of C a r n i t i n e  Uptake  into  Intestinal  Mucosal  Cells The  kinetics  intestinal  mucosal  Gross  of  1984;  1985)  in  c e l l s have  the U n i v e r s i t y and  (Gudjonsson 1983;  of c a r n i t i n e  Olsen  guinea pigs  and  1985a;  1985;  groups  1983;  1985b; H a m i l t o n e t a l . ,  The  1983).  done u s i n g  humans,  t e c h n i q u e s used  included  lumenal  bolus  injection, everted i n t e s t i n a l rings  and  tissue  isolated  enterocyte brush  with  The  intestinal  results  of  concur:  carnitine  rapidly  into  duodenum  dependent  and  liver  (Willner (Huth 1981), 1981;  and  absorb  and  1980),  and are  c e l l s than carnitine  carrier  reported  e t a l . , 1978; Shug,  the Ussing  chamber  t a k e n up  ileal  mediated.  1976),  Rebouche,  1977),  the  groups  more while esters.  sodium This i s similar  i n other tissues  Bremer,  cells  or c a r n i t i n e  including  skeletal  the kidney  the  muscle cortex  the e p i d i d y m a l c e l l s (James e t a l . ,  t h e h e a r t ( S a r t o r e l l i e t a l . , 1982; Bohmer  sacs,  Wisconsin  enterocytes i s active,  potentially  (Christiansen  and  the Minnesota  mucosal  into  uptake  incubation,  acetylcarnitine  does not  uptake  carnitine  and  intralumenal  epithelium.  both  jejunal  Carnitine  to  mucosal  b o r d e r membrane v e s i c l e s  mounted  the  culture,  perfusion,  Both  intestinal dose  vascular  l e d by  of Wisconsin  Shaw e t a l , 1 9 8 2 ;  rabbits.  and  by  from  (Gross et a l . ,  e x p e r i m e n t s were  and  release  studied  of the U n i v e r s i t y  L i e t a l . , 1983;  rats,  been  of Minnesota  e t a l . , 1984;  v i t r o and _in v i v o  uptake  e t a l . , 1977)  and  the  cerebral  Bahl et a l . , cortex  (Huth  et  11  al.,  1981).  carnitine The inside  Both  groups  concentrations Gross  and  the mucosal  esterified  cell,  agree  mucosal  i s very  Carnitine  CPT  1981,  Carnitine  rat intestinal one  third  (Gudjonsson  metabolism  ( W i n d m u e l l e r and  major of  energy  CAT  of  within  the mucosal  that  rats  cell.  (CAT,  were e l e v a t e d  and  Ruark,  also  1985). from  the A l l  the  Intestine  found  that  cells  found  i s based  on  1980),  the  fact  cell.  of c a r n i t i n e Hahn and  i n the  after  to  wet liver.  protein),  i n the As  mucosa  the  energy  glutamine the that The  lesser f a t i s not high  a  activity  f o r the  high  acetylcarnitine  c o l l e a g u e s (1985)  found  acyltransferases  intestinal  birth  activity  i n the  nmol/min/mg  Spaeth,  the  (umol/min/g  more a c t i v e  the a c t i v i t i e s of c a r n i t i n e protein)  is  production within  i s probably responsible  esterification  (nmol/min/mg  once  the c a r n i t i n e  of c a r n i t i n e  mucosa  source f o r the mucosal  degree  that  a u t h o r s have  i n the  probably reflects  i n t h e mucosa  occurs.  et a l . , 1985a).  of i n t e s t i n a l  o f CPT  of  the a c t i v i t y  metabolism  activity  release  a p p r o x i m a t e l y 40%  the l i v e r  Sachan  Carrington  acetyltransferase  h o w e v e r , was than  the  high  slow.  Hanson and  only  50%  Other  Acyltransferases  i n adult  w e i g h t ) was  that  at very  demonstrated  of a c e t y l c a r n i t i n e  investigators  of  also  a s much a s  (Hahn e t a l . , 1985;  cells  that  passive diffusion  to a c e t y l c a r n i t i n e .  enterocyte  In  showed  Olsen groups  shown a h i g h d e g r e e  2.6.5  also  and  mucosa of d e v e l o p i n g  decreased  to adult  levels  12  at  weaning.  activity  They a l s o  was  found  much g r e a t e r  that  palmitoylcarnitine  that  the  2.6.6  palmitate  Carnitine  other  was  colleagues  for  degradation  that  II.  carnitine  carnitine  but  i n the  Mcintosh,  1975).  and  germ-free r a t s  i n the  to  intestinal tract  b e f o r e Rebouche  n o r m a l and  implies  i s metabolized  endogenous i n t e s t i n a l f l o r a of  This  mucosal c e l l  ( B r o o k s and  years  I  Intestine  carnitine  several  CPT  CPT  there.  i n the  that  of  i n the  oxidized  tissues  (1984) used  demonstrate that for  evidence  d e g r a d a t i o n of  suspected  mucosal c e l l  i s formed  i s not  compounds i n r a t  Bacterial  than  Degradation  T h e r e i s no  that  lower  is  to responsible  gastrointestinal  tract. In  Rebouche's e x p e r i m e n t s ,  m e t a b o l i t e s were f o u n d either  o r a l or  i n the  hours a f t e r  accumulated  i n the  Carnitine Three  who  Hahn and  with  labelled  had  received  colleagues rats  who  had  carnitine.  injection, labelled carnitine  had  Malabsorption  situations  and  of  possible  Frohlich  serum c a r n i t i n e  bypass surgery  rats  and  i n t e s t i n a l mucosa.  have been s t u d i e d . that  of  phenomenon i n s u c k l i n g  subcutaneously  Twenty-four  2.6.7  feces  intravenous c a r n i t i n e .  (1985) showed a s i m i l a r been i n j e c t e d  labelled carnitine  and  carnitine coworkers  l e v e l s were r e d u c e d  continued  to  decrease  malabsorption (1980) r e p o r t e d  after  jejunoileal  post  operatively.  13  Whether d e c r e a s e d contributed  to  carnitine  this  carnitine  Serum c a r n i t i n e  was  carnitine  was  38%  appears l i k e l y carnitine  carnitine  that  and  a l . , 1974).  in  these  of  lowest and  increase  surgery.  patients. of in  an  oral  serum  Thus, i t results  for  might  Carnitine  be  carnitine  in  and  patients  treatment,  atrophy the  and  reduced to  the  most  levels.  serum  and  With  carnitine  l e v e l s were  also  some  patients  malabsorption  (Bohmer  serum c a r n i t i n e  reduced  synthesis,  intestinal  with  serum c a r n i t i n e  carnitine  During  absorption  carnitine  of  levels the  malabsorption  Fasting  i s elevated  utilization  i s intimately  t y p e s of  from  serum  contributed.  lipolysis  availability  The  drug  due  Metabolism  Adipocyte  suffering  with Crohn's d i s e a s e  Although  have  decreased  Serum c a r n i t i n e  villous  cases could  precursors  the  the  in patients  et  carnitine  the  (1976) f o u n d  infections.  repletion  with a history  acid  in similar  j e j u n o i l e a l bypass surgery  were r e s t o r e d .  decreased  2.7  before  in patients  c a s e s had  nutritional  itself  than  Mansour  levels  schistosomiasis  levels  less  coworkers  upon a d m i n i s t r a t i o n  surgery,  ingestion  malabsorption.  Mikhail  severe  After  precursor  Seccombe and  absorption  measured  carnitine.  carnitine  i s unknown.  (1984) measured  dose o f  or  involved esters  after  fasting  i s increased. in fatty acid  i n the  rat  liver  and  fatty  As oxidation, are  altered  14 and  the  is  percentage  changed The  results  carnitine data  is  of  in  rise  as  the to  of a  increased  effects  liver  are  shown i n T a b l e  due  to  the  than  CPT  II  (Bremer,  decrease of  higher  in  the  the  of  total liver  of  fatty  amounts of  accumulation  of  Aas  amount of  increased  acids  and  serum  levels  fasting  the  All  carnitine.  The  fasting carnitine  carnitine  hepatic  esters  a c t i v i t y of  1971;  carnitine  of  on  1.  during  Daae,  free  of  longer-chain  long-chain  a c t i v i t y of  1981;  the  1978). the  oxidation  also  result  rat  esterification in  on  percent  The  a  Hoppel, studies  esters.  The  carnitine  and  content  given  increase gives  (Brass  of  Norum, in  long-chain  the  is  CPT  I  1965).  liver  is  carnitine  esters. Serum c a r n i t i n e 41%  to  69%  1978).  after  This  In  to  72  increase  acetylcarnitine used  a  esterification in  buffer  and the  hour was  was  fasting mainly  likely  fasting  the  (Frohlich  increased  (Brass  form  result  intramitochondrial  humans, serum c a r n i t i n e  upon  period  in  the  rats  of  carnitine  acyl-CoA/CoA  a l . , 1978;  Hoppel  Hoppel,  of  e s t e r i f i c a t i o n also  et  and  from  being  ratio.  increased  and  Genuth,  1976).  2.8  The  Effect  of  Tetradecylglycidic  Acid  on  Carnitine  Metabolism Tetradecylglycidic structural CPT  I  analogue  i n h i b i t o r by  acid  (Figure McNeil  (TDGA, M c N - 3 8 0 2 ) i s a 2).  I t was  Laboratories  first  fatty  reported  (Tutweiler  et  acid as  al,  a  Effects  of Fasting  Length of F a s t (hours)  on t h e R e l a t i v e  Carnitine  TABLE 1 Amounts o f C a r n i t i n e  and C a r n i t i n e  Esters(%  Acetyl  S h o r t and Mediumchain  of  and C a r n i t i n e  Esters  i n Rat  Liver  Total) Reference  Free  fed  fast  24  fed  fast  26  51  fed  fast  Longchain  fed  fast  2  20  Bohmer and B r e m e r ,  1968  24  73  45  -  -  25  42  2  13  Bohmer,  48  60  49  -  -  38  38  3  16  Bohmer e t a l . ,  24  48  30  17  26  51  50  1  19  Brass  and H o p p e l ,  1981  24  53  41  -  -  43  44  3  18  Brass  and H o p p e l ,  1978  48  53  43  -  -  43  43  3  14  Brass  and H o p p e l ,  1978  72  53  47  -  -  43  45  3  11  Brass  and H o p p e l ,  1978  48  -  29  -  -  -  58  -  14  French  56  31  13  23  27  28  3  18  Pearson  36-48  1967  et a l . ,  1966  1985  and Tubbs ,  1967  16  CH3(CH2 )-j3  FIGURE 2 The  structure  of  tetradecylglycidic  acid.  17  1978; and by  Tutweiler  and D e l l e v i g n e ,  specifically irreversibly  Dellevigne, long-chain  inhibits  1979).  cross  Concentrations  increased  (Tutweiler  decreased  fatty  e t a l . , 1978).  acid oxidation,  remained An  also  short  constant  increase noted.  showed t h a t  Free  of the  state r e s u l t i n g i n and D e l l e v i g n e ,  fasted  increased  and medium-chain a c y l c a r n i t i n e l e v e l s  and l o n g - c h a i n  i n the t o t a l Frost  the e f f e c t s of o r a l  of r a t s previously  carnitine  and W e l l s  TDGA p r o d u c e d  (1982),  of the l i v e r  studying  no change i n l i v e r  of the hepatic  primarily  due t o an i n c r e a s e  neonatal  was rats,  weight but  c a r n i t i n e pool.  This  was  i n f r e e c a r n i t i n e and was  t o be a c o m p e n s a t o r y o f t h e TDGA.  a c y l c a r n i t i n e decreased.  c a r n i t i n e content  the s i z e  inhibition  carnitine esters  (Tutweiler  demonstrated  increased  hypothesized  mitochondrial  gluconeogenesis i s i n h i b i t e d  on t h e l i v e r s  (1985).  significantly,  acids.  e t a l . , 1984).  TDGA a d m i n i s t r a t i o n 48 h o u r s  the o x i d a t i o n of  As a r e s u l t  i n the f a s t i n g  F r e n c h and c o l l e a g u e s  for  ( T u t w e i l e r and  of f r e e c a r n i t i n e i s  i n b o t h man and r a t s  Mandarino  i n rats  i n the c y t o s o l of the  of long-chain  when TDGA i s a d m i n i s t e r e d  1979;  inhibits  the inner  and t h e c o n c e n t r a t i o n  potently  and jLn v i t r o  o r medium-chain f a t t y  accumulates  h e p a t o c y t e as i t cannot  hypoglycemia  vivo  I t ,therefore,  Long-chain acyl-CoA  decrease  CPT I i i i  TDGA v e r y  i n a c t i v a t i n g the a c t i v e s i t e  but not s h o r t  membrane.  1979).  mechanism t o overcome t h e  CHAPTER  Experimental  Design  3  and  Rationale  18  The for is  study  several often  reasons.  used  absorption carnitine  i n the intestine  For example, although  therapeutically,  under  various  absorption  investigate might  of c a r n i t i n e  the claim  be t h e c a u s e  that  be d e f i n e d defective  o f some c a s e s and Rebouche,  might  a role  i n f a t absorption;  jejunum.  nature,  are possibly  intestinal fatty in  lumen.  acids  This  carnitine  fatty  be  carnitine  the in  (Brooks,  before  was t o d e t e r m i n e w h e t h e r  a series  following present  i n rat bile?  might  of the shuttle  encountered  and i n t o t h e  has been p r o p o s e d f o r hypothesized  stream,  lumen and sperm  content  of t h e i n t e s t i n e  absorption  of  The o b j e c t i v e or not b i l e  i n the i n t e s t i n a l  of experiments which  questions:  micelles  1980).  the intestinal  found  amphiphilic  barriers  between t h e blood  c a n be i n v e s t i g a t e d .  carnitine  of t h e i r  the enterocyte  epididymal  carnitine  are absorbed i n  carnitine  that  Normal  carnitine  where i t has been  endogenous c a r n i t i n e  studied  study  acids  epithelium,  mitochondria  because  to a role  i n the epididymis,  epididymal  both  i n t h e mixed  of f a t across  carnitine  absorption  Finally,  some o f t h e m e m b r a n e  i s similar  transport  The  esters,  involved  carnitine  1984).  Alternatively,  across  the transport  lymph.  to  Carnitine  of  i n order to  of systemic  (Engel  the  carnitine  i s n o t known.  deficiency play  oral  the efficiency  conditions  should  i s important  of the present  This  were d e s i g n e d  1) A r e c a r n i t i n e  dietary  contributes  lumen.  was  to done  t o answer t h e  and c a r n i t i n e  2) What i s t h e o r i g i n  must  esters  of the c a r n i t i n e  19  in  bile?  3) Does c a r n i t i n e  fluid?.  The r a t i o n a l e  described are  below.  described  of  and p r o t o c o l  i n Chapter  results  and C a r n i t i n e of several  sources.  carnitine present  Sachan  esters  i n the diet.  there the  may  urine  is  into  was c o l l e c t e d serum  measured.  from route  body  pool  eleven  were  were n o t  (1985) and carnitine i n  subcutaneously or  studies  suggest  excretion  that besides  1984; C e d e r b l a d and 1962).  Possibly,  v i a the b i l e ,  by b a c t e r i a  of carnitine  fed adult  and c a r n i t i n e  Long-chain  concentrations  than  carnitine  where  of the  i t i s  large  et a l . , 1984).  the presence  carnitine  other  that  labelled  for carnitine  and E n g e l ,  carnitine  some t y p e s o f  i t was i n j e c t e d  or degraded  from  used  of Rat B i l e that  from  found  (1984) found  the intestine,  (Rebouche  clarify  indicate  be d e r i v e d  1976; Yue and F r i t z ,  reabsorbed  intestine To  Data  (Rebouche  excreted  either  and  studies  and Ruark  after  be a n o t h e r  Lindstedt,  experiment i s  Content  Hahn and c o l l e a g u e s  intestinal tract  intravenously.  Ester  i n r a t i n t e s t i n a l contents  Rebouche and a s s o c i a t e s the  f o r each  4.  t h e i n t e s t i n a l lumen might  dietary  i n human b i l e - r i c h d u o d e n a l  The s p e c i f i c methods and m a t e r i a l s  3.1 T h e C a r n i t i n e The  exist  carnitine  determined  rats.  ester  ester  in ratbile, Bile  flow,  concentrations  and  i n randomly  bile bile were  acetylcarnitine selected  samples.  20 3.2  The  Origin  of  Carnitine or  very  was  are  from  The  amount and  the  this  type  to  liver  see  or  of  serum.  A  with  the  carnitine  source  of  The  Rat  of  Effects  carnitine  72  increased  esters  shorter-chain  in  esters  in  the of  carnitine the  to  rats  in  which  serum  differently.  bile  to  was  The  were  changes o c c u r r i n g rats  bile  affect  TDGA t r e a t m e n t . the  only  is  carnitine  and  and  serum  carnitine  on  fasting  these  the  the  in  injected  further  investigate  bile.  Hour F a s t  on  the  of  whilst  carnitine  esters  decreased.  Hoppel,  1978).  Carnitine  Content  The  concentrations carnitine  samples.  long-chain  the  amount In  e s t e r i f i c a t i o n increased  Five  collection.  selected  proportion  liver,  carnitine  Long-chain  the  the  overall  ester  a  of  hepatic  Bile  Fasting  bile  of  in  that  liver  group  either  long-chain  in  conditions  reflected  labelled  of  Because  present  the  third  be  bile.  origin  carnitine  intravenously  3.2.1  The  were  i f they  in  suggests  imposing  studied  Bile  amounts of  are  esterification in  conditions  examined  by  Rat  b i l e could  Large  liver.  investigated  carnitine  the  esters  degree,  the  in  present  carnitine  limited  derived  in  origin.  esters  long-chain a  found  extrahepatic  carnitine  Carnitine  rats  were f a s t e d  b i l e flow,  S e r u m was  72  serum,  hours  and  collected  in and  the  and prior  carnitine  i n a l l samples.  were d e t e r m i n e d also  the  (Brass  carnitine  were measured esters  for  of  randomly the  to  21 carnitine results from  the  of this  rats  3.2.2  and c a r n i t i n e fasting  ester  concentrations  experiment  were  Carnitine  of Tetradecylglycidic  Content  production  both are  i s decreased  i s increased  Acid  those  T r e a t m e n t on  long-chain  and t h e  (French  esterified carnitine  carnitine  concentration  of  free  e t a l . , 1985).  I n t h e serum,  and t o t a l c a r n i t i n e  concentrations  decreased. Six  rats  were  TDGA, a c c o r d i n g (1985).  fasted  B i l e was c o l l e c t e d ester  samples.  Long-chain  randomly  were  carnitine  samples.  was a l s o compared  which  the rats  were  3.2.3  The R e c o v e r y  of French  were  The serum  to those  with  and c o l l e a g u e s  measured  esters  measured.  fasted  and i n t u b a t e d  and t h e b i l e f l o w ,  concentrations  selected  concentration studies  f o r 72 h o u r s  to the protocol  carnitine  in  with  of Rat B i l e  A s TDGA i n h i b i t s C P T I , h e p a t i c  carnitine  compared  The  i n the fed state.  The E f f e c t s  ester  measured.  were  c a r n i t i n e and  i n a l l of the  determined i n  carnitine  The r e s u l t s  from  i n the previous  these  study i n  f o r 72 h o u r s .  of Intravenously  Injected  [^^C]Carnitine  Rat B i l e Bile  injection esters serum  was c o l l e c t e d of labelled  from  s i x rats  carnitine.  following  The t y p e s  of  intravenous carnitine  i n t h e b i l e and t h e s p e c i f i c a c t i v i t y o f both t h e and t h e b i l e c a r n i t i n e ,  were  determined.  Possible  22 associations  between the  extrahepatic  sources  3.3  The  Carnitine  The  al.,  Mikhail of  four  malabsorption  1984;  Frohlich  and  et  and  the  of  might  exist  I f an  carnitine  during  malabsorptive  states  Before  investigating  this further,  should  Seventeen collected  from  cholelithiasis. carnitine  and  be  or  that  i n humans (Seccombe  Bohmer e t  al.,1974;  depletion  excessive  bile  might  result  losses.  the  carnitine  samples  content  determined. duodenal  fluid  ten  patients  with  adult  These  were  suspected  samples were t h e n a n a l y s e d  esters.  et  enterohepatic c i r c u l a t i o n  bile-rich  its  Duodenal F l u i d  have i n d i c a t e d  a l . , 1980;  Mansour,1976).  and  examined.  Human B i l e - R i c h  studies  c a r n i t i n e i s present,  human b i l e  hepatic  c a r n i t i n e were  Content  r e s u l t s of  carnitine  of  bile  for  of  CHAPTER  Methods  and  4  Materials  23  4.1 C h e m i c a l s All  and I s o t o p e s  c h e m i c a l s and i s o t o p e s were p u r c h a s e d  Sigma C h e m i c a l otherwise  Company, S t . L o u i s ,  MO.,  from t h e  63178, e x c e p t where  noted.  r 1 - C 1 A c e t y l - C o e n z y m e A, 57.6 mCi/ mmol, Amersham L t d . , Arlington Heights, I l l i n o i s . 1 4  L - r U - ^ C j A s p a r t a t e , 227 C i / m o l , Amersham L t d . , A r l i n g t o n Heights, I l l i n o i s . Carnitine Acetyltransferase, units/mg p r o t e i n . L-Carnitine  pigeon  C i t r a t e Synthase, Quebec.  H y d r o c h l o r i d e , 58 mCi/mmol, Heights, I l l i n o i s .  (ethylenedinitrilotetraacetic  Italy. Amersham  acid),  disodium  salt.  B o e h r i n g e r Mannheim  (N-2-hydroxyethyl piperazine-N-2-ethane s u l f o n i c BDH C h e m i c a l s Canada L t d . , V a n c o u v e r , B.C.  N-ethylmaleimide  85  B o e h r i n g e r Mannheim B i o c h e m i c a , D o r v a l ,  Glutamate-oxaloacetate transaminase, B i o c h e m i c a , D o r v a l , Quebec. HEPES  b r e a s t muscle,  H y d r o c h l o r i d e , Sigma-Tau Company, Rome,  L-Tmethyl-l^Cl Carnitine Limited, Arlington  EDTA  from  acid,  (NEM).  S o m n o t o l , sodium p e n t o b a r b i t a l (65 mg/ml) i n an aqueous p r o p y l e n e g l y c o l b a s e , M.T.C. P h a r m a c e u t i c a l s , M i s s i s s a u g a , Ont. TDGA  ( t e t r a d e c y l g l y c i d i c a c i d ) , d o n a t e d by D r . John Dean and P r o f e s s o r o f P h a r m a c e u t i c a l S c i e n c e , U n i v e r s i t y of B r i t i s h Columbia.  T r a g a c a n t h Gum,  grade  I I I powder.  McNeill,  24 4.2 H o u s i n g Adult used.  and Care  male W i s t a r r a t s  These were  Developmental University in  groups  shavings  hour  off  (380.3  bred and r a i s e d  ± 4.4 g , mean ± S E ) w e r e either  Medicine or i n the Animal  of B r i t i s h  Columbia  (UBC).  o f two o r t h r e e i n p l a s t i c f o r bedding.  Chow a n d w a t e r 12  of Animals  cycle  a t 4 AM.  They  with  the l i g h t s  A l l surgical  Care  Lighting  o f Somnotol  according  (1980)  was p l a c e d o n i t s b a c k  its  body  beneath cavity bile  with  before the surgery.  to Waynforth  incision  Bile  with  was  the l i v e r ,  on a h e a t i n g  duct.  resistance The b i l e  gently  was f e l t  from  d u c t was t h e n  the  surrounding tissue  was  inserted  around  collected  pad w h i c h  rectally.  and a s h o r t  the duct  maintained  of the xiphoid directly  out of the peritoneal the attachment  carefully length  t o form  Each  A 2 cm  p i e c e o f duodenum, l o c a t e d  was p u l l e d  BW)  some m o d i f i c a t i o n s .  w a s made a t t h e l e v e l  and a s m a l l  until  1968).  an i n t r a p e r i t o n e a l ( I P )  t e m p e r a t u r e , 37 °C, m e a s u r e d  cartilage  variations i n  ( 6 . 5 mg/100 g b o d y w e i g h t ,  rat  lateral  diurnal  between  Collection  Rats were a n a e s t h e t i z e d  immediately  wood  p r o c e d u r e s were c o n d u c t e d  (Bohmer and Bremer,  injection  with  housed  t u r n e d o n a t 4 PM a n d t u r n e d  serum  4.3 R a t B i l e  were  was on a r e v e r s e d  f o r possible  levels  of the  access t o Purina Rat  8 a n d 10 AM t o c o n t r o l carnitine  Centre  The r a t s cages  had f r e e  at a l l times.  at the Centre f o r  of the  dissected  of doubled  two l o o s e  from  thread  ligatures.  25 The  bile  duct  was t h e n  semi-transected (internal  diameter  the  opening  the  liver  was  then  sealed  Bile hours.  was c o l l e c t e d f r o m  Bile  carnitine affect The  with rpm  t h e serum  from  bile  beside  bile  was  the peritoneal  the heating  rat for a total  tubes  pad a t  15  vein  of each  The b l o o d  rat.  to determine  of  g BW.  After  into  Freezing  does n o t  (Seccombe,  1981).  neutrality.  a blood  sample  R a t s were  then  was c e n t r i f u g e d  a t 4 °C a n d t h e s e r u m  on serum  minutes,  a density  to confirm  collection,  carnitine analysis.  drainage,  every  o f two  f o r subsequent  a t - 2 0 °C.  c o l l e c t e d a t 15 m i n u t e  collection  flow  samples were a l i q u o t e d  of b i l e  Somnotol.  f o r 5 minutes  bile  Once b i l e  was c o l l e c t e d i n t o  carnitine concentration  the t a i l  - 2 0 °C u n t i l  cannula  was c a l c u l a t e d a s u l / m i n / 1 0 0  two h o u r s  excess  (towards  of the  into  t u b e was r e p l a c e d  a n a l y s i s and f r o z e n  samples were  to  Bile  pH o f t h e s a m p l e s w e r e m o n i t o r e d  taken  at  flow  each  conical polystyrene  After  out, inserted i n  g r a v i m e t r i c a l l y assuming  t u b e was w e i g h e d ,  disposable  glue.  cannula  as t h e r a t .  determined  1 g/ml.  held  i t was  the pancreas  was r e p l a c e d  tubes  The c o l l e c t i o n  flow  each  test  drawn  of entry  cyanoacrylate  sutured.  while  A polyethylene  beyond  The p o i n t  and t h e i n c i s i o n glass  forceps  was t h e n  forward  the i n t e s t i n e  same h e i g h t  and  with  with  scissors.  0.5 mm)  f o r 3-4 mm).  preweighed the  fine  and pushed  established, cavity  with  held  removed  the effect  killed  a t 3,000 and  I n one c a s e , intervals  was  frozen  the blood  throughout the  of f l u i d  loss,  carnitine concentrations.  due  26  4.4  Human B i l e - R i c h Bile-rich  Duodenal  duodenal  adult  fluid  and  female,  The  samples were c o l l e c t e d  Shaughnessy  patients  Hospital University  instructed  to fast which  nasogastric  tube  duodenum and  was  basal fluid  patients  then  concentrated Toronto, the  gallbladder  4.5  12  was  Originally,  collected  which  was  1958, not  carnitine  (Marquis  of  and  the  10  AM  first  were  the  or  1 PM.  segment  by  of  of  Patients  before  of  A the  fluoroscopy.  collected.  ( I V ) w i t h 80  °C  the  In  The  units  assay Fritz,  release  of  1928). at  15  One  minute  for later  Carnitine  ester  or  of  carnitine  i n Serum and  molitor  1964).  r e p l a c e d by  requires CoA  bioassay using  the  (Fraenkel,  a colorimetric  to c a r n i t i n e  Free  Bile  Assay  Tenebrio  used  and  analysis.  organism,  now  A l l  hydrolysis  by  specific  from  fluid  intervals.  measured  was  bile  two  was  this  very  the  to prevent  carnitine  requiring  In  at  s a m p l e was  (Ivy et a l . ,  then  Principle  1951).  male  (Department  hours  confirmed  This stimulates  f r o z e n a t -20  carnitine  ten,  cholelithiasis.  Columbia).  D e t e r m i n a t i o n of L - C a r n i t i n e  4.5.1  The  Urquhart  conducted  injected  samples were n e u t r a l i z e d stored  from  p a n c r e o z y m i n - c h o l e c y s t o k i n i n (Boots Drugs L t d . ,  Ont.).  samples were  N.  placed into  i t s position  obtained  the G a s t r o e n t e r o l o g y U n i t  least  either  some c a s e s , a were  by  of B r i t i s h  f o r at  was  was  Collection  with suspected  f o r Dr.  Pathology,  procedure  Fluid  (Friedman,  commercial i s measured  CAT  assay 1958).  27  spectrophotometryically acetyl-CoA.  The  after  reaction  i n c u b a t i o n of  i s as  each  sample  with  follows:  CAT L-Carnitine  In  + Acetyl-CoA  1972,  Cederblad  sensitivity  of  introducing  the  labelled  with this  carnitine  and  addition  1 4  of  free  formed  amount o f  This  a product  (McGarry and  of  the  (1977) have suggested  superior  to  Another method  of  exchange  limitation  this,  use  McGarry  (DOWEX) r e s i n i s then  Instead,  P a r v i n and  charcoal  slurry  adsorbs  of  and  sample. of  overcome  One  the to  by  which  free  the  F o s t e r , 1976)  1977),  forward  or  binds  reaction.  that N-ethylmaleimide  the  of  ["^C]acetyl-CoA  supernatant  i n the  to  Parvin (NEM)  and is  tetrathionate.  (1972) i s the  unreacted  be  Pande,  of  incubation i s  acetylcarnitine  can  CoA  by  amount  reversibility of  +  the  range  The  carnitine  dissociation  (Parvin  picomole  after  i s the  tetrathionate  CoA,  the  C] acetyl-CoA.  acetyl-CoA.  N-ethylmaleimide  place  [  Lindstedt increased  into  method  i . e . the  Pande  of  to the  reaction:  the  assay  use  > Acetylcarnitine  and  acetylcarnitine  proportional problem  the  <  slurry  Pande  the  Foster  of  and  Lindstedt  a DOWEX c o l u m n t o s e p a r a t e  from  and  free  Cederblad  [14c]acetylcarnitine. (1976) added  to each  any  reaction  unreacted  ( 1 9 7 7 ) recommend  containing acidified  n u c l e o t i d e compounds and  the  an  In  ion  vial;  the  [^ C]acetyl-CoA. 4  the  ethanol.  addition Charcoal  e t h a n o l i s needed  to  of  a  28  prevent  the  well  acetyl-CoA.  as  charcoal,  4.5.2  retention  however,  Procedure All  of  carnitine  Both  i s faster  and  carnitine  assays  were  t i p tubes  microliters  of  sample,  done by  each  assessment  carnitine  (free  of  each  free  were k e p t to prevent  samples,  standards  (1976) r e p o r t e d that  (1981) found exaggerated  that the  protein  protein  sulfate  and  on  total  The  assays  was  The  were  prepared  control  ice during  the  used  and  curve  water.  was  preparation  hydrolysis  controls  (LaRusso,  was  carnitine.  Subsequently, added  by  sample 200  ( 0 . 0 8 3 M)  was  carnitine  requires  the  prior  For  samples,  the  barium  not  of  the  McGarry  also  barium  hydrolysis  of  hydroxide  was  200  of  ul  zinc  free  hydroxide  The of  very  deproteinized.  addition  for neutralization.  Seccombe  contains  for analysis  u l of  and  proteins  bile  i t was  deproteinized  necessary,  serum  though  1984),  precipitated to each  from  Even  were  Although,  t h i s was  interference  results.  ( 0 . 0 8 7 M)  these  carnitine  were  esters.  to assaying f o r c a r n i t i n e .  little  Fifty  control  and  carnitine  The  standard or of  i n disposable  mm).  carnitine  i n order  Foster  x 75  d i l u t i o n of  for  prior  (12  standard  A l l tubes  All  performed  carnitine  assay  the  expensive.  A  human s e r u m . the  least  plus e s t e r i f i e d carnitine).  in duplicate.  serial  effective;  as  used  conical  the  acetylcarnitine  methods a r e  polystyrene  for  and  assay  of  carnitine added  total esters.  first  and  29  the  samples were  addition sulfate  of the z i n c precipitate  15 m i n u t e s In  sulfate. were  The p r o t e i n  removed  with  supernatants  ( 0 . 8 mM),  NEM  ( 2 mM)  and [ 1 -  The  reaction  was i n i t i a t e d  (containing  4.0  1 unit  a t room  was p r e p a r e d  mixing  1 4  The  ( 2 0 mM),  C]acetyl-CoA  by t h e a d d i t i o n After  pH  ( 2 . 5 uM,  the reaction  activated acid  before  o f 50 u l o f  was  Each batch of  use and c o n t a i n e d  c h a r c o a l , 54.5 ml o f e t h a n o l ,  a n d 4.25 m l d i s t i l l e d  with the slurry,  terminated  the reaction  tubes  water. were  i c e , a n d c e n t r i f u g e d a t 3 , 5 0 0 r p m f o r 15 m i n u t e s  The  supernatants  centrifugation  with  t o c l e a n tubes  of the f i n a l  supernatant  to a disposable mini-counting vial  distilled which  transferred  water.  i s difficult the water  Finally,  placed  at 4  °C.  and  repeated.  An a l i q u o t added  were  7.6,  incubationfor  charcoal slurry.  immediately  g of neutralized  buffer  of enzyme).  temperature,  1.25 m l 8 5 % p h o s p h o r i c  on  i n new p o l y s t y r e n e  uCi/nmol).  600 u l o f a s t i r r e d  After  barium  100 u l o f t h e  c o n t a i n e d HEPES  0.02  slurry  before  by c e n t r i f u g a t i o n f o r  were p l a c e d  activity  with  and  °C  100 u l o f a b u f f e r e d s u b s t r a t e s o l u t i o n . solution  60 m i n u t e s  a t 75  °C.  specific  CAT  f o r 2 hours  preparation f o r the assay,  substrate EDTA  incubated  a t 3,500 rpm a t 4  deproteinized tubes  then  As t h e s u p e r n a t a n t to pipette,  i n the v i a l  fluid  then  c o n t a i n i n g 400 u l  contained ethanol,  the pipettor  t o ensure  5 ml o f s c i n t i l l a t i o n  ( 5 0 0 u l ) was  t i p was  quantitative  rinsed  transfer.  ( A C S ) was a d d e d  before  30  the a  v i a l was  mixed  and  counted.  Beckman LS9000 S c i n t i l l a t i o n  standard  quench  The  analysis beyond assay of  the was  curves  a s s a y s were was  range  of  repeated  acylcarnitine of  concentration.  absolute  g  and  the  4.6  15  was  Principle P a n d e and  assay  for  the  following  two  of  the  with  free  and  carnitine  diluted  derived  an  on  external  and  total  linear  regression  concentrations  curve  (0  to  samples.  by  carnitine  quantity  bile  both  standard  with  100  The  were  uM),  the  concentration  subtracting  the  from  the  total  variation  was  approximately  of  carnitine  concentration  collection  Acetylcarnitine  4.6.1  If  the  carnitine  minute  counter  plotted  Interassay  the  each  then  free  Both BW  for  performed.  concentration  samples were counted  correction.  standard  carnitine  The  in  the  carnitine  bile  5%.  per  were c a l c u l a t e d  100  for  sample.  Analysis Method  Caramancion  (1981) devised  determination  of  carnitine  a  radioisotopic  based  on  the  reactions: CAT  1)  Acetylcarnitine  2)  Acetyl  CoA  First,  + CAT  acetyl-CoA. the  formation  [  +  1 4  CoA  Citrate C]oxaloacetate  causes  Citrate of  > Acetyl-CoA  the  dissociation  synthase  [^ C]citrate 4  +  Carnitine  Synthase > [ of  C]citrate  carnitine  (E.C.4.1.3.7) then from  1 4  acetyl-CoA  and  and  catalyzes  31  [l^C]oxaloacetate.  The amount o f [ ] c i t r a t e  proportional  amount o f a c e t y l c a r n i t i n e i n t h e  Excess  t o the  [ ] o x a l o a c e t a t e i s removed  addition  by t h e  formed i s sample.  subsequent  o f g l u t a m a t e - o x a l o a c e t a t e t r a n s a m i n a s e (GOT,  E . C . 2 . 6 . 1 . 1 )  which  catalyses  the  formation of  r 14  [  C]aspartate.  A cation  exchange  resin  i s used  t o remove  [ "^C]aspartate. 4.6.2  Procedure The  James  a c e t y l c a r n i t i n e assays  (Department  laboratories. kept  Used  Unless  o f each  deproteinized  otherwise noted, hydrolysis  barium  hydroxide  ( 0 . 0 8 3  M).  All 20  the  assay  curve  every  Freshly contained  phosphate  3 0 minutes reduced  ranged  mM d i t h i o t h r e i t o l .  esters. were  sulfate  same v o l u m e o f  by t h e  found use  at  3,500  rpm a t  0 . 0 5 uM t o 2 0 uM.  from  (1981)  that  the  sensitivity  of dialysed  enzymes.  a t 0 - 4 ,°C a g a i n s t 5 0 0 m l o f  (pH7 . 4 ) .  The d i a l y s i n g f l u i d was  f o r two hours.  CoA r e a g e n t  0 . 2 mM CoA,  o f the  minutes  10  t h r e e enzymes were d i a l y s e d  changed  3.2  was i m p r o v e d  mM p o t a s s i u m  carnitine  were  T h e p r e c i p i t a t e was t h e n  for  Pande and Caramancion of  o f the  addition  by c e n t r i f u g a t i o n  °C. T h e s t a n d a r d  Hospital  a l l solutions  of 400 ul of zinc  by a d d i t i o n  M) f o l l o w e d b y t h e  4  by L e i g h t o n  ( 1 0 0 u l ) , standard o r blank  sample  ( 0 . 0 8 7  removed  performed  o f P a t h o l o g y , UBC) i n Shaughnessy  on i c e t o p r e v e n t  Aliquots  were  was p r e p a r e d  1 0 mM p o t a s s i u m 400 ul of this  i n advance and  diphosphate was t h e n  (pH9 ) and  left at  32  25  o  C f o r 30 m i n u t e s  potassium To  phosphate  nM L - [ U -  minutes  800  phosphate l 4  °C.  added, and l e f t this  potassium The  f o r 15 m i n u t e s .  ( 1 2 mm  added  i n the order  Immediately  w i t h 4 0 u l 0.6 M  p l u s 6 0 u l o f 67 mM  was p e r f o r m e d  tubes  x 75 mm).  EDTA  i n conical  To e a c h  tube,  ( p H 7.4) a n d 30 u l f r e s h l y  reduced  unit  dialysed  0.8 u n i t  at  25  then 20  The r e a c t i o n  °C f o r 20 m i n u t e s . added  minutes.  3 2 0 mg  stand  removed  were  then  added  f o r 10 m i n u t e s  by c e n t r i f u g a t i o n  scintillation which  counter standard  (pH 7 . 4 ) .  fluid  were  were  counted  w i t h an e x t e r n a l curve  was t h e n  mM  1.5  CAT a n d 5 u l  tube  incubated  p l u s GOT  were  further  which  temperature.  combined  standard  was a l l o w e d  The r e s i n  was  a t 3000 rpm. p l u s 5 ml o f  i n mini-counting  on a Beckman L S 9 0 0 0  plotted  was  ( A G 50W-X8, B i o R a d ) a n d  f o r 10 m i n u t e s  then  test  CoA r e a g e n t ,  0.3 m l o f t h e s u p e r n a t a n t , 0.6 m l o f w a t e r  vials  chilled  m i x t u r e was t h e n  t o each  a t room  was  10 u l 1 2 0 - 1 5 0  30 u l o f g l u t a m a t e  of the resin  acid  before the  a n d t h e m i x t u r e was i n c u b a t e d f o r a  0.6 m l w a t e r to  synthase,  GOT a n d  the following  EDTA  citrate  mM  borosilicate  g i v e n : 155 u l s a m p l e ,  [^C]oxaloacetate.  56  w a s i n c u b a t e d f o r 10  20 u l o f 1 M p e r c h l o r i c  was n e u t r a l i z e d  assay  solution  alpha-ketoglutarate,  (227 Ci/mol)  Then,  hydroxide  u l of a  ( p H 7 . 4 ) , 12 u n i t s / m l d i a l y s e d  C]aspartate  a t 25  o f 8 0 0 u l o f 0.5 M  (pH 7 . 4 ) .  2 0 0 nM E D T A , 5 mM  potassium  assay,  the addition  prepare  containing  50  before  quench  Scintillation  correction.  The  and t h e c o n c e n t r a t i o n s o f  33  acetylcarnitine samples were  4.7 4.7.1  from  in  the  each  initial  and  determined.  final  Only  bile  bile collection  periods  assessed.  Carnitine  Ester  Principle  of  Thin-layer extensively long-chain consuming relative another  to  Separation Method  chromatography distinguish  carnitine and  may  method and  medium-chain  has  been  carnitine  chloroform  (Pande,  the  esters other  esters  1981).  in  Free  of  on  in  and  soluble  by  bile,  short  of  and  but  i n methanol  the  and  long-chain  quantitatively  carnitine  the  solubility  water-soluble  only  time  determine  the  Theoretically, be  and  i t is  esters  carnitine  are  are  b i l e can  forms  based  used  medium  To  carnitine  devised  esters  been  short,  quantitative.  i t s esters. carnitine  has  Unfortunately,  long-chain  long-chain  carnitine  be  (TLC)  between  esters.  not  amounts of  carnitine  from  sample  separated  extraction  with  chloroform:methanol.  4.7.2  V e r i f i c a t i o n of To  verify  the  carnitine  esters  collected  from  vein.  Waynforth  quantitative  The  separation  chloroform:methanol  anaesthetized  [l-l^C]carnitine jugular  by  Method  rats  after  ( s p e c i f i c a c t i v i t y 2.5 jugular  (1980) w i t h  one  vein  was  exception.  of  extraction, injection  of  bile  was  0.5  uCi  mCi/nmol) i n t o  cannulated The  long-chain  hole  a  according into  the  to vein  34  was 21  made b y i n s e r t i n g gauge h y p o d e r m i c  collected  contained  t h e t i p of a bent  needle  into  (45 degrees)  the blood  ["*" C]carnitine 4  vessel.  The  bile  and [ " ^ C ] c a r n i t i n e  esters. To 1:1  separate  (v/v)  t h e aqueous and l i p i d  chloroform:methanol  sample, mixed  and l e f t  was a d d e d  to stand  u l of water  resulting  aqueous and l i p i d  down u n d e r methanol,  and t h e p r o c e s s  respectively.  were  developed  were  removed  visualized  scraping  lipid  bile  from  (1973).  The dried  applied to  standards.  solvent  system:  v/v/v/v/v) as 30 m i n u t e s ,  The  was d e t e r m i n e d  the s i l i c a  i n 1 cm  by bands.  3.  Only  3% o f t h e l a b e l  phase migrated  with  the long-chain  ester standard. migrated  The r e c o v e r y  The o n l y  with  label  present  the long-chain  i nthe  carnitine  was 9 6 . 5 % . T h e r e f o r e ,  chloroform:methanol  the  and t h e s t a n d a r d s  t o i o d i n e vapour. carnitine  The  ammonia,  After  the s o l v e n t tank  a r e shown i n F i g u r e  with  with  (55:50:10:7.5:2.5  of the labelled  fraction  fraction. of  acid  by e x p o s u r e  i n t h e aqueous  carnitine  separated,  concentrated  t h e p l a t e s and c o u n t i n g  results  found  water,  b y Gumpen a n d N o r u m  distribution  The  formic  along  i n the following  chloroform,  concentrated  were  twice.  The samples were then  plates  plates  was r e p e a t e d  f o l l o w e d by  n i t r o g e n , a n d r e d i s s o l v e d i n 50 u l o f w a t e r a n d  G p l a t e s (500 microns)  described  minutes.  was a d d e d  phases were  silica  methanol,  625 u l o f  t o 50 u l o f e a c h  on i c e f o r 30  Then, a f u r t h e r 440 u l o f c h l o r o f o r m 250  fractions,  ester  the extraction  quantitatively  separates  35  FIGURE 3 V e r i f i c a t i o n of the separation of l o n g - c h a i n c a r n i t i n e e s t e r s f r o m o t h e r t y p e s o f c a r n i t i n e by l i p i d extraction using t h i n - l a y e r chromatography (TLC). 2 0 0 u l o f b i l e was c o l l e c t e d from a r a t i n t r a v e n o u s l y i n j e c t e d w i t h [l^C]carnitine. A f t e r e x t r a c t i o n o f t h e b i l e , s i l i c a TLC was p e r f o r m e d o n t h e r e s u l t i n g a q u e o u s ( s o l i d l i n e ) a n d l i p i d (dashed l i n e ) phases. T h e s i l i c a was t h e n s c r a p e d i n 1 cm b a n d s a n d c o u n t e d i n s c i n t i l l a t i o n f l u i d .  36  long-chain  carnitine esters  carnitine  4.7.3  in  were  then  water, with  Carnitine  fasted  4.8  the  and  200  initial  initial  and  treated  separated  above.  hydroxide,  into  phases ul  of  neutralized  carnitine  concentration  c a r n i t i n e assay i n the  of  collection  from  the  collection  extract.  selected  period  esters  free  aqueous  i n randomly  final with  The i n 50  medium-chain  the  bile  of  periods  bile fed  of  rats  both  TDGA.  Procedure  no  hours  before  the  scheduled  again  before  bile  weight.  of  for carnitine.  were a n a l y s e d  r a t s were weighed  was  barium  and  i n the  cages with  bile  ul  assayed  clean  the  forms  redissolved  c a r n i t i n e found  the  rats  Fasting The  of  c o n t r o l was  described  subtracting  esters  from  as  short  determined  concentration  both  by  of  or  nitrogen,  with  quantity  carnitine  and  phases  s u l f a t e and  derived  samples  standard  under  hydrolyzed  The were  lipid  dried  zinc  other  Used  sample,  a q u e o u s and  the  bile.  Procedure Each  from  food  and  but  on  free  the  The basis  individually  access  surgery.  collection.  reported  placed  The  to  r a t s were  amount of of  the  fresh  into water  72  weighed  carnitine in  initial  body  37  4.9  Tetradecylglycidic After  suspended of  t h e 72 h o u r  were  was t h e n  procedure.  anaesthetized rats  were  i n t u b a t e d w i t h TDGA  hours  vehicle  of  alone.  Bonferoni's significance  course  carnitine  attached  observed  closely  the animals  were  initiated.  Two  was after  additional  and i n t u b a t e d w i t h t h e  vehicle  used.  served  Bile  assessed  these  of v a r i a t i o n  was  as  controls  then  gravimetrically.  experiments  used  was a l s o to test  Where s i m u l t a n e o u s correction o f <0.05.  on t h e T a b l e s  of each  animal  the f l u i d  , unless otherwise noted.  was e x c l u s i v e l y  differences.  Each  content  to attain  were  made,  overall  The c h a n g e s i n t h e  determined from  The  differences are  and F i g u r e s .  of the b i l e  determined.  cases  for statistical  Statistical  were  are reported  I n some  comparisons  was a p p l i e d  experiment  mg  T h e TDGA  that  These animals  o b t a i n e d from  coefficient  indicated  collection  2.5  Data  t h e mean ± S E  T-test  later,  and t h e f l o w r a t e  data  were  f o r 72 h o u r s  the administration  The  To e n s u r e  the animals  and b i l e  4.10 A n a l y s i s  rat.  the esophagus.  injected.  Two  fasted  tragacanth  collected  Administration  i n a towel w h i l e a p o l y e t h y l e n e tube  regurgitated,  this  the  rats  a s y r i n g e was p l a c e d i n t o  not  as  fast,  T D G A / 1 0 0 g BW w a s a d m i n i s t e r e d t o e a c h  solution  for  P r e p a r a t i o n and  i n 0.5 m l t r a g a c a n t h ( F r e n c h e t a l . , 1 9 8 5 ) .  was r e s t r a i n e d to  Acid  by c o m p a r i n g  the f i r s t  and  last  the  38  collection used  to  periods.  confirm  these  Duncan's m u l t i p l e differences.  range  test  was  also  CHAPTER 5  The C a r n i t i n e and C a r n i t i n e E s t e r Content  of Rat  Bile  39  5.1  Results  5.1.1  Bile  Flow  The b i l e shown  i n Figure  collection the  flow  period  subsequent  differences  5.1.2  4.  during The was  flow  collection  of  free  to  increased  The amount sample acyl  of b i l e  and t o t a l  collection. experiment,  5.1.3  Acyl bile  collection constant.  the b i l e  2.  was  carnitine  esters.  from  70  i n each  i n Figure  6.  increased  15  minute  The amounts o f  during  bile during  of a c y l c a r n i t i n e again  the  increased.  i n Bile of the c a r n i t i n e esters  Long-chain  carnitine.  carnitine  the  The  increased  free c a r n i t i n e decreased  the percentage  shown i n T a b l e  while  of the experiment.  c a r n i t i n e again  Carnitine Esters  carnitine in  and t o t a l c a r n i t i n e  remained  i s illustrated  Although  further  periods.  of c a r n i t i n e present  The d i s t r i b u t i o n  of  5.  during  the course  minute  f o r any o f  T h e r e w e r e no  of c a r n i t i n e i n the a c y l form  83% during  than  o f f r e e , a c y l and t o t a l  carnitine concentration  percentage  higher  15  Bile  a r e shown i n F i g u r e  concentrations  of the experiment i s  f o r the i n i t i a l  periods.  among t h e c o l l e c t i o n  C a r n i t i n e Content  bile  rate  significantly  The c o n c e n t r a t i o n s the  the course  Only  c a r n i t i n e e s t e r s made up  Approximately  i n t h e form  of r a t b i l e i s  of short  one q u a r t e r  54% of the  and  total  medium-chain^  of t h i s  was  as  29%  40  15  30  45  60  75  90  105  120  TIME OF BILE COLLECTION (min)  FIGURE 4 B i l e f l o w ( m i c r o l i t e r s / m i n / 1 0 0 g BW) d u r i n g b i l e c o l l e c t i o n from, f e d r a t s (mean ± S E ) . B i l e was c o l l e c t e d i n e i g h t c o n s e c u t i v e 15 m i n u t e s a m p l e s a n d t h e f l o w r a t e was a s s e s s e d gravimetrically. F l o w was g r e a t e r i n t h e f i r s t collection p e r i o d t h a n subsequent p e r i o d s ( a t l e a s t p<0.05). There w e r e no f u r t h e r d i f f e r e n c e s b e t w e e n c o l l e c t i o n p e r i o d s .  41  FIGURE  5  The c o n c e n t r a t i o n 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 i n b i l e f r o m f e d r a t s c o l l e c t e d a t 15 m i n u t e i n t e r v a l s ( m e a n ± S E ) . The c o n c e n t r a t i o n s o f t o t a l a n d a c y l c a r n i t i n e w e r e s i g n i f i c a n t l y g r e a t e r a f t e r 120 m i n u t e s o f b i l e c o l l e c t i o n t h a n a f t e r 15 m i n u t e s ( p < 0 . 0 0 1 ) . The c o n c e n t r a t i o n o f f r e e c a r n i t i n e d i d not change d u r i n g b i l e c o l l e c t i o n .  I  I  I  I  I  15  30  45  60  75  TIME  OF  BILE  I  I  I  90  105  120  COLLECTION (min)  FIGURE 6 The a m o u n t 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 i n b i l e c o l l e c t e d f r o m f e d r a t s a t 15 m i n u t e i n t e r v a l s ( n m o l / 1 5 m i n / 1 0 0 g BW, mean ± S E ) . T h e a m o u n t s o f t o t a l a n d a c y l c a r n i t i n e were s i g n i f i c a n t l y g r e a t e r a f t e r 120 m i n u t e s o f b i l e c o l l e c t i o n t h a n a f t e r 15 m i n u t e s ( p < 0 . 0 0 1 ) . The a m o u n t o f f r e e c a r n i t i n e d e c r e a s e d o v e r t h i s same t i m e interval (p<0.02).  43  TABLE  2  The Amounts o f C a r n i t i n e and C a r n i t i n e E s t e r s I n i t i a l B i l e Sample C o l l e c t e d from Fed Rats Type of Carnitine  i n the (n=4)  nmol/15 min/ 1 0 0 g BW  Free  1.04  ± 0.22  17.2  ±  3.9  S h o r t and Medium-chain  3.29  ± 0.21  53.6  ±  2.9  Long-chain  1.81  ± 0.26  29.2  ±  3.4  Total  6.13  ± 0.18  100  ±  2.9  44  acetylcarnitine  (Table  acetylcarnitine  increased  collection.  a  constant,  5.1.4  Table  of  the  course  total  of  of  the  carnitine,  bile  i t remained  Carnitine  Seventeen  esterified.  of  concentration  during  percentage  concentration  4.  neither  The  however.  Serum The  As  3).  In  carnitine in rat  percent  the  a change  of  one  i n the  of  the  total  serum  i s shown  carnitine  was  additional rat studied,  there  amount of  the  carnitine esterification  c a r n i t i n e nor  during  the  course  of  was  degree  the  experiment.  5.2  Discussion Carnitine  bile  and  serum.  colleagues 34.3  uM  c a r n i t i n e i n the  comparable  study used which rats  Results  of and  an  average  collection r a t s used, those  of  Sprague-Dawley had  published  f o r the  of  other  of  46.3  by  two  uM  Gudjonsson found  rats.  In  In  and  rats weighing 18  rat  and  this  found  a d d i t i o n to  differences exist  in  and  27.3  ( n = l l ) was  period.  Gudjonsson  for  concentrations  Gudjonsson  bile  time  been f a s t e d  were used  account  in similar  (1985b) concur.  experiment,  number  i s present  in  a  the  between  this  colleagues:  Gudjonsson  between  and  hours.  in this  experiment.  lower  concentrations  190  Larger, These of  fed,  240  g  Wistar  differences  c a r n i t i n e found  may in  in  TABLE  3  The C o n c e n t r a t i o n o f A c e t y l c a r n i t i n e the I n i t i a l and F i n a l F i f t e e n M i n u t e Periods  Initial umol/1  Sample % of Total  i n Rat B i l e from Bile Collection  Final umol/1  Sample % of Total  MEAN  7.4  15.6  SE  0.7  1.7  1.3  1.6  9  9  9  9  N *  p<0.01 v e r s u s  initial  12.5*  sample  17.7  TABLE The  MEAN SE  4  C o n c e n t r a t i o n o f C a r n i t i n e and C a r n i t i n e E s t e r s i n Rat Serum A f t e r B i l e C o l l e c t i o n (n=5) Free Carnitine umol/1  Total Carnitine umol/1  % Acyl Carnitine  48.9  58.9  17.0  4.4  5.0  2.0  47  the of  bile  collected  collecting Bile  the  bile  flow  by  Gudjonsson  was  rate  and  colleagues.  t h e same i n b o t h  was  measured  amount o f c a r n i t i n e  i n each  The  studies.  to permit d e t e r m i n a t i o n of 15  minute  collection.  published  by  K u i p e r s and  c o l l e a g u e s (1985)  indicate  bile  decreases from  a p p r o x i m a t e l y 7.5  to  4.0  flow  ul/min/100  g BW  bile  collection  this  to a decrease  Kuipers  and  hour  subsequent reported (Bailey  hours.  that  the  Although  The  1984),  of  ul/min/100 carnitine  (Kuipers  g BW found  bile  g rat.  for.12 hours i n the b i l e  at which  flow  was at  the  in this  higher.  this and  experiment.  in bile  Assuming  Minuk  The  except f o r  i t was  discarding  in  in  et a l . , 1985).  e t a l . , 1982;  found  flow  experiment  the experiment  recommend  retained  amount o f c a r n i t i n e  e s t i m a t e d f o r a 350  in bile  have  1976).  collection  (Reuben  i t was  flow.  for nonanaesthetised rats  some i n v e s t i g a t o r s sample,  bile  however,  this  of  attributed  flow  et a l . ,  collection  minutes  They  increased  affect  throughout  that  sodium  does not  constant throughout  15  bile  but  investigators,  Cooper  rate  that  Data  initiation  dependent  a decrease  collection  reported  bile  was  Sarjeant,  be  of  initial  initial  bile Other  flow  to that  rate  acid  reported  pentobarbital  bile  initiation flow  also  e t a l . , 1975;  The similar  i n the b i l e  a n a e s t h e s i a caused  following  following  nonanaesthetised rats.  coworkers  pentobarbital the  i n the 2 hours  from  method  i n 24  a bile  hours  flow  of  over  24  h o u r s , t h e amount  would  be  a p p r o x i m a t e l y 1%  may 8 of of  the  48  rats 0.4  total  body  pool  u m o l / 2 4 h/100  obtained  from  Gudjonsson there the  g BW).  the  and  every  data  bile  (1-3.5%  The  of  and  of  the  concentration  bile  increased  This  was  due  amount of  the  sample  that to  reported.  The  a n a l y s i s of  between 69%  and  75%  contains The  carnitine  serum  from  colleagues  approximately hepatic (Brass al.,  80%  course  remained  of  the  8,  but  the  that  This  carnitine  other  concurs  study  with  Skeletal  esterified  esterification  and  Hoppel,  1981;  1978;  Pearson  and  Brass  Tubbs,  to  1967;  the  such  sample  Bohmer  is  by  so  a  comparable  Gudjonsson  or  50  1978; et  or  indicated  fluid high  carnitine  Hoppel,  is  degree. 16%  reported  from  This  bile,  a l . , 1978)  ranges  and  true  This  23%  in  the  cause  demonstrated  the  et  in  initial  tissue  muscle  (Pace  g BW  throughout.  reported  body  rat,  experiment.  rat bile  esterified.  is esterified  (1978). 51%  i n the  rats in this  i n the  pool).  the  the  was  (1976).  carnitine  constant  esterification No  size  acylcarnitine;  known w h e t h e r  was  that  body  amount of  carnitine  (1985b).  esterification. and  and  carnitine  colleagues  the  pool  c a r n i t i n e / 2 4 h/100  carnitine  the  or  Lindstedt  i s more r e p r e s e n t a t i v e o f  are  the  umol  i n Chapter  I t i s a l s o not  body  and  i n c r e a s i n g amounts of  unknown.  umol  (1985b) estimated  and  phenomenon i s d i s c u s s e d  both  carnitine  total  free carnitine  final  (1.4  Cederblad  1.4  throughout  to  hours  The  colleagues  i s b e t w e e n 0.4  24  by  Pace  is whilst  to  62%  Pace  et  a l . , 1966).  49  The  c a r n i t i n e i n the outflow  liver,  however,  Only carnitine  one  i s 73% e s t e r i f i e d  quarter  esters  acetylcarnitine  carnitine The bile, or  In other  of other  are rarely  percentage  make up  Hoppel, Tubbs, have and  1981; 1967).  been  also  of long-chain greater  carnitine from  esters  1%  fluids,  carnitine ester. or  carnitine esters  in rat  f o r other  fluids  carnitine  c a r n i t i n e (Brass 1967;  long-chain  (Pace  accounted  Large  medium-chain  reported  1968;  of the t o t a l  during  of  In the l i v e r , long-chain  I n the serum,  1981)  the l i v e r  than  1978; Bohmer,  as  medium-chain  observed.  1.5-4% o f t h e t o t a l  reported  Hoppel,  t i s s u e s and  rat  et a l . , 1985).  are i n the form  short-chain  t i s s u e s of the r a t .  esters  and  i s the predominant  esters  3 0 % , was  (Sandor  of the s h o r t  i n the b i l e  acetylcarnitine.  concentrations  of v a s c u l a r l y perfused  and  1966; P e a r s o n  carnitine esters  e t a l . , 1978) and carnitine.  10%  perfusion  (Brass  Long-chain  f o r 15% of t h e c a r n i t i n e  vascular  and  (Sandor  exported  eta l . ,  1985). The bile,  concentration  i s above  of long-chain  the c r i t i c a l  palmitoylcarnitine  o f 15 uM  (Seim  and Z o g r a f i , 1 9 7 0 ) .  carnitine  e s t e r s , which  the form In  presence  and D a r g e l ,  Therefore,  1978;  long-chain  are not water-soluble,  are  probably  of m i c e l l e s i n b i l e .  summary, t h i s of r e l a t i v e l y  experiment large  in rat  micellar concentration for  Yalkowsky  in  carnitine esters  has demonstrated  the  amounts o f c a r n i t i n e and  50  carnitine in  the  esters  in rat  intestinal  Further,  bile  excretion.  represent  a  i s unique  carnitine esters  esterified  c a r n i t i n e than  establishment  carnitine  i n the  short  medium-chain  rat  and  small  explain  possible  and  a  other  of  bile  as  i n t e s t i n e may  recovery  greater  help  reported  after intravenous  injection  (Hahn e t  a l . , 1985;  Lastly,  i t i s possible  carnitine pool  studies  accounted 1984;  excretion.  for  by  Cederblad  that This  i n which  explain  1985).  source  might  suggestion  losses  Lindstedt,  alone 1976;  of  presence lumen of I t may  of the  also  the  subcutaneous  Rebouche et bile  of  fluids.  the  i n the  or  a l . , 1984).  serve  as  a  route  i s supported  carnitine losses  urinary and  percentage  l a b e l l e d c a r n i t i n e from  intestine  carnitine  more  endogenous  Ruark,  origin.  for  t i s s u e s or  an  carnitine  dietary  route  carnitine esters  of  that  i t contains  body  i n t e s t i n e (Sach'an a n d  the  suggests  s o l e l y of  i n that  long-chain  The  This  lumen i s not  may  Bile  bile.  could  not  by  and  body  be  ( R e b o u c h e and Yue  for  Fritz,  Engel, 1962).  CHAPTER 6  The O r i g i n  of  Carnitine  i n Rat  Bile  51  6.1 of  The E f f e c t s Rat  6.1.1  (Figure  Flow and Weight  flow  7).  course  decreased  The r a t e  their  body  Table carnitine times,  of bile  i n bile  Content  of fasted  were  of free  the acyl  The c o n c e n t r a t i o n s  carnitine  concentration  the course  15 m i n u t e  bile  illustrated  during the  14.1  ±  1.6%  i n Table  where t h e r e  acyl  and t o t a l  however,  total  At a l l  carnitine  than  i n the fed  between t h e  i n the fasted  of acyl  and  fed rats.  i n the fasted  acyl  and t o t a l  and t o t a l  from  6.  rats,  was l e s s  content  of free,  and f e d carnitine  while  the  free  d i d not change.  sample  f e d and f a s t e d  experiment.  lost  of the experiment  amounts o f f r e e ,  carnitine  hours  Bile  carnitine  over  The  rats  and n o r m a l l y  greater  increased  period  of  T h e r e w a s no d i f f e r e n c e ,  states.  72  d i d n o t change  The f a s t e d  one e x c e p t i o n ,  concentrations  between  flow  fasting  weight.  concentrations  The  after  5 shows t h e c o n c e n t r a t i o n s  with  state.  Loss  i n rats  of the experiment.  6.1.1.2 C a r n i t i n e  are  Content  Results  Bile  each  on t h e C a r n i t i n e  Bile  6.1.1.1 B i l e  of  o f a 72 h o u r F a s t  fasted  carnitine  and n o r m a l l y  T h e r e w e r e no except free  of the b i l e  fed rats  differences  i n the f i r s t  carnitine  found i n  upon  d i d not change  collection fasting. during the  52  15  30  45  60  75  90  TIME OF BILE COLLECTION •  FED  •  105  120  (min)  FASTED  FIGURE  7  B i l e f l o w ( m i c r o l i t e r s / m i n / 1 0 0 g BW) d u r i n g b i l e c o l l e c t i o n f r o m f e d a n d 72 h o u r f a s t e d r a t s ( m e a n ± S E ) . B i l e was c o l l e c t e d i n e i g h t c o n s e c u t i v e 15 m i n u t e s a m p l e s a n d t h e f l o w r a t e was a s s e s s e d g f a v i m e t r i c a l l y . A t a l l t i m e p e r i o d s , t h e b i l e f l o w d e c r e a s e d upon f a s t i n g . The l e v e l s o f s i g n i f i c a n c e a r e a s f o l l o w s : * = p < 0 . 0 2 , ** = p < 0 . 0 1 , *** = p < 0 . 0 0 1 .  The  Concentrations  of Free,  1  Acyl  TABLE 5 and T o t a l C a r n i t i n e  Consecutive 2  bile 3  collection  i n the Bile periods 5  o f Fed and F a s t e d  (15 minutes 6 7  Rats  each) 8  FREE FED  MEAN SE N  14.2 1.2 9  13.2 0.9 11  14.3 1.0 11  13.8 1.0 11  14.2 1.0 11  13.2 1.0 11  12.4 1.5 11  12.4 1.5 10  FASTED  MEAN SE N P  11.51 0.7 5 0.1378  11.4 1.1 5 0.2604  10.9 0.6 5 0.0470  11.0 0.5 5 0.0916  11.2 0.7 5 0.0780  12 .7 0.6 5 0.7536  12.9 0.4 5 0.8298  12.9 0.4 5 0.8224  FED  MEAN SE N  32.6 1.5 9  40.2 3.3 10  43.0 3.6 11  53.0 3.8 11  61.5 5.3 11  60.9 4.1 11  60.0 3.7 11  59.4 3.6 10  FASTED  MEAN SE N J>.  70.4 4.9 5 0.0000 *  73.7 1.2 5 0.0000 *'*  81.2 2.0 5 0.0000 *  85.4 2.0 5 0.0000  88.5 1.6 5 0.0048  90.2 1.3 5 0.0004 *  92.1 1.1 5 0.0000  89.3 3.5 5 0.0002 *  FED  MEAN SE N  46.3 1.1 9  53.2 3.3 10  57.0 3.3 10  67.0 3.7 11  71.0 3.9 11  74.2 3.8 11  70.5 3.3 11  72.3 3.0 10  FASTED  MEAN SE N P  ACYL  TOTAL  significant  81.9 85.1 4.5 1.9 5 5 0.0000 0.0000 * * a t o v e r a l l t h e 0.05 l e v e l  92.0 2.3 5 0.0000 using  96.4 99.7 102.2 105.0 102.9 1.6 1.6 1.5 3.6 1.1 5 5 5 5 5 0.0002 0.0002 0.0002 0.0000 0.0000 * * * * * B o n f e r o n i ' s c o r r e c t i o n b a s e d on 48 t e s t s  The  Amounts o f F r e e ,  Acyl  1  and T o t a l  Consecutive 2  TABLE 6 Carnitine  bile 3  i n the Bile  collection 4  periods 5  o f Fed and F a s t e d  Rats  (15 minutes each) 6 7 8  FREE FED  MEAN SE N  1.81 0.16 10  1.38 0.14 11  1.41 0.13 11  1.37 0.37 11  1.45 0.13 11  1.27 0.11 11  1.20 0.16 10  1.20 0.16 9  FASTED  MEAN SE N P  0.84 0.05 5 0.0012  0.81 0.05 5 0.0186  0.76 0.26 5 0.0246  0.75 0.03 5 0.2866  0.75 0.04 5 0.0034  0.84 0.04 5 0.0228  0.82 0.06 5 0.1432  0.95 0.09 5 0.2946  FED  MEAN SE N  3.59 0.13 8  4.35 0.34 12  4.32 0.46 11  4.79 0.37 11  5.20 0.26 10  5.71 0.43 11  5.48 0.30 10  5.45 0.31 9  FASTED  MEAN SE N 2  5.34 0.58 5 0.0036  5.30 0.25 5 0.1096  5.79 0.32 5 0.0622  5.91 0.46 5 0.0986  6.01 0.44 5 0.1148  6.03 0.44 5 0.9600  5.93 0.59 5 0.4590  6.49 0.46 5 0.0770  FED  MEAN SE N  5.27 0.16 8  5.72 0.44 11  5.73 0.50 11  6.17 0.37 11  6.66 0.26 10  6.98 0.42 11  6.62 0.30 10  6.65 0.30 9  *  ACYL  TOTAL  FASTED  significant  MEAN SE N E at overall  6.19 6.11 0.60 0.24 5 5 0.0958 0.5770 t h e 0.05 l e v e l  6.55 6.67 6.76 6.87 6.75 7.44 0.34 0.50 0.47 0.47 0.65 0.54 5 5 5 5 5 5 0.3150 0.4518 0.8422 0.9866 0.8370 0.1874 u s i n g B o n f e r o n i ' s c o r r e c t i o n b a s e d o n 48 t e s t s  55 6.1.1.3 C a r n i t i n e E s t e r s The  amounts  in Bile  of short  decreased  from  53  carnitine  i n bile  and m e d i u m - c h a i n c a r n i t i n e e s t e r s  t o 33% although was  the t o t a l  amount  n o t a f f e c t e d by f a s t i n g .  time  t h e amount  of long-chain  from  29 t o 5 4 % o f t h e t o t a l  carnitine esters  of  A t t h e same increased  c a r n i t i n e i n the b i l e  (Figure  8).  6.1.1.4 Serum Upon did  Carnitine  fasting  not d i f f e r  concentration overall  6.1.2  and  from  7).  h o w e v e r , was was  The lower  and t h e  higher.  were  state.  (Table  slightly  and  from  increased 3%  across  increased  fatty  I n both,- t h e (from  of long-chain  after  by B r a s s  t h e amounts  considerably  of t o t a l  amounts  of (from and  fasting  i n the t r a n s f e r of  fatty  membrane a n d t h u s  carnitine esters.  29%  carnitine.  acid o x i d a t i o n i n the  mitochondrial  and  17% t o 13% i n t h e  t o 11% i n the l i v e r )  carnitine i s involved the inner  of r a t b i l e  reported  1).  no c h a n g e s i n t h e a m o u n t s  More  form  to those  53% t o 47% i n the l i v e r ) ,  carnitine esters  reflects  acids  similar  c a r n i t i n e decreased  54% i n t h e b i l e  there  the  were  (1978) i n r a t l i v e r  free  This  (Table  changes i n the c a r n i t i n e content  long-chain to  carnitine,  o f serum t o t a l c a r n i t i n e  Discussion  Hoppel  bile  the fed state  of free  72 h o u r f a s t  of  from  carnitine esterification  The a  the concentration  Therefore,  i s in  less  56  87-  m  o> o o  6-  c  4-  5-  1 10  3-  o  2-  E c  1-  Lb FREE  SHORT AND LONG-CHAIN MEDIUM-CHAIN Fed  TOTAL  I | Fasted FIGURE 8  The amounts o f c a r n i t i n e and c a r n i t i n e e s t e r s i n t h e b i l e o f fed and f a s t e d r a t s i m m e d i a t e l y a f t e r t h e i i t i a t i o n o f b i l e c o l l e c t i o n (mean ± S E ) . S h o r t and m e d i u m - c h a i n a c y l c a r n i t i n e l e v e l s were d e c r e a s e d a f t e r f a s t i n g ( p < 0 . 0 0 1 ) while long-chain c a r n i t i n e ester l e v e l s increased (p<0.001). The f r e e c a r n i t i n e c o n t e n t o f t h e b i l e d i d n o t change a f t e r fasting.  57  carnitine  i s i n the free  liver  bile  and  carnitine not  esters  i n the The  hours  i s that  liver  not  carnitine serum  d i d not  colleagues fasted  from  41%  total  t o 69%  i n the  from  16  This  levels  also  increased  no  i n t h e amount o f l o n g - c h a i n  bile  Minuk  bile  and  change flow  Sarjeant  short  After  These  a 72  during  unit  of  time.  fasting  (1984).  In  other  increased initially  no  fat  carnitine change i n  and  did  Genuth,  not  long-chain  medium-chain  hour  fast,  carnitine  carnitine  there  esters  i n the  t h e amount o f c a r n i t i n e  per  and  dietary  with  Hoppel  and  51%  was  in  1978).  the c o n c e n t r a t i o n  upon f a s t i n g  d i d not  reduced  Hoppel,  total  to the  t h e amount o f  decreased.  increased  while  72  Gudjonsson  i n the b i l e  esters  and  the  a higher  e t a l . , 1978;  carnitine  Although  but  carnitine  (1985b).  upon f a s t i n g  Instead,  for  I n humans, serum  increased  (Brass  by  1978).  esters  serum  t o 32%)  esterification  reflect  esterification  upon f a s t i n g .  Serum  hours  carnitine  the  the  medium-chain  fasting  and  reported  Hoppel,  might  (Frohlich  Carnitine  change  and  i s comparable  f o r 16-18  and  upon  t o 50%  (Seccombe et a l . , 1978).  increase  between  ( f r o m 54%  bile.  hours, c a r n i t i n e  (Brass  carnitine  1976).  those  fasted  f o r 72  esterification  short  bile  carnitine  esterification  carnitine  content  i n the  change.  i n rats  rats  higher  i n serum  increased  carnitine  difference  (43%).  parallel  esterification  One  t h e amount o f  decreased  changes  do  state.  This  which  Similarly,  bile  found  i s a result  was  also  i n the of  observed  the hepatic  by  carnitine  58  content 1981; only  also  increased  Bohmer,  during  1968) but B r a s s  a r e s u l t of decreased Carnitine  was a l s o  (1985b) i n t h e b i l e They  found,  uM f o u n d fasting  period  total  c a r n i t i n e compared esters  this  was  fasted  colleagues 16-18 h o u r s .  c a r n i t i n e , much l e s s t h a n  m i g h t be r e s p o n s i b l e  and medium-chain  carnitine  by G u d j o n s s o n a n d  a f t e r a 72 h o u r f a s t .  Short  that  weight.  o f t w o r a t s who w e r e  study  and H o p p e l ,  and Hoppel found  liver  found  3 0 . 8 uM t o t a l  i n this  f a s t i n g (Brass  forthis  carnitine esters t o 32% i n t h i s  made up 4 3 % c o m p a r e d  The  t h e 81.9  shorter  difference.  made up 3 3 % o f t h e study  and  long-chain  t o t h e 54% i n t h i s  study. In  conclusion,  composition those  t h e changes that  of c a r n i t i n e i n the b i l e ,  occurring  i n the liver  than  occur  i nthe  more c l o s e l y  t h e serum as  parallel  illustrated  below. CARNITINE  SERUM  LIVER  BILE  -amount  no  no  change  no  change  -%  increased  no  change  no  change  decreased  no  change  no  change  increased  no  change  decreased  no  increased  esters  -types  change  of esters  -free -short  and  medium -long These  results also  contributes  change  support  the theory  t o the c a r n i t i n e content  that  bile  increased carnitine  of the i n t e s t i n a l  59  lumen.  Sachan  lowered  the  esters  in  decrease for  72  in  free  rich  in  the  l u m e n by the  esters  bile  between  to  reflects  the  carnitine the  bile  of  24  the  esters. the  was  who  were  in  by  12%. lack  10%  The of  the  This  bile  41% fasted  and decrease  carnitine which  suggests  intestinal  a  dietary  long-chain in  fast  There  rats  by  hour  carnitine  decreased  carnitine  and  a  54%.  increased  relative increase due  that  medium-chain  carnitine  probably  long-chain  contents.  and  in  Lumen f r e e  is likely  association  short  esters  carnitine  and  (1985) found  intestinal  carnitine  carnitine esters  rat  these  hours.  long-chain  Ruark  amount of  the in  and  is  an  carnitine  60 6.2 T h e E f f e c t s Carnitine 6.2.1  of Tetradecylglycidic  Content  bile  treatment increase  and Weight  Loss  of fasted  rats  flow  i s shown i n F i g u r e above  the fasting  period.  stimulated  by t h e t r a g a c a n t h  lost  hours.  fasted  6.2.1.2 C a r n i t i n e  change acyl the  weights  i n total  and t o t a l  flow to  i n t h e second  rats,  bile  alone.  flow  TDGA  upon f a s t i n g the weight  bile was  treated f o r 72  loss  of the  of the B i l e  caused  an i n c r e a s e  i n the bile,  carnitine carnitine  i n the concentration  a decrease  concentrations concentrations  i n acyl (Table  a n d no  8).  Free,  a l l increased  during  experiment. TDGA c a u s e d  carnitine  i n each  no c h a n g e s 15 m i n u t e  i n t h e amounts o f a c y l bile  T h e r e was, h o w e v e r , more f r e e treated Both  from  bile  TDGA  only.  Content  TDGA t r e a t m e n t carnitine  except  vehicle  i s not different  who w e r e  free  body  and w i t h o u t  TDGA c a u s e d  I n two a d d i t i o n a l  14% o f t h e i r  This  9.  with  levels  collection  of  on t h e  Results  The  rats  Treatment  of Rat B i l e  6.2.1.1 B i l e F l o w  rats  Acid  rats  throughout  c o l l e c t i o n (Table 9 ) .  carnitine  i n the t h i r d through  t h e amounts o f a c y l the experiment  and t o t a l  eigth  i n the bile  o f TDGA  collection  periods.  and t o t a l  carnitine  but free  carnitine  increased d i d n o t change.  61  98-  TIME OF •  FASTED  BILE  C O L L E C T I O N (min) p | TDGA-treated  FIGURE 9 B i l e f l o w ( m i c r o l i t e r s / m i n / 1 0 0 g BW) d u r i n g b i l e c o l l e c t i o n f r o m 72 h o u r f a s t e d a n d TDGA t r e a t e d r a t s ( m e a n ± S E ) . B i l e was c o l l e c t e d i n e i g h t c o n s e c u t i v e 15 m i n u t e s a m p l e s a n d t h e flow ratewas assessed g r a v i m e t r i c a l l y . A t most t i m e i n t e r v a l s t h e b i l e f l o w w a s i n c r e a s e d a f t e r TDGA t r e a t m e n t . The l e v e l s of s i g n i f i c a n c e a r e as f o l l o w s : * = p<0.05, ** = p<0.02, *** = p<0.01, **** = p < 0 . 0 0 1 .  62  TABLE 7 The  C o n c e n t r a t i o n s o f C a r n i t i n e and i t s E s t e r s i n t h e Serum o f F e d , F a s t e d a n d TDGA T r e a t e d R a t s  Free Carnitine umol/1  Total Carnitine umol/1  % Acyl Carnitine  Fed MEAN SE  48.9 4.4  58.9 5.0  17.0 2.0  Fasted MEAN SE  35.4 2.6  71.8 5.4  50.4 2.6  **  TDGA t r e a t e d MEAN SE  35.0 3.3  48.1 3.0  27.6 3.6  @@  Treatment  * p<0.05 versus fed ** p<0.001 v e r s u s f e d @ p<0.01 versus fasted @@ P < 0 . 0 0 1 v e r s u s f a s t e d  *  The  Concentrations  1  of Free,  TABLE 8 A c y l and T o t a l C a r n i t i n e TDGA-Treated Rats  Consecutive b i l e 2 3  collection 4 5  i n the Bile  periods 6  o f F a s t e d and  (15 minutes each) 7 8  FREE FASTED  TDGA  MEAN SE N MEAN SE N p  11.5 0.7 5 21.2 0.2 6 0.0000  *  11.4 1.1 5  *  10.9 0.6 5  23.3 1.4 6 0.0004  *  11.0 0.5 5  5  11.2 0.7 5  12.7 0.6 5  12.9 0.4 5  12.9 0.4  26.4 1.0 6 0.0000  26.4 1.4 5 0.0000  22.9 1.0 6 0.0000  24.2 0.7 6 0.0000  25.0 1.1 6 0.0000  25.4 1.0 6 0.0000  81.2 2.0  85.4 2.0  88.5 1.6 5  90.2 1 .3 5  92.1 1. 1 5  89.3 3.5  66.5 2.7 5 0.0000  62.9 3.9 5 0.0010  *  *  *  *  *  ACYL FASTED  TDGA  MEAN SE N  70.4 4.9 5  MEAN SE N p  42.2 3.4 6 0.0008  FASTED  MEAN SE N  81.9 4.5 5 5  TDGA  MEAN SE N P  63.2 2.3 6 0.0036  *  73.7 1.2 5  *  5 49.5 2.6 6 0.0000  5  5  56.0 2.9 6 0.0000  56.6 2.8 6 0.0000  58.5 3.6 6 0.0000  59.9 3.4 6 0.0000  85.1 1.9  92.0 2.3 5  96.4 1.6 5  99.7 1.6 5  102.9 1.5 5  105.0 1.1 5  102.2 3.6 5  71.8 2.8 6 0.0044  79.3 3.0 6 0.0100  79.5 3.1 6 0.0014  82.7 4.3 6 0.0072  84.9 3.8 6 0.0028  91.9 2.9 5 0.0028  89.3 4.4 5 0.0530  *  *  *  *  *  *  TOTAL  significant  at overall  t h e 0.05 l e v e l  *  using Bonferoni's  correction  based  o n 48  tests  The  Amounts o f F r e e ,  1  Acyl  TABLE 9 and T o t a l C a r n i t i n e TDGA-Treated Rats  Consecutive 2  bile 3  collection 4  i n the Bile  periods 5  o f F a s t e d and  (15 minutes each) 7 6 8  FREE FASTED  MEAN SE N  0.84 0.05 5  0.81 0.05 5  0.76 0.26 5  0.75 0.03 5  0.75 0.04 5  0.84 0.04 5  0.82 0.06 5  0.95 0.09 5  TDGA  MEAN SE N P  2.00 0.26 6 0.0032  1.86 0.23 6 0.0028  2.03 0.12 6 0.0012  2.02 0.13 6 0.0000  2.25 0.13 6 0.0000  2.26 0.13 6 0.0000  2.42 0.11 5 0.0000  2.40 0.18 5 0.0000  *  *  *  #  *  *  FASTED  MEAN SE N  5.34 0.58 5  5.30 0.25 5  5.79 0.32 5  5.91 0.46 5  6.01 0.44 5  6.03 0.44 5  5.93 0.59 5  6.49 0.46 5  TDGA  MEAN SE N P  3.92 0.34 6 0.0550  4.28 0.54 6 0.1446  4.84 0.25 6 0.0414  4.98 0.26 6 0.0986  5.47 0.48 6 0.3692  5.48 0.44 6 0.4038  6.33 0.28 5 0.5576  5.73 0.49 5 0.2908  FASTED  MEAN SE N  6.19 0.60 5  6.11 0.24 5  6.55 0.34 5  6.67 0.50 5  6.76 0.47 5  6.87 0.47 5  6.75 0.65 5  7.44 0.54 5  ACYL  TOTAL  TDGA  significant  MEAN 5.93 6.13 0.71 SE 0.40 6 N 6 0 . 6 9 2 8 0 .9806 P a t o v e r a l l t h e 0.05 l e v e l  6.87 7.73 8.13 7.75 8.76 6.99 0.62 0.31 0.58 0.53 0.29 0.35 5 6 5 6 6 6 0.0224 0.4258 0.2986 0.5040 0.6036 0.2386 u s i n g B o n f e r o n i ' s c o r r e c t i o n b a s e d on 48 t e s t s  65  6.2.1.3 C a r n i t i n e In of  the f i r s t  free  esters  Esters bile  carnitine were  (Figure  amounts  and s h o r t  found  esters  carnitine  carnitine  ester  levels  and s h o r t  rats were  collection, the  and medium-chain  further  but there  i n t h e amount o f l o n g - c h a i n  w a s no  carnitine  i n the bile.  Carnitine  TDGA t r e a t m e n t total  carnitine  esterification decreasing  a decrease  i n r a t serum  also  from  caused  was l o w e r  i n the concentration  (Table  upon  7).  TDGA  Carnitine  treatment,  50 t o 2 8 % .  Discussion The  changes i n t h e c a r n i t i n e  administration reported the  amounts  o f t h e TDGA t r e a t e d  had i n c r e a s e d  change  6.2.1.4 Serum  6.2.2  increased  and medium-chain  i n the bile  carnitine  esters  additional  period,  By t h e e n d o f t h e 2 h o u r b i l e  of free  carnitine  of  collection  10). Long-chain  decreased.  i n the Bile  o f TDGA t o f a s t e d  by F r e n c h  and c o l l e a g u e s  same t r e a t m e n t .  esters  decreased  intubation In  the bile,  remained liver,  rats  were  of bile  carnitine  at approximately  levels  after  carnitine  2 hours a f t e r  TDGA  increased.  of e s t e r i f i e d  70% a f t e r  h o w e v e r , TDGA c a u s e d  to those  (1985) f o r r a t l i v e r  levels  the percentage  after  similar  The amount o f l o n g - c h a i n  below f a s t i n g  and f r e e  content  carnitine  TDGA t r e a t m e n t .  a decrease  i n carnitine  Inthe  66  F I G U R E 10 The a m o u n t s o f c a r n i t i n e a n d c a r n i t i n e e s t e r s i n t h e b i l e o f f a s t e d a n d TDGA t r e a t e d r a t s 15 a n d 1 0 5 m i n u t e s a f t e r t h e i n i t i a t i o n o f b i l e c o l l e c t i o n ( m e a n ± S E ) . A f t e r 15 m i n u t e s , f r e e and s h o r t and medium-chain a c y l c a r n i t i n e l e v e l s w e r e i n c r e a s e d i n t h e r a t s t r e a t e d w i t h TDGA. L o n g - c h a i n c a r n i t i n e e s t e r l e v e l s were d e c r e a s e d below fasting levels. A f t e r 105 m i n u t e s , t h e f r e e a n d s h o r t a n d medium-chain c a r n i t i n e e s t e r l e v e l s had i n c r e a s e d further but t h e l o n g - c h a i n a c y l c a r n i t i n e c o n t e n t o f b i l e remained a t t h e 15 m i n u t e l e v e l . The l e v e l s o f s i g n i f i c a n c e a r e a s f o l l o w s : * = p<0.01 a n d ** = p<0.001.  15 Minutes  FREE  SHORT AND  LONG-CHAIN  MEDIUM-CHAIN riFasted  i l l TDGA treated  TOTAL  68  esterification fact  that  from  69% t o 55% (French  esterification  TDGA m i g h t  be d u e t o i n c r e a s e d  medium-chain  carnitine  administration.  r a t serum  acylcarnitine different change  (French after  from  acylcarnitine  of carnitine  French carnitine  were  (1985) found  Frost  and W e l l s  (1982) a l s o  mechanism  t o overcome  The amount  of carnitine  differ  with  administration.  Overall,  those  that  after occur  occurring  a 72 h o u r i n bile  that  fasting  than  the hepatic  levels  with  observed  this  that  TDGA  i ti s a  the inhibition  of the  however,  d i d not  a n d TDGA i n t u b a t i o n ,  carnitine  i n the l i v e r  The serum  f o ruse as energy.  i n the bile,  fast  w a s no  due t o i n c r e a s e d  and h y p o t h e s i z e d  TDGA.  TDGA  after  This i s  where t h e r e  by t h e t i s s u e s  above  rats  decreased.  carnitine.  increased  i n neonatal  TDGA  t h e t o t a l and  i n the bile  may b e d e c r e a s e d  and c o l l e a g u e s  compensatory  changes  both  amount o f a c y l  esters  upon  i n the l i v e r  TDGA i n t u b a t i o n ,  content  treatment. increase  i n the bile  e t a l . , 1985).  the situation  content  by  amounts o f t h e s h o r t and found  concentrations  i n the overall  uptake  esters  The  was n o t a f f e c t e d  These were n o t changed  TDGA a d m i n i s t r a t i o n In  i n the bile  e t a l . , 1985).  more c l o s e l y  the  parallel  t h e serum a s shown  below.  69  CARNITINE  SERUM  LIVER  BILE  -amount  decreased  increased  no  change  -%  decreased  decreased  no  change  no  increased  increased  no  change  increased  decreased  decreased  esters  -types  of  esters  -free -short  and  change  decreased  medium -long  6.3  The  Rat  Bile  Recovery  Labelled immediately this  the  found  the  arrives  there  The due  injected  with  g  he  after  to  the  found  injection  of  C]Carnitine  m e t h o d s and 1.  labelled  s u b c u t a n e o u s and  of  finding  carnitine  Rebouche et  almost  results  This  in  found  in  intravenous  a l . ,  1984)  bile. i n j e c t i o n , 0.5%  rat bile. an  label  larger  (8.7  versus  amounts of  massive  labelled  doses.  and  bile  after  experiment  nonlabelled 0.26  carnitine  colleagues  i n the  i n Gudjonsson's  l a r g e amount of  label  of  Gudjonsson  injected  recovery  the  [  i n t r a v e n o u s l y , was  The  the  after  of  of  Injected  i n Appendix  a l . , 1985;  v i a the  higher  probably  as  et  2.4%  bile.  that  feces  i n the  (1985b) found  BW)  and  injected  reported  2 hours  recovered  hours.  are  (Hahn  Within was  in rat  hypothesis  intestine  injections  Intravenously  carnitine,  experiment  supports  of  nmol  carnitine  4  is  carnitine  carnitine/100 i n the  bile  70  Although bile  the s p e c i f i c a c t i v i t y  and t h e serum  distribution carnitine  was  after  20 m i n u t e s was  different.  esters  i n t h e serum w h i l e  form.  in this  was  e s t e r i f i e d to long-chain  the  actual  carnitine  there  appears  liver  that  enters  but  i s first  the  hepatocyte  stored  of  turning  over  labelled the  pool  long-chain  larger  pool  esterified liver  carnitine oxidized. carnitine  because  i n the skeletal v i a t h e serum.  pool  This  muscle  (Brooks  and F r i t z  (1962),  labelled  carnitine  injected  less  than  The  come  from  could  be  and t r a n s p o r t e d as  are probably slowly  to  long-chain immediately turning  over  1975).  studying  intravenously  1% o f t h e l a b e l l e d  rapidly  pool.  carnitine  has a l a r g e ,  when  either  turnover.  i s unlikely  cells  in  the presence  presumably  tissue,  and M c i n t o s h ,  Yue  that  labelled  esters  (89%),  stable  rapid  muscle  i n muscle  Skeletal  esters  i n the  liver.  reported a large  of  t h e serum,  from  i n the  more  of t h e i r  the injected  esters  originates  in liver;  carnitine  from  carnitine  carnitine  and a s m a l l e r ,  30%  Therefore,  of c a r n i t i n e  enters  (1975) a l s o  of c a r n i t i n e  Alternatively,  the  synthesized  and M c i n t o s h  two p o o l s  one p o o l  that  only  i s not representative  one t h a t  and t h e o t h e r  i n bile  Therefore, the  e s t e r i f i e d to long-chain  or newly  Brooks  the b i l e ;  ester  long-chain  d i s t r i b u t i o n i n the b i l e .  t o be m o r e t h a n  the  carnitine,  esters.  carnitine  i n the  50% of the l a b e l  Of t h e n o n - l a b e l l e d  of l a b e l l e d  similar,  T h e r e w e r e no  was  distribution  of the c a r n i t i n e  the uptake of into  carnitine  dogs,  taken  found  up by t h e  71  skeletal  muscle  chloroform labelled with  extractable  chloroform.  injected reason  to  at  bile  Gudjonsson to  that  and  the  esterified  After  administration  constant.  i n the  the  of  This  that  serum  before  large  amounts of  free  the  is  the  extracted  liver  is  from  another esters  liver.  liver  while  be  carnitine  (1985b) provide  carnitine ester  dramatically  that  of  carnitine esters  muscle.  concept  6%  could  l a b e l l e d long-chain  coworkers  first  increased  however,  long-chain i s the  was  carnitine).  o r i g i n a t e i n the  support  intravenously,  liver,  injection  r e s u l t s suggest  producing  suspect  after  (long-chain  These  c a r n i t i n e than  i n the  evidence  7 hours  c a r n i t i n e i n the  more a c t i v e  found  i n the  carnitine is  entering free  content  further  of  the  bile.  carnitine the  carnitine levels  bile remained  CHAPTER  The  Carnitine  Content  of  7  Human B i l e - R i c h  Duodenal  Fluid  72  7.1  Results The  sample  concentration  i s shown i n T a b l e  acylcarnitine carnitine before  levels  varied  the stimulation most  carnitine  esters  Short  long-chain  10.  was c o n s i s t e n t  carnitine  11.  of carnitine  i n each  (81%),  although  free  The two s a m p l e s  of b i l e flow,  contained  was i n t h e f r e e  i n the duodenal  fluid  and t o t a l collected  very  state.  little  The t y p e s o f  a r e shown i n T a b l e  and medium-chain  carnitine  carnitine  made up 3 7 % o f t h e t o t a l  esters  fluid  The p e r c e n t a g e o f  widely.  of which  duodenal  esters  made up 3 5 % a n d  carnitine.  7.2  Discussion Rudman a n d c o l l e a g u e s  differences  between  t h e serum  cholelithiasis  patients  the  of patients  population  assumed  (1980) r e p o r t e d carnitine  and normal studied  t o be h o m o g e n e o u s w i t h  no s i g n i f i c a n t  concentrations of  controls. i n this  respect  On t h i s  experiment  basis, was  to carnitine  metabolism. The bile-rich  concentrations  free  fluid  varied  human d u o d e n a l  coefficients 119%  of both  of v a r i a t i o n  f o r these  and 140% r e s p e c t i v e l y .  concentration  of only  7%.  carnitine  i n the duodenal  widely.  two s e t s  In rats,  of carnitine  variation  and t o t a l c a r n i t i n e i n The of data  were  the t o t a l  i n t h e b i l e had a c o e f f i c i e n t o f  When  t h e amount o f e s t e r i f i e d  fluid  was e x p r e s s e d  as a  73  TABLE  10  T h e C o n c e n t r a t i o n s o f C a r n i t i n e a n d C a r n i t i n e E s t e r s i n Human Duodenal F l u i d A f t e r S t i m u l a t i o n of B i l e Flow  Subject  Carnitine  Concentration  (uM)  % Acyl Carnitine  Free  Total  1 *  19.1 5.1  94.6 27.3  80 81  2 *  28.3 1.8  89.5 8.8  68 80  3 *  4.6 26.0  38.4 113.4  88 77  4  6.1  35.7  83  5  8.0  42.6  81  6  0.6  4.3  86  7 *  4.0 6.3  25.4 49.6  84 87  8 *  60.9 58.9  412.3 388.8  85 85  9  2.4 * * 7.6  2.5 * * 30.0  4 75  10  3.6 * * 7.8  3.8 * * 23.7  4 67  92.3 33.3  81 2  MEAN * * * SE  16.3 5.0  * S a m p l e s , f r o m t h e same p a t i e n t , c o l l e c t e d 15 m i n u t e s a p a r t . ** S a m p l e s o b t a i n e d b e f o r e b i l e f l o w s t i m u l a t i o n . *** E x c l u d i n g s a m p l e s o b t a i n e d b e f o r e b i l e f l o w s t i m u l a t i o n .  74  T A B L E 11  The R e l a t i v e  Q u a n t i t i e s o f C a r n i t i n e and C a r n i t i n e B i l e - R i c h Human D u o d e n a l F l u i d  Free  MEAN  Carnitine (%)  26.0  S h o r t and MediumChain Carnitine E s t e r s (%) 35.3  Esters i n  Long-chain Carnitine Esters (%) 36.5  SE  3.8  1.5  8.7  N  4  4  4  75  percentage variation The fluid  of the t o t a l , was o n l y  degree  of c a r n i t i n e  i n the f i n a l  also  correspond with  bile  also  from  (206.9  There  the  Bile  humans h a v e  of time that  carnitine  might studied  duodenal  patients  the  have  was  collected  male Yucatan p i g carnitine  fluid,  duodenal  gallbladders,  which  are absent i n  i t remains  to a different  depending  of b i l e  bile.  and, hence, degrees  to fast  Each  i n the  f o r 12-18  sample  extent depending  aspirated  after  #7 a n d #8, t h e r e  In patients,  studied  could  have  on t h e a m o u n t s  along with  two s a m p l e s  the carnitine  on  i n the gallbladder  o f 15 m i n u t e s  In patients  two s a m p l e s .  degrees  Secondly, the samples  not pure  fluid  range  human  as they were i n s t r u c t e d  between  f o r t h e wide  occurred to different  at intervals  differences  of p i g  in bile-rich  s h o w n by c o l l e c t i n g  stimulated.  of a fasted,  Concentration  gastrointestinal was  sample  of r a t  results  one sample  explanations  before the procedure.  been d i l u t e d  This  This  i s concentrated to varying  1971).  of  from  that  These  87% of the t o t a l  concentrations  (Wheeler,  were  those obtained  a r e two p l a u s i b l e  length  hours  period.  from  esterified.  First,  patients  collection  post-mortem.  carnitine  fluid.  bile  the gallbladder  uM) was  of  i n the duodenal  different  analysed f o r carnitine.  immediately  rats.  esterification  ( 8 1 % ) was n o t s i g n i f i c a n t l y  directly  the c o e f f i c i e n t  8%.  bile  of  however,  from bile  the b i l e . each flow  w e r e no  concentrations  # 1 , #2, a n d #3,  of  five  was  major measured  however,  i n  76  there #1  were c o n s i d e r a b l e  had  27.3  94.6  uM  uM  carnitine  carnitine  esterification samples  (80%  To and  of  measured than  4  for  uM  two  contribute  to  it  and  the  In  carnitine  esterified.  fluid,  the  release  (Brooks  and  was  placed  by  was  of  of  distributed  in  evenly  between  carnitine  esters.  percentage  not  different  from  that  the  two  was  from  the  there  was  concluded,  the  do  not  samples.  content  Also, fluid  Before  esterified  of  tissue,  secretions  esters  flow  after,  pancreatic by The  however, i s very  bile  was  because  low  not the  cannula  tissue.  human d u o d e n a l free  is  bile  while  stimulated  rat  was  less  i n j e c t i o n , i s unknown.  The  bile  stimulation  fluid.  i s also  the  carnitine  pre-stimulation  4%  pancreatic  medium-chain The  of  carnitine  1975).  bile-rich  be  carnitine  second).  secretions  the  pancreatic  the  flow  cases,  can  only  which  pancreatic  beyond  Carnitine  both  of  The  Mcintosh,  contaminated  bile  It  pancreozymin-cholecystokinin content  measured  post-stimulation  carnitine  the  patient  only  of  between  for  content  profile  81%  degree  secretions,  before  present.  was  carnitine  81%  carnitine  fluid  ester  from  stimulation,  first  example,  sample but  The  gastrointestinal  the  carnitine  first  latter.  subjects.  that  For  change however  the  carnitine  different  the  gastrointestinal  therefore,  the  the  duodenal  in  in  the  not  that  other  content  in  did  ensure  not  differences.  carnitine,  and  fluid short  long-chain  of  long-chain  of  the  bile  is  carnitine  carnitine  collected  and  esters  from  fed  is rats  77  and  the  not  different  profile  percentage  has  has  of  been  an  the  short  carnitine  levels  Therefore,  dietary  not  i n man  Mansour,  carnitine of  only but  in  esters  bile.  This  ester  imposed  upon  the  due  also  carnitine  rat  is  i s also  a l . , 1984; Bohmer  for  (Gudjonsson in  bile  some e v i d e n c e  that  to  the  decreased  Frohlich et  a l . , 1975).  lead  to  absorption  to from  decreased the  bile.  serum  et a l . ,  humans m i g h t decreased  et  human  is related  1976;  in  the  circulation  carnitine  There  malabsorption  reabsorption  fast  of  (Seccombe et  and  carnitine  rat  enterohepatic  presence  malabsorption  body  fasted  been e s t a b l i s h e d .  Mikhail  medium c a r n i t i n e  demonstrated  carnitine  1980;  of  and  bile collection.  1 9 8 5 b ) and now  that  existence  carnitine  short  r e f l e c t the  before  The  al.,  from  might  patients  of  to  decreased  intestinal  of  CHAPTER 8  Changes i n t h e C a r n i t i n e C o n t e n t of R a t B i l e Collection  During  78  Throughout  this  study,  acylcarnitine  in  The  carnitine  amount of  period  also  increased  phenomenon.  reported bile  effect was  of  with  The  of  Bile Over ml  of  of  the  this  loss  investigated into  a  reported  in  content the bile  still  minute  to  attempt  of  and the  collection  b i l e flow  to  effects  to  the  explain  results  fluid  assessed.  Infusion  two  collected  on  the  by  replacing  hours from  carnitine  vein.  the  effect was  the  of  Appendix  Despite  the  Saline  course  jugular  15  bile collection.  lost  Secondly, collect of  via  the  the  the  the  are  samples  anaesthetic  determined.  of  b i l e was  each  investigated  F i n a l l y , the  were  Effect  in  rat  of  time.  experiment  interrupting  b i l e flow  8.1  3  the  during  were proposed  First,  investigated.  on  found  E a c h was  below.  during  concentration  b i l e increased  Three hypotheses this  the  of  bile collection,  each  fluid  results  350  Carnitine  g  rat. of  of  this  The  the  lost with  Content  almost effect  bile  saline  was infused  experiment  are  2.  infusion  increased  loss  of  t o t a l body  had  no  effect  on  the  composition  the  The  on  of  saline,  during fluid  the  the  carnitine  bile collection.  from  the  carnitine  rats  content  in of  ester  Therefore,  the the  form  of  bile.  79  8.2  The  Effect  Circulation  Interruption  the  Carnitine  Strasberg  and  colleagues,  (1984),  stressed  effects  of  the  circulation  on  substances  whether bile,  or  not  however,  review  of  the  the  Bile of  of  investigating  in bile.  To  acylcarnitine  in a  biliary  enterohepatic  r a t as  determine  content  described  amounts of  carnitine  esters  the  experiment.  I t cannot  that  the  interruption  of  does not  because by  therefore,  The  the  of  circulation  8.3  affects  of  importance  measured  recirculated  the  course  reduced  the  in a  Enterohepatic  of  the  in  3.  Again  bile,  the  Content  interruption  this  b i l e was  Appendix  the  of  on  sampling the  of  the  33% to  alter  repeat  of  concluded,  content  of  c i r c u l a t i o n was  experiment.  this  be  I t would  experiment  P e n t o b a r b i t a l on  during  enterohepatic  carnitine  enterohepatic  in this  Effect  the  the  increased  and  the  be  the  still advisable,  collect  Carnitine  less  bile.  Content  of  Bile In  most  increased then  bile  during  after  for  plateaued  75  content  minutes  c o l l e c t i o n was  r a t was  c o l l e c t i o n , one  already  carnitine  first  Bile  the  responsible  the  the  plateaued.  minutes was  cases,  the  after  a  bile  usually  bile collection  usually  anaesthetized.  increased  would  of  of  initiated  I f the  bile carnitine levels  and 5-10  anaesthetic during  expect  that  the  would  r a t had  been  anaesthetized  for  the have  80  some t i m e . Appendix  This  results  have an e f f e c t  8.4  that  f o r the increase  the course  the exception  conclusions  the anaesthetic  does  i n the b i l e  i n the c a r n i t i n e  of the experiment.  For  of the s a l i n e i n f u s i o n experiment,  c a n be d r a w n  number o f a n i m a l s  results  t o form  instance,  anaesthetic studies  new  from  used.  should  hypotheses  s t i l l than  can, however,  that  further  the effect  more t h o r o u g h l y during  studies.  of the  investigated.  of b i l e .  Future  to determine  the experiment  anaesthetics  The e f f e c t  circulation  of the  use t h e  compare t h e e f f e c t s o f d i f f e r e n t  the enterohepatic  increases  work, because  and d i r e c t  be m o r e c l o s e l y  the c a r n i t i n e composition  examined  this  One  i t seems a p p a r e n t  should  interrupting  of  should  also  be  whether a c y l c a r n i t i n e with  removal  of less  33% of the b i l e . If  the increased  accounted  surgery  of the enterohepatic  itself  manipulation colleagues  c a r n i t i n e i n the bile  cannot  f o r by t h e e f f e c t s o f t h e a n a e s t h e t i c  interruption  the  however,  Summary  small  on  described i n  on t h e c a r n i t i n e e s t e r i f i c a t i o n  during  With no  suggest,  i s not responsible  content  by a n e x p e r i m e n t  4.  The  but  was t e s t e d  should  circulation,  be i n v e s t i g a t e d . duct  i s responsible.  (1984) r e p o r t  that  with  pressure-flow  or the the effect  of  Possibly, physical  of the b i l e  bile  be  duct  r e l a t i o n s h i p s of the b i l e  Strasberg  and  cannulation, are distorted.  81  Such e f f e c t s might account f o r the change i n the c a r n i t i n e content of b i l e during b i l e  collection.  CHAPTER  9  Conclusions  82  The Their  implications  also  9.1  conclusions  study  a r e summarized  and s u g g e s t i o n s  f o r further  below.  research are  presented.  Summary  9.1.1 C a r n i t i n e The to  of t h i s  Content  concentration  that  of r a t serum.  long-chain fluids.  carnitine  of the B i l e of carnitine Bile  esters  I t was e s t i m a t e d  carnitine  passes  through  and c o l l e a g u e s  quantities  of c a r n i t i n e  fluid. degree  to that  content  was a l s o  9.1.2 O r i g i n The  other  r a t tissues  24  of r a t b i l e . similar  varied  hours. similar  considerably, the  The l o n g - c h a i n  to that  carnitine  to  between h e p a t i c  with  ester  of r a t b i l e .  i n b i l e was n o t d e t e r m i n e d  e x p e r i m e n t s were performed  intubated  and  i n the B i l e  Three  In the f i r s t  pool of  i n human b i l e - r i c h d u o d e n a l  of the c a r n i t i n e  sources.  and  or  1% o f t h e t o t a l b o d y  e s t e r i f i c a t i o n was c o n s i s t e n t  conclusively. distinguish  more a c y l c a r n i t i n e  (1985b) r e p o r t e d  found  of Carnitine  origin  that  the quantities  of carnitine  similar  than  similar  i n rat bile.  was a l s o  Although  contained  the b i l e every  Gudjonsson  Carnitine  i n r a t b i l e was  and e x t r a h e p a t i c  two, t h e r a t s  tetradecylglycidic  were e i t h e r  acid;  carnitine fasted  treatments  Birect  t h e amounts o f c a r n i t i n e  and t y p e s  esters  i n the l i v e r  The c h a n g e s  and serum.  to attempt  of  which  carnitine i n the b i l e  or  83  carnitine liver  more c l o s e l y  than In  the  the  Results both  experiment,  injected into  suggest  newly  that  which  9.1.3  Transport  transportation  on  bile  serum.  The  bile  fasted  of  Although there  was  that  the  bile  the  Data  presented  labelled  bile  this  by  energy-rich  and  be  of  the  the  be  bile  long-chain  esterified.  the  (see  Appendix  of  bile  c a r n i t i n e content of  carnitine in of  is of  5). both the  the  water  C a r n i t i n e i n the  an  this.  Bile  i s not  of  route  colleagues after As  the  known.  determined  that  carnitine,  It i s possible,  excretory  feces  bile  have  circulation  R e b o u c h e and  support  serum  Bile  result  colleagues  c a r n i t i n e i n the  administration,  the  from  c a r n i t i n e and  c a r n i t i n e content  i s unknown.  could  i s derived  concerning  c a r n i t i n e i n the and  bile  production.  enterohepatic  that  the  collection.  becomes  concentrations  r a t s might  after  of  and  obtained  formation  Gudjonsson  efficiency  liver  carnitine into  f u n c t i o n of  i s an  in  was  bile  hepatic  Carnitine into  Possible Functions The  rats during  stored  the  of  increased  reabsorption  9.1.4  of  suggested  dependent  or  enters  Some i n f o r m a t i o n  Results  occurring  [ ] c a r n i t i n e  c a r n i t i n e i n the  synthesized  carnitine  The  those  serum.  final  intravenously  parallelled  for  therefore,  carnitine.  (1984),  who  intravenous excretion  c a r n i t i n e e s t e r s would  the  be  of  found  84  inefficient, in  the i n t e s t i n a l Another  to  hydrolysis  ester  might  occur  lumen.  possible  r i dhepatocytes  organic  of the c a r n i t i n e  acidurias  function  of c a r n i t i n e  of excess organic or treatment  with  acids  i n the b i l e i s resulting  from  d r u g s s u c h as v a l p r o i c  acid. The is  involvement  another  the  high  possible  of c a r n i t i n e role.  concentrations  emulsification  transport  enterocytes.  The l o n g  into  the mucosal  indicates within  that  cells  9.2 S u g g e s t i o n s  large  There  quantities  labelled  to  measure i t s u p t a k e further  i n the  w i t h i n the  between u p t a k e o f c a r n i t i n e into  i s involved  the p o r t a l  i n some  blood  process  Work  of C a r n i t i n e of f r e e  and i t s  Esters  and a c e t y l c a r n i t i n e  have  i s no knowledge o f t h e i n t e s t i n a l acylcarnitine  which a r e p r e s e n t i n  and t h e d i e t .  way t o s t u d y  by m i x i n g  interrupting  o r be i n v o l v e d  and i t s r e l e a s e  i n bile  be  problem  delay  esters i n  might a i d i n t h e  fatty acids  for Additional  of l o n g - c h a i n  physiological  in bile  of f a t t y acids  the a b s o r p t i o n  been s t u d i e d .  carnitine  absorption  cells.  9.2.1 The A b s o r p t i o n  handling  found  the c a r n i t i n e  the mucosal  Only  Long-chain  of d i e t a r y  intracellular  i n fatty acid  A s i m p l e , and  this i n anaesthetized  long-chain  carnitine  esters  from t h e r a t i n t e s t i n e bile  flow.  of the w a t e r - i n s o l u b i l i t y  This  would  rats with  while avoid  of long-chain  the  would bile  85  acylcarnitines the  prevent  r e c i r c u l a t i o n of The  further types  and  of  carnitine  The The  studied  effects in  of  these  in  fatty  into  tied  carnitine by  on  fatty  monitoring  intestinal  should  the  lumen  the  rat,  by  should  reabsorption  measured  by  bile.  reported  single  of  caused  and  of  the  a l l  effects  of  Carnitine on  fat  by  in  could  of  the  the  The  then  label  presence  labelled  effect  be  of  determined  from  and  not  could  injecting  i n t e s t i n a l loops.  disappearance  were  experiments  rats  absorption  rats  Absorption  absorption  Future  anaesthetized off  i n Fat  the  absence  of  carnitine. Another carnitine may  reason  i s based  have u s e f u l  with  cystic  absorption  short  the  the  i s usually  role  measured  in  for  the  term  The  results  these  patients  had  range  (Mitchell,  effects et  impaired  carnitine  serum  interest  of  1978b),  fat  that  absorption oral  in acutely  a l . , 1984). in  was  several  are  serum  in  observation  f i b r o s i s (Benke  possible  fibrosis.  for on  be  determined.  acid  of  be  in  bile, a  efficiency  experiments.  this  label  studied  carnitine  investigate acids  who  esters  Involvement  complications  c i r c u l a t i o n of  The  components  possible  absorbed  colleages  quantitated.  dietary  9.2.2  the  enterohepatic  Gudjonsson  of  and  cystic  children  additional  levels  i l l  patients  fibrosis,  fat a  Carnitine with  shown i n A p p e n d i x  carnitine  carnitine  Because  suspected.  in  carnitine  and  was  cystic  6.  Although  the  normal  might  be  86  beneficial  at  investigated  9.2.3  The In  total 24  body  As  only  fluid,  the  could  studies  should  or  be  colleagues  these  carnitine  absorption should  be  and  be  the  that  reduced  s e r u m and  human  humans.  absorption further  various  i t should  be  reduced  lysine  of  and  This  determined  serum  possibilities  reduced  similar  in  carnitine  patients with  each  syndromes,  I t seems s u i t a b l e t h a t  the  bile  serum c a r n i t i n e  malabsorption  decreased  the  general  in carnitine depletion  First,  of  in bile-rich  exists  distinguished.  parenteral  through  1%  (1985) r e p o r t e d  found  p a t i e n t s have  Then,  whether  after  also  various  made o f  levels.  persists  should  calculated that  r a t passed  reported  carnitine  determining  This  Carnitine  have r e p o r t e d  disorders.  not  absorption  the  a l . , 1984).  malabsorptive whether  i t was  result  experiment et  Biliary  and  i n patients with  (Seccombe  intestine.  possibility  some s t u d i e s  one  of  c a r n i t i n e was  malabsorption  levels  the  experiment,  Gudjonsson  results.  of  further.  c a r n i t i n e of  hours.  Although  level  Malabsorption  this  duodenal  the  or  tissue  reduced  methionine  could  be  done  by  tissue carnitine depletion  feeding  with  formulas  lacking in  carnitine.  9.2.4  The  E f f e c t s of  Hepatic  Dysfunction  on  Biliary  Carnitine The  effect  of  liver  disease  on  the  c a r n i t i n e content  of  87  the  bile  should  concentrations  also  of p a t i e n t s  significantly  lower  al.,  This  1980).  synthesis  be s t u d i e d .  than  with  those  could  be d u e  possibility  of u n c o n t r o l l e d  the  bile  also  Biliary To  the  understand  studies  labelled  carnitine.  possible  excretion  9.2.6  should  be  Carnitine  i n Body  completely  pool  controls  to a reduced  (Rudman e t  hepatic  malabsorption.  release  Also,  of c a r n i t i n e  into  investigated.  Carnitine  r a t , body  studies  be  carnitine  a l c o h o l i c c i r r h o s i s were  of c a r n i t i n e or to general  should  serum  of normal  the  9.2.5  The  Pool  Studies  the metabolism  have  been  of c a r n i t i n e i n  attempted  using  None, however, has c o n s i d e r e d of c a r n i t i n e through  repeated  taking  Absorption  into  the b i l e . account  i n Systemic  the These  the  Carnitine  bile.  Deficiency  Syndromes In the is  cases  disorder not always  of systemic i s often  hypothesized  possible  site  1984).  on  serum  intestinal  absorption  the e f f e c t  should  of a bolus  of  therapy  t h e symptoms.  ( G i l b e r t , 1985; E n g e l this  the s i t e  oral carnitine  at a l l e v i a t i n g  that  of defect  In the future,  by m o n i t o r i n g  n o t known and  successful  has been  carnitine deficiency,  It  i s a  and  Rebouche,  be i n v e s t i g a t e d  further  o r a l dose of c a r n i t i n e  carnitine levels i n patients  and  controls.  88 9.3  Implications The  discovery  common a s s u m p t i o n the  i s the  Carnitine content  of  single  the  bile  i n i n t e s t i n a l and  implication  of  precursors  but  dietary also  loss  in patients  oral  lack  and  of  tissue  carnitine depletion these  with  carnitine chronic  r e s u l t , i t might  individuals  carnitine.  with  with  these  patients  due  i t s amino  the  patients  that  only  studies.  to  be  of  of  carnitine  shown whether  or  well  the  levels  do  lack  acid  being  should  malabsorptive  concentrations  that  carnitine  reabsorption  i s s i g n i f i c a n t to  measured  of  the  excretion  not  in  excretion.  Until i t i s conclusively  serum and  supplement  stores  the  i s present  theory  t h i s work i s f o r  carnitine  from  the  It i s possible  carnitine  patient,  carnitine  of  syndromes.  bile.  this bile  route  disputes  consider  absorption  reduced  It also  challenges  carnitine  r e s e a r c h e r s must now  have compromised  If  dietary  bile  carnitine  malabsorption  the  only  in rat  for  Another  from  carnitine  that  intestinal tract.  urine  not  of  e x i s t and advisable  intravenous  or  of  be  syndromes. symptoms  of  to large  doses  References  89  Aas,  M. a n d B r e m e r , J . (1968) Short-chain fatty acid activation in rat liver. A new a s s a y p r o c e d u r e f o r t h e e n z y m e s a n d s t u d i e s on t h e i r i n t r a c e l l u l a r localization. Biochim. Biophys. Acta 164, 157-166.  Aas,  M. a n d D a a e , L.N.W. (1971) Fatty acid activation a c y l t r a n s f e r i n organs from rats i n d i f f e r e n t nutritional states. Biochim. Biophys. Acta 239, 208-216.  Bach,  A.C., S c h i r a r d i n , H., S i h r , M.-0. a n d S t o r c k , D. (1983) F r e e a n d t o t a l c a r n i t i n e i n human s e r u m a f t e r o r a l ingestion of L - c a r n i t i n e . Diabete Metab. 9, 121-124.  Bahl,  J . , N a v i n , T., M a n i o n , A. a n d B r e s s l e r , R. (1981) C a r n i t i n e t r a n s p o r t i n i s o l a t e d r a t h e a r t m y o c y t e s and the e f f e c t s o f 7,8-diOH c h l o r o p r o m a z i n e . C i r c . Res. 48, 378-385.  and  B a i l e y , D.G., P a u l , H. a n d J o h n s o n , G.E. (1975) Pentobarbital i n h i b i t s the b i l i a r y excretion of organic acids: a study of s u c c i n y l s u l f a t h i a z o l e i n the r a t . Can. J . P h y s i o l . Pharm. 53, 470-474. B e n k e , P . J . , M c K a y , B. a n d F o r a d a d a , J . (1984) for cystic fibrosis. Ped. Res. 18, 219A.  L-Carnitine  B i e b e r , L . L . , K r a h l i n g , J . B . , C l a r k e , P.R.H., V a l k n e r , K . J . a n d T o l b e r t , N.E. (1981) Carnitine acyltransferases i n r a t l i v e r peroxixomes. Arch. Biochem. Biophys. 211, 599-604. B o h m e r , T., N o r u m , K.R. a n d B r e m e r , J . (1966) The r e l a t i v e amounts o f l o n g - c h a i n a c y l c a r n i t i n e , a c e t y l c a r n i t i n e and f r e e c a r n i t i n e i n o r g a n s o f r a t s i n d i f f e r e n t n u t r i t i o n a l s t a t e s and w i t h a l l o x a n d i a b e t e s . Biochim. Biophys. Acta 125, 244-251. B o h m e r , T. (1967) Tissue acids(acylcarnitines) metabolism. Biochim.  l e v e l s of a c t i v a t e d f a t t y and t h e r e g u l a t i o n o f f a t t y a c i d Biophys. Acta 144, 259-270.  B o h m e r , T. a n d B r e m e r , J . (1968) Propionylcarnitine physiological variations i n vivo. Biochem. Biophys. Acta 152, 559-567. B o h m e r , T. (1974) Conversion of butyrobetaine to c a r n i t i n e i n the r a t i n vivo. Biochim. Biophys. Acta 343, 551-554. B o h m e r , T., R y d n i n g , A. a n d S o l b e r g , l e v e l s i n human s e r u m i n h e a l t h Chim. A c t a 57, 55-61.  H.E. (1974) and d i s e a s e .  Carnitine Clin.  90  B o h m e r , T., E i k l i d , K. a n d J o n s e n , u p t a k e i n t o human h e a r t c e l l s Biophys. Acta 465, 627-633.  J . (1977) i n culture.  Carnitine Biochim.  B r a s s , E.P. a n d H o p p e l , C.L. (1978) Carnitine metabolism i n t h e f a s t i n g r a t . J . B i o l . Chem. 253, 2688-2693. B r a s s , E.P. and H o p p e l , C.L. (1981) In vivo studies of c a r n i t i n e and f a t t y a c i d m e t a b o l i s m ; problems w i t h t h e use o f a n a e s t h e t i c s . A n a l . Biochem. 110, 77-81. Bremer, J . (1981) The e f f e c t o f f a s t i n g on t h e a c t i v i t y o f l i v e r 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 and i t s i n h i b i t i o n by m a l o n y l - C o A . Biochim. Biophys. Acta 665, 628-631. Bremer, J . (1983) P h y s i o l . Rev.  Carnitine - Metabolism 63, 1420-1480.  and  functions.  B r o o k s , D.E a n d M c i n t o s h , J . E . A . (1975) Turnover of c a r n i t i n e by r a t t i s s u e s . Biochem. J . 148, 439-445. B r o o k s , D.E. (1980) C a r n i t i n e and t h e male r e p r o d u c t i v e t r a c t and i t s r e l a t i o n t o t h e m e t a b o l i s m o f t h e e p i d i d y m i s and spermatozoa. In: Carnitine B i o s y n t h e s i s , M e t a b o l i s m and F u n c t i o n . E d i t e d b y R.A. F r e n k e l and J.D. M c G a r r y . A c a d e m i c P r e s s , L o n d o n , New York, San F r a n c i s c o , pp. 219-236. C a r t e r , H.E., B h a t t a c h a r y a , P.K., W e i d m a n , K.R. a n d F r a e n k e l , G. (1952) C h e m i c a l s t u d i e s on v i t a m i n B t i s o l a t i o n and c h a r a c t e r i z a t i o n o f c a r n i t i n e . Arch. Biochem. Biophys. 38, 405-416. C e d e r b l a d , G. a n d L i n d s t e d t , S. (1972) A method f o r t h e determination of c a r n i t i n e i n the picomole range. C l i n . Chim. A c t a 37, 235-243. C e d e r b l a d , G. a n d L i n d s t e d t , S. (1976) Metabolism of l a b e l l e d c a r n i t i n e i n the r a t . Arch. Biochem. Biophys. 175, 173-180. C h r i s t i a n s e n , R.Z. a n d B r e m e r , J . ( 1 9 7 6 ) Active transport of b u t y r o b e t a i n e and c a r n i t i n e i n t o i s o l a t e d liver cells. Biochim. Biophys. Acta 448, 562-577. C o o p e r , B., E a k i n s , M.N. a n d S l a t e r , T . F . (1976) The e f f e c t o f v a r i o u s a n a e s t h e t i c t e c h n i q u e s on t h e f l o w r a t e , c o n s t i t u e n t s and enzymic c o m p o s i t i o n o f t h e r a t bile. Biochem. Pharm. 25, 1711-1718. E n g e l , A.G. a n d R e b o u c h e , C . J . ( 1 9 8 4 ) Carnitine metabolism and i n b o r n e r r o r s . J . I n h e r i t e d M e t a b . D i s . 7, S38-S43.  91  E n g l a r d , S. (1979) Hydroxylation c a r n i t i n e i n human a n d m o n k e y 1102, 297-300. F r a e n k e l , G. (1951) Effect Arch. Biochem. Biophys.  of gamma-butyrobetaine tissues. FEBS L e t t .  and d i s t r i b u t i o n o f v i t a m i n 34, 457-463.  to  Bt.  F r e n c h , T . J . , G o o d e , A.W., H o l n e s s , M.J. and Sugden, M.C. (1985) C a r n i t i n e m e t a b o l i s m i n l i v e r s and h e a r t s o f 4 8 H - s t a r v e d r a t s : t h e c o n t r i b u t i o n o f f a t and carbohydrate to a c y l c a r n i t i n e formation. Biochem. I n t . 11, 265-272. F r i e d m a n , S. a n d acetylation 491-501.  F r a e n k e l , G. of c a r n i t i n e .  (1955) R e v e r s i b l e enzymic Arch. Biochem. Biophys. 59,  F r i e d m a n , S. (1958) Determination of c a r n i t i n e i n biological materials. Arch. Biochem. Biophys.  75,  F r i t z , I.B. (1955) The e f f e c t s o f m u s c l e e x t r a c t s on o x i d a t i o n o f p a l m i t i c a c i d by l i v e r s l i c e s a n d homogenates. Acta P h y s i o l . Scand. 34, 367-385.  24.  the  F r i t z , I.B. (1968) The m e t a b o l i c c o n s e q u e n c e s o f t h e e f f e c t s o f c a r n i t i n e on l o n g - c h a i n f a t t y a c i d oxidation. I n : C e l l u l a r C o m p a r t m e n t a l i z a t i o n and Control of Fatty Acid Metabolism. E d i t e d by F . C . G r a n . A c a d e m i c P r e s s , L o n d o n , pp. 39-63. F r o h l i c h , J . , Seccombe, I. (1978) Effect carnitine levels i of serum l e v e l s o f B-hydroxybutyrate.  D.W., H a h n , P., D o d e k , P. a n d H y n i e , o f f a s t i n g on f r e e a n d e s t e r i f i e d n human s e r u m a n d u r i n e : correlation f r e e f a t t y a c i d s and Metabolism 27, 555-561.  F r o h l i c h , J . , H a h n , P. a n d C l e a t o r , I . ( 1 9 8 0 ) Changes i n plasma c a r n i t i n e l e v e l s a f t e r j e j u n o i l e a l bypass. Lancet 1, 1 0 8 5 . F r o s t , S.C. a n d W e l l s , M.A. (1982) Effect f a t t y a c i d o x i d a t i o n on n e o n a t a l l i v e r content. Biochem. J . 204, 861-863. G i l b e r t , E.F. 161-169. G r o s s , C . J . and absorption 1320.  (1985)  Carnitine  H e n d e r s o n , L.M. by t h e r a t s m a l l  deficiency.  of i n h i b i t i o n of carnitine Pathology  17,  (1983) Carnitine intestine. Fed. Proc.  42,  G r o s s , C . J . a n d H e n d e r s o n , L.M. (1984) A b s o r p t i o n o f Da n d L - c a r n i t i n e by t h e i n t e s t i n e a n d k i d n e y t u b u l e i n the r a t . B i o c h i m . B i o p h y s . A c t a 772, 209-219.  92  G r o s s , C . J . , H e n d e r s o n , L.M. a n d S a v a i a n o , D.A. A b s o r p t i o n o f L a n d D c a r n i t i n e by i s o l a t e d enterocytes. Fed. Proc. 44, 763.  (1985) guinea  pigs  G u d j o n s s o n , H., L i , B U.K., S t a n c h e r , J . , Shug, A.L. and O l s e n , W.A. (1984) In vivo studies of small intestinal carnintine absorption. Gastroenterology 86, 1101. G u d j o n s s o n , H., L i , B U.K., S h u g , A. a n d O l s e n , W.A. (1985a) In v i v o s t u d i e s of i n t e s t i n a l c a r n i t i n e absorpton i n r a t s . Gastroenterology 88, 1880-1887. G u d j o n s s o n , H., L i , B U.K., Shug, A.L. and O l s e n , W.A. (1985b) Studies of c a r n i t i n e metabolism i n r e l a t i o n intestinal absorption. Am. J . P h y s i o l . 248, G313-G319.  to  G u l e w i t s c h , W. a n d K r i m b e r g , R. (1905) Zur kenntnis der e x t r a k t i v s t o f f e der m u s k e l n , 2 m i t t e i l u n g . Ueber das Carnitin. Z t s c h r f . P h y s i o l . Chem., S t r a s s b . 45, 326-330. G u m p e n , S.A. a n d N o r u m , K.R. (1973) The r e l a t i v e amount o f long-chain a c y l c a r n i t i n e s , s h o r t - c h a i n t i s s u e from r a t s f e d on r a p e s e e d o i l . Biochim. Biophys. Acta 316, 48-55. H a h n , P., C h a n e z , C. a n d H a m i l t o n , J . (1985) Carnitine c a r n i t i n e t r a n s f e r a s e s i n t h e i n t e s t i n a l mucosa of suckling rats. B i o l . Neonate 48, 77-84. H a m i l t o n , J.W., L i , B U.K., S h u g , A. a n d O l s e n , S t u d i e s o f L - c a r n i t i n e a b s o r p t i o n i n man. Gastroenterology 84, 1180.  W.A.  and  (1983)  H a n s o n , P . J . a n d C a r r i n g t o n , J.M. ( 1 9 8 1 ) A c t i v i t y o f 3-oxo acid CoA-transferase, D-3-hydroxybutyrate d e h y d r o g e n a s e , h e x o k i n a s e and 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 n t h e s t o m a c h and s m a l l and l a r g e i n t e s t i n e of the r a t . Biochem. J . 20, 349-355. H a r d i s o n , W.G.M. a n d A p t e r , J . T . (1972) M i c e l l a r theory of biliary cholesterol excretion. Am. J . P h y s i o l . 222, 61-67. H o p p e l , C . L . a n d G e n u t h , S. c a r n i t i n e metabolism. Huth,  (1976) Fasting alters C l i n . Res. 24, 580A.  P. a n d S h u g , A. (1980) P r o p e r t i e s of c a r n i t i n e transport i n r a t kidney cortex s l i c e s . Biochim. Biophys. Acta 602, 621-634.  93  Huth,  P., S c h m i d t , M., H a l l , P., F a r i e l l o , R. a n d S h u g , (1981) T h e u p t a k e o f c a r n i t i n e by s l i c e s o f r a t cerebral cortex. J . Neurochem. 36, 715-723.  A.  I n n i s , S.M. (1985) H e p a t i c t r a n s p o r t o f b i l e s a l t and b i l e composition following total parenteral n u t r i t i o n with a n d w i t h o u t l i p i d e m u l s i o n i n t h e r a t . Am. J . C l i n . Nutr. 4, 1 2 8 3 - 1 2 8 8 . I v y , A.C., O l d b e r g , E . , K l o s t e r , G. a n d L u e t h , H.C. (1928) A hormone mechanism f o r g a l l b l a d d e r c o n t r a c t i o n and evacuation. Am. J . P h y s i o l . 85, 381-383. J a m e s , M., B r o o k s , D. a n d S n o s w e l l , A. (1981) K i n e t i c s of c a r n i t i n e u p t a k e by r a t e p i d i d y m a l c e l l s ; a n d r o g e n d e p e n d e n c e and l a c k o f s t e r e o s p e c i f i c i t y . FEBS L e t t . 126, 53-56. K a r p a t i , G., C a r p e n t e r , S., E n g e l , A.G., W a t t e r s , G., A l l e n , J . , R o t h m a n , S., K l a s s e n , G. a n d M a m e r , O.A. (1975) The s y n d r o m e o f s y s t e m i c c a r n i t i n e d e f i c i e n c y . Neurology 25, 16-24. K u i p e r s , F . , D i j k s t r a , T., H a v i n g a , R., V a n A s s e l t , W., and Vonk, R . J . (1985) Acute e f f e c t s of pentobarbital-anaesthesia on b i l e s e c r e t i o n . Biochem. Pharmacol. 34, 1731-1736. K u t s c h e r , F. Ztschr, Berlin. L a R u s s o , N.F. and w h a t  (1905) Ueber L i e b i g ' s f l e i s c h e x t r a k t . f . Untersuch. d.Nahrungs-u. Genussmittel, 10, 528-537. (1984) Proteins i n bile: t h e y do. Am. J . P h y s i o l .  how t h e y g e t t h e r e 247, G199-G205.  Li,  B U.K., H a m i l t o n , J.W., S h a w , R.A., O l s e n , W.A., Shug, A.L. (1983) Carnitine transport i n rat jejunal m i c r o v i l l o u s membrane v e s i c l e s . Gastroenterology 84, 1230.  Li,  B U.K., G r a v e s , L . L . a n d O l s e n , W.A. (1985) Unidirectional carnitine fluxes i n isolated rat jejunum: evidence of d i f f u s i o n . Fed. Proc. 44,  L i n d s t e d t , G. a n d L i n d s t e d t , S. biosynthesis of c a r n i t i n e . 316-321.  (1965) Studies J . B i o l . Chem.  763.  on t h e 240,  M a n d a r i n o , L . , T s a l i k i a n , E . , B a r t o l d , S., M a r s h , H., C a r n e y , A., B u e r k i n , E . , T u t w e i l e r , G. H a y m o n d , M., H a n d w e r g e r , B. a n d R i z z a , R. (1984) Mechanism of h y p o g l y c e m i a and r e s p o n s e t o t r e a t m e n t w i t h an i n h i b i t o r o f f a t t y a c i d o x i d a t i o n on a p a t i e n t with i n s u l i n resistance to a n t i i n s u l i n receptor antibodies. J . C l i n . E n d o c r i n o l . Metab. 59, 658-664.  94  M a r q u i s , N.R. a n d F r i t z , I . B . (1964) Enzymological determination of f r e e c a r n i t i n e concentrations in rat tissues. J . L i p i d Res. 5, 1 8 4 - 1 8 7 . M c G a r r y , J . D . a n d F o s t e r , D.W. (1976) An i m p r o v e d a n d s i m p l i f i e d r a d i o i s o t o p i c assay f o r the determination f r e e and e s t e r i f i e d c a r n i t i n e . J . L i p i d Res. 17, 277-281.  of  M i k h a i l , M.M. a n d M a n s o u r , M.M. (1976) The r e l a t i o n s h i p between serum c a r n i t i n e l e v e l s and t h e n u t r i t i o n a l status of patients with s c h i s t o s o m i a s i s . C l i n . Chim. Acta 71, 207-214. M i n u k , G.Y. a n d S a r j e a n t , E . J . (1984) Gammaaminobutyric a c i d (GABA) and t h e h e p a t i c b i l e f l o w i n t h e r a t . C l i n . I n v e s t . Med. 7, 1 9 3 - 1 9 7 . M i t c h e l l , M.E. (1978a) C a r n i t i n e m e t a b o l i s m i n human s u b j e c t s I . N o r m a l m e t a b o l i s m . Am. J . C l i n . N u t r . 293-306.  31,  M i t c h e l l , M.E. (1978b) C a r n i t i n e m e t a b o l i s m i n human subjects I I . Values of c a r n i t i n e i n b i o l o g i c a l f l u i d s and t i s s u e s o f " n o r m a l " s u b j e c t s . Am. J . C l i n . N u t r . 31, 481-491. N o r u m , K.R. (1965) A c t i v a t i o n of palmityl-CoA:carnitine p a l m i t y l t r a n s f e r a s e i n the l i v e r s from f a s t e d , f a t - f e d , or d i a b e t i c r a t s . Biochim. Biophys. Acta 98, 652-654. O h t a n i , Y., E n d o , F . a n d M a t s u d a , I . (1982) d e f i c i e n c y and hyperammonemia a s s o c i a t e d acid therapy. J . Ped. 101, 782-785. O s m u d s e n , H. a n d peroxisomal 81-84.  Neat, C E . fatty acid  Carnitine with valproic  (1979) Regulation of oxidation. FEBS L e t t .  107,  P a c e , J . A . , W a n n e m a c h e r , R.W. a n d N e u f e l d , H.A. (1978) Improved r a d i o c h e m i c a l a s s a y f o r c a r n i t i n e and i t s d e r i v a t i v e s i n p l a s m a and t i s s u e e x t r a c t s . C l i n . Chem. 24, 32-35. Paik,  W.K. a n d K i m , S. (1975) Protein methylation: c h e m i c a l e n z y m o l o o g i c a l and b i o l o g i c a l s i g n i f i c a n c e . Adv. E n z y m o l . 42, 227-286.  P a n d e , S.V. a n d P a r v i n , R. (1976) C h a r a c t e r i z a t i o n of c a r n i t i n e a c y l c a r n i t i n e t r a n s l o c a s e system of heart mitochondria. J . B i o l . Chem. 251, 6683-6691.  95  P a n d e , S.V. (1981) Uneven d i s t r i b u t i o n o f p a l m i t o y l c a r n i t i n e i n s o l u t i o n s because of m i g r a t i o n t o air/water interphase. Biochim. Biophys. Acta 663, 669-673. P a n d e , S.V. a n d C a r a m a n c i o n , M.N. (1981) A simple r a d i o i s o t o p i c a s s a y of a c e t y l c a r n i t i n e and a c e t y l - C o A at picomolar l e v e l s . A n a l . Biochem. 112, 30-38. P a r v i n , R. a n d P a n d e , S.V. (1977) Microdetermination of ( - ) 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 activity. A n a l . Biochem. 79, 190-210. P e a r s o n , D . J . a n d T u b b s , P.K. (1967) C a r n i t i n e and derivatives i n rat tissues. Biochem. J . 105, 953-963. R e b o u c h e , C. (1977) C a r n i t i n e movement a c r o s s membranes s t u d i e d i n i s o l a t e d r a t m u s c l e . Biophys. Acta 471, 145-155.  muscle c e l l Biochim.  R e b o u c h e , C . J . a n d E n g e l , A.G. (1983) Kinetic compartmental analysis of c a r n i t i n e metabolism i n the dog. Arch. Biochem. Biophys. 220, 60-70. Rebouche, oral  C . J . and Edmonson, P.F. (1984) Metabolism of c a r n i t i n e i n the r a t . Fed. Proc. 43, 617.  R e b o u c h e , C . J . a n d E n g e l , A.G. (1984) Kinetic compartmental analysis of c a r n i t i n e metabolism i n the human c a r n i t i n e d e f i c i e n c y s y n d r o m e s ; e v i d e n c e f o r alterations i n tissue carnitine transport. J. Clin. Invest. 73, 857-867. R e b o u c h e , C . J . , M a c k , D.L. a n d E d m o n s o n , P . F . (1984) L-carnitine d i s s i m i l a t i o n i n the g a s t r o i n t e s t i n a l of t h e r a t . B i o c h e m i s t r y 23, 6422-6426.  tract  R u d m a n , D., A n s l e y , J . D . a n d S e w e l l , C.W. (1980) Carnitine deficiency i n cirrhosis. In: Carnitine Biosynthesis, M e t a b o l i s m and F u n c t i o n . E d i t e d b y R.A. F r e n k e l a n d J.D. M c G a r r y . A c a d e m i c P r e s s , L o n d o n , New Y o r k , S a n Francisco, pp. 307-320. S a c h a n , D.S. a n d R u a r k , R.A. (1985) D i s t r i b u t i o n of c a r n i t i n e and a c y l c a r n i t i n e i n s m a l l i n t e s t i n e o f c a r n i t i n e - s u p p l e m e n t e d and f a s t e d r a t s . J . Nutr. 865-871.  115,  S a n d o r , A., K i s p a l , G., M e l e g h , B. a n d A l k o n y i , I . (1985) Release of c a r n i t i n e from the perfused r a t l i v e r . Biochim. Biophys. Acta 835, 83-91.  96  S a r t o r e l l i , L.M., C i m a n , M., R i z z o l i , V. a n d S i l i p r a n d i , N. (1982) On t r a n s p o r t m e c h a n i s m s o f c a r n i t i n e a n d i t s derivatives i n r a t heart s l i c e s . I t a l . J . Biochem. 31, 261-268. S c h a n k e r , L . S . a n d S o l o m o n , H.M. (1963) Active o f q u a t e r n a r y ammonium c o m p o u n d s i n t o b i l e . Physiol. 204, 829-832. S c h a r s c h m i d t , B . F . a n d S c h m i d , R. sink. J . Clin. Invest. 62,  (1978) The 1122-1131.  transport Am. J .  micellar  S e c c o m b e , D.W., H a h n , P. a n d N o v a k , M. (1978) The e f f e c t o f d i e t a n d d e v e l o p m e n t on b l o o d l e v e l s o f f r e e a n d e s t e r i f i e d carnitine i n the r a t . Biochim. Biophys. Acta 528, 483-489. S e c c o m b e , D.W. (1981) 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 : t h e i r r o l e i n development. D i s s e r t a t i o n , University of B r i t i s h Columbia, Vancouver, Canada.  Ph.D.  S e c c o m b e , D., B u r g e t , D., F r o h l i c h , J . , H a h n , P., C l e a t o r , I . a n d G o u r l a y , R.H. (1984) Oral 1-carnitine a d m i n i s t r a t i o n a f t e r j e j u n o i l e a l by-pass surgery. I n t . J . Obes. 8, 4 2 7 - 4 3 3 . S e i m , H. a n d D a r g e l , R. (1978) Induktion mitochondrialer volumen anderungen d u r s h homologe o-acylcarnitin-isomere. A c t a B i o l . Med. G e r . 3 7 , 73-82. S h a w , R.D., S h u g , A . L . a n d O l s e n , W.A. (1982) Studies of c a r n i t i n e t r a n s p o r t by r a t s m a l l i n t e s t i n e . Gastroenterology 82, 1177. S h a w , R.D., L i B U.K., H a m i l t o n , J.W., S h u g , A . L . a n d O l s e n , W.A. (1983) Carnitine transport i n the r a t small intestine. Am. J . P h y s i o l . 245, G376-G381. S t r a s b e r g , S.M., H a r v e y , P.R.C. a n d G a l l i n g e r , S. (1984) Anatomy, v i s u a l i z a t i o n and s a m p l i n g of t h e b i l i a r y t r e e i n a n i m a l s a n d man. H e p a t o l o g y 4, 1 S - 3 S . T h u r s t o n , J . H . , C a r r o l l , J . E . , H a u h a r t , R.E. a n d S c h i r o , J.A. (1984) R e p l y t o l e t t e r : V a l p r o a t e t r e a t m e n t and carnitine deficiency. Neurology 34, 1128-1129. T o m i t a , M. a n d S e n d j u , Y. (1927) Uber d i e oxyaminoverbindungen, welche d i e B i u r e t r e a k t i o n zeigen. I I I . Spaltung der gamma-amino-beta-oxybuttersaure i n d i e o p t i s c h - a k - t i v e n komponenten. H o p p e - S e y e r ' s Z. P h y s i o l . Chem. 169, 263-277.  97  T u t w e i l e r , G.F., D e l l e v i g n e , P., M o h r b a c h e r , R. a n d H o , W. (1978) M e t h y l 2 - t e t r a d e c y l g l y c i d a t e ; an o r a l l y e f f e c t i v e hypoglycemic agent which i n h i b i t s long-chain fatty acid oxidation. Fed. Proc. 37, 1308. T u t w e i l e r , G.F. a n d D e l l e v i g n e , P. (1979) Action of the o r a l h y p o g l y c e m i c a g e n t 2 - t e t r a d e c y l g l y c i d i c a c i d on h e p a t i c f a t t y a c i d o x i d a t i o n and g l u c o n e o g e n e s i s . J. B i o l . Chem. 254, 2935-2941. W a y n f o r t h , H.B. (1980) E x p e r i m e n t a l and S u r g i c a l T e c h n i q u e in the Rat. Academic P r e s s , London. W h e e l e r , H.O. (1971) Concentration function gallbladder. Am. J . M e d . 5 1 , 588-595.  of the  W i l l n e r , J . , G i n s b u r g , S. a n d D i M a u r o , S. (1978) transport of c a r n i t i n e into s k e l e t a l muscle. 28, 721-724.  Active Neurology  W i n d m u e l l e r , H.G. a n d S p a e t h , A . E . (1980) Respiratory f u e l s and n i t r o g e n m e t a b o l i s m i n v i v o i n s m a l l i n t e s t i n e of f e d rats. J . B i o l . Chem. 255, 107-112. Y a l k o w s k y , S.H. a n d Z o g r a f i , G. (1970) Potentiometric t i t r a t i o n o f monomeric and m i c e l l a r a c y l c a r n i t i n e s . J . Pharm. S c i . 59, 798-802. Yue,  K.T.N, a n d F r i t z , I . B . ( 1 9 6 2 ) Fate of t r i t i u m - l a b e l l e d c a r n i t i n e a d m i n i s t e r e d t o dogs and rats. Am. J . P h y s i o l . 202, 122-128.  APPENDIX  The  Recovery  of  1  Intravenously Injected Bile  [ ^C]Carnitine i  in  Rat  98  The  o b j e c t i v e of  recovery  of  labelled  vein  in rat  Al.l  Methods  this  the  p u r i t y of TLC  phase was  jugular  [ 14-C ] c a r n i t i n e  system  recommended  composed  of  was  by  verified  by  Amersham.  using  The  n-butanol:acetic  v/v/v).  S u r g i c a l Procedure  The  bile  described of  the  Isotope  the  acid:water(12:3:5, Al.l.2  to determine  bile.  cellulose  mobile  was  c a r n i t i n e i n j e c t e d i n t o the  Al.1.1 P u r i t y of the The  experiment  duct  and  a j u g u l a r v e i n were c a n n u l a t e d  previously.  [l-^CJcarnitine  One  (2.5  ml  of  mCi/nmol) was  the  jugular vein.  Immediately  was  injected, bile  was  changed  every  recovery  of  Subsequent intervals  bile until  label  after  collected.  5 m i n u t e s f o r 60  the  saline  i n the  the  The  the  experiment, a blood  and  serum was  prepared  elapsed.  sample was by  that  be at  taken  tube  the  A n a l y s i s of  Bile disposable  and  10 the  minute termination  from t h e  c e n t r i f u g a t i o n as  samples  mini-counting  scintillation  fluid.  The  (50  vials  tail  described  u l e a c h ) were p l a c e d with  6 ml  of  measured.  Samples  serum  was  rate  earlier.  Al.l.3  uCi  labelled carnitine  so  At  0.05  injected into  collection  could  samples were c o l l e c t e d 2 h o u r s had  slowly  minutes  bile  containing  as  of  aqueous  s a m p l e s were t h e n mixed  and  in  of  vein  99  counted  on  quantitate bile,  the  an  LS9000 S c i n t i l l a t i o n c o u n t e r .  the  labelled  lipid  phase  scintillation the  bile  for  and  fluid  long-chain  from  and  three  In  carnitine  to  esters in  s a m p l e s was  counted.  order  mixed  the  with  The  specific  activities in  by  assaying  the  serum were d e t e r m i n e d  samples  carnitine.  A1.2  Results The  samples Figure  amounts of collected  11.  collected labelled  Also at  the  [ ] c a r n i t i n e  are  plotted  shown a r e  50%,  47%  present  i n the  the  and  30 50%  long-chain  the  i n the  minutes of  the  form  i n the  bile  rat separately  amounts of  of  recovered  maximum a p p r o x i m a t e l y cases.  f o r each  termination  carnitine  recovered  label  i n the  experiments.  The  bile  to  after  labelled i n the  increased  injection  serum  a  in a l l  carnitine  three  in  was  samples  analysed. The  specific  the  serum  the  b i l e was  rats.  a c t i v i t y of  i s shown i n T a b l e less  than  the  12.  i n the  carnitine The  serum  i n the  specific for three  bile  activity of  the  and in  five  100  FIGURE  11  The a p p e a r a n c e o f i n t r a v e n o u s l y i n j e c t e d [ C ] c a r n i t i n e i n 50 u l o f b i l e o f s i x r a t s c o l l e c t e d a t v a r i o u s i n t e r v a l s up to 2 hours a f t e r i n j e c t i o n . App r o x i m a t e l y 0.05 u C i was i n j e c t e d i n t o e a c h r a t . The amount o f [ l ^ C ] c a r n i t i n e f o u n d i n t h e serum a t t h e t e r m i n a t i o n of t h e e x p e r i m e n t i s a l s o i n d i c a t e d f o r each r a t (square symbol). l 4  OPM .i. 100/50 microliters  H  I  m 3  TABLE  The and  12  S p e c i f i c A c t i v i t y of C a r n i t i n e i n t h e Serum B i l e o f R a t s Two H o u r s A f t e r t h e I n t r a v e n o u s I n j e c t i o n of [ l ^ C j c a r n i t i n e  SPECIFIC (DPM/nmol RAT  SERUM  ACTIVITY Carnitine) BILE 232  1  131  2  158  3  299  247  4  254  287  5  300  284  .  214  APPENDIX  The  Effect  of  Saline  Infusion  Carnitine  Content  2  on of  Bile Rat  C o l l e c t i o n and Bile  the  103  The whether  o b j e c t i v e of or  not  collection and  the  the  bile  s a l i n e to  bile  flow  r a t e was  determined. w h e r e no  The  fluid  Unless described  a  27.8  ul/min  Model  collected  (7.0  0.1  ±  f r e e and  a l l procedures  each  rat, a  a  The  bile  peristaltic duct  hours  was  was  i n 15  constant  between  g BW).  this  i n the  and  greater  The  concentration  of  Saline  final  interval  infusion did  intervals.  total  not  from  5  a  the  affect  the  were  cannulated rate  of  Industries,  and  the  bile  collection  no  was  flow  in  period  non-infused  where  infused  of  was  the  free  rat bile  the  rats  non-infused there  period  significant  c a r n i t i n e i n the  bile  amounts of  at  as  samples.  i n the  i n the  of  rats  cannulated  bile  (p<0.01);  concentrations  The  The  i n Chapter  pump ( S c i e n t i f i c  collection  differed  concentration  acylcarnitine  those  infused  then  the  significantly  carnitine  rate  infused  bile.  v e i n was  T h e r e was  was  f o u r t h time  as  bile  flow  were performed  throughout  flow  the  bile  male W i s t a r  minute  except  rats only  of  acyl carnitine  to  jugular  s a l i n e s o l u t i o n was  rats  infused  lost  were compared  Seven a d u l t  ul/min/100  difference  hours  replaced.  In  flow  two  R a t s were  and  4.  f o r two  Bile  rat bile.  assessed  using  603).  determine  a f f e c t s the  fluid  specified,  sterile  of  to  during  the  was  anaesthetized.  ml)  was  replace  results  Chapter  lost  3.2  c a r n i t i n e content  with  experiment  fluid  (approximately  (IV)  and  this  a  c a r n i t i n e i n the  of  rat bile  the at  lower  infused  any  (p<0.01).  bile  carnitine at  bile  of  rats. and the  rat bile  in  time each  104  15  minute  collection  also  d i d not  change w i t h  saline  infusion. On fluid rat  the  b a s i s of  replacement  bile  collected  Therefore,  i t was  experiments,  fluid  these  d i d not  results, affect  i t was  the  carnitine  c o n t i n u o u s l y f o r up decided  that for bile  replacement  was  concluded  not  to  two  that  content hours.  collection necessary.  of  APPENDIX  The  Effect  3  of I n t e r r u p t i o n of the E n t e r o h e p a t i c C i r c u l a t i o n on t h e C a r n i t i n e C o n t e n t o f t h e B i l e  105  The  objective  collection only  from  a n a e s t h e t i z e d and  r a t was  the  i n Chapter  cannula second of  secured  minute  of  tube the  collecting was  The  slightly was  the  by  100  cannula  was  inch  below  the  performed  Carnitine as  then  and  two  while  cannulated inserted The  into  pyloric  Bile  enable  circulation.  duct  inserted  to  duct  greater diameter.  d i s c o n n e c t i n g the u l .  bile  i t s bile  subsequently  r a t one  was  enterohepatic  with cyanoacrylate glue.  intervals  analysis  4.  procedure  a cannulated  A  intestine  and  rat bile  following  interrupting  another  and  the  partially  described  of  of  of  was  described i n Chapter  into  other  end  the  sphincter sampled  interlocking  carnitine  as  ester 4.  at  15  tubes  APPENDIX  The  Effect  of  Pentobarbital  on t h e Bile  4  Carnitine  Content  of  the  106  The f o l l o w i n g effect  e x p e r i m e n t was p e r f o r m e d t o a s s e s s t h e  of pentobarbital  content of r a t b i l e  on t h e c h a n g e s i n t h e a c y l c a r n i t i n e  that  occur a f t e r  the i n i t i a t i o n of  collection. Bile  was c o l l e c t e d  anaesthetized. to  that  f r o m a r a t who had been  The b i l e  o f o t h e r s where b i l e  immediately a f t e r  the b i l e  experiment. in  the c o n t r o l  took  during  carnitine  effect. content  the course of the  r e m a i n e d t h e same l e v e l  b i l e while the t o t a l  was more t h a n d o u b l e t h a t  compared  established  surgery,the a c y l c a r n i t i n e  again increased The f r e e  c o n t e n t was t h e n  c o l l e c t i o n was  the anaesthetic  Despite the delayed of  carnitine  carnitine  of the c o n t r o l s .  as  concentration  APPENDIX 5  Potential  Mechanisms  for  the Rat  Transport Bile  of  Carnitine  Into  107  The been for  mechanism  for carnitine  investigated. the entry  bile.  Of  carnitine  into  Possibly  there  are separate  of the various  forms  of c a r n i t i n e  interest  are the large  quantities  esters.  The  pull  been h y p o t h e s i z e d (Hardison  transport  "micellar  f o r the entry  and A p t e r ,  1 9 7 2 ) may  long-chain  carnitine  esters.  long-chain  carnitine  esters  of  theory"  Both have  not  mechanisms into  the  which  has  also  into  the  bile  be a p p l i c a b l e cholesterol  the capacity  has  long-chain  of c h o l e s t e r o l also  bile  to  and to  the  form  micelles.  A5.1  Micellar  Pull  Theory  Assuming  that  the m i c e l l a r  describes the  the transport  following  would  be  would  passively  equal  concentrations  membrane. associate forming This  enter  bile  would  be  monomeric  the b i l e on b o t h  sink"  decrease  palmitoylcarnitine to  true:  acid  a "micellar  "pulled"  theory  from  sides  into  of the  phospholipid  (Scharschmidt  i n the b i l e  causing  the c a n a l i c u l a r  bile,  mixed and  of  achieving  canalicular  the p a l m i t o y l c a r n i t i n e  and  the  palmitoylcarnitine  the hepatocyte  the concentration  across  accurately  of p a l m i t o y l c a r n i t i n e  Once i n t h e b i l e , with  pull  would  micelles  Schmid,  1978).  monomeric  more c a r n i t i n e membrane f r o m  esters  the  hepatocyte. Some p r o p e r t i e s appropriate long-chain  of long-chain  f o r the " m i c e l l a r carnitine  esters  pull"  carnitine theory.  are amphiphiles  esters First, and  the  seem  108  concentration critical  micellar concentration.  associate as  in  with  forming  explain  bile  how  long-chain than  both  of the  To t e s t either  t h e serum  likely  m i c e l l e s as w e l l  Secondly,  this  could concentrated  and t h e h e p a t o c y t e s  while  the hypothesis,  (1985),  were  into  forming  carnitine,  bile  acid.  one w o u l d  acids  flow.  expect with  bile  a c i d was Despite  flow,  increase  bile  i s a micelle applies to  more a c y l c a r n i t i n e i n t h e taurocholate.  bile  To d i s t i n g u i s h and t h e  a non-micelle  forming  i n two a d d i t i o n a l r a t s .  an i n c r e a s e d " b i l e  flow,  free  and a c y l c a r n i t i n e i n t h e b i l e  rats  remained  t h e same  BW),  the rate of  Taurocholate  dehydrocholate,  infused  to  of each r a t .  the e f f e c t s of the m i c e l l e formation  increased  (IV) with  and a l b u m i n ( 4 % )  I f the m i c e l l a r theory  t h e two r a t s i n f u s e d  between  acids  during  jugular vein  and d e h y d r o c h o l i c  bile  (1972),  of the b i l e  a cannulated  dependent  infused  (0.24 umol/min/100 g  and A p t e r  Solutions  bile-acid  r a t s were  or dehydrocholate  collection. infused  I n f u s i o n on t h e C a r n i t i n e  ( 1 . 2 u m o l / m i n / 1 0 0 g BW), a c c o r d i n g  to Hardison  Taurocholic  Acid  Bile  taurocholate  according  bile  therefore  carnitine i snot.  Content  of  i s above i t s  a c y l c a r n i t i n e s a r e more  A5.2 The E f f e c t s o f B i l e  Innis  I t would  a c i d and p h o s p h o l i p i d  m i c e l l e s o f i t s own.  the b i l e  free  of p a l m i t o y l c a r n i t i n e i n b i l e  the concentration  of taurocholate  as i n t h e b i l e  of s a l i n e  of  infused  infused  109  rats.  Hence,  each  15 m i n u t e s  infused  t h e amount o f c a r n i t i n e was t h r e e  resulted  the carnitine  disputes  bile  was i n f u s e d .  of the b i l e  This  esters  into  investigating  that  were  carnitine  rats  acid  both  taurocholate  i n saline  increased.  of b i l e  long-chain  might  that  carnitine  i n fasting  This  have  been  i s formed  appears  concentrated of the With  the  a t t h e same  levels  carnitine  at the  t o be a  increased  the concentration  bile  i s true.  infusion,  remained  of  The r e s u l t s  the latter  With  found i n  might  b u t t h e amount o f  when b i l e  of c a r n i t i n e .  although the  i s independent  i t sformation.  controls  of r a t b i l e ,  amount o f c a r n i t i n e  i n the bile  membrane, t h e r e  infusion,  of  was h i g h e r  and d e h y d r o c h o l a t e  Therfore,  concentration  i n bile  suggests  infused  cannalicular  For instance,  the bile  after  infusion  f o r m a t i o n and  content  c o l l e c t i o n d i d not d i f f e r .  bile  concentration  made.  the overall  Alternatively,  the fasted  the carnitine  of c a r n i t i n e  15 m i n u t e  due t o t h e  the b i l e .  While  observations  to micelle  also  suggests  increased  f o r the transport  Observations  acid  concentration  theory  each  bile  content  Other  indicate  and c a r n i t i n e  the micellar  i n fed rats,  as  flow  than  than  in  f o r the saline  rather  concentration  flow.  than  higher  volume  carnitine  other  bile  when d e h y d r o c h o l a t e  increased  A5.3  i n the bile i n  controls. Increased  that  times  found  constant bile  flow,  as i n  of the carnitine i n  110  the  bile,  thus,  concentration as  i n fed  bile  of  the  r a t s but  might In  remains  constant.  initially  subsequent  these  s t u d i e s , the  was  carnitine  esterification  each  showed  carnitine doses  not  of  a  esters  suggests  that  into  bile  not  is still  actively  or  quaternary  did  fold  i n the  carnitine.  are  overall the  bile  Free the  transport  e s t e r s have m o l e c u l a r  mixed  or  ammonium c o m p o u n d s a r e  carnitine  found  1963),  greater  esters  e x p l a i n why  long-chain  the  from  than  colleagues i n the  the same  the  in bile.  formation.  The  not  into  on  the  course  (1985b),  amount with  did  but  of bolus  not  change.  one  the  bile.  actively  g/mol.  Other  transported  As  with short-chain  l e s s than  molecular long-chain  then  another.  carnitine is  actively  could  total  f r e e and a c y l c a r n i t i n e  weights  amounts of  carnitine esters  micelle  300  to  imposed  e s p e c i a l l y those  have g r e a t e r  large  free  injection  of  unknown w h e t h e r  weights  are  be  throughout  n e c e s s a r i l y dependent  Solomon,  of  carnitine levels  molecular  esters  and  after  and  might  might  treatments  passively transported  long-chain  ratio  increase  (Schanker  and  bile  however,  reabsorption  increase  Gudjonsson  two  This  It  water  a f f e c t e d by  experiment.  however,  formed  fasting,  occur.  carnitine  of  In  300  weights,  g/mol, this  carnitine  transported be  available for  APPENDIX  The Serum C a r n i t i n e  Content  6  of C y s t i c  Fibrosis  Patients  Ill  TABLE  13  The C o n c e n t r a t i o n o f F r e e and T o t a l C a r n i t i n e nf Cystic. F i b r o s i s P a t i en t s Patient Sample Free Total Carnitine Carnitine # # (uM) (uM) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33  1 2 1 1 1 1 1 1 2 1 2 1 1 1 1 2 3 1 1 1 2 3 1 1 1 1 1 1 1 2 1 1 1 1 2 3 1 2 3 1 1 1 1 1 1  38.0 45.0 52.1 71.8 33.2 35.4 31.0 32.2 43.5 50.3 51.6 8.0 18.9 49.9 40.2 34.9 7.5 47.3 61.9 48.7 49.5 49.5 28.4 54.6 33.2 49.5 39.7 56.2 49.7 47.0 40.3 39.0 25.4 43.0 38.1 36.7 38.6 37.7 44.7 29.8 50.7 38.3 40.5 45.6 39.7  54.5 53.2 57.2 84.9 42.2 47.5 42.0 50.1 49.8 58.6 64.4 10.1 24.5 45.2 54.3 49.3 11.5 52.6 68.3 69.5 61.5 61.5 50.1 60.6 42.2 56.0 44.9 66.1 57.5 60.6 46.8 48.8 36.8 65.4 55.4 55.9 49.2 49.3 66.0 47.2 79.0 53.4 52.0 52.0 47.7  i n t h e Serum Acyl Carnitine (%) 30 15 9 15 21 25 26 36 13 14 20 21 23 45 46 29 35 10 9 18 20 20 43 9 21 19 12 15 14 22 14 20 31 33 31 34 21 24 32 37 36 28 15 15 17  

Cite

Citation Scheme:

    

Usage Statistics

Country Views Downloads
China 28 38
United States 6 0
Germany 3 2
Japan 2 0
Saudi Arabia 1 0
France 1 0
United Kingdom 1 0
City Views Downloads
Beijing 23 0
Ashburn 6 0
Unknown 6 2
Shenzhen 5 38
Tokyo 2 0

{[{ mDataHeader[type] }]} {[{ month[type] }]} {[{ tData[type] }]}
Download Stats

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0096708/manifest

Comment

Related Items