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 1986

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

Item Metadata

Download

Media
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
Citation
1.0096708.ris

Full Text

THE CARNITINE AND CARNITINE ESTER CONTENT OF RAT BILE AND HUMAN DUODENAL FLUID By JENNIFER JANE HAMILTON B . S c , The U n i v e r s i t y of Guelph, 1983 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES D i v i s i o n of Human N u t r i t i o n We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA August 1986 ©J' C o p y r i g h t : J e n n i f e r Jane Hamilton, 1986 i i ABSTRACT C a r n i t i n e i s n e c e s s a r y f o r t h e b e t a - o x i d a t i o n o f l o n g - c h a i n f a t t y a c i d s . C a r n i t i n e e s t e r s a r e p r o d u c e d t o t r a n s p o r t f a t t y a c i d s a c r o s s t h e o t h e r w i s e i m p e r m e a b l e i n n e r m i t o c h o n d r i a l membrane. A l t h o u g h c a r n i t i n e h a s been i n v e s t i g a t e d i n many t i s s u e s , t h e c o n t e n t and p o s s i b l e r o l e s o f c a r n i t i n e i n t h e g a s t r o i n t e s t i n a l t r a c t h ave n o t been c o m p l e t e l y i n v e s t i g a t e d . The k i n e t i c s o f c a r n i t i n e a b s o r p t i o n have been d e t e r m i n e d b u t t h e t r e a t m e n t and s o u r c e s o f c a r n i t i n e i n t h e i n t e s t i n a l t r a c t a r e unknown. T h i s s t u d y i n v e s t i g a t e d t h e p o s s i b l e c o n t r i b u t i o n o f b i l e t o t h e c a r n i t i n e c o n t e n t o f t h e i n t e s t i n a l l u m e n . F i r s t , t h 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 b i l e were m e a s u r e d . T h e n , t h e o r i g i n o f c a r n i t i n e i n t h e b i l e was s t u d i e d . F i n a l l y , t h e c a r n i t i n e c o n t e n t o f human b i l e - r i c h d u o d e n a l f l u i d was i n v e s t i g a t e d . B i l e was c o l l e c t e d f r o m e l e v e n a n a e s t h e t i z e d ( p e n t o b a r b i t a l ) a d u l t m a le r a t s . B i l e f l o w was m e a s u r e d g r a v i m e t r i c a l l y and c a r n i t i n e and i t s e s t e r s were d e t e r m i n e d u s i n g a r a d i o c h e m i c a l c a r n i t i n e a s s a y . S p e c i f i c t y p e s o f c a r n i t i n e e s t e r s were q u a n t i t a t e d a f t e r f i r s t e x t r a c t i n g t h e s a m p l e s w i t h c h l o r o f o r m and m e t h a n o l . The o r i g i n o f c a r n i t i n e 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 m e a s u r i n g t h e c a r n i t i n e c o n t e n t o f b i l e f r o m r a t s who were f a s t e d f o r 72 h o u r s o r o r a l l y a d m i n i s t e r e d w i t h t e t r a d e c y l g l y c i d i c a c i d , an i n h i b i t o r o f 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 . T h e s e t r e a t m e n t s d i f f e r e n t l y a f f e c t t h e amounts and t y p e s o f c a r n i t i n e e s t e r s f o u n d i n t h e l i v e r i i i and e x t r a h e p a t i c a l l y . The b i l e c a r n i t i n e was compared t o t h e serum and l i v e r c a r n i t i n e a f t e r t h e s e t r e a t m e n t s t o see i f s i m i l a r i t i e s e x i s t e d . The o r i g i n o f c a r n i t i n e i n t h e b i l e was f u r t h e r s t u d i e d by a d m i n i s t e r i n g ^ C ] c a r n i t i n e i n t r a v e n o u s l y and d e t e r m i n i n g i t s r e c o v e r y i n t h e b i l e . Human b i l e - r i c h d u o d e n a l f l u i d was c o l l e c t e d f r o m t e n p a t i e n t s w i t h s u s p e c t e d c h o l e l i t h i a s i s by d u o d e n a l a s p i r a t i o n u s i n g n a s o g a s t r i c i n t u b a t i o n a f t e r 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 i n j e c t i o n . L a r g e q u a n t i t i e s o f t o t a l c a r n i t i n e and l o n g - c h a i n c a r n i t i n e e s t e r s were f o u n d i n r a t b i l e r e l a t i v e t o o t h e r t i s s u e s . I t a p p e a r s t h a t c a r n i t i n e e n t e r s t h e b i l e b o t h d i r e c t l y f r o m t h e n e w l y s y n t h e s i z e d o r s t o r e d h e p a t i c c a r n i t i n e p o o l and a l s o f o l l o w i n g h e p a t i c u p t a k e o f c a r n i t i n e f r o m s e rum. The l a t t e r i s f i r s t e s t e r i f i e d i n t h e l i v e r b e f o r e e n t e r i n g t h e b i l e . The t y p e s and amounts o f c a r n i t i n e e s t e r s i n t h e b i l e , t h u s , a p p e a r t o r e f l e c t t h e m e t a b o l i c s t a t e a t t h e t i m e o f b i l e f o r m a t i o n . C a r n i t i n e was a l s o f o u n d i n human b i l e - r i c h d u o d e n a l f l u i d . The p e r c e n t a g e o f l o n g - c h a i n c a r n i t i n e e s t e r s was s i m i l a r t o t h a t f o u n d i n r a t b i l e . The d i s c o v e r y o f c a r n i t i n e i n r a t b i l e c h a l l e n g e s t h e common a s s u m p t i o n t h a t o n l y d i e t a r y c a r n i t i n e i s p r e s e n t i n t h e i n t e s t i n a l t r a c t . I t a l s o d i s p u t e s t h e t h e o r y t h a t u r i n e i s t h e s o l e r o u t e f o r c a r n i t i n e e x c r e t i o n . I n a d d i t i o n , i t s u g g e s t s t h a t c a r n i t i n e d e p l e t i o n c o u l d r e s u l t i f c a r n i t i n e i n t h e b i l e i s n o t r e a b s o r b e d i n t h e i n t e s t i n e . T h i s m i g h t o c c u r w i t h g e n e r a l m a l a b s o r p t i o n s y n d r o m e s . i v TABLE OF CONTENTS A b s t r a c t i i L i s t o f T a b l e s i x L i s t o f F i g u r e s x i A c k n o w l e d g e m e n t s x i i L i s t o f A b b r e v i a t i o n s x i i i C h a p t e r 1 PURPOSE AND ORGANIZATION 1 C h a p t e r 2 LITERATURE REVIEW 2 2.1 D i s c o v e r y o f C a r n i t i n e 2 2.2 C h e m i s t r y o f C a r n i t i n e 2 2.3 F u n c t i o n o f C a r n i t i n e 4 2.4 S y n t h e s i s o f C a r n i t i n e 6 2.5 P r e s e n c e and D i s t r i b u t i o n o f C a r n i t i n e 6 2.6 C a r n i t i n e and t h e I n t e s t i n e 8 2.6.1 C a r n i t i n e i n Food 8 2.6.2 O r a l A d m i n i s t r a t i o n o f C a r n i t i n e 8 2.6.3 C a r n i t i n e i n t h e C o n t e n t s o f t h e G a s t r o i n t e s t i n a l T r a c t . 9 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 t h e I n t e s t i n a l M ucosa 10 2.6.5 C a r n i t i n e A c y l t r a n s f e r a s e s i n t h e I n t e s t i n e 11 2.6.6 C a r n i t i n e D e g r a d a t i o n i n t h e I n t e s t i n e 12 2.6.7 C a r n i t i n e M a l a b s o r p t i o n 12 2.7 C a r n i t i n e M e t a b o l i s m D u r i n g F a s t i n g 13 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 A c i d on C a r n i t i n e M e t a b o l i s m 14 V C h a p t e r 3 EXPERIMENTAL DESIGN AND RATIONALE 18 3.1 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 C o n t e n t o f R a t B i l e 19 3.2 The O r i g i n o f C a r n i t i n e i n R a t B i l e 20 3.2.1 The E f f e c t s o f a 72 Hour F a s t on t h e C o n t e n t o f R a t B i l e 20 3.2.2 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 A c i d 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 B i l e 21 3.2.3 The R e c o v e r y 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 R a t B i l e . . . . 21 3.3 The C a r n i t i n e C o n t e n t o f B i l e - R i c h Human D u o d e n a l F l u i d 22 C h a p t e r 4 METHODS AND MATERIALS 23 4.1 C h e m i c a l s and I s o t o p e s 23 4.2 H o u s i n g and C a r e o f A n i m a l s 24 4.3 R a t B i l e C o l l e c t i o n 24 4.4 Human B i l e - R i c h D u o d e n a l F l u i d C o l l e c t i o n . . . . 26 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 i n Serum and B i l e 26 4.5.1 P r i n c i p l e o f t h e C a r n i t i n e A s s a y 26 4.5.2 P r o c e d u r e Used 28 4.6 A c e t y l c a r n i t i n e A n a l y s i s 30 4.6.1 P r i n c i p l e o f Method 30 4.6.2 P r o c e d u r e Used 31 4.7 C a r n i t i n e E s t e r S e p a r a t i o n 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 Me t h o d 33 4.7.3 P r o c e d u r e Used 36 4.8 F a s t i n g P r o c e d u r e s 36 v i 4.9 T e t r a d e c y l g l y c i d i c A c i d P r e p a r a t i o n and A d m i n i s t r a t i o n 37 4.10 A n a l y s i s o f D a t a 37 C h a p t e r 5 THE CARNITINE AND CARNITINE ESTER CONTENT OF RAT B I L E 39 5.1 R e s u l t s 39 5.1.1 B i l e F l o w . 39 5.1.2 C a r n i t i n e C o n t e n t o f B i l e 39 5.1.3 C a r n i t i n e E s t e r s i n B i l e 39 5.1.4 Serum C a r n i t i n e 44 5.2 D i s c u s s i o n 44 C h a p t e r 6 THE ORIGIN OF CARNITINE IN RAT B I L E 51 6.1 The E f f e c t s o f a 72 Hour F a s 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 B i l e 51 6.1.1 R e s u l t s 51 6.1.1.1 B i l e F l o w and W e i g h t L o s s 51 6.1.1.2 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 . . . 51 6.1.1.3 C a r n i t i n e E s t e r s i n B i l e 55 6.1.1.4 Serum C a r n i t i n e 55 6.1.2 D i s c u s s i o n 55 6.2 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 A c i d 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 B i l e 60 6.2.1 R e s u l t s 60 6.2.1.1 B i l e F l o w and W e i g h t L o s s 60 6.2.1.2 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 . . . 60 6.2.1.3 C a r n i t i n e E s t e r s i n B i l e 65 6.2.1.4 Serum C a r n i t i n e 65 6.2.2 D i s c u s s i o n 65 v i i 6.3 The R e c o v e r y o f I n t r a v e n o u s l y I n j e c t e d [ 1 4 c ] C a r n i t i n e i n R a t B i l e 69 C h a p t e r 7 THE CARNITINE CONTENT OF HUMAN B I L E - R I C H DUODENAL FLUID 72 7.1 R e s u l t s 72 7.2 D i s c u s s i o n 72 C h a p t e r 8 CHANGES IN THE CARNITINE CONTENT OF RAT B I L E DURING COLLECTION 78 8.1 The E f f e c t o f S a l i n e I n f u s 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 78 8.2 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 C o n t e n t o f t h e B i l e 79 8.3 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 C o n t e n t o f t h e B i l e 79 8.4 Summary 80 C h a p t e r 9 CONCLUSIONS 82 9 .1 Summary 82 9.1.1 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 82 9.1.2 O r i g i n o f C a r n i t i n e i n t h e B i l e 82 9.1.3 The T r a n s p o r t o f C a r n i t i n e i n t o B i l e . . . 83 9.1.4 P o s s i b l e F u n c t i o n s o f C a r n i t i n e i n t h e B i l e 83 9.2 S u g g e s t i o n s f o r A d d i t i o n a l Work 84 9.2.1 The A b s o r p t i o n o f C a r n i t i n e and i t s E s t e r s 84 9.2.2 The I n v o l v e m e n t o f C a r n i t i n e i n F a t A b s o r p t i o n 85 9.2.3 The M a l a b s o r p t i o n o f B i l i a r y C a r n i t i n e 86 v i i i 9.2.4 The E f f e c t s o f H e p a t i c D y s f u n c t i o n on B i l i a r y C a r n i t i n e 86 9.2.5 B i l i a r y C a r n i t i n e i n Body P o o l S t u d i e s 87 9.2.6 C a r n i t i n e A b s o r p t i o n i n 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 Syndromes 87 9.3 I m p l i c a t i o n s 88 R e f e r e n c e s 89 A p p e n d i x 1 The R e c o v e r y o f I n t r a v e n o u s l y I n j e c t e d [ 1 4 C ] C a r n i t i n e i n R a t B i l e 98 A l . l M e t h o d s 98 A l . 1 . 1 P u r i t y o f I s o t o p e 98 A l . l . 2 S u r g i c a l P r o c e d u r e 98 A l . l . 3 A n a l y s i s o f S a m p l e s 98 A1.2 R e s u l t s 99 A p p e n d i x 2 The 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 o f R a t B i l e 103 A p p e n d i x 3 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 C o n t e n t o f R a t B i l e 105 A p p e n d i x 4 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 C o n t e n t o f R a t B i l e 106 A p p e n d i x 5 P o t e n t i a l M e c h a n i s m s f o r t h e T r a n s p o r t o f C a r n i t i n e i n t o R a t B i l e 107 A5.1 M i c e l l a r P u l l T h e o r y 107 A5.2 The E f f e c t s o f B i l e A c i d I n f u s 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 108 A5.3 O t h e r O b s e r v a t i o n s 109 A p p e n d i x 6 C a r n i t i n e C o n t e n t o f Serum o f C y s t i c F i b r o s i s P a t i e n t s I l l L I S T OF TABLES The 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 L i v e r 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 R a t s 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 i n R a t B i l e A f t e r 15 and 120 M i n u t e s o f 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 and C a r n i t i n e E s t e r s i n R a t Serum A f t e r 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 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 o f F e d and F a s t e d R a t s The Amounts 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 o f Fed and F a s t e d R a t s 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 and 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 and T o t a l C a r n i t i n e i n t h e B i l e o f F a s t e d and T D G A - T r e a t e d R a t s The Amounts 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 o f F a s t e d and T D G A - 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 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 Human D u o d e n a l F l u i d A f t e r S t i m u l a t i o n o f B i l e F l o w 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 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 o f C a r n i t i n e 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 T a b l e 13. The C o n c e n t r a t i o n s o f F r e e and T o t a l C a r n i t i n e i n t h e Serum o f C y s t i c F i b r o s i s P a t i e n t s I l l x i L I S T OF FIGURES F i g . 1. The S t r u c t u r e o f C a r n i t i n e 3 F i g . 2. The S t r u c t u r e o f T e t r a d e c y l g l y c i d i c A c i d 16 F i g . 3. T h i n - l a y e r C h r o m a t o g r a p h i c V e r i f i c a t i o n o f t h e S e p a r a t i o n o f 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 E x t r a c t i o n 35 F i g . 4. B i l e F l o w D u r i n g B i l e C o l l e c t i o n f r o m Fed R a t s 40 F i g . 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 Fed 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 41 F i g . 6. The Amounts 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 Fed R a t s a t 15 M i n u t e I n t e r v a l s 42 F i g . 7. B i l e F l o w D u r i n g B i l e C o l l e c t i o n f r o m Fed and 72 Hour F a s t e d R a t s 52 F i g . 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 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 56 F i g . 9. B i l e F l o w R a t e s 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 Hour 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 61 F i g . 10. 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 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 and 105 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 66 F i g . 1 1. 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 [ 1 4 C ] C a r n i t i n e i n 50 u l o f B i l e o f 6 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 t o 2 H o u r s A f t e r I n j e c t i o n 100 ACKNOWLEDGEMENTS The a u t h o r w o u l d l i k e t o t h a n k t h e f o l l o w i n g p e o p l e who h e l p e d w i t h t h i s p r o j e c t : - D r . P e t e r Hahn f o r h i s s u p e r v i s i o n and g r e a t f r i e n d s h i p . - c o m m i t t e e members, D r . S h e i l a I n n i s and Dr J o s e p h L e i c h t e r f o r t h e i r t i m e and g u i d a n c e . - L e i g h t o n James and D r . D a v i d Seccombe f o r t h e i r k i n d a s s i s t a n c e . - M a r t a T a l l e r , L i v i a S r u b i s k i , Wendy Cannon, V i c k y L o u i e and Nada Hahn f o r t h e i r t e c h n i c a l a d v i c e , and e v e r s t i m u l a t i n g c o n v e r s a t i o n . - t h e C y s t i c F i b r o s i s F o u n d a t i o n o f Canada f o r f i n a n c i a l s u p p o r t . - b o t h h e r h u s b a n d , Drew M e i k l e , and h e r p a r e n t s f o r m o r a l s u p p o r t . x i i i L I S T OF ABBREVIATIONS BW body w e i g h t °C d e g r e e s C e l s i u s c - c e n t i - C i C u r i e CAT 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 CoA coenzyme A CPT 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 CV c o e f f i c i e n t o f v a r i a t i o n DPM d i s i n t e g r a t i o n s p e r m i n u t e EDTA e t h y l e n e d i a m i n e t e t r a a c e t a t e g gram GOT 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 h h o u r HEPES N - 2 - h y d r o x y e t h y l p i p e r a z i n e - N ' - 2 - e t h a n e s u l p h o n i c a c i d I P i n t r a p e r i t o n e a l l y IV i n t r a v e n o u s l y 1 l i t e r m- m i l l i - M m o l a r min m i n u t e mol mole n- n a n o - NEM N - e t h y l m a l e i m i d e SE s t a n d a r d e r r o r TDGA t e t r a d e c y l g l y c i d i c a c i d x i v TLC t h i n l a y e r c h r o m a t o g r a p h y rpm r o t a t i o n s p e r m i n u t e u- m i c r o - UBC U n i v e r s i t y o f B r i t i s h C o l u m b i a v volume CHAPTER 1 Purpose and O r g a n i z a t i o n 1 The metabolism of c a r n i t i n e has been widely s t u d i e d i n many organs. The presence and r o l e of c a r n i t i n e i n the i n t e s t i n e , however, has been l a r g e l y i gnored. The o b j e c t i v e of t h i s work was to i n v e s t i g a t e the content and o r i g i n of c a r n i t i n e i n r a t b i l e and measure i t s presence i n human duodenal f l u i d . T h i s might r e v e a l an endogenous source of c a r n i t i n e i n the i n t e s t i n e and help to e l u c i d a t e a p o t e n t i a l metabolic r o l e f o r c a r n i t i n e i n t h i s organ. A review of the l i t e r a t u r e r e l e v a n t to t h i s study i s presented i n Chapter Two. In Chapter Three, the experimental design and r a t i o n a l e f o r each experiment are d e s c r i b e d . The methods used i n t h i s work are des c r i b e d i n Chapter Four. The experimental r e s u l t s are reported and dis c u s s e d i n Chapters F i v e , Six and Seven. Chapter E i g h t i n c l u d e s a d i s c u s s i o n of the r e s u l t s of other r e l a t e d i n v e s t i g a t i o n s . In Chapter Nine the experimental r e s u l t s are summarized and c o n c l u s i o n s are made. Suggestions f o r f u r t h e r s t u d i e s and the p o s s i b l e i m p l i c a t i o n s of t h i s work are a l s o presented. CHAPTER 2 L i t e r a t u r e R e v i e w 2 2.1 D i s c o v e r y o f C a r n i t i n e I n 1905 c a r n i t i n e was i n d e p e n d e n t l y d i s c o v e r e d by two l a b o r a t o r i e s as a component o f L i e b i g ' s meat e x t r a c t ( G u l e w i t s c h and K r i m b e r g ; K u t s c h e r ) . The s t r u c t u r e o f c a r n i t i n e was d e t e r m i n e d i n 1927 ( T o m i t a and S e n d j u ) and i n 1 1952 i t was i d e n t i f i e d a s a g r o w t h f a c t o r f o r t h e mealworm, T e n e b r i o m o l i t o r ( C a r t e r e t a l . , 1 9 5 2 ) . By 1955 t h e r o l e o f c a r n i t i n e i n l i p i d m e t a b o l i s m was known ( F r i e d m a n and F r a e n k e l ; F r i t z ) . 2.2 C h e m i s t r y o f C a r n i t i n e The s t r u c t u r e o f c a r n i t i n e , g a m m a - t r i m e t h y l a m i n o - 3 - h y d r o x y b u t y r a t e , i s shown i n F i g u r e 1. L - c a r n i t i n e i s t h e b i o l o g i c a l l y a c t i v e i s o m e r . F a t t y a c i d s a r e bound e n z y m i c a l l y t o t h e h y d r o x y l g r o u p o f c a r n i t i n e t o f o r m s h o r t - c h a i n ( 2 - 6 c a r b o n s ) , m e d i u m - c h a i n ( 8 - 1 2 c a r b o n s ) and l o n g - c h a i n (14 o r more c a r b o n s ) c a r n i t i n e e s t e r s . F r e e c a r n i t i n e and s h o r t 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 a r e u n i f o r m l y d i s t r i b u t e d i n a q u e o u s s o l u t i o n s b u t l o n g - c h a i n c a r n i t i n e e s t e r s m i g r a t e t o t h e a i r / w a t e r i n t e r p h a s e ( P a n d e , 1981) . Y a l k o w s k y and Z o g r a f i ( 1 9 7 0 ) showed t h a t c a r n i t i n e e s t e r s w i t h b e t w e e n 8 and 16 c a r b o n s a r e a m p h i p h i l e s and f o r m m i c e l l e s . The c r i t i c a l m i c e l l a r c o n c e n t r a t i o n d e c r e a s e s w i t h i n c r e a s i n g m o l e c u l a r w e i g h t . Pande ( 1 9 8 1 ) showed t h a t an u n e v e n 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 a l s o o c c u r s a t c o n c e n t r a t i o n s b e l o w t h e c r i t i c a l m i c e l l a r 3 + C H 3 C H 3 - N - C H 2 - C H - C H 2 - C O O - C H 3 OH FIGURE 1 The s t r u c t u r e o f c a r n i t i n e . 4 c o n c e n t r a t i o n . L o n g - c h a i n c a r n i t i n e e s t e r s a r e evenly- d i s t r i b u t e d i n s o l u t i o n s o f m e t h a n o l and c h l o r o f o r m ( P a n d e , 1981) . 2.3 F u n c t i o n o f C a r n i t i n e The p r i n c i p a l f u n c t i o n o f c a r n i t i n e i s t o t r a n s p o r t l o n g - c h a i n f a t t y a c i d s a c r o s s t h e i n n e r m i t o c h o n d r i a l membrane t o t h e m i t o c h o n d r i a l m a t r i x where b e t a - o x i d a t i o n o c c u r s ( F r i t z , 1 9 6 8 ) . I t i s n e c e s s a r y b e c a u s e f a t t y a c y l - C o A f o r m e d i n t h e c y t o s o l c a n n o t c r o s s t h e i n n e r m i t o c h o n d r i a l membrane w h i c h i s i m p e r m e a b l e t o CoA. The f o r m a t i o n o f f a t t y a c y l c a r n i t i n e i s c a t a l y z e d by 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 (CPT I , E.C.2.3.1.21) w h i c h i s l o c a t e d on t h e o u t e r s i d e o f t h e i n n e r m i t o c h o n d r i a l membrane. The t r a n s p o r t o f t h e c a r n i t i n e e s t e r a c r o s s t h e membrane i s f a c i l i t a t e d 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 ( P a n d e and P a r v i n , 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 e s t e r i s c a t a l y z e d by CPT I I w h i c h i s l o c a t e d on t h e i n s i d e o f t h e membrane. C a r n i t i n e c a n t h e n r e t u r n t o t h e o u t e r s i d e o f t h e membrane and r e p e a t t h i s p r o c e s s . C a r n i t i n e i s a l s o i n v o l v e d i n t h e b e t a - o x i d a t i o n o f s h o r t and m e d i u m - c h a i n f a t t y a c i d s . U s u a l l y t h e s e a r e a c t i v a t e d t o a c y l - C o A w i t h i n t h e m i t o c h o n d r i a and c a r n i t i n e i s n o t needed f o r membrane t r a n s p o r t ( A a s and B r e m e r , 1 9 6 8 ) . S h o r t e r - c h a i n f a t t y a c i d s may a r i s e as a r e s u l t o f o x i d a t i o n o f v e r y l o n g - c h a i n f a t t y a c i d s . P e r o x i s o m e s p a r t i a l l y s h o r t e n t h e s e f a t t y a c i d s i n d e p e n d e n t l y o f c a r n i t i n e 5 (Osmundsen and N e a t , 1 9 7 9 ) . The s h o r t e n e d e s t e r s a r e t h e n bound t o c a r n i t i n e and t r a n s f e r r e d t o t h e m i t o c h o n d r i a a s c a r n i t i n e e s t e r s f o r c o m p l e t e o x i d a t i o n ( B i e b e r e t a l . , 1 9 8 1 ) . P e r o x i s o m a l and m i t o c h o n d r i a l c a r n i t i n e a c e t y l - and o c t a n o y l - t r a n s f e r a s e s a r e r e q u i r e d f o r t h i s p r o c e s s ( B r e m e r , 1 9 8 3 ) . S h o r t e r - c h a i n c a r n i t i n e e s t e r s may a l s o be i n v o l v e d i n t h e m a i n t e n a n c e o f t h e i n n e r m i t o c h o n d r i a l a c y l - C o A / C o A r a t i o ( P e a r s o n and T u b b s , 1 9 6 7 ) . A c e t y l c a r n i t i n e i s f o r m e d f r o m c a r n i t i n e and a c e t y l - C o A f r e e i n g CoA w i t h i n t h e m i t o c h o n d r i a l m a t r i x f o r f u r t h e r f a t t y a c i d o x i d a t i o n ( B r e m e r , 1 9 8 3 ) . C a r n i t i n e may a l s o p l a y a r o l e i n c e r t a i n d i s e a s e o r t o x i c i t y s t a t e s . As a r e s u l t o f some i n b o r n e r r o r s o f m e t a b o l i s m , o r g a n i c a c i d s t h a t u n d e r g o i n t r a m i t o c h o n d r , i a l c h a i n s h o r t e n i n g a c c u m u l a t e a t t h e p o i n t o f t h e m e t a b o l i c d e f e c t c a u s i n g an a b n o r m a l l y h i g h a c y l - C o A / C o A r a t i o ( E n g e l and R e b o u c h e , 1 9 8 4 ) . An a p p a r e n t c a r n i t i n e d e f i c i e n c y r e s u l t s a s t h e c a r n i t i n e i s u s e d t o a c c e p t t h e o r g a n i c a c i d f r o m t h e CoA. The c a r n i t i n e e s t e r s t h e n l e a v e t h e c e l l and a r e e x c r e t e d i n t h e u r i n e . The same s i t u a t i o n o c c u r s d u r i n g v a l p r o i c a c i d t h e r a p y ( O h t a n i e t a l . , 1 9 8 2 ) . T h u r s t o n and c o l l e a g u e s ( 1 9 8 4 ) have h y p o t h e s i z e d t h a t h i g h l e v e l s o f v a l p r o y l - C o A a r e f o r m e d w i t h i n t h e m i t o c h o n d r i a and t h a t c a r n i t i n e i s a g a i n u s e d t o b u f f e r t h e a c y l - C o A / C o A r a t i o . I n b o t h o f t h e s e c o n d i t i o n s , c a r n i t i n e a d m i n i s t r a t i o n a l l o w s 6 t h e e x c r e t i o n o f t h e a c c u m u l a t e d a c i d f r o m t h e m i t o c h o n d r i a and a l l e v i a t e s 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 . 2.4 S y n t h e s i s o f C a r n i t i n e I n t h e s y n t h e s i s o f c a r n i t i n e , p r o t e i n - b o u n d l y s i n e and f r e e m e t h i o n i n e f i r s t f o r m t r i m e t h y l l y s i n e ( P a i k and K i m , 1 9 7 5 ) . T h e r e a r e s e v e r a l a d d i t i o n a l s t e p s i n t h e p r o c e s s b e f o r e t h e e v e n t u a l 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 h y d r o x y l a s e ( E . C . 1 . 1 4 . 1 1 . 1 ) t o p r o d u c e c a r n i t i n e ( L i n d s t e d t and L i n d s t e d t , 1 9 6 5 ) . I n humans, t h i s f i n a l s t e p i n c a r n i t i n e s y n t h e s i s o c c u r s i n t h e l i v e r , k i d n e y and b r a i n ( E n g l a r d , 1 9 7 9 ) , b u t i n r a t s , i s l i m i t e d t o t h e l i v e r (Bohmer, 1 9 7 4 ) . 2.5 P r e s e n c e and D i s t r i b u t i o n o f C a r n i t i n e M u s c l e c o n t a i n s 9 5 - 9 8 % o f t h e t o t a l body c a r n i t i n e i n man and dogs ( R e b o u c h e and E n g e l , 1983; Rebouche and E n g e l , 1984) and 9 8 % i n r a t s ( C e d e r b l a d and L i n d s t e d t , 1 9 7 6 ) . The r e m a i n i n g c a r n i t i n e i s i n t h e s e r u m , l i v e r and k i d n e y s . B o t h d o u b l e ( C e d e r b l a d and L i n d s t e d t , 1976) and t r i p l e c o m p a r t m e n t m o d e l s ( R e b o u c h e and E n g e l , 1983) have been h y p o t h e s i z e d t o e x p l a i n t h e d i s t r i b u t i o n o f c a r n i t i n e i n mammals. The two c o m p a r t m e n t m o d e l i n c l u d e s a s m a l l serum p o o l o f r a p i d l y t u r n i n g o v e r c a r n i t i n e and a l a r g e s t a b l e m u s c l e p o o l . The t h r e e c o m p a r t m e n t model d e f i n e s t h e l i v e r and k i d n e y c a r n i t i n e a s a s e p a r a t e p o o l h a v i n g a v e r y h i g h t u r n o v e r r a t e ( R e b o u c h e and E n g e l , 1 9 8 3 ) . B o t h m o d e l s have 7 been mathematically t e s t e d using data obtained from r a d i o l a b e l l e d t r a c e r experiments but i n both cases, the c a l c u l a t e d c a r n i t i n e e x c r e t i o n was g r e a t e r than the amount of c a r n i t i n e found i n the u r i n e . S i m i l a r r e s u l t s were a l s o found by Yue and F r i t z (1962) and Brooks and Mcintosh (1975). No other means of c a r n i t i n e breakdown or e x c r e t i o n , other than u r i n a r y , i s known. Methods f o r the assessment of c a r n i t i n e s t a t u s are c o n t r o v e r s i a l . There i s no c o r r e l a t i o n between muscle and serum c a r n i t i n e c o n c e n t r a t i o n s (Cederblad and L i n s t e d t , 1976). Both muscle and serum c a r n i t i n e l e v e l s are low only i n the case of severe muscle d e p l e t i o n ( M i t c h e l l , 1978b). C a r n i t i n e d e f i c i e n c y i s very r a r e and although not always rec o g n i z e d , the symptoms are d i s t i n c t . The importance of s u b c l i n i c a l c a r n i t i n e d e f i c i e n c i e s i s not known. The degree of e s t e r i f i c a t i o n of serum c a r n i t i n e i s a u s e f u l index of metabolic s t a t e . For i n s t a n c e , h i g h l y e s t e r i f i e d serum c a r n i t i n e i s i n d i c a t i v e of f a s t i n g (Brass and Hoppel, 1978), a high d i e t a r y f a t content (Seccombe et a l . , 1978) or some types of metabolic a c i d u r i a (Engel and Rebouche, 1984). The s p e c i f i c c a r n i t i n e e s t e r s present give a d d i t i o n a l i n f o r m a t i o n about the metabolic s t a t e of the i n d i v i d u a l . 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 c a r n i t i n e content of food has been s t u d i e d by many l a b o r a t o r i e s ; the r e s u l t s have been reviewed ( M i t c h e l l , 1978a). C a r n i t i n e i s most abundant i n meats but i s a l s o present i n p l a n t s . 2.6.2 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 a b s o r p t i o n of c a r n i t i n e was f i r s t demonstrated by K a r p a t i and c o l l e a g u e s (1975). Upon a d m i n i s t e r i n g o r a l c a r n i t i n e to a c a r n i t i n e d e f i c i e n t p a t i e n t , the serum c a r n i t i n e c o n c e n t r a t i o n i n c r e a s e d . Since then, s i m i l a r o b s e r v a t i o n s have been made by many other authors. G e n e r a l l y , a f t e r treatment with o r a l c a r n i t i n e , the symptoms a s s o c i a t e d with c a r n i t i n e d e f i c i e n c y disappeared, and serum and u r i n e c a r n i t i n e l e v e l s i n c r e a s e d ( G i l b e r t , 1985; Engel and Rebouche, 1984). In 1983, Bach and c o l l e a g u e s s t u d i e d c a r n i t i n e a b s o r p t i o n using twelve apparently healthy students. Two grams of L - c a r n i t i n e were administered o r a l l y to s i x of the students; the remainder r e c e i v e d a placebo. Free and t o t a l serum c a r n i t i n e were monitored during the subsequent 24 hours. C a r n i t i n e l e v e l s peaked a f t e r 3.5 hours and had not returned to b a s e l i n e l e v e l s at the end of the 24 hour p e r i o d . Other i n v e s t i g a t o r s have repo r t e d that serum c a r n i t i n e l e v e l s are s t i l l e l e v a t e d i n humans 4 hours a f t e r an o r a l dose (Seccombe et a l . , 1984; F r o h l i c h et a l . , 1978). S i m i l a r o b s e r v a t i o n s have been made i n r a t s . 9 Gudjonsson and c o l l e a g u e s (1984) observed that a f t e r an i n t r a l u m i n a l dose of l a b e l l e d c a r n i t i n e , the amount of l a b e l i n the serum had not yet peaked 8 hours l a t e r . S i m i l a r l y , Gross and Henderson (1984) found 35% of an i n t r a l u m i n a l dose of l a b e l l e d c a r n i t i n e i n the serum 7 hours a f t e r a d m i n i s t r a t i o n . 2.6.3 C a r n i t i n e i n the Contents of the G a s t r o i n t e s t i n a l T r a c t Sachan and Ruark (1985) s t u d i e d fed and f a s t e d a d u l t r a t s to determine the content of endogenous c a r n i t i n e i n the i n t e s t i n a l lumen and t i s s u e . Although the d i e t s contained only f r e e c a r n i t i n e and lo n g - c h a i n c a r n i t i n e e s t e r s , short and medium-chain c a r n i t i n e e s t e r s were found i n the i n t e s t i n a l lumen. With f a s t i n g , t h i s f r a c t i o n decreased. The authors hypothesized that these short and medium-chain c a r n i t i n e e s t e r s may have been r e l e a s e d from i n t e s t i n a l t i s s u e , formed due to i n t r a l u m i n a l e s t e r i f i c a t i o n or produced by i n t e s t i n a l microorganisms. I n v e s t i g a t i o n s of the i n t e s t i n a l contents of s u c k l i n g r a t s by Hahn and coworkers (1985) found that the degree of c a r n i t i n e e s t e r i f i c a t i o n i n c r e a s e d throughout the l e n g t h of the g a s t r o i n t e s t i n a l t r a c t . I t was hypothesized that t h i s was due to p r e f e r e n t i a l a b s o r p t i o n of f r e e c a r n i t i n e . 10 2.6.4 K i n e t i c s o f C a r n i t i n e U p t a k e i n t o I n t e s t i n a l M u c o s a l C e l l s The k i n e t i c s o f c a r n i t i n e u p t a k e and r e l e a s e f r o m i n t e s t i n a l m u c o s a l c e l l s h ave been s t u d i e d by g r o u p s l e d by G r o s s o f t h e U n i v e r s i t y o f M i n n e s o t a ( G r o s s e t a l . , 1983; 1984; 1985) and O l s e n o f t h e U n i v e r s i t y o f W i s c o n s i n ( G u d j o n s s o n e t a l . , 1984; 1985a; 1985b; H a m i l t o n e t a l . , 1983; L i e t a l . , 1983; 1985; Shaw e t a l , 1982; 1 9 8 3 ) . B o t h i n v i t r o and _in v i v o e x p e r i m e n t s were done u s i n g humans, r a t s , g u i n e a p i g s and r a b b i t s . The t e c h n i q u e s u s e d i n c l u d e d i n t e s t i n a l l u m e n a l and v a s c u l a r p e r f u s i o n , i n t r a l u m e n a l b o l u s dose i n j e c t i o n , e v e r t e d i n t e s t i n a l r i n g s and s a c s , e n t e r o c y t e t i s s u e c u l t u r e , m u c o s a l i n c u b a t i o n , i s o l a t e d b r u s h b o r d e r membrane v e s i c l e s and t h e U s s i n g chamber mounted w i t h i n t e s t i n a l e p i t h e l i u m . The r e s u l t s o f b o t h t h e M i n n e s o t a and W i s c o n s i n g r o u p s c o n c u r : c a r n i t i n e and a c e t y l c a r n i t i n e a r e t a k e n up more r a p i d l y i n t o j e j u n a l m u c o s a l c e l l s t h a n i l e a l c e l l s w h i l e t h e duodenum does n o t a b s o r b c a r n i t i n e o r c a r n i t i n e e s t e r s . C a r n i t i n e u p t a k e i n t o e n t e r o c y t e s i s a c t i v e , s o d i u m d e p e n d e n t and p o t e n t i a l l y c a r r i e r m e d i a t e d . T h i s i s s i m i l a r t o c a r n i t i n e u p t a k e r e p o r t e d i n o t h e r t i s s u e s i n c l u d i n g t h e l i v e r ( C h r i s t i a n s e n and B r e m e r , 1 9 7 6 ) , t h e s k e l e t a l m u s c l e ( W i l l n e r e t a l . , 1978; R e b o u c h e , 1 9 7 7 ) , t h e k i d n e y c o r t e x ( H u t h and Shug, 1 9 8 0 ) , t h e e p i d i d y m a l c e l l s ( James e t a l . , 1 9 8 1 ) , t h e h e a r t ( S a r t o r e l l i e t a l . , 1982; B a h l e t a l . , 1981; Bohmer e t a l . , 1977) and t h e c e r e b r a l c o r t e x ( H u t h e t 11 a l . , 1 9 8 1 ) . B o t h g r o u p s a l s o showed t h a t a t v e r y h i g h c a r n i t i n e c o n c e n t r a t i o n s p a s s i v e d i f f u s i o n o c c u r s . The G r o s s and O l s e n g r o u p s a l s o d e m o n s t r a t e d t h a t once i n s i d e t h e m u c o s a l c e l l , as much as 5 0 % o f t h e c a r n i t i n e i s e s t e r i f i e d t o a c e t y l c a r n i t i n e . O t h e r a u t h o r s h a v e a l s o shown a h i g h d e g r e e o f a c e t y l c a r n i t i n e p r o d u c t i o n w i t h i n t h e e n t e r o c y t e (Hahn e t a l . , 1985; S a c h a n and R u a r k , 1 9 8 5 ) . A l l i n v e s t i g a t o r s a g r e e t h a t t h e r e l e a s e o f c a r n i t i n e f r o m t h e m u c o s a l c e l l s i s v e r y s l o w . 2.6.5 C a r n i t i n e A c y l t r a n s f e r a s e s i n t h e I n t e s t i n e I n 1 9 8 1 , Hanson and C a r r i n g t o n f o u n d t h a t t h e a c t i v i t y o f CPT i n a d u l t r a t i n t e s t i n a l mucosa ( u m o l / m i n / g wet w e i g h t ) was o n l y one t h i r d t h e a c t i v i t y f o u n d i n t h e l i v e r . 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 (CAT, nmol/min/mg p r o t e i n ) , h o w e v e r , was a p p r o x i m a t e l y 4 0 % more a c t i v e i n t h e mucosa t h a n t h e l i v e r ( G u d j o n s s o n e t a l . , 1 9 8 5 a ) . As t h e e n e r g y m e t a b o l i s m o f i n t e s t i n a l c e l l s i s b a s e d on g l u t a m i n e m e t a b o l i s m ( W i n d m u e l l e r and S p a e t h , 1 9 8 0 ) , t h e l e s s e r a c t i v i t y o f CPT p r o b a b l y r e f l e c t s t h e f a c t t h a t f a t i s n o t a m a j o r e n e r g y s o u r c e f o r t h e m u c o s a l c e l l . The h i g h a c t i v i t y o f CAT i n t h e mucosa i s p r o b a b l y r e s p o n s i b l e f o r t h e h i g h d e g r e e o f e s t e r i f i c a t i o n o f c a r n i t i n e t o a c e t y l c a r n i t i n e w i t h i n t h e m u c o s a l c e l l . Hahn and c o l l e a g u e s ( 1 9 8 5 ) f o u n d t h a t t h e a c t i v i t i e s o f c a r n i t i n e a c y l t r a n s f e r a s e s (nmol/min/mg p r o t e i n ) i n t h e i n t e s t i n a l mucosa o f d e v e l o p i n g r a t s were e l e v a t e d a f t e r b i r t h and d e c r e a s e d t o a d u l t l e v e l s 12 at weaning. They a l s o found that mucosal c e l l CPT I a c t i v i t y was much grea t e r than that of CPT I I . T h i s i m p l i e s that p a l m i t o y l c a r n i t i n e i s formed i n the mucosal c e l l but that the p a l m i t a t e i s not o x i d i z e d t h e r e . 2.6.6 C a r n i t i n e Degradation i n the I n t e s t i n e There i s no evidence that c a r n i t i n e i s metabolized to other compounds i n r a t t i s s u e s (Brooks and Mcintosh, 1975). B a c t e r i a l degradation of c a r n i t i n e i n the i n t e s t i n a l t r a c t was suspected f o r s e v e r a l years before Rebouche and c o l l e a g u e s (1984) used normal and germ-free r a t s to demonstrate that endogenous i n t e s t i n a l f l o r a i s r e s p o n s i b l e f o r degradation of c a r n i t i n e i n the lower g a s t r o i n t e s t i n a l t r a c t . In Rebouche's experiments, l a b e l l e d c a r n i t i n e and m e t a b o l i t e s were found i n the feces of r a t s who had r e c e i v e d e i t h e r o r a l or intravenous c a r n i t i n e . Hahn and c o l l e a g u e s (1985) showed a s i m i l a r phenomenon i n s u c k l i n g r a t s who had been i n j e c t e d subcutaneously with l a b e l l e d c a r n i t i n e . Twenty-four hours a f t e r i n j e c t i o n , l a b e l l e d c a r n i t i n e had accumulated i n the i n t e s t i n a l mucosa. 2.6.7 C a r n i t i n e Malabsorption Three s i t u a t i o n s of p o s s i b l e c a r n i t i n e malabsorption have been s t u d i e d . F r o h l i c h and coworkers (1980) repo r t e d that serum c a r n i t i n e l e v e l s were reduced a f t e r j e j u n o i l e a l bypass surgery and continued to decrease post o p e r a t i v e l y . 13 Whether decreased c a r n i t i n e or c a r n i t i n e p recursor i n g e s t i o n c o n t r i b u t e d to t h i s i s unknown. Seccombe and coworkers (1984) measured c a r n i t i n e a b s o r p t i o n i n s i m i l a r p a t i e n t s . Serum c a r n i t i n e was measured upon a d m i n i s t r a t i o n of an o r a l dose of c a r n i t i n e . A f t e r surgery, the i n c r e a s e i n serum c a r n i t i n e was 38% l e s s than before surgery. Thus, i t appears l i k e l y that j e j u n o i l e a l bypass surgery r e s u l t s i n c a r n i t i n e malabsorption. M i k h a i l and Mansour (1976) found decreased serum c a r n i t i n e l e v e l s i n p a t i e n t s s u f f e r i n g from i n t e s t i n a l s c h i s t o s o m i a s i s i n f e c t i o n s . The p a t i e n t s with the most severe cases had the lowest serum c a r n i t i n e l e v e l s . With n u t r i t i o n a l r e p l e t i o n and drug treatment, serum c a r n i t i n e l e v e l s were r e s t o r e d . Serum c a r n i t i n e l e v e l s were a l s o decreased i n p a t i e n t s with Crohn's disease and some p a t i e n t s with a h i s t o r y of v i l l o u s atrophy and malabsorption (Bohmer et a l . , 1974). Although the reduced serum c a r n i t i n e l e v e l s i n these cases could be due to reduced a b s o r p t i o n of the p r e c u r s o r s f o r c a r n i t i n e s y n t h e s i s , c a r n i t i n e malabsorption i t s e l f might have c o n t r i b u t e d . 2.7 C a r n i t i n e Metabolism During F a s t i n g Adipocyte l i p o l y s i s i s e l e v a t e d a f t e r f a s t i n g and f a t t y a c i d a v a i l a b i l i t y and u t i l i z a t i o n i s i n c r e a s e d . As c a r n i t i n e i s i n t i m a t e l y i n v o l v e d i n f a t t y a c i d o x i d a t i o n , the types of c a r n i t i n e e s t e r s i n the r a t l i v e r are a l t e r e d 14 and t h e p e r c e n t a g e o f c a r n i t i n e e s t e r i f i c a t i o n i n t h e serum i s c h a n g e d ( B r a s s and H o p p e l , 1 9 7 8 ) . The r e s u l t s o f s t u d i e s on t h e e f f e c t s o f f a s t i n g 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 l i v e r a r e shown i n T a b l e 1. A l l d a t a i s g i v e n as a p e r c e n t o f t o t a l l i v e r c a r n i t i n e . The i n c r e a s e i n t h e o x i d a t i o n o f f a t t y a c i d s d u r i n g f a s t i n g g i v e s r i s e t o i n c r e a s e d amounts o f l o n g e r - c h a i n c a r n i t i n e e s t e r s . The a c c u m u l a t i o n o f l o n g - c h a i n c a r n i t i n e e s t e r s i s a l s o due t o t h e h i g h e r a c t i v i t y o f h e p a t i c a c t i v i t y o f CPT I t h a n CPT I I ( B r e m e r , 1981; Aas and Daae, 1971; Norum, 1 9 6 5 ) . The d e c r e a s e i n t h e amount o f f r e e c a r n i t i n e i n t h e l i v e r i s a r e s u l t o f t h e i n c r e a s e d l e v e l s o f l o n g - c h a i n c a r n i t i n e e s t e r s . Serum c a r n i t i n e e s t e r i f i c a t i o n i n r a t s i n c r e a s e d f r o m 4 1 % t o 69% a f t e r a 72 h o u r f a s t i n g p e r i o d ( B r a s s and H o p p e l , 1 9 7 8 ) . T h i s i n c r e a s e was m a i n l y i n t h e f o r m o f a c e t y l c a r n i t i n e and was l i k e l y t h e r e s u l t o f c a r n i t i n e b e i n g u s e d t o b u f f e r t h e i n t r a m i t o c h o n d r i a l a c y l - C o A / C o A r a t i o . I n humans, serum c a r n i t i n e e s t e r i f i c a t i o n a l s o i n c r e a s e d upon f a s t i n g ( F r o h l i c h e t a l . , 1978; H o p p e l and G e n u t h , 1 9 7 6 ) . 2.8 The E f f e c t o f T e t r a d e c y l g l y c i d i c A c i d on C a r n i t i n e M e t a b o l i s m T e t r a d e c y l g l y c i d i c a c i d (TDGA, McN-3802) i s a f a t t y a c i d s t r u c t u r a l a n a l o g u e ( F i g u r e 2 ) . I t was f i r s t r e p o r t e d a s a CPT I i n h i b i t o r by M c N e i l L a b o r a t o r i e s ( T u t w e i l e r e t a l , TABLE 1 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 L i v e r L e n g t h 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 ( % o f T o t a l ) o f F a s t R e f e r e n c e ( h o u r s ) F r e e A c e t y l S h o r t and L o n g - Medium- c h a i n c h a i n 24 f e d f a s t f e d 26 f a s t 51 f e d f a s t f e d 2 f a s t 20 Bohmer and B r e m e r , 1968 24 73 45 - - 25 42 2 13 Bohmer, 1967 48 60 49 - - 38 38 3 16 Bohmer e t a l . , 1966 24 48 30 17 26 51 50 1 19 B r a s s and H o p p e l , 1981 24 53 41 - - 43 44 3 18 B r a s s and H o p p e l , 1978 48 53 43 - - 43 43 3 14 B r a s s and H o p p e l , 1978 72 53 47 - - 43 45 3 11 B r a s s and H o p p e l , 1978 48 - 29 - - - 58 - 14 F r e n c h e t a l . , 1985 36-48 56 31 13 23 27 28 3 18 P e a r s o n and Tubbs , 1967 16 CH3(CH2 )-j3 FIGURE 2 The s t r u c t u r e o f t e t r a d e c y l g l y c i d i c a c i d . 17 1978; T u t w e i l e r and D e l l e v i g n e , 1979). TDGA very p o t e n t l y and s p e c i f i c a l l y i n h i b i t s CPT I i i i v i vo and jLn v i t r o i n r a t s by i r r e v e r s i b l y i n a c t i v a t i n g the a c t i v e s i t e ( T u t w e i l e r and D e l l e v i g n e , 1979). I t , t h e r e f o r e , i n h i b i t s the o x i d a t i o n of long - c h a i n but not short or medium-chain f a t t y a c i d s . Long-chain acyl-CoA accumulates i n the c y t o s o l of the hepatocyte as i t cannot c r o s s the inner m i t o c h o n d r i a l membrane. Concent r a t i o n s of lon g - c h a i n c a r n i t i n e e s t e r s decrease and the c o n c e n t r a t i o n of f r e e c a r n i t i n e i s in c r e a s e d ( T u t w e i l e r et a l . , 1978). As a r e s u l t of the decreased f a t t y a c i d o x i d a t i o n , gluconeogenesis i s i n h i b i t e d when TDGA i s administered i n the f a s t i n g s t a t e r e s u l t i n g i n hypoglycemia i n both man and r a t s ( T u t w e i l e r and D e l l e v i g n e , 1979; Mandarino et a l . , 1984). French and c o l l e a g u e s demonstrated the e f f e c t s of o r a l TDGA a d m i n i s t r a t i o n on the l i v e r s of r a t s p r e v i o u s l y f a s t e d f o r 48 hours (1985). Free c a r n i t i n e i n c r e a s e d s i g n i f i c a n t l y , short and medium-chain a c y l c a r n i t i n e l e v e l s remained constant and lon g - c h a i n a c y l c a r n i t i n e decreased. An i n c r e a s e i n the t o t a l c a r n i t i n e content of the l i v e r was a l s o noted. F r o s t and Wells (1982), studying neonatal r a t s , showed that TDGA produced no change i n l i v e r weight but in c r e a s e d the s i z e of the h e p a t i c c a r n i t i n e p o o l . T h i s was p r i m a r i l y due to an i n c r e a s e i n f r e e c a r n i t i n e and was hypothesized to be a compensatory mechanism to overcome the i n h i b i t i o n of the TDGA. CHAPTER 3 E x p e r i m e n t a l D e s i g n and R a t i o n a l e 18 The s t u d y o f c a r n i t i n e i n t h e i n t e s t i n e i s i m p o r t a n t f o r s e v e r a l r e a s o n s . F o r e x a m p l e , a l t h o u g h o r a l c a r n i t i n e i s o f t e n u s e d t h e r a p e u t i c a l l y , t h e e f f i c i e n c y o f c a r n i t i n e a b s o r p t i o n u n d e r v a r i o u s c o n d i t i o n s i s n o t known. N o r m a l c a r n i t i n e a b s o r p t i o n s h o u l d be d e f i n e d i n o r d e r t o i n v e s t i g a t e t h e c l a i m t h a t d e f e c t i v e c a r n i t i n e a b s o r p t i o n m i g h t be t h e c a u s e o f some c a s e s 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 ( E n g e l and R e b o u c h e , 1 9 8 4 ) . F i n a l l y , c a r n i t i n e m i g h t p l a y a r o l e i n f a t a b s o r p t i o n ; b o t h a r e a b s o r b e d i n t h e j e j u n u m . C a r n i t i n e e s t e r s , b e c a u s e o f t h e i r a m p h i p h i l i c n a t u r e , a r e p o s s i b l y i n v o l v e d i n t h e m i x e d m i c e l l e s o f t h e i n t e s t i n a l l u m e n . A l t e r n a t i v e l y , c a r n i t i n e m i g h t s h u t t l e f a t t y a c i d s a c r o s s some o f t h e membrane b a r r i e r s e n c o u n t e r e d i n t h e t r a n s p o r t o f f a t a c r o s s t h e e n t e r o c y t e and i n t o t h e l y m p h . T h i s i s s i m i l a r t o a r o l e t h a t h a s been p r o p o s e d f o r c a r n i t i n e i n t h e e p i d i d y m i s , where i t h a s been h y p o t h e s i z e d t o t r a n s p o r t f a t t y a c i d s b e t w e e n t h e b l o o d s t r e a m , e p i d i d y m a l e p i t h e l i u m , e p i d i d y m a l lumen and sperm m i t o c h o n d r i a ( B r o o k s , 1 9 8 0 ) . The e n dogenous c a r n i t i n e c o n t e n t o f t h e i n t e s t i n e must be s t u d i e d b e f o r e t h e i n t e s t i n a l a b s o r p t i o n o f d i e t a r y c a r n i t i n e c a n be i n v e s t i g a t e d . The o b j e c t i v e o f t h e p r e s e n t s t u d y was t o d e t e r m i n e w h e t h e r o r n o t b i l e c o n t r i b u t e s t o t h e c a r n i t i n e f o u n d i n t h e i n t e s t i n a l l u m e n . T h i s was done i n a s e r i e s o f e x p e r i m e n t s w h i c h were d e s i g n e d t o answer t h e f o l l o w i n g q u e s t i o n s : 1) A r e 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 p r e s e n t i n r a t b i l e ? 2) What i s t h e o r i g i n o f t h e c a r n i t i n e 19 i n b i l e ? 3) Does c a r n i t i n e e x i s t i n human b i l e - r i c h d u o d e n a l f l u i d ? . The r a t i o n a l e and p r o t o c o l f o r e a c h e x p e r i m e n t i s d e s c r i b e d b e l o w . The s p e c i f i c methods and m a t e r i a l s u s e d a r e d e s c r i b e d i n C h a p t e r 4. 3.1 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 C o n t e n t o f R a t B i l e The r e s u l t s o f s e v e r a l s t u d i e s i n d i c a t e t h a t c a r n i t i n e o f t h e i n t e s t i n a l lumen m i g h t be d e r i v e d f r o m o t h e r t h a n d i e t a r y s o u r c e s . S a c h a n and R u a r k f o u n d some t y p e s o f c a r n i t i n e e s t e r s i n r a t i n t e s t i n a l c o n t e n t s t h a t were n o t p r e s e n t i n t h e d i e t . Hahn and c o l l e a g u e s ( 1 9 8 5 ) and Rebouche and a s s o c i a t e s ( 1 9 8 4 ) f o u n d l a b e l l e d c a r n i t i n e i n t h e i n t e s t i n a l t r a c t a f t e r i t was i n j e c t e d s u b c u t a n e o u s l y o r i n t r a v e n o u s l y . D a t a f r o m body p o o l s t u d i e s s u g g e s t t h a t t h e r e may be a n o t h e r r o u t e f o r c a r n i t i n e e x c r e t i o n b e s i d e s t h e u r i n e ( R e b o u c h e and E n g e l , 1984; C e d e r b l a d and L i n d s t e d t , 1976; Yue and F r i t z , 1 9 6 2 ) . P o s s i b l y , c a r n i t i n e i s e x c r e t e d i n t o t h e i n t e s t i n e , v i a t h e b i l e , where i t i s e i t h e r r e a b s o r b e d o r d e g r a d e d by b a c t e r i a o f t h e l a r g e i n t e s t i n e ( R e b o u c h e e t a l . , 1 9 8 4 ) . To c l a r i f y t h e p r e s e n c e o f c a r n i t i n e i n r a t b i l e , b i l e was c o l l e c t e d f r o m e l e v e n f e d a d u l t r a t s . B i l e f l o w , b i l e and serum c a r n i t i n e and c a r n i t i n e e s t e r c o n c e n t r a t i o n s were m e a s u r e d . L o n g - c h a i n c a r n i t i n e e s t e r and a c e t y l c a r n i t i n e c o n c e n t r a t i o n s were d e t e r m i n e d i n r a n d o m l y s e l e c t e d s a m p l e s . 20 3.2 The O r i g i n o f C a r n i t i n e i n R a t B i l e C a r n i t i n e f o u n d i n t h e b i l e c o u l d be o f e i t h e r h e p a t i c o r e x t r a h e p a t i c o r i g i n . L a r g e amounts o f l o n g - c h a i n c a r n i t i n e e s t e r s a r e p r e s e n t i n b i l e . B e c a u s e t h e s e l o n g - c h a i n c a r n i t i n e e s t e r s a r e p r e s e n t i n t h e serum t o o n l y a v e r y l i m i t e d d e g r e e , t h i s s u g g e s t s t h a t c a r n i t i n e i s d e r i v e d f r o m t h e l i v e r . The o r i g i n o f t h e c a r n i t i n e i n b i l e was i n v e s t i g a t e d by i m p o s i n g c o n d i t i o n s on r a t s w h i c h a f f e c t c a r n i t i n e e s t e r i f i c a t i o n i n t h e l i v e r and serum d i f f e r e n t l y . The c o n d i t i o n s s t u d i e d were f a s t i n g and TDGA t r e a t m e n t . The amount and t y p e o f c a r n i t i n e e s t e r s i n t h e b i l e were e x a m i n e d t o s e e i f t h e y r e f l e c t e d t h e c h a n g e s o c c u r r i n g i n t h e l i v e r o r s e r u m. A t h i r d g r o u p o f r a t s was i n j e c t e d i n t r a v e n o u s l y w i t h l a b e l l e d c a r n i t i n e t o f u r t h e r i n v e s t i g a t e t h e s o u r c e o f c a r n i t i n e i n t h e b i l e . 3.2.1 The E f f e c t s o f a 72 Hour F a s 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 B i l e F a s t i n g i n c r e a s e d t h e p r o p o r t i o n o f l o n g - c h a i n c a r n i t i n e e s t e r s i n t h e l i v e r , w h i l s t t h e amount o f s h o r t e r - c h a i n c a r n i t i n e e s t e r s d e c r e a s e d . I n t h e s e r u m , t h e o v e r a l l c a r n i t i n e e s t e r i f i c a t i o n i n c r e a s e d ( B r a s s and H o p p e l , 1 9 7 8 ) . F i v e r a t s were f a s t e d f o r 72 h o u r s p r i o r t o b i l e c o l l e c t i o n . The b i l e f l o w , c a r n i t i n e and c a r n i t i n e e s t e r c o n c e n t r a t i o n s were m e a s u r e d i n a l l s a m p l e s . L o n g - c h a i n c a r n i t i n e e s t e r s were d e t e r m i n e d i n r a n d o m l y s e l e c t e d s a m p l e s . Serum was a l s o c o l l e c t e d and t h e 21 c a r n i t i n e and c a r n i t i n e e s t e r c o n c e n t r a t i o n s m e a s u r e d . The r e s u l t s o f t h i s f a s t i n g e x p e r i m e n t were compared w i t h t h o s e f r o m r a t s i n t h e f e d s t a t e . 3.2.2 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 A c i d 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 B i l e As TDGA i n h i b i t s CPT I , h e p a t i c l o n g - c h a i n c a r n i t i n e e s t e r p r o d u c t i o n i s d e c r e a s e d and t h e 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 i s i n c r e a s e d ( F r e n c h e t a l . , 1 9 8 5 ) . I n t h e serum, b o t h e s t e r i f i e d c a r n i t i n e and t o t a l c a r n i t i n e c o n c e n t r a t i o n s a r e d e c r e a s e d . S i x r a t s were f a s t e d f o r 72 h o u r s and i n t u b a t e d w i t h TDGA, a c c o r d i n g t o t h e p r o t o c o l o f F r e n c h and c o l l e a g u e s ( 1 9 8 5 ) . B i l e was c o l l e c t e d and t h e b i l e f l o w , c a r n i t i n e and c a r n i t i n e e s t e r c o n c e n t r a t i o n s were m e a s u r e d i n a l l o f t h e s a m p l e s . L o n g - c h a i n c a r n i t i n e e s t e r s were d e t e r m i n e d i n r a n d o m l y s e l e c t e d s a m p l e s . The serum c a r n i t i n e c o n c e n t r a t i o n was a l s o m e a s u r e d . The r e s u l t s f r o m t h e s e s t u d i e s were compared t o t h o s e i n t h e p r e v i o u s s t u d y i n w h i c h t h e r a t s were f a s t e d f o r 72 h o u r s . 3.2.3 The R e c o v e r y 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 R a t B i l e B i l e was c o l l e c t e d f r o m s i x r a t s f o l l o w i n g i n t r a v e n o u s i n j e c t i o n o f l a b e l l e d c a r n i t i n e . The t y p e s o f c a r n i t i n e e s t e r s 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 b o t h t h e serum and t h e b i l e c a r n i t i n e , were d e t e r m i n e d . P o s s i b l e 22 a s s o c i a t i o n s between the b i l e and the h e p a t i c and e x t r a h e p a t i c sources of c a r n i t i n e were examined. 3.3 The C a r n i t i n e Content of Human B i l e - R i c h Duodenal F l u i d The r e s u l t s of four s t u d i e s have i n d i c a t e d that c a r n i t i n e malabsorption might e x i s t i n humans (Seccombe et a l . , 1984; F r o h l i c h et a l . , 1980; Bohmer et al.,1974; M i k h a i l and Mansour,1976). I f an e n t e r o h e p a t i c c i r c u l a t i o n of c a r n i t i n e i s present, c a r n i t i n e d e p l e t i o n might r e s u l t during malabsorptive s t a t e s or e x c e s s i v e b i l e l o s s e s . Before i n v e s t i g a t i n g t h i s f u r t h e r , the c a r n i t i n e content of human b i l e should be determined. Seventeen b i l e - r i c h duodenal f l u i d samples were c o l l e c t e d from ten a d u l t p a t i e n t s with suspected c h o l e l i t h i a s i s . These samples were then analysed f o r c a r n i t i n e and i t s e s t e r s . CHAPTER 4 Methods and M a t e r i a l s 23 4.1 Chemicals and Isotopes A l l chemicals and i s o t o p e s were purchased from the Sigma Chemical Company, St. L o u i s , MO., 63178, except where otherwise noted. r1- 1 4C1 Acetyl-Coenzyme A, 57.6 mCi/ mmol, Amersham L t d . , A r l i n g t o n Heights, I l l i n o i s . L - r U - ^ C j A s p a r t a t e , 227 Ci/mol, Amersham L t d . , A r l i n g t o n Heights, I l l i n o i s . 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 , from pigeon breast muscle, 85 units/mg p r o t e i n . L - C a r n i t i n e H y d r o c h l o r i d e , Sigma-Tau Company, Rome, I t a l y . L - T m e t h y l - l ^ C l C a r n i t i n e H y d r o c h l o r i d e , 58 mCi/mmol, Amersham L i m i t e d , A r l i n g t o n Heights, I l l i n o i s . C i t r a t e Synthase, Boehringer Mannheim Biochemica, D o r v a l , Quebec. EDTA ( e t h y l e n e d i n i t r i l o t e t r a a c e t i c a c i d ) , disodium s a l t . Glutamate-oxaloacetate transaminase, Boehringer Mannheim Biochemica, D o r v a l , Quebec. HEPES (N-2-hydroxyethyl piperazine-N-2-ethane s u l f o n i c a c i d , BDH Chemicals Canada L t d . , Vancouver, B.C. N-ethylmaleimide (NEM). Somnotol, sodium p e n t o b a r b i t a l (65 mg/ml) i n an aqueous propylene g l y c o l base, M.T.C. Pharmaceuticals, 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 ) , donated by Dr. John M c N e i l l , Dean and P r o f e s s o r of Pharmaceutical Science, U n i v e r s i t y of B r i t i s h Columbia. Tragacanth Gum, grade I I I powder. 24 4.2 H o u s i n g and C a r e o f A n i m a l s A d u l t male W i s t a r r a t s ( 3 8 0 . 3 ± 4.4 g, mean ± SE) were u s e d . T h e s e were b r e d and r a i s e d e i t h e r a t t h e C e n t r e f o r D e v e l o p m e n t a l M e d i c i n e o r i n t h e A n i m a l C a r e C e n t r e o f t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a ( U B C ) . The r a t s were h o u s e d i n g r o u p s o f two o r t h r e e i n p l a s t i c c a g e s w i t h wood s h a v i n g s f o r b e d d i n g . They had f r e e a c c e s s t o P u r i n a R a t Chow and w a t e r a t a l l t i m e s . L i g h t i n g was on a r e v e r s e d 12 h o u r c y c l e w i t h t h e l i g h t s t u r n e d on a t 4 PM and t u r n e d o f f a t 4 AM. A l l s u r g i c a l p r o c e d u r e s were c o n d u c t e d b e t w e e n 8 and 10 AM t o c o n t r o l f o r p o s s i b l e d i u r n a l v a r i a t i o n s i n serum c a r n i t i n e l e v e l s (Bohmer and B r e m e r , 1 9 6 8 ) . 4.3 R a t B i l e C o l l e c t i o n R a t s were a n a e s t h e t i z e d w i t h an i n t r a p e r i t o n e a l ( I P ) i n j e c t i o n o f S o m n o t o l ( 6 . 5 mg/100 g body w e i g h t , BW) i m m e d i a t e l y b e f o r e t h e s u r g e r y . B i l e was c o l l e c t e d a c c o r d i n g t o W a y n f o r t h ( 1 9 8 0 ) w i t h some m o d i f i c a t i o n s . E a c h r a t was p l a c e d on i t s b a c k on a h e a t i n g pad w h i c h m a i n t a i n e d i t s body t e m p e r a t u r e , 37 °C, m e a s u r e d r e c t a l l y . A 2 cm l a t e r a l i n c i s i o n was made a t t h e l e v e l o f t h e x i p h o i d c a r t i l a g e and a s m a l l p i e c e o f duodenum, l o c a t e d d i r e c t l y b e n e a t h t h e l i v e r , was p u l l e d g e n t l y o u t o f t h e p e r i t o n e a l c a v i t y u n t i l r e s i s t a n c e was f e l t f r o m t h e a t t a c h m e n t o f t h e b i l e d u c t . The b i l e d u c t was t h e n c a r e f u l l y d i s s e c t e d f r o m t h e s u r r o u n d i n g t i s s u e and a s h o r t l e n g t h o f d o u b l e d t h r e a d was i n s e r t e d a r o u n d t h e d u c t t o f o r m two l o o s e l i g a t u r e s . 25 The b i l e d u c t was t h e n h e l d w i t h f o r c e p s w h i l e i t was s e m i - t r a n s e c t e d w i t h f i n e s c i s s o r s . A p o l y e t h y l e n e c a n n u l a ( i n t e r n a l d i a m e t e r 0.5 mm) was t h e n drawn o u t , i n s e r t e d i n t h e o p e n i n g and p u s h e d f o r w a r d b eyond t h e p a n c r e a s ( t o w a r d s t h e l i v e r f o r 3-4 mm). The p o i n t o f e n t r y o f t h e c a n n u l a was t h e n s e a l e d w i t h c y a n o a c r y l a t e g l u e . Once b i l e f l o w was e s t a b l i s h e d , t h e i n t e s t i n e was r e p l a c e d i n t o t h e p e r i t o n e a l c a v i t y and t h e i n c i s i o n s u t u r e d . B i l e was c o l l e c t e d i n t o p r e w e i g h e d g l a s s t e s t t u b e s h e l d b e s i d e t h e h e a t i n g pad a t t h e same h e i g h t as t h e r a t . B i l e was c o l l e c t e d f r o m e a c h r a t f o r a t o t a l o f two h o u r s . The c o l l e c t i o n t u b e was r e p l a c e d e v e r y 15 m i n u t e s , and f l o w d e t e r m i n e d g r a v i m e t r i c a l l y a s s u m i n g a d e n s i t y o f 1 g/ml. B i l e f l o w was c a l c u l a t e d as u l / m i n / 1 0 0 g BW. A f t e r e a c h t u b e was w e i g h e d , b i l e s a m p l e s were a l i q u o t e d i n t o d i s p o s a b l e c o n i c a l p o l y s t y r e n e t u b e s f o r s u b s e q u e n t c a r n i t i n e a n a l y s i s and f r o z e n a t -20 °C. F r e e z i n g does n o t a f f e c t t h e serum c a r n i t i n e c o n c e n t r a t i o n ( S e c c o m b e , 1 9 8 1 ) . The pH o f t h e s a m p l e s were m o n i t o r e d t o c o n f i r m n e u t r a l i t y . A f t e r two h o u r s o f b i l e c o l l e c t i o n , a b l o o d s a m p l e was t a k e n f r o m t h e t a i l v e i n o f e a c h r a t . R a t s were t h e n k i l l e d w i t h e x c e s s S o m n o t o l . The b l o o d was c e n t r i f u g e d a t 3,000 rpm f o r 5 m i n u t e s a t 4 °C and t h e serum removed and f r o z e n a t -20 °C u n t i l c a r n i t i n e a n a l y s i s . I n one c a s e , t h e b l o o d s a m p l e s were 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 t h r o u g h o u t t h e b i l e c o l l e c t i o n t o d e t e r m i n e t h e e f f e c t o f f l u i d l o s s , due t o b i l e d r a i n a g e , on serum c a r n i t i n e c o n c e n t r a t i o n s . 26 4.4 Human B i l e - R i c h D u o d e n a l F l u i d C o l l e c t i o n B i l e - r i c h d u o d e n a l f l u i d was o b t a i n e d f r o m t e n , male and f e m a l e , a d u l t p a t i e n t s w i t h s u s p e c t e d c h o l e l i t h i a s i s . The s a m p l e s were c o l l e c t e d by t h e G a s t r o e n t e r o l o g y U n i t o f S h a u g h n e s s y H o s p i t a l f o r D r . N. U r q u h a r t ( D e p a r t m e n t o f P a t h o l o g y , U n i v e r s i t y o f B r i t i s h C o l u m b i a ) . P a t i e n t s were i n s t r u c t e d t o f a s t f o r a t l e a s t 12 h o u r s b e f o r e t h e p r o c e d u r e w h i c h was e i t h e r c o n d u c t e d a t 10 AM o r 1 PM. A n a s o g a s t r i c t u b e was p l a c e d i n t o t h e f i r s t segment o f t h e duodenum and i t s p o s i t i o n was c o n f i r m e d by f l u o r o s c o p y . I n some c a s e s , a b a s a l f l u i d s a m p l e was c o l l e c t e d . The p a t i e n t s were t h e n i n j e c t e d ( I V ) w i t h 80 u n i t s o f c o n c e n t r a t e d 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 ( B o o t s D r u g s L t d . , T o r o n t o , O n t . ) . T h i s s t i m u l a t e s t h e r e l e a s e o f b i l e f r o m t h e g a l l b l a d d e r ( I v y e t a l . , 1 9 2 8 ) . One o r two f l u i d s a m p l e s were t h e n 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 . A l l s a m p l e s were n e u t r a l i z e d t o p r e v e n t e s t e r h y d r o l y s i s and s t o r e d f r o z e n a t -20 °C f o r l a t e r c a r n i t i n e a n a l y s i s . 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 i n Serum and B i l e 4.5.1 P r i n c i p l e o f t h e C a r n i t i n e A s s a y O r i g i n a l l y , c a r n i t i n e was m e a s u r e d by b i o a s s a y u s i n g t h e c a r n i t i n e r e q u i r i n g o r g a n i s m , T e n e b r i o m o l i t o r ( F r a e n k e l , 1 9 5 1 ) . I n 1958, t h i s was r e p l a c e d by a c o l o r i m e t r i c a s s a y w h i c h was n o t v e r y s p e c i f i c t o c a r n i t i n e ( F r i e d m a n , 1 9 5 8 ) . The c a r n i t i n e a s s a y now u s e d r e q u i r e s c o m m e r c i a l CAT ( M a r q u i s and F r i t z , 1 9 6 4 ) . F r e e CoA i s m e a s u r e d 27 s p e c t r o p h o t o m e t r y i c a l l y a f t e r i n c u b a t i o n o f e a c h s a m p l e w i t h a c e t y l - C o A . The r e a c t i o n i s a s f o l l o w s : CAT L - C a r n i t i n e + A c e t y l - C o A < > A c e t y l c a r n i t i n e + CoA I n 1972, C e d e r b l a d and L i n d s t e d t i n c r e a s e d t h e s e n s i t i v i t y o f t h e a s s a y i n t o t h e p i c o m o l e r a n g e by i n t r o d u c i n g t h e u s e o f [ 1 4 C ] a c e t y l - C o A . The amount o f l a b e l l e d a c e t y l c a r n i t i n e f o r m e d a f t e r i n c u b a t i o n i s p r o p o r t i o n a l t o t h e amount o f c a r n i t i n e i n t h e s a m p l e . One p r o b l e m w i t h t h i s method i s t h e r e v e r s i b i l i t y o f t h e r e a c t i o n : i . e . t h e d i s s o c i a t i o n o f a c e t y l c a r n i t i n e t o f r e e c a r n i t i n e and a c e t y l - C o A . T h i s c a n be overcome by t h e a d d i t i o n o f t e t r a t h i o n a t e ( M c G a r r y and F o s t e r , 1976) o r N - e t h y l m a l e i m i d e ( P a r v i n and P a n d e , 1 9 7 7 ) , w h i c h b i n d s t o t h e f r e e CoA, a p r o d u c t o f t h e f o r w a r d r e a c t i o n . P a r v i n and Pande ( 1 9 7 7 ) have s u g g e s t e d t h a t N - e t h y l m a l e i m i d e (NEM) i s s u p e r i o r t o t e t r a t h i o n a t e . A n o t h e r l i m i t a t i o n o f t h e C e d e r b l a d and L i n d s t e d t method ( 1 9 7 2 ) i s t h e u s e o f a DOWEX c o l u m n t o s e p a r a t e t h e u n r e a c t e d [ " ^ C ] a c e t y l - C o A f r o m t h e [ 1 4 c ] a c e t y l c a r n i t i n e . I n p l a c e o f t h i s , M c G a r r y and F o s t e r ( 1 9 7 6 ) added an i o n e x c h a n g e (DOWEX) r e s i n s l u r r y t o e a c h r e a c t i o n v i a l ; t h e s u p e r n a t a n t i s t h e n f r e e o f any u n r e a c t e d [ ^ 4 C ] a c e t y l - C o A . I n s t e a d , P a r v i n and Pande ( 1 9 7 7 ) recommend t h e a d d i t i o n o f a c h a r c o a l s l u r r y c o n t a i n i n g a c i d i f i e d e t h a n o l . C h a r c o a l a d s o r b s n u c l e o t i d e compounds and e t h a n o l i s n e e d e d t o 28 p r e v e n t t h e r e t e n t i o n o f c a r n i t i n e and a c e t y l c a r n i t i n e a s w e l l a s a c e t y l - C o A . B o t h methods a r e e f f e c t i v e ; t h e c h a r c o a l , h o w e v e r , i s f a s t e r and l e a s t e x p e n s i v e . 4.5.2 P r o c e d u r e u s e d A l l c a r n i t i n e a s s a y s were p e r f o r m e d i n d i s p o s a b l e p o l y s t y r e n e c o n i c a l t i p t u b e s (12 x 75 mm). F i f t y m i c r o l i t e r s o f e a c h s a m p l e , s t a n d a r d o r c o n t r o l were u s e d f o r t h e a s s e s s m e n t o f e a c h o f f r e e c a r n i t i n e and t o t a l c a r n i t i n e ( f r e e p l u s e s t e r i f i e d c a r n i t i n e ) . The a s s a y s were done i n d u p l i c a t e . A c a r n i t i n e s t a n d a r d c u r v e was p r e p a r e d by s e r i a l d i l u t i o n o f c a r n i t i n e and w a t e r . The c o n t r o l was human serum. A l l t u b e s were k e p t on i c e d u r i n g p r e p a r a t i o n f o r t h e a s s a y i n o r d e r t o p r e v e n t t h e h y d r o l y s i s o f c a r n i t i n e e s t e r s . A l l s a m p l e s , s t a n d a r d s and c o n t r o l s were d e p r o t e i n i z e d p r i o r t o a s s a y i n g f o r c a r n i t i n e . A l t h o u g h , M c G a r r y and F o s t e r ( 1 9 7 6 ) r e p o r t e d t h a t t h i s was n o t n e c e s s a r y , Seccombe ( 1 9 8 1 ) f o u n d t h a t i n t e r f e r e n c e f r o m serum p r o t e i n s e x a g g e r a t e d t h e r e s u l t s . E v e n t h o u g h b i l e c o n t a i n s v e r y l i t t l e p r o t e i n ( L a R u s s o , 1 9 8 4 ) , i t was a l s o d e p r o t e i n i z e d . The p r o t e i n was p r e c i p i t a t e d by t h e a d d i t i o n o f 200 u l z i n c s u l f a t e ( 0 . 0 8 7 M) t o e a c h s a m p l e f o r a n a l y s i s o f f r e e c a r n i t i n e . S u b s e q u e n t l y , 200 u l o f b a r i u m h y d r o x i d e ( 0 . 0 8 3 M) was added f o r n e u t r a l i z a t i o n . The a s s a y o f t o t a l c a r n i t i n e r e q u i r e s t h e p r i o r h y d r o l y s i s o f c a r n i t i n e e s t e r s . F o r t h e s e s a m p l e s , t h e b a r i u m h y d r o x i d e was added f i r s t and 29 t h e s a m p l e s were t h e n i n c u b a t e d f o r 2 h o u r s a t 75 °C b e f o r e a d d i t i o n o f t h e z i n c s u l f a t e . The p r o t e i n and b a r i u m s u l f a t e p r e c i p i t a t e were removed by c e n t r i f u g a t i o n f o r 15 m i n u t e s a t 3,500 rpm a t 4 °C. I n p r e p a r a t i o n f o r t h e a s s a y , 100 u l o f t h e d e p r o t e i n i z e d s u p e r n a t a n t s were p l a c e d i n new p o l y s t y r e n e t u b e s w i t h 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 . The s u b s t r a t e s o l u t i o n c o n t a i n e d HEPES b u f f e r ( 2 0 mM), pH 7.6, EDTA ( 0 . 8 mM), NEM (2 mM) and [ 1 - 1 4 C ] a c e t y l - C o A ( 2 . 5 uM, s p e c i f i c a c t i v i t y 0.02 u C i / n m o l ) . The r e a c t i o n was i n i t i a t e d by t h e a d d i t i o n o f 50 u l o f CAT ( c o n t a i n i n g 1 u n i t o f e n z y m e ) . A f t e r i n c u b a t i o n f o r 60 m i n u t e s a t room t e m p e r a t u r e , t h e r e a c t i o n was t e r m i n a t e d w i t h 600 u l o f a s t i r r e d c h a r c o a l s l u r r y . E a c h b a t c h o f s l u r r y was p r e p a r e d i m m e d i a t e l y b e f o r e u se and c o n t a i n e d 4.0 g o f n e u t r a l i z e d a c t i v a t e d c h a r c o a l , 54.5 ml o f e t h a n o l , 1.25 ml 8 5 % p h o s p h o r i c a c i d and 4.25 ml d i s t i l l e d w a t e r . A f t e r m i x i n g w i t h t h e s l u r r y , t h e r e a c t i o n t u b e s were p l a c e d on i c e , and c e n t r i f u g e d a t 3,500 rpm f o r 15 m i n u t e s a t 4 °C. The s u p e r n a t a n t s were t r a n s f e r r e d t o c l e a n t u b e s and c e n t r i f u g a t i o n r e p e a t e d . An a l i q u o t o f t h e f i n a l s u p e r n a t a n t ( 5 0 0 u l ) was t h e n a d ded t o a d i s p o s a b l e m i n i - c o u n t i n g v i a l c o n t a i n i n g 400 u l d i s t i l l e d w a t e r . As t h e s u p e r n a t a n t c o n t a i n e d e t h a n o l , w h i c h i s d i f f i c u l t t o p i p e t t e , t h e p i p e t t o r t i p was r i n s e d w i t h t h e w a t e r i n t h e v i a l t o e n s u r e q u a n t i t a t i v e t r a n s f e r . F i n a l l y , 5 ml o f s c i n t i l l a t i o n f l u i d (ACS) was added b e f o r e 30 t h e v i a l was m i x e d and c o u n t e d . The s a m p l e s were c o u n t e d on a Beckman LS9000 S c i n t i l l a t i o n c o u n t e r w i t h an e x t e r n a l s t a n d a r d q u e n c h c o r r e c t i o n . The s t a n d a r d c u r v e s f o r b o t h t h e f r e e and t o t a l c a r n i t i n e a s s a y s were t h e n p l o t t e d and l i n e a r r e g r e s s i o n a n a l y s i s was p e r f o r m e d . I f c a r n i t i n e c o n c e n t r a t i o n s were beyond t h e r a n g e o f t h e s t a n d a r d c u r v e (0 t o 100 uM), t h e a s s a y was r e p e a t e d w i t h d i l u t e d s a m p l e s . The c o n c e n t r a t i o n o f a c y l c a r n i t i n e was d e r i v e d by s u b t r a c t i n g t h e 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 f r o m t h e t o t a l c a r n i t i n e c o n c e n t r a t i o n . I n t e r a s s a y v a r i a t i o n was a p p r o x i m a t e l y 5%. B o t h t h e a b s o l u t e q u a n t i t y o f c a r n i t i n e i n t h e b i l e p e r 100 g BW and t h e c a r n i t i n e c o n c e n t r a t i o n were c a l c u l a t e d f o r e a c h 15 m i n u t e b i l e c o l l e c t i o n s a m p l e . 4.6 A c e t y l c a r n i t i n e A n a l y s i s 4.6.1 P r i n c i p l e o f M e t h o d Pande and C a r a m a n c i o n ( 1 9 8 1 ) d e v i s e d a r a d i o i s o t o p i c a s s a y f o r t h e d e t e r m i n a t i o n o f c a r n i t i n e b a s e d on t h e f o l l o w i n g two r e a c t i o n s : CAT 1) A c e t y l c a r n i t i n e + CoA > A c e t y l - C o A + C a r n i t i n e C i t r a t e S y n t h a s e 2) A c e t y l CoA + [ 1 4 C ] o x a l o a c e t a t e > [ 1 4 C ] c i t r a t e F i r s t , CAT c a u s e s t h e d i s s o c i a t i o n o f c a r n i t i n e and a c e t y l - C o A . C i t r a t e s y n t h a s e ( E . C . 4 . 1 . 3 . 7 ) t h e n c a t a l y z e s t h e f o r m a t i o n o f [ ^ 4 C ] c i t r a t e f r o m a c e t y l - C o A and 3 1 [ l ^ C ] o x a l o a c e t a t e . The amount o f [ ] c i t r a t e f o r m e d i s p r o p o r t i o n a l t o t h e amount o f a c e t y l c a r n i t i n e i n t h e s a m p l e . E x c e s s [ ] o x a l o a c e t a t e i s removed by t h e s u b s e q u e n t a d d i t i o n 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 ) w h i c h c a t a l y s e s t h e f o r m a t i o n o f r 1 4 [ C ] a s p a r t a t e . A c a t i o n e x c h a n g e r e s i n i s u s e d t o remove [ " ^ C ] a s p a r t a t e . 4 . 6 . 2 P r o c e d u r e Used The a c e t y l c a r n i t i n e a s s a y s were p e r f o r m e d by L e i g h t o n James ( D e p a r t m e n t o f P a t h o l o g y , UBC) i n S h a u g h n e s s y H o s p i t a l l a b o r a t o r i e s . U n l e s s o t h e r w i s e n o t e d , a l l s o l u t i o n s were k e p t on i c e t o p r e v e n t h y d r o l y s i s o f t h e c a r n i t i n e e s t e r s . A l i q u o t s o f e a c h s a m p l e ( 1 0 0 u l ) , s t a n d a r d o r b l a n k were d e p r o t e i n i z e d by a d d i t i o n o f 4 0 0 u l o f z i n c s u l f a t e ( 0 . 0 8 7 M) f o l l o w e d by t h e a d d i t i o n o f t h e same volume o f b a r i u m h y d r o x i d e ( 0 . 0 8 3 M). The p r e c i p i t a t e was t h e n removed by c e n t r i f u g a t i o n f o r 1 0 m i n u t e s a t 3 , 5 0 0 rpm a t 4 °C. The s t a n d a r d c u r v e r a n g e d f r o m 0 . 0 5 uM t o 2 0 uM. Pande and C a r a m a n c i o n ( 1 9 8 1 ) f o u n d t h a t t h e s e n s i t i v i t y o f t h e a s s a y was i m p r o v e d by t h e u s e o f d i a l y s e d enzymes. A l l t h r e e enzymes were d i a l y s e d a t 0 - 4 ,°C a g a i n s t 5 0 0 ml o f 2 0 mM p o t a s s i u m p h o s p h a t e (pH 7 . 4 ) . The d i a l y s i n g f l u i d was ch a n g e d e v e r y 3 0 m i n u t e s f o r two h o u r s . F r e s h l y r e d u c e d CoA r e a g e n t was p r e p a r e d i n a d v a n c e and c o n t a i n e d 0 . 2 mM CoA, 1 0 mM p o t a s s i u m d i p h o s p h a t e (pH 9 ) and 3 . 2 mM d i t h i o t h r e i t o l . 4 0 0 u l o f t h i s was t h e n l e f t a t 32 o 25 C f o r 30 m i n u t e s b e f o r e t h e a d d i t i o n o f 800 u l o f 0.5 M p o t a s s i u m p h o s p h a t e (pH 7 . 4 ) . c o n t a i n i n g 200 nM EDTA, 5 mM a l p h a - k e t o g l u t a r a t e , 56 mM p o t a s s i u m p h o s p h a t e (pH 7 . 4 ) , 12 u n i t s / m l d i a l y s e d GOT and 50 nM L - [ U - l 4 C ] a s p a r t a t e (227 C i / m o l ) was i n c u b a t e d f o r 10 m i n u t e s a t 25 °C. T h e n , 20 u l o f 1 M p e r c h l o r i c a c i d was a d d e d , and l e f t f o r 15 m i n u t e s . I m m e d i a t e l y b e f o r e t h e a s s a y , t h i s was n e u t r a l i z e d w i t h 40 u l 0.6 M c h i l l e d p o t a s s i u m h y d r o x i d e p l u s 60 u l o f 67 mM EDTA (pH 7 . 4 ) . The a s s a y was p e r f o r m e d i n c o n i c a l b o r o s i l i c a t e t e s t t u b e s (12 mm x 75 mm). To e a c h t u b e , t h e f o l l o w i n g was added i n t h e o r d e r g i v e n : 155 u l s a m p l e , 10 u l 120-150 mM EDTA (pH 7.4) and 30 u l f r e s h l y r e d u c e d CoA r e a g e n t , 1.5 u n i t d i a l y s e d c i t r a t e s y n t h a s e , 0.8 u n i t CAT and 5 u l [ ^ C ] o x a l o a c e t a t e . The r e a c t i o n m i x t u r e was t h e n i n c u b a t e d a t 25 °C f o r 20 m i n u t e s . 30 u l o f g l u t a m a t e p l u s GOT were t h e n added and t h e m i x t u r e was i n c u b a t e d f o r a f u r t h e r 20 m i n u t e s . 320 mg o f t h e r e s i n (AG 50W-X8, B i o R a d ) and 0.6 ml w a t e r were t h e n added t o e a c h t u b e w h i c h was a l l o w e d t o s t a n d f o r 10 m i n u t e s a t room t e m p e r a t u r e . The r e s i n was removed by c e n t r i f u g a t i o n f o r 10 m i n u t e s a t 3000 rpm. 0.3 ml o f t h e s u p e r n a t a n t , 0.6 ml o f w a t e r p l u s 5 ml o f s c i n t i l l a t i o n f l u i d were t h e n c o m b i n e d i n m i n i - c o u n t i n g v i a l s w h i c h were c o u n t e d on a Beckman LS9000 S c i n t i l l a t i o n c o u n t e r w i t h an e x t e r n a l s t a n d a r d q u e n c h c o r r e c t i o n . The s t a n d a r d c u r v e was t h e n p l o t t e d and t h e c o n c e n t r a t i o n s o f To p r e p a r e 800 u l o f a s o l u t i o n 33 a c e t y l c a r n i t i n e i n e a c h s a m p l e d e t e r m i n e d . O n l y b i l e s a m p l e s f r o m t h e i n i t i a l and f i n a l b i l e c o l l e c t i o n p e r i o d s were a s s e s s e d . 4.7 C a r n i t i n e E s t e r S e p a r a t i o n 4.7.1 P r i n c i p l e o f M e thod T h i n - l a y e r c h r o m a t o g r a p h y ( T L C ) has been u s e d e x t e n s i v e l y t o d i s t i n g u i s h b e t w e e n s h o r t , medium and l o n g - c h a i n c a r n i t i n e e s t e r s . U n f o r t u n a t e l y , i t i s t i m e c o n s u m i n g and may n o t be q u a n t i t a t i v e . To d e t e r m i n e t h e r e l a t i v e amounts o f l o n g - c h a i n c a r n i t i n e e s t e r s i n b i l e , a n o t h e r method has been d e v i s e d b a s e d on t h e s o l u b i l i t y o f c a r n i t i n e and i t s e s t e r s . F r e e c a r n i t i n e and s h o r t 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 a r e w a t e r - s o l u b l e b u t l o n g - c h a i n c a r n i t i n e e s t e r s a r e o n l y s o l u b l e i n m e t h a n o l and c h l o r o f o r m ( P a n d e , 1 9 8 1 ) . T h e o r e t i c a l l y , t h e l o n g - c h a i n c a r n i t i n e e s t e r s i n b i l e c a n be q u a n t i t a t i v e l y s e p a r a t e d f r o m t h e o t h e r f o r m s o f c a r n i t i n e by e x t r a c t i o n w i t h c h l o r o f o r m : m e t h a n o l . 4.7.2 V e r i f i c a t i o n o f M e thod To v e r i f y t h e q u a n t i t a t i v e s e p a r a t i o n o f l o n g - c h a i n c a r n i t i n e e s t e r s by c h l o r o f o r m : m e t h a n o l e x t r a c t i o n , b i l e was c o l l e c t e d f r o m a n a e s t h e t i z e d r a t s a f t e r i n j e c t i o n o f 0.5 u C i [ l - l ^ C ] c a r n i t i n e ( s p e c i f i c a c t i v i t y 2.5 m C i / n m o l ) i n t o a j u g u l a r v e i n . The j u g u l a r v e i n was c a n n u l a t e d a c c o r d i n g t o W a y n f o r t h ( 1 9 8 0 ) w i t h one e x c e p t i o n . The h o l e i n t o t h e v e i n 34 was made by i n s e r t i n g t h e t i p o f a b e n t ( 4 5 d e g r e e s ) 21 gauge h y p o d e r m i c n e e d l e i n t o t h e b l o o d v e s s e l . The b i l e c o l l e c t e d c o n t a i n e d [ " * " 4 C ] c a r n i t i n e and [ " ^ C ] c a r n i t i n e e s t e r s . To s e p a r a t e t h e a q u e o u s and l i p i d f r a c t i o n s , 625 u l o f 1:1 ( v / v ) c h l o r o f o r m : m e t h a n o l was added t o 50 u l o f e a c h s a m p l e , m i x e d and l e f t t o s t a n d on i c e f o r 30 m i n u t e s . T h e n , a f u r t h e r 440 u l o f c h l o r o f o r m was added f o l l o w e d by 250 u l o f w a t e r and t h e p r o c e s s was r e p e a t e d t w i c e . The r e s u l t i n g a q u e o u s and l i p i d p h a s e s were s e p a r a t e d , d r i e d down u n d e r n i t r o g e n , and r e d i s s o l v e d i n 50 u l o f w a t e r and m e t h a n o l , r e s p e c t i v e l y . The s a m p l e s were t h e n a p p l i e d t o s i l i c a G p l a t e s ( 5 0 0 m i c r o n s ) a l o n g w i t h s t a n d a r d s . The p l a t e s were d e v e l o p e d i n t h e f o l l o w i n g s o l v e n t s y s t e m : m e t h a n o l , c h l o r o f o r m , w a t e r , c o n c e n t r a t e d ammonia, c o n c e n t r a t e d f o r m i c a c i d ( 5 5 : 5 0 : 1 0 : 7 . 5 : 2 . 5 v / v / v / v / v ) a s d e s c r i b e d by Gumpen and Norum ( 1 9 7 3 ) . A f t e r 30 m i n u t e s , t h e p l a t e s were removed f r o m t h e s o l v e n t t a n k and t h e s t a n d a r d s were v i s u a l i z e d by e x p o s u r e t o i o d i n e v a p o u r . The d i s t r i b u t i o n o f t h e l a b e l l e d c a r n i t i n e was d e t e r m i n e d by s c r a p i n g t h e p l a t e s and c o u n t i n g t h e s i l i c a i n 1 cm b a n d s . The r e s u l t s a r e shown i n F i g u r e 3. O n l y 3% o f t h e l a b e l f o u n d i n t h e a q u e o u s p h a s e m i g r a t e d w i t h t h e l o n g - c h a i n c a r n i t i n e e s t e r s t a n d a r d . The o n l y l a b e l p r e s e n t i n t h e l i p i d f r a c t i o n m i g r a t e d w i t h t h e l o n g - c h a i n c a r n i t i n e e s t e r f r a c t i o n . The r e c o v e r y was 9 6 . 5 % . T h e r e f o r e , t h e e x t r a c t i o n o f b i l e w i t h c h l o r o f o r m : m e t h a n o l q u a n t i t a t i v e l y s e p a r a t e s 35 FIGURE 3 V e r i f i c a t i o n o f t h e s e p a r a t i o n o f 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 e x t r a c t i o n u s i n g t h i n - l a y e r c h r o m a t o g r a p h y ( T L C ) . 2 0 0 u l o f b i l e was c o l l e c t e d f r o m 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 ] c a r n i t i n e . 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 on 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 ) and l i p i d ( d a s h e d l i n e ) p h a s e s . The s i l i c a was t h e n s c r a p e d i n 1 cm bands and 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 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 t h e o t h e r f o r m s o f c a r n i t i n e i n b i l e . 4.7.3 P r o c e d u r e U s e d E a c h s a m p l e , s t a n d a r d o r c o n t r o l was s e p a r a t e d i n t o a q u e o u s and l i p i d p h a s e s as d e s c r i b e d a b o v e . The p h a s e s were t h e n d r i e d u n d e r n i t r o g e n , r e d i s s o l v e d i n 50 u l o f w a t e r , h y d r o l y z e d w i t h 200 u l b a r i u m h y d r o x i d e , n e u t r a l i z e d w i t h z i n c s u l f a t e and a s s a y e d f o r c a r n i t i n e . The q u a n t i t y o f s h o r t 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 were d e r i v e d by s u b t r a c t i n g t h e 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 e t e r m i n e d i n t h e c a r n i t i n e a s s a y f r o m t h e c o n c e n t r a t i o n o f c a r n i t i n e f o u n d i n t h e a q u e o u s e x t r a c t . C a r n i t i n e e s t e r s were a n a l y s e d i n r a n d o m l y s e l e c t e d b i l e s a m p l e s f r o m t h e i n i t i a l b i l e c o l l e c t i o n p e r i o d o f f e d r a t s and b o t h t h e i n i t i a l and f i n a l c o l l e c t i o n p e r i o d s o f b o t h f a s t e d and r a t s t r e a t e d w i t h TDGA. 4.8 F a s t i n g P r o c e d u r e The r a t s were w e i g h e d and p l a c e d i n d i v i d u a l l y i n t o c l e a n c a g e s w i t h no f o o d b u t f r e e a c c e s s t o f r e s h w a t e r 72 h o u r s b e f o r e t h e s c h e d u l e d s u r g e r y . The r a t s were w e i g h e d a g a i n b e f o r e b i l e c o l l e c t i o n . The amount o f c a r n i t i n e i n t h e b i l e was r e p o r t e d on t h e b a s i s o f t h e i n i t i a l body w e i g h t . 37 4.9 T e t r a d e c y l g l y c i d i c A c i d P r e p a r a t i o n and A d m i n i s t r a t i o n A f t e r t h e 72 h o u r f a s t , r a t s were i n t u b a t e d w i t h TDGA s u s p e n d e d i n 0.5 ml t r a g a c a n t h ( F r e n c h e t a l . , 1 9 8 5 ) . 2.5 mg o f TDGA/100 g BW was a d m i n i s t e r e d t o e a c h r a t . E a c h a n i m a l was r e s t r a i n e d i n a t o w e l w h i l e a p o l y e t h y l e n e t u b e a t t a c h e d t o a s y r i n g e was p l a c e d i n t o t h e e s o p h a g u s . The TDGA s o l u t i o n was t h e n i n j e c t e d . To e n s u r e t h a t t h e f l u i d was n o t r e g u r g i t a t e d , t h e a n i m a l s were o b s e r v e d c l o s e l y a f t e r t h i s p r o c e d u r e . Two h o u r s l a t e r , t h e a n i m a l s were a n a e s t h e t i z e d and b i l e c o l l e c t i o n i n i t i a t e d . Two a d d i t i o n a l r a t s were f a s t e d f o r 72 h o u r s and i n t u b a t e d w i t h t h e t r a g a c a n t h v e h i c l e a l o n e . T h e s e a n i m a l s s e r v e d as c o n t r o l s f o r t h e a d m i n i s t r a t i o n v e h i c l e u s e d . B i l e was t h e n c o l l e c t e d and t h e f l o w r a t e a s s e s s e d g r a v i m e t r i c a l l y . 4.10 A n a l y s i s o f D a t a The d a t a o b t a i n e d f r o m t h e s e e x p e r i m e n t s a r e r e p o r t e d a s t h e mean ± SE , u n l e s s o t h e r w i s e n o t e d . I n some c a s e s t h e c o e f f i c i e n t o f v a r i a t i o n was a l s o d e t e r m i n e d . The T - t e s t was e x c l u s i v e l y u s e d t o t e s t f o r s t a t i s t i c a l d i f f e r e n c e s . Where s i m u l t a n e o u s c o m p a r i s o n s were made, B o n f e r o n i ' s c o r r e c t i o n was a p p l i e d t o a t t a i n o v e r a l l s i g n i f i c a n c e o f <0.05. S t a t i s t i c a l d i f f e r e n c e s a r e i n d i c a t e d on t h e T a b l e s and F i g u r e s . The c h a n g e s i n t h e c o u r s e o f e a c h e x p e r i m e n t were d e t e r m i n e d by c o m p a r i n g 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 f r o m t h e f i r s t and l a s t 38 c o l l e c t i o n p e r i o d s . Duncan's m u l t i p l e r a n g e t e s t was a l s o u s e d t o c o n f i r m t h e s e d i f f e r e n c e s . CHAPTER 5 The Carni t ine and Carni t ine Ester Content of Rat B i l e 39 5.1 R e s u l t s 5.1.1 B i l e F l o w The b i l e f l o w d u r i n g t h e c o u r s e o f t h e e x p e r i m e n t i s shown i n F i g u r e 4. The f l o w r a t e f o r t h e i n i t i a l 15 m i n u t e c o l l e c t i o n p e r i o d was s i g n i f i c a n t l y h i g h e r t h a n f o r any o f t h e s u b s e q u e n t c o l l e c t i o n p e r i o d s . T h e r e were no f u r t h e r d i f f e r e n c e s among t h e c o l l e c t i o n p e r i o d s . 5.1.2 C a r n i t i n e C o n t e n t o f B i l e 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 a r e shown i n F i g u r e 5. A c y l and t o t a l c a r n i t i n e c o n c e n t r a t i o n s i n c r e a s e d d u r i n g b i l e c o l l e c t i o n w h i l e t h e f r e e c a r n i t i n e c o n c e n t r a t i o n r e m a i n e d c o n s t a n t . The p e r c e n t a g e o f c a r n i t i n e i n t h e a c y l f o r m i n c r e a s e d f r o m 70 t o 8 3 % d u r i n g t h e c o u r s e o f t h e e x p e r i m e n t . The amount o f c a r n i t i n e p r e s e n t i n e a c h 15 m i n u t e s a m p l e o f b i l e i s i l l u s t r a t e d i n F i g u r e 6. The amounts o f a c y l and t o t a l c a r n i t i n e a g a i n i n c r e a s e d d u r i n g b i l e c o l l e c t i o n . A l t h o u g h f r e e c a r n i t i n e d e c r e a s e d d u r i n g t h e e x p e r i m e n t , t h e p e r c e n t a g e o f a c y l c a r n i t i n e a g a i n i n c r e a s e d . 5.1.3 C a r n i t i n e E s t e r s i n B i l e The d i s t r i b u t i o n o f t h e c a r n i t i n e e s t e r s o f r a t b i l e i s shown i n T a b l e 2. L o n g - c h a i n c a r n i t i n e e s t e r s made up 29% o f t h e b i l e c a r n i t i n e . A p p r o x i m a t e l y 5 4% o f t h e t o t a l c a r n i t i n e was i n t h e f o r m o f s h o r t 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 . O n l y one q u a r t e r o f t h i s was a s 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 and t h e f l o w r a t e was a s s e s s e d g r a v i m e t r i c a l l y . F l o w was g r e a t e r i n t h e f i r s t c o l l e c t i o n p e r i o d t h a n s u b s e q u e n t p e r i o d s ( a t l e a s t p < 0 .05). T h e r e were 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 (mean ± S E ) . The c o n c e n t r a t i o n s o f t o t a l and 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 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 n o t c h a n g e 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 I I I 15 30 4 5 60 75 90 105 120 TIME OF BILE COLLECTION (min) FIGURE 6 The amounts 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 min/100 g BW, mean ± S E ) . The amounts o f t o t a l and 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 amount 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 i n t e r v a l ( p < 0 . 0 2 ) . 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 t h e I n i t i a l B i l e Sample C o l l e c t e d f r o m Fed R a t s (n=4) Type o f nmol/15 m i n / C a r n i t i n e 100 g BW F r e e 1.04 ± 0.22 17.2 ± 3.9 S h o r t and M e d i u m - c h a i n 3.29 ± 0.21 53.6 ± 2.9 L o n g - c h a i n 1.81 ± 0.26 29.2 ± 3.4 T o t a l 6.13 ± 0.18 100 ± 2.9 44 a c e t y l c a r n i t i n e ( T a b l e 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 i n c r e a s e d d u r i n g t h e c o u r s e o f t h e b i l e c o l l e c t i o n . As a p e r c e n t a g e o f t o t a l c a r n i t i n e , i t r e m a i n e d c o n s t a n t , h o w e v e r . 5.1.4 Serum C a r n i t i n e The c o n c e n t r a t i o n o f c a r n i t i n e i n r a t serum i s shown i n T a b l e 4. S e v e n t e e n p e r c e n t o f t h e t o t a l c a r n i t i n e was e s t e r i f i e d . I n t h e one a d d i t i o n a l r a t s t u d i e d , t h e r e was n e i t h e r a c h a n g e i n t h e amount o f c a r n i t i n e n o r t h e d e g r e e o f c a r n i t i n e e s t e r i f i c a t i o n d u r i n g t h e c o u r s e o f t h e e x p e r i m e n t . 5.2 D i s c u s s i o n C a r n i t i n e i s p r e s e n t i n s i m i l a r c o n c e n t r a t i o n s i n r a t b i l e and s e r u m . R e s u l t s p u b l i s h e d by G u d j o n s s o n and c o l l e a g u e s ( 1 9 8 5 b ) c o n c u r . G u d j o n s s o n f o u n d 27.3 and 34.3 uM c a r n i t i n e i n t h e b i l e o f two r a t s . I n t h i s e x p e r i m e n t , an a v e r a g e o f 46.3 uM ( n = l l ) was f o u n d i n a c o m p a r a b l e c o l l e c t i o n t i m e p e r i o d . I n a d d i t i o n t o t h e number o f r a t s u s e d , o t h e r d i f f e r e n c e s e x i s t b e t w e e n t h i s s t u d y and t h o s e o f G u d j o n s s o n and c o l l e a g u e s : G u d j o n s s o n u s e d S p r a g u e - D a w l e y r a t s w e i g h i n g b e t w e e n 190 and 240 g w h i c h had been f a s t e d f o r 18 h o u r s . L a r g e r , f e d , W i s t a r r a t s were u s e d i n t h i s e x p e r i m e n t . T h e s e d i f f e r e n c e s may a c c o u n t f o r t h e l o w e r 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 f o u n d i n 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 i n R a t B i l e f r o m t h e 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 B i l e C o l l e c t i o n P e r i o d s I n i t i a l S a mple F i n a l S ample umol/1 % o f T o t a l u m ol/1 % o f T o t a l MEAN 7.4 15.6 12 . 5 * 17.7 SE 0.7 1.7 1.3 1.6 N 9 9 9 9 * p<0.01 v e r s u s i n i t i a l s a m p l e TABLE 4 The 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 Ra t Serum A f t e r B i l e C o l l e c t i o n (n=5) F r e e T o t a l % A c y l C a r n i t i n e C a r n i t i n e C a r n i t i n e u m ol/1 umol/1 MEAN SE 48.9 4.4 58.9 5.0 17.0 2.0 47 t h e b i l e c o l l e c t e d by G u d j o n s s o n and c o l l e a g u e s . The method o f c o l l e c t i n g b i l e was t h e same i n b o t h s t u d i e s . B i l e f l o w r a t e was m e a s u r e d t o p e r m i t d e t e r m i n a t i o n o f t h e amount o f c a r n i t i n e i n e a c h 15 m i n u t e c o l l e c t i o n . D a t a p u b l i s h e d by K u i p e r s and c o l l e a g u e s ( 1 9 8 5 ) i n d i c a t e t h a t b i l e f l o w d e c r e a s e s f r o m a p p r o x i m a t e l y 7.5 t o 4.0 u l / m i n / 1 0 0 g BW i n t h e 2 h o u r s f o l l o w i n g 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 f r o m n o n a n a e s t h e t i s e d r a t s . They a t t r i b u t e d t h i s t o a d e c r e a s e i n t h e b i l e a c i d d e p e n d e n t b i l e f l o w . K u i p e r s and c o w o r k e r s a l s o r e p o r t e d t h a t s o d i u m p e n t o b a r b i t a l a n a e s t h e s i a c a u s e d a d e c r e a s e i n b i l e f l o w i n t h e h o u r f o l l o w i n g b i l e c o l l e c t i o n b u t i n c r e a s e d f l o w i n s u b s e q u e n t h o u r s . O t h e r i n v e s t i g a t o r s , h o w e v e r , have r e p o r t e d t h a t p e n t o b a r b i t a l d o e s n o t a f f e c t b i l e f l o w ( B a i l e y e t a l . , 1975; C o o p e r e t a l . , 1 9 7 6 ) . The b i l e f l o w r a t e t h r o u g h o u t t h i s e x p e r i m e n t was s i m i l a r t o t h a t r e p o r t e d f o r n o n a n a e s t h e t i s e d r a t s a t 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 ( K u i p e r s e t a l . , 1 9 8 5 ) . The f l o w r a t e was c o n s t a n t t h r o u g h o u t t h e e x p e r i m e n t e x c e p t f o r t h e i n i t i a l 15 m i n u t e s o f c o l l e c t i o n a t w h i c h i t was h i g h e r . A l t h o u g h some i n v e s t i g a t o r s recommend d i s c a r d i n g t h i s i n i t i a l b i l e s a m p l e , ( R e u b e n e t a l . , 1982; M i n u k and S a r j e a n t , 1 9 8 4 ) , i t was r e t a i n e d i n t h i s e x p e r i m e n t . The amount o f c a r n i t i n e f o u n d i n b i l e i n 24 h o u r s may be e s t i m a t e d f o r a 350 g r a t . A s s u m i n g a b i l e f l o w o f 8 u l / m i n / 1 0 0 g BW f o r . 1 2 h o u r s o v e r 24 h o u r s , t h e amount o f c a r n i t i n e f o u n d i n t h e b i l e w o u l d be a p p r o x i m a t e l y 1% o f t h e 48 r a t s t o t a l body p o o l e v e r y 24 h o u r s ( 1 . 4 umol o r 0.4 umol/24 h/100 g BW). The c a r n i t i n e body p o o l s i z e was o b t a i n e d f r o m t h e d a t a o f C e d e r b l a d and L i n d s t e d t ( 1 9 7 6 ) . G u d j o n s s o n and c o l l e a g u e s ( 1 9 8 5 b ) e s t i m a t e d t h a t i n t h e r a t , t h e r e i s b e t w e e n 0.4 and 1.4 umol c a r n i t i n e / 2 4 h/100 g BW i n t h e b i l e ( 1 - 3 . 5 % o f t h e t o t a l c a r n i t i n e body p o o l ) . The c o n c e n t r a t i o n and t h e amount o f c a r n i t i n e i n t h e b i l e i n c r e a s e d t h r o u g h o u t t h e c o u r s e o f t h e e x p e r i m e n t . T h i s was due t o i n c r e a s i n g amounts o f a c y l c a r n i t i n e ; t h e amount o f f r e e c a r n i t i n e r e m a i n e d c o n s t a n t t h r o u g h o u t . T h i s phenomenon i s d i s c u s s e d i n C h a p t e r 8, b u t t h e c a u s e i s unknown. I t i s a l s o n o t known w h e t h e r t h e i n i t i a l s a m p l e o r t h e f i n a l s a m p l e i s more r e p r e s e n t a t i v e o f t r u e b i l e , so b o t h a r e r e p o r t e d . The a n a l y s i s o f c a r n i t i n e i n t h e r a t b i l e i n d i c a t e d t h a t b e t w e e n 6 9 % and 8 0 % was e s t e r i f i e d . T h i s i s c o m p a r a b l e t o t h e 7 5 % c a r n i t i n e e s t e r i f i c a t i o n r e p o r t e d by G u d j o n s s o n and c o l l e a g u e s ( 1 9 8 5 b ) . No o t h e r body t i s s u e o r f l u i d c o n t a i n s c a r n i t i n e t h a t i s e s t e r i f i e d t o s u c h a h i g h d e g r e e . The serum f r o m t h e r a t s i n t h i s s t u d y d e m o n s t r a t e d 16% e s t e r i f i c a t i o n . T h i s c o n c u r s w i t h t h e 2 3 % r e p o r t e d by P a c e and c o l l e a g u e s ( 1 9 7 8 ) . S k e l e t a l m u s c l e c a r n i t i n e i s a p p r o x i m a t e l y 5 1 % e s t e r i f i e d ( P a c e e t a l . , 1978) w h i l s t h e p a t i c c a r n i t i n e e s t e r i f i c a t i o n r a n g e s f r o m 50 t o 6 2 % ( B r a s s and H o p p e l , 1981; B r a s s and H o p p e l , 1978; P a c e e t a l . , 1978; P e a r s o n and T u b b s , 1967; Bohmer e t a l . , 1 9 6 6 ) . 49 The c a r n i t i n e i n t h e o u t f l o w o f v a s c u l a r l y p e r f u s e d r a t l i v e r , h o w e v e r , i s 7 3 % e s t e r i f i e d ( S a n d o r e t a l . , 1 9 8 5 ) . O n l y one q u a r t e r o f t h e s h o r t 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 i n t h e b i l e a r e i n t h e f o r m o f a c e t y l c a r n i t i n e . I n o t h e r t i s s u e s and f l u i d s , a c e t y l c a r n i t i n e i s t h e p r e d o m i n a n t c a r n i t i n e e s t e r . L a r g e c o n c e n t r a t i o n s o f o t h e r s h o r t - c h a i n o r 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 a r e r a r e l y o b s e r v e d . The p e r c e n t a g e o f l o n g - c h a i n c a r n i t i n e e s t e r s i n r a t b i l e , 3 0 % , was a l s o g r e a t e r t h a n r e p o r t e d f o r o t h e r f l u i d s o r t i s s u e s o f t h e r a t . I n t h e l i v e r , l o n g - c h a i n c a r n i t i n e e s t e r s make up 1.5-4% o f t h e t o t a l c a r n i t i n e ( B r a s s and H o p p e l , 1 981; 1978; Bohmer, 1968; 1967; 1966; P e a r s o n and T u b b s , 1 9 6 7 ) . I n t h e s e r u m , l o n g - c h a i n c a r n i t i n e e s t e r s h a v e been r e p o r t e d a s 1% ( P a c e e t a l . , 1978) and 10% ( B r a s s and H o p p e l , 1981) o f t h e t o t a l c a r n i t i n e . L o n g - c h a i n c a r n i t i n e e s t e r s a c c o u n t e d f o r 15% o f t h e c a r n i t i n e e x p o r t e d f r o m t h e l i v e r d u r i n g v a s c u l a r p e r f u s i o n ( S a n d o r e t a l . , 1 9 8 5 ) . The c o n c e n t r a t i o n o f l o n g - c h a i n c a r n i t i n e e s t e r s i n r a t b i l e , i s a b o v e t h e c r i t i c a l m i c e l l a r c o n c e n t r a t i o n f o r p a l m i t o y l c a r n i t i n e o f 15 uM ( S e i m and D a r g e l , 1978; Y a l k o w s k y and Z o g r a f i , 1 9 7 0 ) . T h e r e f o r e , l o n g - c h a i n c a r n i t i n e e s t e r s , w h i c h a r e n o t w a t e r - s o l u b l e , a r e p r o b a b l y i n t h e f o r m o f m i c e l l e s i n b i l e . I n summary, t h i s e x p e r i m e n t has d e m o n s t r a t e d t h e p r e s e n c e o f r e l a t i v e l y l a r g e amounts o f c a r n i t i n e and 50 c a r n i t i n e e s t e r s i n r a t b i l e . T h i s s u g g e s t s t h a t c a r n i t i n e i n t h e i n t e s t i n a l l umen i s n o t s o l e l y o f d i e t a r y o r i g i n . F u r t h e r , b i l e may r e p r e s e n t a p o s s i b l e r o u t e f o r c a r n i t i n e e x c r e t i o n . B i l e i s u n i q u e i n t h a t i t c o n t a i n s more l o n g - c h a i n c a r n i t i n e e s t e r s and a g r e a t e r p e r c e n t a g e o f e s t e r i f i e d c a r n i t i n e t h a n o t h e r body t i s s u e s o r f l u i d s . The e s t a b l i s h m e n t o f b i l e a s an e n d o g e n o u s s o u r c e o f c a r n i t i n e i n t h e i n t e s t i n e may h e l p e x p l a i n t h e p r e s e n c e o f s h o r t 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 i n t h e lumen o f t h e r a t s m a l l i n t e s t i n e (Sach'an and R u a r k , 1 9 8 5 ) . I t may a l s o e x p l a i n t h e r e c o v e r y o f l a b e l l e d c a r n i t i n e f r o m t h e i n t e s t i n e r e p o r t e d a f t e r i n t r a v e n o u s o r s u b c u t a n e o u s i n j e c t i o n (Hahn e t a l . , 1985; Rebouche e t a l . , 1 9 8 4 ) . L a s t l y , i t i s p o s s i b l e t h a t b i l e m i g h t s e r v e a s a r o u t e f o r c a r n i t i n e e x c r e t i o n . T h i s s u g g e s t i o n i s s u p p o r t e d by body p o o l s t u d i e s i n w h i c h c a r n i t i n e l o s s e s c o u l d n o t be a c c o u n t e d f o r by u r i n a r y l o s s e s a l o n e ( R e b o u c h e and E n g e l , 1984; C e d e r b l a d and L i n d s t e d t , 1976; Yue and F r i t z , 1 9 6 2 ) . CHAPTER 6 The O r i g i n of C a r n i t i n e i n Rat B i l e 51 6.1 The E f f e c t s o f a 72 h o u r F a s 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 B i l e 6.1.1 R e s u l t s 6.1.1.1 B i l e F l o w and W e i g h t L o s s B i l e f l o w d e c r e a s e d i n r a t s a f t e r f a s t i n g 72 h o u r s ( F i g u r e 7 ) . The r a t e o f b i l e f l o w d i d n o t change d u r i n g t h e c o u r s e o f t h e e x p e r i m e n t . The f a s t e d r a t s l o s t 14.1 ± 1.6% o f t h e i r body w e i g h t . 6.1.1.2 C a r n i t i n e C o n t e n t o f B i l e T a b l e 5 shows t h e 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 o f f a s t e d and n o r m a l l y f e d r a t s . A t a l l t i m e s , w i t h one e x c e p t i o n , t h e a c y l and t o t a l c a r n i t i n e c o n c e n t r a t i o n s were g r e a t e r i n t h e f a s t e d t h a n i n t h e f e d s t a t e . T h e r e was no d i f f e r e n c e , h o w e v e r , between t h e c o n c e n t r a t i o n s o f f r e e c a r n i t i n e i n t h e f a s t e d and f e d s t a t e s . The c o n c e n t r a t i o n s o f a c y l and t o t a l c a r n i t i n e i n c r e a s e d o v e r t h e c o u r s e o f t h e e x p e r i m e n t w h i l e t h e f r e e c a r n i t i n e c o n c e n t r a t i o n d i d n o t c h a n g e . The amounts 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 f o u n d i n e a c h 15 m i n u t e b i l e s a m p l e f r o m f a s t e d and n o r m a l l y f e d r a t s a r e i l l u s t r a t e d i n T a b l e 6. T h e r e were no d i f f e r e n c e s b e t w e e n f e d and f a s t e d r a t s , e x c e p t i n t h e f i r s t c o l l e c t i o n p e r i o d where t h e r e was l e s s f r e e c a r n i t i n e upon f a s t i n g . The 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 ch a n g e d u r i n g t h e e x p e r i m e n t . 52 15 30 45 60 75 90 105 120 TIME OF BILE COLLECTION (min) • FED • 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 and 72 h o u r f a s t 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 and 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 as f o l l o w s : * = p<0.02, ** = p<0.01, *** = p<0.001. TABLE 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 t h e B i l e o f F e d and F a s t e d R a t s C o n s e c u t i v e b i l e c o l l e c t i o n p e r i o d s (15 m i n u t e s e a c h ) 1 2 3 5 6 7 8 FREE FED MEAN 14.2 13.2 14.3 13.8 14.2 13.2 12.4 12.4 SE 1.2 0.9 1.0 1.0 1.0 1.0 1.5 1.5 N 9 11 11 11 11 11 11 10 FASTED MEAN 11.51 11.4 10.9 11.0 11.2 12 .7 12.9 12.9 SE 0.7 1.1 0.6 0.5 0.7 0.6 0.4 0.4 N 5 5 5 5 5 5 5 5 P 0.1378 0.2604 0.0470 0.0916 0.0780 0.7536 0.8298 0.8224 ACYL FED MEAN 32.6 40.2 43.0 53.0 61.5 60.9 60.0 59.4 SE 1.5 3.3 3.6 3.8 5.3 4.1 3.7 3.6 N 9 10 11 11 11 11 11 10 FASTED MEAN 70.4 73.7 81.2 85.4 88.5 90.2 92.1 89.3 SE 4.9 1.2 2.0 2.0 1.6 1.3 1.1 3.5 N 5 5 5 5 5 5 5 5 J>. 0.0000 0.0000 0.0000 0.0000 0.0048 0.0004 0.0000 0.0002 * * ' * * * * TOTAL FED MEAN 46.3 53.2 57.0 67.0 71.0 74.2 70.5 72.3 SE 1.1 3.3 3.3 3.7 3.9 3.8 3.3 3.0 N 9 10 10 11 11 11 11 10 FASTED MEAN 81.9 85.1 92.0 96.4 99.7 102.9 105.0 102.2 SE 4.5 1.9 2.3 1.6 1.6 1.5 1.1 3.6 N 5 5 5 5 5 5 5 5 P 0.0000 0.0000 0.0000 0.0002 0.0002 0.0002 0.0000 0.0000 * * * * * * * s i g n i f i c a n t a t o v e r a l l t h e 0.05 l e v e l 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 TABLE 6 The Amounts 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 o f Fed and F a s t e d R a t s C o n s e c u t i v e b i l e c o l l e c t i o n p e r i o d s ( 1 5 m i n u t e s e a c h ) 1 2 3 4 5 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 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 P 0.0012 * 0.0186 0.0246 0.2866 0.0034 0.0228 0.1432 0.2946 ACYL FED MEAN 3.59 4.35 4.32 4.79 5.20 5.71 5.48 5.45 SE 0.13 0.34 0.46 0.37 0.26 0.43 0.30 0.31 N 8 12 11 11 10 11 10 9 FASTED MEAN 5.34 5.30 5.79 5.91 6.01 6.03 5.93 6.49 SE 0.58 0.25 0.32 0.46 0.44 0.44 0.59 0.46 N 5 5 5 5 5 5 5 5 2 0.0036 0.1096 0.0622 0.0986 0.1148 0.9600 0.4590 0.0770 TOTAL FED MEAN 5.27 5.72 5.73 6.17 6.66 6.98 6.62 6.65 SE 0.16 0.44 0.50 0.37 0.26 0.42 0.30 0.30 N 8 11 11 11 10 11 10 9 FASTED MEAN 6.19 6.11 6.55 6.67 6.76 6.87 6.75 7.44 SE 0.60 0.24 0.34 0.50 0.47 0.47 0.65 0.54 N 5 5 5 5 5 5 5 5 E 0.0958 0.5770 0.3150 0.4518 0.8422 0.9866 0.8370 0.1874 s i g n i f i c a n t a t o v e r a l l t h e 0.05 l e v e l 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 55 6.1.1.3 C a r n i t i n e E s t e r s i n B i l e The amounts o f s h o r t 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 d e c r e a s e d f r o m 53 t o 3 3 % a l t h o u g h t h e t o t a l amount o f c a r n i t i n e i n b i l e was n o t a f f e c t e d by f a s t i n g . A t t h e same t i m e t h e amount o f l o n g - c h a i n c a r n i t i n e e s t e r s i n c r e a s e d f r o m 29 t o 54% o f t h e t o t a l c a r n i t i n e i n t h e b i l e ( F i g u r e 8 ) . 6.1.1.4 Serum C a r n i t i n e Upon f a s t i n g t h e c o n c e n t r a t i o n o f serum t o t a l c a r n i t i n e d i d n o t d i f f e r f r o m t h e f e d s t a t e ( T a b l e 7 ) . 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 , h o w e v e r , was l o w e r and t h e o v e r a l l c a r n i t i n e e s t e r i f i c a t i o n was h i g h e r . 6.1.2 D i s c u s s i o n The c h a n g e s i n t h e c a r n i t i n e c o n t e n t o f r a t b i l e a f t e r a 72 h o u r f a s t were s i m i l a r t o t h o s e r e p o r t e d by B r a s s and H o p p e l ( 1 9 7 8 ) i n r a t l i v e r ( T a b l e 1 ) . I n bo t h , - t h e amounts o f f r e e c a r n i t i n e d e c r e a s e d s l i g h t l y ( f r o m 17% t o 13% i n t h e b i l e and f r o m 5 3 % t o 4 7 % i n t h e l i v e r ) , t h e amounts o f l o n g - c h a i n c a r n i t i n e e s t e r s i n c r e a s e d c o n s i d e r a b l y ( f r o m 2 9 % t o 54% i n t h e b i l e and f r o m 3% t o 1 1 % i n t h e l i v e r ) and t h e r e were no c h a n g e s i n t h e amounts o f t o t a l c a r n i t i n e . T h i s r e f l e c t s i n c r e a s e d f a t t y a c i d o x i d a t i o n i n t h e f a s t i n g s t a t e . More c a r n i t i n e i s i n v o l v e d i n t h e t r a n s f e r o f f a t t y a c i d s a c r o s s t h e i n n e r m i t o c h o n d r i a l membrane and t h u s i s i n t h e f o r m o f l o n g - c h a i n c a r n i t i n e e s t e r s . T h e r e f o r e , l e s s 56 m o> o o c 1 10 o E c 8- 7- 6 - 5 - 4- 3 - 2- 1-Lb FREE SHORT AND LONG-CHAIN MEDIUM-CHAIN TOTAL Fed I | Fasted FIGURE 8 The amounts of 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 fe d 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 i t i a t i o n of b i l e c o l l e c t i o n (mean ± SE). Shor t and medium-chain a c y l c a r n i t i n e l e v e l s were decreased a f t e r f a s t i n g (p< 0 . 0 0 1 ) w h i l e 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 i n c r e a s e d (p< 0 . 0 0 1 ) . The f r e e c a r n i t i n e c o n t e n t of the b i l e d i d not change a f t e r f a s t i n g . 57 c a r n i t i n e i s i n t h e f r e e s t a t e . One d i f f e r e n c e b e t w e e n t h e l i v e r and b i l e i s t h a t t h e amount o f s h o r t 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 d e c r e a s e d i n t h e b i l e ( f r o m 54% t o 32%) b u t n o t i n t h e l i v e r ( 4 3 % ) . The c h a n g e s i n serum c a r n i t i n e upon f a s t i n g f o r 72 h o u r s do n o t p a r a l l e l t h o s e i n t h e b i l e . Serum c a r n i t i n e e s t e r i f i c a t i o n i n c r e a s e d f r o m 16 t o 50% and t h e t o t a l c a r n i t i n e d i d n o t c h a n g e . T h i s i s c o m p a r a b l e t o t h e 5 1 % serum c a r n i t i n e e s t e r i f i c a t i o n r e p o r t e d by G u d j o n s s o n and c o l l e a g u e s i n r a t s f a s t e d f o r 16-18 h o u r s ( 1 9 8 5 b ) . I n o t h e r r a t s f a s t e d f o r 72 h o u r s , c a r n i t i n e e s t e r i f i c a t i o n i n c r e a s e d f r o m 4 1 % t o 69% ( B r a s s and H o p p e l , 1 9 7 8 ) . T h e s e i n i t i a l l y h i g h e r c a r n i t i n e l e v e l s m i g h t r e f l e c t a h i g h e r d i e t a r y f a t c o n t e n t (Seccombe e t a l . , 1 9 7 8 ) . I n humans, serum c a r n i t i n e e s t e r i f i c a t i o n a l s o i n c r e a s e d upon f a s t i n g w i t h no c h a n g e i n t o t a l c a r n i t i n e ( F r o h l i c h e t a l . , 1978; H o p p e l and G e n u t h , 1 9 7 6 ) . C a r n i t i n e e s t e r i f i c a t i o n i n t h e b i l e d i d n o t i n c r e a s e upon f a s t i n g . I n s t e a d , t h e amount o f l o n g - c h a i n c a r n i t i n e e s t e r s i n c r e a s e d w h i l e s h o r t 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 d e c r e a s e d . A f t e r a 72 h o u r f a s t , t h e r e was no c h a n g e i n t h e amount o f l o n g - c h a i n c a r n i t i n e e s t e r s i n t h e s erum ( B r a s s and H o p p e l , 1 9 7 8 ) . A l t h o u g h t h e c o n c e n t r a t i o n o f c a r n i t i n e i n t h e b i l e i n c r e a s e d upon f a s t i n g t h e amount o f c a r n i t i n e f o u n d i n t h e b i l e d i d n o t c h a n g e p e r u n i t t i m e . T h i s i s a r e s u l t o f r e d u c e d b i l e f l o w d u r i n g f a s t i n g w h i c h was a l s o o b s e r v e d by M i n u k and S a r j e a n t ( 1 9 8 4 ) . S i m i l a r l y , t h e h e p a t i c c a r n i t i n e 58 c o n t e n t a l s o i n c r e a s e d d u r i n g f a s t i n g ( B r a s s and H o p p e l , 1981; Bohmer, 1968) b u t B r a s s and H o p p e l f o u n d t h a t t h i s was o n l y a r e s u l t o f d e c r e a s e d l i v e r w e i g h t . C a r n i t i n e was a l s o f o u n d by G u d j o n s s o n and c o l l e a g u e s ( 1 9 8 5 b ) i n t h e b i l e o f two r a t s who were f a s t e d 16-18 h o u r s . They f o u n d , 30.8 uM t o t a l c a r n i t i n e , much l e s s t h a n t h e 81.9 uM f o u n d i n t h i s s t u d y a f t e r a 72 h o u r f a s t . The s h o r t e r f a s t i n g p e r i o d m i g h t be r e s p o n s i b l e f o r t h i s d i f f e r e n c e . S h o r t 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 made up 3 3 % o f t h e t o t a l c a r n i t i n e compared t o 3 2 % i n t h i s s t u d y and l o n g - c h a i n c a r n i t i n e e s t e r s made up 4 3 % compared t o t h e 54% i n t h i s s t u d y . I n c o n c l u s i o n , t h e c h a n g e s t h a t o c c u r i n t h e c o m p o s i t i o n o f c a r n i t i n e i n t h e b i l e , more c l o s e l y p a r a l l e l t h o s e o c c u r r i n g i n t h e l i v e r t h a n t h e serum a s i l l u s t r a t e d b e l o w . CARNITINE SERUM LIVER B I L E -amount -% e s t e r s - t y p e s o f e s t e r s - f r e e - s h o r t and medium - l o n g no c h a n g e i n c r e a s e d d e c r e a s e d i n c r e a s e d no c h a n g e no c h a n g e no change no c h a n g e no c h a n g e i n c r e a s e d no c h a n g e no change no c h a n g e d e c r e a s e d i n c r e a s e d T h e s e r e s u l t s a l s o s u p p o r t t h e t h e o r y t h a t b i l e c a r n i t i n e c o n t r i b u t e s t o t h e c a r n i t i n e c o n t e n t o f t h e i n t e s t i n a l 59 l u m e n . S a c h a n and R u a r k ( 1 9 8 5 ) f o u n d t h a t a 24 h o u r f a s t l o w e r e d t h e amount o f s h o r t 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 i n t h e r a t i n t e s t i n a l l umen by 54%. T h e r e was a 4 1 % d e c r e a s e i n t h e s e e s t e r s i n t h e b i l e o f r a t s who were f a s t e d f o r 72 h o u r s . Lumen f r e e c a r n i t i n e d e c r e a s e d by 10% and l o n g - c h a i n c a r n i t i n e e s t e r s i n c r e a s e d by 12%. The d e c r e a s e i n f r e e c a r n i t i n e p r o b a b l y r e f l e c t s t h e l a c k o f d i e t a r y c a r n i t i n e and t h e r e l a t i v e i n c r e a s e i n l o n g - c h a i n c a r n i t i n e e s t e r s i s l i k e l y due t o t h e c a r n i t i n e i n t h e b i l e w h i c h i s r i c h i n l o n g - c h a i n c a r n i t i n e e s t e r s . T h i s s u g g e s t s an a s s o c i a t i o n b e t w e e n t h e b i l e and t h e i n t e s t i n a l c a r n i t i n e c o n t e n t s . 60 6.2 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 A c i d 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 B i l e 6.2.1 R e s u l t s 6.2.1.1 B i l e F l o w and W e i g h t L o s s The b i l e f l o w o f f a s t e d r a t s w i t h and w i t h o u t TDGA t r e a t m e n t i s shown i n F i g u r e 9. TDGA c a u s e d b i l e f l o w t o i n c r e a s e a b o v e t h e f a s t i n g l e v e l s e x c e p t i n t h e s e c o n d b i l e c o l l e c t i o n p e r i o d . I n two a d d i t i o n a l r a t s , b i l e f l o w was s t i m u l a t e d by t h e t r a g a c a n t h v e h i c l e a l o n e . TDGA t r e a t e d r a t s l o s t 14% o f t h e i r body w e i g h t s upon f a s t i n g f o r 72 h o u r s . T h i s i s n o t d i f f e r e n t f r o m t h e w e i g h t l o s s o f t h e r a t s who were f a s t e d o n l y . 6.2.1.2 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 TDGA t r e a t m e n t c a u s e d an i n c r e a s e i n t h e 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 i n t h e b i l e , a d e c r e a s e i n a c y l and no ch a n g e i n t o t a l c a r n i t i n e c o n c e n t r a t i o n s ( T a b l e 8 ) . F r e e , a c y l and t o t a l c a r n i t i n e c o n c e n t r a t i o n s a l l i n c r e a s e d d u r i n g t h e e x p e r i m e n t . TDGA c a u s e d no c h a n g e s i n t h e amounts o f a c y l and t o t a l c a r n i t i n e i n e a c h 15 m i n u t e b i l e c o l l e c t i o n ( T a b l e 9 ) . T h e r e was, h o w e v e r , more f r e e c a r n i t i n e i n t h e b i l e o f TDGA t r e a t e d r a t s i n t h e t h i r d t h r o u g h e i g t h c o l l e c t i o n p e r i o d s . B o t h t h e amounts o f a c y l and t o t a l c a r n i t i n e i n c r e a s e d t h r o u g h o u t t h e e x p e r i m e n t b u t f r e e c a r n i t i n e d i d n o t c h a n g e . 61 9- 8- TIME OF BILE COLLECTION (min) • FASTED 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 and TDGA t r e a t 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 and t h e f l o w r a t e w a s a s s e s s e d 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 was 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 o f 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<0.001. 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 and TDGA T r e a t e d R a t s T r e a t m e n t F r e e C a r n i t i n e u m ol/1 T o t a l C a r n i t i n e u mol/1 % A c y l C a r n i t i n e F ed MEAN SE 48.9 4.4 58.9 5.0 17.0 2.0 F a s t e d 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 * p<0.05 v e r s u s f e d ** p<0.001 v e r s u s f e d @ p<0.01 v e r s u s f a s t e d @@ P<0.001 v e r s u s f a s t e d TABLE 8 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 o f F a s t e d and TDGA - T r e a t e d R a t s C o n s e c u t i v e b i l e c o l l e c t i o n p e r i o d s ( 1 5 m i n u t e s e a c h ) 1 2 3 4 5 6 7 8 FREE FASTED MEAN 11.5 11.4 10.9 11.0 11.2 12.7 12.9 12.9 SE 0.7 1.1 0.6 0.5 0.7 0.6 0.4 0.4 N 5 5 5 5 5 5 5 5 TDGA MEAN 21.2 23.3 22.9 24.2 25.0 25.4 26.4 26.4 SE 0.2 1.4 1.0 0.7 1.1 1.0 1.0 1.4 N 6 6 6 6 6 6 6 5 p 0.0000 0.0004 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 * * * * * * * * ACYL FASTED MEAN 70.4 73.7 81.2 85.4 88.5 90.2 92.1 89.3 SE 4.9 1.2 2.0 2.0 1.6 1 .3 1. 1 3.5 N 5 5 5 5 5 5 5 5 TDGA MEAN 42.2 49.5 56.0 56.6 58.5 59.9 66.5 62.9 SE 3.4 2.6 2.9 2.8 3.6 3.4 2.7 3.9 N 6 6 6 6 6 6 5 5 p 0.0008 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0010 * * * * * * * * TOTAL FASTED MEAN 81.9 85.1 92.0 96.4 99.7 102.9 105.0 102.2 SE 4.5 1.9 2.3 1.6 1.6 1.5 1.1 3.6 N 5 5 5 5 5 5 5 5 TDGA MEAN 63.2 71.8 79.3 79.5 82.7 84.9 91.9 89.3 SE 2.3 2.8 3.0 3.1 4.3 3.8 2.9 4.4 N 6 6 6 6 6 6 5 5 P 0.0036 0.0044 0.0100 0.0014 * 0.0072 0.0028 0.0028 0.0530 s i g n i f i c a n t a t o v e r a l l t h e 0.05 l e v e l 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 TABLE 9 The Amounts 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 o f F a s t e d and TDGA-Treated R a t s C o n s e c u t i v e b i l e c o l l e c t i o n p e r i o d s ( 1 5 m i n u t e s e a c h ) 1 2 3 4 5 6 7 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 2.00 0.26 6 1.86 0.23 6 2.03 0.12 6 2.02 0.13 6 2.25 0.13 6 2.26 0.13 6 2.42 0.11 5 2.40 0.18 5 P 0.0032 0.0028 0.0012 * 0.0000 * 0.0000 * 0.0000 # 0.0000 * 0.0000 * ACYL 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 3.92 0.34 6 4.28 0.54 6 4.84 0.25 6 4.98 0.26 6 5.47 0.48 6 5.48 0.44 6 6.33 0.28 5 5.73 0.49 5 P 0.0550 0.1446 0.0414 0.0986 0.3692 0.4038 0.5576 0.2908 TOTAL 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 TDGA MEAN SE N 5.93 0.40 6 6.13 0.71 6 6.87 0.31 6 6.99 0.35 6 7.73 0.58 6 7.75 0.53 6 8.76 0.29 5 8.13 0.62 5 P 0.6928 0.9806 0.5040 0.6036 0.2386 0.2986 0.0224 0.4258 s i g n i f i c a n t a t o v e r a l l t h e 0.05 l e v e l 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 E s t e r s i n t h e B i l e I n t h e f i r s t b i l e c o l l e c t i o n p e r i o d , i n c r e a s e d amounts o f f r e e c a r n i t i n e and s h o r t 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 were f o u n d i n t h e b i l e o f t h e TDGA t r e a t e d r a t s ( F i g u r e 1 0 ) . 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 . By t h e end o f t h e 2 h o u r b i l e c o l l e c t i o n , t h e amounts o f f r e e c a r n i t i n e and s h o r t 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 had i n c r e a s e d f u r t h e r b u t t h e r e was no a d d i t i o n a l c hange i n t h e amount o f l o n g - c h a i n c a r n i t i n e e s t e r s i n t h e b i l e . 6.2.1.4 Serum C a r n i t i n e TDGA t r e a t m e n t c a u s e d a d e c r e a s e i n t h e c o n c e n t r a t i o n o f t o t a l c a r n i t i n e i n r a t serum ( T a b l e 7 ) . C a r n i t i n e e s t e r i f i c a t i o n a l s o was l o w e r upon TDGA t r e a t m e n t , d e c r e a s i n g f r o m 50 t o 28%. 6.2.2 D i s c u s s i o n The c h a n g e s i n t h e c a r n i t i n e c o n t e n t o f b i l e a f t e r a d m i n i s t r a t i o n o f TDGA t o f a s t e d r a t s were s i m i l a r t o t h o s e r e p o r t e d by F r e n c h and c o l l e a g u e s ( 1 9 8 5 ) f o r r a t l i v e r a f t e r t h e same t r e a t m e n t . The amount o f l o n g - c h a i n c a r n i t i n e e s t e r s d e c r e a s e d b e l o w f a s t i n g l e v e l s 2 h o u r s a f t e r TDGA i n t u b a t i o n and f r e e c a r n i t i n e l e v e l s i n c r e a s e d . I n t h e b i l e , t h e p e r c e n t a g e o f e s t e r i f i e d c a r n i t i n e r e m a i n e d a t a p p r o x i m a t e l y 7 0 % a f t e r TDGA t r e a t m e n t . I n t h e l i v e r , h o w e v e r , TDGA c a u s e d a d e c r e a s e i n c a r n i t i n e 66 FIGURE 10 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 f a s t e d and TDGA t r e a t e d r a t s 15 and 105 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 (mean ± 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 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 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 b e l o w f a s t i n g l e v e l s . A f t e r 105 m i n u t e s , t h e f r e e and s h o r t 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 l e v e l s had i n c r e a s e d f u r t h e r b u t 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 r e m a i n e d 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 and ** = p<0.001. 15 Minutes FREE SHORT AND LONG-CHAIN TOTAL MEDIUM-CHAIN r i F a s t e d i l l TDGA treated 68 e s t e r i f i c a t i o n f r o m 6 9 % t o 5 5 % ( F r e n c h e t a l . , 1 9 8 5 ) . The f a c t t h a t e s t e r i f i c a t i o n i n t h e b i l e was n o t a f f e c t e d by TDGA m i g h t be due t o i n c r e a s e d amounts o f t h e s h o r t 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 f o u n d i n t h e b i l e upon TDGA a d m i n i s t r a t i o n . T h e s e were n o t c h a n g e d i n t h e l i v e r a f t e r TDGA a d m i n i s t r a t i o n ( F r e n c h e t a l . , 1 9 8 5 ) . I n r a t serum a f t e r TDGA i n t u b a t i o n , t h e t o t a l and a c y l c a r n i t i n e c o n c e n t r a t i o n s were b o t h d e c r e a s e d . T h i s i s d i f f e r e n t f r o m t h e s i t u a t i o n i n t h e b i l e where t h e r e was no ch a n g e i n t h e o v e r a l l amount o f a c y l c a r n i t i n e . The serum a c y l c a r n i t i n e c o n t e n t may be d e c r e a s e d due t o i n c r e a s e d u p t a k e o f c a r n i t i n e e s t e r s by t h e t i s s u e s f o r u s e as e n e r g y . F r e n c h and c o l l e a g u e s ( 1 9 8 5 ) f o u n d t h a t t h e h e p a t i c c a r n i t i n e c o n t e n t i n c r e a s e d a b o v e f a s t i n g l e v e l s w i t h TDGA t r e a t m e n t . F r o s t and W e l l s ( 1 9 8 2 ) a l s o o b s e r v e d t h i s i n c r e a s e i n n e o n a t a l r a t s and h y p o t h e s i z e d t h a t i t i s a c o m p e n s a t o r y mechanism t o overcome t h e i n h i b i t i o n o f t h e TDGA. The amount o f c a r n i t i n e i n t h e b i l e , h o w e v e r , d i d n o t d i f f e r w i t h TDGA a d m i n i s t r a t i o n . O v e r a l l , a f t e r a 72 h o u r f a s t and TDGA i n t u b a t i o n , t h e c h a n g e s t h a t o c c u r i n b i l e c a r n i t i n e more c l o s e l y p a r a l l e l t h o s e o c c u r r i n g i n t h e l i v e r t h a n t h e serum a s shown b e l o w . 69 CARNITINE SERUM LIV E R B I L E -amount -% e s t e r s - t y p e s o f e s t e r s - f r e e - s h o r t and medium - l o n g d e c r e a s e d d e c r e a s e d no c h a n g e d e c r e a s e d i n c r e a s e d d e c r e a s e d d e c r e a s e d no c h a n g e no change i n c r e a s e d i n c r e a s e d no c h a n g e i n c r e a s e d d e c r e a s e d 6.3 The R e c o v e r y 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 R a t B i l e L a b e l l e d c a r n i t i n e , i n j e c t e d i n t r a v e n o u s l y , was a l m o s t i m m e d i a t e l y f o u n d i n r a t b i l e . The methods and r e s u l t s o f t h i s e x p e r i m e n t a r e r e p o r t e d i n A p p e n d i x 1. T h i s f i n d i n g s u p p o r t s t h e h y p o t h e s i s t h a t t h e l a b e l l e d c a r n i t i n e f o u n d i n t h e i n t e s t i n e and f e c e s a f t e r s u b c u t a n e o u s and i n t r a v e n o u s i n j e c t i o n s (Hahn e t a l . , 1985; Rebouche e t a l . , 1984) a r r i v e s t h e r e v i a t h e b i l e . W i t h i n 2 h o u r s o f i n j e c t i o n , 0.5% o f l a b e l l e d c a r n i t i n e was r e c o v e r e d i n t h e r a t b i l e . G u d j o n s s o n and c o l l e a g u e s ( 1 9 8 5 b ) f o u n d 2.4% o f an i n j e c t e d l a b e l i n t h e b i l e a f t e r 4 h o u r s . The h i g h e r r e c o v e r y i n G u d j o n s s o n ' s e x p e r i m e n t i s p r o b a b l y due t o t h e l a r g e amount o f n o n l a b e l l e d c a r n i t i n e i n j e c t e d w i t h t h e l a b e l ( 8 . 7 v e r s u s 0.26 nmol c a r n i t i n e / 1 0 0 g BW) as he f o u n d l a r g e r amounts o f c a r n i t i n e i n t h e b i l e a f t e r i n j e c t i o n o f m a s s i v e d o s e s . 70 A l t h o u g h t h e s p e c i f i c a c t i v i t y o f t h e c a r n i t i n e i n t h e b i l e and t h e serum a f t e r 20 m i n u t e s was s i m i l a r , t h e e s t e r d i s t r i b u t i o n was d i f f e r e n t . T h e r e were no l o n g - c h a i n c a r n i t i n e e s t e r s i n t h e serum w h i l e 5 0 % o f t h e l a b e l i n b i l e was i n t h i s f o r m . Of t h e n o n - l a b e l l e d c a r n i t i n e , o n l y 3 0 % was e s t e r i f i e d t o l o n g - c h a i n e s t e r s . T h e r e f o r e , t h e d i s t r i b u t i o n o f l a b e l l e d c a r n i t i n e i s n o t r e p r e s e n t a t i v e o f t h e a c t u a l c a r n i t i n e d i s t r i b u t i o n i n t h e b i l e . T h e r e f o r e , t h e r e a p p e a r s t o be more t h a n one p o o l o f c a r n i t i n e i n t h e l i v e r t h a t e n t e r s t h e b i l e ; one t h a t e n t e r s f r o m t h e s e r u m , b u t i s f i r s t e s t e r i f i e d t o l o n g - c h a i n c a r n i t i n e e s t e r s i n t h e h e p a t o c y t e and t h e o t h e r t h a t o r i g i n a t e s f r o m e i t h e r s t o r e d o r n e w l y s y n t h e s i z e d c a r n i t i n e i n t h e l i v e r . B r o o k s and M c i n t o s h ( 1 9 7 5 ) a l s o r e p o r t e d t h e p r e s e n c e o f two p o o l s o f c a r n i t i n e i n l i v e r ; a l a r g e ( 8 9 % ) , r a p i d l y t u r n i n g o v e r p o o l and a s m a l l e r , more s t a b l e p o o l . The l a b e l l e d l o n g - c h a i n c a r n i t i n e e s t e r s p r e s u m a b l y come f r o m t h e l a r g e r p o o l b e c a u s e o f t h e i r r a p i d t u r n o v e r . A l t e r n a t i v e l y , t h e i n j e c t e d l a b e l l e d c a r n i t i n e c o u l d be e s t e r i f i e d i n t h e s k e l e t a l m u s c l e t i s s u e , and t r a n s p o r t e d t o t h e l i v e r v i a t h e s e r u m . T h i s i s u n l i k e l y as l o n g - c h a i n c a r n i t i n e e s t e r s i n m u s c l e c e l l s a r e p r o b a b l y i m m e d i a t e l y o x i d i z e d . S k e l e t a l m u s c l e h a s a l a r g e , s l o w l y t u r n i n g o v e r c a r n i t i n e p o o l ( B r o o k s and M c i n t o s h , 1 9 7 5 ) . Yue and F r i t z ( 1 9 6 2 ) , when s t u d y i n g t h e u p t a k e o f l a b e l l e d c a r n i t i n e i n j e c t e d i n t r a v e n o u s l y i n t o d o g s , f o u n d t h a t l e s s t h a n 1% o f t h e l a b e l l e d c a r n i t i n e t a k e n up by t h e 71 s k e l e t a l m u s c l e i n t h e 7 h o u r s a f t e r i n j e c t i o n was c h l o r o f o r m e x t r a c t a b l e ( l o n g - c h a i n c a r n i t i n e ) . 6% o f t h e l a b e l l e d c a r n i t i n e i n t h e l i v e r , h o w e v e r , c o u l d be e x t r a c t e d w i t h c h l o r o f o r m . T h e s e r e s u l t s s u g g e s t t h a t t h e l i v e r i s more a c t i v e a t p r o d u c i n g 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 i n j e c t e d c a r n i t i n e t h a n i s t h e m u s c l e . T h i s i s a n o t h e r r e a s o n t o s u s p e c t t h a t l a b e l l e d 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 o u n d i n t h e b i l e o r i g i n a t e i n t h e l i v e r . G u d j o n s s o n and c o w o r k e r s ( 1 9 8 5 b ) p r o v i d e f u r t h e r e v i d e n c e t o s u p p o r t t h e c o n c e p t t h a t serum c a r n i t i n e i s f i r s t e s t e r i f i e d i n t h e l i v e r b e f o r e e n t e r i n g t h e b i l e . A f t e r a d m i n i s t r a t i o n o f l a r g e amounts o f f r e e c a r n i t i n e i n t r a v e n o u s l y , t h e c a r n i t i n e e s t e r c o n t e n t o f t h e b i l e i n c r e a s e d d r a m a t i c a l l y w h i l e f r e e c a r n i t i n e l e v e l s r e m a i n e d c o n s t a n t . CHAPTER 7 The C a r n i t i n e C o n t e n t o f Human B i l e - R i c h D u o d e n a l F l u i d 72 7.1 R e s u l t s The c o n c e n t r a t i o n o f c a r n i t i n e i n e a c h d u o d e n a l f l u i d s a m p l e i s shown i n T a b l e 10. The p e r c e n t a g e o f a c y l c a r n i t i n e was c o n s i s t e n t ( 8 1 % ) , a l t h o u g h f r e e and t o t a l c a r n i t i n e l e v e l s v a r i e d w i d e l y . The two s a m p l e s c o l l e c t e d b e f o r e t h e s t i m u l a t i o n o f b i l e f l o w , c o n t a i n e d v e r y l i t t l e c a r n i t i n e most o f w h i c h was i n t h e f r e e s t a t e . The t y p e s o f c a r n i t i n e e s t e r s i n t h e d u o d e n a l f l u i d a r e shown i n T a b l e 11. S h o r t 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 made up 3 5 % and l o n g - c h a i n c a r n i t i n e e s t e r s made up 37% o f t h e t o t a l c a r n i t i n e . 7.2 D i s c u s s i o n Rudman and c o l l e a g u e s ( 1 9 8 0 ) r e p o r t e d no s i g n i f i c a n t d i f f e r e n c e s b e t ween t h e serum c a r n i t i n e c o n c e n t r a t i o n s o f c h o l e l i t h i a s i s p a t i e n t s and n o r m a l c o n t r o l s . On t h i s b a s i s , t h e p o p u l a t i o n o f p a t i e n t s s t u d i e d i n t h i s e x p e r i m e n t was assumed t o be homogeneous w i t h r e s p e c t t o c a r n i t i n e m e t a b o l i s m . The c o n c e n t r a t i o n s o f b o t h f r e e and t o t a l c a r n i t i n e i n b i l e - r i c h human d u o d e n a l f l u i d v a r i e d w i d e l y . The c o e f f i c i e n t s o f v a r i a t i o n f o r t h e s e two s e t s o f d a t a were 119% and 140% r e s p e c t i v e l y . I n r a t s , t h e t o t a l c o n c e n t r a t i o n o f c a r n i t i n e i n t h e b i l e had a c o e f f i c i e n t o f v a r i a t i o n o f o n l y 7%. When t h e amount o f e s t e r i f i e d c a r n i t i n e i n t h e d u o d e n a l f l u i d was e x p r e s s e d as a 73 TABLE 10 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 C a r n i t i n e E s t e r s i n Human D u o d e n a l F l u i d A f t e r S t i m u l a t i o n o f B i l e F l o w S u b j e c t C a r n i t i n e C o n c e n t r a t i o n (uM) % A c y l C a r n i t i n e F r e e T o t a l 1 * 19.1 94.6 80 5.1 27.3 81 2 * 28.3 89.5 68 1.8 8.8 80 3 * 4.6 38.4 88 26.0 113.4 77 4 6.1 35.7 83 5 8.0 42.6 81 6 0.6 4.3 86 7 * 4.0 25.4 84 6.3 49.6 87 8 * 60.9 412.3 85 58.9 388.8 85 9 2.4 ** 2.5 ** 4 7.6 30.0 75 10 3.6 ** 3.8 ** 4 7.8 23.7 67 MEAN *** 16.3 92.3 81 SE 5.0 33.3 2 * 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 TABLE 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 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 F r e e C a r n i t i n e S h o r t and Medium- L o n g - c h a i n (%) C h a i n C a r n i t i n e C a r n i t i n e E s t e r s (%) E s t e r s (%) MEAN SE N 26.0 3.8 4 35.3 1.5 4 36.5 8.7 4 75 p e r c e n t a g e o f t h e t o t a l , h o w e v e r , t h e c o e f f i c i e n t o f v a r i a t i o n was o n l y 8%. The d e g r e e o f c a r n i t i n e e s t e r i f i c a t i o n i n t h e d u o d e n a l f l u i d ( 8 1 % ) was n o t s i g n i f i c a n t l y d i f f e r e n t f r o m t h a t o f r a t b i l e i n t h e f i n a l b i l e c o l l e c t i o n p e r i o d . T h e s e r e s u l t s a l s o c o r r e s p o n d w i t h t h o s e o b t a i n e d f r o m one s a m p l e o f p i g b i l e a l s o a n a l y s e d f o r c a r n i t i n e . T h i s s a m p l e was c o l l e c t e d d i r e c t l y f r o m t h e g a l l b l a d d e r o f a f a s t e d , m a le Y u c a t a n p i g i m m e d i a t e l y p o s t - m o r t e m . 87% o f t h e t o t a l c a r n i t i n e ( 2 0 6 . 9 uM) was e s t e r i f i e d . T h e r e a r e two p l a u s i b l e e x p l a n a t i o n s f o r t h e w i d e r a n g e o f c a r n i t i n e c o n c e n t r a t i o n 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 . F i r s t , humans have g a l l b l a d d e r s , w h i c h a r e a b s e n t i n r a t s . B i l e i s c o n c e n t r a t e d t o v a r y i n g d e g r e e s d e p e n d i n g on t h e l e n g t h o f t i m e t h a t i t r e m a i n s i n t h e g a l l b l a d d e r ( W h e e l e r , 1 9 7 1 ) . C o n c e n t r a t i o n o f b i l e a n d , h e n c e , c a r n i t i n e m i g h t have o c c u r r e d t o d i f f e r e n t d e g r e e s i n t h e p a t i e n t s s t u d i e d a s t h e y were i n s t r u c t e d t o f a s t f o r 12-18 h o u r s b e f o r e t h e p r o c e d u r e . S e c o n d l y , t h e s a m p l e s s t u d i e d were d u o d e n a l f l u i d , n o t p u r e b i l e . E a c h s a m p l e c o u l d have been d i l u t e d t o a d i f f e r e n t e x t e n t d e p e n d i n g on t h e amounts o f g a s t r o i n t e s t i n a l f l u i d a s p i r a t e d a l o n g w i t h t h e b i l e . T h i s was shown by c o l l e c t i n g two s a m p l e s f r o m e a c h o f f i v e p a t i e n t s a t i n t e r v a l s o f 15 m i n u t e s a f t e r b i l e f l o w was s t i m u l a t e d . I n p a t i e n t s #7 and #8, t h e r e were no m a j o r d i f f e r e n c e s b e t w e e n t h e c a r n i t i n e c o n c e n t r a t i o n s m e a s u r e d i n t h e two s a m p l e s . I n p a t i e n t s , #1, #2, and #3, h o w e v e r , 76 t h e r e were c o n s i d e r a b l e d i f f e r e n c e s . F o r e x a m p l e , p a t i e n t #1 had 94.6 uM c a r n i t i n e i n t h e f i r s t s a m p l e b u t o n l y 27.3 uM c a r n i t i n e i n t h e l a t t e r . The d e g r e e o f c a r n i t i n e e s t e r i f i c a t i o n d i d n o t c hange however between t h e two s a m p l e s ( 8 0 % f o r t h e f i r s t and 8 1 % f o r t h e s e c o n d ) . To e n s u r e t h a t t h e c a r n i t i n e m e a s u r e d was f r o m t h e b i l e and n o t o t h e r g a s t r o i n t e s t i n a l s e c r e t i o n s , t h e c a r n i t i n e c o n t e n t o f d u o d e n a l f l u i 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 was m e a s u r e d i n two s u b j e c t s . I n b o t h c a s e s , t h e r e was l e s s t h a n 4 uM c a r n i t i n e p r e s e n t . I t c a n be c o n c l u d e d , t h e r e f o r e , t h a t g a s t r o i n t e s t i n a l s e c r e t i o n s do n o t c o n t r i b u t e t o t h e c a r n i t i n e c o n t e n t o f t h e s a m p l e s . A l s o , t h e c a r n i t i n e e s t e r p r o f i l e o f t h e p r e - s t i m u l a t i o n f l u i d i s d i f f e r e n t f r o m t h e p o s t - s t i m u l a t i o n f l u i 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 , c a r n i t i n e was o n l y 4% e s t e r i f i e d w h i l e a f t e r , i t was 8 1 % e s t e r i f i e d . The c a r n i t i n e c o n t e n t o f p a n c r e a t i c f l u i d , t h e r e l e a s e o f w h i c h i s a l s o s t i m u l a t e d by 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 i n j e c t i o n , i s unknown. The c a r n i t i n e c o n t e n t o f p a n c r e a t i c t i s s u e , h o w e v e r , i s v e r y l o w ( B r o o k s and M c i n t o s h , 1 9 7 5 ) . The r a t b i l e was n o t c o n t a m i n a t e d by p a n c r e a t i c s e c r e t i o n s b e c a u s e t h e c a n n u l a was p l a c e d beyond t h e p a n c r e a t i c t i s s u e . C a r n i t i n e i n b i l e - r i c h human d u o d e n a l f l u i d i s d i s t r i b u t e d e v e n l y b e t w e e n f r e e c a r n i t i n e , s h o r t 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 and l o n g - c h a i n c a r n i t i n e e s t e r s . The p e r c e n t a g e o f l o n g - c h a i n c a r n i t i n e e s t e r s i s n o t d i f f e r e n t f r o m t h a t o f t h e b i l e c o l l e c t e d f r o m f e d r a t s 77 and t h e p e r c e n t a g e o f s h o r t and medium c a r n i t i n e e s t e r s i s n o t d i f f e r e n t f r o m t h a t o f f a s t e d r a t b i l e . T h i s e s t e r p r o f i l e m i g h t r e f l e c t t h e s h o r t f a s t i m p o s e d upon t h e p a t i e n t s b e f o r e b i l e c o l l e c t i o n . The e x i s t e n c e o f an e n t e r o h e p a t i c c i r c u l a t i o n f o r c a r n i t i n e h a s been d e m o n s t r a t e d i n t h e r a t ( G u d j o n s s o n e t a l . , 1985b) and t h e p r e s e n c e o f c a r n i t i n e i n t h e human b i l e has now been e s t a b l i s h e d . T h e r e i s a l s o some e v i d e n c e t h a t c a r n i t i n e m a l a b s o r p t i o n i n man i s r e l a t e d t o d e c r e a s e d serum c a r n i t i n e l e v e l s (Seccombe e t a l . , 1984; F r o h l i c h e t a l . , 1980; M i k h a i l and M a n s o u r , 1976; Bohmer e t a l . , 1 9 7 5 ) . T h e r e f o r e , m a l a b s o r p t i o n i n humans m i g h t l e a d t o d e c r e a s e d body c a r n i t i n e n o t o n l y due t o d e c r e a s e d a b s o r p t i o n o f d i e t a r y c a r n i t i n e b u t a l s o t o d e c r e a s e d i n t e s t i n a l r e a b s o r p t i o n o f c a r n i t i n e f r o m t h e b i l e . CHAPTER 8 Changes i n the C a r n i t i n e Content of Rat B i l e D u r i n g C o l l e c t i o n 78 T h r o u g h o u t t h i s s t u d y , t h e c o n c e n t r a t i o n o f a c y l c a r n i t i n e i n b i l e i n c r e a s e d d u r i n g r a t b i l e c o l l e c t i o n . The amount o f c a r n i t i n e f o u n d i n e a c h 15 m i n u t e c o l l e c t i o n p e r i o d a l s o i n c r e a s e d w i t h t i m e . T h r e e h y p o t h e s e s were p r o p o s e d t o a t t e m p t t o e x p l a i n t h i s phenomenon. E a c h was i n v e s t i g a t e d and t h e r e s u l t s a r e r e p o r t e d b e l o w . F i r s t , t h e e f f e c t o f t h e f l u i d l o s t v i a t h e b i l e d u r i n g t h e e x p e r i m e n t was a s s e s s e d . S e c o n d l y , t h e e f f e c t o f i n t e r r u p t i n g t h e b i l e f l o w t o c o l l e c t t h e s a m p l e s was i n v e s t i g a t e d . F i n a l l y , 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 on b i l e f l o w were d e t e r m i n e d . 8.1 The E f f e c t o f S a l i n e I n f u s 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 B i l e O v er t h e c o u r s e o f two h o u r s o f b i l e c o l l e c t i o n , a l m o s t 3 ml o f b i l e was c o l l e c t e d f r o m e a c h 350 g r a t . The e f f e c t o f t h i s l o s s on t h e c a r n i t i n e c o m p o s i t i o n o f t h e b i l e was i n v e s t i g a t e d by r e p l a c i n g t h e f l u i d l o s t w i t h s a l i n e i n f u s e d i n t o a j u g u l a r v e i n . The r e s u l t s o f t h i s e x p e r i m e n t a r e r e p o r t e d i n A p p e n d i x 2. D e s p i t e t h e i n f u s i o n o f s a l i n e , t h e c a r n i t i n e e s t e r c o n t e n t s t i l l i n c r e a s e d d u r i n g b i l e c o l l e c t i o n . T h e r e f o r e , t h e l o s s o f t o t a l body f l u i d f r o m t h e r a t s i n t h e f o r m o f b i l e had no e f f e c t 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 . 79 8.2 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 C o n t e n t o f t h e B i l e S t r a s b e r g and c o l l e a g u e s , i n a r e v i e w o f b i l i a r y s a m p l i n g ( 1 9 8 4 ) , s t r e s s e d t h e i m p o r t a n c e o f i n v e s t i g a t i n g t h e e f f e c t s o f t h e 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 s u b s t a n c e s m e a s u r e d i n b i l e . To d e t e r m i n e w h e t h e r o r n o t t h i s a f f e c t s t h e a c y l 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 , b i l e was r e c i r c u l a t e d i n a r a t a s d e s c r i b e d i n A p p e n d i x 3. A g a i n t h e amounts o f c a r n i t i n e e s t e r s i n c r e a s e d d u r i n g t h e c o u r s e o f t h e e x p e r i m e n t . I t c a n n o t be c o n c l u d e d , h o w e v e r , t h a t t h e 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 does n o t a l t e r 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 , b e c a u s e 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 was s t i l l r e d u c e d by 3 3 % i n t h i s e x p e r i m e n t . I t w o u l d be a d v i s a b l e , t h e r e f o r e , t o r e p e a t t h i s e x p e r i m e n t and c o l l e c t l e s s b i l e . 8.3 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 C o n t e n t o f B i l e I n most c a s e s , t h e c a r n i t i n e c o n t e n t o f b i l e u s u a l l y i n c r e a s e d d u r i n g t h e f i r s t 75 m i n u t e s o f b i l e c o l l e c t i o n and t h e n p l a t e a u e d . B i l e c o l l e c t i o n was u s u a l l y i n i t i a t e d 5-10 m i n u t e s a f t e r t h e r a t was a n a e s t h e t i z e d . I f t h e a n a e s t h e t i c was r e s p o n s i b l e f o r t h e i n c r e a s e d b i l e c a r n i t i n e d u r i n g t h e b i l e c o l l e c t i o n , one w o u l d e x p e c t t h a t t h e l e v e l s w o u l d have a l r e a d y p l a t e a u e d a f t e r a r a t had been a n a e s t h e t i z e d f o r 80 some t i m e . T h i s was t e s t e d by an e x p e r i m e n t d e s c r i b e d i n A p p e n d i x 4. The r e s u l t s s u g g e s t , h o w e v e r , t h a t t h e a n a e s t h e t i c does h a v e an e f f e c t 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 i n t h e b i l e b u t i s n o t r e s p o n s i b l e f o r t h e i n c r e a s e i n t h e c a r n i t i n e c o n t e n t d u r i n g t h e c o u r s e o f t h e e x p e r i m e n t . 8.4 Summary W i t h t h e e x c e p t i o n o f t h e s a l i n e i n f u s i o n e x p e r i m e n t , no c o n c l u s i o n s c a n be drawn f r o m t h i s w o r k , b e c a u s e o f t h e s m a l l number o f a n i m a l s u s e d . One c a n , h o w e v e r , use t h e r e s u l t s t o f o r m new h y p o t h e s e s and d i r e c t f u r t h e r s t u d i e s . F o r i n s t a n c e , i t seems a p p a r e n t t h a t t h e e f f e c t o f t h e a n a e s t h e t i c s h o u l d be more c l o s e l y i n v e s t i g a t e d . F u t u r e s t u d i e s s h o u l d compare t h e e f f e c t s o f d i f f e r e n t a n a e s t h e t i c s on t h e c a r n i t i n e c o m p o s i t i o n o f b i l e . The e f f e c t o f i n t e r r u p t i n g 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 s h o u l d a l s o be e x a m i n e d more t h o r o u g h l y t o d e t e r m i n e w h e t h e r a c y l c a r n i t i n e s t i l l i n c r e a s e s d u r i n g t h e e x p e r i m e n t w i t h r e m o v a l o f l e s s t h a n 3 3 % o f t h e b i l e . I f t h e i n c r e a s e d c a r n i t i n e i n t h e b i l e c a n n o t be a c c o u n t e d 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 o r t h e 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 , t h e e f f e c t o f s u r g e r y i t s e l f s h o u l d be i n v e s t i g a t e d . P o s s i b l y , p h y s i c a l m a n i p u l a t i o n o f t h e b i l e d u c t i s r e s p o n s i b l e . S t r a s b e r g and c o l l e a g u e s ( 1 9 8 4 ) r e p o r t t h a t w i t h b i l e d u c t c a n n u l a t i o n , t h e p r e s s u r e - f l o w r e l a t i o n s h i p s o f t h e b i l e a r e d i s t o r t e d . 81 Such e f f e c t s might account for 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 c o l l e c t i o n . CHAPTER 9 C o n c l u s i o n s 82 The c o n c l u s i o n s o f t h i s s t u d y a r e s u m m a r i z e d b e l o w . T h e i r i m p l i c a t i o n s and s u g g e s t i o n s f o r f u r t h e r r e s e a r c h a r e a l s o p r e s e n t e d . 9.1 Summary 9.1.1 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 The c o n c e n t r a t i o n o f c a r n i t i n e i n r a t b i l e was s i m i l a r t o t h a t o f r a t s e r u m . B i l e c o n t a i n e d more a c y l c a r n i t i n e and l o n g - c h a i n c a r n i t i n e e s t e r s t h a n o t h e r r a t t i s s u e s o r f l u i d s . I t was e s t i m a t e d t h a t 1% o f t h e t o t a l body p o o l o f c a r n i t i n e p a s s e s t h r o u g h t h e b i l e e v e r y 24 h o u r s . G u d j o n s s o n and c o l l e a g u e s ( 1 9 8 5 b ) r e p o r t e d s i m i l a r q u a n t i t i e s o f c a r n i t i n e i n r a t b i l e . C a r n i t i n e was a l s o f o u n d i n human b i l e - r i c h d u o d e n a l f l u i d . A l t h o u g h t h e q u a n t i t i e s v a r i e d c o n s i d e r a b l y , t h e d e g r e e o f c a r n i t i n e 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 and s i m i l a r t o t h a t o f r a t b i l e . The l o n g - c h a i n c a r n i t i n e e s t e r c o n t e n t was a l s o s i m i l a r t o t h a t o f r a t b i l e . 9.1.2 O r i g i n o f C a r n i t i n e i n t h e B i l e The o r i g i n o f t h e c a r n i t i n e i n b i l e was n o t d e t e r m i n e d c o n c l u s i v e l y . T h r e e e x p e r i m e n t s were p e r f o r m e d t o a t t e m p t t o d i s t i n g u i s h b e t w e e n h e p a t i c and e x t r a h e p a t i c c a r n i t i n e s o u r c e s . I n t h e f i r s t t wo, t h e r a t s were e i t h e r f a s t e d o r i n t u b a t e d w i t h 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 m e n t s w h i c h B i r e c t t h e amounts o f c a r n i t i n e and t y p e s o f c a r n i t i n e e s t e r s i n t h e l i v e r and s e r u m . The c h a n g e s i n t h e b i l e 83 c a r n i t i n e more c l o s e l y p a r a l l e l l e d t h o s e o c c u r r i n g i n t h e l i v e r t h a n t h e s e r um. I n t h e f i n a l e x p e r i m e n t , [ ] c a r n i t i n e was i n t r a v e n o u s l y i n j e c t e d i n t o r a t s d u r i n g b i l e c o l l e c t i o n . R e s u l t s s u g g e s t t h a t c a r n i t i n e i n t h e b i l e i s d e r i v e d f r o m b o t h n e w l y s y n t h e s i z e d o r s t o r e d h e p a t i c c a r n i t i n e and serum c a r n i t i n e w h i c h e n t e r s t h e l i v e r and becomes e s t e r i f i e d . 9.1.3 The T r a n s p o r t o f C a r n i t i n e i n t o B i l e Some i n f o r m a t i o n was o b t a i n e d c o n c e r n i n g t h e t r a n s p o r t a t i o n o f c a r n i t i n e i n t o t h e b i l e ( s e e A p p e n d i x 5 ) . R e s u l t s s u g g e s t e d t h a t t h e c a r n i t i n e c o n t e n t o f b i l e i s b o t h d e p e n d e n t on b i l e f o r m a t i o n and t h e c a r n i t i n e c o n t e n t o f t h e s e r u m . The i n c r e a s e d 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 i n t h e b i l e o f f a s t e d r a t s m i g h t be t h e r e s u l t o f w a t e r r e a b s o r p t i o n a f t e r b i l e p r o d u c t i o n . 9.1.4 P o s s i b l e F u n c t i o n s o f C a r n i t i n e i n t h e B i l e The f u n c t i o n o f c a r n i t i n e i n t h e b i l e i s n o t known. A l t h o u g h G u d j o n s s o n and c o l l e a g u e s have d e t e r m i n e d t h a t t h e r e i s an e n t e r o h e p a t i c c i r c u l a t i o n o f c a r n i t i n e , t h e e f f i c i e n c y o f t h i s i s unknown. I t i s p o s s i b l e , t h e r e f o r e , t h a t t h e b i l e c o u l d be an e x c r e t o r y r o u t e f o r c a r n i t i n e . D a t a p r e s e n t e d by Rebouche and c o l l e a g u e s ( 1 9 8 4 ) , who f o u n d l a b e l l e d c a r n i t i n e i n t h e f e c e s a f t e r i n t r a v e n o u s a d m i n i s t r a t i o n , s u p p o r t t h i s . As t h e e x c r e t i o n o f e n e r g y - r i c h l o n g - c h a i n c a r n i t i n e e s t e r s w o u l d be 84 i n e f f i c i e n t , h y d r o l y s i s of the c a r n i t i n e e s t e r might occur i n the i n t e s t i n a l lumen. Another p o s s i b l e f u n c t i o n of c a r n i t i n e i n the b i l e i s to r i d hepatocytes of excess organic a c i d s r e s u l t i n g from organic a c i d u r i a s or treatment with drugs such as v a l p r o i c a c i d . The involvement of c a r n i t i n e i n f a t t y a c i d a b s o r p t i o n i s another p o s s i b l e r o l e . Long-chain c a r n i t i n e e s t e r s i n the high c o n c e n t r a t i o n s found i n b i l e might a i d i n the e m u l s i f i c a t i o n of d i e t a r y f a t t y a c i d s or be i n v o l v e d i n the i n t r a c e l l u l a r t r a n s p o r t of f a t t y a c i d s w i t h i n the e n t e r o c y t e s . The long delay between uptake of c a r n i t i n e i n t o the mucosal c e l l s and i t s r e l e a s e i n t o the p o r t a l blood i n d i c a t e s that the c a r n i t i n e i s i n v o l v e d i n some process w i t h i n the mucosal c e l l s . 9.2 Suggestions f o r A d d i t i o n a l Work 9.2.1 The Absorption of C a r n i t i n e and i t s E s t e r s Only the a b s o r p t i o n of f r e e and a c e t y l c a r n i t i n e have been s t u d i e d . There i s no knowledge of the i n t e s t i n a l h a n d l i n g of long-chain a c y l c a r n i t i n e which are present i n l a r g e q u a n t i t i e s i n b i l e and the d i e t . A simple, and p h y s i o l o g i c a l way to study t h i s i n an a e s t h e t i z e d r a t s would be by mixing l a b e l l e d l o n g - c h a i n c a r n i t i n e e s t e r s with b i l e to measure i t s uptake from the r a t i n t e s t i n e while i n t e r r u p t i n g f u r t h e r b i l e flow. T h i s would avoid the problem of the w a t e r - i n s o l u b i l i t y of long-chain 85 a c y l c a r n i t i n e s and p r e v e n t p o s s i b l e c o m p l i c a t i o n s c a u s e d by t h e r e c i r c u l a t i o n o f t h e a b s o r b e d l a b e l i n t h e b i l e . 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 o f b i l e , r e p o r t e d by G u d j o n s s o n and c o l l e a g e s who s t u d i e d a s i n g l e r a t , s h o u l d be f u r t h e r q u a n t i t a t e d . The e f f i c i e n c y o f r e a b s o r p t i o n o f a l l t y p e s o f c a r n i t i n e e s t e r s s h o u l d be m e a s u r e d and t h e e f f e c t s o f d i e t a r y c o m p o n e n t s d e t e r m i n e d . 9.2.2 The I n v o l v e m e n t o f C a r n i t i n e i n F a t A b s o r p t i o n The e f f e c t s o f c a r n i t i n e on f a t a b s o r p t i o n were n o t s t u d i e d i n t h e s e e x p e r i m e n t s . F u t u r e e x p e r i m e n t s c o u l d i n v e s t i g a t e t h i s i n a n a e s t h e t i z e d r a t s by i n j e c t i n g l a b e l l e d f a t t y a c i d s i n t o t i e d o f f i n t e s t i n a l l o o p s . The e f f e c t o f c a r n i t i n e on f a t t y a c i d a b s o r p t i o n c o u l d t h e n be d e t e r m i n e d by m o n i t o r i n g t h e d i s a p p e a r a n c e o f t h e l a b e l f r o m t h e i n t e s t i n a l lumen o f r a t s i n t h e p r e s e n c e and a b s e n c e o f c a r n i t i n e . A n o t h e r r e a s o n f o r t h e i n t e r e s t i n f a t a b s o r p t i o n and c a r n i t i n e i s b a s e d on t h e o b s e r v a t i o n t h a t o r a l c a r n i t i n e may h ave u s e f u l s h o r t t e r m e f f e c t s i n a c u t e l y i l l p a t i e n t s w i t h c y s t i c f i b r o s i s ( B e n k e e t a l . , 1 9 8 4 ) . B e c a u s e f a t a b s o r p t i o n i s u s u a l l y i m p a i r e d i n c y s t i c f i b r o s i s , a p o s s i b l e r o l e f o r c a r n i t i n e was s u s p e c t e d . C a r n i t i n e was m e a s u r e d i n t h e serum o f s e v e r a l c h i l d r e n w i t h c y s t i c f i b r o s i s . The r e s u l t s a r e shown i n A p p e n d i x 6. A l t h o u g h t h e s e p a t i e n t s had serum c a r n i t i n e l e v e l s i n t h e n o r m a l r a n g e ( M i t c h e l l , 1 9 7 8 b ) , a d d i t i o n a l c a r n i t i n e m i g h t be 86 b e n e f i c i a l a t t h e l e v e l o f t h e i n t e s t i n e . T h i s s h o u l d be i n v e s t i g a t e d f u r t h e r . 9.2.3 The M a l a b s o r p t i o n o f B i l i a r y C a r n i t i n e I n t h i s e x p e r i m e n t , i t was c a l c u l a t e d t h a t 1% o f t h e t o t a l body c a r n i t i n e o f t h e r a t p a s s e d t h r o u g h t h e b i l e e a c h 24 h o u r s . G u d j o n s s o n and c o l l e a g u e s ( 1 9 8 5 ) r e p o r t e d s i m i l a r r e s u l t s . As c a r n i t i n e was a l s o f o u n d i n b i l e - r i c h human d u o d e n a l f l u i d , t h e p o s s i b i l i t y e x i s t s t h a t g e n e r a l m a l a b s o r p t i o n c o u l d r e s u l t i n c a r n i t i n e d e p l e t i o n i n humans. A l t h o u g h some s t u d i e s have r e p o r t e d r e d u c e d serum c a r n i t i n e l e v e l s i n p a t i e n t s w i t h v a r i o u s m a l a b s o r p t i o n s y n d r o m e s , o n l y one e x p e r i m e n t r e p o r t e d d e c r e a s e d c a r n i t i n e a b s o r p t i o n ( Seccombe e t a l . , 1 9 8 4 ) . I t seems s u i t a b l e t h a t f u r t h e r s t u d i e s s h o u l d be made o f p a t i e n t s w i t h v a r i o u s m a l a b s o r p t i v e d i s o r d e r s . F i r s t , i t s h o u l d be d e t e r m i n e d w h e t h e r o r n o t t h e s e p a t i e n t s have r e d u c e d serum o r t i s s u e c a r n i t i n e l e v e l s . T h e n , t h e p o s s i b i l i t i e s o f r e d u c e d c a r n i t i n e a b s o r p t i o n and r e d u c e d l y s i n e and m e t h i o n i n e a b s o r p t i o n s h o u l d be d i s t i n g u i s h e d . T h i s c o u l d be done by d e t e r m i n i n g w h e t h e r serum and t i s s u e c a r n i t i n e d e p l e t i o n p e r s i s t s a f t e r p a r e n t e r a l f e e d i n g w i t h f o r m u l a s l a c k i n g i n c a r n i t i n e . 9.2.4 The E f f e c t s o f H e p a t i c D y s f u n c t i o n on B i l i a r y C a r n i t i n e The e f f e c t o f l i v e r d i s e a s e on t h e c a r n i t i n e c o n t e n t o f 87 t h e b i l e s h o u l d a l s o be s t u d i e d . The serum c a r n i t i n e c o n c e n t r a t i o n s o f p a t i e n t s w i t h a l c o h o l i c c i r r h o s i s were s i g n i f i c a n t l y l o w e r t h a n t h o s e o f n o r m a l c o n t r o l s (Rudman e t a l . , 1 9 8 0 ) . T h i s c o u l d be due t o a r e d u c e d h e p a t i c s y n t h e s i s o f c a r n i t i n e o r t o g e n e r a l m a l a b s o r p t i o n . A l s o , t h e p o s s i b i l i t y o f u n c o n t r o l l e d r e l e a s e o f c a r n i t i n e i n t o t h e b i l e s h o u l d a l s o be i n v e s t i g a t e d . 9.2.5 B i l i a r y C a r n i t i n e i n Body P o o l S t u d i e s To u n d e r s t a n d c o m p l e t e l y t h e m e t a b o l i s m o f c a r n i t i n e i n t h e r a t , body p o o l s t u d i e s have been a t t e m p t e d u s i n g l a b e l l e d c a r n i t i n e . None, h o w e v e r , has c o n s i d e r e d t h e p o s s i b l e e x c r e t i o n o f c a r n i t i n e t h r o u g h t h e b i l e . T h e s e s t u d i e s s h o u l d be r e p e a t e d t a k i n g i n t o a c c o u n t t h e b i l e . 9.2.6 C a r n i t i n e A b s o r p t i o n i n 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 S y n dromes I n c a s e s 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 , t h e s i t e o f t h e d i s o r d e r i s o f t e n n o t known and o r a l c a r n i t i n e t h e r a p y i s n o t a l w a y s s u c c e s s f u l a t a l l e v i a t i n g t h e symptoms. I t has been h y p o t h e s i z e d t h a t i n t e s t i n a l a b s o r p t i o n i s a p o s s i b l e s i t e o f d e f e c t ( G i l b e r t , 1985; E n g e l and R e b o u c h e , 1 9 8 4 ) . I n t h e f u t u r e , t h i s s h o u l d be i n v e s t i g a t e d f u r t h e r by m o n i t o r i n g t h e e f f e c t o f a b o l u s o r a l d ose o f c a r n i t i n e on serum c a r n i t i n e l e v e l s i n p a t i e n t s and c o n t r o l s . 88 9 . 3 I m p l i c a t i o n s The d i s c o v e r y of c a r n i t i n e i n r a t b i l e c h a l l e n g e s the common assumption that only d i e t a r y c a r n i t i n e i s present i n the i n t e s t i n a l t r a c t . I t a l s o d i s p u t e s the theory that u r i n e i s the s i n g l e route f o r c a r n i t i n e e x c r e t i o n . C a r n i t i n e r e s e a r c h e r s must now c o n s i d e r the c a r n i t i n e content of the b i l e i n i n t e s t i n a l and e x c r e t i o n s t u d i e s . Another i m p l i c a t i o n of t h i s work i s f o r p a t i e n t s with malabsorption syndromes. I t i s p o s s i b l e that these p a t i e n t s have compromised c a r n i t i n e s t o r e s not only due to l a c k of a b s o r p t i o n of d i e t a r y c a r n i t i n e and i t s amino a c i d p r e c u r s o r s but a l s o from l a c k of r e a b s o r p t i o n of c a r n i t i n e from the b i l e . U n t i l i t i s c o n c l u s i v e l y shown whether or not t h i s b i l e l o s s i s s i g n i f i c a n t to the w e l l being of the p a t i e n t , serum and t i s s u e c a r n i t i n e l e v e l s should be measured i n p a t i e n t s with c h r o n i c malabsorptive syndromes. I f reduced c a r n i t i n e c o n c e n t r a t i o n s do e x i s t and symptoms of c a r n i t i n e d e p l e t i o n r e s u l t , i t might be a d v i s a b l e to supplement these i n d i v i d u a l s with intravenous or l a r g e doses of o r a l c a r n i t i n e . R e f e r e n c e s 89 A a s , M. and B r e m e r , J . ( 1 9 6 8 ) S h o r t - c h a i n f a t t y a c i d a c t i v a t i o n i n r a t l i v e r . A new a s s a y p r o c e d u r e f o r t h e enzymes and 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 l o c a l i z a t i o n . B i o c h i m . B i o p h y s . A c t a 164, 157-166. A a s , M. and Daae, L.N.W. ( 1 9 7 1 ) F a t t y a c i d a c t i v a t i o n and a c y l t r a n s f e r i n o r g a n s f r o m 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 . B i o c h i m . B i o p h y s . A c t a 239, 2 0 8 - 2 1 6 . B a c h , A.C., S c h i r a r d i n , H., S i h r , M.-0. and S t o r c k , D. ( 1 9 8 3 ) F r e e and t o t a l c a r n i t i n e i n human serum a f t e r o r a l i n g e s t i o n o f L - c a r n i t i n e . D i a b e t e M e t a b . 9, 121-124. B a h l , J . , N a v i n , T., M a n i o n , A. and B r e s s l e r , R. ( 1 9 8 1 ) 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 t h e 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 . R e s . 48, 3 7 8 - 3 8 5 . B a i l e y , D.G., P a u l , H. and J o h n s o n , G.E. ( 1 9 7 5 ) P e n t o b a r b i t a l i n h i b i t s t h e b i l i a r y e x c r e t i o n o f o r g a n i c a c i d s : a s t u d y o f s u c c i n y l s u l f a t h i a z o l e i n t h e r a t . Can. J . P h y s i o l . Pharm. 5 3 , 4 7 0 - 4 7 4 . B e n k e , P . J . , McKay, B. and F o r a d a d a , J . ( 1 9 8 4 ) L - C a r n i t i n e f o r c y s t i c f i b r o s i s . P e d . R e s . 18, 219A. 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 . and T o l b e r t , N.E. ( 1 9 8 1 ) C a r n i t i n e a c y l t r a n s f e r a s e s i n r a t l i v e r p e r o x i x o m e s . A r c h . B i o c h e m . B i o p h y s . 2 1 1 , 5 9 9 - 6 0 4 . Bohmer, T., Norum, K.R. and B r e m e r , J . ( 1 9 6 6 ) The r e l a t i v e a mounts 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 . B i o c h i m . B i o p h y s . A c t a 125, 2 4 4 - 2 5 1 . Bohmer, T. ( 1 9 6 7 ) T i s s u e l e v e l s o f a c t i v a t e d f a t t y a c i d s ( a c y l c a r n i t i n e s ) 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 m e t a b o l i s m . B i o c h i m . B i o p h y s . A c t a 144, 2 5 9 - 2 7 0 . Bohmer, T. and B r e m e r , J . ( 1 9 6 8 ) P r o p i o n y l c a r n i t i n e p h y s i o l o g i c a l v a r i a t i o n s i n v i v o . B i o c h e m . B i o p h y s . A c t a 152, 5 5 9 - 5 6 7 . Bohmer, T. ( 1 9 7 4 ) C o n v e r s i o n o f b u t y r o b e t a i n e t o c a r n i t i n e i n t h e r a t i n v i v o . B i o c h i m . B i o p h y s . A c t a 3 4 3 , 5 5 1 - 5 5 4 . Bohmer, T., R y d n i n g , A. and S o l b e r g , H.E. ( 1 9 7 4 ) C a r n i t i n e l e v e l s i n human serum i n h e a l t h and d i s e a s e . C l i n . C h i m . A c t a 5 7 , 5 5 - 6 1 . 90 Bohmer, T., E i k l i d , K. and J o n s e n , J . ( 1 9 7 7 ) C a r n i t i n e u p t a k e i n t o human h e a r t c e l l s i n c u l t u r e . B i o c h i m . B i o p h y s . A c t a 4 6 5 , 6 2 7 - 6 3 3 . B r a s s , E.P. and H o p p e l , C.L. ( 1 9 7 8 ) C a r n i t i n e m e t a b o l i s m i n t h e f a s t i n g r a t . J . B i o l . Chem. 253, 2 6 8 8 - 2 6 9 3 . B r a s s , E.P. and H o p p e l , C.L. ( 1 9 8 1 ) I n v i v o s t u d i e s o f 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 ; p r o b l e m s 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 . B i o c h e m . 110, 7 7 - 8 1 . B r e m e r , J . ( 1 9 8 1 ) 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 . B i o c h i m . B i o p h y s . A c t a 6 6 5 , 6 2 8 - 6 3 1 . B r e m e r , J . ( 1 9 8 3 ) C a r n i t i n e - M e t a b o l i s m and f u n c t i o n s . P h y s i o l . Rev. 6 3 , 1420-1480. B r o o k s , D.E and M c i n t o s h , J.E.A. ( 1 9 7 5 ) T u r n o v e r o f c a r n i t i n e by r a t t i s s u e s . B i o c h e m . J . 148, 4 3 9 - 4 4 5 . B r o o k s , D.E. ( 1 9 8 0 ) 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 s p e r m a t o z o a . I n : C a r n i t i n e 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 by 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 Y o r k , San F r a n c i s c o , pp. 219- 2 3 6 . C a r t e r , H.E., B h a t t a c h a r y a , P.K., Weidman, K.R. and F r a e n k e l , G. ( 1 9 5 2 ) 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 . A r c h . B i o c h e m . B i o p h y s . 38, 4 0 5 - 4 1 6 . C e d e r b l a d , G. and L i n d s t e d t , S. ( 1 9 7 2 ) A method f o r t h e d e t e r m i n a t i o n o f c a r n i t i n e i n t h e p i c o m o l e r a n g e . C l i n . C h i m. A c t a 3 7 , 2 3 5 - 2 4 3 . C e d e r b l a d , G. and L i n d s t e d t , S. ( 1 9 7 6 ) M e t a b o l i s m o f l a b e l l e d c a r n i t i n e i n t h e r a t . A r c h . B i o c h e m . B i o p h y s . 175, 173-180. C h r i s t i a n s e n , R.Z. and B r e m e r , J . ( 1 9 7 6 ) A c t i v e t r a n s p o r t o f 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 l i v e r c e l l s . B i o c h i m . B i o p h y s . A c t a 4 48, 5 6 2 - 5 7 7 . C o o p e r , B., E a k i n s , M.N. and S l a t e r , T.F. ( 1 9 7 6 ) 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 e n z y m i c c o m p o s i t i o n o f t h e r a t b i l e . B i o c h e m . Pharm. 25, 1711-1718. E n g e l , A.G. and R e b o u c h e , C . J . ( 1 9 8 4 ) C a r n i t i n e m e t a b o l i s m 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, S3 8 - S 4 3 . 91 E n g l a r d , S. ( 1 9 7 9 ) 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 t o c a r n i t i n e i n human and monkey t i s s u e s . FEBS L e t t . 1 102, 297-300. F r a e n k e l , G. ( 1 9 5 1 ) E f f e c t and d i s t r i b u t i o n o f v i t a m i n B t . A r c h . B i o c h e m . B i o p h y s . 34, 4 5 7 - 4 6 3 . F r e n c h , T . J . , Goode, A.W., H o l n e s s , M.J. and S u g d e n , M.C. ( 1 9 8 5 ) 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 c a r b o h y d r a t e t o a c y l c a r n i t i n e f o r m a t i o n . B i o c h e m . I n t . 11 , 2 6 5 - 2 7 2 . F r i e d m a n , S. and F r a e n k e l , G. ( 1 9 5 5 ) R e v e r s i b l e e n z y m i c a c e t y l a t i o n o f c a r n i t i n e . A r c h . B i o c h e m . B i o p h y s . 59, 4 9 1 - 5 0 1 . F r i e d m a n , S. ( 1 9 5 8 ) D e t e r m i n a t i o n o f c a r n i t i n e i n b i o l o g i c a l m a t e r i a l s . A r c h . B i o c h e m . B i o p h y s . 7 5 , 24. F r i t z , I . B . ( 1 9 5 5 ) 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 t h e 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 and h o m o g e n a t e s . A c t a P h y s i o l . S c a n d . 34, 3 6 7 - 3 8 5 . F r i t z , I . B . ( 1 9 6 8 ) 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 o x i d a t i o n . 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 C o n t r o l o f F a t t y A c i d M e t a b o l i s m . 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. 3 9 - 6 3 . F r o h l i c h , J . , Seccombe, D.W., Hahn, P., Dodek, P. and H y n i e , I . ( 1 9 7 8 ) E f f e c t o f f a s t i n g on f r e e and e s t e r i f i e d c a r n i t i n e l e v e l s i n human serum and u r i n e : c o r r e l a t i o n o f serum l e v e l s o f f r e e f a t t y a c i d s and B - h y d r o x y b u t y r a t e . M e t a b o l i s m 27, 5 5 5 - 5 6 1 . F r o h l i c h , J . , Hahn, P. and C l e a t o r , I . ( 1 9 8 0 ) Changes i n p l a s m a 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 b y p a s s . L a n c e t 1, 1085. F r o s t , S.C. and W e l l s , M.A. ( 1 9 8 2 ) E f f e c t o f i n h i b i t i o n o f 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 c a r n i t i n e c o n t e n t . B i o c h e m . J . 204, 8 6 1 - 8 6 3 . G i l b e r t , E.F. ( 1 9 8 5 ) C a r n i t i n e d e f i c i e n c y . P a t h o l o g y 17, 161-169. G r o s s , C . J . and H e n d e r s o n , L.M. ( 1 9 8 3 ) C a r n i t i n e a b s o r p t i o n by t h e r a t s m a l l i n t e s t i n e . F e d . P r o c . 4 2 , 1320. G r o s s , C . J . and H e n d e r s o n , L.M. ( 1 9 8 4 ) A b s o r p t i o n o f D- and L - c a r n i t i n e by t h e i n t e s t i n e and k i d n e y t u b u l e i n t h e r a t . B i o c h i m . B i o p h y s . A c t a 7 7 2 , 2 0 9 - 2 1 9 . 92 G r o s s , C . J . , H e n d e r s o n , L.M. and S a v a i a n o , D.A. ( 1 9 8 5 ) A b s o r p t i o n o f L and D c a r n i t i n e by i s o l a t e d g u i n e a p i g s e n t e r o c y t e s . F e d . P r o c . 44, 7 6 3 . 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. ( 1 9 8 4 ) I n v i v o s t u d i e s o f s m a l l i n t e s t i n a l c a r n i n t i n e a b s o r p t i o n . G a s t r o e n t e r o l o g y 8 6 , 1 1 0 1 . G u d j o n s s o n , H., L i , B U.K., Shug, A. and O l s e n , W.A. ( 1 9 8 5 a ) I n v i v o s t u d i e s o f i n t e s t i n a l c a r n i t i n e a b s o r p t o n i n r a t s . G a s t r o e n t e r o l o g y 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. ( 1 9 8 5 b ) S t u d i e s o f c a r n i t i n e m e t a b o l i s m i n r e l a t i o n t o i n t e s t i n a l a b s o r p t i o n . Am. J . P h y s i o l . 248, G313-G319. G u l e w i t s c h , W. and K r i m b e r g , R. ( 1 9 0 5 ) Z u r k e n n t n i s d e r e x t r a k t i v s t o f f e d e r m u s k e l n , 2 m i t t e i l u n g . U e b e r das C a r n i t i n . Z t s c h r f . P h y s i o l . Chem., S t r a s s b . 4 5 , 3 2 6 - 3 3 0 . Gumpen, S.A. and Norum, K.R. ( 1 9 7 3 ) The r e l a t i v e amount o f l o n g - c h a i n 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 f r o m r a t s f e d on r a p e s e e d o i l . B i o c h i m . B i o p h y s . A c t a 3 1 6 , 4 8 - 5 5 . Hahn, P., C h a n e z , C. and H a m i l t o n , J . ( 1 9 8 5 ) C a r n i t i n e and 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 o f s u c k l i n g r a t s . B i o l . N e o n a t e 4 8 , 77-84. H a m i l t o n , J.W., L i , B U.K., Shug, A. and O l s e n , W.A. ( 1 9 8 3 ) 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. G a s t r o e n t e r o l o g y 84, 1180. H a n s o n , P . J . and 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 a c i d C o A - t r a n s f e r a s e , D - 3 - h y d r o x y b u t y r a t e 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 o f t h e r a t . B i o c h e m . J . 20, 3 4 9 - 3 5 5 . H a r d i s o n , W.G.M. and A p t e r , J .T. ( 1 9 7 2 ) M i c e l l a r t h e o r y o f b i l i a r y c h o l e s t e r o l e x c r e t i o n . Am. J . P h y s i o l . 222, 6 1 - 6 7 . H o p p e l , C.L. and G e n u t h , S. ( 1 9 7 6 ) F a s t i n g a l t e r s c a r n i t i n e m e t a b o l i s m . C l i n . R e s . 24, 580A. H u t h , P. and Shug, A. ( 1 9 8 0 ) P r o p e r t i e s o f c a r n i t i n e t r a n s p o r t i n r a t k i d n e y c o r t e x s l i c e s . B i o c h i m . B i o p h y s . A c t a 6 0 2 , 621-634. 93 H u t h , P., S c h m i d t , M., H a l l , P., F a r i e l l o , R. and Shug, A. ( 1 9 8 1 ) The 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 c e r e b r a l c o r t e x . J . Neurochem. 36, 7 1 5 - 7 2 3 . I n n i s , S.M. ( 1 9 8 5 ) 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 c o m p o s i t i o n f o l l o w i n g t o t a l p a r e n t e r a l n u t r i t i o n w i t h and 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 . N u t r . 4, 1283-1288. I v y , A.C., O l d b e r g , E., K l o s t e r , G. and L u e t h , H.C. ( 1 9 2 8 ) 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 e v a c u a t i o n . Am. J . P h y s i o l . 8 5 , 3 8 1 - 3 8 3 . James, M., B r o o k s , D. and S n o s w e l l , A. ( 1 9 8 1 ) K i n e t i c s o f 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 . , Rothman, S., K l a s s e n , G. and Mamer, O.A. ( 1 9 7 5 ) The syndrome 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 . N e u r o l o g y 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., Van A s s e l t , W., and Vonk, R . J . ( 1 9 8 5 ) A c u t e e f f e c t s o f p e n t o b a r b i t a l - a n a e s t h e s i a on b i l e s e c r e t i o n . B i o c h e m . P h a r m a c o l . 34, 1731-1736. K u t s c h e r , F. ( 1 9 0 5 ) U e b e r L i e b i g ' s f l e i s c h e x t r a k t . Z t s c h r , f . U n t e r s u c h . d . N a h r u n g s - u . G e n u s s m i t t e l , B e r l i n . 10, 5 2 8 - 5 3 7 . L a R u s s o , N.F. ( 1 9 8 4 ) P r o t e i n s i n b i l e : how t h e y g e t t h e r e and what t h e y do. Am. J . P h y s i o l . 247, G199-G205. L i , B U.K., H a m i l t o n , J.W., Shaw, R.A., O l s e n , W.A., Shug, A.L. ( 1 9 8 3 ) C a r n i t i n e t r a n s p o r t i n r a t j e j u n a l m i c r o v i l l o u s membrane v e s i c l e s . G a s t r o e n t e r o l o g y 8 4 , 1230. L i , B U.K., G r a v e s , L . L . and O l s e n , W.A. ( 1 9 8 5 ) U n i d i r e c t i o n a l c a r n i t i n e f l u x e s i n i s o l a t e d r a t j e j u n u m : e v i d e n c e o f d i f f u s i o n . F e d . P r o c . 44, 763. L i n d s t e d t , G. and L i n d s t e d t , S. ( 1 9 6 5 ) S t u d i e s on t h e b i o s y n t h e s i s o f c a r n i t i n e . J . B i o l . Chem. 240, 3 1 6 - 3 2 1 . 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. Haymond, M., H a n d w e r g e r , B. and R i z z a , R. ( 1 9 8 4 ) M e c h a n i s m o f 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 w i t h i n s u l i n r e s i s t a n c e t o a n t i i n s u l i n r e c e p t o r a n t i b o d i e s . J . C l i n . E n d o c r i n o l . M e t a b . 59, 658-664. 94 M a r q u i s , N.R. and F r i t z , I . B . ( 1 9 6 4 ) E n z y m o l o g i c a l d e t e r m i n a t i o n o f f r e e c a r n i t i n e c o n c e n t r a t i o n s i n r a t t i s s u e s . J . L i p i d R e s . 5, 184-187. M c G a r r y , J.D. and F o s t e r , D.W. ( 1 9 7 6 ) An i m p r o v e d and s i m p l i f i e d r a d i o i s o t o p i c a s s a y f o r t h e d e t e r m i n a t i o n o f 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 R e s . 17, 2 7 7 - 2 8 1 . M i k h a i l , M.M. and M a n s o u r , M.M. ( 1 9 7 6 ) The r e l a t i o n s h i p b e t w e e n 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 s t a t u s o f p a t i e n t s w i t h s c h i s t o s o m i a s i s . C l i n . Chim. A c t a 7 1 , 207-214. M i n u k , G.Y. and S a r j e a n t , E . J . ( 1 9 8 4 ) G a m m a a m i n o b u t y r i c 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, 193-197. M i t c h e l l , M.E. ( 1 9 7 8 a ) 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 . 3 1 , 2 9 3 - 3 0 6 . M i t c h e l l , M.E. ( 1 9 7 8 b ) 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 I . V a l u e s o f 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 . 3 1 , 4 8 1 - 4 9 1 . Norum, K.R. ( 1 9 6 5 ) A c t i v a t i o n o f p a l m i t y l - C o A : c a r n i t i n e p a l m i t y l t r a n s f e r a s e i n t h e l i v e r s f r o m f a s t e d , f a t - f e d , o r d i a b e t i c r a t s . B i o c h i m . B i o p h y s . A c t a 9 8 , 6 5 2 - 6 5 4 . O h t a n i , Y., Endo, F. and M a t s u d a , I . ( 1 9 8 2 ) C a r n i t i n e d e f i c i e n c y and hyperammonemia a s s o c i a t e d w i t h v a l p r o i c a c i d t h e r a p y . J . P e d . 101, 7 8 2 - 7 8 5 . Osmudsen, H. and N e a t , C E . ( 1 9 7 9 ) R e g u l a t i o n o f p e r o x i s o m a l f a t t y a c i d o x i d a t i o n . FEBS L e t t . 107, 8 1 - 8 4 . P a c e , J.A., Wannemacher, R.W. and N e u f e l d , H.A. ( 1 9 7 8 ) I m p r o v e d 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, 3 2 - 3 5 . P a i k , W.K. and K i m , S. ( 1 9 7 5 ) P r o t e i n m e t h y l a t i o n : 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 . 4 2 , 2 2 7 - 2 8 6 . P a n d e , S.V. and P a r v i n , R. ( 1 9 7 6 ) 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 a c y l c a r n i t i n e t r a n s l o c a s e s y s t e m o f h e a r t m i t o c h o n d r i a . J . B i o l . Chem. 2 5 1 , 6 6 8 3 - 6 6 9 1 . 95 P a n d e , S.V. ( 1 9 8 1 ) 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 b e c a u s e o f m i g r a t i o n t o a i r / w a t e r i n t e r p h a s e . B i o c h i m . B i o p h y s . A c t a 6 6 3 , 66 9 - 6 7 3 . P a n d e , S.V. and C a r a m a n c i o n , M.N. ( 1 9 8 1 ) A s i m p l e r a d i o i s o t o p i c a s s a y o f 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 a t p i c o m o l a r l e v e l s . A n a l . B i o c h e m . 112, 3 0 - 3 8 . P a r v i n , R. and P a n d e , S.V. ( 1 9 7 7 ) M i c r o d e t e r m i n 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 a c e t y l t r a n s f e r a s e a c t i v i t y . A n a l . B i o c h e m . 79, 190-210. P e a r s o n , D.J. and T u b b s , P.K. ( 1 9 6 7 ) C a r n i t i n e and d e r i v a t i v e s i n r a t t i s s u e s . B i o c h e m . J . 105, 9 5 3 - 9 6 3 . R e b o u c h e , C. ( 1 9 7 7 ) C a r n i t i n e movement a c r o s s m u s c l e c e l l 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 . B i o c h i m . B i o p h y s . A c t a 4 7 1 , 145-155. R e b o u c h e , C . J . and E n g e l , A.G. ( 1 9 8 3 ) K i n e t i c c o m p a r t m e n t a l a n a l y s i s o f c a r n i t i n e m e t a b o l i s m i n t h e d o g . A r c h . B i o c h e m . B i o p h y s . 220, 60- 7 0 . R e b o u c h e , C . J . and Edmonson, P.F. ( 1 9 8 4 ) M e t a b o l i s m o f o r a l c a r n i t i n e i n t h e r a t . F e d . P r o c . 4 3 , 617. R e b o u c h e , C . J . and E n g e l , A.G. ( 1 9 8 4 ) K i n e t i c c o m p a r t m e n t a l a n a l y s i s o f c a r n i t i n e m e t a b o l i s m i n t h e 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 a l t e r a t i o n s i n t i s s u e c a r n i t i n e t r a n s p o r t . J . C l i n . I n v e s t . 7 3 , 8 5 7 - 8 6 7 . R e b o u c h e , C . J . , Mack, D.L. and Edmonson, P.F. ( 1 9 8 4 ) L - c a r n i t i n e d i s s i m i l a t i o n i n t h e g a s t r o i n t e s t i n a l t r a c t o f t h e r a t . B i o c h e m i s t r y 2 3 , 6 4 2 2 - 6 4 2 6 . Rudman, D., A n s l e y , J.D. and S e w e l l , C.W. ( 1 9 8 0 ) C a r n i t i n e d e f i c i e n c y i n c i r r h o s i s . I n : C a r n i t i n e 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 by 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 Y o r k , San F r a n c i s c o , pp. 3 0 7 - 3 2 0 . S a c h a n , D.S. and R u a r k , R.A. ( 1 9 8 5 ) D i s t r i b u t i o n o f 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 . N u t r . 115, 8 6 5 - 8 7 1 . S a n d o r , A., K i s p a l , G., M e l e g h , B. and A l k o n y i , I . ( 1 9 8 5 ) R e l e a s e o f c a r n i t i n e f r o m t h e p e r f u s e d r a t l i v e r . B i o c h i m . B i o p h y s . A c t a 8 3 5 , 8 3 - 9 1 . 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. and S i l i p r a n d i , N. ( 1 9 8 2 ) On t r a n s p o r t mechanisms o f 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 r a t h e a r t s l i c e s . I t a l . J . B i o c h e m . 3 1 , 261-268. S c h a n k e r , L.S. and S o l o m o n , H.M. ( 1 9 6 3 ) A c t i v e t r a n s p o r t o f q u a t e r n a r y ammonium compounds i n t o b i l e . Am. J . P h y s i o l . 204, 82 9 - 8 3 2 . S c h a r s c h m i d t , B.F. and S c h m i d , R. ( 1 9 7 8 ) The m i c e l l a r s i n k . J . C l i n . I n v e s t . 6 2 , 1 1 2 2 - 1 1 3 1 . Seccombe, D.W., Hahn, P. and Novak, M. ( 1 9 7 8 ) The e f f e c t o f d i e t and 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 and e s t e r i f i e d c a r n i t i n e i n t h e r a t . B i o c h i m . B i o p h y s . A c t a 5 2 8 , 4 8 3 - 4 8 9 . Seccombe, D.W. ( 1 9 8 1 ) 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 d e v e l o p m e n t . Ph.D. D i s s e r t a t i o n , U n i v e r s i t y o f B r i t i s h C o l u m b i a , V a n c o u v e r , C a n a d a . Seccombe, D., B u r g e t , D., F r o h l i c h , J . , Hahn, P., C l e a t o r , I . and G o u r l a y , R.H. ( 1 9 8 4 ) O r a l 1 - c a r n i t i n e 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 b y - p a s s s u r g e r y . I n t . J . Obes. 8, 4 2 7 - 4 3 3 . S e i m , H. and D a r g e l , R. ( 1 9 7 8 ) I n d u k t i o n m i t o c h o n d r i a l e r v o l u m e n a n d e r u n g e n d u r s h homologe o - a c y l c a r n i t i n - i s o m e r e . A c t a B i o l . Med. G e r . 37, 73- 8 2 . Shaw, R.D., Shug, A.L. and O l s e n , W.A. ( 1 9 8 2 ) S t u d i e s o f 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 . G a s t r o e n t e r o l o g y 8 2 , 1177. Shaw, R.D., L i B U.K., H a m i l t o n , J.W., Shug , A.L. and O l s e n , W.A. ( 1 9 8 3 ) C a r n i t i n e t r a n s p o r t i n t h e r a t s m a l l i n t e s t i n e . Am. J . P h y s i o l . 2 4 5, G376-G381. S t r a s b e r g , S.M., H a r v e y , P.R.C. and G a l l i n g e r , S. ( 1 9 8 4 ) Anatomy, v i s u a l i z a t i o n and s a m p l i n g o f t h e b i l i a r y t r e e i n a n i m a l s and man. H e p a t o l o g y 4, 1S-3S. 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. and S c h i r o , J.A. ( 1 9 8 4 ) 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 c a r n i t i n e d e f i c i e n c y . N e u r o l o g y 34, 1 1 2 8 - 1 1 2 9 . T o m i t a , M. and S e n d j u , Y. ( 1 9 2 7 ) Uber d i e o x y a m i n o v e r b i n d u n g e n , w e l c h e d i e B i u r e t r e a k t i o n z e i g e n . I I I . S p a l t u n g d e r g a m m a - a m i n o - b e t a - o x y b u t t e r s a u r e i n d i e o p t i s c h - a k - t i v e n k o m p o n e n t e n . H o p p e - S e y e r ' s Z. P h y s i o l . Chem. 169, 263 - 2 7 7 . 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. and Ho, W. ( 1 9 7 8 ) 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 h y p o g l y c e m i c a g e n t w h i c h i n h i b i t s l o n g - c h a i n f a t t y a c i d o x i d a t i o n . F e d . P r o c . 3 7, 1308. T u t w e i l e r , G.F. and D e l l e v i g n e , P. ( 1 9 7 9 ) A c t i o n o f t h e 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, 2 9 3 5 - 2 9 4 1 . W a y n f o r t h , H.B. ( 1 9 8 0 ) 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 i n t h e R a t . A c a d e m i c P r e s s , L o n d o n . W h e e l e r , H.O. ( 1 9 7 1 ) C o n c e n t r a t i o n f u n c t i o n o f t h e g a l l b l a d d e r . Am. J . Med. 5 1 , 5 8 8 - 5 9 5 . W i l l n e r , J . , G i n s b u r g , S. and D i M a u r o , S. ( 1 9 7 8 ) A c t i v e t r a n s p o r t o f c a r n i t i n e i n t o s k e l e t a l m u s c l e . N e u r o l o g y 28, 721-724. W i n d m u e l l e r , H.G. and S p a e t h , A.E. ( 1 9 8 0 ) R e s p i r a t o r y 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 o f f e d r a t s . J . B i o l . Chem. 2 5 5 , 107-112. Y a l k o w s k y , S.H. and Z o g r a f i , G. ( 1 9 7 0 ) P o t e n t i o m e t r i c 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 . 5 9 , 7 9 8 - 8 0 2 . Yue, K.T.N, and F r i t z , I . B . ( 1 9 6 2 ) F a t e o f 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 r a t s . Am. J . P h y s i o l . 2 02, 122-128. APPENDIX 1 The R e c o v e r y o f I n t r a v e n o u s l y I n j e c t e d [ i ^ C ] C a r n i t i n e i n R a t B i l e 98 The o b j e c t i v e of t h i s experiment was to determine the recovery of l a b e l l e d c a r n i t i n e i n j e c t e d i n t o the j u g u l a r v e i n i n r a t b i l e . A l . l Methods Al.1.1 P u r i t y of the Isotope The p u r i t y of the [ 14-C ] c a r n i t i n e was v e r i f i e d by using the c e l l u l o s e TLC system recommended by Amersham. The mobile phase was composed of n - b u t a n o l : a c e t i c acid:water(12:3:5, v / v / v ) . A l . l . 2 S u r g i c a l Procedure The b i l e duct and a j u g u l a r v e i n were cannulated as d e s c r i b e d p r e v i o u s l y . One ml of s a l i n e c o n t a i n i n g 0.05 uCi of [ l - ^ C J c a r n i t i n e (2.5 mCi/nmol) was slowly i n j e c t e d i n t o the j u g u l a r v e i n . Immediately a f t e r the l a b e l l e d c a r n i t i n e was i n j e c t e d , b i l e was c o l l e c t e d . The c o l l e c t i o n tube was changed every 5 minutes f o r 60 minutes so that the r a t e of recovery of the l a b e l i n the b i l e could be measured. Subsequent b i l e samples were c o l l e c t e d at 10 minute i n t e r v a l s u n t i l 2 hours had e l a p s e d . At the t e r m i n a t i o n of the experiment, a blood sample was taken from the t a i l v e i n and serum was prepared by c e n t r i f u g a t i o n as d e s c r i b e d e a r l i e r . A l . l . 3 A n a l y s i s of Samples B i l e and serum samples (50 u l each) were placed i n d i s p o s a b l e mini-counting v i a l s with 6 ml of aqueous s c i n t i l l a t i o n f l u i d . The samples were then mixed and 99 c o u n t e d on an LS9000 S c i n t i l l a t i o n c o u n t e r . I n o r d e r t o q u a n t i t a t e t h e l a b e l l e d l o n g - c h a i n c a r n i t i n e e s t e r s i n t h e b i l e , t h e l i p i d p h a s e f r o m t h r e e s a m p l e s was m i x e d w i t h s c i n t i l l a t i o n f l u i d and c o u n t e d . The s p e c i f i c a c t i v i t i e s i n t h e b i l e and serum were d e t e r m i n e d by a s s a y i n g t h e s a m p l e s f o r c a r n i t i n e . A1.2 R e s u l t s The amounts o f [ ] c a r n i t i n e r e c o v e r e d i n t h e b i l e s a m p l e s c o l l e c t e d a r e p l o t t e d f o r e a c h r a t s e p a r a t e l y i n F i g u r e 1 1 . A l s o shown a r e t h e amounts o f l a b e l i n t h e serum c o l l e c t e d a t t h e t e r m i n a t i o n o f t h e e x p e r i m e n t s . The l a b e l l e d c a r n i t i n e r e c o v e r e d i n t h e b i l e i n c r e a s e d t o a maximum a p p r o x i m a t e l y 30 m i n u t e s a f t e r i n j e c t i o n i n a l l c a s e s . 5 0 % , 4 7 % and 50% o f t h e l a b e l l e d c a r n i t i n e was p r e s e n t i n t h e l o n g - c h a i n f o r m i n t h e t h r e e s a m p l e s a n a l y s e d . The s p e c i f i c a c t i v i t y o f t h e c a r n i t i n e i n t h e b i l e and t h e serum i s shown i n T a b l e 12. The s p e c i f i c a c t i v i t y i n t h e b i l e was l e s s t h a n i n t h e serum f o r t h r e e o f t h e f i v e r a t s . 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 [ l 4 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 t o 2 h o u r s 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 o f 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 e a c h r a t ( s q u a r e s y m b o l ) . OPM . i . 100/50 microliters H I m 3 TABLE 12 The S p e c i f i c A c t i v i t y o f C a r n i t i n e 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 [ l ^ C j c a r n i t i n e S P E C I F I C A C T I V I T Y (DPM/nmol C a r n i t i n e ) RAT SERUM B I L E 1 131 232 2 158 . 214 3 299 247 4 254 287 5 300 284 APPENDIX 2 The 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 o f R a t B i l e 103 The o b j e c t i v e o f t h i s e x p e r i m e n t was t o d e t e r m i n e w h e t h e r o r n o t t h e b i l e f l u i d l o s t d u r i n g two h o u r s o f b i l e c o l l e c t i o n ( a p p r o x i m a t e l y 3.2 m l ) a f f e c t s t h e b i l e f l o w r a t e and t h e c a r n i t i n e c o n t e n t o f r a t b i l e . R a t s were i n f u s e d ( I V ) w i t h s a l i n e t o r e p l a c e t h e f l u i d l o s t as b i l e . The b i l e f l o w r a t e was a s s e s s e d and f r e e and a c y l c a r n i t i n e d e t e r m i n e d . The r e s u l t s were compared t o t h o s e i n C h a p t e r 5 where no f l u i d was r e p l a c e d . U n l e s s s p e c i f i e d , a l l p r o c e d u r e s were p e r f o r m e d as d e s c r i b e d C h a p t e r 4. S e v e n a d u l t male W i s t a r r a t s were a n a e s t h e t i z e d . I n e a c h r a t , a j u g u l a r v e i n was c a n n u l a t e d and a s t e r i l e s a l i n e s o l u t i o n was i n f u s e d a t a r a t e o f 27.8 u l / m i n u s i n g a p e r i s t a l t i c pump ( S c i e n t i f i c I n d u s t r i e s , M o d e l 6 0 3 ) . The b i l e d u c t was t h e n c a n n u l a t e d and b i l e was c o l l e c t e d f o r two h o u r s i n 15 m i n u t e s a m p l e s . B i l e f l o w was c o n s t a n t t h r o u g h o u t t h e c o l l e c t i o n p e r i o d ( 7 . 0 ± 0.1 u l / m i n / 1 0 0 g BW). T h e r e was no s i g n i f i c a n t d i f f e r e n c e b e t w e e n t h i s and t h e b i l e f l o w i n n o n - i n f u s e d r a t s e x c e p t i n t h e f i n a l c o l l e c t i o n p e r i o d where t h e b i l e f l o w was s i g n i f i c a n t l y g r e a t e r i n t h e i n f u s e d r a t s ( p < 0 . 0 1 ) . The c o n c e n t r a t i o n o f t o t a l c a r n i t i n e i n t h e b i l e o f t h e i n f u s e d r a t s o n l y d i f f e r e d f r o m t h e n o n - i n f u s e d r a t b i l e a t t h e f o u r t h t i m e i n t e r v a l ( p < 0 . 0 1 ) ; t h e r e was a l o w e r c a r n i t i n e c o n c e n t r a t i o n i n t h e b i l e o f t h e i n f u s e d r a t s . S a l i n e i n f u s i o n d i d n o t a f f e c t t h e f r e e c a r n i t i n e and a c y l c a r n i t i n e c o n c e n t r a t i o n s o f r a t b i l e a t any o f t h e t i m e i n t e r v a l s . The amounts o f c a r n i t i n e i n t h e r a t b i l e i n e a c h 104 15 m i n u t e c o l l e c t i o n a l s o d i d n o t c h a n g e w i t h s a l i n e i n f u s i o n . On t h e b a s i s o f t h e s e r e s u l t s , i t was c o n c l u d e d t h a t f l u i d r e p l a c e m e n t d i d n o t a f f e c t t h e c a r n i t i n e c o n t e n t o f r a t b i l e c o l l e c t e d c o n t i n u o u s l y f o r up t o two h o u r s . T h e r e f o r e , i t was d e c i d e d t h a t f o r b i l e c o l l e c t i o n e x p e r i m e n t s , f l u i d r e p l a c e m e n t was n o t n e c e s s a r y . APPENDIX 3 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 C o n t e n t o f t h e B i l e 105 The o b j e c t i v e o f t h e f o l l o w i n g p r o c e d u r e was t o e n a b l e c o l l e c t i o n o f r a t b i l e f r o m a c a n n u l a t e d b i l e d u c t w h i l e o n l y p a r t i a l l y i n t e r r u p t i n g 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 . A r a t was a n a e s t h e t i z e d and i t s b i l e d u c t c a n n u l a t e d a s d e s c r i b e d i n C h a p t e r 4. The c a n n u l a was t h e n i n s e r t e d i n t o a n o t h e r c a n n u l a o f s l i g h t l y g r e a t e r d i a m e t e r . The o t h e r end o f t h e s e c o n d t u b e was s u b s e q u e n t l y i n s e r t e d i n t o t h e i n t e s t i n e o f t h e r a t one i n c h b e l o w t h e p y l o r i c s p h i n c t e r and s e c u r e d w i t h c y a n o a c r y l a t e g l u e . B i l e was s a m p l e d a t 15 m i n u t e i n t e r v a l s by d i s c o n n e c t i n g t h e two i n t e r l o c k i n g t u b e s and c o l l e c t i n g 100 u l . C a r n i t i n e and c a r n i t i n e e s t e r a n a l y s i s was p e r f o r m e d a s d e s c r i b e d i n C h a p t e r 4. APPENDIX 4 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 C o n t e n t o f t h e B i l e 1 0 6 The f o l l o w i n g experiment was performed to a s s e s s the e f f e c t of p e n t o b a r b i t a l on the changes i n the a c y l c a r n i t i n e c o n t e n t of r a t b i l e t h a t o ccur a f t e r the i n i t i a t i o n of c o l l e c t i o n . B i l e was c o l l e c t e d from a r a t who had been a n a e s t h e t i z e d . The b i l e c a r n i t i n e c o n t e n t was then compared to t h a t of o t h e r s where b i l e c o l l e c t i o n was e s t a b l i s h e d i m m e d i a t e l y a f t e r the a n a e s t h e t i c took e f f e c t . D e s p i t e the d e l a y e d s u r g e r y , t h e a c y l c a r n i t i n e c o n t e n t of the b i l e a g a i n i n c r e a s e d d u r i n g the cou r s e of the exp e r i m e n t . The f r e e c a r n i t i n e remained the same l e v e l as i n the c o n t r o l b i l e w h i l e the t o t a l c a r n i t i n e c o n c e n t r a t i o n was more than double t h a t of the c o n t r o l s . APPENDIX 5 P o t e n t i a l M e c h a n i s m s f o r t h e T r a n s p o r t o f C a r n i t i n e I n t o R a t B i l e 107 The mechanism f o r c a r n i t i n e t r a n s p o r t i n t o b i l e has n o t been i n v e s t i g a t e d . P o s s i b l y t h e r e a r e s e p a r a t e mechanisms f o r t h e e n t r y o f t h e v a r i o u s f o r m s o f c a r n i t i n e i n t o t h e b i l e . Of i n t e r e s t a r e t h e l a r g e q u a n t i t i e s o f l o n g - c h a i n c a r n i t i n e e s t e r s . The " m i c e l l a r p u l l t h e o r y " w h i c h h a s a l s o been h y p o t h e s i z e d f o r t h e e n t r y o f c h o l e s t e r o l i n t o t h e b i l e ( H a r d i s o n and A p t e r , 1972) may a l s o be a p p l i c a b l e t o l o n g - c h a i n c a r n i t i n e e s t e r s . B o t h c h o l e s t e r o l and t h e l o n g - c h a i n c a r n i t i n e e s t e r s h ave t h e c a p a c i t y t o f o r m m i c e l l e s . A5.1 M i c e l l a r P u l l T h e o r y A s s u m i n g t h a t t h e m i c e l l a r p u l l t h e o r y a c c u r a t e l y d e s c r i b e s t h e t r a n s p o r t o f p a l m i t o y l c a r n i t i n e i n t o t h e b i l e , t h e f o l l o w i n g w o u l d be t r u e : m onomeric p a l m i t o y l c a r n i t i n e w o u l d p a s s i v e l y e n t e r t h e b i l e f r o m t h e h e p a t o c y t e a c h i e v i n g e q u a l c o n c e n t r a t i o n s on b o t h s i d e s o f t h e c a n a l i c u l a r membrane. Once i n t h e b i l e , t h e p a l m i t o y l c a r n i t i n e w o u l d a s s o c i a t e w i t h b i l e a c i d and p h o s p h o l i p i d m i x e d m i c e l l e s f o r m i n g a " m i c e l l a r s i n k " ( S c h a r s c h m i d t and S c h m i d , 1 9 7 8 ) . T h i s w o u l d d e c r e a s e t h e c o n c e n t r a t i o n o f monomeric p a l m i t o y l c a r n i t i n e i n t h e b i l e c a u s i n g more c a r n i t i n e e s t e r s t o be " p u l l e d " a c r o s s t h e c a n a l i c u l a r membrane f r o m t h e h e p a t o c y t e . Some p r o p e r t i e s o f l o n g - c h a i n c a r n i t i n e e s t e r s seem a p p r o p r i a t e f o r t h e " m i c e l l a r p u l l " t h e o r y . F i r s t , l o n g - c h a i n c a r n i t i n e e s t e r s a r e a m p h i p h i l e s and t h e 108 c o n c e n t r a 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 b i l e i s a b o v e i t s c r i t i c a l m i c e l l a r c o n c e n t r a t i o n . I t w o u l d t h e r e f o r e l i k e l y a s s o c i a t e w i t h b i l e 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 as w e l l a s f o r m i n g m i c e l l e s o f i t s own. S e c o n d l y , t h i s c o u l d e x p l a i n how l o n g - c h a i n a c y l c a r n i t i n e s a r e more c o n c e n t r a t e d i n t h e b i l e t h a n b o t h t h e serum and t h e h e p a t o c y t e s w h i l e f r e e c a r n i t i n e i s n o t . A5.2 The E f f e c t s o f B i l e A c i d I n f u s 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 To t e s t t h e h y p o t h e s i s , r a t s were i n f u s e d ( I V ) w i t h e i t h e r t a u r o c h o l a t e ( 1 . 2 u m o l / m i n / 1 0 0 g BW), a c c o r d i n g t o I n n i s ( 1 9 8 5 ) , o r d e h y d r o c h o l a t e ( 0 . 2 4 umol/min/100 g BW), a c c o r d i n g t o H a r d i s o n and A p t e r ( 1 9 7 2 ) , d u r i n g b i l e c o l l e c t i o n . S o l u t i o n s o f t h e b i l e a c i d s and a l b u m i n ( 4 % ) were i n f u s e d i n t o a c a n n u l a t e d j u g u l a r v e i n o f e a c h r a t . T a u r o c h o l i c and d e h y d r o c h o l i c a c i d s i n c r e a s e t h e r a t e o f b i l e - a c i d d e p e n d e n t b i l e f l o w . T a u r o c h o l a t e i s a m i c e l l e f o r m i n g b i l e a c i d . I f t h e m i c e l l a r t h e o r y a p p l i e s t o c a r n i t i n e , one w o u l d e x p e c t more a c y l c a r n i t i n e i n t h e b i l e o f t h e two r a t s i n f u s e d w i t h t a u r o c h o l a t e . To d i s t i n g u i s h b e t w e e n t h e e f f e c t s o f t h e m i c e l l e f o r m a t i o n and t h e i n c r e a s e d b i l e f l o w , d e h y d r o c h o l a t e , a n o n - m i c e l l e f o r m i n g b i l e a c i d was i n f u s e d i n two a d d i t i o n a l r a t s . D e s p i t e an i n c r e a s e d " b i l e f l o w , t h e c o n c e n t r a t i o n o f f r e e and a c y l c a r n i t i n e i n t h e b i l e o f t a u r o c h o l a t e i n f u s e d r a t s r e m a i n e d t h e same as i n t h e b i l e o f s a l i n e i n f u s e d 109 r a t s . H e n c e , t h e amount o f c a r n i t i n e f o u n d i n t h e b i l e i n e a c h 15 m i n u t e s was t h r e e t i m e s h i g h e r t h a n f o r t h e s a l i n e i n f u s e d c o n t r o l s . I n c r e a s e d b i l e f l o w and c a r n i t i n e c o n c e n t r a t i o n a l s o r e s u l t e d when d e h y d r o c h o l a t e was i n f u s e d . T h i s s u g g e s t s t h a t 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 i n c r e a s e d due t o t h e i n c r e a s e d b i l e v o lume r a t h e r t h a n t o m i c e l l e f o r m a t i o n and d i s p u t e s t h e m i c e l l a r t h e o r y f o r t h e t r a n s p o r t o f l o n g - c h a i n c a r n i t i n e e s t e r s i n t o t h e b i l e . A5.3 O t h e r O b s e r v a t i o n s W h i l e i n v e s t i g a t i n g t h e c a r n i t i n e c o n t e n t o f r a t b i l e , o t h e r o b s e r v a t i o n s were made. F o r i n s t a n c e , a l t h o u g h t h e c o n c e n t r a t i o n o f c a r n i t i n e i n b i l e was h i g h e r i n f a s t i n g t h a n i n f e d r a t s , t h e o v e r a l l amount o f c a r n i t i n e f o u n d i n e a c h 15 m i n u t e c o l l e c t i o n d i d n o t d i f f e r . T h i s m i g h t i n d i c a t e t h a t c a r n i t i n e i n t h e b i l e i s i n d e p e n d e n t o f b i l e f l o w . A l t e r n a t i v e l y , t h e b i l e m i g h t have been c o n c e n t r a t e d i n t h e f a s t e d r a t s a f t e r i t s f o r m a t i o n . The r e s u l t s o f t h e b i l e a c i d i n f u s i o n s u g g e s t s t h a t t h e l a t t e r i s t r u e . W i t h b o t h t a u r o c h o l a t e and d e h y d r o c h o l a t e i n f u s i o n , t h e c o n c e n t r a t i o n o f b i l e c a r n i t i n e r e m a i n e d a t t h e same l e v e l s a s i n s a l i n e i n f u s e d c o n t r o l s b u t t h e amount o f c a r n i t i n e i n c r e a s e d . T h e r f o r e , when b i l e i s f o r m e d a t t h e c a n n a l i c u l a r membrane, t h e r e a p p e a r s t o be a c o n s t a n t c o n c e n t r a t i o n o f c a r n i t i n e . W i t h i n c r e a s e d b i l e f l o w , as i n b i l e a c i d i n f u s i o n , t h e c o n c e n t r a t i o n o f t h e c a r n i t i n e i n 110 t h e b i l e , t h u s , r e m a i n s c o n s t a n t . I n f a s t i n g , h o w e v e r , t h e c o n c e n t r a t i o n o f t h e i n i t i a l l y f o r m e d b i l e m i g h t be t h e same as i n f e d r a t s b u t s u b s e q u e n t w a t e r r e a b s o r p t i o n f r o m t h e b i l e m i g h t o c c u r . I n t h e s e s t u d i e s , t h e o v e r a l l r a t i o o f f r e e t o t o t a l c a r n i t i n e was n o t a f f e c t e d by t h e t r e a t m e n t s i m p o s e d b u t c a r n i t i n e e s t e r i f i c a t i o n d i d i n c r e a s e t h r o u g h o u t t h e c o u r s e o f e a c h e x p e r i m e n t . G u d j o n s s o n and c o l l e a g u e s ( 1 9 8 5 b ) , h o w e v e r , showed a two f o l d i n c r e a s e i n t h e amount o f c a r n i t i n e e s t e r s i n t h e b i l e a f t e r i n j e c t i o n w i t h b o l u s d o s e s o f c a r n i t i n e . F r e e c a r n i t i n e l e v e l s d i d n o t c h a n g e . T h i s s u g g e s t s t h a t t h e t r a n s p o r t o f f r e e and a c y l c a r n i t i n e i n t o b i l e a r e n o t n e c e s s a r i l y d e p e n d e n t on one a n o t h e r . I t i s s t i l l unknown w h e t h e r o r n o t c a r n i t i n e i s a c t i v e l y o r p a s s i v e l y t r a n s p o r t e d i n t o t h e b i l e . O t h e r q u a t e r n a r y ammonium compounds a r e a c t i v e l y t r a n s p o r t e d ( S c h a n k e r and S o l o m o n , 1 9 6 3 ) , e s p e c i a l l y t h o s e w i t h m o l e c u l a r w e i g h t s g r e a t e r t h a n 300 g /mol. As s h o r t - c h a i n c a r n i t i n e e s t e r s h a v e m o l e c u l a r w e i g h t s l e s s t h a n 300 g / m o l , and l o n g - c h a i n e s t e r s h a v e g r e a t e r m o l e c u l a r w e i g h t s , t h i s m i g h t e x p l a i n why l a r g e amounts o f l o n g - c h a i n c a r n i t i n e e s t e r s a r e f o u n d i n b i l e . The a c t i v e l y t r a n s p o r t e d l o n g - c h a i n c a r n i t i n e e s t e r s c o u l d t h e n be a v a i l a b l e f o r m i x e d m i c e l l e f o r m a t i o n . APPENDIX 6 The Serum C a r n i t i n e C o n t e n t o f C y s t i c F i b r o s i s P a t i e n t s I l l 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 i n t h e Serum n f C y s t i c . F i b r o s i s P a t i e n t s P a t i e n t Sample F r e e T o t a l A c y l # # C a r n i t i n e C a r n i t i n e C a r n i t i n e (uM) (uM) (%) 1 1 38.0 54.5 30 2 45.0 53.2 15 2 1 52.1 57.2 9 3 1 71.8 84.9 15 4 1 33.2 42.2 21 5 1 35.4 47.5 25 6 1 31.0 42.0 26 7 1 32.2 50.1 36 2 43.5 49.8 13 8 1 50.3 58.6 14 2 51.6 64.4 20 9 1 8.0 10.1 21 10 1 18.9 24.5 23 11 1 49.9 45.2 45 12 1 40.2 54.3 46 2 34.9 49.3 29 3 7.5 11.5 35 13 1 47.3 52.6 10 14 1 61.9 68.3 9 15 1 48.7 69.5 18 2 49.5 61.5 20 3 49.5 61.5 20 16 1 28.4 50.1 43 17 1 54.6 60.6 9 18 1 33.2 42.2 21 19 1 49.5 56.0 19 20 1 39.7 44.9 12 21 1 56.2 66.1 15 22 1 49.7 57.5 14 2 47.0 60.6 22 23 1 40.3 46.8 14 24 1 39.0 48.8 20 25 1 25.4 36.8 31 26 1 43.0 65.4 33 2 38.1 55.4 31 3 36.7 55.9 34 27 1 38.6 49.2 21 2 37.7 49.3 24 3 44.7 66.0 32 28 1 29.8 47.2 37 29 1 50.7 79.0 36 30 1 38.3 53.4 28 31 1 40.5 52.0 15 32 1 45.6 52.0 15 33 1 39.7 47.7 17

Cite

Citation Scheme:

    

Usage Statistics

Country Views Downloads
China 23 33
Japan 2 0
France 1 0
United States 1 0
City Views Downloads
Beijing 23 0
Tokyo 2 0
Unknown 1 1
Ashburn 1 0

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

Share

Share to:

Comment

Related Items