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UBC Theses and Dissertations

GCMS analysis of valproic acid metabolites and the effects of salicylate on valproic acid metabolism Kassam, Jeanine Posset 1985

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GCMS ANALYSIS OF VALPROIC ACID METABOLITES AND THE EFFECTS OF SALICYLATE ON VALPROIC ACID METABOLISM by J E A N I N E POSSET KASSAM 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 o f P h a r m a c e u t i c a l C h e m i s t r y F a c u l t y o f P h a r m a c e u t i c a l S c i e n c e s We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF B R I T I S H COLUMBIA J u n e 1985 s O e a n i n e P o s s e t K a s s a m , 1985 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by h i s or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 - i i -ABSTRACT An a s s a y has been d e v e l o p e d f o r t h e s i m u l t a n e o u s q u a n t i t a t i v e a n a l y s i s o f VPA a n d t w e l v e o f i t s m e t a b o l i t e s n a m e l y 2 - e n e V P A , 3 - e n e V P A , 4 - e n e V P A , ( E ) - 2 , 4 - d i e n e V P A , ( E , E ) - 2 , 3 ' - d i e n e V P A , 3-OH V P A , 4-OH V P A , 5-OH V P A , 3 - k e t o V P A , 4 - k e t o V P A , 2 - p r o p y l s u c c i n i c a c i d and 2 - p r o p y l g l u t a r i c a c i d . The a s s a y i s a c c o m p l i s h e d i n a s i n g l e r u n u s i n g t h e H e w l e t t - P a c k a r d 5987A G C - M S . VPA a n d i t s m e t a b o l i t e s w e r e m e a s u r e d u s i n g s e l e c t e d i o n m o n i t o r i n g o f t h e c h a r a c t e r i s t i c ( M - 5 7 ) + i o n s o f t h e t e r t b u t y l d i m e t h y l s i l y l (tBDMS) d e r i v a t i v e s . The 4-OH VPA was m e a s u r e d as t h e u n d e r i v a t i z e d Y - l a c t o n e . [ 2 H 6 ^ - V P A , [ 2 H 3 3 - 2 - e n e VPA a n d 3 - o c t a n o n e s e r v e d as i n t e r n a l s t a n d a r d s . I n a t y p i c a l a s s a y 1 mL o f u r i n e o r s e r u m i s a d j u s t e d t o pH 13 t o h y d r o l y z e t h e c o n j u g a t e s , t h e n a c i d i f i e d t o pH 2 and e x t r a c t e d w i t h e t h y l a c e t a t e . The s o l v e n t i s t h e n d r i e d , c o n c e n t r a t e d t o 2 0 0 uL a n d d e r i v a t i z e d w i t h tBDMS r e -a g e n t . The c h r o m a t o g r a p h i c r u n t i m e w i t h a 25 m x 0 . 3 2 mm I . D . b o n d e d p h a s e OV 1701 c a p i l l a r y c o l u m n was 22 m i n u t e s . A l l m e t a b o l i t e s w e r e w e l l r e s o l v e d and e x t r a c t s o f c o n t r o l s e r u m o r u r i n e showed no i n t e r f e r e n c e s . The tBDMS and TMS d e r i v a t i v e s o f VPA m e t a b o l i t e s w e r e c o m p a r e d . M i x e d tBDMS-TMS d e r i v a t i v e s w e r e a l s o i n v e s t i g a t e d . T h i s a s s a y h a s been a p p l i e d t o t h e a n a l y s i s o f u r i n e and s e r u m s a m p l e s f r o m p e d i a t r i c p a t i e n t s o n VPA t h e r a p y . The e f f e c t s o f a c e t y l s a l i c y l i c a c i d (ASA) on t h e m e t a b o l i s m o f VPA w e r e i n v e s t i g a t e d . U r i n e s a m p l e s o f s i x p e d i a t r i c p a t i e n t s u n d e r v a l p r o i c a c i d t h e r a p y a n d a n o r m a l a d u l t s u b j e c t t a k i n g v a l p r o i c a c i d w e r e o b t a i n e d b e f o r e and a f t e r a d m i n i s t r a t i o n o f A S A . The a n a l y s e s showed t h a t t h e e x c r e t i o n o f c o n j u g a t e d and unchanged VPA was enhanced i n a l l s u b j e c t s f o l l o w i n g a d m i n i s t r a t i o n of ASA. The 3 - k e t o VPA, the second major u r i n a r y m e t a b o l i t e a f t e r V P A - g l u c u r o n i d e , was m a r k e d l y r e d u c e d . The average e x c r e t i o n of 2-ene VPA and 3-OH VPA was a l s o l o w e r e d . These r e s u l t s suggest t h a t t h e p - o x i d a t i o n pathway which i n c l u d e s 2-ene VPA, 3-OH VPA and 3 - k e t o VPA was i n h i b i t e d by s a l i c y l a t e a t some s t a g e p r i o r t o t h e f o r m a t i o n o f 2-ene VPA. S i n c e serum samples of t h e normal s u b j e c t were a v a i l a b l e t h e k i n e -t i c s o f VPA m e t a b o l i t e s b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA were d e t e r m i n e d . It was found t h a t the f o r m a t i o n c l e a r a n c e , the f r a c t i o n m e t a b o l i z e d and the e l i m i n a t i o n c l e a r a n c e of t h e e - o x i d a t i o n metabo-l i t e s were d e c r e a s e d f o l l o w i n g ASA a d m i n i s t r a t i o n . These r e s u l t s c o n -f i r m e d t h e u r i n a r y d a t a o b t a i n e d w i t h t h e seven s u b j e c t s . S a l i c y l a t e was found t o i n h i b i t the p - o x i d a t i o n of VPA. Other m e t a b o l i c pathways o f VPA were not s i g n i f i c a n t l y a f f e c t e d by ASA a d m i n i s t r a t i o n . The mechanism by which s a l i c y l a t e a f f e c t s the g - o x i d a t i o n of v a l p r o i c a c i d i s d i s c u s s e d . - i v -TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES v i LIST OF FIGURES i x LIST OF ABBREVIATIONS x i i I . INTRODUCTION 1 A . P h a r m a c o k i n e t i c s 2 B. Mechanism o f a c t i o n 3 C. M e t a b o l i s m 4 1 . I d e n t i f i c a t i o n of VPA m e t a b o l i t e s 7 2 . A n t i c o n v u l s a n t a c t i v i t y o f VPA m e t a b o l i t e s 8 D. S i d e - e f f e c t s and t o x i c i t y 9 E. I n t e r a c t i o n between VPA and ASA 12 F. . A n a l y s i s of v a l p r o i c a c i d and m e t a b o l i t e s 14 I I . EXPERIMENTAL 17 A . Reagents and m a t e r i a l s 17 1 . VPA m e t a b o l i t e s 17 2 . I n t e r n a l s t a n d a r d s 17 3 . Reagents 18 B. Drug study 19 C. A n a l y s i s 20 1 . Stock s o l u t i o n s of i n t e r n a l s t a n d a r d s 20 2 . P r e p a r a t i o n of u r i n e and serum s t a n d a r d s 21 3 . E x t r a c t i o n and d e r i v a t i z a t i o n of s t a n d a r d s and p a t i e n t samples 22 4 . D e t e r m i n a t i o n of u r i n a r y c r e a t i n i n e 24 5 . I n s t r u m e n t a t i o n 24 I I I . RESULTS AND DISCUSSION 26 A. A n a l y s i s o f VPA and i t s m e t a b o l i t e s 26 1 . Sample e x t r a c t i o n 26 2 . D e r i v a t i z a t i o n 29 3 . S e l e c t e d i o n chromatograms 38 4 . Q u a n t i t a t i v e a n a l y s i s 49 - V -TABLE OF CONTENTS (CONT'D) Page 5 . Comparison of tBDMS and TMS d e r i v a t i v e s 62 6 . Mixed d e r i v a t i v e s 65 B. ASA - VPA I n t e r a c t i o n 69 1 . A n a l y s i s of VPA and i t s m e t a b o l i t e s i n u r i n e samples 69 1.1 U r i n a r y p r o f i l e s of VPA m e t a b o l i t e s -e f f e c t of ASA 69 1 . 2 U r i n a r y p r o f i l e s of m e t a b o l i t e s grouped i n t o pathways - E f f e c t of ASA 85 2 . P h a r m a c o k i n e t i c study 101 3 . D i s c u s s i o n 127 SUMMARY AND CONCLUSIONS 135 REFERENCES 138 - v i -L I S T OF TABLES T a b l e P a g e I C h a r a c t e r i s t i c i o n s ( m / z ) i n t h e m a s s s p e c t r a o f 37 t h e tBDMS d e r i v a t i v e s o f v a l p r o i c a c i d m e t a b o l i t e s I I S e r u m VPA a n d m e t a b o l i t e c o n c e n t r a t i o n s f o r 3 4 61 p e d i a t r i c p a t i e n t s on VPA m o n o t h e r a p y I I I C o m p a r i s o n o f t h e r e l a t i v e s e n s i t i v i t y o f tBDMS 6 4 a n d TMS d e r i v a t i v e s o f VPA and m e t a b o l i t e s when m e a s u r e d by SIM IV C o m p a r i s o n o f d e r i v a t i v e r e a c t i o n t i m e s , 64 c h r o m a t o g r a p h y t i m e s a n d s t a b i l i t y o f tBDMS and TMS d e r i v a t i v e s o f VPA and i t s m e t a b o l i t e s V U r i n a r y p r o f i l e s o f VPA and i t s m e t a b o l i t e s 75 b e f o r e a n d a f t e r a d m i n i s t r a t i o n o f ASA i n p a t i e n t BP VI U r i n a r y p r o f i l e s o f VPA and i t s m e t a b o l i t e s 76 b e f o r e and a f t e r a d m i n i s t r a t i o n o f ASA i n p a t i e n t CP V I I U r i n a r y p r o f i l e s o f VPA and i t s m e t a b o l i t e s 77 b e f o r e a n d a f t e r a d m i n i s t r a t i o n o f ASA i n p a t i e n t SS V I I I U r i n a r y p r o f i l e s o f VPA and i t s m e t a b o l i t e s 78 b e f o r e and a f t e r a d m i n i s t r a t i o n o f ASA i n p a t i e n t TR IX U r i n a r y p r o f i l e s o f VPA and i t s m e t a b o l i t e s 79 b e f o r e a n d a f t e r a d m i n i s t r a t i o n o f ASA i n p a t i e n t VS X U r i n a r y p r o f i l e s o f VPA and i t s m e t a b o l i t e s 80 b e f o r e and a f t e r a d m i n i s t r a t i o n o f ASA i n p a t i e n t WN X I U r i n a r y p r o f i l e s o f VPA and i t s m e t a b o l i t e s 81 b e f o r e a n d a f t e r a d m i n i s t r a t i o n o f ASA i n a h e a l t h y v o l u n t e e r X I I M e a n , d i f f e r e n c e and p e r c e n t c h a n g e o f u r i n a r y 83 c o n c e n t r a t i o n s o f VPA and i t s m e t a b o l i t e s b e f o r e and a f t e r a d m i n i s t r a t i o n o f ASA (n=7) X I I I M e a n , d i f f e r e n c e a n d p e r c e n t c h a n g e o f u r i n a r y 8 4 p r o f i l e s o f VPA and i t s m e t a b o l i t e s b e f o r e and a f t e r a d m i n i s t r a t i o n o f ASA (n=7) - v i i -LIST OF TABLES (CONT'D) T a b l e Page XIV U r i n a r y p r o f i l e s e x p r e s s e d as d i f f e r e n t VPA 90 m e t a b o l i c pathways b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA i n p a t i e n t BP XV U r i n a r y p r o f i l e s e x p r e s s e d as d i f f e r e n t VPA 91 m e t a b o l i c pathways b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA i n p a t i e n t CP XVI U r i n a r y p r o f i l e s e x p r e s s e d as d i f f e r e n t VPA 92 m e t a b o l i c pathways b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA i n p a t i e n t SS XVII U r i n a r y p r o f i l e s e x p r e s s e d as d i f f e r e n t VPA 93 m e t a b o l i c pathways b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA i n p a t i e n t TR XVIII U r i n a r y p r o f i l e s e x p r e s s e d as d i f f e r e n t VPA 94 m e t a b o l i c pathways b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA i n p a t i e n t VS XIX U r i n a r y p r o f i l e s e x p r e s s e d as d i f f e r e n t VPA 95 m e t a b o l i c pathways b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA i n p a t i e n t WN XX U r i n a r y p r o f i l e s e x p r e s s e d as d i f f e r e n t VPA 96 m e t a b o l i c pathways b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA i n a h e a l t h y v o l u n t e e r XXI Mean, d i f f e r e n c e and p e r c e n t change of 98 u r i n a r y c o n c e n t r a t i o n s of VPA and i t s m e t a b o l i t e s e x p r e s s e d as m e t a b o l i c pathways b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA (n=7) XXII Mean, d i f f e r e n c e and p e r c e n t change i n 99 u r i n a r y p r o f i l e s of VPA m e t a b o l i t e s pathways e x p r e s s e d as p e r c e n t of t o t a l e x c r e t e d b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA (n=7) XXII Comparison between the average amounts of 100 m e t a b o l i t e s e x c r e t e d a t steady s t a t e f o r s i x p a t i e n t s and one v o l u n t e e r and f o r s i x p a t i e n t s o n l y b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA XXIV C u m u l a t i v e e x c r e t e d amounts o f VPA and i t s 106 m e t a b o l i t e s over an 8 hour p e r i o d i n a normal s u b j e c t b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA - v i i i -LIST OF TABLES (CONT'D) T a b l e Page XXV C u m u l a t i v e e x c r e t e d amounts of m e t a b o l i c pathways 107 f o r VPA and i t s m e t a b o l i t e s over an 8 hour p e r i o d i n a normal s u b j e c t b e f o r e and a f t e r a d m i n i s t r a t i o n o f ASA XXVI AUC v a l u e s of VPA and i t s m e t a b o l i t e s over an 8 108 hour p e r i o d i n a normal s u b j e c t b e f o r e and a f t e r a d m i n i s t r a t i o n o f ASA XXVII AUC v a l u e s of m e t a b o l i c pathways f o r VPA and i t s 109 m e t a b o l i t e s over an 8 hour p e r i o d i n a normal s u b j e c t b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA XXVIII M e t a b o l i c c l e a r a n c e s ( C l m ) o f VPA m e t a b o l i t e s 116 i n a normal s u b j e c t b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA XXIX Average serum steady s t a t e c o n c e n t r a t i o n s of 117 p a r e n t drug and m e t a b o l i t e s combined i n pathways (Cm-} s s ) and m e t a b o l i t e - p a r e n t drug c o n c e n t r a t i o n r a t i o ( C m / C p ) i n a normal s u b j e c t b e f o r e (Day 1) and a f t e r a d m i n i s t r a t i o n of ASA (Day 2) XXX F o r m a t i o n c l e a r a n c e s (CIf^ and C l ^ ) i n L/h of 118 VPA m e t a b o l i t e s i n a normal s u b j e c t b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA XXXI F r a c t i o n m e t a b o l i z e d (fm) by each pathway i n a 119 normal s u b j e c t b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA XXXII P h a r m a c o k i n e t i c parameters determined f o r pathway R' 124 i n a normal s u b j e c t t a k i n g v a l p r o i c a c i d b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA XXXIII P h a r m a c o k i n e t i c parameters determined f o r the sum 124 o f the o x i d a t i v e pathways (pathways 1 t o 5) i n a normal s u b j e c t t a k i n g v a l p r o i c a c i d b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA XXXIV P h a r m a c o k i n e t i c parameters d e t e r m i n e d f o r pathways 125 1 t o 5 i n a normal s u b j e c t t a k i n g v a l p r o i c a c i d b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA XXXV P h a r m a c o k i n e t i c parameters d e t e r m i n e d f o r 2 - e n e VPA 126 and 4-ene VPA i n a normal s u b j e c t t a k i n g v a l p r o i c a c i d b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA i x -LIST OF FIGURES F i g u r e Page 1 Human m e t a b o l i s m of v a l p r o i c a c i d 6 2 S e l e c t e d i o n chromatogram (m/z 118) of the tBDMS 28 d e r i v a t i v e of a c e t i c a c i d i n u r i n e e x t r a c t 3 Mass spectrum o f 4-OH VPA l a c t o n e i n u r i n e e x t r a c t 30 4 Mass spectrum of tBDMS d e r i v a t i v e of 4-ene VPA i n 30 u r i n e e x t r a c t 5 Mass spectrum of tBDMS d e r i v a t i v e of 2-ene VPA i n 31 u r i n e e x t r a c t 6 Mass spectrum of tBDMS d e r i v a t i v e of 3-ene VPA i n 31 u r i n e e x t r a c t 7 Mass spectrum of tBDMS d e r i v a t i v e of ( E , E ) - 2 , 3 ' ~ 32 d i e n e VPA i n u r i n e e x t r a c t 8 Mass spectrum of tBDMS d e r i v a t i v e of ( E ) - 2 , 4 - d i e n e 32 VPA i n u r i n e e x t r a c t 9 Mass spectrum of tBDMS d e r i v a t i v e of VPA i n u r i n e 33 e x t r a c t 10 Mass spectrum of tBDMS d e r i v a t i v e of 4 - k e t o VPA 33 i n u r i n e e x t r a c t 11 Mass spectrum of tBDMS d e r i v a t i v e of 3 - k e t o VPA 34 i n u r i n e e x t r a c t 12 Mass spectrum of di-tBDMS d e r i v a t i v e of 3 - k e t o 34 VPA i n u r i n e e x t r a c t 13 Mass spectrum of tBDMS d e r i v a t i v e of 3-OH VPA 35 i n u r i n e e x t r a c t 14 Mass spectrum of di-tBDMS d e r i v a t i v e of 5-OH VPA 35 i n u r i n e e x t r a c t 15 Mass spectrum of di-tBDMS d e r i v a t i v e of 2 - p r o p y l 36 s u c c i n i c a c i d i n u r i n e e x t r a c t 16 ' Mass spectrum o f di-tBDMS d e r i v a t i v e of 2 - p r o p y l 36 g l u t a r i c a c i d i n u r i n e e x t r a c t 17 S e l e c t e d i o n chromagram of 4-OH VPA l a c t o n e s from 40 a p a t i e n t u r i n e sample - X -LIST OF FIGURES (CONT'D) F i g u r e Page 18 S e l e c t e d i o n chromatograms of tBDMS d e r i v a t i v e s o f ( E ) - 2 , 4 - d i e n e VPA and ( E , E ) - 2 , 3 ' - d i e n e VPA from a p a t i e n t u r i n e sample 41 19 S e l e c t e d i o n chromatograms of tBDMS d e r i v a t i v e s of 4 - e n e VPA, 3-ene VPA, ( Z ) - 2 - e n e VPA and ( E ) - 2 -ene VPA from a p a t i e n t u r i n e sample 42 20 S e l e c t e d i o n chromatogram of tBDMS d e r i v a t i v e o f VPA from a p a t i e n t u r i n e sample 43 21 S e l e c t e d i o n chromatograms of tBDMS d e r i v a t i v e s o f 3 - k e t o VPA and 4 - k e t o VPA from a p a t i e n t u r i n e sample 44 22 S e l e c t e d i o n chromatograms of tBDMS d e r i v a t i v e s o f 5-0H VPA and 2 - p r o p y l s u c c i n i c a c i d from a p a t i e n t u r i n e sample 45 23 . S e l e c t e d i o n chromatogram of tBDMS d e r i v a t i v e of 2 - p r o p y l g l u t a r i c a c i d from a p a t i e n t u r i n e sample 46 24 S e l e c t e d i o n chromatograms of tBDMS d e r i v a t i v e s o f v a l p r o i c a c i d m e t a b o l i t e s from a p a t i e n t u r i n e sample 47 25 S e l e c t e d i o n chromatograms of tBDMS d e r i v a t i v e s of v a l p r o i c a c i d m e t a b o l i t e s from a p a t i e n t serum sample 48 26 4-OH VPA l a c t o n e c a l i b r a t i o n c u r v e 50 27 4-Ene VPA tBDMS d e r i v a t i v e c a l i b r a t i o n c u r v e 51 28 3-Ene VPA tBDMS d e r i v a t i v e c a l i b r a t i o n c u r v e 52 29 ( E ) - 2 - E n e VPA tBDMS d e r i v a t i v e c a l i b r a t i o n c u r v e 53 30 VPA tBDMS d e r i v a t i v e c a l i b r a t i o n curve 54 31 3 - K e t o VPA tBDMS d e r i v a t i v e c a l i b r a t i o n c u r v e 55 32 4 - K e t o VPA tBDMS d e r i v a t i v e c a l i b r a t i o n c u r v e 56 33 3-0H VPA TMS d e r i v a t i v e c a l i b r a t i o n c u r v e 57 34 5-OH VPA tBDMS d e r i v a t i v e c a l i b r a t i o n c u r v e 58 - x i LIST OF FIGURES (CONT'D) F i g u r e Page 35 2 - P r o p y l s u c c i n i c a c i d tBDMS d e r i v a t i v e c a l i b r a t i o n 59 c u r v e 36 2 - P r o p y l g l u t a r i c a c i d tBDMS d e r i v a t i v e c a l i b r a t i o n 60 c u r v e 37 S e l e c t e d i o n chromatograms of TMS d e r i v a t i v e s of 63 v a l p r o i c a c i d m e t a b o l i t e s from a p a t i e n t u r i n e sample 38 Mass chromatogram and mass spectrum of tBDMS-TMS 66 mixed d e r i v a t i v e o f 3-OH VPA i n u r i n e e x t r a c t 39 Mass chromatogram and mass spectrum of tBDMS-TMS 67 mixed d e r i v a t i v e o f 4-OH VPA i n u r i n e e x t r a c t 40 U r i n a r y p r o f i l e s of VPA m e t a b o l i t e s i n seven 70 ' s u b j e c t s b e f o r e (day 1) and a f t e r (day 2) a d m i n i s t r a t i o n of ASA 41 M e t a b o l i c pathways f o r VPA and i t s m e t a b o l i t e s 86 42 U r i n a r y p r o f i l e s o f VPA m e t a b o l i t e s e x p r e s s e d 87 as m e t a b o l i c pathways i n seven s u b j e c t s b e f o r e (day 1) and a f t e r (day 2) a d m i n i s t r a t i o n of ASA 43 VPA serum c o n c e n t r a t i o n v e r s u s time curve i n 111 a normal s u b j e c t b e f o r e and a f t e r a d m i n i s t r a t i o n o f ASA 44 ( E ) - 2 - E n e VPA serum c o n c e n t r a t i o n v e r s u s time 112 c u r v e i n a normal s u b j e c t b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA 45 3 - K e t o VPA serum c o n c e n t r a t i o n v e r s u s time 113 c u r v e i n a normal s u b j e c t b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA 46 4-Ene VPA serum c o n c e n t r a t i o n v e r s u s time 114 c u r v e i n a normal s u b j e c t b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA VPA [ 2 H 6 ] - V P A VPA-D 6 3 - OH VPA 4 - OH VPA 5 - OH VPA 3 - Keto VPA 4 - Keto VPA 2-Ene VPA [ 2 H 3 ] - 2 - E n e VPA 2 - Ene V P A-D3 3 - Ene VPA 4 - Ene VPA ( E ) - 2 , 4 - D i e n e VPA ( E , E ) - 2 , 3 ' - D i e n e VPA 2-PSA 2-PGA TMS MSTFA tBDMS tBDMCS MTBSTFA GC-MS SIM m - x i i -LIST OF ABBREVIATIONS V a l p r o i c a c i d ( 2 - p r o p y l p e n t a n o i c a c i d ) [ 2 Hs ] - V a l p r o i c a c i d [ 2 H 6 ] - v a l p r o i c a c i d 2 - P r o p y l - 3 - h y d r o x y p e n t a n o i c a c i d 2 - P r o p y l - 4 - h y d r o x y p e n t a n o i c a c i d 2 - P r o p y l - 5 - h y d r o x y p e n t a n o i c a c i d 2 - P r o p y l - 3 - k e t o p e n t a n o i c a c i d 2 - P r o p y l - 4 - k e t o p e n t a n o i c a c i d 2 - P r o p y l - 2 - p e n t e n o i c a c i d [ 3 , 5 , 5 - 2 H ] - 3 - h e p t e n e - 4 - c a r b o x y l i c a c i d [ 3 , 5 , 5 , 2 H ] - 3 h e p t e n e - 4 - c a r b o x y l i c a c i d 2 - P r o p y l - 3 - p e n t e n o i c a c i d 2 - P r o p y l - 4 - p e n t e n o i c a c i d 2 - P r o p y l - ( E ) - 2 , 4 - p e n t a d i e n o i c a c i d 2 - [ ( E ) - l ' - p r o p e n y l ] - ( E ) - 2 - p e n t e n o i c a c i d 2 - P r o p y l s u c c i n i c a c i d 2 - P r o p y l g l u t a r i c a c i d T r i m e t h y l s i l y l N - m e t h y l - N - t r i m e t h y l s i l y l t r i f l u o r o a c e t a m i d e T e r t i a r y b u t y l d i m e t h y l s i l y l T e r t i a r y b u t y l d i m e t h y l c h l o r o s i 1 y l N - t e r t - b u t y l d i m e t h y l s i l y l - N - m e t h y l -t r i f l u o r o a c e t a m i d e Gas chromatography - mass s p e c t r o m e t r y S e l e c t e d i o n m o n i t o r i n g m e t e r , m u l t i p l e t (NMR) - x i i i -LIST OF ABBREVIATIONS (CONT'D) q q u a d r u p l e t s s i n g l e t SD Standard d e v i a t i o n GLC Gas l i q u i d chromatography HPLC High performance l i q u i d chromatography [ 2H4]-4 - H e p t a n o n e [3,3,5,5 , - 2 H]-4 - h e p t a n o n e bp b o i l i n g p o i n t NMR N u c l e a r magnetic resonance b r s broad s i n g l e t DMAP 4 - D i m e t h y l a m i n o p y r i d i n e ASA A c e t y l s a l i c y l i c a c i d SA S a l i c y l i c a c i d Degrees ° Degrees C e l s i u s - xiv -ACKNOWLEDGEMENT The a u t h o r i s v e r y g r a t e f u l t o Dr. F . S . Abbott f o r h i s a b l e g u i d a n c e and s u p e r v i s i o n t h r o u g h o u t the c o u r s e of t h i s s t u d y . The a u t h o r e x p r e s s e s her g r a t i t u d e t o D r . J . Orr f o r h i s g r e a t h e l p i n t h e p h a r m a c o k i n e t i c s t u d y . The a u t h o r i s a l s o i n d e b t e d t o Ms. S h e i l a F e r g u s o n , Cathy Hannah, and S u k b h i n d e r Panesar f o r t h e i r h e l p i n t h e a s s a y development. Thanks a l s o go to Andrew Acheampong f o r h i s h e l p f u l s u g g e s t i o n s . The t e c h n i c a l a s s i s t a n c e of Mr. R. Burton was g r e a t l y a p p r e c i a t e d . The f i n a n c i a l s u p p o r t p r o v i d e d by a s c h o l a r s h i p from the government of t h e I v o r y - C o a s t i s g r a t e f u l l y acknowledged. - 1 -I . INTRODUCTION V a l p r o i c A c i d (VPA) i s a major a n t i c o n v u l s a n t drug i n t r o d u c e d i n N o r t h America i n 1978. A l t h o u g h VPA was f i r s t d e s c r i b e d i n 1881 ( 1 ) , i t s a n t i c o n v u l s a n t p r o p e r t i e s were only d i s c o v e r e d i n 1963 by Meunier and c o - w o r k e r s ( 2 ) . V a l p r o i c a c i d i s used i n t r e a t m e n t of p e t i t mal (absence s e i z u r e s ) , p r i m a r y g e n e r a l i z e d t o n i c c l o n i c s e i z u r e s and s i m p l e f e b r i l e c o n v u l s i o n s . In the body VPA i s t r a n s f o r m e d i n t o many m e t a b o l i t e s . Some of these m e t a b o l i t e s are thought to have a n t i e p i l e p t i c p r o p e r t i e s and/or t o be i n v o l v e d i n cases of h e p a t o t o x i c i t y . The s t r u c t u r e of v a l p r o i c a c i d i s r e l a t e d t o the f a t t y a c i d s as shown i n the f i g u r e b e l o w . CH 3 - CH 2 - CH 2 \ H - COOH V a l p r o i c A c i d / CH 3 - CH 2 - CH 2 Most e p i l e p t i c p a t i e n t s undergo l o n g - t e r m a n t i c o n v u l s a n t t h e r a p y and are l i k e l y t o r e q u i r e m e d i c a t i o n f o r o t h e r i l l n e s s e s . F o r e x a m p l e , VPA i s one of the drugs o f c h o i c e i n s i m p l e f e b r i l e c o n v u l s i o n s i n young c h i l d r e n . VPA i s o f t e n c o - a d m i n i s t e r e d w i t h a n t i p y r e t i c drugs i n t r e a t i n g the f e v e r a s s o c i a t e d w i t h t h i s c o n d i t i o n ( 3 - 5 ) . I t i s t h e r e -f o r e i m p o r t a n t t o determine the consequences of i n t e r a c t i o n s between a n t i c o n v u l s a n t s such as VPA and o t h e r commonly used d r u g s . - 2 -The purpose of t h i s work i s to study the e f f e c t of s a l i c y l a t e on the m e t a b o l i s m of v a l p r o i c a c i d . A. P h a r m a c o k i n e t i c s The p h a r m a c o k i n e t i c s of VPA have been e x t e n s i v e l y s t u d i e d and data r e p o r t e d i n s e v e r a l r e v i e w s ( 6 - 9 ) . V a l p r o i c a c i d a d m i n i s t e r e d as t h e sodium s a l t or as f r e e a c i d i s r a p i d l y absorbed from the g a s t r o i n t e s t i n a l t r a c t . Peak plasma l e v e l s are reached i n 1-2 hours f o l l o w i n g an o r a l dose ( 1 0 ) . I t s a b s o l u t e b i o a v a i l a b i l i t y i s c l o s e to 100% ( 1 0 , 1 1 ) . There i s a s i g n i f i c a n t r e l a t i o n s h i p between the d e c r e a s e i n number of s e i z u r e s and i n c r e a s i n g VPA serum l e v e l . The r e l a t i o n s h i p between VPA dose and serum l e v e l appears c u r v i l i n e a r ( 1 2 ) . The t h e r a p e u t i c e f f e c t i s o b t a i n e d when the c o n c e n t r a t i o n o f VPA i n plasma i s i n the range of 50-100 ug/mL ( 1 3 ) . The a p p a r e n t volume of d i s t r i b u t i o n of v a l p r o i c a c i d i s i n the range o f 0 . 1 - 0 . 4 L/kg ( 9 , 1 1 , 1 4 ) , due to h i g h plasma p r o t e i n b i n d i n g . T h i s r e l a t i v e l y s m a l l v a l u e i n d i c a t e s t h a t the drug i s d i s t r i b u t e d o n l y i n the c i r c u l a t i o n and the r a p i d l y exchangeable e x t r a c e l l u l a r w a t e r ( 9 ) . VPA i s p r e s e n t a t low c o n c e n t r a t i o n s i n b r a i n , l i v e r , s k e l e t a l m u s c l e , s p l e e n , l u n g , and h e a r t of Rhesus monkey (15) and of man ( 1 6 ) . V a l p r o i c a c i d i s h i g h l y bound t o plasma p r o t e i n s (>90%), m a i n l y t o the a l b u m i n f r a c t i o n ( 1 7 , 1 8 ) . The b i n d i n g of v a l p r o i c a c i d i s c o n c e n -t r a t i o n dependent and the f r e e f r a c t i o n i n plasma i s i n c r e a s e d when t o t a l plasma c o n c e n t r a t i o n s are g r e a t e r t h a n 100 ug/mL ( 1 9 ) . Serum - 3 -p r o t e i n b i n d i n g of VPA i s d e c r e a s e d s i g n i f i c a n t l y i n p a t i e n t s w i t h m i l d and s e v e r e c h o l e s t a s i s (20) and i n uremic c o n d i t i o n s ( 2 1 ) . Plasma h a l f - l i f e v a l u e s f o r v a l p r o i c a c i d are i n the range o f 12-16 hours i n h e a l t h y v o l u n t e e r s and 6-10 hours i n e p i l e p t i c p a t i e n t s ( 1 4 , 2 2 ) . The plasma c l e a r a n c e o f VPA a v e r a g e s 0 . 1 2 mL/min i n h e a l t h y v o l u n t e e r s but i n c r e a s e s t o 0 . 2 3 - 0 . 2 9 mL/min i n p a t i e n t s r e c e i v i n g o t h e r a n t i e p i l e p t i c drugs such as p h e n y t o i n , c a r b a m a z e p i n e , p r i m i d o n e , and p h e n o b a r b i t a l ( 2 3 , 2 4 ) . These d i f f e r e n c e s i n c l e a r a n c e c o u l d be e x p l a i n e d by the i n d u c t i o n of the h e p a t i c d r u g - m e t a b o l i z i n g enzymes by t h e s e a n t i e p i l e p t i c d r u g s . B. Mechanism of A c t i o n The p r e c i s e mode of a c t i o n of VPA i s s t i l l u n c e r t a i n . E x t e n s i v e animal and c l i n i c a l s t u d i e s have e s t a b l i s h e d t h a t VPA a d m i n i s t r a t i o n i n c r e a s e s the l e v e l of the i n h i b i t o r y t r a n s m i t t e r gamma-aminobutyric a c i d (GABA) i n the b r a i n ( 2 5 - 3 0 ) . I n c r e a s e d GABA l e v e l s seem t o be due t o the c o m p e t i t i v e i n h i b i t i o n of GABA m e t a b o l i s m by the enzyme GABA-amino t r a n s f e r a s e (GABA-T) ( 2 6 , 2 7 ) . VPA has a l s o been r e p o r t e d t o have a more p o t e n t a c t i o n on s u c c i n i c s e m i - a l d e h y d e dehydrogenase, the second enzyme i n the d e g r a d a t i o n of GABA ( 3 1 , 3 2 ) . Some i n v e s t i g a t o r s have r e p o r t e d t h a t the i n c r e a s e of GABA l e v e l s i s a l s o due to the e l e v a t i o n of t h e GABA b i o s y n t h e t i c enzyme g l u t a m i c a c i d d e c a r b o x y l a s e (GAD) a f t e r a d m i n i s t r a t i o n of VPA to mice ( 3 3 - 3 5 ) . F u r t h e r m o r e , II L o s c h e r r e p o r t e d t h a t the change i n GAD l e v e l s i n mouse b r a i n was s p e c i f i c t o the synaptosomal f r a c t i o n ( 3 5 ) . In a r e c e n t r e v i e w , S t r o l i n B e n e d e t t i e t a l . (36) mentioned a l a c k of r e p r o d u c i b i l i t y of - 4 -L o s c h e r s r e s u l t s r e g a r d i n g the s t i m u l a t i o n of GAD. S t r o l i n B e n e d e t t i e t a l • (36) a l s o r e p o r t e d t h a t when VPA i s l o c a l l y a p p l i e d by i o n t o p h o r e s i s , there i s a very weak or no e f f e c t on the e l e c t r i c a c t i -v i t y of n e u r o n s . When VPA i s a d m i n i s t e r e d i n t r a p e r i t o n e a l l y a t doses o f 100-400 mg/kg, t h e r e i s a marked i n h i b i t o r y e f f e c t on the s p o n t a n e -ous a c t i v i t y of n e u r o n s . T h i s suggests t h a t a m e t a b o l i t e ( s ) may be p a r t l y r e s p o n s i b l e f o r the a n t i c o n v u l s a n t a c t i v i t y of VPA ( 3 6 ) . VPA i s a l s o known t o i n t e r a c t w i t h s e r o t o n i n , g l y c i n e , a s p a r t a t e and c y c l i c n u c l e o t i d e s ( 3 7 ) . F u r t h e r m o r e , p r e l i m i n a r y data i n d i c a t e t h a t v a l p r o a t e i n t e r a c t s w i t h p o t a s s i u m f l u x e s i m p o r t a n t f o r the c o n t r o l of e p i l e p t o g e n e s i s ( 3 8 ) . C. M e t a b o l i s m V a l p r o i c a c i d i s e l i m i n a t e d i n humans and a n i m a l s m a i n l y by m e t a -b o l i s m ( 3 9 , 4 0 ) . Renal e x c r e t i o n of the unchanged drug r e p r e s e n t s o n l y 1-4% o f the a d m i n i s t e r e d dose ( 1 4 ) . The m e t a b o l i s m of VPA has been e x t e n s i v e l y s t u d i e d i n man ( 4 1 - 4 4 ) , r a t ( 4 5 - 4 8 ) , r a b b i t ( 4 5 ) , dog ( 4 3 , 4 5 ) , and mouse ( 4 3 ) . VPA m e t a b o l i s m i s q u i t e complex and the human m e t a b o l i s m o f VPA i s i l l u s t r a t e d i n F i g u r e 1 . M u l t i p l e m e t a b o l i c p a t h -ways have been proposed f o r the d e g r a d a t i o n of v a l p r o i c a c i d ( 4 9 ) . Two of t h e s e , g l u c u r o n i d a t i o n and B - o x i d a t i o n , are p r e d o m i n a n t . C o n j u g a -t i o n w i t h g l u c u r o n i c a c i d i s a major m e t a b o l i c pathway f o r VPA and i t s m e t a b o l i t e s . In man, about 15-20% of the a d m i n i s t e r e d o r a l dose o f VPA was r e c o v e r e d i n the u r i n e as VPA g l u c u r o n i d e ( 5 0 ) . R e c e n t l y , v a l -p r o y l c a r n i t i n e , a new c o n j u g a t e of v a l p r o i c a c i d has been d i s c o v e r e d and c h a r a c t e r i z e d i n the u r i n e of two p e d i a t r i c p a t i e n t s . The v a l -p r o y l c a r n i t i n e c o n j u g a t e was d e t e c t e d by f a s t atom bombardment-mass - 5 -s p e c t r o m e t r y (FAB-MS) and thermospray h i g h performance l i q u i d c h r o m a t o -graphy-mass s p e c t r o m e t r y (HPLC-MS) ( 5 1 ) . G l y c i n e c o n j u g a t i o n , a l t h o u g h a minor m e t a b o l i c r o u t e f o r VPA, i s of some importance f o r t h e u n s a t u r a t e d m e t a b o l i t e s ( 4 9 ) . The p r o d u c t s of B - o x i d a t i o n , namely 2 - e n e VPA, 3-OH VPA and 3 - k e t o VPA, are the major m e t a b o l i t e s o f VPA i n p l a s m a . O x i d a t i o n i n t h e . w - p o s i t i o n y i e l d s 5-OH VPA, and 2 - p r o p y l -g l u t a r i c a c i d . The (w-1) o x i d a t i o n pathway l e a d s t o the f o r m a t i o n of 4 - OH VPA, 4 - k e t o VPA and 2 - p r o p y l s u c c i n i c a c i d . By a d m i n i s t r a t i o n of 5 - 0H VPA t o r a t , Granneman e t a l . (49) showed t h a t 2 - p r o p y l g l u t a r i c a c i d was the t e r m i n a l p r o d u c t of the w - o x i d a t i o n pathway. In a s i m i l a r manner, a d m i n i s t r a t i o n o f 4-OH VPA r e s t r i c t e d (w-1) m e t a b o l i t e s t o 4 - k e t o VPA and 2 - p r o p y l s u c c i n i c a c i d . The h y d r o x y l a t e d m e t a b o l i t e s 5 - OH VPA and 4-OH VPA were not m e t a b o l i c a l l y dehydrated to form u n s a t u r a t e d m e t a b o l i t e s . C o n v e r s e l y , a d m i n i s t r a t i o n of 3-ene VPA and 4 - e n e VPA t o r a t s produced n e g l i g i b l e amounts o f 4-OH VPA and 5-OH VPA, t h e r e f o r e d e m o n s t r a t i n g t h a t 3 - and 4-ene VPA b e l o n g to a d i f f e r e n t m e t a b o l i c r o u t e than the w and (w-1) o x i d a t i o n pathways. 3-Ene VPA and 4-ene VPA o r i g i n a t e through d e h y d r o g e n a t i o n . These m e t a b o l i t e s can undergo m u l t i p l e m e t a b o l i c r e a c t i o n s to form p o l y u n s a t u r a t e d metabo-l i t e s of v a l p r o i c a c i d ( F i g u r e 1 ) . R e c e n t l y , P r i c k e t t and B a i l l i e (52) showed t h a t i n c u b a t i o n of v a l p r o i c a c i d w i t h r a t l i v e r microsomes l e d to the f o r m a t i o n o f 3 - , 4 -and 5-OH VPA. 3-OH VPA which was thought t o a r i s e o n l y from the B - o x i d a t i o n pathway was thus shown t o be a p r o d u c t of the (w-2) o x i d a t i o n which i s c a t a l y z e d by the cytochrome P-450 i n r a t l i v e r microsomes ( 5 2 ) . CH,—CH,—CH CHCOOGLu VALPROIC ACID OH C H 2 - C H 2 - C H 2 CHCOOH C H 3 - C H 2 - C H 2  5-OH VPA I HOOC—CH 2—CH 2 CHCOOH C H 3 - C H 2 - C H 2 I 2-Propy.glutar ic acid CH^"— CHg—CH 2 CHj— CH2~— CH 2 ^CHCOOH 1 OH CH,—CH—CH, 3 ' \ C H 3 — C H ^ C H ^ 4-OH VPA CHCOOH I C H 3 — C - C H 2 CHCOOH C H 3 - C H 2 - C H 2 4-Keto VPA I C H 3 - C H 2 - C H 2 - C H ( ^ COOH COOH HOOC-CH 2-C H 3 — C H 2 - C H 2 CHCOOH 2-Propyl ma Ionic ac id 2 - P r o p y l 5 u c c i n i c ac id F i g u r e 1. Human M e t a b o l i s m o f V a l p r o i c A c i d . C H 2 = C H - C H 2 C H - j - O ^ - C H ^ 4-ene VPA CHCOOH C H 2 - C H - C H 2 C H p C H - C H 2  4,4 ' -d iene VPA CHCOOH CH,-CH=CH C H j - C H g - C H ^ 3-ene VPA CHCOOH C H 3 - C H 2 - % C H ^ C H g - C H ^ 2-ene VPA C—COOH 1 / 0 H C H 3 — CH 2 —CH CH-COOH C H ^ C H - C H C-COOH CHj—CH 2 —CH^ (E) 2,4-diene VPA CH-—CH=CH 3 \ ^ C - C O O H C H — C H 2 — C H 2(E),3'(E)-d1ene VPA 3-OH VPA C H 3 - C H 2 - C ^ C H 3 - C H 2 - C H 2 CHCOOH 3-Keto VPA - 7 -1 . I d e n t i f i c a t i o n o f VPA M e t a b o l i t e s The i d e n t i f i c a t i o n of VPA m e t a b o l i t e s i s c o m p l i c a t e d by the c h e m i -c a l l a b i l i t y of some of the m e t a b o l i t e s under the a c i d i c c o n d i t i o n s used f o r the e x t r a c t i o n . F o r e x a m p l e , 3 - k e t o VPA d e c a r b o x y l a t e s t o 3 - heptanone and 4-OH VPA and 5-0H VPA form y and 6 l a c t o n e s r e s p e c t i v e -l y . The major m e t a b o l i t e s i n plasma of e p i l e p t i c p a t i e n t s on VPA t h e r a p y were found t o be 2-ene VPA (10% o f VPA c o n c e n t r a t i o n ) , 3-OH VPA (1-3%) and 3 - k e t o VPA (4-8%) ( 4 0 , 5 3 ) . VPA g l u c u r o n i d e and 3 - k e t o VPA (>20%) r e p r e s e n t the major u r i n a r y m e t a b o l i t e s ( 5 4 ) . R e c e n t l y , s e v e r a l d o u b l y u n s a t u r a t e d VPA m e t a b o l i t e s have been r e p o r t e d i n u r i n e and plasma of e p i l e p t i c p a t i e n t s r e c e i v i n g t h e r a p e u t i c doses o f VPA ( 5 5 ) . Two d i e n e s were i d e n t i f i e d as the 4 , 4 ' - d i e n e VPA and ( E ) - 2 , 4 - d i e n e VPA. The presence of the 4 , 4 ' - d i e n e VPA i s c o n s i d e r e d abnormal s i n c e i t i s n e v er found i n p a t i e n t s w i t h o u t severe t o x i c m a n i f e s t a t i o n s ( 5 5 , 5 6 ) . In p a t i e n t s w i t h r e v e r s i b l e or no s i d e - e f f e c t s the diene m e t a b o l i t e s i n u r i n e and serum r e p r e s e n t a p p r o x i m a t e l y 0 . 5 t o 5% o f the sum of v a l -p r o i c a c i d p l u s i t s m e t a b o l i t e s ( 5 5 ) . In one case of f a t a l h e p a t i c f a i l u r e the sum of 4 d i f f e r e n t d i e n e s - V P A was g r e a t l y i n c r e a s e d i n u r i n e and serum (14-25%) w i t h the abnormal 4 , 4 ' - d i e n e VPA r e p r e s e n t i n g 4 - 7% o f the sum of VPA and i t s m e t a b o l i t e s ( 5 6 ) . T h i s s u g g e s t s t h a t appearance of s i d e - e f f e c t s and h e p a t i c d y s f u n c t i o n a s s o c i a t e d w i t h the p r e s e n c e of 4 - 4 ' d i e n e VPA may i n d i c a t e a m e t a b o l i c a b e r r a t i o n i n which case v a l p r o i c a c i d t h e r a p y s h o u l d be d i s c o n t i n u e d . The m e t a b o l i s m of 4-ene VPA which appears to be a q u a n t i t a t i v e l y i m p o r t a n t m e t a b o l i t e o f VPA i n cases o f VPA - induced h e p a t o t o x i c i t y , has been r e c e n t l y examined i n the i s o l a t e d p e r f u s e d r a t l i v e r ( 5 7 ) . - 8 -In t h i s s t u d y , R e t t e n m e i e r e t a l . (57) have r e p o r t e d the presence o f e i g h t m e t a b o l i t e s of 4-ene VPA. They showed i n d i r e c t e v i d e n c e of the f o r m a t i o n of c h e m i c a l l y r e a c t i v e i n t e r m e d i a t e s such as 3 - o x o - 4 - e n e VPA CoA as the r e a c t i v e s p e c i e s r e s p o n s i b l e f o r the i n h i b i t i o n of f a t t y a c i d B - o x i d a t i o n . 2 . A n t i c o n v u l s a n t A c t i v i t y o f VPA M e t a b o l i t e s VPA shows a l a t e onset of t h e r a p e u t i c e f f e c t s a f t e r a d m i n i s t r a t i o n o f the drug (58-59) and c a r r y - o v e r e f f e c t s when t h e r a p y i s d i s c o n t i n u e d ( 6 0 ) . These e f f e c t s c o u l d be e x p l a i n e d by the f o r m a t i o n of a c t i v e m e t a b o l i t e s and/or t h e i r slow a c c u m u l a t i o n i n the b r a i n . The a n t i c o n v u l s a n t a c t i v i t y of s e v e r a l m e t a b o l i t e s o f VPA has been t e s t e d by measuring the e f f e c t on the t h r e s h o l d s f o r e l e c t r o - and p e n t e t r a z o l e - i n d u c e d c o n v u l s i o n s i n mice ( 6 1 , 6 2 ) . The m e t a b o l i t e s t e s t e d were: 2-ene VPA, 3-ene VPA, 4 - e n e VPA, 3-OH VPA, 4-OH VPA, 5-OH VPA, 3 - k e t o VPA and 2 - p r o p y l g l u t a r i c a c i d . A l l m e t a b o l i t e s i n c r e a s e d the t h r e s h o l d f o r maximal e l e c t r o c o n v u l s i o n s e x c e p t 5-OH VPA which was not a c t i v e . The 2 - e n e VPA and 4 - e n e VPA were the most a c t i v e compounds d i s p l a y i n g 60-90% o f the potency of VPA. However, these r e s u l t s d i d not t a k e i n t o a c c o u n t p o s s i b l e d i f f e r e n c e s i n t i s s u e d i s t r i b u t i o n . The p o s s i b i l i t y of a l o n g h a l f l i f e or of an i r r e v e r s i b l e , c o v a l e n t b i n d i n g of one or more m e t a b o l i t e s must be taken i n t o a c c o u n t when a s s e s s i n g t h e i r a n t i c o n v u l s a n t p o t e n c i e s . More r e c e n t s t u d i e s have shown t h a t a f t e r a c u t e and p r o l o n g e d t r e a t m e n t w i t h VPA, o n l y VPA and 2-ene VPA a r e d e t e c t a b l e i n the b r a i n of dogs and r a t s a l t h o u g h s e v e r a l o t h e r m e t a b o l i t e s are p r e s e n t i n the plasma ( 6 2 ) . F u r t h e r m o r e , 2 - e n e VPA was - 9 -shown t o accumulate i n some b r a i n r e g i o n s and i n mice was found t o be 1 . 3 t i m e s more p o t e n t than VPA when c a l c u l a t i o n s were based on whole b r a i n c o n c e n t r a t i o n s ( 6 3 ) . D. S i d e - E f f e c t s and T o x i c i t y A l t h o u g h v a l p r o i c a c i d i s c o n s i d e r e d t o be a r e l a t i v e l y s a f e a n t i -c o n v u l s a n t , some s i d e - e f f e c t s are commonly o b s e r v e d . Nausea and v o m i t -i n g , g a s t r o i n t e s t i n a l d i s c o m f o r t , and d i a r r h e a have been most f r e q u e n t -l y r e p o r t e d a f t e r a d m i n i s t r a t i o n o f VPA ( 6 4 - 6 6 ) . The i n c i d e n c e of s i d e e f f e c t s has been reduced by the i n t r o d u c t i o n of e n t e r i c - c o a t e d t a b l e t s . N e u r o l o g i c a l and p s y c h i a t r i c d i s o r d e r s ( s e d a t i o n , r e v e r s i b l e t r e m o r , s t u p o r o u s s t a t e , c o n f u s i o n a l e p i s o d e s ) have a l s o been r e p o r t e d ( 6 7 - 7 1 ) . B l o o d d i s o r d e r s , m o s t l y t h r o m b o c y t o p e n i a and p l a t e l e t d y s f u n c t i o n , o c c u r r e d m a i n l y i n c h i l d r e n and a d o l e s c e n t s ( 7 2 - 7 4 ) . P a n c r e a t i t i s has r e c e n t l y been 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 a d m i n i s t r a t i o n ( 7 5 - 7 7 ) . V a l p r o i c a c i d was a l s o shown t o induce hyperammonemia (78-81) w i t h or w i t h o u t a s s o c i a t e d c l i n i c a l s i g n s . V a l p r o a t e was found t o i n h i b i t urea s y n t h e s i s i n r a t h e p a t o c y t e s , t o d e c r e a s e c o n c e n t r a t i o n s of N - a c e t y l g l u t a m a t e , t o decrease the c e l l u l a r c o n c e n t r a t i o n of a c e t y l - C o A (a s u b s t r a t e of N - a c e t y l g l u t a m a t e s y n t h e t a s e ) and t o d e c r e a s e c e l l u l a r c o n c e n t r a t i o n s of g l u t a m a t e , a s p a r t a t e and c i t r u l l i n e ( 8 2 ) . Warter e t  a l . ( 8 3 , 8 4 ) a t t r i b u t e d the cause of the hyperammonemia t o a r e n a l o r i g i n ( a c c e l e r a t e d c a t a b o l i s m or reduced s y n t h e s i s of g l u t a m i n e i n the k i d n e y ) r a t h e r than t o h e p a t i c d y s f u n c t i o n . Plasma c a r n i t i n e c o n -c e n t r a t i o n s were found t o be l o w e r and b l o o d ammonia v a l u e s h i g h e r i n p a t i e n t s t r e a t e d w i t h VPA than i n c o n t r o l p a t i e n t s ( 8 5 ) . A s i g n i f i c a n t - 10 -i n v e r s e r e l a t i o n s h i p was found between pjasma c a r n i t i n e c o n c e n t r a t i o n s and dosage o f VPA. A f t e r o r a l i n g e s t i o n of D , L - c a r n i t i n e f o r f o u r w e e k s , both hyperammonemia and c a r n i t i n e d e f i c i e n c y were c o r r e c t e d . T h i s i n d i c a t e d t h a t reduced c a r n i t i n e may be one of the p o s s i b l e c a u s e s o f hyperammonemia ( 8 5 ) . The main r o l e of c a r n i t i n e i s the t r a n s p o r t of l o n g - c h a i n f a t t y a c i d s i n t o m i t o c h o n d r i a . The t r a n s p o r t i n t o m i t o c h o n -d r i a of s h o r t - c h a i n f a t t y a c i d s such as VPA c o u l d be a f f e c t e d through the a c t i o n of a c e t y l c a r n i t i n e t r a n s f e r a s e . At p r e s e n t t h e r e i s no r e a l e x p l a n a t i o n f o r the c a r n i t i n e d e f i c i e n c y observed i n V P A - t r e a t e d p a t i e n t s . V a l p r o a t e was a l s o shown t o induce h y p e r g l y c i n e m i a and h y p e r g l y c i n u r i a ( 6 7 ) . S e v e r a l forms o f h e p a t i c i n j u r y have been r e p o r t e d i n a s s o c i a t i o n w i t h v a l p r o i c a c i d t h e r a p y . The most common form c o n s i s t s of an i n -c r e a s e of h e p a t i c enzymes which r e t u r n t o normal when VPA dose i s l o w e r e d . The second form c o n s i s t s of h e p a t i t i s which may l e a d to h e p a -t i c n e c r o s i s . The t h i r d f o r m , m a i n l y seen i n c h i l d r e n so f a r , i s a v e r y s e v e r e h e p a t o t o x i c i t y r e s e m b l i n g R e y e ' s syndrome (RS) which i s an a c u t e o f t e n f a t a l d i s e a s e t h a t u s u a l l y f o l l o w s a v i r a l i l l n e s s ( e . g . c h i c k e n pox or i n f l u e n z a ) . I t s c l i n i c a l m a n i f e s t a t i o n s i n c l u d e among many s i g n s a f a t t y d e g e n e r a t i o n of the l i v e r , e n c e p h a l o p a t h y , h y p e r -ammonemia, a n o r e x i a , l e t h a r g y , v o m i t i n g , and severe m e t a b o l i c a c i d o s i s ( 8 6 ) . The mechanism of t h i s s e v e r e h e p a t o t o x i c i t y i s s t i l l unknown. Jeavons has a n a l y z e d 67 c a s e s (87) and d i s c u s s e d the p a r t p l a y e d by v a l p r o i c a c i d . In many c a s e s , p a t i e n t s were a l r e a d y s e v e r e l y i l l and/ or under p o l y t h e r a p y . T o x i c VPA m e t a b o l i t e s are s u s p e c t e d t o be r e s p o n s i b l e f o r the h e p a t i c l e s i o n s which are c h a r a c t e r i z e d by a - 11 -m i c r o v e s i c u l a r s t e a t o s i s accompanied sometimes by c i r r h o s i s or n e c r o s i s ( 8 8 ) . These l e s i o n s resemble those seen i n Jamaican v o m i t i n g s i c k n e s s (JVS) and R e y e ' s syndrome (89-91) and have been reproduced by a d m i n i s t r a t i o n o f 4 - p e n t e n o i c a c i d to r a t s ( 9 2 ) . C o a d m i n i s t r a t i o n o f o t h e r a n t i c o n v u l s a n t s such as p h e n o b a r b i t a l c o u l d a l s o c o n t r i b u t e t o t h e VPA h e p a t o t o x i c i t y by enzyme i n d u c t i o n . The h e p a t o t o x i c i t y i s p o s s i b l y e x p l a i n e d on the b a s i s of an i d i o s y n c r a t i c m e t a b o l i c pathway. Kochen e t a l . (56) have r e p o r t e d an i n c r e a s e of s e v e r a l u n s a t u r a t e d m e t a b o l i t e s of v a l p r o i c a c i d i n a case of f a t a l h e p a t i c f a i l u r e . U n s a t u r a t e d m e t a b o l i t e s i n c l u d i n g 4-ene VPA were i n c r e a s e d t o l e v e l s 4-5 t i m e s the c o n t r o l s and f i v e doubly u n s a t u r a t e d m e t a b o l i t e s have been r e p o r t e d ( 5 5 ) . I t i s not known whether t h e s e u n s a t u r a t e d m e t a b o l i t e s are the cause or the e f f e c t o f the h e p a t o t o x i c i t y syndrome. O v e r a l l the i n c i d e n c e of s e r i o u s t o x i c i t y w i t h VPA remains r e l a t i v e l y low and the f r e q u e n c y of f a t a l h e p a t i c t o x i c i t y has been e s t i m a t e d t o be about 1 i n 2 0 , 0 0 0 . T e s t i n g serum a m i n o t r a n s f e r a s e s i s of some v a l u e i n the f i r s t s i x months of t h e r a p y ' when the f r e q u e n c y of h e p a t o t o x i c i t y i s g r e a t e s t e s p e c i a l l y i n p r e -d i s p o s e d p a t i e n t s w i t h mental r e t a r d a t i o n , s t r u c t u r a l b r a i n damage or m e t a b o l i c d i s o r d e r s . When a b n o r m a l i t i e s of serum a m i n o t r a n s f e r a s e s are d e t e c t e d , VPA dose s h o u l d be lowered or t r e a t m e n t d i s c o n t i n u e d ( 8 6 ) . High doses o f VPA (750 mg/kg) were shown to produce m i c r o v e s i c u l a r s t e a t o s i s i n r a t s as d i d lower doses of VPA (350 mg/kg) a f t e r p r e t r e a t -ment w i t h p h e n o b a r b i t a l ( 9 3 ) . The s t e a t o g e n i c e f f e c t o f VPA seemed m e d i a t e d by a t o x i c m e t a b o l i t e , the f o r m a t i o n of which may be enhanced by the i n d u c i n g e f f e c t s of the h e p a t i c m i x e d - f u n c t i o n o x i d a s e s y s t e m . - 12 -I n c r e a s e d amounts of d i c a r b o x y l i c a c i d s ( a d i p i c , s u b e r i c , s e b a c i c a c i d s ) i n u r i n e from p a t i e n t s and r a t s g i v e n v a l p r o i c a c i d have been r e p o r t e d ( 9 4 ) . D i c a r b o x y l i c (Cs-Cio) a c i d u r i a has a l s o been a s s o c i a t e d w i t h c a r n i t i n e d e f i c i e n c y and Jamaican v o m i t i n g s i c k n e s s ( 9 4 ) . V a l p r o a t e has been shown t o i n h i b i t f a t t y a c i d o x i d a t i o n p o s s i b l y by b e i n g an u n p h y s i o l o g i c a l s u b s t r a t e f o r the B - o x i d a t i o n p r o c e s s . In c o n c e n t r a t i o n s s i m i l a r t o t h a t of v a l p r o a t e t h e r a p y i n man, VPA i n h i -b i t e d p y r u v a t e and p a l m i t a t e o x i d a t i o n , urea s y n t h e s i s and g l u c o n e o -g e n e s i s i n i s o l a t e d r a t h e p a t o c y t e s ( 9 5 ) . VPA a l s o lowered B - h y d r o x y -b u t y r a t e / a c e t o a c e t a t e and l a c t a t e / p y r u v a t e r a t i o s , and a c e t y l - C o A l e v e l s ( 9 6 ) . These changes seem t o be due t o the a c c u m u l a t i o n of v a l p r o y l - C o A and i t s m e t a b o l i t e s i n the m a t r i x of h e p a t i c m i t o c h o n d r i a , t h u s i n t e r f e r i n g w i t h B - o x i d a t i o n of p h y s i o l o g i c a l l o n g - c h a i n f a t t y a c i d s . T h i s i n t e r f e r e n c e i s n o r m a l l y w e l l compensated but i n cases o f m u l t i p l e drug t h e r a p y or i n b o r n e r r o r s of m e t a b o l i s m i t can l e a d t o i m p a i r e d l i v e r f u n c t i o n . There i s some e v i d e n c e t h a t the i n c i d e n c e of m a l f o r m a t i o n s i s i n c r e a s e d i n e p i l e p t i c women under v a l p r o i c a c i d t h e r a p y ( 9 7 - 9 8 ) , but t h e r i s k of f e t a l m a l f o r m a t i o n s i s a l s o h i g h e r i n e p i l e p t i c mothers who are not t a k i n g d r u g s . The most f r e q u e n t m a l f o r m a t i o n i s n e u r a l tube d e f e c t s ( 9 9 , 1 0 0 ) . The r o l e of v a l p r o a t e i n the e t i o l o g y of t h i s m a l f o r m a t i o n i s s t i l l u n c e r t a i n ( 8 7 ) . The r i s k of s p i n a b i f i d a i n p a t i e n t s under VPA t h e r a p y i s e s t i m a t e d a t a p p r o x i m a t e l y 1% ( 9 9 ) . E. I n t e r a c t i o n between VPA and ASA In v i t r o s a l i c y l a t e has been r e p o r t e d to d i s p l a c e v a l p r o i c a c i d from b i n d i n g t o plasma p r o t e i n ( 1 0 1 , 1 0 2 ) . Schobben e t a l . (101) - 13 -r e p o r t e d an i n c r e a s e of v a l p r o a t e e x c r e t i o n i n v o l u n t e e r s when s a l i c y l a t e was a d m i n i s t e r e d . A s t u d y i n Rhesus monkey r e p o r t e d an i n c r e a s e i n f r e e f r a c t i o n o f VPA and an i n c r e a s e d v a l p r o a t e c l e a r a n c e when s a l i c y l a t e was c o a d m i n i s t e r e d ( 1 0 3 ) . Orr e t a l . (104) have o b s e r v e d an i n c r e a s e d f r e e f r a c t i o n o f VPA i n serum and a d e c r e a s e d i n t r i n s i c c l e a r a n c e i n p e d i a t r i c p a t i e n t s . S i n c e ASA i s l a r g e l y e l i m i n a t e d as c o n j u g a t e d m e t a b o l i t e s , a c o m p e t i t i o n between VPA g l u c u r o n i d e and s a l i c y l a t e c o n j u g a t e s was expected ( 1 0 4 , 1 0 5 ) . However, t h e r e was no d e c r e a s e i n t h e renal c l e a r a n c e o f VPA c o n j u g a t e s i n t h e p r e s e n c e of s a l i c y l a t e . These r e s u l t s suggested t h a t o t h e r m e t a b o l i t e s o f VPA must be a f f e c t e d by s a l i c y l a t e c o - a d m i n i s t r a t i o n . The e f f e c t s o f s a l i c y l a t e on VPA m e t a b o l i s m are t h e r e f o r e s t i l l u n c l e a r and a n a l y -s i s of t h e major VPA m e t a b o l i t e s i s r e q u i r e d t o d e t e r m i n e t h e m e t a b o l i c e f f e c t s of s a l i c y l a t e . It has been f r e q u e n t l y r e p o r t e d t h a t s a l i c y l a t e s have a d e l e t e r -i o u s e f f e c t on h e p a t i c f u n c t i o n but i t i s not c l e a r whether t h i s i s due t o s a l i c y l a t e " p e r s e " o r t o an u n d e r l y i n g l i v e r d i s e a s e which i s i n t e n s i f i e d by s a l i c y l a t e . S a l i c y l a t e s are known t o d e c r e a s e l i p o g e n e -s i s by p a r t i a l l y b l o c k i n g i n c o r p o r a t i o n of a c e t a t e i n t o f a t t y a c i d s . F u r t h e r m o r e , s a l i c y l a t e s i n h i b i t l i p o l y s i s i n f a t c e l l s and d i s p l a c e t h e f a t t y a c i d s from b i n d i n g s i t e s on plasma p r o t e i n s , thus i n c r e a s i n g t h e e n t r y and o x i d a t i o n of f a t t y a c i d s w i t h i n t h e t i s s u e s ( 1 0 6 ) . S a l i c y l a t e s have been found t o d e c r e a s e the i n h i b i t o r y e f f e c t s of VPA on f a t t y a c i d o x i d a t i o n by i n h i b i t i n g t h e f o r m a t i o n of v a l p r o y l - C o A and i t s m e t a b o l i t e s , s u s p e c t e d t o be r e s p o n s i b l e f o r the e f f e c t s of VPA on l i p i d m e t a b o l i s m ( 1 0 7 ) . - 14 -Over the l a s t s e v e r a l y e a r s an a s s o c i a t i o n has been o b s e r v e d between R e y e ' s syndrome and ASA i n g e s t i o n ( 1 0 8 , 1 0 9 ) . Both s a l i c y l a t e and v a l p r o i c a c i d have been i n d i c a t e d as c a u s i n g a R e y e - l i k e syndrome. However, t h e r e i s c o n t r o v e r s i a l e v i d e n c e f o r a c a u s a l r e l a t i o n s h i p between ASA and Reye syndrome (110-113). In the case o f VPA, the R e y e - l i k e syndrome might a r i s e from one of the m e t a b o l i t e s ( 9 2 ) . I f s a l i c y l a t e does i n h i b i t VPA m e t a b o l i s m , then the p o t e n t i a l dangers o f g i v i n g both drugs c o n c o m i t a n t l y may i n f a c t be r e d u c e d . F. A n a l y s i s o f V a l p r o i c A c i d and M e t a b o l i t e s G a s - l i q u i d c h r o m a t o g r a p h i c (GLC) methods have been e x t e n s i v e l y used f o r the d e t e r m i n a t i o n of v a l p r o i c a c i d i n body f l u i d s (114-116) . These GLC methods may be c o u p l e d w i t h flame i o n i z a t i o n d e t e c t o r s (117-120) , n i t r o g e n - s e l e c t i v e d e t e c t o r s (121-122) or e l e c t r o n - c a p t u r e d e t e c t o r s ( 1 2 3 ) . D i f f e r e n t e x t r a c t i o n s o l v e n t s have been r e p o r t e d i n the l i t e r a t u r e : c h l o r o f o r m ( 1 1 6 , 1 2 4 , 1 2 5 ) ; carbon t e t r a c h l o r i d e ( 1 2 6 ) ; hexane ( 1 2 7 ) ; e t h y l a c e t a t e ( 5 4 ) ; 10% e t h y l a c e t a t e i n hexane ( 1 2 8 , 1 2 9 ) . The most e f f i c i e n t s o l v e n t f o r the e x t r a c t i o n o f VPA and i t s m e t a b o l i t e s was e t h y l a c e t a t e as r e p o r t e d by Nau e t a l . (54) The o t h e r s o l v e n t s gave poor r e c o v e r i e s f o r the more p o l a r m e t a b o l i t e s of VPA. To improve the gas c h r o m a t o g r a p h i c p r o p e r t i e s of v a l p r o i c a c i d and i t s m e t a b o l i t e s methyl e s t e r d e r i v a t i v e s ( 1 2 7 ) , t r i m e t h y 1 s i l y l e s t e r (TMS) d e r i v a t i v e s ( 4 0 , 5 4 ) , p h e n y l a c y l e s t e r d e r i v a t i v e s (130) and t e r t - b u t y l d i m e t h y l s i l y l e s t e r d e r i v a t i v e s (tBDMS) (129) have been a s s a y e d . - 15 -Enzyme immunoassays ( 1 3 1 , 132) and h i g h performance l i q u i d chroma-t o g r a p h i c a s s a y s (133-134) have a l s o been r e p o r t e d f o r the a n a l y s i s o f v a l p r o i c a c i d . The l a r g e number of v a l p r o i c a c i d m e t a b o l i t e s , some p r e s e n t a t ve ry low c o n c e n t r a t i o n s , and t h e i r s t r u c t u r a l s i m i l a r i t y to endogenous f a t t y a c i d s r e q u i r e v e r y a c c u r a t e methods of a n a l y s i s . Good r e s u l t s u s i n g c a p i l l a r y GLC have been r e p o r t e d i n the l i t e r a t u r e f o r the a n a l y s i s of hydroxy c a r b o x y l i c a c i d s (135) and f a t t y a c i d s ( 1 3 6 ) . C a p i l l a r y GC-MS appears to be the b e s t method f o r the a n a l y s i s o f VPA and i t s m e t a b o l i t e s ( 5 4 ) . A s e n s i t i v e assay u s i n g s e l e c t e d i o n m o n i t o r i n g and t r i m e t h y l s i l y l d e r i v a t i v e s f o r the s i m u l t a n e o u s a n a l y s i s of VPA, 2 - , 3 - , and 4-ene VPA, 3 - , 4 - , and 5-OH VPA, 3 - k e t o VPA, and 2 - p r o p y l g l u t a r i c a c i d has been r e p o r t e d by Nau e t a l • ( 5 4 ) . Some known m e t a b o l i t e s such as 2 - p r o p y l s u c c i n i c a c i d , 4 - k e t o VPA, and d i e n e s - V P A were not d e t e r m i n e d . Wool l a r d e t a l . (137) have r e p o r t e d an i n c r e a s e d s e n s i t i v i t y of the t e r t - b u t y l d i m e t h y l s i l y l (tBDMS) d e r i v a t i v e s of f a t t y a c i d s compared t o t h e TMS d e r i v a t i v e s . Good r e s u l t s o b t a i n e d w i t h t h e tBDMS d e r i v a t i v e s have been r e p o r t e d i n the l i t e r a t u r e ( 1 3 8 - 1 4 0 ) . T h i s l a b o r a t o r y has s u c c e s s f u l l y used tBDMS d e r i v a t i v e s f o r the a n a l y s i s and p h a r m a c o k i n e t i c s of VPA (129,141) and the i d e n t i f i c a t i o n of VPA m e t a b o l i t e s i n serum and u r i n e u s i n g GC-MS ( 4 4 ) . - 16 -OBJECTIVES 1. In t h i s s t u d y s e n s i t i v e and s p e c i f i c assays f o r t w e l v e VPA metabo-l i t e s are t o be d e v e l o p e d u s i n g c a p i l l a r y gas chromatography-mass s p e c t r o m e t r y . Assay development w i l l emphasize the r e s o l u t i o n of 4 - e n e VPA from VPA and w i l l seek t o i n c r e a s e t h e s e n s i t i v i t y and s e l e c t i v i t y of t h e assay f o r a l l m e t a b o l i t e s . The tBDMS d e r i v a -t i v e s o f VPA m e t a b o l i t e s w i l l t h e r e f o r e be i n v e s t i g a t e d t o t a k e advantage of i n t e n s e [ M - 5 7 ] + i o n s f o r s e l e c t e d i o n m o n i t o r i n g . The assay w i l l t e n t a t i v e l y be a p p l i e d t o p a t i e n t samples and w i l l p r o v i d e the o p p o r t u n i t y t o o b t a i n p h a r m a c o k i n e t i c data f o r VPA m e t a b o l i t e s . 2 . VPA m e t a b o l i t e s are t o be measured i n u r i n e samples from c h i l d r e n on v a l p r o i c a c i d t h e r a p y b e f o r e and a f t e r t h e a d m i n i s t r a t i o n o f ASA. The purpose o f t h i s s t u d y i s t o i n v e s t i g a t e t h e e f f e c t s of s a l i c y l a t e on VPA m e t a b o l i s m and t o d e t e r m i n e which m e t a b o l i c pathway(s) i f any i s i n v o l v e d i n t h i s i n t e r a c t i o n . 3 . VPA m e t a b o l i t e s are t o be measured i n serum and u r i n e samples from a h e a l t h y v o l u n t e e r a d m i n i s t e r e d v a l p r o i c a c i d . A f t e r r e a c h i n g s t e a d y s t a t e on VPA t h i s v o l u n t e e r w i l l be a d m i n i s t e r e d ASA. Serum and u r i n e samples w i l l be a n a l y z e d f o r VPA and m e t a b o l i t e s b e f o r e and a f t e r ASA i n g e s t i o n . T h i s study w i l l p r o v i d e pharmaco-k i n e t i c data on VPA m e t a b o l i t e s i n a d d i t i o n t o s u p p o r t i n g data o b t a i n e d from t h e p a t i e n t s t u d i e s . - 17 -I I . EXPERIMENTAL A . REAGENTS AND MATERIALS 1 . VPA M e t a b o l i t e s V a l p r o i c a c i d ( d i - n - p r o p y l a c e t i c a c i d ) was o b t a i n e d from K and K F i n e C h e m i c a l s , ICN P h a r m a c e u t i c a l ( P l a i n v i e w , NY). The m e t a b o l i t e s used f o r the s t a n d a r d c a l i b r a t i o n c u r v e s were s y n t h e s i z e d as r e p o r t e d e l s e w h e r e ( 4 4 ) . These m e t a b o l i t e s are namely 2 - e n e VPA, 3 - e n e VPA, 4 - e n e VPA, 3 - O H VPA, 4-OH VPA, 5-OH VPA, 3 - k e t o VPA, 4 - k e t o VPA, 2 - p r o p y l g l u t a r i c a c i d and 2 - p r o p y l s u c c i n i c a c i d . The d i u n s a t u r a t e d m e t a b o l i t e 2 , 4 - d i e n e VPA was a k i n d g i f t from D r . T. B a i l l i e ( U n i v e r s i t y of W a s h i n g t o n , School o f Pharmacy, S e a t t l e , U . S . A . ) . 2 . I n t e r n a l S t a n d a r d s The 3 - o c t a n o n e (99%) was o b t a i n e d from A l d r i c h Chemical Company ( M i l w a u k e e , WI) . The i n t e r n a l s t a n d a r d , [ 2 H 3 ] - 2 - e n e VPA ( [ 3 , 5 , 5 -2 H ] - 3 - h e p t e n e - 4 - c a r b o x y l i c a c i d ) was p r e p a r e d by d e h y d r a t i o n and h y d r o l y s i s of the c y a n o h y d r i n o b t a i n e d from [ 3 , 3 , 5 , 5 - 2 H ] - 4 - h e p t a n o n e . [ 2 H 4 ] - 4 - H e p t a n o n e was p r e p a r e d from 4 - h e p t a n o n e a f t e r s e v e r a l exchanges w i t h s o l u t i o n s of Na02H i n 2 H 2 0 . P e r c e n t d e u t e r i u m i n c o r p o r a t e d was 95%. A m i x t u r e of c i s - and t r a n s - [ 2 H 3 ] - 2 - e n e VPA ( 1:4 r a t i o ) was o b t a i n e d ; bp 8 8 - 9 1 ° C , 0 . 4 mm Hg, ^H-NMR ( C D C I 3 ) r e l a t i v e t o t e t r a m e t h y l s i l a n e 0 . 8 - 1 . 1 ( m , 6 ) , 1 .2 - 1 . 7 ( m , 2 ) , 2 . 0 - 2 . 6 ( q , 2 H ) , 1 0 - 1 1 . 8 ( b r s , 1 , COOH). - 18 -The s y n t h e s i s of the i n t e r n a l s t a n d a r d [ 2 H6]-VPA has p r e v i o u s -l y been r e p o r t e d (141) . 3 . Reagents C h e m i c a l s were r e a g e n t grade and o b t a i n e d from the f o l l o w i n g s o u r c e s : 1 . MERCK SHARP AND DOHME CANADA LTD. ( M o n t r e a l , Quebec). 1-Bromopropane 3 , 3 , 3 - d 3 , >95 atom % d e u t e r i u m l a b e l l e d as i n d i c a t e d . Deuterium o x i d e , p u r i t y 9 9 . 8 atom % d e u t e r i u m . 2 . CALEDONE (Georgetown, O n t a r i o ) . E t h y l a c e t a t e d i s t i l l e d - i n - g l a s s g r a d e . Methanol d i s t i l l e d - i n - g l a s s g r a d e . 3 . ALDRICH CHEMICAL COMPANY ( M i l w a u k e e , W I . , USA). 4 - H e p t a n o n e . t-BDMS r e a g e n t : t e r t - b u t y l d i m e t h y l s i l y l c h l o r i d e , 97% p u r i t y . 4 - D i m e t h y l a m i n o p y r i d i n e (DMAP), 99% p u r i t y . P y r i d i n e MTBSTFA: N - t e r t - b u t y l d i m e t h y l s i l y l - N - m e t h y l t r i f l u o r o a c e t a m i d e . 4 . BDH CHEMICALS ( C a n a d a ) . Anhydrous sodium s u l f a t e . Sodium h y d r o x i d e . - 19 -5 . FISCHER SCIENTIFIC LTD. (Canada) H y d r o c h l o r i c A c i d 6 . PIERCE CHEMICAL COMPANY ( R o c k f o r d , I l l i n o i s ) . TMS r e a g e n t : N - M e t h y l - N - t r i m e t h y l s i l y l t r i f l u o r o a c e t a m i d e . The tBDMCS r e a g e n t was p r e p a r e d i n p y r i d i n e t o c o n t a i n 5% DMAP. 50 mg DMAP were d i s s o l v e d i n 1 mL dry p y r i d i n e and t h i s m i x t u r e was then added to 1 g of tBDMCS. B. DRUG STUDY The study i n c l u d e d 6 e p i l e p t i c c h i l d r e n and one h e a l t h y v o l u n t e e r . The s i x e p i l e p t i c c h i l d r e n (2 m a l e s , 4 f e m a l e s ) were between the ages o f 4 and 13 y e a r s and weighed between 1 3 . 4 and 56 k g . These c h i l d r e n were a l l under v a l p r o i c a c i d t h e r a p y f o r a t l e a s t two months p r i o r t o the s t u d y . VPA was a d m i n i s t e r e d i n s y r u p form (Dekapene R ) i n the dose range of 18-49 mg/kg/day. F i v e of the c h i l d r e n were r e c e i v i n g VPA o n l y . One c h i l d (VS) was a l s o r e c e i v i n g carbamazepine and a c e t a z o l a m i d e w h i c h were d i s c o n t i n u e d 24 hours p r i o r t o the s t u d y . The p a t i e n t s were a d m i t t e d to the h o s p i t a l 12 hours p r i o r t o the f i r s t study day and a l o w - f a t c a l o r i e - c o n t r o l l e d d i e t was i n i t i a t e d . On the morning of the f i r s t day of the s t u d y , a f t e r an o v e r n i g h t f a s t , each c h i l d r e c e i v e d h i s usual 0800 h dose of VPA. T o t a l v o i d u r i n e samples were c o l l e c t e d a t i n t e r v a l s t h r o u g h o u t t h e 12 h o u r - s a m p l i n g p e r i o d . The second d a i l y dose of VPA was a d m i n i s t e r e d a t 2000 h and a regimen of a c e t y l s a l i c y l i c a c i d i n doses r a n g i n g from - 20 -11-16 mg/kg was i n i t i a t e d e v e r y 6 hours f o r 4 doses i n 4 p a t i e n t s on day 2 a t 0900 h . One p a t i e n t r e c e i v e d a s i n g l e dose of ASA o f 2 9 . 6 mg/kg and the s i x t h p a t i e n t r e c e i v e d a s i n g l e low dose o f 16.4 mg/kg ASA. U r i n e samples were c o l l e c t e d on day 2 as on day 1 . The u r i n e samples were k e p t f r o z e n ( - 2 0 ° C ) u n t i l a n a l y z e d . The h e a l t h y male v o l u n t e e r (age 44, w e i g h t 62 kg) had t a k e n 600 mg v a l p r o i c a c i d t w i c e a day ( a t 0800 h and 2000 h) f o r 4 days and was a t s t e a d y - s t a t e on the morning of the f i r s t day of the study a f t e r an o v e r n i g h t f a s t . B l o o d and u r i n e were c o l l e c t e d b e f o r e the morning 600 mg dose o f VPA. B l o o d samples were then c o l l e c t e d a t 0 . 5 , 1 , 1 . 5 , 2 , 2 . 5 , 3 , 5 , 7 and 8 h o u r s . T o t a l u r i n e samples were c o l l e c t e d a t i n t e r v a l s t h r o u g h o u t t h e 8 hour s a m p l i n g p e r i o d . The second 600 mg d a i l y dose of VPA was a d m i n i s t e r e d a t 2000 h . From 2100 h 650 mg of a c e t y l s a l i c y l i c a c i d were a d m i n i s t e r e d e v e r y 6 hours f o r 4 doses (9 pm, 3 am, 9 am, 3 pm). On the second day of the study b l o o d and u r i n e were c o l l e c t e d b e f o r e the 0800 h dose o f VPA. B l o o d and u r i n e samples were then c o l l e c t e d on day 2 as on day 1 . U r i n e and serum samples were kept f r o z e n ( - 2 0 ° C ) u n t i l a n a l y s i s . C. ANALYSIS 1 . S t o c k S o l u t i o n s o f I n t e r n a l S t a n d a r d s S t o c k s o l u t i o n c o n c e n t r a t i o n s of i n t e r n a l s t a n d a r d [ 2 H 3 ] - 2 - e n e VPA were 100 ug/mL e i t h e r i n 3N NaOH f o r a n a l y s i s of p a t i e n t samples o r i n d i s t i l l e d w a t e r f o r a n a l y s i s of r e f e r e n c e s t a n d a r d s . Stock s o l u t i o n c o n c e n t r a t i o n s of the i n t e r n a l s t a n d a r d s [ 2 H6]-VPA and 3 - o c t a n o n e were r e s p e c t i v e l y 100 ug/mL and 1 mg/mL i n d i s t i l l e d w a t e r . - 21 -2 . P r e p a r a t i o n of U r i n e and Serum S t a n d a r d s Two s e t s of r e f e r e n c e s t a n d a r d s were p r e p a r e d . One s e t i n 3N NaOH was p r e p a r e d by the a d d i t i o n of 3-OH VPA, 4-OH VPA l a c t o n e , 5-OH VPA l a c t o n e , and 3 - k e t o VPA e t h y l e s t e r t o the a l k a l i n e s o l u t i o n and a l l o w i n g these t o d i s s o l v e w i t h g e n t l e s h a k i n g over 5 d a y s . The o t h e r r e f e r e n c e s e t was made i n u r i n e or serum and c o n t a i n e d 4 - e n e VPA, 3-ene VPA, 2-ene VPA, VPA, 4 - k e t o VPA, 2 - p r o p y l s u c c i n i c a c i d and 2 - p r o p y l g l u t a r i c a c i d . F o r each c a l i b r a t i o n p o i n t 0 . 1 mL o f s t a n d a r d i n a l k a l i n e s o l u t i o n was added t o 0 . 9 mL of i t s c o r r e s p o n d i n g s t a n d a r d i n u r i n e or serum a t the time of a n a l y s i s . The c o n c e n t r a t i o n s thus o b t a i n e d i n the m i x t u r e were 2 , 5 , 1 0 , 1 5 , 30 and 45 ng/ml f o r 4-OH VPA and 5-0H VPA; 0 . 1 8 , 0 . 4 5 , 0 . 9 0 , 4 . 5 , 9 , and 1 3 . 5 ug/mL f o r 4-ene VPA, 3 - e n e VPA, 4 - k e t o VPA and 2 - p r o p y l s u c c i n i c a c i d ; 0 . 3 6 , 0 . 9 0 , 1 . 8 , 9 , 18, and 27 pg/mL f o r 2-ene VPA; 1 8 , 3 6 , 5 4 , 7 2 , 108 and 144 ug/mL f o r VPA; 5 , 1 0 , 2 5 , 5 0 , 100 and 150 ng/ml f o r 3 - k e t o VPA, 0 . 3 6 , 0 . 9 0 , 1 . 8 0 , 4 . 5 , 1 5 , 18 ug/mL f o r 2 - p r o p y l g l u t a r i c a c i d and f i n a l l y 2 , 5 , 1 0 , 1 5 , 2 0 , and 25 ug/mL f o r 3-OH VPA. The c a l i b r a t i o n c u r v e s were o b t a i n e d by a p l o t of the a r e a r a t i o of the m e t a b o l i t e o r VPA peak t o t h a t of the i n t e r n a l s t a n d a r d v e r s u s t h e c o n c e n t r a t i o n of the m e t a b o l i t e . [ ^ l - V P A was used as the i n t e r n a l s t a n d a r d f o r VPA. The [ 2 H 3 ] - ( E ) - 2 - e n e VPA was used as the i n t e r n a l s t a n d a r d f o r a l l the o t h e r m e t a b o l i t e s . The 3 - o c t a n o n e was i n i t i a l l y i n v e s t i g a t e d as an i n t e r n a l s t a n d a r d f o r 4-OH VPA l a c t o n e but was e v e n t u a l l y used t o m o n i t o r the e v a p o r a t i o n of the e x t r a c t . A s t a n d a r d c u r v e was run p r i o r t o each b a t c h of p a t i e n t s a m p l e s . - 22 -3 . E x t r a c t i o n and D e r i v a t i z a t i o n o f S t a n d a r d s and P a t i e n t Samples The e x t r a c t i o n and d e r i v a t i z a t i o n procedure of s t a n d a r d s and p a t i e n t s samples i s i l l u s t r a t e d i n scheme 1 . To 0 . 1 mL of s t a n d a r d i n a l k a l i n e s o l u t i o n were added 0 . 9 mL o f i t s c o r r e s p o n d i n g s t a n d a r d i n u r i n e or serum and 100 uL of each of t h r e e i n t e r n a l s t a n d a r d s o l u t i o n s . T h i s m i x t u r e a t pH 1 2 . 5 - 1 3 was i n c u b a t e d 1 hour a t 6 0 ° C t o h y d r o l y z e c o n j u g a t e d m e t a b o l i t e s {mainly V P A - g l u c u r o n i d e ) . A f t e r c o o l i n g the m i x t u r e was brought t o pH 2 w i t h 110 uL o f 4N HC1 and l e f t a t room temperature f o r 15 m i n . The m i x t u r e was then e x t r a c t e d w i t h 3 mL o f e t h y l a c e t a t e by g e n t l e r o t a t i o n f o r 20 m i n . T h i s l a t t e r procedure was r e p e a t e d i n the case of serum t o ensure complete e x t r a c t i o n . The o r g a n i c phase was t r a n s f e r e d and v o r t e x - m i x e d w i t h anhydrous sodium s u l f a t e , and c e n t r i f u g e d (5 m i n , 2 , 0 0 0 rpm, 2 5 ° ) . The s u p e r n a t a n t ( a p p r o x i m a t e l y 2 . 4 mL) was t r a n s f e r e d to a c o n i c a l tube and c o n c e n -t r a t e d t o 200 uL under a n i t r o g e n s t r e a m . To o b t a i n tBDMS d e r i v a t i v e s 50 uL o f tBDMCS r e a g e n t c o n t a i n i n g 5% c a t a l y s t (DMAP) were added to the c o n c e n t r a t e d e x t r a c t i n a c o n i c a l r e a c t i o n v i a l and the m i x t u r e heated a t 6 0 ° C f o r 4 h o u r s . TMS d e r i v a t i v e s were o b t a i n e d by a d d i t i o n o f 50 uL of MSTFA r e -agent to the c o n c e n t r a t e d e x t r a c t , the m i x t u r e was then heated a t 60° f o r 20 - 30 m i n . The tBDMS-TMS mixed d e r i v a t i v e s were o b t a i n e d by a d d i t i o n of 50 uL of MSTFA r e a g e n t to the tBDMS d e r i v a t i v e s p r e p a r e d from the r e a g e n t i n p y r i d i n e and h e a t i n g a t 60° f o r a f u r t h e r 10 m i n . In a l l c a s e s , 1 uL of d e r i v a t i z e d e x t r a c t was i n j e c t e d i n t o the gas chromatograph - mass s p e c t r o m e t e r . - 23 -URINE INTERNAL STANDARDS (pH 13, 100 uL) SERUM | (1 mL) | 2-ENE VPA - D 3, 3-0CTAN0NE, VPA-D 6 I INCUBATE 1 H, 60* I ADJUST TO pH 2.0, 4N HCL I EXTRACT WITH ETHYL ACETATE (3 mL) I DRY OVER ANHYDROUS N a 2 S 0 4 I CONCENTRATE TO 200 uL ( N 2 ) I DERIVATIZE WITH t-BDMCS REAGENT {5% CATALYST), 60', 4H OR MSTFA REAGENT, 60", 20 - 30 MIN. SCHEME 1. EXTRACTION AND DERIVATIZATION PROCEDURE - 24 -For p a t i e n t samples 1 mL u r i n e or serum was taken and 100 uL o f i n t e r n a l s t a n d a r d [ 2 H 3 ] - 2 - e n e VPA i n 3N NaOH and 100 uL o f each of the o t h e r two i n t e r n a l s t a n d a r d s o l u t i o n s were added. A l l o t h e r s t e p s were c a r r i e d out i n e x a c t l y the same manner as the c a l i b r a t i o n p r o c e -d u r e . In p a t i e n t samples where the a v a i l a b l e volume of u r i n e or serum was l e s s than 1 mL, a known volume was p i p e t t e d and d i l u t e d w i t h d i s t i l l e d water t o a f i n a l volume o f 1 mL. C o r r e c t i o n s were made f o r the d i l u t i o n f a c t o r i n c a l c u l a t i o n s . 4 . D e t e r m i n a t i o n o f u r i n a r y c r e a t i n i n e U r i n a r y c r e a t i n i n e l e v e l s were determined by the q u a n t i t a t i v e f o r m a t i o n of a r e d complex between a l k a l i n e p i c r i c a c i d and c r e a t i n i n e ( J a f f e r e a c t i o n ) ( 1 4 2 ) . A s t a n d a r d c u r v e was p r e p a r e d and a n a l y z e d t o g e t h e r w i t h the p a t i e n t s ' s a m p l e s . The s p e c t r o p h o t o m e t r y a n a l y s i s was c a r r i e d out a t 500 nm. 5 . I n s t r u m e n t a t i o n The assay was performed on a H e w l e t t - P a c k a r d 5987A gas chromato-graph - mass s p e c t r o m e t e r . O p e r a t i n g c o n d i t i o n s were s o u r c e t e m p e r a -t u r e 2 0 0 ° , t r a n s f e r l i n e temperature 2 4 0 ° , i n j e c t i o n p o r t temperature 240° and h e l i u m ( c a r r i e r gas) f l o w r a t e 1 mL/min. The mass s p e c t r o -meter was o p e r a t e d w i t h an e l e c t r o n i o n i z a t i o n energy of 70 e V . The c a p i l l a r y column used was an 0V 1701 bonded p h a s e , 25 m x 0 . 3 2 mm I . D . w i t h a f i l m t h i c k n e s s o f 0 . 2 5 u (Quadrex S c i e n t i f i c , New Haven, C o n n e c t i c u t ) . For tBDMS and mixed d e r i v a t i v e s the column oven i n i t i a l t e m p e r a t u r e was 50°C ( z e r o h o l d t i m e ) and programmed a t 3 0 ° / m i n t o - 25 -1 0 0 ° C , t h e n 8 ° / m i n from 1 0 0 ° C t o a f i n a l temperature o f 260°C ( w i t h a 1 min h o l d t i m e ) . T o t a l run time was a p p r o x i m a t e l y 22 m i n . F o r TMS d e r i v a t i v e s , the i n i t i a l oven temperature was 7 5 ° C (10 min h o l d t i m e ) and the r a t e of i n c r e a s e of temperature was 8 ° / m i n from 7 5 ° C t o 200°C then 3 0 ° / m i n to a f i n a l temperature o f 260°C ( w i t h a 5 min h o l d t i m e ) . The t o t a l time r e q u i r e d was a p p r o x i m a t e l y 30 m i n . - 26 -I I I . RESULTS AND DISCUSSION A. A n a l y s i s o f VPA and i t s M e t a b o l i t e s The q u a n t i t a t i v e and s i m u l t a n e o u s d e t e r m i n a t i o n of v a l p r o i c a c i d and t w e l v e of i t s m e t a b o l i t e s , namely 2-ene VPA, 3 - e n e VPA, 4 - e n e VPA, ( E ) - 2 , 4 - d i e n e VPA, ( E , E ) - 2 , 3 ' - d i e n e VPA, 3-OH VPA, 4-OH VPA, 5-OH VPA, 3 - k e t o VPA, 4 - k e t o VPA, 2 - p r o p y l s u c c i n i c a c i d (2-PSA) and 2 - p r o p y l -g l u t a r i c a c i d (2-PGA) i s p r e s e n t e d h e r e . The method used a c a p i l l a r y GCMS assay i n the s e l e c t e d i o n m o n i t o r i n g mode. The c h o i c e of e x t r a c -t i o n c o n d i t i o n s and d e r i v a t i z a t i o n p r o c e d u r e s are d i s c u s s e d . S e l e c t e d i o n chromatograms were o b t a i n e d f o r p a t i e n t u r i n e and serum s a m p l e s . The tBDMS and TMS d e r i v a t i v e s of v a l p r o i c a c i d and i t s m e t a b o l i t e s were compared. Mixed tBDMS-TMS d e r i v a t i v e s were a l s o i n v e s t i g a t e d . 1 . Sample e x t r a c t i o n The most e f f i c i e n t s o l v e n t f o r the r e c o v e r y of VPA and i t s metabo-l i t e s was e t h y l a c e t a t e as r e p o r t e d by Nau e t a l . (54) and the r e c o v e r y was i n the range of 75-90%. Other s o l v e n t s such as hexane, 10% e t h y l a c e t a t e i n hexane, c h l o r o f o r m , and d i c h l o r o m e t h a n e were s u i t a b l e f o r VPA and some of the u n s a t u r a t e d m e t a b o l i t e s but r e c o v e r i e s were poor f o r the more p o l a r m e t a b o l i t e s . F o r u r i n e s a m p l e s , 3 mL o f e t h y l a c e t a t e gave o p t i m a l r e s u l t s . In the case of serum,two e x t r a c t i o n s , each f o r 20 min u s i n g g e n t l e r o t a t i o n , p r e v e n t e d f o r m a t i o n of i n t r a c t a b l e e m u l s i o n s and gave good r e c o v e r i e s o f VPA and i t s m e t a b o l i t e s . A l k a l i n e h y d r o l y s i s (pH 1 2 - 1 3 , 1 h o u r , 6 0 ° C ) o f the c o n j u g a t e d m e t a b o l i t e s was the p r e f e r r e d method - 27 -s i n c e i t was easy t o perform and ensures h y d r o l y s i s o f B - g l u c u r o n i d a s e r e s i s t a n t forms s h o u l d they be p r e s e n t ( 1 4 3 ) . An i n i t i a l approach t o the e x t r a c t i o n and a n a l y s i s of m e t a b o l i t e s f o l l o w i n g a l k a l i n e h y d r o l y s i s was t o a d j u s t t h e s o l u t i o n pH t o 2 and heat f o r 2 h o u r s . Under t h e s e c o n d i t i o n s 4-OH VPA and 5-OH VPA are c o n v e r t e d t o t h e i r Y and 6" l a c t o n e s r e s p e c t i v e l y and 3 - k e t o VPA t o 3-heptanone ( 1 4 4 ) . T h i s same approach was used by L o s c h e r i n a GC method f o r t h e a n a l y s i s o f VPA m e t a b o l i t e s . ( 4 0 ) . In a s i m i l a r way we attempted t o measure 3 - k e t o VPA v i a t h e 3-heptanone u s i n g GCMS and by m o n i t o r i n g m/z 114. [ 2 H 4 ] - 3 - H e p t a n -one was added at the a c i d i f i c a t i o n s t e p t o s e r v e as i n t e r n a l s t a n d a r d . In many c a s e s 3-heptanone was e i t h e r l o s t by e v a p o r a t i o n d u r i n g t h e 2 h o u r - h e a t i n g p e r i o d i n s p i t e of s e a l e d r e a c t i o n tubes o r was p r e s e n t a t low c o n c e n t r a t i o n s because of i n c o m p l e t e d e c a r b o x y l a t i o n of 3 - k e t o VPA. The tBDMS d e r i v a t i v e of a c e t i c a c i d r e s u l t i n g from t h e h y d r o l y s i s of e t h y l a c e t a t e under the a c i d i c c o n d i t i o n s used i n t e r f e r e d w i t h the i n t e r n a l s t a n d a r d [ 2 H/i . ]-3-heptanone peak at m/z 118 ( F i g u r e 2 ) . The e x t r a c t i o n and a n a l y s i s of i n t a c t 3 - k e t o VPA was then i n v e s t i -g a t e d . It was found t h a t b r i n g i n g t h e sample t o pH 2 f o r 15 m i n u t e s at room t e m p e r a t u r e gave o p t i m a l r e s u l t s . W h i l e some 3 - k e t o VPA i s d e c a r b o x y l a t e d t o form 3-heptanone a l a r g e p a r t remains i n t a c t . S i m i l a r r e s u l t s were r e p o r t e d by Kochen e t a l . ( 5 6 ) . The 3 - k e t o VPA upon d e r i v a t i z a t i o n can be assayed as t h e mono or d i d e r i v a t i v e depend-i n g on t h e d e r i v a t i z i n g reagent u s e d . These e x t r a c t i o n c o n d i t i o n s a l s o c o n v e r t e d 4-OH VPA t o i t sY - l a c t o n e i s o m e r s which were e x t r a c t e d and a n a l y z e d by m o n i t o r i n g m/z 100. - 28 -A C E T I C ACID t - B D M S D E R I V A T I V E (a) 2.24 T m/z 118 R E T E N T I O N TIME (MIN.) F i g u r e 2. S e l e c t e d i o n chromatogram (m/z 118) o f the tBDMS d e r i v a t i v e o f a c e t i c a c i d ( a ) . - 29 -2 . D e r i v a t i z a t i o n The c h o i c e of tBDMS d e r i v a t i v e s of VPA and m e t a b o l i t e s f o r s e l e c t -ed i o n m o n i t o r i n g was based on t h e r e p o r t e d i n c r e a s e d s e n s i t i v i t y o f tBDMS d e r i v a t i v e s compared t o TMS (137) and the p r e v i o u s s u c c e s s f u l use o f tBDMS d e r i v a t i v e s f o r measuring VPA and [ 2 H6]-VPA i n t h i s l a b o r a t o r y ( 1 2 9 , 141) . The mass s p e c t r a of the tBDMS d e r i v a t i v e s of VPA m e t a b o l i t e s c o n -t a i n t y p i c a l and i n t e n s e ( M - 5 7 ) + fragment i o n s c o r r e s p o n d i n g t o t h e l o s s of t h e t - b u t y l fragment as seen i n t h e mass s p e c t r a o f VPA and i t s m e t a b o l i t e s p r e s e n t e d i n F i g u r e s 3 t o 1 6 . Other c h a r a c t e r i s t i c i o n s from mass s p e c t r a o f m e t a b o l i t e s o b t a i n e d by GCMS are g i v e n i n Table I . The m a j o r i t y of our a n a l y s e s have been a c c o m p l i s h e d u s i n g tBDMS reagent prepared i n p y r i d i n e c a t a l y z e d by 5% d i m e t h y l a m i n o p y r i d i n e (DMAP). T h i s reagent was f i r s t t e s t e d by Kuldeep S i n g h , a P h . D . s t u d e n t i n our l a b o r a t o r y . The c a t a l y s t reduced r e a c t i o n t i m e s but gave i n t e r f e r i n g background peaks when i t exceeded 5% by weight of t h e r e a g e n t m i x t u r e . The r e a c t i o n t i m e o f 4 hours a t 60°C was found t o c o m p l e t e l y d e r i v a t i z e VPA and t o g i v e r e p r o d u c i b l e peaks f o r the m e t a -b o l i t e s . T h i s reagent gave p r i m a r i l y t h e mono t-BDMS d e r i v a t i v e o f 3 - k e t o VPA (m/z 215) ( F i g u r e 11) and f u r t h e r s t o r a g e at 0° produced l i t t l e o r no f o r m a t i o n o f t h e d i - d e r i v a t i v e ( F i g u r e 1 2 ) . The 5-OH VPA was r e a d i l y d e r i v a t i z e d and formed a d i - d e r i v a t i v e (m/z 331) ( F i g u r e • 1 4 ) . On t h e o t h e r hand 4-OH VPA remained l a r g e l y as t h e ^ - l a c t o n e and was a n a l y z e d as such (m/z 100) ( F i g u r e 3 ) . When u s i n g t h e tBDMS reagent i n p y r i d i n e 3-OH VPA does not d e r i v a -t i z e r e a d i l y and chromatographs p o o r l y . Longer r e a c t i o n t i m e s were - 30 -100, 111 o z < o z ZD E D < UJ > LU CC 50 CH 3-CH 2-CH 2 \ .100 CH-C=0 CH2~CH~CH2 I o .41 .55 • •III, .111 A .69 • " LL L I I i 1 4 \ . m/z F i g u r e 3. Mass spectrum o f 4-OH VPA l a c t o n e i n u r i n e e x t r a c t . ioo1 UJ o z < Q z ZD CD < 50. LU > UJ C H 2 « = C H — C H ? / C H C O O - S i - CH3 X199 (M-57) C H 3 — C H 2 - C H 2 C(CH 3) 3 75 I. . l i l l . l •• ill .III,,.!...! j i J_ 241^  m/z F i g u r e 4. Mass spectrum of t-BDMS d e r i v a t i v e of 4-ene VPA i n u r i n e e x t r a c t . - 31 -100, LU O z < o z CD < 50.| LU > UJ cr i 9 9 ( M - 5 7 ) + CH,—CH,—CH. 3 2 ^ , C H 3 — C H 2 — C H ^ C—COOi-tBDMS 73 •J 'In i 125 / — ,A\, l . . . I, I, m/z F i g u r e 5. Mass spectrum of tBDMS d e r i v a t i v e of 2 - e n e VPA i n u r i n e e x t r a c t . 100, UJ o z < o z ID CD < 50. UJ > UJ DC CH,—CH—CH 3 \ C H 3 - C H 2 - C H 2 ^CHCOO-tBDMS (M~57) + 73 z 5 5 115 J_LI 241 Ii L. m/z F i g u r e 6. Mass spectrum of tBDMS d e r i v a t i v e of 3-ene VPA i n u r i n e e x t r a c t . - 32 -100-, LU O z < o z m 50 LU > LU DC 197 (M-57)+ CH,— CH=CH 3 V —COO-tBDMS CH — C H 2 — C H 75 123 153 239 m/z F i g u r e 7 . Mass spectrum of tBDMS d e r i v a t i v e of ( E , E ) - 2 , 3 ' - d i e n e VPA i n u r i n e e x t r a c t 100 UJ o z < o z 5 50 UJ > UJ rr CH^=CH-CH ^C - C 0 0-tBDMS 197 (M-57)+ CHj—CH 2 —CH^ 75 j.n J i n j j 123 , 153 • ill i i i — J — i — i — » 239 i m/z F i g u r e 8 . Mass spectrum of tBDMS d e r i v a t i v e of ( E ) - 2 , 4 - d i e n e VPA i n u r i n e e x t r a c t . - 33 -100, LU o z < o z CD < LU > < -J U J cr C H 3 — C H 2 — C H 2 C H 3 — C H g - C H 2 Z 7 5 :CHC00-tBDMS ^ ( M - 5 7 ) + 50. 129. 1 I  .II ...I.N.I I,, ill, • I .1 . L 243. • i m/z F i g u r e 9. Mass spectrum of tBDMS d e r i v a t i v e of VPA i n u r i n e e x t r a c t . U J o z < o z CD < U J > U J cr 100, 50. 75 43 187 1L± ,215 ( M - 5 7 ) + o C H 3 — C — C H 2 / C H 3 - C H 2 — C H 2 CHCOO-tBDMS 281 i i I J I m/z F i g u r e 10. Mass spectrum of tBDMS d e r i v a t i v e of 4 - k e t o VPA i n u r i n e e x t r a c t . - 34 -UJ O z < o z z> CD < UJ > < - J UJ CL 100. 50. 75 73 CH3-CH2-C'X CH3-CH2-CH^ CHCOO -tBDMS 111 II, , J 171 Hi I •! 215 ( M - 5 7 ) 4 187 _i HI m/z F i g u r e 11. Mass spectrum of tBDMS d e r i v a t i v e of 3 - k e t o VPA i n u r i n e e x t r a c t . UJ o z < a z CD < UJ > < UJ cr 100. 50. CH —CH =C' CH3—CH^CH^ 0-tBDMS CHCOO -tBDMS 329 ( M - 5 7 ) + 75 73 147 H i .U iL 216 255 287 i tl Jl 8 ,1 371 , I, m/z F i g u r e 12. Mass spectrum of di-tBDMS d e r i v a t i v e of 3 - k e t o VPA i n u r i n e e x t r a c t . - 35 -LU 1 0 0 . o z < D 3 m < LU > LU 5 0 . 7 5 7 3 . / ° H CH 3-CH £—CH^ CHj—CH 2-CH 2 CH-COO-tBDMS 1 5 9 2 1 7 ( M - 5 7 ) + 1 2 9 J U L 1 9 9 i _ l , 2 5 9 1 • m/z F i g u r e 1 3 . Mass spectrum of tBDMS d e r i v a t i v e of 3-OH VPA i n u r i n e e x t r a c t . \3 JlL 0-tBDMS C H , — C H , — C H , C H 3 — C H 2 - C H 2 C H C O O-tBDMS 3 3 1 ( M - 5 7 ) + 147 J i_ 3 7 3 \ m/z F i g u r e 14. Mass spectrum of di-tBDMS d e r i v a t i v e of 5-OH VPA i n u r i n e e x t r a c t . - 36 -UJ o z < a z ZD CD < UJ > UJ 100 50 t B D M S - O O C - C H , C H 3 — C H 2 — C H 2 CHCOO - t B D M S 147 73 75 O i U L l L i i i 171 331 (M-57) + 257 I, iik li,.L i.u. , 373 L_ m/z F i g u r e 15. Mass spectrum of di-tBDMS d e r i v a t i v e of 2 - p r o p y l s u c c i n i c a c i d i n u r i n e e x t r a c t . UJ 100 o z < o z 5 50 UJ > < _ J UJ n t B D M S - 0 O C - C H 2 - C H 2 C H 3 - C H 2 - C H 2 ^ c H c o o - t B D M S 345(M-57) + 73 j_k_L 75 129 1 8 5 I . . i t ^ 301 ill I213 2 7 1 . H I . , i L .i • M , t i . I L_i 3J7 m/z F i g u r e 16. Mass spectrum of di-tBDMS d e r i v a t i v e of 2 - p r o p y l g l u t a r i c a c i d i n u r i n e e x t r a c t . Table I. Characteristic ions (m/z) in the mass spectra of the tBDMS derivatives of valproic acid metabolites COMPOUND BASE peak (%) (M-15)+ CH3 (M-57) + C4H9 (M-99) + C4H9 + ( C H 2 ) 3 (M -101) f C4H9 + C 0 2 (M-131)f tBDMSO (M-132) + tBDMSOH (M-189) + C4H9 + tBDMSOH (M-217)+ C4H9 + HCOOtBDMS Other Ions (E,E)-2,3'-Diene 197 254 (0) 239 (3) 197 (100) — 153 (3) 123 (9) — 75 (29) (E)-2-4-Diene a 197 254 (0) 239 (3) 197 (100) — 153 (2) 123 (7) — 75 (18) 4-Enea 199 256 (0) 241 (3) 199 (100) 157 (6) 75 129 171 (62) (7) (3) 3-Ene 199 255 (0) 241 (4) 199 (100) 157 (2) 155 (10) — — 73 75 115 (76) (72) (40) (Z)-2-Ene 199 256 (0) 241 (2) 199 (100) — — — — 73 75 (8) (42) (E)-2-Ene 199 256 (0) 241 (4) 199 (100) — 155 (4) 125 (9) — 73 75 (24) (18) VPA 201 258 (0) 243 (1) 201 (100) 159 (3) — — — — 73 75 129 (25) (82) (13) 3-Keto (mono-derivative) 75 272 (0) 215 (32) 171 (53) 73 187 (21) (5) 3-Keto (dideri vati 329 ve) 386 (0) 371 (4) 329 (100) 287 (8) — 255 (28) — 73 75 147 (56) (64) (62) 4-Keto 75 272 (0) 215 (93) 171 (6) — — 73 187 (30) (45) 3-OH 75 274 (0) 259 (1) 217 (30) — — — — 75 129 159 (100M15) (25) 5-OH ( d i -deri vative) 331 a 388 (0) 373 (4) 331 (100) — — 257 (4) 199 (13) 171 (7) 147 75 73 (91) (64) (88) 2-PSA (dideri vati 331 ve) 388 (0) 373 (3) 331 (100) — — 257 (6) — 171 (17) 73 75 147 (60) (70) (83) 2-PGAa 345 (di d e r i v a t i ve) 402 (0) 387 (4) 345 (100) — 301 (12) 271 (3) 213 (3) 185 (18) 73 75 129 (41) (25) (17) results s i m i l a r to those reported by Rettenmeier et a l . (57). - 38 -n e c e s s a r y t o get r e p r o d u c i b l e peaks f o r t h e 3-OH VPA m o n o - d e r i v a t i v e ( F i g u r e 1 3 ) . The di-tBDMS d e r i v a t i v e s of 3-OH VPA and 4-OH VPA were r a r e l y s e e n . More r e c e n t l y , MTBSTFA a very r e a c t i v e tBDMS reagent has become a v a i l a b l e ( 1 4 5 ) . With t h i s r e a g e n t , r e a c t i o n t i m e s f o r t h e d e r i v a t i z a -t i o n o f VPA m e t a b o l i t e s were s h o r t e n e d (20-30 m i n ) , and 3-OH VPA was more r e a d i l y d e r i v a t i z e d . However, 3 - k e t o VPA forms both t h e mono and d i - d e r i v a t i v e s and on s t a n d i n g at 0° c o n t i n u e s t o c o n v e r t from mono (m/z 215) t o t h e d i - d e r i v a t i v e (m/z 3 2 9 ) . T h i s s u g g e s t s t h a t s h o r t r e a c t i o n t i m e s w i t h t h i s reagent may be i n a d e q u a t e f o r r e p r o d u c i b l e d e r i v a t i v e f o r m a t i o n o f 3 - k e t o VPA. I d e a l l y , r e a c t i o n t i m e s s h o u l d be s u f f i c i e n t t o ensure complete f o r m a t i o n of the 3 - k e t o VPA di-tBDMS d e r i v a t i v e . With t h e MTBSTFA r e a g e n t , 4-OH VPA s t i l l remains l a r g e l y as t h e y - 1 a c t o n e . _ S i m i l a r r e s u l t s were found when MSTFA was used to form TMS d e r i v a t i v e s . The s t a b i l i t y of tBDMS d e r i v a t i v e s prepared u s i n g t h e p y r i d i n e reagent was d e t e r m i n e d by t h e r e p e a t e d a n a l y s i s of t h e same s e t o f s t a n d a r d u r i n e samples over a one-week p e r i o d . The samples were f i r s t a n a l y z e d on t h e day of p r e p a r a t i o n , then t h r e e and seven days l a t e r w h i l e b e i n g s t o r e d at room t e m p e r a t u r e . I t was found t h a t the r e l a t i v e s t a n d a r d d e v i a t i o n of t h e s l o p e s of t h e c a l i b r a t i o n c u r v e s d i d not exceed 6%. When d e r i v a t i z e d samples were t i g h t l y capped and s t o r e d at - 2 0 ° C they were found t o be s t a b l e f o r s e v e r a l weeks. 3 . S e l e c t e d Ion Chromatograms S e l e c t e d i o n chromatograms f o r t h e u n d e r i v a t i z e d 4-OH VPA l a c t o n e s (m/z 100) and f o r t h e tBDMS d e r i v a t i v e s o f ( E ) - 2 , 4 - d i e n e VPA and - 39 -( E , E ) - 2 , 3 ' - d i e n e VPA (m/z 1 9 7 ) ; 4 - e n e , 3 - e n e , ( Z ) - 2 - e n e and ( E ) - 2 - e n e VPA (m/z 1 9 9 ) ; VPA (m/z 2 0 1 ) ; 3 - k e t o and 4 - k e t o VPA (m/z 2 1 5 ) ; 5-0H VPA and 2 - p r o p y l s u c c i n i c a c i d (m/z 3 3 1 ) ; and 2 - p r o p y l g l u t a r i c a c i d (m/z 345) a r e i l l u s t r a t e d i n F i g u r e s 17 t o 2 3 . The SIM chromatograms f o r tBDMS d e r i v a t i v e s o f VPA m e t a b o l i t e s o b t a i n e d f o r a p a t i e n t u r i n e and serum sample are i l l u s t r a t e d t o g e t h e r i n F i g u r e 24 and 25 r e s p e c t i v e l y . A l l peaks of i n t e r e s t were s u i t a b l y r e s o l v e d and serum and u r i n e c o n -t r o l s showed no i n t e r f e r i n g background p e a k s . The i o n s s e l e c t e d f o r each m e t a b o l i t e are shown i n F i g u r e s 24 and 2 5 . Ions were chosen based on the p r o d u c t i o n o f s t a b l e b a s e l i n e s , the s e n s i t i v i t y of d e t e c t i o n and t h e s e l e c t i v i t y a c h i e v e d . An OV-1701 c a p i l l a r y column was chosen f o r the a n a l y s i s based on t h e r e s o l u t i o n o f 4 - e n e VPA from VPA w i t h i n a r e a s o n a b l e GC run t i m e . T h i s was a c c o m p l i s h e d i n a p p r o x i m a t e l y 16 m i n . The two major d i e n e - V P A m e t a b o l i t e s were r e a d i l y seen a t m/z 197 i n serum and u r i n e . Other apparent d i e n e s were p r e s e n t but the l a c k of a u t h e n t i c s t a n d a r d s made t h e i r i d e n t i f i c a t i o n i m p o s s i b l e . Both tBDMS mono (m/z 215) and d i - d e r i v a t i v e (m/z 32,9) o f 3 - k e t o VPA are shown. The tBDMS d i - d e r i v a t i v e o f 3 - k e t o VPA i s seen e x t e n -s i v e l y o n l y w i t h MTBSTFA. r e a g e n t . The chromatogram f o r 3-OH VPA o b t a i n e d w i t h tBDMS r e a g e n t i n p y r i d i n e i s not always r e l i a b l e . B e t t e r r e s u l t s were o b t a i n e d w i t h MTBSTFA. No peak f o r 4-OH VPA m o n o - d e r i v a -t i v e i s e v i d e n t s i n c e under t h e c o n d i t i o n s of e x t r a c t i o n 4-OH VPA e x i s t s l a r g e l y as t h e y - l a c t o n e isomers (m/z 100, peaks 1 ) . - 40 -4-OH VPA LACTONES 5.81 (a) 5.89 (b) RETENTION TIME (MIN ) F i g u r e 17. S e l e c t e d i o n chromatogram o f 4-OH VPA l a c t o n e s (2 isomers a and b) from a p a t i e n t u r i n e s a m p l e . C o n d i t i o n s : c a p i l l a r y column (25 m x 0 . 3 2 mm I . D . ) w i t h 0V 1701 bonded phase ( 0 . 2 5 p ) ; t e m p e r a t u r e program: 3 0 ° / m i n from 50° t o 1 0 0 ° t h e n 8 ° / m i n from 1 0 0 ° t o 2 6 0 ° , h o l d l . m i n a t 2 6 0 ° . - 41 -(b) 8 D6 DIENES VPA t-BDMS DERIVATIVES m/z 197 15" RETENTION TIME (MIN ) F i g u r e 18. S e l e c t e d i o n chromatograms of tBDMS d e r i v a t i v e s of ( E ) - 2 , 4 - d i e n e VPA (a) and ( E , E ) - 2 , 3 ' - d i e n e VPA (b) f r o m a p a t i e n t u r i n e s a m p l e . C o n d i t i o n s : c a p i l l a r y column (25 m x 0 . 3 2 mm I . D . ) w i t h OV 1701 bonded phase ( 0 . 2 5 u); t e m p e r a t u r e program: 3 0 ° / m i n f r o m 50° t o 1 0 0 ° t h e n 8 ° / m i n from 1 0 0 ° t o 2 6 0 ° , h o l d 1 min at 2 6 0 ° . - 42 -4-ENE VPA , 3-ENE VPA, CIS-2-ENE VPA AND TRANS 2-ENE VPA t-BDMS DERIVATIVES (a)i 6 3 4 1 (b) 6 . 4 3 ( c ) 6 . 5 5 m/z 199 10 RETENTION TIME (MIN ) F i g u r e 19. S e l e c t e d i o n chromatograms of tBDMS d e r i v a t i v e s of 4 - e n e VPA ( a ) , 3-ene VPA ( b ) , ( Z ) - 2 - e n e VPA (c) and ( E ) - 2 - e n e VPA (d) f r o m a p a t i e n t u r i n e s a m p l e . C o n d i t i o n s : c a p i l l a r y column (25 m x 0 . 3 2 mm I . D . ) w i t h OV 1701 bonded phase ( 0 . 2 5 y ) ; t e m p e r a t u r e program: 3 0 ° / m i n f r o m 50° t o 1 0 0 ° t h e n 8 ° / m i n from 1 0 0 ° t o 2 6 0 ° , h o l d 1 min at 2 6 0 ° . - 43 -6.30 V P A t - B D M S DERIVATIVE m/z 201 RETENTION TIME (MIN ) F i g u r e 2 0 . S e l e c t e d i o n chromatogram o f tBDMS d e r i v a t i v e of VPA from a p a t i e n t u r i n e s a m p l e . C o n d i t i o n s : c a p i l l a r y column (25 m x 0 . 3 2 mm I . D . ) w i t h OV 1701 bonded phase ( 0 . 2 5 p ) ; t e m p e r a t u r e program: 3 0 ° / m i n from 50° t o 1 0 0 ° t h e n 8 ° / m i n from 1 0 0 ° t o 2 6 0 ° , h o l d 1 min a t 2 6 0 ° . - 44 -3 - K E T O V P A AND 4 - K E T O V P A t - B D M S DERIVATIVES ( a ) 8.91 m/z 215 (b) 9.46 6 10 RETENTION TIME (MIN ) F i g u r e 2 1 . S e l e c t e d i o n chromatograms of tBDMS d e r i v a t i v e s of 3 - k e t o VPA (a) and 4 - k e t o VPA (b) from a p a t i e n t u r i n e s a m p l e . C o n d i t i o n s : c a p i l l a r y column (25 m x 0 . 3 2 mm I . D . ) w i t h OV 1701 bonded phase ( 0 . 2 5 y ) ; t e m p e r a t u r e program: 3 0 ° / m i n from 50° t o 1 0 0 ° t h e n 8 ° / m i n from 1 0 0 ° t o 2 6 0 ° , h o l d 1 min a t 2 6 0 ° . - 45 -5 - O H VPA AND 2 - P R O P Y L S U C C I N I C ACID RETENTION TIME (MIN ) F i g u r e 22. S e l e c t e d i o n chromatograms of tBDMS d e r i v a t i v e s of 5-OH VPA (a) and 2 - p r o p y l s u c c i n i c a c i d (b) from a p a t i e n t u r i n e sample C o n d i t i o n s : c a p i l l a r y column (25 m x 0 . 3 2 mm I . D . ) w i t h 0V 1701 bonded phase ( 0 . 2 5 y ) ; t e m p e r a t u r e program: 30°/rnin f r o m 50° t o 1 0 0 ° t h e n 8 ° / m i n from 1 0 0 ° t o 2 6 0 ° , h o l d 1 min at 2 6 0 ° . t - B D M S DERIVATIVES (a) 1&57 m/z 331 ( b ) 13.7 - 46 -2-PROPYLGLUTARIC ACID t-BDMS DERIVATIVE 15 m/z 345 02 15" 14 RETENTION TIME (MIN ) F i g u r e 2 3 . S e l e c t e d i o n chromatogram o f tBDMS d e r i v a t i v e of 2 - p r o p y l g l u t a r i c a c i d from a p a t i e n t u r i n e s a m p l e . C o n d i t i o n s : c a p i l l a r y column (25 m x 0 . 3 2 mm I .D.) w i t h OV 1701 bonded phase ( 0 . 2 5 y ) ; t e m p e r a t u r e program: 3 0 ° / m i n from 50° t o 1 0 0 ° t h e n 8 ° / m i n from 1 0 0 ° t o 2 6 0 ° , h o l d 1 min a t 2 6 0 ° . - 47 -S E L E C T E D ION C H R O M A T O G R A M S OF tBDMS DERIVATIVES OF VALPRO IC ACID METABOL ITES FROM A PATIENT URINE S A M P L E m/z 128 m/z 1 0 0 ^ JUv. 1 2 -10 UL. -9 - i 1 3 --4-\ -8 3 - , m/z 217 14 m/z 215 Jl m/z 207 m/z 202 m/z 2 0 r -4 m/z 199" m/z 197 JU 17- -18 _A 4 -19 m/z 345 I J \ m / z 331 7-16 m/z 317 — i 16 r— 2 T 6 8 10 — F 12 — i — 14 TIME (min) Peak numbers c o r r e s p o n d t o : 1 = 4-OH l a c t o n e s , 2 a = 3 - 0 c t a n o n e , 3 = ( E ) - 2 , 4 - D i e n e VPA, 4 = ( E , E ) - 2 , 3 - D i e n e VPA, 5 = 4-Ene VPA, 60= 3-Ene VPA, 7 = ( Z ) - 2 - E n e VPA, 8 = ( E ) - 2 - E n e VPA, 9 = VPA, 10^ = ( Z ) - [ 2 H 3 ] - 2 - E n e VPA, l l a = ( E ) - [ 2 H 3 ] - 2 - E n e VPA, 12a = [ 2 H f i ] - V P A , 13 = 3 - K e t o VPA, 14 = 4 - K e t o VPA, 15 = 3-OH VPA, 16 = A d i p i c a c i d , 17 = 5-OH VPA, 18 = 2 - P r o p y l s u c c i n i c a c i d , 19 = 2 - P r o p y l g l u t a r i c a c i d , (a = i n t e r n a l s t a n d a r d ) . F i g u r e 2 4 . S e l e c t e d i o n chromatograms o f tBDMS d e r i v a t i v e s o f v a l p r o i c a c i d m e t a b o l i t e s from a p a t i e n t u r i n e s a m p l e . - 48 -SELECTED ION CHROMATOGRAMS OF VPA METABOLITES FROM A PATIENT SERUM SAMPLE (tBDMS DERIVATIVES). m/z 128. m/z 100. i 2 J 13 t ^9 _14 m/z 215 m/z 207 J 1 _8 m/z 202 m/z 201 _16 m/z 345 _15 m/z 331 m/z 199 -m/z 197 6 8 TO TIME (MIN) 12 14 T 6 Peak numbers c o r r e s p o n d t o : 1 = 4-OH VPA l a c t o n e s , 2 a = 3 - 0 c t a n o n e , 3 = ( E ) - 2 , 4 - D i e n e VPA, 4 = ( E , E ) - 2 , 3 ' - D i e n e VPA, 5 = 4-Ene VPA, 6 = 3-Ene VPA, 7 = (Z)-2-Ene VPA, 8 = ( E ) - 2 - E n e VPA, 9 = VPA, 10* = (z ) - [ 2 H 3 ] - 2 - E n e VPA, l i a = ( E ) - [ 2 H 3 ] - 2 - E n e VPA, 12a = [ 2 H e ] _ V P A , 13 = 3 - K e t o VPA, 14 = 4 - K e t o VPA, 15 = 5-OH VPA, 16 = 2 - P r o p y l g l u t a r i c a c i d , (a = i n t e r n a l s t a n d a r d . ) F i g u r e 2 5 . S e l e c t e d i o n chromatograms of tBDMS d e r i v a t i v e s o f v a l p r o i c a c i d m e t a b o l i t e s from a p a t i e n t serum s a m p l e . - 49 -In F i g u r e 25 t h e i o n chromatogram f o r 3-OH VPA (m/z 217) was not i n c l u d e d s i n c e the amount o f 3-OH VPA i n serum was lower than the l i m i t s of d e t e c t i o n . In serum 4-OH VPA i s a g a i n d e t e c t e d as the l a c t o n e and s m a l l amounts o f 5-OH VPA and 2-PGA (m/z 345) are e v i d e n t . 2-PSA was not d e t e c t e d i n serum s a m p l e s . 4 . Q u a n t i t a t i v e A n a l y s i s C a l i b r a t i o n c u r v e s f o r VPA u s i n g [ 2 H s ] - V P A as an i n t e r n a l s t a n d a r d and f o r the m e t a b o l i t e s u s i n g [ 2 H 3 ] - 2 - e n e VPA t r a n s as an i n t e r n a l s t a n d a r d were l i n e a r over the ranges s e l e c t e d w i t h c o e f f i -c i e n t s of d e t e r m i n a t i o n , r 2 , e x c e e d i n g 0 . 9 9 . C a l i b r a t i o n c u r v e s o b t a i n e d f o r VPA and i t s m e t a b o l i t e s are shown i n F i g u r e s 26 t o 3 6 . The 3-OH VPA was a n a l y z e d as the TMS d e r i v a t i v e . The r e p r o d u c i b i l i t y of the method was checked over a p e r i o d of f i v e months. The r e l a t i v e s t a n d a r d d e v i a t i o n o f the s l o p e s o b t a i n e d was found t o be l e s s than 8% f o r VPA, 3 - k e t o VPA, 4 - k e t o VPA, 4 - e n e VPA, 3-ene VPA, 2-ene VPA, and 5-OH VPA. The r e l a t i v e s t a n d a r d d e v i a t i o n of the s l o p e s exceeded 10% f o r 2-PSA and 4-OH VPA. In the case o f 2-PGA some improvement i n r e p r o d u c i b i l i t y may be o b t a i n e d by u s i n g a d i c a r b o x y l i c a c i d as an i n t e r n a l s t a n d a r d . Because of the v a r i a b i l i t y observed s t a n d a r d c u r v e s were run p r i o r t o each b a t c h o f p a t i e n t s a m p l e s . Our method has been i n use f o r o v e r a y e a r and p a t i e n t s r e s u l t s a r e shown i n T a b l e I I . The v a l u e s o b t a i n e d f o r 3 - k e t o VPA, 2-ene VPA, 4-OH VPA, 5-OH VPA are i n the same range as those o b t a i n e d by L o s c h e r (40) and Nau e t a l . ( 5 4 ) . - 50 -CONCENTRATION (MG/L) gure 2 6 . 4-OH VPA l a c t o n e c a l i b r a t i o n c u r v e . Peak a r e a r a t i o s were c a l c u l a t e d u s i n g [ 2 H 3 ] - 2 - e n e VPA as i n t e r n a l s t a n d a r d . C o n d i t i o n s : c a p i l l a r y column (25 m x 0 . 3 2 mm I . D . ) w i t h 0V 1701 bonded phase ( 0 . 2 5 y ) ; t e m p e r a t u r e program: 3 0 ° / m i n from 50° t o 1 0 0 ° , then 8 ° / m i n from 1 0 0 ° t o 2 6 0 ° , h o l d 1 min a t 2 6 0 ° . 2.5, - 51 -2 . CONCENTRATION (MG/L) F i g u r e 27. 4-Ene VPA tBDMS d e r i v a t i v e c a l i b r a t i o n c u r v e . Peak a r e a r a t i o s were c a l c u l a t e d u s i n g [ 2 H 3 ] - 2 - e n e VPA as i n t e r n a l s t a n d a r d . C o n d i t i o n s : c a p i l l a r y c o l u m n , ( 2 5 m x 0 . 3 2 mm I . D . ) w i t h OV 1701 bonded phase ( 0 . 2 5 y ) ; t e m p e r a t u r e program: 3 0 ° / m i n from 50° t o 1 0 0 ° , then 8 ° / m i n from 1 0 0 ° t o 2 6 0 ° , h o l d 1 min a t 2 6 0 ° . - 52 -1.25, CONCENTRATION (MG/L) F i g u r e 2 8 . 3-Ene VPA tBDMS d e r i v a t i v e c a l i b r a t i o n c u r v e . Peak a r e a r a t i o s were c a l c u l a t e d u s i n g [ 2 H 3 ] - 2 - e n e VPA as i n t e r n a l s t a n d a r d . C o n d i t i o n s : c a p i l l a r y column (25 m x 0 . 3 2 mm I . D . ) w i t h 0V 1701 bonded phase ( 0 . 2 5 y ) ; t e m p e r a t u r e program: 3 0 ° / m i n from 50° t o 1 0 0 ° , then 8 ° / m i n from 1 0 0 ° t o 2 6 0 ° , h o l d 1 min a t 2 6 0 ° . - 53 -CONCENTRATION (MG/L) gure 2 9 . ( E ) - 2 - E n e VPA tBDMS d e r i v a t i v e c a l i b r a t i o n c u r v e . Peak area r a t i o s were c a l c u l a t e d u s i n g [ 2 H 3 ] - 2 - e n e VPA as i n t e r n a l s t a n d a r d . C o n d i t i o n s : c a p i l l a r y column (25 m x 0 . 3 2 mm I . D . ) w i t h 0V 1701 bonded phase ( 0 . 2 5 y ) ; t e m p e r a t u r e program: 3 0 ° / m i n from 50° t o 1 0 0 ° , then 8 ° / m i n from 1 0 0 ° t o 2 6 0 ° , h o l d 1 min a t 2 6 0 ° . - 54 -CONCENTRATION (MG/L) gure 3 0 . VPA tBDMS d e r i v a t i v e c a l i b r a t i o n c u r v e . Peak area r a t i o s were c a l c u l a t e d u s i n g [2Hs]-VPA as i n t e r n a l s t a n d a r d . C o n d i t i o n s : c a p i l l a r y column (25 m x 0 . 3 2 mm I . D . ) w i t h OV 1701 bonded phase ( 0 . 2 5 p ) ; temperature program: 3 0 ° / m i n from 50° t o 1 0 0 ° , then 8 ° / m i n from 100* t o 2 6 0 ° , h o l d 1 min a t 2 6 0 ° . - 55 -CONCENTRATION (MG/L) gure 3 1 . 3 - K e t o VPA tBDMS d e r i v a t i v e c a l i b r a t i o n c u r v e . Peak a r e a r a t i o s were c a l c u l a t e d u s i n g [ 2 H 3 ] - 2 - e n e VPA as i n t e r n a l s t a n d a r d . C o n d i t i o n s : c a p i l l a r y column (25 m x 0 . 3 2 mm I . D . ) w i t h 0V 1701 bonded phase ( 0 . 2 5 u ) ; temperature program: 3 0 ° / m i n from 50° t o 1 0 0 ° , then 8 ° / m i n from 100° t o 2 6 0 ° , h o l d 1 min a t 2 6 0 ° . - 56 -1 , CONCENTRATION (MG/L) F i g u r e 3 2 . 4 - K e t o VPA tBDMS d e r i v a t i v e c a l i b r a t i o n c u r v e . Peak a r e a r a t i o s were c a l c u l a t e d u s i n g [ z H 3 ] - 2 - e n e VPA as i n t e r n a l s t a n d a r d . C o n d i t i o n s : c a p i l l a r y column (25 m x 0 . 3 2 mm I . D . ) w i t h OV 1701 bonded phase ( 0 . 2 5 u ) ; temperature program: 3 0 ° / m i n from 50° t o 1 0 0 ° , then 8 ° / m i n from 100* t o 2 6 0 ° , h o l d 1 min a t 2 6 0 ° . - 57 CONCENTRATION (MG/L) gure 3 3 . 3-OH VPA TMS d e r i v a t i v e c a l i b r a t i o n c u r v e . Peak area r a t i o s were c a l c u l a t e d u s i n g [ 2 H 3 ] - 2 - e n e VPA as i n t e r n a l s t a n d a r d . C o n d i t i o n s : c a p i l l a r y column (25 m x 0 . 3 2 mm I . D . ) w i t h OV 1701 bonded phase ( 0 . 2 5 u ) ; temperature program: 3 0 ° / m i n from 50° t o 1 0 0 ° , then 8 ° / m i n from 100* t o 2 6 0 ° , h o l d 1 min a t 2 6 0 ° . - 58 -CONCENTRATION (MG/L) F i g u r e 3 4 . 5-OH VPA tBDMS d e r i v a t i v e c a l i b r a t i o n c u r v e . Peak a r e a r a t i o s were c a l c u l a t e d u s i n g [ 2 H 3 ] - 2 - e n e VPA as i n t e r n a l s t a n d a r d . C o n d i t i o n s : c a p i l l a r y column (25 m x 0 . 3 2 mm I . D . ) w i t h OV 1701 bonded phase ( 0 . 2 5 u ) ; t e m p e r a t u r e program: 3 0 ° / m i n from 50° t o 1 0 0 ° , then 8 ° / m i n from 100* t o 2 6 0 ° , h o l d 1 min a t 2 6 0 ° . - 59 -0.8 , CONCENTRATION (MG/L) F i g u r e 3 5 . 2 - P r o p y l s u c c i n i c a c i d tBDMS d e r i v a t i v e c a l i b r a t i o n c u r v e . Peak a r e a r a t i o s were c a l c u l a t e d u s i n g [ 2 H 3 ] - 2 - e n e VPA as i n t e r n a l s t a n d a r d . C o n d i t i o n s : c a p i l l a r y column (25 m x 0 . 3 2 mm I . D . ) w i t h OV 1701 bonded phase ( 0 . 2 5 u ) ; temperature program: 3 0 ° / m i n from 50° t o 1 0 0 ° , then 8 ° / m i n from 100° t o 2 6 0 ° , h o l d 1 min a t 2 6 0 ° . - 60 -rL = 0 .9986 s l o p e = 0.0277 10 CONCENTRATION (MG/L) 2b gure 3 6 . 2 - P r o p y l g l u t a r i c a c i d tBDMS d e r i v a t i v e c a l i b r a t i o n c u r v e . Peak a r e a r a t i o s were c a l c u l a t e d u s i n g [ 2 H 3 ] - 2 - e n e VPA as i n t e r n a l s t a n d a r d . C o n d i t i o n s : c a p i l l a r y column (25 m x 0 . 3 2 mm I . D . ) w i t h OV 1701 bonded phase ( 0 . 2 5 u ) ; t e m p e r a t u r e program: 3 0 ° / m i n from 50° t o 1 0 0 ° , t h e n 8 ° / m i n from 100* t o 2 6 0 ° , h o l d 1 min a t 2 6 0 ° . - 61 -T a b l e I I Serum VPA and m e t a b o l i t e c o n c e n t r a t i o n s 8 f o r 34 p e d i a t r i c p a t i e n t s on VPA monotherapy. Compound Mean (yg/mL) Range % of VPA 4-OH VPA 0 . 3 8 T r b - 1 . 7 8 0 . 8 ( E ) - 2 , 4 - D i e n e VPAC 0 . 2 0 0 . 0 2 - 0 . 5 8 0 . 4 ( E , E ) - 2 , 3 ' - D i e n e VPAC 2 . 9 5 0 . 5 0 - 7 . 2 9 6 . 4 4-Ene VPA 0.67 0 . 1 6 - 1 . 2 2 1 . 4 3-Ene VPA 0 . 9 4 0 . 2 5 - 1 . 8 6 2 . 0 ( Z ) - 2 - E n e VPA 0 . 1 9 0 . 0 6 - 0 . 4 0 0 . 4 ( E ) - 2 - E n e VPA 5 . 5 3 0 . 9 5 - 1 1 . 3 11.9 VPA 4 6 . 3 8 11 .8-105 100 3 - K e t o VPA 3 . 5 9 0 . 2 9 - 1 5 . 6 0 7.7 4 - K e t o VPA 0 . 4 0 0 . 0 1 - 1 . 2 9 0 . 9 5-OH VPA 0 . 1 8 Tr - 1 . 2 5 0 . 4 2 - P r o p y l s u c c i n i c a c i d 0 . 0 4 T r - 0 . 4 4 0 . 1 2 - P r o p y l g l u t a r i c a c i d 0 . 2 0 Tr - 1 . 2 3 0 . 4 a . A l l a n a l y s e s were done u s i n g t h e tBDMS r e a g e n t . b . T r = t r a c e c . The ( E ) - 2 - e n e VPA s t a n d a r d c u r v e was used f o r q u a n t i t a t i o n . - 62 -5 . Comparison o f tBDMS and TMS D e r i v a t i v e s VPA m e t a b o l i t e s were a l s o d e r i v a t i z e d u s i n g MSTFA and the s e l e c t e d i o n chromatograms f o r a p a t i e n t u r i n e sample u s i n g the same column as f o r t h e tBDMS d e r i v a t i v e s a r e shown i n F i g u r e 3 7 . In o r d e r t o s e p a r a t e 4 - e n e VPA and VPA a l o n g e r c h r o m a t o g r a p h i c t i m e and d i f f e r e n t c o n d i -t i o n s than used f o r the tBDMS d e r i v a t i v e s were r e q u i r e d . Using our e x t r a c t i o n and d e r i v a t i z a t i o n c o n d i t i o n s , 4-OH VPA l a c t o n e s are p r e s e n t (peaks 1 , m/z 100) as w e l l as i s o m e r s o f t h e 4-OH VPA m o n o - d e r i v a t i v e (peaks 1 6 , m/z 2 1 7 ) . The i s o m e r s o f 3-OH VPA appeared as mono TMS d e r i v a t i v e s i n c o n t r a s t t o tBDMS d e r i v a t i v e s and gave sharp peaks (peaks 1 5 , m/z 2 1 7 ) . 3 - K e t o VPA was p r e s e n t as both t h e m o n o - d e r i v a -t i v e (peak 14) and t h e d i - d e r i v a t i v e (peaks 17) and upon s t a n d i n g the m o n o - d e r i v a t i v e 3 - k e t o VPA was found t o d e c r e a s e w i t h a c o r r e s p o n d i n g i n c r e a s e of t h e d i - d e r i v a t i v e i s o m e r s . In o r d e r t o compare t h e r e l a t i v e s e n s i t i v i t y of d e t e c t i o n of t h e tBDMS and TMS d e r i v a t i v e s a c a l i b r a t i o n s t a n d a r d , a p a t i e n t u r i n e s a m p l e , and a p a t i e n t serum sample were each e x t r a c t e d and d i v i d e d , w i t h one p a r t d e r i v a t i z e d by MTBSTFA reagent and the o t h e r h a l f by MSTFA. D i e t h y l 1 - m e t h y l b u t y l m a l o n a t e s e r v e d as a n o n d e r i v a t i z e d i n t e r n a l s t a n d a r d . The samples were run on t h r e e d i f f e r e n t days over a p e r i o d of one week. A l l samples were s t o r e d a t - 1 8 ° C and found t o be s t a b l e . The r e s u l t s a r e g i v e n i n T a b l e I I I . Except f o r 3 - k e t o VPA, t h e tBDMS d e r i v a t i v e s i n a l l c a s e s gave g r e a t e r s e n s i t i v i t y . This was p a r t i c u l a r l y t r u e and has s p e c i a l s i g n i f i c a n c e f o r the u n s a t u r a t e d m e t a b o l i t e s 3 - and 4 - e n e VPA which are f r e q u e n t l y p r e s e n t i n c o n c e n t r a -t i o n s near t h e l o w e r d e t e c t i o n l i m i t s . B a r r i n g d i f f e r e n c e s i n the - 63 -S E L E C T E D ION CHROMATOGRAMS OF TMS DERIVATIVES OF VALPROIC ACID METABOLITES FROM A PATIENT URINE SAMPLE m/z 207 m/z 202 m/z 201 m/z 199 m/z 197 m/z 100 m/z 303 19 m/z 289 m/z 287 m/z 217 m/z 215 T 1 1 1 1 1 I 1 8 10 12 14 16 18 20 22 TIME (min) Peak numbers c o r r e s p o n d t o : 1 = 4-OH VPA l a c t o n e s , 2 = ( E , E ) - 2 , 3 ' - D i e n e VPA, 3 , 4 = Dienes VPA, 5 = ( E ) - 2 - E n e VPA, 6 = ( Z ) - 2 - E n e VPA, 7 .= 3-Ene VPA, 8 = 4-Ene VPA. 9 = VPA, 10* = ( E ) - [ 2 H 3 ] - 2 - E n e VPA, l l a = ( Z ) - [ 2 H 3 ] -2 - Ene VPA, 12a = [2Hg]-VPA, 13 = 4 - K e t o VPA, 14 = 3 - K e t o VPA, TMS mono-d e r i v a t i v e , 15 = 3-OR VPA (2 i s o m e r s ) , 16 = 4-OH VPA (2 i s o m e r s ) , 17 = 3 - Keto VPA TMS d i d e r i v a t i v e (2 i s o m e r s ) , 18 = 5-OH VPA, 19 = 2 - P r o p y l -s u c c i n i c a c i d , 20 = 2 - P r o p y l g l u t a r i c a c i d , (a = i n t e r n a l s t a n d a r d ) . F i g u r e 37. S e l e c t e d i o n chromatograms o f TMS d e r i v a t i v e s o f v a l p r o i c a c i d m e t a b o l i t e s f r o m a p a t i e n t u r i n e s a m p l e . - 64 -TABLE I I I Comparison of the r e l a t i v e s e n s i t i v i t y of tBDMS and TMS d e r i v a t i v e s o f VPA and m e t a b o l i t e s when measured by SIM. Peaks were the ( M - 5 7 ) + and ( M - 1 5 ) + i o n s r e s p e c t i v e l y . METABOLITE R e l a t i v e s e n s i t i v i t y tBDMS/TMSa (n = 12 f o r each m e t a b o l i t e ) Dienes VPA, ( Z ) - 2 - E n e VPA 10 - 20 X 3-Ene VPA, 4-Ene VPA VPA, ( E ) - 2 - E n e VPA, 4 - K e t o VPA, 2-PSA 5 - 1 0 2-PGA 5-OH VPA, 3 - K e t o VPA . 3 - 2 a Determined from the peak area r a t i o s of the r e s p e c t i v e ( M - 5 7 ) + o r ( M - 1 5 ) + m/z to the m/z 160 o f d i e t h y l 1 - m e t h y l b u t y l m a l o n a t e . TABLE IV Comparison of d e r i v a t i v e r e a c t i o n t i m e s , chromatography t i m e s and s t a b i l i t y of tBDMS and TMS d e r i v a t i v e s of VPA and i t s m e t a b o l i t e s . tBDMS TMS d e r i v a t i v e s d e r i v a t i v e s D e r i v a t i v e R e a c t i o n Chromatography t i m e s adequate r e s o l u t i o n S t a b i l i t y ( 4 ° ) t imes ( 5 0 ° ) 4 H f o r 16 - 20 1 week minimum 20 - 30 min min 22 - 30 min 1 - 2 days - 65 -chromatography and the degree of d e r i v a t i z a t i o n , the g r e a t e r s e n s i t i v i -t y o b s e r v e d w i t h the i o n m o n i t o r i n g of the tBDMS d e r i v a t i v e s i s very s i m i l a r t o the comparisons seen by Wool l a r d f o r TMS and tBDMS d e r i v a -t i v e s of C 1 3 and C 2 0 s t r a i g h t - c h a i n f a t t y a c i d s (137) . The tBDMS and TMS d e r i v a t i v e s were a l s o compared i n terms of d e r i v a t i z a t i o n and chromatography t i m e s and s t a b i l i t y ( T a b l e I V ) . 6 . Mixed D e r i v a t i v e s Mixed tBDMS-TMS d e r i v a t i v e s as d e s c r i b e d by W o o l l a r d (137) f o r hydroxy f a t t y a c i d s were o b t a i n e d f o r 3-OH VPA and 4-OH VPA. The c h r o -m a t o g r a p h i c c o n d i t i o n s were i d e n t i c a l to t h a t used f o r the tBDMS d e r i -v a t i v e s . Ion chromatograms and the mass s p e c t r a are shown i n F i g u r e s 38 and 3 9 . The i s o m e r s of 3-OH VPA appear as a s i n g l e peak. The most c h a r a c t e r i s t i c i o n i s m/z 131 and c o n f i r m s the p o s i t i o n of the TMS s u b s t i t u e n t . The 4-OH VPA isomers appeared as 2 peaks and gave s e v e r a l c h a r a c t e r i s t i c i o n s . The base peak was m/z 199 ( M - T M S O H-C4H9). I f a f a i r l y u n r e a c t i v e tBDMS r e a g e n t i s to be used t h e n , f o r m a t i o n of mixed d e r i v a t i v e s has p o t e n t i a l f o r the a n a l y s i s of VPA m e t a b o l i t e s . These i n i t i a l r e s u l t s s u g g e s t t h a t a c o m b i n a t i o n of r e a g e n t s m i g h t p r o v i d e o p t i m a l d e r i v a t i v e s f o r the GCMS-SIM a n a l y s i s of VPA metabo-l i t e s i . e . s e n s i t i v e and s e l e c t i v e i o n s f o r the u n s a t u r a t e s (tBDMS) and mixed d e r i v a t i v e s f o r the hydroxy m e t a b o l i t e s t h a t are p o o r l y d e r i v a -t i z e d u s i n g tBDMS r e a g e n t a l o n e . W i t h tBDMS r e a g e n t the d e t e c t i o n l i m i t i s t y p i c a l l y 0 . 1 ug/mL. Compared t o p r e v i o u s r e p o r t e d a s s a y s , our method o f f e r s the f o l l o w i n g - 66 -3 - O H VPA t - B D M S : TMS MIXED DERIVATIVE 9 . 3 8 (a) g r o RETENTION TIME (MIN.) 10CL UJ o z < o z < UJ > UJ cr 5 Q 0-TMS CH3-CH2-CH-CH-C0-0-TBDMS C 3 H 7 , 73 " in • i .131 '0-TMS II CH3-CH2-CH .199 (b) il L I 289 Dl - 573* I 331 [n-i l _ l 15]* mix F i g u r e 38. Mass chromatogram (a) and mass spectrum (b) o f tBDMS-TMS mixed d e r i v a t i v e o f 3-OH VPA i n u r i n e e x t r a c t . - 67 -4-OH VPA t - B D M S : TMS MIXED DERIVATIVE 9.69 9,32 m/z 2 8 9 g i (a) 10 RETENTION TIME (MIN.) 0-TftS CH3-CH-CH2-CH-CO-O-TBDMS C 3 H 7 100, 199 UJ O z < o z ffl < IU > CC 50 7 s •0-TMS » 1^47 CH3-CH «;7 m/z 289 EM - 573* (b) 33^  W-15]+ Figure 39. Mass chromatogram (a) and mass spectrum (b) of tBDMS-TMS mixed derivative of 4-OH VPA in urine extract. - 68 -a d v a n t a g e s : s h o r t e r chromatography t i m e s , a l a r g e r number of metabo-l i t e s a n a l y z e d , a b e t t e r s e n s i t i v i t y e s p e c i a l l y f o r the u n s a t u r a t e d m e t a b o l i t e s and an improved s t a b i l i t y of the d e r i v a t i v e s . The s e n s i t i -v i t y and s e l e c t i v i t y of t h i s c a p i l l a r y GCMS method make i t a method o f c h o i c e f o r d e t e r m i n a t i o n of m e t a b o l i c p r o f i l e s of VPA i n e p i l e p t i c p a t i e n t s and f o r i n t e r a c t i o n and p h a r m a c o k i n e t i c s t u d i e s . - 69 -B . ASA - VPA I n t e r a c t i o n 1 . A n a l y s i s o f VPA a n d i t s m e t a b o l i t e s i n u r i n e s a m p l e s T h i s s t u d y was c o n d u c t e d i n s i x p e d i a t r i c p a t i e n t s and one a d u l t h e a l t h y v o l u n t e e r . The m e t a b o l i t e s i n v e s t i g a t e d w e r e n a m e l y : 4-OH V P A , ( E ) - 2 , 4 - d i e n e V P A , ( E , E ) - 2 , 3 ' - d i e n e V P A , 4 - e n e V P A , 3 - e n e V P A , ( E ) - 2 - e n e V P A , 3 - k e t o V P A , 4 - k e t o V P A , 3-OH V P A , 5-OH V P A , 2 - p r o p y l -s u c c i n i c a c i d a n d 2 - p r o p y l g l u t a r i c a c i d . U n c h a n g e d VPA and i t s c o n -j u g a t e s w e r e a l s o i n v e s t i g a t e d , b u t t h e y w e r e n o t d i f f e r e n t i a t e d i n t h e a n a l y s i s . The u r i n e s a m p l e s s t u d i e d came f r o m a p r e v i o u s s t u d y done a few y e a r s ago by O r r e t a l . ( 1 0 4 ) a n d we t h o u g h t t h a t s p o n t a n e o u s h y d r o l y s i s o v e r s u c h a l o n g p e r i o d o f t i m e w o u l d g i v e u n r e l i a b l e d a t a on t h e p r o p o r t i o n o f u n c h a n g e d d r u g and t h e c o n j u g a t e s . To a l l e v i a t e a p o s s i b l e e r r o r i n t h e u r i n e v o l u m e s o f t h e s i x p e d i a t r i c p a t i e n t s , r e s u l t s o f r e c o v e r e d m e t a b o l i t e s w e r e n o r m a l i z e d u s i n g u r i n a r y c r e a t i n i n e l e v e l s a n d e x p r e s s e d a s ug/mg c r e a t i n i n e . The d a t a o b t a i n e d w e r e a n a l y z e d f i r s t on t h e b a s i s o f e a c h i n d i v i d u a l m e t a b o l i t e , and s e c o n d l y upon g r o u p i n g m e t a b o l i t e s i n t o s e v e r a l d i f f e r e n t m e t a b o l i c p a t h w a y s . 1 . 1 U r i n a r y p r o f i l e s o f VPA m e t a b o l i t e s - e f f e c t o f ASA The g r a p h i c a l r e s u l t s f o r i n d i v i d u a l m e t a b o l i t e s b e f o r e ( d a y 1) a n d a f t e r ( d a y 2 ) a d m i n i s t r a t i o n o f ASA a r e shown i n F i g u r e 4 0 . The r e s u l t s o b t a i n e d f o r 4 - e n e VPA a r e i l l u s t r a t e d i n F i g u r e 4 0 a . The a m o u n t o f 4 - e n e VPA e x c r e t e d b e f o r e a n d a f t e r a d m i n i s t r a t i o n o f ASA i n a l l s u b j e c t s b u t VS shows s l i g h t v a r i a t i o n s i n t h e r a n g e 0 . 1 5 - 0 . 4 0 % . The ( E ) - 2 - e n e VPA was l o w e r e d i n s i x s u b j e c t s and u n c h a n g e d i n one (TR) ( F i g u r e 4 0 b ) . F i g u r e 4 0 c shows t h a t 4-OH VPA was e l e v a t e d a f t e r 70 -F i g u r e 4 0 . U r i n a r y p r o f i l e s o f VPA m e t a b o l i t e s i n seven s u b j e c t s b e f o r e (day 1) and a f t e r (day 2) a d m i n i s t r a t i o n o f ASA. - 71 -100-80 60 40 204 VPA ( e ) 50 40 30 ce x 20J, 101 3-KETO VPA (f) Day 1 Day 2 Day 1 Day 2 4-KETO VPA (g) VS 10 81 o 3-0H VPA ( h ) Day 1 Day 2 Day 1 Day 2 F i g u r e 4 0 . ( C o n t i n u e d ) % OF TOTAL EXCRETED % OF TOTAL EXCRETED 0 3 00 = T> corn t/raz ro % OF TOTAL EXCRETED o ox* a_—G "O/JLLi-CrD 33 c: o o I—I o - 73 -a d m i n i s t r a t i o n of ASA (day 2) i n f o u r s u b j e c t s out of s e v e n . S u b j e c t s VS and FA d i s p l a y e d a s l i g h t d e c r e a s e and s u b j e c t BP a l a r g e d e c r e a s e i n the amount of 4-OH VPA e x c r e t e d on day 2 . No net e f f e c t was o b s e r v e d i n the change o f 3-ene VPA between day 1 and day 2 i n t h e seven s u b j e c t s ( F i g u r e 4 0 d ) . The t o t a l of c o n j u g a t e d and unchanged VPA r e c o v e r e d was r a i s e d on day 2 i n s i x s u b j e c t s but v i r t u a l l y unchanged f o r p a t i e n t TR ( F i g u r e 4 0 e ) . The e f f e c t of ASA i n g e s t i o n on 3 - k e t o VPA e x c r e t i o n i s c h a r a c t e r i z e d by a net r e d u c t i o n o f m e t a b o l i t e e x c r e t e d on day 2 i n a l l s u b j e c t s e x c e p t VS which remained unchanged ( F i g u r e 4 0 f ) . The r e c o v e r y o f 4 - k e t o VPA on day 2 i s s l i g h t l y reduced i n a l l s u b j e c t s b u t one (CP) ( F i g u r e 4 0 g ) . 3-OH VPA e x c r e t i o n on day 2 was e l e v a t e d i n two s u b j e c t s and d e c r e a s e d i n the r e s t of them w i t h an o v e r a l l d e c r e a s e i n the mean of % o f t o t a l m e t a b o l i t e s e x c r e t e d ( F i g u r e 4 0 h ) . The e x c r e t i o n o f 5-OH VPA shows o n l y s l i g h t v a r i a t i o n s between day 1 and day 2 ( F i g u r e 4 0 i ) . The d i f f e r e n c e of 2 - p r o p y l s u c c i n i c a c i d e x c r e t e d b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA i s not p a r t i c u l a r l y s i g n i f i c a n t e x c e p t a l a r g e d e c r e a s e i n p a t i e n t VS ( F i g u r e 4 0 j ) . The amount of 2 - p r o p y l g l u t a r i c a c i d r e c o v e r e d on day 2 was e l e v a t e d i n f o u r s u b j e c t s and d e c r e a s e d i n the o t h e r s u b j e c t s ( F i g u r e 4 0 k ) . The major d i e n e p r e s e n t i n u r i n e the ( E , E ) - 2 , 3 ' - d i e n e VPA was l o w e r e d i n a l l s u b j e c t s e x c e p t TR and WN ( F i g u r e 4 0 1 ) . The ( E ) - 2 , 4 - d i e n e VPA was i n c r e a s e d i n two s u b j e c t s , unchanged i n two o t h e r s u b j e c t s and dropped i n the r e m a i n i n g t h r e e s u b j e c t s ( F i g u r e 40m). U r i n a r y p r o f i l e s o f VPA and i t s m e t a b o l i t e s b e f o r e and a f t e r a d m i n i -s t r a t i o n o f ASA f o r the seven s u b j e c t s of t h i s study are p r e s e n t e d i n T a b l e s V t o X I . In p a t i e n t BP VPA r e c o v e r e d on day 2 showed a marked - 74 -i n c r e a s e of % t o t a l e x c r e t e d of 1 3 . 8 and 3 - k e t o a decrease of 17.7 ( T a b l e V ) . For p a t i e n t CP r e c o v e r e d VPA i n c r e a s e d from 4 4 . 5 % t o 57.3% w h i l e 3 - k e t o d e c r e a s e d from 23 t o 13.4%; 3-OH VPA was markedly i n c r e a s e d ( T a b l e V I ) . P a t i e n t SS showed a marked d e c r e a s e o f 3 - k e t o VPA (from 18.9% t o 1.9% o f t o t a l e x c r e t e d ) and an i n c r e a s e of r e c o v e r e d VPA o f 1 9 . 2 i n % of t o t a l e x c r e t e d a f t e r a d m i n i s t r a t i o n of ASA ( T a b l e V I I ) . For p a t i e n t TR, VPA r e c o v e r e d on day 2 was s l i g h t l y i n c r e a s e d (+ 1% of t o t a l e x c r e t e d ) , w h i l e 3 - k e t o VPA was d e c r e a s e d by 6 . 3 % (of t o t a l e x c r e t e d ) (Table V I I I ) . The 3 - k e t o VPA r e c o v e r e d on day 2 f o r p a t i e n t VS was v i r t u a l l y unchanged (+ 0.1% of t o t a l e x c r e t e d ) , the r e c o v e r y of VPA i n c r e a s e d from 7 4 . 2 % t o 8 2 . 4 % , ( T a b l e I X ) . P a t i e n t WN showed a marked d e c r e a s e of e x c r e t e d 3 - k e t o (from 2 9 . 3 % t o 4.4%) a f t e r a d m i n i -s t r a t i o n of ASA, VPA was i n c r e a s e d by 2 2 . 4 i n % o f t o t a l e x c r e t e d ( T a b l e X ) . The normal s u b j e c t d i s p l a y e d a l a r g e i n c r e a s e i n % VPA r e c o v e r e d (+ 27.7) and a l a r g e d e c r e a s e i n 3 - k e t o r e c o v e r e d (- 2 2 . 1 ) a f t e r a d m i n i s t r a t i o n of ASA ( T a b l e X I ) . T a b l e X I I summarizes the mean, d i f f e r e n c e and p e r c e n t change of u r i n a r y c o n c e n t r a t i o n s o f VPA and i t s m e t a b o l i t e s b e f o r e and a f t e r a d m i n i s t r a t i o n o f ASA i n the seven s u b j e c t s of the s t u d y . VPA c o n -j u g a t e s ( m a i n l y g l u c u r o n i d e ) and 3 - k e t o VPA r e p r e s e n t the most abundant m e t a b o l i t e s i n u r i n e w i t h l e v e l s r e s p e c t i v e l y a t 611 and 260 ug/mg c r e a t i n i n e on day 1 ( T a b l e X I I ) . The average i n c r e a s e o f VPA f o r t h e 7 s u b j e c t s a f t e r a d m i n i s t r a t i o n of ASA was 5 2 . 4 % . The average decrease o f 3 - k e t o VPA on day 2 was 6 8 . 7 % . The m e t a b o l i t e s 4-OH VPA and 2 - p r o -p y l g l u t a r i c a c i d are q u a n t i t a t i v e l y i m p o r t a n t and r e p r e s e n t r e s p e c t i v e -l y 5 and 11.9% of t o t a l m e t a b o l i t e s e x c r e t e d . The average i n c r e a s e s - 75 -Tabl e V U r i n a r y P r o f i l e s o f VPA and I t s M e t a b o l i t e s B e f o r e and A f t e r A d m i n i s t r a t i o n of ASA i n P a t i e n t BP Day 1 Day 2 Day 2 - Day 1 VPA a.l one VPA + ASA change i n yg/mg c r e a t i n i n e change i n % t o t a l e x c r e t e d yg/mg c r e a t -i n i n e % o f t o t a l e x c r e t e d yg/mg c r e a t -i n i ne % o f t o t a l e x c r e t e d 4-OH VPA 110 7 . 6 85 5 . 5 - 25 - 2.1 ( E ) - 2 , 4 - D i e n e VPA 0 . 5 8 0 . 0 4 1 .26 0 . 0 8 + 0.7 + 0 . 0 4 ( E , E ) - 2 , 3 ' - D i e n e VPA 9 . 6 8 0 . 7 5 . 5 0 . 4 - 4 . 1 8 - 0 . 3 4-Ene VPA 4 0 . 3 3 . 9 0 . 2 - 0 . 1 - 0 . 1 3-Ene VPA 1 . 7 5 0 . 1 1 . 4 0 . 8 - 0 . 3 5 - 0 . 0 2 ( E ) - 2 - E n e - V P A 28 1 . 9 18 1 . 2 - 10 - 0.7 VPAa 801 55 1092 6 8 . 8 + 291 + 1 3 . 8 3 - K e t o VPA 312 2 1 . 4 58 3 . 7 - 254 - 17.7 4 - K e t o VPA 44 3 31 2 - 13 - 1 3-OH VPAb 4 . 3 0 . 3 52 3 . 4 + 47.7 + 3 . 1 5-OH VPA 20 1 . 4 34 2 . 2 + 14 - 0 . 8 2 - P r o p y l s u c c i n i c a c i d 3 . 3 0 . 2 4 . 4 0 . 3 + 1.1 + 0.1 2 - P r o p y l g l u t a r i c a c i d 118 8 . 1 201 12.7 + 83 + 4 . 6 Sum VPA + m e t a b o l i t e s 1457 100 1587 100 + 130 a . VPA d a t a i n c l u d e VPA c o n j u g a t e s and unchanged VPA. b . 3-OH VPA v a l u e s have been o b t a i n e d by u s i n g TMS d e r i v a t i v e s , a l l o t h e r m e t a b o l i t e s have been measured by u s i n g t-BDMCS r e a g e n t . - 76 -T a b l e VI U r i n a r y P r o f i l e s o f VPA and I t s M e t a b o l i t e s B e f o r e and A f t e r A d m i n i s t r a t i o n of ASA i n P a t i e n t CP Day 1 Day 2 Day 2 - Day 1 VPA a l o n e VPA + ASA ug/mg c r e a t -i n i n e % o f t o t a l e x c r e t e d ug/mg c r e a t -i n i n e % o f t o t a l e x c r e t e d change i n ug/mg c r e a t i n i n e change i n % t o t a l e x c r e t e d 4-OH VPA 58 4 . 1 8 2 . 2 5 . 9 + 2 4 . 2 + 1 .8 ( E ) - 2 , 4 - D i e n e VPA 1 0 . 0 3 0 . 8 3 0 . 0 6 - 0.17 + 0 . 0 3 ( E , E ) - 2 , 3 ' - D i e n e VPA 8 . 3 0 . 6 3 . 6 8 0 . 2 6 - 4 . 6 2 - 0 . 3 4 4-Ene VPA 2 . 9 0 . 2 3 . 7 0 . 3 + 0 . 8 + 0.1 3-Ene VPA 1.07 0 . 0 8 1 . 0 6 0 . 0 9 - 0 . 0 1 + 0.01 ( E ) - 2 - E n e - V P A 2 0 . 4 1 . 5 1 3 . 8 1 - 6 . 6 - 0 . 5 VPA a 621 4 4 . 5 799 5 7 . 3 + 178 + 12.8 3 - K e t o VPA 321 23 187 13.4 - 134 - 9 . 6 4 - K e t o VPA 46 3 . 3 53 3 . 9 + 7 + 0 . 6 3-OH VPAb 0 . 2 5 0 . 0 1 4 . 6 0 . 3 3 + 4 . 3 5 + 0 . 3 2 5-OH VPA 5 5 . 3 4 5 1 . 6 3 . 7 - 3 . 7 - 0 . 3 2 - P r o p y l s u c c i n i c a c i d 5 . 6 0 . 4 6 . 2 0 . 5 + 0 . 6 + 0.1 2 - P r o p y l g l u t a r i c a c i d 261 18.6 185 13.3 - 76 - 5 . 3 Sum VPA + m e t a b o l i t e s 1402 100 1389 100 - 13 a . VPA data i n c l u d e VPA c o n j u g a t e s and unchanged VPA. b . 3-OH VPA v a l u e s have been o b t a i n e d by u s i n g TMS d e r i v a t i v e s , a l l o t h e r m e t a b o l i t e s have been measured by u s i n g t-BDMCS r e a g e n t . - 77 -T a b l e VII U r i n a r y P r o f i l e s o f VPA and I t s M e t a b o l i t e s B e f o r e and A f t e r A d m i n i s t r a t i o n of ASA i n P a t i e n t SS Day 1 Day 2 Day 2 - Day 1 VPA a l o n e VPA + ASA change i n ug/mg c r e a t i n i n e change i n % t o t a l e x c r e t e d ug/mg c r e a t -i n i n e % o f t o t a l e x c r e t e d ug/mg c r e a t -i n i n e % o f t o t a l e x c r e t e d 4-OH VPA 1 2 . 9 3 . 3 2 5 . 8 4 . 8 + 12.9 + 1 . 5 ( E ) - 2 , 4 - D i e n e VPA 0 . 4 4 0 . 1 0 0 . 4 4 0 . 8 0 - 0 . 0 2 ( E , E ) - 2 , 3 ' - D i e n e VPA 4 . 1 7 1 2 . 4 2 0 . 4 4 - 1.75 - 0 . 5 6 4-Ene VPA 0 . 5 7 0 . 1 5 1 . 3 9 0 . 2 5 + 0 . 8 2 + 0.10 3-Ene VPA 0 . 1 8 0 . 0 5 0 . 3 2 0 . 0 6 + 0.14 + 0.01 ( E ) - 2 - E n e VPA 7 . 4 8 1 . 9 4 . 5 8 0 . 8 - 2 . 9 - 1.1 VPAa 192 4 5 . 8 360 65 + 168 + 19.2 3 - K e t o VPA 7 9 . 5 1 8 . 9 10.1 1 . 9 - 6 9 . 4 - 17 4 - K e t o VPA 1 3 . 9 3 . 6 9 . 1 1.7 - 4 . 8 - 1 . 9 3-OH VPAb 2 6 . 1 6 . 2 8 . 9 1 . 6 - 17.2 - 4 . 6 5-OH VPA 1 4 . 9 3 . 8 16.7 3 . 1 + 1 . 8 - 0.7 2 - P r o p y l s u c c i n i c a c i d 1 .37 0 . 4 2 . 4 4 0 . 4 5 + 1.07 + 0 . 0 5 2 - P r o p y l g l u t a r i c a c i d 6 6 . 4 15.8 112 2 0 . 2 + 4 5 . 6 + 4 . 4 Sum VPA + m e t a b o l i t e s 420 100 554 100 + 134 a . VPA d a t a i n c l u d e VPA c o n j u g a t e s and unchanged VPA. b. 3-OH VPA v a l u e s have been o b t a i n e d by u s i n g TMS d e r i v a t i v e s , a l l o t h e r m e t a b o l i t e s have been measured by u s i n g t-BDMCS r e a g e n t . - 78 -T a b l e V I I I U r i n a r y P r o f i l e s o f VPA and I t s M e t a b o l i t e s B e f o r e and A f t e r A d m i n i s t r a t i o n of ASA i n P a t i e n t TR Day 1 Day 2 Day 2 - Day 1 VPA a 1 one VPA + ASA yg/mg c r e a t -i n i n e % o f t o t a l e x c r e t e d yg/mg c r e a t -i n i n e % o f t o t a l e x c r e t e d change i n yg/mg c r e a t i n i n e change i n % t o t a l e x c r e t e d 4-OH VPA 87 6 . 3 83 10.1 - 4 + 3 . 8 ( E ) - 2 , 4 - D i e n e VPA 1 . 6 3 0.11 1 . 0 8 0 . 1 3 - 0 . 5 5 + 0 . 0 2 ( E , E ) - 2 , 3 ' - D i e n e VPA 10 0.71 7 . 5 0 . 9 1 - 2 . 5 - 0 . 2 4-Ene VPA 2 . 8 0 . 2 2 . 6 0 . 3 - 0 . 2 - 0 .1 3-Ene VPA 0 . 9 0.07 1 . 2 0 . 1 + 0 . 3 - 0 . 0 3 ( E ) - 2 - E n e VPA 21 1 . 5 12 1 . 5 - 9 0 VPAa 611 4 3 . 8 367 4 4 . 8 - 244 + 1 3 - K e t o VPA 228 1 6 . 4 83 10.1 - 145 - 6 . 3 4 - K e t o VPA 67 5 34 4 . 2 - 33 - 0 . 8 3-OH VPAb 38 2.7 0 . 5 0 . 0 6 - 3 7 . 5 - 2 . 6 4 5-OH VPA 80 5.7 48 5 . 9 - 32 + 0 . 2 2 - P r o p y l s u c c i n i c a c i d 8 0 . 6 8 1 0 + 0 . 4 2 - P r o p y l g l u t a r i c a c i d 240 17.2 170 2 0 . 8 - 70 + 3 . 6 Sum VPA + m e t a b o l i t e s 1395 100 818 100 - 577 a . VPA d a t a i n c l u d e VPA c o n j u g a t e s and unchanged VPA. b . 3-OH VPA v a l u e s have been o b t a i n e d by u s i n g TMS d e r i v a t i v e s , a l l o t h e r m e t a b o l i t e s have been measured by u s i n g t-BDMCS r e a g e n t . - 79 -T a b l e IX U r i n a r y P r o f i l e s o f VPA and I t s M e t a b o l i t e s B e f o r e and A f t e r A d m i n i s t r a t i o n of ASA i n P a t i e n t VS Day 1 Day 2 Day 2 - Day 1 VPA a l o n e VPA + ASA change i n yg/mg c r e a t i n i n e change i n % t o t a l e x c r e t e d yg/mg c r e a t -i ni ne % o f t o t a l e x c r e t e d yg/mg c r e a t -i n i n e % o f t o t a l e x c r e t e d 4-OH VPA 3 7 . 5 4 . 2 83 4 . 1 + 4 5 . 5 - 0 . 1 ( E ) - 2 , 4 - D i e n e VPA 2 . 0 9 0 . 2 2 1.76 0 . 0 9 - 0 . 3 3 - 0 . 1 3 ( E , E ) - 2 , 3 ' - D i e n e VPA 8 . 9 1 5 . 2 0 . 2 6 - 3 . 7 - 0.74 4-Ene VPA 5 . 9 0.7 16.7 0 . 9 + 1 0 . 8 + 0 . 2 3-Ene VPA 1 . 2 0 . 1 1 . 6 0 . 0 8 + 0 . 4 - 0 . 0 2 ( E ) - 2 - E n e VPA 17 .9 2 13 0 . 7 - 4 . 9 - 1.3 VPAa 676 7 4 . 2 1606 8 2 . 4 + 930 + 8 . 2 3 - K e t o VPA 1 4 . 3 1 . 6 3 2 . 2 1.7 + 17.9 + 0.1 4 - K e t o VPA 3 . 3 0 . 4 6 0 . 3 + 2.7 - 0 . 1 3-OH VPAb 1 4 . 2 1 . 5 6 3 . 5 0 . 1 8 - 10.7 - 1 . 3 8 5-OH VPA 3 0 . 1 3 . 3 72 3 . 7 + 4 1 . 9 + 0 . 4 2 - P r o p y l s u c c i n i c a c i d 7 2 . 5 8 9 0 . 6 4 . 6 + 18.1 - 3 . 4 2 - P r o p y l g l u t a r i c a c i d 2 6 . 5 2 . 9 2 0 . 1 1 - 6 . 4 - 1 .9 Sum VPA + metabol i t e s 910 100 1949 100 + 1039 a . VPA d a t a i n c l u d e VPA c o n j u g a t e s and unchanged VPA. b . 3-OH VPA v a l u e s have been o b t a i n e d by u s i n g TMS d e r i v a t i v e s , a l l o t h e r m e t a b o l i t e s have been measured by u s i n g t-BDMCS r e a g e n t . - 80 -T a b l e X U r i n a r y P r o f i l e s o f VPA and I t s M e t a b o l i t e s B e f o r e and A f t e r A d m i n i s t r a t i o n of ASA i n P a t i e n t WN Day 1 Day 2 Day 2 - Day 1 VPA a l o n e VPA + ASA change i n ug/mg c r e a t i n i n e change i n % t o t a l e x c r e t e d ug/mg c r e a t -i n i n e % o f t o t a l e x c r e t e d ug/mg c r e a t -i n i n e % o f t o t a l e x c r e t e d 4-OH VPA 7 8 . 4 6 . 8 7 6 . 9 7.7 - 1 . 5 + 0 . 9 ( E ) - 2 , 4 - D i e n e VPA 0 . 7 9 0.07 0 . 7 9 0 . 0 8 0 + 0.01 ( E , E ) - 2 , 3 ' - D i e n e VPA 4 . 2 0 . 4 3 . 8 0 . 4 - 0 . 4 0 4-Ene VPA 2 . 5 2 0 . 2 3 . 0 4 0 . 3 + 0 . 5 2 + 0.1 3-Ene VPA 1 . 2 2 0 . 1 0 . 7 9 0 . 0 8 - 0 . 4 3 - 0 . 0 2 ( E ) - 2 - E n e VPA 14.1 1 . 3 9 . 5 6 0 . 9 5 - 4 . 5 4 - 0 . 3 5 VPA a 403 3 4 . 9 591 5 7 . 3 + 188 + 2 2 . 4 3 - K e t o VPA 336 2 9 . 3 4 4 . 5 4 . 4 - 2 9 1 . 5 - 2 4 . 9 4 - K e t o VPA 45 4 . 2 38 3 . 7 - 7 - 0 . 5 3-OH VPAb 5 5 . 8 4 . 9 2 2 . 4 2 . 2 - 3 3 . 4 - 2.7 5-OH VPA 3 4 . 7 3 . 2 4 0 . 4 4 + 5.7 + 0 . 8 2 - P r o p y l s u c c i n i c a c i d 4 . 3 0 . 4 5 . 4 0 . 5 + 1.1 + 0.1 2 - P r o p y l g l u t a r i c a c i d 170 14.9 193 18.7 + 23 + 3 . 9 Sum VPA + m e t a b o l i t e s 1150 100 1030 100 - 120 a . VPA data i n c l u d e VPA c o n j u g a t e s and unchanged VPA. b . 3-OH VPA v a l u e s have been o b t a i n e d by u s i n g TMS d e r i v a t i v e s , a l l o t h e r m e t a b o l i t e s have been measured by u s i n g t-BDMCS r e a g e n t . - 81 -Table XI U r i n a r y P r o f i l e s o f VPA and I t s M e t a b o l i t e s B e f o r e and A f t e r A d m i n i s t r a t i o n of ASA i n a H e a l t h y V o l u n t e e r Day 1 Day 2 Day 2 - Day 1 VPA a l o n e VPA + ASA yg/mg c r e a t -i n i ne % o f t o t a l e x c r e t e d yg/mg c r e a t -i ni ne % o f t o t a l e x c r e t e d change i n yg/mg c r e a t i n i n e change i n % t o t a l e x c r e t e d 4-OH VPA 42 2 . 5 49 2 . 4 + 7 - 0 . 1 ( E ) - 2 , 4 - D i e n e VPA 0 . 8 0 . 0 5 0 . 7 0 . 3 - 0 . 1 - 0 . 0 2 ( E , E ) - 2 f 3 ' - D i e n e VPA 4 . 8 0 . 3 3 . 2 0 . 1 5 - 1 . 6 - 0 .15 4-Ene VPA 3 . 5 0 . 2 4 . 4 0 . 2 + 0 . 9 0 3-Ene VPA 1 . 4 0 . 0 8 1.7 0 . 0 8 + 0 . 3 0 ( E ) - 2 - E n e VPA 9 . 1 0 . 5 8 . 6 0 . 4 - 0 . 5 - 0.1 VPAa 970 5 5 . 6 1704 8 3 . 3 + 734 + 27.7 3 - K e t o VPA 530 3 0 . 5 173 8 . 4 - 357 - 2 2 . 1 4 - K e t o VPA 2 8 . 4 1.7 24 1 . 2 - 4 . 4 - 0 . 5 3-OH VPAb 49 2 . 9 5 . 8 0 . 3 - 4 3 . 2 - 2 . 6 5-OH VPA 6 0 . 3 10 0 . 5 + 4 + 0 . 2 2 - P r o p y l s u c c i n i c a c i d 3 . 1 0 . 2 9 0 . 4 + 5 . 9 + 0 . 2 2 - P r o p y l g l u t a r i c a c i d 9 . 5 5 . 5 5 2 . 3 2 . 6 + 4 2 . 8 - 2 . 9 Sum VPA + m e t a b o l i t e s 1658 100 2046 100 + 388 a . VPA data i n c l u d e VPA c o n j u g a t e s and unchanged VPA. b . 3-OH VPA v a l u e s have been o b t a i n e d by u s i n g TMS d e r i v a t i v e s , a l l o t h e r m e t a b o l i t e s have been measured by u s i n g t-BDMCS r e a g e n t . - 82 -were 16% (4-OH VPA) and 7.6% (2-PGA) a f t e r t h e a d m i n i s t r a t i o n of ASA. The ( E ) - 2 - e n e VPA, 4 - k e t o VPA, 3-OH VPA, 5-OH VPA and 2 - p r o p y l s u c c i n i c a c i d r e p r e s e n t between 1 and 3% of t o t a l m e t a b o l i t e s e x c r e t e d on day 1 . The m i n o r m e t a b o l i t e s ( l e s s than 1%) a r e ( E ) - 2 , 4 d i e n e VPA, ( E , E ) - 2 , 3 ' - d i e n e VPA, 4 - e n e VPA and 3-ene VPA ( T a b l e X I I ) . A f t e r ASA a d m i n i s t r a t i o n , ( E , E ) - 2 , 3 ' - d i e n e VPA d e c r e a s e d w h i l e 4 - e n e VPA was e l e v a t e d compared t o day 1. The changes i n ( E , E ) - 2 , 3 ' - d i e n e VPA, ( E ) - 2 - e n e VPA and 3 - k e t o VPA r e c o v e r e d on day 2 were found t o be s i g n i f i c a n t l y d i f f e r e n t from day 1 . T a b l e X I I I summarizes t h e mean, d i f f e r e n c e and p e r c e n t change of u r i n a r y p r o f i l e s o f VPA and i t s m e t a b o l i t e s b e f o r e and a f t e r a d m i n i -s t r a t i o n <of ASA i n t h e seven s u b j e c t s of t h i s s t u d y . The u r i n a r y p r o -f i l e s o f VPA m e t a b o l i t e s showed an average d e c r e a s e o f 2-ene VPA ( -3 8 % ) , 3 - k e t o VPA ( - 69.3%) and 3-OH VPA ( - 56.6%) and an average i n c r e a s e o f 29.7% f o r VPA. The changes i n 4-ene VPA, ( E ) - 2 - e n e VPA, VPA, 3 - k e t o VPA e x c r e t e d on day 2 were found s i g n i f i c a n t l y d i f f e r e n t from day 1 . These r e s u l t s suggested an enhanced e x c r e t i o n o f VPA c o n -j u g a t e s and a d e c r e a s e d e l i m i n a t i o n o f 8 - o x i d a t i o n m e t a b o l i t e s (2-ene VPA, 3-OH VPA and 3 - k e t o VPA) f o l l o w i n g ASA a d m i n i s t r a t i o n . T h i s l a s t o b s e r v a t i o n prompted us t o i n v e s t i g a t e VPA m e t a b o l i t e p r o f i l e s on day 1 and day 2 by g r o u p i n g them i n t o s e v e r a l m e t a b o l i c pathways. - 83 -Table XII Mean, d i f f e r e n c e and p e r c e n t change of u r i n a r y c o n c e n t r a t i o n s o f VPA and i t s m e t a b o l i t e s (ug/mg c r e a t i n i n e ) b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA (n=7). Day 1 VPA a l o n e Day 2 VPA + ASA Day 2 - Day 1 mean ug/mg c r e a t i n e (SD) mean ug/mg c r e a t i n e (SD) A % change 4-OH VPA 6 0 . 8 ( 3 3 . 2 ) 6 9 . 3 ( 2 2 . 9 ) + 8 . 5 + 14 ( E ) - 2 , 4 - D i e n e VPA 1 . 0 5 ( 0 . 6 0 ) 0 . 9 8 ( 0 . 4 3 ) - 0.07 - 6.7 ( E , E ) - 2 , 3 ' - D i e n e VPA 7.15 ( 2 . 6 5 ) 4.47 ( 1 . 7 1 ) * - 2 . 6 8 - 3 7 . 5 4-Ene VPA 3.17 ( 1 . 6 1 ) 5 . 1 0 ( 5 . 2 1 ) + 1 . 9 + 6 0 . 8 3-Ene VPA 1.10 ( 0 . 4 9 ) 1.15 ( 0 . 4 8 ) + 0 . 0 5 + 4 . 5 ( E ) - 2 - E n e VPA 1 6 . 9 ( 7 . 2 ) 11.4 ( 4 . 3 ) * - 5 . 5 - 3 2 . 5 VPA a 611 (254) 931 ( 5 5 6 ) * * + 320 + 5 2 . 4 3 - K e t o VPA 260 (173) 81 ( 7 2 ) * - 179 - 68.7 4 - K e t o VPA 3 6 . 8 ( 1 9 . 4 ) 2 7 . 9 ( 1 6 . 5 ) - 8 . 9 - 2 4 . 2 3-OH VPAb 2 6 . 8 ( 2 1 . 7 ) 14 ( 1 8 . 2 ) - 1 2 . 8 - 4 7 . 8 5-OH VPA 3 4 . 4 ( 2 5 . 6 ) 39 ( 2 1 . 2 ) + 4 . 6 + 13.4 2 - P r o p y l s u c c i n i c a c i d 14 ( 2 5 . 9 ) 18 ( 3 2 . 1 ) + 4 + 2 8 . 6 2 - P r o p y l g l u t a r i c a c i d 127 (100) 133 (73) + 6 + 4 . 7 Sum VPA + m e t a b o l i t e s 1199 (418) 1339 (566) + 140 + 11.7 a . VPA d a t a i n c l u d e VPA c o n j u g a t e s and unchanged VPA. b. 3-OH VPA v a l u e s have been o b t a i n e d by u s i n g TMS d e r i v a t i v e s , a l l o t h e r m e t a b o l i t e s have been measured by u s i n g t-BDMCS r e a g e n t . * S i g n i f i c a n t l y d i f f e r e n t from day 1 ( p < 0 . 0 5 ) . S i g n i f i c a n c e determined by S t u d e n t ' s p a i r e d t t e s t . Approaches s i g n i f i c a n c e (p = 0 . 0 7 3 7 ) . - 84 -T a b l e X I I I Mean, d i f f e r e n c e and p e r c e n t change of u r i n a r y p r o f i l e s o f VPA and i t s m e t a b o l i t e s (% t o t a l e x c r e t e d ) b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA (n=7). Day 1 VPA a l o n e mean % t o t a l e x c r e t e d (SD) Day 2 VPA + ASA Day 2 - Day 1 mean % t o t a l e x c r e t e d (SD) % change 4-OH VPA 5 ( 1 . 9 ) 5 . 8 ( 2 . 5 ) + 0 . 8 0 + 16 ( E ) - 2 , 4 - D i e n e VPA 0 . 0 9 ( 0 . 0 6 ) 0 . 2 2 ( 0 . 2 7 ) + 0.13 + 144 ( E , E ) - 2 , 3 ' - D i e n e VPA 0.67 ( 0 . 2 7 ) 0 . 4 0 ( 0 . 2 5 ) * * - 0.27 - 40 4-Ene VPA 0 . 2 8 ( 0 . 1 9 ) 0 . 3 5 ( 0 . 2 5 ) * + 0 . 0 7 + 25 3-Ene VPA 0 . 0 8 ( 0 . 0 2 ) 0 . 1 8 ( 0 . 2 7 ) + 0.10 + 125 ( E ) - 2 - E n e VPA 1.51 ( 0 . 5 2 ) 0 . 9 4 ( 0 . 3 5 ) * - 0.57 - 38 VPAa 5 0 . 5 ( 1 2 . 6 ) 6 5 . 5 ( 1 4 ) * + 15 + 29.7 3 - K e t o VPA 2 0 . 2 ( 9 . 7 ) 6 . 2 ( 4 . 5 ) * - 14 - 6 9 . 3 4 - K e t o VPA 3 . 0 ( 1 . 5 ) 2 . 4 ( 1 . 5 ) - 0 . 6 - 20 3-OH VPAb 2 . 6 5 ( 2 . 3 ) 1 .15 ( 1 . 3 ) - 1 . 5 - 5 6 . 6 5-OH VPA 3 . 1 ( 1 . 8 ) 3 . 3 ( 1 . 7 ) + 0 . 2 + 6 . 4 2 - P r o p y l s u c c i n i c a c i d 1 .46 ( 2 . 9 ) 1.11 ( 1 . 6 ) - 0 . 3 5 - 24 2 - P r o p y l g l u t a r i c a c i d 11 .9 ( 6 . 2 ) 1 2 . 8 ( 8 . 1 ) + 0 . 9 0 + 7 . 6 Sum VPA + m e t a b o l i t e s 100 100 a . VPA d a t a i n c l u d e VPA c o n j u g a t e s and unchanged VPA. b. 3-OH VPA v a l u e s have been o b t a i n e d by u s i n g TMS d e r i v a t i v e s , a l l o t h e r m e t a b o l i t e s have been measured by u s i n g t-BDMCS r e a g e n t . * S i g n i f i c a n t l y d i f f e r e n t from day 1 ( p < 0 . 0 5 ) . S i g n i f i c a n c e determined by S t u d e n t ' s p a i r e d t t e s t . * * Approaches s i g n i f i c a n c e (p = 0 . 0 6 9 3 ) . - 85 -1 . 2 U r i n a r y p r o f i l e s o f m e t a b o l i t e s grouped i n t o pathways - e f f e c t o f  ASA Based on the p r e s e n t knowledge of the metabolism of VPA i n man VPA m e t a b o l i t e s were grouped i n t o s i x d i f f e r e n t pathways ( F i g u r e 4 1 ) . The g r a p h i c a l r e s u l t s f o r m e t a b o l i t e pathways b e f o r e and a f t e r a d m i n i -s t r a t i o n of ASA are shown i n F i g u r e 4 2 . Pathway 1 c o r r e s p o n d s to the B - o x i d a t i o n pathway and i n c l u d e s 2-ene VPA, 3-OH VPA and 3 - k e t o VPA. The sum of m e t a b o l i t e s of pathway 1 was c o n s i s t e n t l y reduced i n a l l s u b j e c t s ( F i g u r e 4 2 a ) . Pathway 2 i n v o l v e s 3-ene VPA and the major d i e n e ( E , E ) - 2 , 3 ' - d i e n e VPA. On day 2 , the sum of these two m e t a b o l i t e s was reduced i n a l l s u b j e c t s e x c e p t TR ( F i g u r e 4 2 b ) . Pathway 3 c o n t a i n s 4 - e n e VPA and the ( E ) - 2 , 4 - d i e n e VPA. The r e s u l t of ASA a d m i n i s t r a t i o n on the r e c o v e r y of t h e s e two m e t a b o l i t e s i s a s l i g h t i n c r e a s e of the mean o f % of t o t a l e x c r e t e d ( F i g u r e 4 2 c ) , a l l s u b j e c t s d i s p l a y e d a v e r y s l i g h t i n c r e a s e or no change between day 1 and day 2 . Pathway 4 c o r -responds t o the (w-1) o x i d a t i o n pathway and i n c l u d e s 4-OH VPA, 4 - k e t o VPA and 2 - p r o p y l s u c c i n i c a c i d . The e x c r e t i o n of the sum of these t h r e e m e t a b o l i t e s was unchanged or s l i g h t l y i n c r e a s e d i n f i v e s u b j e c t s and d e c r e a s e d i n the r e m a i n i n g two w i t h v i r t u a l l y no e f f e c t on the average mean o f % of t o t a l e x c r e t e d a f t e r a d m i n i s t r a t i o n of ASA ( F i g u r e 4 2 d ) . Pathway 5 c o r r e s p o n d s t o the w - o x i d a t i o n pathway and i n v o l v e s 5-OH VPA and 2 - p r o p y l g l u t a r i c a c i d . Four s u b j e c t s d i s p l a y e d a s m a l l e l e v a t i o n and the r e m a i n i n g t h r e e a s m a l l decrease i n the e l i m i n a t i o n of the sum o f 5-OH VPA and 2 - p r o p y l g l u t a r i c a c i d ( F i g u r e 4 2 e ) . The l a s t i n v e s t i -g a t e d pathway d e f i n e d as R' i n c l u d e s the sum of c o n j u g a t e d and unchanged VPA e x c r e t e d . S i n c e the f r a c t i o n of unchanged VPA e x c r e t e d Pathway 5 Pathway 4 Pathway 1 F i g u r e 4 1 . M e t a b o l i c pathways f o r VPA and i t s m e t a b o l i t e s . - 87 -F i g u r e 4 2 . U r i n a r y p r o f i l e s o f VPA m e t a b o l i t e s e x p r e s s e d as m e t a b o l i c pathways i n seven s u b j e c t s b e f o r e (day 1.) and a f t e r (day 2) a d m i n i s t r a t i o n o f ASA. - 88 -F i g u r e 4 2 . ( C o n t i n u e d ) . - 89 -i s very s m a l l , pathway R' i n v o l v e s l a r g e l y V P A - g l u c u r o n i d e . In a l l s u b j e c t s e x c r e t i o n o f VPA was e l e v a t e d ( F i g u r e 4 2 f ) . U r i n a r y p r o f i l e s of VPA and i t s m e t a b o l i t e s e x p r e s s e d as metabo-l i t e pathways b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA f o r the seven s u b -j e c t s of t h i s study are p r e s e n t e d i n T a b l e s XIV t o XX. The m e t a b o l i c p r o f i l e f o r p a t i e n t BP shows a decrease i n % of t o t a l e x c r e t e d o f 1 5 . 6 f o r pathway 1 and an i n c r e a s e of 14 f o r pathway R1 (Table X I V ) . P a t h -way 1 d e c r e a s e d from 24.4% of t o t a l e x c r e t e d on day 1 t o 14.8% on day 2 f o r p a t i e n t C P , and pathway R' i n c r e a s e d from 4 4 . 5 % t o 57.3% (Table X V ) . For p a t i e n t SS ( T a b l e X V I ) , pathway 1 was d e c r e a s e d by 2 2 . 7 (change i n % o f t o t a l e x c r e t e d ) and pathway R' was i n c r e a s e d by 1 9 . 2 . Pathway R' was o n l y s l i g h t l y i n c r e a s e d (+ 1.1% of t o t a l e x c r e t e d ) f o r p a t i e n t TR, a l t h o u g h pathway 1 was lowered by 8 . 5 ( T a b l e X V I I ) . P a t h -way 1 was s l i g h t l y d e c r e a s e d (2.6% of t o t a l e x c r e t e d ) and pathway R' s l i g h t l y i n c r e a s e d (8.2% o f t o t a l e x c r e t e d ) f o r p a t i e n t VS ( T a b l e X V I I I ) . P a t i e n t WN d i s p l a y e d a marked decrease i n pathway 1 (- 27.6% o f t o t a l e x c r e t e d ) and a marked i n c r e a s e i n the sum of VPA unchanged and c o n j u g a t e d (+ 2 2 . 3 % of t o t a l e x c r e t e d ) as i l l u s t r a t e d i n T a b l e XIX. The normal s u b j e c t (Table XX) showed s i m i l a r v a r i a t i o n s ; - 26.3% o f t o t a l e x c r e t e d f o r pathway 1 , and 24.8% i n c r e a s e i n % of t o t a l e x c r e t e d f o r pathway R 1 . T a b l e XXI i l l u s t r a t e s the mean, d i f f e r e n c e and p e r c e n t change of u r i n a r y c o n c e n t r a t i o n s n o r m a l i z e d by c r e a t i n i n e l e v e l s o f VPA m e t a b o l i c pathways f o r the seven s u b j e c t s of t h i s s t u d y . Pathways 2 and 3 r e p r e s e n t q u a n t i t a t i v e l y minor m e t a b o l i c pathways. On the average pathway 1 was d e c r e a s e d by 64% and pathway R' was i n c r e a s e d by 52.4% a f t e r a d m i n i s t r a t i o n of ASA. The changes i n pathways 1 and 2 on day 2 - 90 -Table XIV U r i n a r y P r o f i l e s Expressed as D i f f e r e n t VPA M e t a b o l i c Pathways B e f o r e and A f t e r A d m i n i s t r a t i o n of ASA i n P a t i e n t BP Day 1 Day 2 Day 2 - Day 1 VPA a l one VPA + ASA yg/mg c r e a t -i ni ne % o f t o t a l e x c r e t e d yg/mg c r e a t -i ni ne % o f t o t a l e x c r e t e d Change i n yg/mg c r e a t i n i n e Change i n % o f t o t a l e x c r e t e d Pathway l a 3 4 4 . 3 2 3 . 6 128 8 - 2 1 6 . 3 - 15.6 Pathway 2 b 11.4 0 . 8 6 . 9 0 . 5 - 4 . 5 - 0 . 3 Pathway 3 C 4 . 6 0 . 3 5 . 2 0 . 3 + 0 . 6 0 Pathway 4 d 157.3 11 120.4 7 . 5 - 3 6 . 9 - 3 . 5 Pathway 5 e 138 9 . 5 235 15 + 97 + 5 . 5 Pathway R ' f 801 55 1092 69 + 291 + 14 a . Pathway 1 i n c l u d e s 2-ene VPA, 3-OH VPA and 3 - k e t o VPA. b . Pathway 2 i n c l u d e s 3-ene VPA and ( E , E ) - 2 , 3 ' - d i e n e VPA. c . Pathway 3 i n c l u d e s 4 - e n e VPA and ( E ) - 2 , 4 - d i e n e VPA. d . Pathway 4 i n c l u d e s 4-OH VPA, 4 - k e t o VPA and 2 - p r o p y l s u c c i n i c a c i d . e . Pathway 5 i n c l u d e s 5-0H VPA and 2 - p r o p y l g l u t a r i c a c i d . f . Pathway R' i n c l u d e s VPA c o n j u g a t e d and u n c o n j u g a t e d . - 91 -T a b l e XV U r i n a r y P r o f i l e s E x p r e s s e d as D i f f e r e n t VPA M e t a b o l i c Pathways B e f o r e and A f t e r A d m i n i s t r a t i o n of ASA i n P a t i e n t CP Day 1 Day 2 Day 2 - Day 1 VPA a l o n e VPA + ASA Change i n ug/mg c r e a t i n i n e Change i n % o f t o t a l e x c r e t e d ug/mg c r e a t -i n i n e % o f t o t a l e x c r e t e d ug/mg c r e a t -i n i n e % of t o t a l e x c r e t e d Pathway l a 3 4 1 . 7 2 4 . 4 205 1 4 . 8 - 136.7 - 9 . 2 Pathway 2 b 9 . 4 0.7 4 . 7 0 . 3 - 4 . 7 - 0 . 4 Pathway 3 C 3 . 9 0 . 2 4 . 5 0 . 3 + 0 . 6 + 0.1 Pathway 4 d 1 0 9 . 6 7.7 141 10.2 + 3 1 . 4 + 2 . 5 Pathway 5 e 3 1 6 . 3 2 2 . 6 2 3 6 . 6 17 - 79.7 - 5 . 6 Pathway R ' f 621 4 4 . 5 799 5 7 . 3 + 178 + 1 2 . 8 a . Pathway 1 i n c l u d e s 2 - e n e VPA, 3-0H VPA and 3 - k e t o VPA. b . Pathway 2 i n c l u d e s 3-ene VPA and ( E , E ) - 2 , 3 ' - d i e n e VPA. c . Pathway 3 i n c l u d e s 4 - e n e VPA and ( E ) - 2 , 4 - d i e n e VPA. d . Pathway 4 i n c l u d e s 4-OH VPA, 4 - k e t o VPA and 2 - p r o p y l s u c c i n i c a c i d . e . Pathway 5 i n c l u d e s 5-OH VPA and 2 - p r o p y l g l u t a r i c a c i d . f . Pathway R' i n c l u d e s VPA c o n j u g a t e d and u n c o n j u g a t e d . - 92 -T a b l e XVI U r i n a r y P r o f i l e s E x p r e s s e d as D i f f e r e n t VPA M e t a b o l i c Pathways B e f o r e and A f t e r A d m i n i s t r a t i o n of ASA i n P a t i e n t SS Day 1 Day 2 Day 2 - Day 1 VPA a l o n e VP A + ASA Change i n ug/mg c r e a t i n i n e Change i n % o f t o t a l e x c r e t e d ug/mg c r e a t -i n i n e % o f t o t a l e x c r e t e d ug/mg c r e a t -i n i n e % o f t o t a l e x c r e t e d Pathway l a Pathway 2 b Pathway 3 C Pathway 4 d Pathway 5 e Pathway R , f 113 27 4 . 3 5 1 1 0 . 2 2 8 . 2 6 . 7 8 1 . 3 1 9 . 4 192 4 5 . 8 2 3 . 6 4 . 3 2 . 7 0 . 5 1 . 8 0 . 3 3 7 . 3 6 . 7 129 2 3 . 2 360 65 - 8 9 . 4 - 22.7 - 1 . 6 5 - 0 . 5 + 0 . 8 + 0.1 + 9 . 1 0 + 47.7 + 4 . 2 + 168 + 19.2 a . Pathway 1 i n c l u d e s 2-ene VPA, 3-OH VPA and 3 - k e t o VPA. b . Pathway 2 i n c l u d e s 3-ene VPA and ( E , E ) - 2 , 3 ' - d i e n e VPA. c . Pathway 3 i n c l u d e s 4 - e n e VPA and ( E ) - 2 , 4 - d i e n e VPA. d . Pathway 4 i n c l u d e s 4-OH VPA, 4 - k e t o VPA and 2 - p r o p y l s u c c i n i c a c i d . e . Pathway 5 i n c l u d e s 5-OH VPA and 2 - p r o p y l g l u t a r i c a c i d . f . Pathway R1 i n c l u d e s VPA c o n j u g a t e d and u n c o n j u g a t e d . - 93 -T a b l e XVII U r i n a r y P r o f i l e s E x p r e s s e d as D i f f e r e n t VPA M e t a b o l i c Pathways B e f o r e and A f t e r A d m i n i s t r a t i o n of ASA i n P a t i e n t TR Day 1 Day 2 Day 2 - Day 1 VPA a l o n e VPA + ASA Change i n ug/mg c r e a t i n i n e Change i n % o f t o t a l e x c r e t e d ug/mg c r e a t -i n i n e % o f t o t a l e x c r e t e d ug/mg c r e a t -i n i n e % o f t o t a l e x c r e t e d Pathway l a Pathway 2 b Pathway 3 C Pathway 4 d Pathway 5 e Pathway R ' f 287 2 0 . 5 1 0 . 9 0 . 8 4 . 4 0 . 3 162 12 320 23 611 4 3 . 7 9 5 . 5 12 8 . 7 1 3 . 7 0 . 5 125 1 5 . 3 218 2 6 . 6 367 4 4 . 8 - 191.5 - 8 . 5 - 2 . 2 + 0 . 2 - 0 .7 + 0 . 2 - 37 + 3 . 3 - 102 + 3 . 6 - 244 + 1.1 a . Pathway 1 i n c l u d e s 2 - e n e VPA, 3-OH VPA and 3 - k e t o VPA. b . Pathway 2 i n c l u d e s 3-ene VPA and ( E , E ) - 2 , 3 ' - d i e n e VPA. c . Pathway 3 i n c l u d e s 4 - e n e VPA and ( E ) - 2 , 4 - d i e n e VPA. d . Pathway 4 i n c l u d e s 4-OH VPA, 4 - k e t o VPA and 2 - p r o p y l s u c c i n i c a c i d . e . Pathway 5 i n c l u d e s 5-OH VPA and 2 - p r o p y l g l u t a r i c a c i d . f . Pathway R1 i n c l u d e s VPA c o n j u g a t e d and u n c o n j u g a t e d . - 94 -T a b l e X V I I I U r i n a r y P r o f i l e s E x p r e s s e d as D i f f e r e n t VPA M e t a b o l i c Pathways B e f o r e and A f t e r A d m i n i s t r a t i o n of ASA i n P a t i e n t VS Day 1 VPA a l o n e Day 2 VPA + ASA Day 2 - Day 1 ug/mg c r e a t -i n i n e % o f t o t a l e x c r e t e d ug/mg c r e a t -i n i n e % o f t o t a l e x c r e t e d Change i n ug/mg c r e a t i n i n e Change i n % o f t o t a l e x c r e t e d Pathway l a Pathway 2 b Pathway 3 C Pathway 4 d Pathway 5 € Pathway R . f 4 6 . 4 10.1 8 113.3 5 6 . 6 676 5 . 2 1.1 0 . 9 12.6 6 . 2 7 4 . 2 4 8 . 7 6 . 8 1 8 . 5 179.6 9 2 . 1 1606 2 . 6 0 . 3 1 9 4 . 7 8 2 . 4 + 2 . 3 - 3 . 3 + 1 0 . 5 + 6 6 . 3 + 3 5 . 5 + 930 - 2 . 6 - 0 . 8 + 0 . 1 - 3 . 6 - 1 . 5 + 8 . 2 a . b . c . d . e . f . Pathway 1 i n c l u d e s 2-ene VPA, 3-OH VPA and 3 - k e t o VPA. Pathway 2 i n c l u d e s 3-ene VPA and ( E , E ) - 2 , 3 ' - d i e n e VPA. Pathway 3 i n c l u d e s 4 - e n e VPA and ( E ) - 2 , 4 - d i e n e VPA. Pathway 4 i n c l u d e s 4-OH VPA, 4 - k e t o VPA and 2 - p r o p y l s u c c i n i c a c i d . Pathway 5 i n c l u d e s 5-OH VPA and 2 - p r o p y l g l u t a r i c a c i d . Pathway R' i n c l u d e s VPA c o n j u g a t e d and u n c o n j u g a t e d . - 95 -T a b l e XIX U r i n a r y P r o f i l e s E x p r e s s e d as D i f f e r e n t VPA M e t a b o l i c Pathways B e f o r e and A f t e r A d m i n i s t r a t i o n of ASA i n P a t i e n t WN Day 1 Day 2 Day 2 - Day 1 VPA a l o n e VPA + ASA ug/mg c r e a t -i n i n e % o f t o t a l e x c r e t e d ug/mg c r e a t -i n i n e % o f t o t a l e x c r e t e d Change i n ug/mg c r e a t i n i n e Change i n % o f t o t a l e x c r e t e d Pathway l a 406 3 5 . 3 7 6 . 5 7 . 4 - 3 2 9 . 5 - 27 .6 Pathway 2 b 5 . 4 0 . 5 4 . 6 0 . 4 - 0 . 8 - 0 .1 Pathway 3 C 3 . 3 0 . 3 3 . 8 0 . 4 + 0 . 5 + 0.1 Pathway 4 d 128 11.1 120 11.6 - 8 + 0 . 6 Pathway 5 e 205 17.8 233 2 2 . 6 + 28 + 4 . 8 Pathway R ' f 403 35 591 5 7 . 3 + 188 + 2 2 . 3 a . Pathway 1 i n c l u d e s 2-ene VPA, 3-OH VPA and 3 - k e t o VPA. b . Pathway 2 i n c l u d e s 3-ene VPA and ( E , E ) - 2 , 3 ' - d i e n e VPA. c . Pathway 3 i n c l u d e s 4 - e n e VPA and ( E ) - 2 , 4 - d i e n e VPA. d . Pathway 4 i n c l u d e s 4-OH VPA, 4 - k e t o VPA and 2 - p r o p y l s u c c i n i c a c i d . e . Pathway 5 i n c l u d e s 5-OH VPA and 2 - p r o p y l g l u t a r i c a c i d . f . Pathway R' i n c l u d e s VPA c o n j u g a t e d and u n c o n j u g a t e d . - 96 -T a b l e XX U r i n a r y P r o f i l e s E x p r e s s e d as D i f f e r e n t VPA M e t a b o l i c Pathways B e f o r e and A f t e r A d m i n i s t r a t i o n of ASA i n a H e a l t h y V o l u n t e e r Day 1 VPA a l o n e Day 2 VPA + ASA Day 2 - D a y ! ug/mg c r e a t -i n i n e % o f t o t a l e x c r e t e d ug/mg c r e a t -i n i n e % o f t o t a l e x c r e t e d Change i n ug/mg c r e a t i n i n e Change i n % o f t o t a l e x c r e t e d Pathway l a Pathway 2 b Pathway 3 C Pathway 4 d Pathway 5 e Pathway R ' f 588 6 . 2 4 . 3 7 3 . 5 1 5 . 5 970 3 5 . 5 0 . 4 0 . 3 4 . 4 0 . 9 5 8 . 5 187.4 4 . 9 5 . 1 82 6 2 . 3 1704 9 . 2 0 . 2 0 . 3 4 3 8 3 . 3 - 4 0 0 . 6 - 1 . 3 + 0 . 8 + 8 . 5 + 4 6 . 8 + 734 - 2 6 . 3 - 0 . 2 0 - 0 . 4 + 2.1 + 2 4 . 8 a . Pathway 1 i n c l u d e s 2 - e n e VPA, 3-OH VPA and 3 - k e t o VPA. b. Pathway 2 i n c l u d e s 3-ene VPA and ( E , E ) - 2 , 3 ' - d i e n e VPA. c . Pathway 3 i n c l u d e s 4 - e n e VPA and ( E ) - 2 , 4 - d i e n e VPA. d . Pathway 4 i n c l u d e s 4-OH VPA, 4 - k e t o VPA and 2 - p r o p y l s u c c i n i c a c i d . e . Pathway 5 i n c l u d e s 5-OH VPA and 2 - p r o p y l g l u t a r i c a c i d . f . Pathway R' i n c l u d e s VPA c o n j u g a t e d and u n c o n j u g a t e d . - 97 -were found s i g n i f i c a n t l y d i f f e r e n t from day 1. The mean, d i f f e r e n c e and p e r c e n t change of t h e % o f t o t a l e x c r e t e d are i l l u s t r a t e d i n T a b l e X X I I . Pathways 2 and 3 r e p r e s e n t l e s s t h a n 1% o f % t o t a l e x c r e t e d b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA. Pathway 4 was v i r t u a l l y not a f f e c t e d by ASA i n g e s t i o n . Pathway R' and 1 r e p r e s e n t r e s p e c t i v e l y 5 0 . 5 % and 2 4 . 5 % o f t o t a l e x c r e t e d . U r i n a r y e x c r e t i o n of m e t a b o l i t e s of pathway R' was i n c r e a s e d by 2 9 . 7 % . The sum o f t h e ^ - o x i d a t i o n metabo-l i t e s (pathway 1) was d e c r e a s e d by 66% a f t e r ASA i n g e s t i o n . Pathway 5 i s o n l y m i l d l y a f f e c t e d by ASA a d m i n i s t r a t i o n (12.7% i n c r e a s e ) . The changes i n pathways 1 , 3 and R' on day 2 were found s i g n i f i c a n t l y d i f -f e r e n t from day 1 . S i n c e t h i s s t u d y i n c l u d e d on one hand a h e a l t h y a d u l t and on the o t h e r hand s i x p e d i a t r i c p a t i e n t s , i t was of i n t e r e s t t o d e t e r m i n e i f t h e study was not b i a s e d by t h e r e s u l t s of t h e normal s u b j e c t . T a b l e XXIII shows a comparison between t h e average amount of m e t a b o l i t e s e x c r e t e d f o r f i v e p a t i e n t s and one v o l u n t e e r and f o r f i v e p a t i e n t s o n l y . T h i s t a b l e shows t h a t pathways 1 , 2 , 3 , 4 and 5 are v i r t u a l l y unchanged i n t h e two groups and t h a t t h e % change i n pathway R' i s o n l y s l i g h t l y l o w e r i n the group of p e d i a t r i c p a t i e n t s (46% com-pared t o 52%). The r e s u l t s o b t a i n e d f o r t h e two groups of s u b j e c t s a r e , t h e r e f o r e , v e r y s i m i l a r . - 98 -T a b l e XXI Mean, d i f f e r e n c e and p e r c e n t change of u r i n a r y c o n c e n t r a t i o n s (ug/mg c r e a t i n i n e ) o f VPA and i t s m e t a b o l i t e s e x p r e s s e d as m e t a b o l i c pathways b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA (n=7). Day 1 VPA a l o n e mean ug/mg c r e a t i n i n e (SD) Day 2 VPA + ASA Day 2 - Day 1 mean ug/mg c r e a t i n i n e (SD) A % change Pathway l a 304 (181) 109 ( 6 8 ) * - 195 - 64 Pathway 2 b 8 . 2 ( 2 . 9 ) 5 . 6 ( 2 ) * - 2 . 6 - 31.7 Pathway 3 C 4 . 2 ( 2 . 1 ) 6 . 1 ( 5 . 6 ) + 1 . 9 + 4 5 . 2 Pathway 4 d 110 (47) 115 (45) + 5 + 4 . 5 Pathway 5 e 162 (123) 172 (76) + 10 + 6 . 2 Pathway R , f 611 (254) 931 ( 5 5 6 ) * * + 320 + 5 2 . 4 a . Pathway 1 i n c l u d e s 2 - e n e VPA, 3-OH VPA and 3 - k e t o VPA. b . Pathway 2 i n c l u d e s 3-ene VPA and ( E , E ) - 2 , 3 ' - d i e n e VPA. c . Pathway 3 i n c l u d e s 4 - e n e VPA and ( E ) - 2 , 4 - d i e n e VPA. d . Pathway 4 i n c l u d e s 4-OH VPA, 4 - k e t o VPA and 2 - p r o p y l s u c c i n i c a c i d . e . Pathway 5 i n c l u d e s 5-OH VPA and 2 - p r o p y l g l u t a r i c a c i d . f . Pathway R' i n c l u d e s VPA c o n j u g a t e d and u n c o n j u g a t e d . * S i g n i f i c a n t l y d i f f e r e n t from day 1 ( p < 0 . 0 5 ) . S i g n i f i c a n c e d e t e r m i n e d by S t u d e n t ' s p a i r e d t t e s t . * * Approaches s i g n i f i c a n c e (p = 0 . 0 7 3 7 ) . - 99 -T a b l e XXII Mean, d i f f e r e n c e and p e r c e n t change i n u r i n a r y p r o f i l e s o f VPA m e t a b o l i c pathways e x p r e s s e d as p e r c e n t of t o t a l e x c r e t e d b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA (n=7). Day 1 VPA a l o n e mean % t o t a l e x c r e t e d (SD) Day 2 VPA + ASA Day 2 - Day 1 mean % t o t a l e x c r e t e d (SD) A % change Pathway l a 2 4 . 5 ( 1 0 . 3 ) 8 . 3 ( 4 . 2 ) * - 1 6 . 2 - 66 Pathway 2 b 0.76 ( 0 . 2 5 ) 0 . 4 6 ( 0 . 2 6 ) - 0 . 3 0 - 3 9 . 5 Pathway 3 C 0 . 3 6 ( 0 . 2 4 ) 0 . 4 4 ( 0 . 2 6 ) * + 0 . 0 8 + 22 Pathway 4 d 9 . 4 ( 3 . 1 ) 9 . 2 ( 3 . 7 ) - 0 . 2 - 2 Pathway 5 e 1 4 . 2 ( 8 . 7 ) 16 ( 9 . 2 ) + 1 . 8 + 12.7 Pathway R ' f 5 0 . 5 ( 1 2 . 6 ) 6 5 . 5 ( 1 4 ) * + 15 + 2 9 . 7 a . Pathway 1 i n c l u d e s 2 - e n e VPA, 3-OH VPA and 3 - k e t o VPA. b . Pathway 2 i n c l u d e s 3-ene VPA and ( E , E ) - 2 , 3 ' - d i e n e VPA. c . Pathway 3 i n c l u d e s 4 - e n e VPA and ( E ) - 2 , 4 - d i e n e VPA. d . Pathway 4 i n c l u d e s 4-OH VPA, 4 - k e t o VPA and 2 - p r o p y l s u c c i n i c a c i d . e . Pathway 5 i n c l u d e s 5-OH VPA and 2 - p r o p y l g l u t a r i c a c i d . f . Pathway R' i n c l u d e s VPA c o n j u g a t e d and u n c o n j u g a t e d . * S i g n i f i c a n t l y d i f f e r e n t from day 1 ( p < 0 . 0 5 ) . S i g n i f i c a n c e d e t e r m i n e d by S t u d e n t ' s p a i r e d t t e s t . - 100 -T a b l e X X I I I Comparison between the average amounts of m e t a b o l i t e s e x c r e t e d (ug/mg c r e a t i n i n e ) a t s t e a d y s t a t e f o r s i x p a t i e n t s and one v o l u n t e e r and f o r s i x p a t i e n t s o n l y b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA. S i x p a t i e n t s and one v o l u n t e e r S i x p a t i e n t s o n l y Day 1 VPA a l o n e Day 2 VPA+ASA Day 2 - Day 1 % change Day 1 VPA a l o n e Day 2 VPA+ASA Day 2 - Day 1 % change Pathway l a 304 109 - 64 256 96 - 63 Pathway 2 8 . 2 5 . 6 - 32 8 . 6 5.7 - 34 Pathway 3 4 . 2 6 . 1 + 45 4 . 2 6 . 2 + 48 Pathway 4 110 115 + 5 116 121 + 4 Pathway 5 162 172 + 6 186 191 + 3 Pathway R! 611 931 + 52 551 803 + 46 a . Pathways 1 , 2 , 3 , 4 , 5 and R' have been d e f i n e d i n T a b l e X I V . - 101 -2 . P h a r m a c o k i n e t i c study To study the e f f e c t s of ASA on VPA, i t was i m p o r t a n t t o a l s o examine the e f f e c t s of ASA on the f o r m a t i o n and e l i m i n a t i o n pathways of VPA m e t a b o l i t e s . A l i n e a r model w i t h e l i m i n a t i o n from the c e n t r a l compartment was thought a p p r o p r i a t e f o r t h i s p u r p o s e . Scheme 2 i l l u s t r a t e s t h i s model i n which Cp i s the steady s t a t e c o n c e n t r a t i o n of p a r e n t drug i n the c e n t r a l compartment; C m i , Cm2, (^3, C m 4 , and Cm5 are the steady s t a t e c o n c e n t r a t i o n s of m e t a b o l i t e s r e a r r a n g e d r e s p e c t i v e l y i n pathways 1 , 2 , 3 , 4 and 5 i n the c e n t r a l compartment; C l p i s the t o t a l body c l e a r a n c e of p a r e n t d r u g ; C l f j , C l f 2 , C I f 3 , C l f 4 , CIf5 a r e t h e f o r m a t i o n c l e a r a n c e s c o r r e s p o n d i n g t o pathways 1 t o 5 ; C l r i s the r e n a l c l e a r a n c e of p a r e n t drug o t h e r than C l f j t o C I f 5 , and C l b l - - | e i s the e l i m i n a t i o n c l e a r a n c e v i a the b i l e . C l m i , C l m 2 , C l m 3 , C^nrt* C 1 m 5 a r e t n e e l i m i n a t i o n c l e a r a n c e s of m e t a b o l i t e s of pathways 1 t o 5 ( C l m i s a l s o c a l l e d m e t a b o l i t e c l e a r a n c e ) . C l r c i s the e l i m i n a t i o n c l e a r a n c e of VPA c o n j u g a t e s ; k 0 i s the d o s i n g r a t e of p a r e n t d r u g . Two o t h e r parameters c o n s i d e r e d i n t h i s a n a l y s i s are the f r a c t i o n s m e t a b o l i z e d ( f m i » f m 2 » tm3» ^m4» ^m5^ a n c l t n e c o n c e n t r a t i o n o f m e t a b o l i t e to p a r e n t drug r a t i o s ( C m / C p ) . T h i s model was based upon a model r e c e n t l y r e p o r t e d by Levy e t a l • to c h a r a c t e r i z e t h e i n d u c t i o n of clobazam by carbamazepine ( 1 4 6 ) . In the model i n Scheme 2 two assumptions were made: 1 . U r i n e i s the e x c r e t i o n organ f o r most of the m e t a b o l i t e s e x c e p t the g l u c u r o n i d e s which a l s o undergo b i l i a r y e x c r e t i o n . 2 . U r i n a r y V P A - g l u c u r o n i d e and unchanged VPA are grouped: Scheme 2. P h a r m a c o k i n e t i c model a p p l i e d i n t h e s t u d y o f VPA/ASA i n t e r a c t i o n i n a normal s u b j e c t . - 103 -C l r * = CI r c + CI r VPA where CI r c i s t h e m e t a b o l i t e c l e a r a n c e of t h e c o n j u g a t e s and CI r vp/\ i s t h e r e n a l c l e a r a n c e o f unchanged VPA. S i n c e the u r i n a r y e x c r e t i o n of f r e e v a l p r o i c a c i d i s small (14) t h e e r r o r i n t r o d u c e d by not measuring i t i s m i n i m a l . The t o t a l body c l e a r a n c e o f VPA, C l p , i s d e f i n e d by t h e f o l l o w i n g e q u a t i o n : C l p = C l r « + C l f i + C l f2 + C l f 3 + C l f 4 + C l f 5 + C l b i l e The b i l i a r y c l e a r a n c e was not d e t e r m i n e d s i n c e no b i l e o r f e c e s was c o l 1e c t e d . The m e t a b o l i t e c l e a r a n c e f o r a g i v e n m e t a b o l i c pathway at s t e a d y -s t a t e i s g i v e n by C 1 m i , s s = m1'» urine (1) AUC m i where mi u r i n e r e p r e s e n t s the e x c r e t e d amount o f m e t a b o l i t e s of the pathway i n v e s t i g a t e d and AUCml- i s t h e sum of t h e areas under t h e c u r v e f o r t h e m e t a b o l i t e s i n t h a t pathway d u r i n g t h e same t i m e i n t e r v a l . The f o r m a t i o n c l e a r a n c e f o r each m e t a b o l i c pathway at s t e a d y - s t a t e c o n c e n t r a t i o n i s g i v e n b y : - 104 -C 1 f i , s s = C m i » s s • C 1 m i (2) C P V P A , s s By r e a r r a n g i n g the terms of t h i s e q u a t i o n t h e m e t a b o l i t e - t o parent drug c o n c e n t r a t i o n r a t i o (Cm/Cp) i s g i v e n b y : Cm = CLfx (3) Cp CI no-where Cm-j j S S i s t h e average s t e a d y - s t a t e c o n c e n t r a t i o n of sum of m e t a b o l i t e s of a g i v e n pathway and Cp yp/^ s s i s the average s t e a d y - s t a t e c o n c e n t r a t i o n of parent drug i n t h e c e n t r a l compartment. The average c o n c e n t r a t i o n o f t h e m e t a b o l i t e s i n each pathway i s o b t a i n e d by e q u a t i o n (4) and t h e average c o n c e n t r a t i o n of parent drug by e q u a t i o n (5) where T i s t h e time i n t e r v a l . C m i . s s = AUC mi , s s (4) T and C P ) S S = AUC V P A . s s (5) T The f r a c t i o n m e t a b o l i z e d i n each g i v e n pathway ( f m i ) i s t h e r a t i o o f f o r m a t i o n c l e a r a n c e over t o t a l body c l e a r a n c e of parent drug ( e q u a t i o n 6 ) : fmi = C l f i . s s (6) C 1 p ' , s s T o t a l body c l e a r a n c e of p a r e n t drug ( C l p ) S S ) j S o b t a i n e d by C l p . s s = Do§e (7) AUC,. - 105 -T h i s model was chosen on the b a s i s t h a t the r a t i o of steady s t a t e c o n c e n t r a t i o n s of m e t a b o l i t e and p a r e n t drug i s equal t o a . r a t i o of c l e a r a n c e s ( e q u a t i o n 3) and cannot be e x p r e s s e d as a r a t i o of r a t e c o n s t a n t s because the volume of d i s t r i b u t i o n of the c e n t r a l c o m p a r t -ments f o r the p a r e n t drug and m e t a b o l i t e s may not be e q u a l . Serum c o n c e n t r a t i o n s o f VPA and i t s m e t a b o l i t e s were o b t a i n e d o n l y f o r the a d u l t v o l u n t e e r s i n c e the serum samples of the s i x p e d i a t r i c p a t i e n t s were no l o n g e r a v a i l a b l e . Due t o t h e i r very low c o n c e n t r a -t i o n s i n serum the ( E ) - 2 , 4 - d i e n e VPA and the 3-OH VPA were not measured i n the serum s a m p l e s . A l l o t h e r m e t a b o l i t e s determined f o r u r i n e samples were measured. To c a l c u l a t e m e t a b o l i t e c l e a r a n c e s C l m i -( e q u a t i o n 1) the amount of e x c r e t e d m e t a b o l i t e s and AUC v a l u e s were n e c e s s a r y . C u m u l a t i v e e x c r e t e d amounts of i n d i v i d u a l m e t a b o l i t e s and of m e t a b o l i t e s grouped i n t o d i f f e r e n t m e t a b o l i c pathways b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA are l i s t e d r e s p e c t i v e l y i n T a b l e s XXIV and XXV. The e x c r e t e d amount of unchanged and c o n j u g a t e d VPA has i n c r e a s e d by 91% and the sum of m e t a b o l i t e s of pathway 1 (2-ene VPA, 3-OH VPA and 3 - k e t o VPA) has d e c r e a s e d by 66% a f t e r a d m i n i s t r a t i o n of ASA ( T a b l e XXV). AUC v a l u e s f o r i n d i v i d u a l m e t a b o l i t e s and f o r m e t a b o l i t e s grouped i n t o pathways are i l l u s t r a t e d r e s p e c t i v e l y i n T a b l e s XXVI and X X V I I . A l l AUC v a l u e s f o r i n d i v i d u a l m e t a b o l i t e s were i n c r e a s e d on day 2 e x c e p t the ( E , E ) - 2 , 3 ' - d i e n e VPA and 3 - k e t o VPA which were d e c r e a s e d r e s p e c t i v e l y by 11 and 77% (Table X X V I ) . The AUC f o r unchanged and c o n j u g a t e d VPA was i n c r e a s e d by 20%, the AUCs f o r pathways 1 and 2 were unchanged, and the AUCs f o r pathways 3 , 4 and 5 were markedly - 106 -T a b l e XXIV C u m u l a t i v e e x c r e t e d amounts o f VPA and i t s m e t a b o l i t e s o v e r an 8 hour p e r i o d i n a normal s u b j e c t b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA. Dose o f VPA was 600 mg. CUMULATIVE EXCRETED AMOUNTS Day 1 VPA a l o n e Day 2 VPA + ASA Day 2 - Day 1 % change mq (% of dose) mg (% of dose) 4-OH VPA 3 . 4 ( 0 . 6 ) 4 . 3 ( 0 . 7 ) + 2 6 . 5 ( E ) - 2 , 4 - D i e n e VPA 0 . 0 7 ( 0 . 0 1 ) 0 . 0 6 ( 0 . 0 1 ) - 1 4 . 3 ( E , E ) - 2 , 3 ' - D i e n e VPA 0 . 3 9 ( 0 . 0 6 ) 0 . 2 8 ( 0 . 0 5 ) - 2 8 . 2 4-Ene VPA 0 . 2 8 ( 0 . 0 5 ) 0 . 3 9 ( 0 . 0 6 ) + 3 9 . 3 3-Ene VPA 0.11 ( 0 . 0 2 ) 0 . 1 5 ( 0 . 0 2 ) + 3 6 . 4 ( E ) - 2 - E n e VPA 0 . 7 4 ( 0 . 1 2 ) 0 . 7 6 ( 0 . 1 3 ) + 2.7 VPAa 7 8 . 4 (13) 150 (25) + 91 3 - K e t o VPA 4 3 . 1 ( 7 . 2 ) 1 5 . 2 ( 2 . 5 ) - 6 4 . 7 4 - K e t o VPA 2 . 3 ( 0 . 4 ) 2 .10 ( 0 . 3 5 ) - 8.7 3-OH VPA 4 . 0 ( 0 . 7 ) 0 . 5 1 ( 0 . 0 8 ) - 8 7 . 2 5-OH VPA 0 . 5 ( 0 . 0 8 ) 0 . 8 9 ( 0 . 1 5 ) + 78 2 - P r o p y l s u c c i n i c a c i d 0 . 2 5 ( 0 . 0 4 ) 0 . 7 9 ( 0 . 1 3 ) + 216 2 - P r o p y l g l u t a r i c a c i d 7 . 7 ( 1 . 3 ) 4 . 6 ( 0 . 7 7 ) - 4 0 . 3 Sum VPA + m e t a b o l i t e s 141 ( 2 3 . 5 ) 180 (30) + 2 7 . 5 a . VPA i n c l u d e s VPA g l u c u r o n i d e and unchanged VPA. - 107 -T a b l e XXV C u m u l a t i v e e x c r e t e d amounts of m e t a b o l i c pathways f o r VPA and i t s m e t a b o l i t e s o v e r an 8 hour p e r i o d i n a normal s u b j e c t b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA. C u m u l a t i v e e x c r e t e d amount (mg) Day 1 (VPA a l o n e ) mg (% o f dose) Day 2 (VPA + ASA) mg (% o f dose) Day 2 - Day 1 % change m i 3 4 7 . 8 0 (8) 16.47 ( 2 . 7 ) - 66 m 2 b 0 . 5 0 ( 0 . 0 8 ) 0 . 4 3 ( 0 . 0 7 ) - 14 m 3 c 0 . 3 5 ( 0 . 0 5 ) 0 . 4 5 ( 0 . 0 7 ) + 29 5 . 9 5 (1) 7 . 1 9 ( 1 . 2 ) + 21 m 5 e 8 . 1 9 ( 1 . 4 ) 5 . 4 9 ( 0 . 9 ) - 33 V P A f 7 8 . 4 0 (13) 150 (25) + 91 T o t a l m e t a b o l i t e s 141.2 ( 2 3 . 5 ) 180 (30) + 27 a . mi i n c l u d e s 2-ene VPA, 3-OH VPA and 3 - k e t o VPA. b . mz i n c l u d e s 3-ene VPA and ( E , E ) - 2 , 3 ' - d i e n e VPA. c . m3 i n c l u d e s 4 - e n e VPA and ( E ) - 2 , 4 - d i e n e VPA. d . 1114 i n c l u d e s 4-OH VPA, 4 - k e t o VPA and 2 - p r o p y l s u c c i n i c a c i d . e . m5 i n c l u d e s 5-OH VPA and 2 - p r o p y l g l u t a r i c a c i d . f . VPA i n c l u d e s c o n j u g a t e s and unchanged d r u g . - 108 -T a b l e XXVI AUC v a l u e s (mg.h/L) of VPA and i t s m e t a b o l i t e s 9 over an 8 hour p e r i o d i n a normal s u b j e c t b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA. Day 1 Day 2 Day 2 - Day 1 VPA a l o n e VPA + ASA -mg.h/L % o f t o t a l AUCs mg.h/L % of t o t a l AUCs % change 4-OH VPA 0 . 3 9 0 . 0 8 0.47 0 . 0 8 + 20 ( E , E ) - 2 , 3 ' - D i e n e VPA 6 . 2 2 1.27 5 . 5 5 0 . 9 5 - 11 4-Ene VPA 0 . 7 8 0 . 1 6 1 . 0 5 0 . 1 8 + 35 3-Ene VPA 1 . 4 5 0 . 3 0 2 . 0 3 0 . 3 5 + 40 ( E ) - 2 - E n e VPA 17.52 3 . 6 2 0 . 9 3 . 6 + 19 VPA 458 9 3 . 7 549 9 4 . 3 + 20 3 - K e t o VPA 4 . 3 0 0 . 8 8 0 . 9 7 0.17 - 77 4 - K e t o VPA 0 . 3 9 0 . 0 8 0 . 6 6 0.11 + 69 5-OH VPA 0 . 1 9 0 . 0 4 0 . 4 1 0 . 0 7 + 116 2 - P r o p y l s u c c i n i c a c i d 0 . 0 1 0 . 0 0 2 0.11 0 . 0 2 '• + 1000 2 - P r o p y l g l u t a r i c a c i d 0 . 2 5 0 . 0 5 0 . 5 1 0 . 0 8 + 104 Sum VPA + m e t a b o l i t e s 489 100 582 100 + 19 a . The ( E ) - 2 , 4 - d i e n e VPA and 3-OH VPA were not measured i n serum. - 109 -T a b l e XXVII AUC v a l u e s (mg.h/L) of m e t a b o l i c pathways f o r VPA and i t s m e t a b o l i t e s o v e r an 8 hour p e r i o d i n a normal s u b j e c t b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA. Day 1 VPA a l o n e Day 2 VPA + ASA Day 2 - Day 1 mg.h/L % o f t o t a l AUCs mg.h/L % o f t o t a l AUCs % change m i a 2 1 . 8 4 . 5 2 1 . 9 3 . 8 + 0 . 5 m2 7.67 1 . 6 7 . 5 8 1 . 3 - 1 m3 0 . 7 8 0.16 1 . 0 5 0 . 1 8 + 35 m 4 0 . 7 9 0.16 1 . 2 4 0.21 + 57 n»5 0 . 4 4 0 . 0 9 0 . 9 2 0.16 + 109 VPA 458 9 3 . 7 549 9 4 . 3 + 20 Sum VPA + m e t a b o l i t e s 489 100 582 100 + 19 a . mj_, m 2 , 1113, 1114, m$ and VPA have been d e f i n e d i n T a b l e X I V . - 110 -i n c r e a s e d . Over a l l the AUC of t o t a l m e t a b o l i t e s was i n c r e a s e d by 19% f o l l o w i n g ASA ( T a b l e X X V I I ) . The serum VPA c o n c e n t r a t i o n v e r s u s time curve f o r the normal s u b j e c t i s i l l u s t r a t e d i n F i g u r e 4 3 . The f i g u r e shows t h a t t o t a l VPA serum c o n c e n t r a t i o n s were e l e v a t e d a f t e r a d m i n i s t r a t i o n of ASA (day 2 ) . The 2-ene VPA serum c o n c e n t r a t i o n v e r s u s time c u r v e ( F i g u r e 44) shows an i n c r e a s e d AUC a f t e r a d m i n i s t r a t i o n of ASA. The AUC v a l u e f o r 3 - k e t o VPA was markedly d e c r e a s e d on day 2 as shown i n F i g u r e 4 5 . The 4-ene VPA serum c o n c e n t r a t i o n v e r s u s time curve b e f o r e and a f t e r a d m i n i s t r a -t i o n of ASA shows an i n c r e a s e d AUC on day 2 ( F i g u r e 4 6 ) . Serum c o n c e n -t r a t i o n v e r s u s time c u r v e s f o r the o t h e r m e t a b o l i t e s were not i l l u s t r a t e d s i n c e they r e p r e s e n t minor m e t a b o l i t e s i n serum. U s i n g e x c r e t e d amounts of m e t a b o l i t e s and AUC v a l u e s , the metabo-l i t e s c l e a r a n c e s ( C l m i - ) f o r the o x i d a t i v e pathways were c a l c u l a t e d and are l i s t e d i n T a b l e X X V I I I . A l l m e t a b o l i t e c l e a r a n c e s were d e c r e a s e d a f t e r a d m i n i s t r a t i o n of ASA, pathways 1 and 5 b e i n g t h e most a f f e c t e d ( r e s p e c t i v e d e c r e a s e s o f 66 and 6 8 % ) . The sum of m e t a b o l i t e c l e a r a n c e s ( £ C l m i - ) was decreased by 54% on day 2 . To c a l c u l a t e f o r m a t i o n c l e a r a n c e s ( e q u a t i o n 2) one needs the serum c o n c e n t r a t i o n s of m e t a b o l i t e s (Cm 1-) and the m e t a b o l i t e - t o p a r e n t drug r a t i o ( C m / C p ) . These v a l u e s are l i s t e d i n T a b l e XXIX. A l l serum c o n c e n t r a t i o n s were i n c r e a s e d e x c e p t m e t a b o l i t e s i n pathways 1 and 2 w h i c h are v i r t u a l l y unchanged. The m e t a b o l i t e - t o - p a r e n t drug r a t i o s are d e c r e a s e d f o r pathways 1 and 2 (17 and 18%) and i n c r e a s e d f o r pathways 3 , 4 and 5 r e s p e c t i v e l y by 1 2 , 29 and 70%. - I l l -F i g u r e 43. VPA serum c o n c e n t r a t i o n v e r s u s t i m e c u r v e i n a normal s u b j e c t b e f o r e and a f t e r a d m i n i s t r a t i o n o f ASA. 1 2 0 J 5 3 5 § "15 i5 TIME (hours) o — — o Day 1 (VPA a l o n e ) m ^ Day 2 (VPA + ASA) - 112 -F i g u r e 44. ( E ) - 2 - E n e VPA serum c o n c e n t r a t i o n v e r s u s t i m e c u r v e i n a normal s u b j e c t b e f o r e and a f t e r a d m i n i s t r a t i o n o f ASA. S 4 6 § 15 T2 TIME (hours) o o Day 1 (VPA a l o n e ) Day 2 (VPA + ASA) - 113 -F i g u r e 4 5 . 3 - K e t o VPA serum c o n c e n t r a t i o n v e r s u s t i m e c u r v e i n a normal s u b j e c t b e f o r e and a f t e r a d m i n i s t r a t i o n o f ASA. 1.2 TIME (hours) o o Day 1 (VPA a l o n e ) • - • Day 2 (VPA + ASA) - 114 -F i g u r e 46. 4-Ene VPA serum c o n c e n t r a t i o n v e r s u s t i m e c u r v e i n a normal s u b j e c t b e f o r e and a f t e r a d m i n i s t r a t i o n o f ASA. 5 3 g § i5 i"2 TIME (hours) o o Day 1 (VPA a l o n e ) # Day 2 (VPA + ASA) - 115 -F o r m a t i o n c l e a r a n c e s f o r the o x i d a t i v e pathways are l i s t e d i n T a b l e XXX. F o r m a t i o n c l e a r a n c e s f o r pathways 3 and 4 are unchanged but are d e c r e a s e d by 7 1 , 27 , and 44% r e s p e c t i v e l y f o r pathways 1 , 2 and 5 . The sum of f o r m a t i o n c l e a r a n c e s f o r the o x i d a t i v e pathways e C l f 1 -i s d e c r e a s e d by 60% f o l l o w i n g a d m i n i s t r a t i o n of ASA. The c l e a r a n c e of unchanged and c o n j u g a t e d VPA C l r < i s i n c r e a s e d by 60%. The c a l c u l a t e d body c l e a r a n c e C l p ' i s g i v e n to be the sum of f o r m a t i o n c l e a r a n c e s ( C I ) and r e n a l c l e a r a n c e of VPA ( C l r > ) . C l p « was i n c r e a s e d by 6% a f t e r a d m i n i s t r a t i o n of ASA. The f r a c t i o n m e t a b o l i z e d ( f m i ) f o r a g i v e n pathway i s the r a t i o of the f o r m a t i o n c l e a r a n c e to C l p ' ( e q u a t i o n 6 ) . These v a l u e s f o r day 1 and day 2 are l i s t e d i n T a b l e XXXI. For pathways 3 and 4 fm i s v i r t u a l l y unchanged, but i s decreased i n the o t h e r o x i d a t i v e pathways w i t h an o v e r a l l drop o f 62% a f t e r a d m i n i s t r a t i o n of ASA. The f r a c t i o n of unchanged and c o n j u g a t e d VPA f r ' was i n c r e a s e d by 50% on day 2 . To i n v e s t i g a t e the e f f e c t s of ASA on the d i f f e r e n t m e t a b o l i c p a t h -ways the p h a r m a c o k i n e t i c parameters d e f i n e d p r e v i o u s l y were r e p o r t e d f o r pathway R ' , f o r the sum of the o x i d a t i v e pathways and f o r i n d i v i d u a l pathways 1 t o 5 . Pathway R' The p h a r m a c o k i n e t i c parameters f o r pathway R' are l i s t e d i n T a b l e X X X I I . P r i o r t o a d m i n i s t r a t i o n of ASA the average s t e a d y - s t a t e c o n c e n -t r a t i o n of VPA was 5 7 . 2 ug/mL a s s o c i a t e d w i t h a c l e a r a n c e (Clr<) o f 0.1712 L / h . A d m i n i s t r a t i o n of ASA r e s u l t e d i n a 20% i n c r e a s e i n the s t e a d y - s t a t e serum VPA c o n c e n t r a t i o n (Table XXXII) and a 60% i n c r e a s e - 116 -Table XXVIII M e t a b o l i t e c l e a r a n c e s (Clm) o f VPA m e t a b o l i t e s i n a normal s u b j e c t b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA. M e t a b o l i t e C l e a r a n c e s ( L / h r ) a Day 1 (VPA a l o n e ) Day 2 (VPA + ASA) Day 2 - Day 1 % change c l m l 2.1926 0.7520 - 66 C V 0 . 0 6 5 2 0.0567 - 13 Clm3 0.4487 0.4286 - 4 C 1 m 4 7.5316 5.7984 - 23 C 1 m 5 18.6136 5.9674 - 68 28.8517 13.0031 - 54 a . The m e t a b o l i t e c l e a r a n c e C l m ^ i s c a l c u l a t e d u s i n g t h e amount of m e t a b o l i t e s of a g i v e n pathway r e c o v e r e d i n u r i n e over t h e c o r r e s p o n d i n g AUC. - 117 -T a b l e XXIX Average serum steady s t a t e c o n c e n t r a t i o n s of p a r e n t drug and m e t a b o l i t e s combined i n pathways ( C m j , s s ) and m e t a b o l i t e - p a r e n t drug c o n c e n t r a t i o n r a t i o (Cm/Cp) i n a normal s u b j e c t b e f o r e (Day 1) and a f t e r a d m i n i s t r a t i o n of ASA (Day 2 ) . C m i , ss (m g/D Cm/Cp . Day 1 VPA a l o n e Day 2 VPA+ASA Day 2 - Day 1 % change Day 1 VPA a l o n e Day 2 VPA+ASA Day 2 - Day 1 % change m^a 2 . 7 2 2 . 7 4 + 1 0 . 0 4 8 0 . 0 4 0 - 17 ITI2 0 . 9 6 0 . 9 5 - 1 0.017 0.014 - 18 IT>3 0 . 0 9 8 0.131 + 34 0.0017 0 . 0 0 1 9 + 12 ni4 0 . 0 9 9 0 . 1 5 5 + 57 0.0017 0 . 0 0 2 3 + 29 ms 0 . 0 5 5 0.115 + 109 0.0010 0.0017 + 70 m T 3 . 9 3 2 4 . 0 9 1 + 4 0 . 0 6 9 4 0 . 0 5 9 9 - 14 VPA b 5 7 . 2 6 8 . 6 + 20 — — — a . m^, m2, 1113, 1TI4, 1115, have been d e f i n e d i n T a b l e XXV. b . C, ,^ , ss f o r VPA i s d e f i n e d as Cp c o n c e n t r a t i o n of p a r e n t drug s i n c e VPA c o n j u g a t e s were not measured i n serum. - 118 -T a b l e XXX F o r m a t i o n c l e a r a n c e s ( C l f j and C l f c ) i n L/h of VPA m e t a b o l i t e s i n a normal s u b j e c t b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA. F o r m a t i o n C l e a r a n c e s ( L / h ) a Day 1 Day 2 Day 2 - Day 1 (VPA a l o n e ) (VPA + ASA) % Change C l f c b NAC NA — c i f i d 0.1044 0.0300 - 71 Cl f 2 0.0011 0.0008 - 27 Cl f 3 0.0008 0.0008 0 C l f 4 0.0130 0.0131 + 1 C l f 5 0.0179 0.0100 - 44 eClfl- 0.1372 0.0547 - 60 C l r ' e 0.1712 0.2732 + 60 ci p. f 0.3084 0.3279 + 6 a . C l f - j - Cm . C l m j Cp b. Clfc l s n o t a v a i l a b l e s i n c e the c o n j u g a t e s were not measured i n serum. c . Not a v a i l a b l e . d . Clfi - Clfc r e p r e s e n t the f o r m a t i o n c l e a r a n c e s of m e t a b o l i t e s r e s p e c t i v e l y i n v o l v e d i n pathways 1 t o 5. e . C l r ' = C l r c + C l r and c a l c u l a t e d u s i n g the t o t a l r e c o v e r e d VPA i n urine/AUC VPA. f . Clp" = eClfi + C l r < - 119 -Table XXXI Fraction of VPA metabolized (fm) by each pathway in a normal subject before and after administration of ASA. Fraction Metabolized (fmi)a Day 1 (VPA alone) Day 2 (VPA + ASA) Day 2 - Day 1 % Change fmj 0.338 0.091 - 73 fm2 0.004 0.003 - 25 fm3 0.003 0.003 0 flTl4 0.042 0.040 - 5 fm5 0.058 0.030 - 48 fmj 0.445 0.167 - 62 f r 1 - CI r• c i p . 0.555 0.833 + 50 a - fmi = C l f j where ( C l p ' = e C l f - j + C l r O CTpT - 120 -i n the r e n a l c l e a r a n c e of VPA and i t s g l u c u r o n i d e ; C l r i i n c r e a s e d from 0.1712 L/h t o 0.2732 L/h ( T a b l e X X X I I ) . The f r a c t i o n f r « c o r r e s p o n d i n g t o t h e r a t i o of r e n a l c l e a r a n c e o f VPA t o t h e c a l c u l a t e d body c l e a r a n c e of parent drug ( C l r ' / C l p ' ) showed an i n c r e a s e of 50%. These r e s u l t s i n d i c a t e t h a t more VPA was p r e s e n t i n serum a f t e r a d m i n i s t r a t i o n of ASA, more drug was m e t a b o l i z e d to g l u c u r o n i d e and more drug was c l e a r e d from t h e body by r e n a l e l i m i n a t i o n . O x i d a t i v e pathways (pathways 1-5) The p h a r m a c o k i n e t i c parameters f o r the sum of the o x i d a t i v e p a t h -ways 1 t o 5 are g i v e n i n T a b l e X X X I I I . The sum of m e t a b o l i t e c l e a r -ances ( e C l f - j ) f o r the o x i d a t i v e pathways decreased by 54% a f t e r a d m i n i s t r a t i o n of ASA. The sum o f s t e a d y s t a t e c o n c e n t r a t i o n of a l l o x i d a t i v e VPA m e t a b o l i t e s was 3 . 9 3 ug/mL on day 1 and was s l i g h t l y i n c r e a s e d t o 4 . 0 9 ug/mL on day 2 . The sum o f m e t a b o l i t e s - t o parent drug r a t i o ( C m j / C p ) was d e c r e a s e d by 14% a f t e r a d m i n i s t r a t i o n of ASA. The t o t a l f o r m a t i o n c l e a r a n c e f o r t h e o x i d a t i v e m e t a b o l i t e s ( C l f j ) showed an o v e r a l l d e c l i n e of 60%. The f r a c t i o n m e t a b o l i z e d ( f m j ) was d e c r e a s e d by 62%. These r e s u l t s i n d i c a t e an o v e r a l l marked i n h i b i t i o n by s a l i c y l a t e o f VPA m e t a b o l i s m v i a pathways 1 - 5 . Pathway 1 ( g - o x i d a t i o n ) The m e t a b o l i t e c l e a r a n c e and t h e f o r m a t i o n c l e a r a n c e of e - o x i d a -t i o n p r o d u c t s were d e c r e a s e d r e s p e c t i v e l y by 66 and 71% and t h e f r a c -t i o n m e t a b o l i z e d was m a r k e d l y reduced (73%, Table XXXIV). These r e -s u l t s i n d i c a t e t h a t s a l i c y l a t e s t r o n g l y i n h i b i t s t h e e - o x i d a t i o n - 121 -pathway. The s t e a d y - s t a t e c o n c e n t r a t i o n of t h e p - o x i d a t i o n m e t a b o l i t e s was v i r t u a l l y unchanged. As t h e serum c o n c e n t r a t i o n of 2-ene VPA i n c r e a s e d , t h e serum c o n c e n t r a t i o n o f 3 - k e t o VPA was l o w e r e d ( F i g u r e s 44 and 45) and the o v e r a l l e f f e c t was no change i n serum c o n c e n t r a t i o n of pathway 1 The r a t i o o f ^ - o x i d a t i o n m e t a b o l i t e s - t o p a r e n t d r u g d i s p l a y e d a 17% d e c l i n e . Pathway 2 The a d m i n i s t r a t i o n of ASA r e s u l t e d i n a d e c r e a s e of the m e t a b o l i t e c l e a r a n c e , t h e f o r m a t i o n c l e a r a n c e and t h e f r a c t i o n m e t a b o l i z e d by 1 3 , 27 and 25% r e s p e c t i v e l y . These r e s u l t s i n d i c a t e an i n h i b i t o r y e f f e c t o f s a l i c y l a t e on t h e f o r m a t i o n and t h e e l i m i n a t i o n of t h e sum o f 3-ene VPA and ( E , E ) - 2 , 3 ' - d i e n e VPA. The s t e a d y - s t a t e c o n c e n t r a t i o n of t h e s e two m e t a b o l i t e s was v i r t u a l l y unchanged; t h e m e t a b o l i t e s - t o p a r e n t drug c o n c e n t r a t i o n r a t i o was d e c r e a s e d by 18%. Pathway 3 The m e t a b o l i t e c l e a r a n c e of t h e sum of 4-ene VPA and ( E ) - 2 , 4 - d i e n e VPA was s l i g h t l y d e c r e a s e d a f t e r ASA a d m i n i s t r a t i o n (4%). The f o r m a -t i o n c l e a r a n c e and the f r a c t i o n m e t a b o l i z e d were unchanged. S a l i c y l a t e a p p a r e n t l y i n h i b i t e d t h e e l i m i n a t i o n of t h e m e t a b o l i t e s of pathway 3 s i n c e t h e s t e a d y - s t a t e c o n c e n t r a t i o n of t h e m e t a b o l i t e s was i n c r e a s e d by 34% and t h e metabol i t e - t o parent drug c o n c e n t r a t i o n r a t i o was i n c r e a s e d by 12%. - 122 -Pathway 4 The m e t a b o l i t e c l e a r a n c e of the sum of 4-OH VPA, 4 - k e t o VPA and 2 - p r o p y l s u c c i n i c a c i d showed a 23% d e c r e a s e a f t e r ASA a d m i n i s t r a t i o n ( T a b l e XXXIV) whereas the f o r m a t i o n c l e a r a n c e and the f r a c t i o n m e t a b o l i z e d were v i r t u a l l y unchanged. SA a p p a r e n t l y i n h i b i t e d the e l i m i n a t i o n of t h e s e m e t a b o l i t e s . A g r e a t e r amount of these m e t a b o l i t e s was p r e s e n t i n serum s i n c e t h e i r s t e a d y - s t a t e c o n c e n t r a t i o n showed a marked 57% i n c r e a s e . The m e t a b o l i t e s - t o - p a r e n t drug c o n c e n t r a t i o n r a t i o was enhanced (29%). Pathway 5 The c o a d m i n i s t r a t i o n of ASA i n h i b i t e d the m e t a b o l i t e c l e a r a n c e , the f o r m a t i o n c l e a r a n c e and the f r a c t i o n m e t a b o l i z e d of the sum o f 5-OH VPA and 2 - p r o p y l g l u t a r i c a c i d i n the r e s p e c t i v e p r o p o r t i o n s of 68%, 44% and 48%. S i n c e the r e l a t i v e i n h i b i t i o n of the f o r m a t i o n c l e a r a n c e was l e s s than t h a t of the m e t a b o l i t e c l e a r a n c e , more m e t a b o l i t e was p r e s e n t i n the serum. The s t e a d y - s t a t e c o n c e n t r a t i o n of these m e t a b o l i t e s showed 109% i n c r e a s e and the m e t a b o l i t e s - t o - p a r e n t drug c o n c e n t r a t i o n r a t i o was enhanced by 70% ( T a b l e XXXIV). U n s a t u r a t e d m e t a b o l i t e s 2-ene VPA and 4-ene VPA S i n c e 2 - e n e VPA and 4 - e n e VPA d i s p l a y some a n t i c o n v u l s a n t a c t i v i t y and 4 - e n e VPA has been i m p l i c a t e d i n VPA h e p a t o t o x i c i t y i t was of i n t e r e s t t o study the i n d i v i d u a l f a t e of these m e t a b o l i t e s a f t e r a d m i n i s t r a t i o n of ASA. The r e s u l t s i n terms of m e t a b o l i t e c l e a r a n c e , steady s t a t e serum c o n c e n t r a t i o n , m e t a b o l i t e - t o - p a r e n t drug c o n c e n t r a -t i o n r a t i o , f o r m a t i o n c l e a r a n c e and f r a c t i o n m e t a b o l i z e d are l i s t e d i n - 123 -T a b l e XXXV. These r e s u l t s i n d i c a t e t h a t s a l i c y l a t e i n h i b i t e d t h e f o r m a t i o n , t h e e l i m i n a t i o n and t h e f r a c t i o n m e t a b o l i z e d of 2-ene VPA. The s t e a d y s t a t e c o n c e n t r a t i o n o f 2 - e n e VPA showed a 19% i n c r e a s e a f t e r a d m i n i s t r a t i o n of ASA s u g g e s t i n g a p o s s i b i l i t y of enhancement of the t h e r a p e u t i c e f f e c t s i n c e 2-ene VPA d i s p l a y s a n t i c o n v u l s a n t p r o p e r t i e s . The m e t a b o l i t e - t o p a r e n t drug c o n c e n t r a t i o n r a t i o was unchanged. ASA a d m i n i s t r a t i o n s t i m u l a t e d t h e f o r m a t i o n o f 4 - e n e VPA, t h e f r a c t i o n m e t a b o l i z e d showing a 10% i n c r e a s e . The m e t a b o l i t e c l e a r a n c e was v i r t u a l l y unchanged. The serum c o n c e n t r a t i o n showed a 34% i n c r e a s e and the m e t a b o l i t e - t o p a r e n t drug c o n c e n t r a t i o n r a t i o d i s p l a y e d a 12% i n c r e a s e s u g g e s t i n g a p o s s i b l e i n c r e a s e d r i s k o f s i d e - e f f e c t s due t o g r e a t e r amounts of 4-ene VPA i n serum. It i s d i f f i c u l t t o make d e f i n i -t i v e c o n c l u s i o n s s i n c e t h i s study has been c a r r i e d out i n o n l y one s u b j e c t . More e x p e r i m e n t s are needed to c o n c l u d e on t h e complex e f f e c t o f s a l i c y l a t e on t h e m e t a b o l i s m of v a l p r o i c a c i d . - 124 -T a b l e XXXII P h a r m a c o k i n e t i c parameters determined f o r pathway R' i n a normal s u b j e c t t a k i n g v a l p r o i c a c i d b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA. C l r ' a C P Day 1 0.1712 5 7 . 2 0 . 5 5 5 Day 2 0.2732 6 8 . 6 0 . 8 3 3 % change + 60 + 20 + 50 a . C l r « = C l r r + C l r and c a l c u l a t e d u s i n g the t o t a l r e c o v e r e d VPA i n urine/AUC VPA. b . f r » = C l r « T a b l e XXXIII P h a r m a c o k i n e t i c parameters determined f o r the sum of the o x i d a t i v e pathways (pathways 1 t o 5) i n a normal s u b j e c t t a k i n g v a l p r o i c a c i d b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA. £ C l m i CmT CmT/Cp e c i f j f m T (L/h) (mg/L) (L/h] Day 1 28.8517 3 . 9 3 2 0 . 0 6 9 4 0.1372 0 . 4 4 5 Day 2 13.0031 4 . 0 9 1 0 . 0 5 9 9 0.0547 0.167 % change - 54 + 4 - 14 - 60 - 62 - 125 -T a b l e XXXIV P h a r m a c o k i n e t i c parameters determined f o r pathways 1 t o 5 i n a normal s u b j e c t t a k i n g v a l p r o i c a c i d b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA. Clm % (L/h) cm (mg/L) Cjn/Cp C l f (L/h) fm Pathway 1 Day 1 2.1926 2.72 0 . 0 4 8 0.1044 0 . 3 3 8 Day 2 0.7520 2.74 0 . 0 4 0 0 . 0 3 0 0 0.091 % change - 66 + 1 - 17 - 71 - 73 Pathway 2 Day 1 0 . 0 6 5 2 0 . 9 6 0.017 0.0011 0 . 0 0 4 Day 2 0.0567 0 . 9 5 0.014 0 . 0 0 0 8 0 . 0 0 3 % change - 13 - 1 - 18 - 27 - 25 Pathway 3 Day 1 0.4487 0 . 0 9 8 0.0017 0 . 0 0 0 8 0 . 0 0 3 Day 2 0.4286 0.131 0.0019 0.0008 0 . 0 0 3 % change - 4 + 34 + 12 0 0 Pathway 4 Day 1 7 .5316 0 . 0 9 9 0.0017 0.0130 0 . 0 4 2 Day 2 5.7984 0.155 0.0023 0.0131 0 . 0 4 0 % change - 23 + 57 + 29 + 1 - 5 Pathway 5 Day 1 18.6136 0 . 0 5 5 0.0010 0.0179 0 . 0 5 8 Day 2 5.9674 0.115 0.0017 0.0100 0 . 0 3 0 % change - 68 + 109 + 70 - 44 - 48 - 126 -T a b l e XXXV P h a r m a c o k i n e t i c parameters determined f o r 2-Ene VPA and 4-Ene VPA i n a normal s u b j e c t t a k i n g v a l p r o i c a c i d b e f o r e and a f t e r a d m i n i s t r a t i o n of ASA. Clm ( L / h r ) Cm (mg/L) Cm/Cp C l f (L/h) fm Day 1 (VPA a l o n e ) 0.0422 2 . 1 9 0 . 0 3 8 0.0016 0 . 0 0 5 2 2-Ene VPA Day 2 (VPA + ASA) 0 . 0 3 6 4 2 . 6 1 0 . 0 3 8 0.0014 0 . 0 0 4 3 % change - 14 + 19 0 - 12 - 17 Day 1 (VPA a l o n e ) 0 . 3 5 9 0 0 . 0 9 8 0.0017 0 . 0 0 0 6 0 . 0 0 2 0 4-Ene VPA Day 2 (VPA + ASA) 0.3714 0.131 0.0019 0.0007 0 . 0 0 2 2 % change + 3 + 34 + 12 + 17 + 10 - 127 -3 . D i s c u s s i o n In p r e v i o u s s t u d i e s s a l i c y l a t e has been r e p o r t e d t o d i s p l a c e VPA from i t s p r o t e i n b i n d i n g s i t e s ( 1 0 1 - 1 0 3 ) . The i n t e r a c t i o n of s a l i c y l i c a c i d w i t h v a l p r o i c a c i d i n p e d i a t r i c e p i l e p t i c p a t i e n t s i n d i c a t e d e l e v a t e d f r e e VPA serum l e v e l s and lowered f r e e VPA c l e a r a n c e s ( 1 0 5 ) . Furthermore t o t a l VPA and f r e e VPA h a l f - l i v e s were d e c r e a s e d . S i n c e serum c o n c e n t r a t i o n s of f r e e and t o t a l VPA were i n c r e a s e d w i t h no d e c r e a s e i n r e n a l c l e a r a n c e of VPA c o n j u g a t e s ( 1 0 4 ) , i t was suggested t h a t a m e t a b o l i c i n h i b i t i o n may a l s o be i n v o l v e d i n t h e i n t e r a c t i o n o f s a l i c y l a t e w i t h VPA. To understand the i n t e r a c t i o n of s a l i c y l a t e on VPA a s h o r t r e v i e w o f f a t t y a c i d m e t a b o l i s m may be u s e f u l . Review of f a t t y a c i d m e t a b o l i s m V a l p r o i c a c i d i s a s h o r t - c h a i n branched f a t t y a c i d m o s t l y e l i m i n -a t e d by m e t a b o l i s m . g - O x i d a t i o n r e p r e s e n t s one o f t h e major pathways f o r t h e d e g r a d a t i o n of f a t t y a c i d s . U n t i l very r e c e n t l y t h e c e l l u l a r s i t e o f g - o x i d a t i o n was t h o u g h t t o be e x c l u s i v e l y t h e i n n e r membrane o f t h e m i t o c h o n d r i o n . S i n c e 1976, a n o t h e r c l a s s of s u b c e l l u l a r p a r t i c l e s c a l l e d peroxisomes o r m i c r o b o d i e s have been shown t o c o n t a i n a l l t h e enzymes n e c e s s a r y f o r t h e g - o x i d a t i o n of l o n g c h a i n f a t t y a c i d s . Peroxisomes have been observed i n r a t l i v e r ( 1 4 7 ) , mouse l i v e r (148) and human l i v e r ( 1 4 9 ) . In r a t s t h e h e p a t i c peroxisomal g - o x i d a t i o n c a p a c i t y r e p r e s e n t s a p p r o x i m a t e l y h a l f o f t h e m i t o c h o n d r i a l c a p a c i t y ( 1 5 0 ) . M i t o c h o n d r i a and peroxisomes a r e b i o c h e m i c a l l y d i s t i n c t . The m i t o c h o n d r i a a r e more a c t i v e towards g - o x i d a t i o n o f s h o r t e r c h a i n f a t t y a c i d s whereas peroxisomes f a v o u r g - o x i d a t i o n of l o n g c h a i n f a t t y a c i d s - 128 -( C i o - C 2 2 ) « C a r n i t i n e i s not r e q u i r e d f o r the t r a n s p o r t of l o n g c h a i n m o n o c a r b o x y l i c a c i d s i n t o the p e r o x i s o m e s . The h y p o l i p i d e m i c drug c l o f i b r a t e a d m i n i s t e r e d t o a n i m a l s can i n c r e a s e B - o x i d a t i o n of l o n g c h a i n f a t t y a c i d s f i v e t o t e n - f o l d i n p e r o x i s o m e s . A c y l - C o A i s t h e s u b s t r a t e f o r B - o x i d a t i o n . A c y l - C o A d e r i v a t i v e s of f a t t y a c i d s are not a b l e to p e n e t r a t e the m i t o c h o n d r i a l membrane. They are t r a n s p o r t e d i n t o the m i t o c h o n d r i a t o the s i t e of o x i d a t i o n as e s t e r s of the base c a r n i t i n e w i t h the i n t e r v e n t i o n of the enzyme c a r n i t i n e - a c y l t r a n s f e r -a s e . A c y l - C o A d e r i v a t i v e s of l o n g c h a i n f a t t y a c i d s e n t e r f r e e l y i n t o the peroxisomes and are then i n c o m p l e t e l y B - o x i d i z e d to o c t a n o y l - C o A and hexanoyl-CoA which t o g e t h e r w i t h the produced a c e t y l - C o A l e a v e the peroxisomes f o r f i n a l d e g r a d a t i o n i n the m i t o c h o n d r i a ( 1 5 1 ) . The enzymes of B - o x i d a t i o n have v e r y h i g h a f f i n i t i e s f o r t h e i r s u b s t r a t e s thus making i t v e r y d i f f i c u l t to d e t e c t the i n t e r m e d i a t e s o f B - o x i d a -t i o n . The two major p r o d u c t s of B - o x i d a t i o n are a c e t y l - C o A and a c e t o -a c e t a t e . A c e t o a c e t a t e w i t h i t s r e d u c t i o n p r o d u c t B - h y d r o x y b u t y r a t e , and i t s d e c a r b o x y l a t i o n p r o d u c t , acetone c o n s t i t u t e a group of com-pounds known as "ketone b o d i e s " . A c e t y l - C o A i s the s u b s t r a t e f o r two competing r e a c t i o n s w i t h o x a l o a c e t a t e t o form c i t r a t e i n the TCA c y c l e o r w i t h a c e t o a c e t y l - C o A t o form ketone b o d i e s ( k e t o g e n e s i s ) . There i s a n e c e s s a r y i n t e r r e l a t i o n between B - o x i d a t i o n and c a r b o h y d r a t e metabo-l i s m s i n c e the end p r o d u c t of B - o x i d a t i o n , a c e t y l - C o A i s an a c t i v a t o r of p y r u v a t e c a r b o x y l a s e , a key enzyme i n g l u c o n e o g e n e s i s . In c o n d i -t i o n s such as d i a b e t e s , B - 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 are i n c r e a s e d and the e x c e s s of a c e t y l - C o A produced l e a d s t o the f o r m a t i o n of e x c e s -s i v e ketone b o d i e s , a c o n d i t i o n known as k e t o s i s which can a l s o o c c u r - 129 -when t h e r e i s a reduced s u p p l y of c a r b o h y d r a t e such as i n s t a r v a t i o n or n u t r i t i o n a l i m b a l a n c e . The w and (w-1) o x i d a t i o n r e a c t i o n s o c c u r i n the e n d o p l a s m i c r e t i -culum and l e a d t o the f o r m a t i o n of d i c a r b o x y l i c a c i d s v i a hydroxy i n t e r m e d i a t e s . One of the enzymes i n v o l v e d i n these pathways i s a mixed f u n c t i o n oxygenase. The u r i n a r y data o b t a i n e d w i t h the seven s u b j e c t s i n d i c a t e t h a t B - o x i d a t i o n i s i n h i b i t e d by the c o a d m i n i s t r a t i o n of ASA. The f a c t t h a t t h e t h r e e m e t a b o l i t e s of t h i s pathway ( i . e . 2-ene VPA, 3-OH VPA and 3 - k e t o VPA) are d e c r e a s e d s u g g e s t s t h a t the ASA i n t e r a c t i o n o c c u r s a t a stage p r i o r to the f o r m a t i o n of 2-ene VPA. S i n c e VPA i s B - o x i d i z e d i t i s assumed t h a t s i m i l a r t o o t h e r m o n o c a r b o x y l i c a c i d s , VPA e x i s t s as t h e C o A - d e r i v a t i v e i n t r a m i t o c h o n d r i a l l y . Indeed v a l p r o y l - C o A has been i s o l a t e d from r a t l i v e r h e p a t o c y t e s and c h a r a c t e r i z e d by h i g h - p e r f o r m -ance l i q u i d chromatography ( 1 0 7 ) . B e c k e r and H a r r i s (107) r e p o r t e d t h a t VPA i n h i b i t e d f a t t y a c i d s y n t h e s i s from endogenous s u b s t r a t e s by h e p a t o c y t e s i s o l a t e d from m e a l - f e d female r a t s and t h a t s a l i c y l a t e p a r t i a l l y p r o t e c t e d a g a i n s t VPA i n h i b i t i o n of f a t t y a c i d s y n t h e s i s . VPA was a l s o shown t o d e c r e a s e p r o d u c t i o n of ketone b o d i e s from f a t t y a c i d s by h e p a t o c y t e s i s o l a t e d from 48 hour f a s t e d r a t s . S a l i c y l a t e a l m o s t t o t a l l y r e v e r s e d t h i s VPA i n h i b i t i o n of ketone b o d i e s produced from o l e a t e . S a l i c y l a t e a l s o p a r t i a l l y p r o t e c t e d a g a i n s t t h e VPA i n h i b i t i o n of g l u c o n e o g e n e s i s from l a c t a t e . T h i s study a l s o r e p o r t e d t h a t i n c r e a s i n g c o n c e n t r a t i o n s o f VPA caused a marked d e c r e a s e i n the c e l l u l a r c o n t e n t of u n e s t e r i f i e d - C o A and a c e t y l - C o A and an i n c r e a s e i n medium-chain a c y l - C o A e s t e r s (composed l a r g e l y of v a l p r o y l - C o A ) , where-- 130 -as the c o n c e n t r a t i o n of l o n g - c h a i n a c y l - C o A e s t e r s remained unchanged. S a l i c y l a t e e x e r t e d a s i g n i f i c a n t p r o t e c t i v e e f f e c t a g a i n s t both the d e p l e t i o n of CoA and a c e t y l - C o A and the a c c u m u l a t i o n of medium-chain a c y l - C o A e s t e r s caused by VPA. F o r m a t i o n of v a l p r o y l - C o A seems a p r e r e q u i s i t e f o r the e f f e c t of VPA on f a t t y a c i d m e t a b o l i s m . S i n c e v a l p r o y l - C o A i s formed by i s o l a t e d r a t l i v e r m i t o c h o n d r i a i t i s assumed t o be a B - o x i d a t i o n p r o d u c t ( 1 0 7 ) , t h e r e f o r e an i n t e r m e d i a t e m e t a b o l i t e between VPA and the f o r m a t i o n o f 2-ene VPA i n the B - o x i d a t i o n pathway. M a h l e r ' s enzyme, a f a t t y a c i d a c t i v a t i n g enzyme (FAAE) which c a t a l y z e s the f o r m a t i o n of a c y l - C o A from a wide v a r i e t y of f a t t y a c i d s i n the presence of CoA and A T P , i s known t o a c t i v a t e C 4 t o C 1 2 s t r a i g h t c h a i n f a t t y a c i d s and s e v e r a l b r a n c h e d - c h a i n f a t t y a c i d s ( 1 5 2 ) . S a l i c y l a t e has been r e p o r t e d t o i n h i b i t hexanoyl-CoA f o r m a t i o n by M a h l e r ' s enzyme ( 1 5 3 ) . S i n c e s a l i -c y l a t e l o w e r s the degree of v a l p r o i c a c i d i n h i b i t i o n of m e t a b o l i c p r o c e s s e s and reduces the a c c u m u l a t i o n of medium-chain a c y l - C o A e s t e r s (composed l a r g e l y of v a l p r o y l - C o A as s t a t e d b e f o r e ) , s a l i c y l a t e i s e x p e c t e d t o i n h i b i t the f o r m a t i o n of v a l p r o y l - C o A . The r e s u l t s we o b t a i n e d w i t h u r i n e samples of seven s u b j e c t s and serum samples of the a d u l t h e a l t h y v o l u n t e e r s u p p o r t the h y p o t h e s i s t h a t v a l p r o i c a c i d must be f i r s t a c t i v a t e d t o a CoA e s t e r i n the m i t o c h o n d r i a b e f o r e b e i n g f u r t h e r m e t a b o l i z e d . I f we admit t h a t f o r m a t i o n of v a l p r o y l - C o A , s i m i l a r t o the f o r m a t i o n of h e x a n o y l - C o A , i s dependent on M a h l e r ' s e n z y m e , s a l i c y l a t e may a l s o i n h i b i t the f o r m a t i o n of v a l p r o y l - C o A by the same mechanism. I f f o r m a t i o n of v a l p r o y l - C o A i s lowered then the s u b -- 131 -sequent p r o d u c t s of the B - o x i d a t i o n pathway i . e . 2-ene VPA, 3-OH VPA and 3 - k e t o VPA would be d e c r e a s e d . Peroxisomes have been r e p o r t e d t o be i n a c t i v e w i t h b u t y r y l - C o A and l e s s a c t i v e w i t h o c t a n o y l - C o A than w i t h l a u r o y l - C o A or p a l m i t o y l - C o A ( 1 4 7 ) . A l t h o u g h peroxisomes appear s p e c i a l i z e d f o r the B - o x i d a t i o n of l o n g c h a i n f a t t y a c i d s , they e x h i b i t some a c t i v i t y towards s h o r t e r c h a i n f a t t y a c i d s such as o c t a n o y l - C o A and they may be r e s p o n s i b l e f o r p a r t of the B - o x i d a t i o n of v a l p r o i c a c i d {CQ f a t t y a c i d ) . In the e x p e r i m e n t s r e p o r t e d by B e c k e r and H a r r i s ( 1 0 7 ) , no procedure was r e p o r t e d to d i f f e r e n t i a t e m i t o c h o n d r i a l and p e r o x i s o m a l B - o x i d a t i o n , and the m i t o c h o n d r i a l p r e p a r a t i o n s may have been c o n t a m i n a t e d by p e r o -x i s o m e s . I t has been r e p o r t e d t h a t the a d m i n i s t r a t i o n of ASA t o r a t s caused a p r o l i f e r a t i o n of h e p a t i c peroxisomes (154, 155) and a s e v e n - f o l d i n c r e a s e i n p e r o x i s o m a l enzyme a c t i v i t y ( 1 5 5 ) . In t h i s s t u d y m i t o c h o n d r i a l and p e r o x i s o m a l B - o x i d a t i o n were d i f f e r e n t i a t e d on the b a s i s t h a t p e r o x i s o m a l p a l m i t o y l - C o A o x i d a t i o n i s c y a n i d e i n s e n s i -t i v e . The a p p a r e n t time f o r r e a c h i n g h a l f - w a y to the maximal p e r o x i -somal i n d u c t i o n by ASA a d m i n i s t r a t i o n was 3 . 5 days and the h a l f - l i f e a f t e r removal of the drug was 2 . 0 d a y s . There was no s i g n i f i c a n t e f f e c t of ASA a d m i n i s t r a t i o n on the m i t o c h o n d r i a l enzyme a c t i v i t y . SA shows the same e f f e c t s on peroxisome p r o l i f e r a t i o n as the h y p o l i p i d e m i c drug c l o f i b r a t e (156, 1 5 7 ) . F o r c l o f i b r a t e and SA t o e x e r t an i n c r e a s e i n p e r o x i s o m a l B - o x i d a t i o n a c t i v i t y , seven to t e n days are n e c e s s a r y to induce peroxisome p r o l i f e r a t i o n ( 1 5 5 - 1 5 7 ) . Heinemeyer e t a l • r e p o r t e d the e f f e c t s of c l o f i b r a t e on v a l p r o i c a c i d o x i d a t i o n and g l u c u r o n i d a t i o n i n male r a t s a d m i n i s t e r e d c l o f i b r a t e a t - 132 -t h e dose of 500 mg/kg f o r seven days ( 1 5 8 ) . C l o f i b r a t e was found t o enhance the e x c r e t i o n o f VPA and 2 - p r o p y l g l u t a r i c a c i d g l u c u r o n i d e s ; B - o x i d a t i o n was s t i m u l a t e d ( i n c r e a s e d e x c r e t i o n of 2-ene VPA and 3 - k e t o V P A ) ; the r e c o v e r y o f 4-OH VPA was a l s o i n c r e a s e d 5 t i m e s . In our study ASA a d m i n i s t r a t i o n (one day) was too s h o r t t o induce peroxisome p r o l i f e r a t i o n . To t e s t the p o t e n t i a l e f f e c t of ASA on p e r o x i s o m a l i n d u c t i o n and the consequences on VPA m e t a b o l i s m , ASA s h o u l d be c o a d m i n i s t e r e d w i t h VPA f o r seven t o ten d a y s . As v a l p r o i c a c i d i s B - o x i d i z e d by both m i t o c h o n d r i a and p e r o x i s o m e s , the c o a d m i n i s t r a t i o n of ASA may have complex e f f e c t s on VPA m e t a b o l i s m depending on the r e l a t i v e p r o p o r t i o n of 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 B - o x i d a t i o n and on the degree of p e r o x i s o m a l i n d u c t i o n by ASA. P o s s i b l e c l i n i c a l s i g n i f i c a n c e o f ASA/VPA i n t e r a c t i o n The main c o n c e r n w i t h v a l p r o i c a c i d t h e r a p y i s i t s p o t e n t i a l t o induce r a r e but o f t e n f a t a l c a s e s of h e p a t o t o x i c i t y . The i n h i b i t i o n of f a t t y a c i d B - o x i d a t i o n r e p o r t e d w i t h v a l p r o i c a c i d has a l s o been observed w i t h i t s u n s a t u r a t e d m e t a b o l i t e s 4-ene VPA and 2 , 4 - d i e n e VPA which are p o t e n t i n d u c e r s of m i c r o v e s i c u l a r s t e a t o s i s i n young r a t s ( 1 5 9 ) . I t has been suggested t h a t VPA i n h i b i t s f a t t y a c i d B - o x i d a t i o n by s e q u e s t e r i n g of CoA (107, 159) whereas 4 - e n e VPA CoA e s t e r i s a d i r e c t and p o t e n t i n h i b i t o r of a s p e c i f i c enzyme(s) i n the B - o x i d a t i o n s e q u e n c e . C o a d m i n i s t r a t i o n of ASA a t t h e r a p e u t i c doses f o r one day showed an i n h i b i t i o n of v a l p r o i c a c i d B - o x i d a t i o n t h a t can be e x p l a i n e d by the i n h i b i t i o n by ASA of M a h l e r ' s enzyme r e s p o n s i b l e f o r v a l p r o y l CoA f o r m a t i o n . I f M a h l e r ' s enzyme (152) were i n v o l v e d i n the f o r m a t i o n - 133 -o f 4-ene VPA CoA, then the f o r m a t i o n of t h i s s t e a t o g e n i c p r o d u c t may a l s o be i n h i b i t e d by ASA. Thus c o a d m i n i s t r a t i o n of ASA under the c o n d i t i o n s s t u d i e d here might d e c r e a s e the p o t e n t i a l h e p a t o t o x i c i t y of VPA. When p h y s i o l o g i c a l B - o x i d a t i o n i s i n h i b i t e d , t h e r e i s i n c r e a s e d e x c r e t i o n of d i c a r b o x y l i c a c i d s ( 1 6 0 , 161) . Long c h a i n f a t t y a c i d s are n o r m a l l y degraded by p e r o x i s o m a l B - o x i d a t i o n i n t o s h o r t e r c h a i n f a t t y a c i d s which then e n t e r the m i t o c h o n d r i a t o be f u r t h e r m e t a b o l i z e d . When m i t o c h o n d r i a l B - o x i d a t i o n i s i m p a i r e d t h e s e s h o r t e r c h a i n f a t t y a c i d s are degraded by w - o x i d a t i o n to form d i c a r b o x y l i c a c i d s , but e n h a n c e d . e x c r e t i o n of a d i p i c a c i d has a l s o been r e p o r t e d i n h e a l t h y p a t i e n t s r e c e i v i n g VPA ( 1 6 2 ) . In p a t i e n t s r e c e i v i n g i n c r e a s i n g doses o f v a l p r o i c a c i d , t h e r e i s a l a r g e i n c r e a s e i n g l u c u r o n i d a t i o n and a s i g n i f i c a n t decrease o f B - o x i d a t i o n ( 1 6 3 ) , temporary s e q u e s t r a t i o n of f r e e CoA r e s u l t s i n enhanced exposure of g l u c u r o n y l t r a n s f e r a s e t o VPA. In our study the i n h i b i t i o n o f VPA B - o x i d a t i o n r e s u l t e d i n an i n c r e a s e d g l u c u r o n i d a t i o n . S i n c e VPA i s h i g h l y bound t o serum albumin (90%) s m a l l r e d u c t i o n s i n the e x t e n t of b i n d i n g r e s u l t i n l a r g e i n c r e a s e s i n t h e f r e e f r a c t i o n and h e p a t i c e x t r a c t i o n . S a l i c y l a t e has been shown t o d i s p l a c e VPA from i t s b i n d i n g s i t e s i n c r e a s i n g the f r e e f r a c t i o n o f VPA ( 1 0 4 ) . Our study showed t h a t serum VPA m e t a b o l i t e s were a l s o i n c r e a s e d t h e r e f o r e e x p o s i n g the l i v e r t o h i g h e r doses o f 4 - e n e VPA, the s u s -p e c t e d h e p a t o t o x i n which may be i n v o l v e d i n the o c c u r r e n c e of VPA i n d u c e d Reye syndrome ( 8 9 , 9 1 ) . In some c a s e s a c a u s a l r e l a t i o n s h i p has been e s t a b l i s h e d between s a l i c y l a t e i n g e s t i o n and Reye syndrome (108, 110, 113) . F o r m a t i o n of - 134 -4-ene VPA was enhanced by c o a d m i n i s t r a t i o n of ASA. T h u s , exposure t o g r e a t e r amounts o f 4 - e n e VPA i n serum due to p r o t e i n b i n d i n g d i s p l a c e -ment by ASA s u g g e s t s t h a t c a u t i o n s h o u l d apply f o r the c o n c o m i t t a n t a d m i n i s t r a t i o n of t h e s e two drugs p a r t i c u l a r l y i n h i g h r i s k p a t i e n t s , m o s t l y m e n t a l l y r e t a r d e d c h i l d r e n w i t h i n b o r n e r r o r s of m e t a b o l i s m . - 135 -SUMMARY AND CONCLUSIONS 1. A simultaneous quantitative analysis of valproic add and twelve of its metabolites has been developed using capillary GC-NS. 2. The 4-ene VPA peak was well resolved from that of VPA. All other peaks of interest were also well resolved and there were no inter-fering background peaks. 3. All calibration curves for standards had correlation coefficients greater than 0.99. The lower detection limit for metabolites was on the order of 0.1 ug/mL. 4. This assay is the f irst one reported for the simultaneous analysis of twelve VPA metabolites 1n patient samples. 5. The use of TMS and tBDMS derivatives for VPA metabolite analysis was compared. The tBDMS derivatives were found superior 1n terms of the sensitivity obtained for the unsaturated metabolites, the shorter chromatography time of the analysis and the stability of the derivatives. - 136 -6. P r e l i m i n a r y d a t a o b t a i n e d w i t h mixed TMS-tBDMS d e r i v a t i v e s suggest t h a t t h e s e may be a good a l t e r n a t i v e t o the tBDMS a l o n e f o r t h e a n a l y s i s of t h e more p o l a r m e t a b o l i t e s such as 3-HO VPA. 7 . To study t h e e f f e c t s of ASA on VPA m e t a b o l i s m , u r i n a r y m e t a b o l i c p r o f i l e s o f seven s u b j e c t s ( s i x p e d i a t r i c p a t i e n t s and one normal a d u l t s u b j e c t ) were d e t e r m i n e d . The r e s u l t s o b t a i n e d showed t h a t t h e e x c r e t i o n o f c o n j u g a t e d and unchanged VPA was enhanced i n a l l s u b j e c t s f o l l o w i n g a d m i n i s t r a t i o n of ASA. T h i s f i n d i n g i s c o n -s i s t e n t w i t h t h e f a c t t h a t s a l i c y l a t e d i s p l a c e s VPA from i t s b i n d i n g s i t e s r e s u l t i n g i n h i g h e r l e v e l s of f r e e VPA i n serum. The u r i n a r y m e t a b o l i c p r o f i l e s showed t h a t t h e e x c r e t i o n o f 5 - k e t o VPA was markedly r e d u c e d . The mean e x c r e t i o n of 2-ene VPA and 3-OH VPA f o r t h e seven s u b j e c t s was a l s o l o w e r e d . 8. Grouping m e t a b o l i t e s i n t o pathways showed t h a t t h e B - o x i d a t i o n of v a l p r o i c a c i d was i n h i b i t e d by s a l i c y l a t e p r o b a b l y a t some s t a g e p r i o r t o t h e f o r m a t i o n of 2-ene VPA. 9 . The k i n e t i c s o f VPA and i t s m e t a b o l i t e s were determined f o r one normal a d u l t v o l u n t e e r b e f o r e and a f t e r a d m i n i s t r a t i o n o f ASA. From serum and u r i n e d a t a , t h e f o r m a t i o n c l e a r a n c e , t h e f r a c t i o n m e t a b o l i z e d and t h e e l i m i n a t i o n c l e a r a n c e of t h e B - o x i d a t i o n meta-- 137 -b o l i t e s were a l l i n h i b i t e d by SA. The o t h e r o x i d a t i v e VPA m e t a -b o l i c pathways were l e s s (or not} a f f e c t e d by ASA a d m i n i s t r a t i o n . The e v i d e n c e o b t a i n e d from the ASA-VPA i n t e r a c t i o n suggests t h a t SA i n h i b i t s the B - o x i d a t i o n pathway by b l o c k i n g M a h l e r ' s enzyme, the enzyme r e s p o n s i b l e f o r the f o r m a t i o n of v a l p r o y l - C o A which i s a p r e r e q u i s i t e B - o x i d a t i o n i n t e r m e d i a r y m e t a b o l i t e . - 138 -REFERENCES 1 . 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