STIMULATION OF 5-AMINOLEVULINIC ACID METABOLISM BY UROPORPHYRINOGEN I IN RAT LIVER HOMOGENATES by ALLY S H I V J I B . S c , The U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1979 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE " i n THE FACULTY OF GRADUATE STUDIES I n The De p a r t m e n t o f P a t h o l o g y We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA September 1983 © A l l y S h i v j i , 1983 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s for an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree that t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g of t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e H e a d o f my D e p a r t m e n t or by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook Place Vancouver, Canada V6T 1W5 - i i -ABSTRACT S t u d i e s were c a r r i e d o u t t o i n v e s t i g a t e t h e e v e n t s t h a t p r e v e n t t h e e x c e s s i v e a c c u m u l a t i o n a n d e x c r e t i o n o f 5 - a m i n o l e v u l i n i c a c i d a n d p o r p h o b i l i n o g e n , i n e r y t h r o p o i e t i c p o r p h y r i a and p o r p h y r i a c u t a n e a t a r d a . The e f f e c t o f e x c e s s u r o p o r p h y r i n o g e n s I and I I I r c o p r o p o r p h y r i n o g e n I I I , a n d t h e c o r r e s p o n d i n g p o r p h y r i n s , on t h e r a t e o f 4-(14-C) 5 - a m i n o l e v u l i n i c a c i d m e t a b o l i s m , were s t u d i e d . E x p e r i m e n t s were c a r r i e d o u t w i t h m i t o c h o n d r i a f r e e r a t l i v e r h omogenates, p r e p a r e d f r o m n o r m a l and h e x a c h l o r o b e n z e n e t r e a t e d r a t s . The c o n s u m p t i o n o f 5 - a m i n o l e v u l i n i c a c i d was f o l l o w e d by m e a s u r i n g i t s c o n c e n t r a t i o n i n c o n t e n t o f a l i q u o t s , removed a t v a r i o u s i n t e r v a l s . The r a t e s o f p o r p h o b i l i n o g e n and p o r p h y r i n a c c u m u l a t i o n were a l s o m e a s u r e d t o o b t a i n a d d i t i o n a l i n f o r m a t i o n on t h e n a t u r e o f i n t e r a c t i o n s , i f a n y , b e t w e e n t h e enzymes. The p a t t e r n o f p o r p h y r i n s y n t h e s i s was e x a m i n e d by h i g h p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h i c a n a l y s i s . I n c o m p a r i n g t h e b i o c h e m i c a l e v e n t s t a k i n g p l a c e i n t h e n o r m a l and p o r p h y r i c r a t l i v e r p r e p a r a t i o n s , t h e l a t t e r h a d an i m p a i r e d a b i l i t y t o d e c a r b o x y l a t e u r o p o r p h y r i n o g e n , a s w e l l as a h i g h e r c a p a c i t y ( a p p r o x i m a t e l y f i v e f o l d ) t o m e t a b o l i z e 5 - a m i n o l e v u l i n i c a c i d . Of t h e compounds s t u d i e d , o n l y u r o p o r p h y r i n o g e n I h a d an e f f e c t on t h e pathway. I t s t i m u l a t e d t h e a c t i v i t i e s o f 5 - a m i n o l e v u l i n i c a c i d d e h y d r a s e an d u r o p o r p h y r i n o g e n I s y n t h a s e i n t h e n o r m a l a s w e l l a s - i i i -p o r p h y r i c l i v e r p r e p a r a t i o n s . A s a r e s u l t , 5 - a m i n o l e v u l i n i c a c i d m e t a b o l i s m a n d p o r p h y r i n s y n t h e s i s w e r e s t i m u l a t e d . H i g h p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h i c a n a l y s i s o f p o r p h y r i n s a m p l e s s h o w e d a n a c c e l e r a t e d g r o w t h o f u r o p o r p h y r i n a n d h e p t a c a r b o x y l i c a c i d p o r p h y r i n f r a c t i o n s ( r e l a t i v e t o o t h e r p o r p h y r i n s ) , u p o n a d d i t i o n o f u r o p o r p h y r i n o g e n I . T h e p a t t e r n o f p o r p h y r i n s y n t h e s i s r e m a i n e d u n a l t e r e d w h e n u r o p o r p h y r i n o g e n I w a s a d d e d t o p r e p a r a t i o n s o f p o r p h y r i c l i v e r s . T h e r e s u l t s o f t h e s e e x p e r i m e n t s , t o g e t h e r w i t h o t h e r e x p e r i m e n t a l a n d c l i n i c a l d a t a , s u g g e s t t h a t u r o p o r p h y r i n o g e n I , w h i c h d i r e c t l y s t i m u l a t e s 5 - a m i n o l e v u l i n i c a c i d d e h y d r a s e a n d u r o p o r p h y r i n o g e n I s y n t h a s e i n v i t r o , p r o b a b l y s t i m u l a t e s t h e p a t h w a y i n t h e same w a y , i n e r y t h r o p o i e t i c p o r p h y r i a a n d p o r p h y r i a c u t a n e a t a r d a . H e n c e , i n c r e a s e i n t h e c o n v e r s i o n o f 5 - a m i n o l e v u l i n i c a c i d a n d p o r p h o b i l i n o g e n t o p o r p h y r i n o g e n s , b r o u g h t a b o u t b y u r o p o r p h y r i n o g e n I , p r e v e n t s t h e a c c u m u l a t i o n o f p o r p h y r i n p r e c u r s o r s i n t h e s e t w o c o n d i t i o n s . - i v -TABLE OF CONTENTS A b s t r a c t i i L i s t o f T a b l e s v i L i s t o f F i g u r e s v i i Acknowledgements . i x SECTION PAGE I INTRODUCTION 1 A. B i o c h e m i s t r y o f t h e heme b i o s y n t h e t i c pathway 2 1. P r o p e r t i e s o f p o r p h y r i n s r e f e r r e d t o i n t h i s p r o j e c t . 2 2. Heme b i o s y n t h e s i s 6 3. R e g u l a t i o n o f heme b i o s y n t h e s i s 17 4. E x c r e t i o n o f i n t e r m e d i a t e s 19 B. P o r p h y r i a s 20 1. C l a s s i f i c a t i o n o f p o r p h y r i a s 23 2. C l i n i c a l a nd b i o c h e m i c a l a s p e c t s o f p o r p h y r i a s 24 3. A c u t e p o r p h y r i a s 31 4. N o n - a c u t e p o r p h y r i a s 34 5. P o r p h y r i a c u t a n e a t a r d a 36 I I METHODS A. M e t a b o l i c s t u d y 45 B. F r a c t i o n a t i o n 47 C. Q u a n t i t a t i o n . . 53 D. A n a l y s i s o f p o r p h y r i n s u s i n g HPLC 56 -v-E . E f f e c t o f p o r p h y r i n s a n d p o r p h y r i n o g e n s 57 F. E f f e c t o f e x c e s s p o r p h o b i l i n o g e n 60 G. M e t a b o l i c s t u d i e s w i t h h e x a c h l o r o b e n z e n e t r e a t e d r a t l i v e r s 60 I I I RESULTS A. Methods 61 B. M e t a b o l i c s t u d y 1. Normal l i v e r 62 2. H e x a c h l o r o b e n z e n e t r e a t e d r a t s 74 IV DISCUSSION 85 V BIBLIOGRAPHY 92 - v i -L I S T OF TABLES T a b l e 1. A c t i v i t i e s o f t h e enzymes o f heme b i o s y n t h e t i c pathway i n n o r m a l human l i v e r s 22 T a b l e I I . The p o r p h y r i a s 27 - v i i -L I S T OF FIGURES F i g u r e 1. S t r u c t u r e o f p o r p h y r i n s m e n t i o n e d i n t h i s s t u d y 3 F i g u r e 2. A b s o r p t i o n s p e c t r u m o f e t i o t y p e p o r p h y r i n s d i s s o l v e d i n d i l u t e HC1 7 F i g u r e 3. Heme b i o s y n t h e t i c pathway 9 F i g u r e 4. 5 - a m i n o l e v u l i n i c a c i d 11 F i g u r e 5. P o r p h o b i l i n o g e n . 11 F i g u r e 6. The h e r e d i t a r y p o r p h y r i a s 25 F i g u r e 7. The heme pathway i n p o r p h y r i a c u t a n e a t a r d a 41 F i g u r e 8. O u t l i n e o f p r o c e d u r e s f o r t h e m e t a b o l i c s t u d y 46 F i g u r e 9. O u t l i n e o f t h e f r a c t i o n a t i o n and q u a n t i t a t i o n p r o c e d u r e s 48 F i g u r e 10. Sephadex G-15 e l u t i o n p a t t e r n d u r i n g sample f r a c t i o n a t i o n 50 F i g u r e 11. M e t a b o l i s m o f ALA i n n o r m a l r a t l i v e r p r e p a r a t i o n s . . . 64 F i g u r e 12. P r o f i l e o f p o r p h y r i n s y n t h e s i s i n n o r m a l r a t l i v e r . . . 6 6 F i g u r e 13. M e t a b o l i s m o f ALA i n t h e p r e s e n c e o f e x c e s s U r o ' g e n I ( n o r m a l l i v e r ) 69 F i g u r e 14. P r o f i l e o f p o r p h y r i n s y n t h e s i s i n t h e p r e s e n c e o f e x c e s s U r o ' g e n I 71 F i g u r e 15. The p a t t e r n o f p o r p h y r i n s y n t h e s i s i n t h e p r e s e n c e an d a b s e n c e o f e x c e s s U r o ' g e n 1 72 F i g u r e 16. U r i n a r y p o r p h y r i n e x c r e t i o n p a t t e r n o f HCB t r e a t e d r a t s 75 F i g u r e 17. C r y o s t a t s e c t i o n s o f r a t l i v e r s s t a i n e d w i t h t h e f a t s t a i n 76 - v i i i -F i g u r e 18. C r y o s t a t s e c t i o n s o f r a t l i v e r s u n d e r uv l i g h t 78 F i g u r e 19. H e m a t o x y l i n and e o s i n s e c t i o n s o f t h e r a t l i v e r s 79 F i g u r e 20. M e t a b o l i s m o f ALA i n p r e p a r a t i o n s o f n o r m a l and HCB t r e a t e d l i v e r s 80 F i g u r e 21. P a t t e r n o f p o r p h y r i n s y n t h e s i s i n p r e p a r a t i o n s o f HCB t r e a t e d r a t l i v e r 82 F i g u r e 22. M e t a b o l i s m o f ALA i n HCB t r e a t e d r a t l i v e r p r e p a r a t i o n s , i n t h e p r e s e n c e o f e x c e s s U r o ' g e n I....84 F i g u r e 23. S c h e m a t i c r e p r e s e n t a t i o n o f t h e e f f e c t s o f U r o ' g e n on t h e pathway 89 - i x -ACKNOWLEDGMENTS The a u t h o r w o u l d l i k e t o e x t e n d e x p r e s s i o n o f t h a n k s t o : C h a r l e s Ramey f o r h i s e x p e r t t e c h n i c a l a d v i c e , s u g g e s t i o n s , h e l p f u l d i s c u s s i o n s , t i m e r e n d e r e d f o r p h o t o c o p y i n g o f t h e s i and u s e o f e q u i p m e n t . D r . P. E . R e i d f o r u s e o f c h e m i c a l s and e q u i p m e n t , r a t l i v e r and h e l p f u l a d v i c e . D r . D. B r o o k s and h i s g r o u p f o r u s e o f e q u i p m e n t , h e l p , i n f o r m a t i v e c o n v e r s a t i o n s a t c o f f e e , a nd t h e a n n u a l p i c n i c s . D r . J . B e n b a s a t f o r h e l p f u l d i s c u s s i o n s . D r . G. Quamme f o r u s e o f l i q u i d s c i n t i l l a t i o n c o u n t e r s . The R a d i o a c t i v e P r o t e c t i o n B r a n c h f o r s u g g e s t i o n s and u s e o f equ i p m e n t . Committee members, D r . D' D o l p h i n and D r . J . F r d h l i c h , f o r a d v i c e , g u i d a n c e , a n d h e l p f u l s u g g e s t i o n s . D r . H. Hughes f o r r e v i e w i n g t h e t h e s i s . My s u p e r v i s o r , D r . M. B e r n s t e i n , w i t h o u t whom t h i s t h e s i s w o u l d n o t h a v e m a t e r i a l i z e d , f o r h i s c o n s t a n t e n c o u r a g e m e n t , g u i d a n c e , a n d h e l p . I N T R O D U C T I O N T h e p o r p h y r i a s a r e m e t a b o l i c d i s t u r b a n c e s o f t h e h e m e b i o s y n t h e t i c p a t h w a y . T h e h e m e p a t h w a y p l a y s a n i m p o r t a n t r o l e i n p r o v i d i n g t h e p r o s t h e t i c g r o u p f o r t h e s y n t h e s i s o f h e m o p r o t e i n s t h a t c a r r y o u t b i o l o g i c a l p r o c e s s e s s u c h a s o x y g e n t r a n s p o r t b y h e m o g l o b i n , e l e c t r o n t r a n s p o r t b y m i t o c h o n d r i a l c y t o c h r o m e s , d r u g d e t o x i f i c a t i o n b y m i c r o s o m a l c y t o c h r o m e s , h y d r o g e n p e r o x i d e d e c o m p o s i t i o n b y c a t a l a s e s e t c L"1,2D. M a j o r s i t e s o f h e m e b i o s y n t h e s i s a r e t h e b o n e m a r r o w , w h e r e a p p r o x i m a t e l y 3 0 0 mg o f heme i s s y n t h e s i z e d d a i l y , a n d t h e l i v e r w h e r e a p p r o x i m a t e l y 3 0 - 1 0 0 mg i s s y n t h e s i z e d e a c h d a y L31. S m a l l a m o u n t s o f h e m e a r e a l s o s y n t h e s i z e d b y o t h e r t i s s u e s s u c h a s t h e a d r e n a l s , k i d n e y s , a s w e l l a s f i b r o b l a s t s C 4 , 5 3 . T h e r e a r e s t i l l m a n y a s p e c t s o f t h i s p a t h w a y , n o t w e l l u n -d e r s t o o d , t h a t b e c o m e m a n i f e s t u n d e r c e r t a i n c o n d i t i o n s . F o r e x a m p l e , i n a l l p o r p h y r i a s , t h e a c t i v i t y o f t h e r a t e l i m i t i n g a n d r e g u l a t o r y e n z y m e , 5 - a m i n o l e v u l i n i c a c i d s y n t h a s e ( A L A - S ) , i s i n c r e a s e d t o e n s u r e a d e q u a t e s y n t h e s i s o f heme C 6 D . C o n -s e q u e n t l y , 5 - a m i n o l e v u l i n i c a c i d ( A L A ) a n d p o r p h o b i l i n o g e n ( P B G ) , t h e p o r p h y r i n p r e c u r s o r s , a c c u m u l a t e a n d a r e e x c r e t e d i n l a r g e q u a n t i t i e s C7D. T h e s e i n t e r m e d i a t e s a p p e a r t o i n -t e r f e r e w i t h t h e n o r m a l c e l l u l a r f u n c t i o n s o f t h e n e u r o n s a n d p r e c i p i t a t e t h e c h a r a c t e r i s t i c s y m p t o m s t h a t a r e a s s o c i a t e d w i t h n e u r o l o g i c a l d y s f u n c t i o n s C 2 , 7 D . H o w e v e r , i n e r y t h r o p o i e t i c p o r p h y r i a ( E P ) a n d p o r p h y r i a c u t a n e a t a r d a ( P C T ) , d e s p i t e i n c r e a s e d A L A - S a c t i v i t y C 8 - 1 2 3 , t h e p o r p h y r i n p r e c u r s o r s a r e n o t s t o r e d o r e x c r e t e d i n e x c e s s i v e a m o u n t s - 2 -C 1 3 , 1 4 3 . The r e a s o n f o r t h i s o b s e r v a t i o n i s n o t w e l l u n d e r -s t o o d . To g a i n more i n s i g h t i n t o t h e r e g u l a t o r y mechanisms o f t h e pathway, e x p e r i m e n t s were c a r r i e d o u t t o s t u d y t h e e f f e c t s o f some i n t e r m e d i a t e s t h a t a c c u m u l a t e i n EP and PCT. BIOCHEMISTRY OF THE HEME BIOSYNTHETIC PATHWAY ' 1 ) PROPERTIES OF PORPHYRINS REFERRED TO IN THIS PROJECT S y n t h e s i s o f heme p r o c e e d s t h r o u g h a s e r i e s o f c y c l i c t e t r a p y r r o l e s known a s p o r p h y r i n o g e n s o r h e x a h y d r o p o r p h y r i n s C 1 5 , 1 6 3 . T h e s e c o l o u r l e s s n o n - f l u o r e s c i n g compounds a r e e a s -i l y o x i d i z e d t o t h e i r c o r r e s p o n d i n g p o r p h y r i n s , i n t h e p r e s e n c e o f o x y g e n , by l i g h t o r p o r p h y r i n s ( a u t o - o x i d a t i o n ) C 1 5 , 1 6 3 . The b a s i c s t r u c t u r e o f a l l p o r p h y r i n s i s t h e p o r p h y r i n r i n g ( a l s o known a s p o r p h i n e ) , w h i c h c o n s i s t s o f f o u r p y r r o l e s ( d e s i g n a t e d A,B,C, and D) j o i n e d by methene b r i d g e s («,(},#, a n d S ) ( f i g . l a ) C 1 6 - 1 8 D . The n a t u r a l l y o c -c u r r i n g p o r p h y r i n s d i f f e r by t h e t y p e and number o f s u b s t i t u e n t s i n p l a c e o f some, o r a l l , o f t h e e i g h t h -h y d r o g e n s o f t h e p y r r o l e s ( f i g . 1 ) C 2 , 1 7 , 1 9 3 . T h e s e p o r p h y r i n s h a v e a h i g h l y c o n j u g a t e d d o u b l e bond s y s t e m ( e l e v e n d o u b l e b onds) t h a t s t a b i l i z e s t h e m o l e c u l e s and i m p a r t s a r o m a t i c c h a r a c t e r t o them C 1 6 , 1 7 3 . L i k e o t h e r a r o m a t i c s y s -tems, t h e p o r p h y r i n r i n g i s h y d r o p h o b i c a n d s t a b l e t o a d v e r s e c h e m i c a l c o n d i t i o n s s u c h as s t r o n g a c i d s a n d b a s e s C 1 6 , 1 7 , 1 9 3 . E t i o p o r p h y r i n , w i t h f o u r m e t h y l and f o u r e t h y l s u b s t i t u e n t s - 3 -Figure 1. Structure of Porphyrins Mentioned i n this Study A. Porphyrin (porphine) B. Porphyrinogen C. Uroporphyrin III (URO III) D. Uroporphyrin I (URO I) E. Heptacarboxylic acid porphyrin III (HEPTA III) F. Hexacarboxylic acid porphyrin III (HEXA III) -4-P P G. Pentacarboxylic acid porphyrin III (PENTA III) V M P P I. Protoporphyrin IX (PROTO) P P K . Harderoporphyrin (HARDERO) P P H. Coproporphyrin III (COPRO III) P P L. Isocoproporphyrin (ISOCOPRO) - 5 -Legend for the substituents: A P M V ( = R : Acetate : Propionate : Methyl ): V i n y l : H, V i n y l , E t h y l , or Hydroxyethyl Brackets contain the respective abbreviations The following abbreviations are used i n denoting porphyrinogens (hexahydroporphyrins), the reduced forms of the porphyrins: Uro'gen Hepta 1 gen Hexa'gen Penta 1 gen Copro'gen Proto'gen Hardero'gen Isocopro 1 gen Uroporphyrinogen Heptacarboxylic acid porphyrinogen Hexacarboxylic acid porphyrinogen Pentacarboxylic acid porphyrinogen Coproporphyrinogen Protoporphyrinogen Harderoporphyrinogen Isocoproporphyrinogen -6-a t t h e p e r i p h e r a l p o s i t i o n s C173, h a s a s p e c t r a l p a t t e r n , i n t h e v i s i b l e r e g i o n , s i m i l a r t o t h a t shown i n f i g . 2. P o r p h y r i n s a s s o c i a t e d w i t h heme b i o s y n t h e s i s h a v e s i x o r more o f t h e p y r r o l i c p o s i t i o n s s u b s t i t u t e d w i t h a l k y l s u b s t i t u e n t s ( w i t h t h e more h y d r o p h o b i c ones a l s o c o n t a i n i n g v i n y l g r o u p s ) w h i c h g i v e r i s e t o s p e c t r a l c h a r a c t e r i s t i c s r e s e m b l i n g t h o s e o f e t i o p o r p h y r i n C16,17,193. As a r e s u l t , t h e y a r e r e f e r r e d t o a s e t i o - t y p e p o r p h y r i n s C16,17,193. P o r p h y r i n s , w h i c h h a ve a v e r y s t r o n g a b s o r p t i o n b a n d a r o u n d 400 nm, e x h i b i t a v e r y i n t e n s e r e d f l u o r e s c e n c e upon e x c i t a t i o n w i t h u l t r a v i o l e t (uv) l i g h t , c o r r e s p o n d i n g t o t h i s b and (known as t h e S o r e t band) C2,16,193. T h i s p r o p e r t y i s o f c o n s i d e r a b l e v a l u e i n d e t e c t i n g s m a l l q u a n t i t i e s o f p o r p h y r i n s i n b i o l o g i c a l mate-r i a l s C16,19,203. P o r p h y r i n s a r e h i g h l y c o l o u r e d compounds w h i c h i m p a r t a r e d d i s h c o l o u r t o t h e u r i n e o f p o r p h y r i c p a t i e n t s C2,173. 2) HEME BIOSYNTHESIS The heme pathway c o n s i s t s o f a s e t o f i r r e v e r s i b l e r e a c -t i o n s i n w h i c h ALA i s u t i l i z e d t o f o r m heme C6,213. T h e s e r e a c t i o n s a r e p e c u l i a r l y c o m p a r t m e n t a l i z e d b e t w e e n t h e m i t o -c h o n d r i a and t h e c y t o p l a s m . The pathway, a b o u t e q u a l l y d i s t r i b u t e d b etween t h e two compartments, b e g i n s and ends i n t h e m i t o c h o n d r i a . The r e a c t i o n s i n s i d e t h e m i t o c h o n d r i a a r e m a i n l y o x i d a t i o n - r e d u c t i o n , whereas t h e e x t r a m i t o c h o n d r i a l ones a r e c o n d e n s a t i o n a n d d e c a r b o x y l a t i o n C23. The.pathway i s -7-500 600 700 X ( n m ) Figure 2. Absorbance spectrum of etiotype porphyrins dissolved i n d i l u t e HC1. The intense peak around AOOnm i s the Soret band. The r e l a t i v e i n t e n s i t i e s of the secondary absorption bands are c h a r a c t e r i s t i c for a given type of porphyrin. - 8 -s c h e m a t i c a l l y o u t l i n e d i n f i g . 3. 5 - a m i n o l e v u l i n i c a c i d s y n t h a s e ( A L A - S ) T h e l a r g e a n d c o m p l e x h e me m o l e c u l e i s s y n t h e s i z e d f r o m s i m p l e p r e c u r s o r s , g l y c i n e a n d s u c c i n y l c o e n z y m e A C 2 D . T h e m a i n s o u r c e o f s u c c i n y l c o e n z y m e A a p p e a r s t o b e t h e t r i c a r b o x y l i c a c i d c y c l e C 2 1 3 . T h e f i r s t s t e p , c a t a l y z e d b y A L A - S C 2 , 3 D , i n v o l v e s c o n d e n s a t i o n o f g l y c i n e ( a c t i v a t e d b y p y r i d o x a l p h o s p h a t e ) w i t h t h e c o e n z y m e t o f i r s t f o r m a m i n o - B - k e t o a d i p i c a c i d w h i c h i s q u i c k l y d e c a r b o x y l a t e d t o f o r m A L A ( f i g . 4 ) C 2 , 3 3 . T h i s p a r t o f t h e p a t h w a y , s i t u a t e d i n t h e m i t o c h o n d r i a , i s t h e o n l y r e a c t i o n t h a t i s e n d o t h e r m i c a n d i n v o l v e s a c o f a c t o r ( p y r i d o x a l p h o s p h a t e ) C 2 , 3 , 2 2 3 . T h e e n z y m e , e i t h e r s i t u a t e d i n t h e m i t o c h o n d r i a l m a t r i x o r l o o s e l y b o u n d t o t h e i n n e r m e m b r a n e C 2 , 2 3 ] , i s p r e s e n t i n a m u c h s m a l l e r q u a n t i t y i n c o m p a r i s o n t o t h e r e s t o f t h e e n z y m e s i n t h e p a t h w a y C 6 1 . C o n s e q u e n t l y , t h e f o r m a t i o n o f A L A i s t h e r a t e l i m i t i n g e v e n t i n t h e p a t h w a y , a n d t h e s y n t h a s e p l a y s a n i m p o r t a n t r o l e o f r e g u l a t i n g t h e b i o s y n t h e s i s o f h e me C 2 , 3 3 . Two f o r m s o f A L A - S e x i s t , a l a r g e r c y t o s o l i c f o r m o f a p p r o x i -m a t e l y 1 7 8 , 0 0 0 d a l t o n s C 2 4 , 2 5 3 , a n d a s m a l l e r m i t o c h o n d r i a l f o r m o f a b o u t 1 1 3 , 0 0 0 d a l t o n s C 2 4 D . T h e c y t o s o l i c f o r m i s t h o u g h t t o r e p r e s e n t t h e z y m o g e n , w h e r e a s t h e m i t o c h o n d r i a l o n e , t h e m o d i f i e d a c t i v a t e d f o r m C 2 4 , 2 6 3 . A l t e r n a t i v e l y , t h e s e t w o f o r m s may b e i s o e n z y m e s C 2 1 D . T h e s o l u b l e e n z y m e a p p e a r s t o h a v e s u l p h y d r y l ( S - H ) g r o u p s a t t h e a c t i v e s i t e F i g u r e 3. Heme b i o s y n t h e t i c pathway. The l o c a t i o n and the sequence of e v e n t s i n the pathway. ( — — — ) : n e g a t i v e f eedback e f f e c t o f heme on ALA-S a c t i v i t y : B ( 6 ) : p y r i d o x a l phosphate. I Heme «- Proto IX A) Glycine, SuccinylCoA ALA-S ALA A Fe +2 Copro'gen oxidase Proto'gen oxidase Proto'gen T X Copro'gen III +• Mitochondrion A A Cytoplasm ALA-D SYN COSYN Uro'gen-D _ ALA • PBG • Pre-uro'gen • Uro'gen ]]i • Copro'gen IM_ -11-F i g u r e 4. 5_AMINOLEVULINlC ACID Figure 5. PORPHOBILINOGEN -12-t h a t p a r t i c i p a t e i n t h e r e a c t i o n C23. The a c t i v i t y o f ALA-S i s s t r o n g l y i n h i b i t e d by heme o r hemin (an i r o n (Fe+++) c h e l a t e o f p r o t o p o r p h y r i n ( f e r r i p r o t o p o r p h y r i n ) ) C2,33. ALA d e h y d r a s e (ALA-D) A f t e r b e i n g r e l e a s e d f r o m t h e enzyme s u r f a c e , ALA d i f f u s e s i n t o t h e c y t o p l a s m , where two m o l e c u l e s a r e c o n d e n s e d t o f o r m a m o n o p y r r o l e , PBG ( f i g . 5) [ 2 , 3 , 2 3 3 . ALA d e h y d r a s e (ALA-D) c a t a l y z e s t h i s r e a c t i o n ; and l i k e most o f t h e o t h e r enzymes, i t s a c t i v i t y i s much h i g h e r t h a n t h a t o f ALA-S C233. The d e h y d r a s e i s made up o f s e v e r a l s u b u n i t s and r e q u i r e s Zn(++) w h i c h , i n t h e r a t , i s p r o b a b l y t i g h t l y bound t o t h e m a c r o m o l e c u l e , b u t n o t i n o t h e r mammals C27,283. The enzyme p o s e s s e s s u l p h y d r y l g r o u p s t h a t p l a y t h e v i t a l r o l e o f m a i n t a i n i n g t h e p r o p e r enzyme c o n f o r m a t i o n ? and a r e t h e r e f o r e , n e c e s s a r y f o r a c t i v i t y C23. L e a d i s among some o f t h e compounds t h a t i n h i b i t t h i s enzyme, b u t a d d i t i o n o f t h i o l compounds r e v e r s e s t h i s i n h i b i t i o n C2,33. A l t h o u g h c o p r o p o r p h y r i n I I I (COPRO I I I ) , p r o t o p o r p h y r i n IX (PROTO), and heme ( f i g . 1H-J) have b e e n shown t o i n h i b i t ALA-D, t h i s enzyme d o e s n o t a p p e a r t o have any r e g u l a t o r y f u n c t i o n s C33. -13-Uroporphyrinocren I synthase / Uroporphyrinogen III cosynthase (SYN / COSYN) These two enzymes act sequentially to polymerize four mol-ecules of PBG to form a c y c l i c tetrapyrrole, uroporphyrinogen III (Uro'gen III) ( f i g . Ic) C233. Recent evidence indicates that Uro'gen I synthase (SYN), also known as PBG deaminase, f i r s t catalyzes the condensation of four PBG molecules to form a l i n e a r tetrapyrrole, pre-Uro'gen (hydroxymethylbilane) C29,303. Uro'gen III cosynthase (COSYN) then c y c l i z e s t h i s polypyrrole to form the assymetrical Uro'gen III C29D. These two enzymes appear to have physical contact to f a c i l i t a t e the synthesis of Uro'gen III C313. The a c t i v i t y of COSYN can be destroyed, s e l e c t i v e l y , by subjecting the enzyme to mild heat (55 C) C25,32D. In the absence of t h i s enzyme a c t i v i t y , pre-Uro'gen c y c l i z e s spontaneously to form only Uro'gen I ( f i g . ID), which cannot be converted to Uro'gen III C2,32D. Normally, the reaction carried out by SYN i s the rate l i m i t i n g step i n the cyt o s o l i c part of the pathway; with the a c t i v i t y being comparable to that of ALA-S C63. The large excess of COSYN a c t i v i t y appears to ensure formation of mostly Uro'gen III C333. COSYN i s strongly i n h i b i t e d by excess Fe(++), URO I and I I I , COPRO I and I I I , and the corresponding porphyrinogens C34,353. - 1 4 -U r o p o r p h v r i n o c r e n d e c a r b o x y l a s e ( U r o ' g e n D) U r o p o r p h y r i n o g e n , a n e i g h t c a r b o x y l p o r p h y r i n o g e n , i s c o n v e r t e d t o a f o u r c a r b o x y l c o m p o u n d , c o p r o p o r p h y r i n o g e n ( C o p r o ' g e n ) , b y U r o ' g e n d e c a r b o x y l a s e ( U r o ' g e n D) C 2 3 , 2 5 3 . T h e d e c a r b o x y l a t i o n o f U r o ' g e n I I I i s a s e q u e n t i a l p r o c e s s t h a t s t a r t s f r o m r i n g D a n d p r o c e e d s i n a c l o c k w i s e d i r e c t i o n t h r o u g h i n t e r m e d i a t e s s u c h a s s e v e n , s i x , a n d f i v e c a r b o x y l p o r p h y r i n o g e n s ( H e p t a ' g e n , H e x a ' g e n , a n d P e n t a ' g e n , r e s p e c t i v e l y ) C 2 5 , 3 6 , 3 7 3 . F i g u r e 1 s h o w s t h e o x i d i z e d f o r m s o f t h e s e p o r p h y r i n o g e n s . W i t h U r o ' g e n I , o n t h e o t h e r h a n d , t h e a c e t a t e s i d e c h a i n s a r e d e c a r b o x y l a t e d i n a r a n d o m f a s h i o n C 3 8 3 . L i k e o t h e r s u l p h y d r y l c o n t a i n i n g e n z y m e s , U r o ' g e n D i s s e n s i t i v e t o s u l p h y d r y l r e a g e n t s . H o w e v e r , t h e a c t i v i t y c a n b e r e s t o r e d w i t h t h e a d d i t i o n o f c o m p o u n d s s u c h a s d i t h i o t h r e i t o l a n d c y s t e i n e C 2 3 . I r o n h a s b e e n o b s e r v e d t o i n h i b i t t h e e n z y m e C 3 9 3 . O x y g e n i n h i b i t s e n z y m e a c t i v i t y b y o x i d i z i n g s u b s t r a t e s t o p o r p h y r i n s , w h i c h a r e n o t m e t a b o l i z e d b y U r o ' g e n D C 2 3 3 . C o p r o p o r p h y r i n o g e n o x i d a s e ( C o p r o ' g e n o x i d a s e ) S u b s e q u e n t e v e n t s o f t h e p a t h w a y t a k e p l a c e i n s i d e t h e m i -t o c h o n d r i a . C o p r o p o r p h y r i n o g e n I I I e n t e r s t h e m i t o c h o n d r i a a n d u n d e r g o e s o x i d a t i v e d e c a r b o x y l a t i o n t o f o r m p r o t o p o r p h y r i n o g e n I X ( P r o t o ' g e n I X ) , a d i c a r b o x y l i c a c i d c o m p o u n d c o n t a i n i n g t w o v i n y l s i d e c h a i n s C 2 1 , 4 0 3 . -15-C o p r © p o r p h y r i n o g e n o x i d a s e ( C o p r o ' g e n o x i d a s e ) c a t a l y z e s t h e c o n v e r s i o n o f t h e two p r o p i o n i c a c i d s i d e c h a i n s , on r i n g s A and B, t o v i n y l g r o u p s [41,423. The r e a c t i o n a p p e a r s t o p r o -c e e d t h r o u g h a t h r e e c a r b o x y l i c a c i d i n t e r m e d i a t e , h a r d e r o p o r p h y r i n o g e n ( f i g . I K ) , by t h e a c t i o n o f t h e enzyme on r i n g A p r i o r t o r i n g B [32,37,403. P e n t a ' g e n c a n a l s o s e r v e a s a s u b s t r a t e o f C o p r o ' g e n o x i d a s e , b u t i t s m e t a b o l i s m y i e l d s d e h y d r o i s o c o p r o p o r p h y r i n o g e n ( f i g . I D [ 3 3 . U r o ' g e n D s u b s e -q u e n t l y c o n v e r t s t h i s compound t o h a r d e r o p o r p h y r i n o g e n , w h i c h i s m e t a b o l i z e d b y C o p r o ' g e n o x i d a s e t o f o r m P r o t o ' g e n [ 6,23,433. T h i s a l t e r n a t e pathway i s b e l i e v e d be o f o n l y m i -n o r s i g n i f i c a n c e [ 6 3 . The o x i d a s e , l o o s e l y b ound t o t h e i n n e r m i t o c h o n d r i a l membrane [ 6 3 , r e q u i r e s o x y g e n f o r c a r r y i n g o u t t h i s s t e p [ 4 0 , 4 2 1 . I n c e r t a i n o r g a n i s m s , C o p r o ' g e n o x i d a s e c a r r i e s o u t t h i s r e a c t i o n u n d e r a n a e r o b i c c o n d i t i o n s , b u t o n l y i n t h e p r e s e n c e o f ATP, L - m e t h i o n i n e , and e i t h e r NAD+ o r NADP+ [ 4 4 3 . The enzyme h a s a h i g h s u b s t r a t e s p e c i f i c i t y , a n d u t i l -i z e s o n l y C o p r o ' g e n I I I C6,21,40,423. P r o d u c t i o n o f C o p r o ' g e n I w o u l d r e s u l t i n i t s a c c u m u l a t i o n and e v e n t u a l e x c r e t i o n . C o p r o ' g e n o x i d a s e i s a l s o i n h i b i t e d by l e a d C243. P r o t o p o r p h y r i n o g e n o x i d a s e ( P r o t o ' g e n o x i d a s e ) A l t h o u g h p o r p h y r i n o g e n s r e a d i l y o x i d i z e n o n - e n z y m a t i c a l l y [ 4 1 3 , s t u d i e s h a v e shown t h a t t h e o x i d a t i o n o f P r o t o ' g e n t o PR0T0 i s c a t a l y z e d b y P r o t o ' g e n o x i d a s e [ 40,45,463. T h i s s u l p h y d r y l enzyme i s most a c t i v e i n t h e p r e s e n c e o f s u l p h y d r y l -16-containing compounds C45,463. Proto'gen oxidase i s in h i b i t e d by excess heme (ferroprotoporphyrin), and to a lesser degree, by hemin (ferriprotoporphyrin), a heme molecule conjugated to a f e r r i c ion C23. Ferrochelatase In the f i n a l step, chelation of ferrous ion to PROTO, to form heme, i s carried out by ferrochelatase C2,3,21,233, an enzyme firml y attached to the inside of the inner mitochondrial membrane C6,213. It i s believed that formation of Fe(++), by the reduction of Fe(+++), takes place at a l o -cation close to, or at the inner mitochondrial membrane to f a c i l i t a t e the chelation C23. Unlike a l o t of the enzymes, ferrochelatase does not have a s t r i c t substrate s p e c i f i c i t y ; i t can carry out chelation of other bivalent metal ions to various dicarboxylic porphyrins C2,233. Lipids are necessary for a c t i v i t y ; t h e i r removal results i n i n a c t i v a t i o n of the enzyme C2,233. S p e c i f i c roles have not been established for the requirement of l i p i d s ; therefore, t h e i r functions remain speculative. The enzyme contains essential SH groups, and consequently, requires appropriate agents to remain viable i n v i t r o C213. Enzyme a c t i v i t y i s i n h i b i t e d by excess heme, EDTA, or agents that can react with the SH groups C21,233. - 1 7 -3) R E G U L A T I O N O F HEME B I O S Y N T H E S I S A L A - S , t h e r a t e l i m i t i n g e n z y m e i n t h e p a t h w a y C 4 7 3 , c o n t r o l s t h e f l u x o f A L A i n t o t h e p a t h w a y C 4 8 3 a n d d e t e r m i n e s t h e r a t e o f heme s y n t h e s i s . A L A - S i s a n i n d u c i b l e e n z y m e C 2 , 4 7 3 w h i c h , i n t h e r a t , h a s a h a l f l i f e o f a p p r o x i m a t e l y 70 m i n C l l , 2 4 3 . T h e a c t i v i t y o f t h i s e n z y m e i s c o n t r o l l e d m a i n l y a t t h e l e v e l o f i t s s y n t h e s i s b y a n e n d p r o d u c t i n d u c t i o n / r e p r e s s i o n m e c h a n i s m C 2 , 4 8 3 . I t i s n o t c l e a r w h e t h e r h e m e i n h i b i t s A L A - S s y n t h e s i s a t t h e l e v e l o f t r a n s c r i p t i o n C 4 9 3 o r t r a n s l a t i o n C 5 0 3 . E n d p r o d u c t i n h i b i t i o n a l s o p l a y s a p a r t i n r e g u l a t i n g A L A - S a c t i v i t y , b u t t o a s m a l l e r e x t e n t C 2 , 4 8 3 . I n t r a m i t o c h o n d r i a l h e m e c o n c e n t r a t i o n a p p e a r s t o a f f e c t A L A - S a c t i v i t y d i r e c t l y t h r o u g h f e e d b a c k i n h i b i t i o n ; w h e r e a s t h e e x t r a m i t o c h o n d r i a l h e m e l e v e l i n f l u -e n c e s A L A - S a c t i v i t y b y a f f e c t i n g i t s s y n t h e s i s C 2 3 . T h e c u r r e n t n o t i o n i s t h a t t h e r e g u l a t i o n o f t h e p a t h w a y i s m e d i a t e d t h r o u g h a p o o l o f f r e e h e m e , w h i c h i s i n e q u i l i b r i u m w i t h t h e h e m e t h a t i s b e i n g u s e d f o r h e m o p r o t e i n s y n t h e s i s C 1 1 3 . F a c t o r s t h a t r e d u c e t h e s i z e o f t h i s r e g u l a t o r y p o o l d e r e p r e s s t h e s y n t h e s i s o f A L A - S C 2 3 . T h e s i z e o f t h e f r e e h e m e p o o l i s a f f e c t e d b y t w o m e c h a n i s m s C 2 , 6 , 5 1 , 5 2 3 : a ) I n c r e a s e d h e m e r e q u i r e m e n t s . D u r i n g h y p o x i a o r d r u g d e t o x i f i c a t i o n , t h e r e i s s t i m u l a t i o n o f h e m o p r o t e i n s y n -t h e s i s ( h e m o g l o b i n s y n t h e s i s , i n d u c t i o n o f d r u g m e t a b o l i z i n g e n z y m e s , o r i n c r e a s e d t u r n o v e r o f m i c r o s o m a l c y t o c h r o m e s ) w h i c h r a p i d l y c o n s u m e s f r e e h e m e . b ) D e c r e a s e d h e m e b i o s y n t h e s i s . E n z y m e d e f e c t s , g e n e t i c a l l y -18-determined or chemically induced, can s i g n i f i c a n t l y de-crease heme biosynthesis and a f f e c t the size of the heme regulatory pool. Under these conditions, ALA-S i s induced to increase the biosynthesis of heme i n an e f f o r t to meet the demands, as well as restore the regulatory pool of heme. ALA-S can also be induced by a number of hormones i . e . steroid hormones, i n s u l i n , growth hormone etc C533. The biosynthetic pathways i n the l i v e r and the bone marrow are i d e n t i c a l , yet the mechanism(s) c o n t r o l l i n g the induction of ALA-S, i n the two tissu e s , appear to d i f f e r C2,52,543. This i s demonstrated by the a b i l i t y of d i f f e r e n t agents to induce ALA-S i n these tissues C2,3,54,553. The biochemical basis and significance for the difference i s not known, but i t i s suspected that the erythroid c e l l s have a d i f f e r e n t enzyme or a factor that may be responsible for the o v e r a l l regulation of the pathway C553. This i s in f e r r e d from the observation that the population of the hepatocytes i s normally stable, and therefore, the enzyme concentrations inside the c e l l s remain constant C553. Under t h i s condition, ALA-S i s probably i n -volved i n o v e r a l l control of heme biosynthesis. In the erythroid t i s s u e , on the other hand, pathway enzymes appear sequentially according to the pathway, during the course of d i f f e r e n t i a t i o n C55,563. Although ALA-S may be the rate l i m i t i n g enzyme, i t s role i n the o v e r a l l regulation here i s not yet known C553. Furthermore, repression of ALA-S has not been d i r e c t l y demonstrated C553. Some studies have shown heme to regulate i t s own synthesis, by i n t e r f e r i n g with iron u t i l -- 1 9 -I z a t i o n L"2,33. T h i s mode o f heme r e g u l a t i o n i s t h o u g h t t o b e m o r e i m p o r t a n t t h a n t h e e n d p r o d u c t i n h i b i t i o n o f A L A - S , i n t h e e r y t h r o i d t i s s u e C 2 3 . A l t h o u g h h e m e b i o s y n t h e s i s i s r e g u l a t e d p r i m a r i l y a t t h e l e v e l o f A L A - S , t h e l o c a t i o n o f p a t h w a y e n z y m e s a n d t h e a b i l i t y o f o t h e r a g e n t s t o i n f l u e n c e t h e p a t h w a y s u g g e s t e x -i s t e n c e o f s e c o n d a r y c o n t r o l s i t e s C 2 , 3 , 2 1 3 . F o r e x a m p l e , m e m b r a n e p e r m e a b i l i t y i n t h e p a s s a g e o f i n t e r m e d i a t e s b e t w e e n m i t o c h o n d r i a a n d t h e c y t o p l a s m h a s ( l o n g b e e n s u g g e s t e d a s p o s s i b l e r e g u l a t o r y s i t e s L " 2 , 3 , 2 1 3 . I n c r e a s e d e r y t h r o p o i e s i s a t h i g h a l t i t u d e s a n d t h e e f f e c t o f o x y g e n c o n c e n t r a t i o n o n e n z y m e a c t i v i t i e s s u g g e s t a r e g u l a t o r y r o l e f o r o x y g e n t e n s i o n C 2 , 5 4 3 . P r e s e n c e o f o t h e r e n z y m e s o r p a t h w a y s s u c h a s P B G o x y g e n a s e o r h e m e o x y g e n a s e , may r e g u l a t e h e m e b i o s y n t h e s i s b y m o d u l a t i n g t h e c o n c e n t r a t i o n o f t h e i r r e s p e c t i v e s u b s t r a t e s C 2 , 2 1 D . T h e s e p o s s i b l e r e g u l a t o r y a g e n t s a n d o t h e r s n o t d i s c u s s e d h e r e , s t i l l r e q u i r e f u r t h e r e v a l u a t i o n t o e s t a b l i s h t h e i r r o l e i n t h e p h y s i o l o g i c c o n t r o l o f h e m e b i o s y n t h e s i s . 4 ) E X C R E T I O N O F I N T E R M E D I A T E S Heme p r e c u r s o r p o o l s i n t h e l i v e r a r e n o r m a l l y q u i t e s m a l l ( t r a c e a m o u n t s ) C 5 7 , 5 8 3 . I n t e r m e d i a t e s c o n s t a n t l y e s c a p e f r o m t h e p a t h w a y , a n d r e a d i l y l e a v e t h e c e l l s C 6 , 5 8 , 5 9 3 . T h e s e h e m e p r e c u r s o r s a r e r a p i d l y e x c r e t e d f r o m t h e b o d y C 6 3 . T h e s m a l l s i z e o f t h e c e l l u l a r p r e c u r s o r p o o l s a n d t h e l o w l e v e l s o f e x c r e t i o n i n d i c a t e t h a t t h e p a t h w a y , n o r m a l l y , o p e r a t e s -20-v e r y e f f i c i e n t l y C3,6,23H. The r o u t e o f e x c r e t i o n i s l a r g e l y d e t e r m i n e d by t h e s o l u b i l i t y o f t h e s e compounds i n w a t e r ; w h i c h i n t h e c a s e o f p o r p h y r i n s , i s d e p e n d a n t on t h e number o f c a r b o x y l g r o u p s p r e s e n t C6,59,603. The h y d r o p h i l i c i n t e r m e d i a t e s (ALA, PBG, URO, and Uro'gen) a r e u s u a l l y e x c r e t e d e x c l u s i v e l y i n t h e u r i n e , where a s t h e h y d r o p h o b i c o n e s , s u c h a s PR0T0 and o t h e r d i c a r b o x y l i c p o r p h y r i n s ( r e s u l t i n g f r o m m e t a b o l i s m o f PR0T0 by m i c r o o r g a n i s m s i n t h e GI t r a c t ) a r e e x c r e t e d i n b i l e C6,59,61D. C0PR0 i s r e a d i l y t a k e n up by h e p a t o c y t e s and e x c r e t e d t h r o u g h t h e GI t r a c t . C o p r o ' g e n , on t h e o t h e r h a n d , i s o n l y e x c r e t e d t h r o u g h t h e k i d n e y s . C o n s e q u e n t l y , COPRO i s d e t e c t e d i n t h e u r i n e a s w e l l a s t h e f e c e s [.2,6,6011. T h i s e x c r e t i o n p a t t e r n i s n o t s t r i c t l y a d h e r e d t o , a s i n c e r t a i n p o r p h y r i c c o n d i t i o n s , URO i s a l s o e x c r e t e d i n t h e f e c e s C2D. F u r t h e r m o r e , l i v e r d i s e a s e a n d h o r m o n a l changes c a n m o d i f y t h e p o r p h y r i n e x c r e t i o n p a t t e r n s C63. I n most c a s e s , t h e c h a n g e s i n e x c r e t i o n p a t t e r n a r e m a i n l y due t o ch a n g e s i n p o r p h y r i n p r o d u c t i o n , r a t h e r t h a n c h a n g e s i n t h e i r e x c r e t i o n r o u t e s C2,63. PORPHYRIAS P o r p h y r i a s a r e c o n d i t i o n s o f m e t a b o l i c d i s t u r b a n c e s i n v o l v -i n g t h e heme pathway. T h e y a r e c h a r a c t e r i z e d b y d e c r e a s e d a c t i v i t y o f enzyme(s) o t h e r t h a n ALA-S. S t i m u l a t i o n o f t h e pathway, i n an e f f o r t t o meet heme r e q u i r e m e n t s , l e a d s t o ex--21-c e s s i v e p r o d u c t i o n , s t o r a g e , and e x c r e t i o n o f i n t e r m e d i a t e s [ 6 , 6 2 3 . N o r m a l l y , a c t i v i t i e s o f a l l t h e pathway enzymes, e x c e p t SYN, g r e a t l y e x c e e d t h a t o f ALA-S ( t a b l e I) [ 6 , 5 5 3 . F u r t h e r m o r e , t h e c a p a c i t y t o consume PBG i n v i v o i s g r e a t e r t h a n t h e a b i l i t y t o p r o d u c e ALA; e v e n t h o u g h t h e enzyme measurements ( t a b l e I ) do n o t show t h i s [ 6 3 . T h e r e f o r e , most o f t h e ALA i s u s e d up f o r p o r p h y r i n s y n t h e s i s [ 6 3 . S u b s t r a t e c o n c e n t r a t i o n s i n t h e l i v e r h a v e b e e n shown t o be much s m a l l e r t h a n t h e r e -s p e c t i v e Km v a l u e s o f t h e enzymes [ 6 3 . T h i s s i t u a t i o n p r o b a b l y f a c i l i t a t e s t h e pathway when t h e heme demands a r e i n c r e a s e d . The pathway c a n s y n t h e s i z e more heme by s i m p l y s u p p l y i n g t h e v a r i o u s enzymes w i t h more s u b s t r a t e . N o r m a l l y , a s m a l l i n c r e m e n t i n t h e a c t i v i t y o f ALA-S c a u s e s a s m a l l i n -c r e a s e i n heme s y n t h e s i s . A c c o m p a n y i n g t h i s i s a s l i g h t e l e v a t i o n i n t h e e x c r e t i o n o f heme p r e c u r s o r s t h a t a r e n o r -m a l l y p r e s e n t i n u r i n e a n d f e c e s [6,63,643. However, when ALA s y n t h e s i s i s e l e v a t e d m a r k e d l y , SYN i s u n a b l e t o cope w i t h t h e r e s u l t i n g PBG p r o d u c t i o n . C o n s e q u e n t l y , PBG a c c u m u l a t e s i n t h e l i v e r and i s e x c r e t e d i n much l a r g e r q u a n t i t i e s compared t o o t h e r i n t e r m e d i a t e s [ 6,58,62,653. S i m i l a r l y , enzyme d e f e c t s i n p o r p h y r i c i n d i v i d u a l s become r a t e l i m i t i n g , u n d e r c e r t a i n c o n d i t i o n s , and p r e v e n t t h e pathway f r o m s y n t h e s i z i n g s u f f i c i e n t amounts o f heme, by i m p e d i n g t h e f l o w o f s u b s t r a t e s . The e x t e n t o f t h e enzyme d e f e c t i s v a r i a b l e ; a s m a l l d e c r e a s e i n a c t i v i t y may n o t a f -f e c t heme s y n t h e s i s u n d e r n o r m a l c i r c u m s t a n c e s , b u t c a n become r a t e l i m i t i n g when heme demands a r e i n c r e a s e d [ 2 3 . A l a r g e -22-T a b l e _ I . A c t i v i t i e s o f t h e enzymes o f heme b i o s y n t h e t i c pathway i n nor m a l human l i v e r s 1 [ 6 , 5 3 ] . ENZYME LIVER nmol ALA/g l i v e r / h 5- A m i n o l e v u l i n i c a c i d s y n t h e t a s e 6- A m i n o l e v u l i n i c a c i d d e h y d r a s e U r o p o r p h y r i n o g e n I s y n t h e t a s e U r o p o r p h y r i n o g e n d e c a r b o x y l a s e C o p r o p o r p h y r i n o g e n o x i d a s e F e r r o c h e l a t a s e 0.09 - 0.38 9.80 - 17.50 0.08 - 0.22 7.80 - 19.50 7.60 - 9.50 3.50 - 17.60 1 The a c t i v i t i e s a r e t h e range o f a c t i v i t i e s r e p o r t e d . A c t i v i t y i s e x p r e s s e d a s nmoles o f ALA e q u i v a l e n t s formed o r used p e r h r p e r mg o f p r o t e i n . - 2 3 -r e d u c t i o n i n a c t i v i t y may r e n d e r t h a t s t e p r a t e l i m i t i n g e v e n u n d e r n o r m a l c o n d i t i o n s . The i n d u c t i o n o f ALA-S s y n t h e s i s i s c o n t i n g e n t upon t h e m a g n i t u d e o f t h e d e f e c t and t h e i m p e n d i n g heme r e q u i r e m e n t s . S t i m u l a t i o n o f t h e pathway i s marked b y i n c r e a s e d a c c u m u l a t i o n and e x c r e t i o n o f ALA, PBG, and t h e r e -s p e c t i v e enzyme s u b s t r a t e ( s ) , a l o n g w i t h o t h e r n o r m a l l y e x c r e t e d p r e c u r s o r s L"63. The p a t t e r n o f heme p r e c u r s o r o v e r p r o d u c t i o n and e x c r e t i o n , t h u s o b s e r v e d , i s u n i q u e and c h a r a c t e r i s t i c o f e a c h enzyme d e f e c t . C LASSIFICATION OF PORPHYRIAS I n t h e p a s t , i t was assumed t h a t t h e d e f e c t was c o n f i n e d t o t h e t i s s u e i n w h i c h heme p r e c u r s o r s a c c u m u l a t e d ; t h e r e f o r e , i t was c u s t o m a r y t o c l a s s i f y p o r p h y r i a s i n t o e r y t h r o p o i e t i c a n d h e p a t i c , b a s e d on t h e o c c u r r e n c e o f i n t e r m e d i a t e s i n t h e s e t i s s u e s C 2 , 6 6 3 . W i t h t h e d i s c o v e r y o f c h e m i c a l l y i n d u c e d f o r m s , a n a d d i t i o n a l c l a s s o f c h e m i c a l p o r p h y r i a was i n t r o d u c e d C663 . The c o n c e p t o f e r y t h r o p o i e t i c and h e p a t i c p o r p h y r i a s i s s t i l l w i d e l y u s e d , e v e n t h o u g h i t h a s b e e n w e l l e s t a b l i s h e d t h a t enzyme a b n o r m a l i t i e s a r e a l s o p r e s e n t i n o t h e r c e l l t y p e s . I t i s s t i l l a m y s t e r y a s t o why p o r p h y r i n a c c u m u l a t i o n i s c o n f i n e d t o s p e c i f i c t i s s u e s , e v e n t h o u g h t h e pathway i s a p p a r e n t l y i d e n t i c a l i n a l l c e l l t y p e s C553 . An a l t e r n a t e c l a s s i f i c a t i o n s y s t e m , u s e d b y some, d i v i d e s t h e h e r e d i t a r y f o r m s i n t o a c u t e and n o n - a c u t e , d e p e n d i n g upon t h e c l i n i c a l symptoms. The l o c a t i o n o f t h e v a r i o u s d e f e c t s a l o n g - 2 4 -the pathway are schematically represented in f i g . 6 . Table II shows the various porphyrias grouped according to both clas-sifications. . CLINICAL AND BIOCHEMICAL ASPECTS OF PORPHYRIAS Pathway intermediates, in excessive quantities, play a major role in precipitating the characteristic c l i n i c a l symp-toms. The acute and non-acute porphyrias differ in the type of precursors that accumulate and get excreted. Characteristically, in the acute disorders, ALA and PBG accu-mulate and get excreted in large quantities. In the non-acute ones, on the other hand, excretion of these compounds is within the normal range C 2 3 3 . The reason for the absence of excessive porphyrin precursor accumulation and excretion in the non-acute porphyrias is poorly understood. Symptoms in the active phase of acute porphyrias are believed to be neurological manifestations arising from the pharmacological effects of ALA and PBG on the nervous system C 2 , 2 3 3 . In experimental systems, ALA has been observed to inhibit Na(+)/K(+) dependant ATPase in red blood cells and the brain, bind to #-amino butyric acid receptor sites, cause changes in motor activ i t i e s C 2 , 6 7 3 etc. High concentrations of ALA and PBG, both interfere with functions of neurons, cause epileptic attacks, and death C 2 , 6 7 3 . These studies, and the observation of acute attacks in patients who excrete ex-cessive quantities of ALA and PBG, strongly implicate these -25-Figure 6. The hereditary porphyrias. The positions of the enzyme defects, along the pathway, and the corresponding i n t e r -mediates that accumulate. - 2 6 -THE PORPHYRIAS Non-acute Acute glycine.succinyl CoA PCT PBG T f L c oP r o 'gen I _ uro'gln 111 - HEPTA ' typelll&l« | ~ HEXA f - PENTA L copro'gen 111, copro'gen 111 [~~ uro'gen I AL*A > ALA ~] ^ ~ J PSDP AIPJ • PBG J pre-yro'gen > uro'gen I ? proto H o 2I0 60 120 180 time (min) 240 60 120 180 time (min) 240 Figure 20. Metabolism of ALA in preparations of normal and HCB treated rat l i v e r s . (A) ALA metabolism; (B) PBG accumulation; (C) prophyrin synthesis. (•_•_•): 1.5uM ALA (normal l i v e r ) ; ( • - • - • ) : 3.5 mM ALA; ( • - • - • ) : 4.0mM ALA (porphyric rat l i v e r ) . CO o I -81-n o r m a l l i v e r p r e p a r a t i o n s c o n t a i n i n g e x c e s s U r o ' g e n I ; i . e . an i n c r e a s e d r a t e o f p o r p h y r i n s y n t h e s i s , p a r t w a y t h r o u g h t h e i n c u b a t i o n p e r i o d . The p r o f i l e o f p o r p h y r i n s y n t h e s i s ob-t a i n e d ( f i g . 21) d i f f e r e d f r o m t h e n o n - p o r p h y r i c p a t t e r n i n t h a t , URO and HEPTA c o n s t i t u t e d t h e major f r a c t i o n s . I n a d -d i t i o n , t h e q u a n t i t i e s o f COPRO and PROTO were r e l a t i v e l y much s m a l l e r . T h e s e o b s e r v a t i o n s a l l a g r e e w i t h t h e c o n c e p t o f i m p a i r e d U r o ' g e n D a c t i v i t y . U r o ' g e n I s t i m u l a t e d t h e pathway i n t h e same way a s b e f o r e . F i g u r e s 22A and 22B show ALA m e t a b o l i s m and PBG a c c u m u l a t i o n i n t h e p r e s e n c e and a b s e n c e o f U r o ' g e n I . P o r p h y r i n s y n t h e s i s was a l s o a c c e l e r a t e d ; h o w e v e r , t h e s t i m u l a t i o n was o b s e r v e d f r o m t h e b e g i n i n g , r a t h e r t h a n p a r t w a y t h r o u g h t h e i n c u b a t i o n ( o b s e r v e d i n t h e a b s e n c e o f exogenous U r o ' g e n I ) . C o n s e -q u e n t l y , t h e r a t e o f p o r p h y r i n s y n t h e s i s was c o n s t a n t f o r t h e d u r a t i o n o f t h e e x p e r i m e n t , a n d t h e s i z e o f t h e f r a c t i o n i n -c r e a s e d l i n e a r l y w i t h t i m e ( f i g . 2 2 C ) . HPLC a n a l y s i s r e v e a l e d no c h a n g e s i n t h e p a t t e r n o f p o r p h y r i n p r o d u c t i o n , upon a d d i -t i o n o f U r o ' g e n I . -82-time Figure 21. Pattern of porphyrin synthesis i n preparation of HCB treated rat l i v e r . Figure 22. Metabolism of ALA i n HCB treated rat l i v e r preparations, i n the presence of excess Uro'gen I. I a (A) ALA metabolism; (B) PBG accumulation; (C) porphyrin synthesis. I (•-•-•): 3.5mM ALA; ( • - • - r ) : 3.5mM ALA and 0.65uM Uro'gen I. -85-DISCUSSIQN The a c t i v i t y of the enzyme that converted ALA to PBG, ALA-D, determined the rate of ALA metabolism. Under the ex-perimental conditions, a minimum of l.OmM ALA was required to es t a b l i s h a constant rate of metabolism for a period of 4 hrs. Concentrations greater than l.OmM did not increase the rate of metabolism s i g n i f i c a n t l y , which suggested that ALA-D was op-erating at p r a c t i c a l l y maximum a c t i v i t y . The rapid accumula-t i o n of PBG refl e c t e d a large difference between the rates of PBG production and consumption. Since the rate of porphyrinogen synthesis remained unchanged, when the concen-t r a t i o n of ALA was increased from l.OmM to 2.0mM, i t was as-sumed that SYN, l i k e ALA-D, operated at maximum v e l o c i t y . These observations were consistent with the information given i n table I, that the a c t i v i t y of ALA-D greatly exceeds that of the rate l i m i t i n g SYN. Uro'gen has been shown to be decarboxylated i n a series of steps to form Copro'gen. It has been demonstrated that the decarboxylation of Hepta'gen i s the rate l i m i t i n g step of Uro'gen D C383. HPLC analysis showed that the bulk of the porphyrin f r a c t i o n was comprised of COPRO and PROTO, which suggested that Uro'gen D, present i n a r e l a t i v e excess to SYN, probably decarboxylated Uro'gen to Copro'gen very rapidly. Furthermore, URO and HEPTA fra c t i o n s were always larger than those of HEXA, and PENTA, which supported the fi n d i n g that decarboxylation of Hepta'gen i s the slowest. Presence of PROTO indicated mitochondrial damage. Injury to mitochondria, -86-p r o b a b l y d u r i n g h o m o g e n i z a t i o n , c o n t a m i n a t e d t h e s u p e r n a t a n t w i t h t h e m i t o c h o n d r i a l enzyme, C o p r o ' g e n o x i d a s e . As a r e -s u l t , C o p r o ' g e n was m e t a b o l i z e d t o PROTO, w h i c h g r a d u a l l y a c -c u m u l a t e d t o e x c e e d C o p r o ' g e n i n t h e f i n a l h o u r . I t i s e v i d e n t t h a t t h e s e r e s u l t s a g r e e d w i t h t h e e s t a b l i s h e d c h a r -a c t e r i s t i c s o f t h e pathway. L i v e r p r e p a r a t i o n s f r o m p o r p h y r i c r a t s h a d a much h i g h e r c a p a c i t y t o m e t a b o l i z e ALA. T h e s e r e s u l t s were s i m i l a r t o t h o s e o b t a i n e d by San M a r t i n de V i a l e e t a l . C1433, b u t t h e y d i s a g r e e d w i t h Rajamanickam e t a l . C1393. I n t h e l a t t e r s t u d y , t h e r a t s were k e p t on a d i e t o f HCB f o r a maximum pe-r i o d o f 29 d a y s , w h i c h was s u b s t a n t i a l l y s h o r t e r t h a n o u r s . I t i s p o s s i b l e t h a t l o n g t e r m c h r o n i c a d m i n i s t r a t i o n o f HCB (> 3 months) may l e a d t o an e l e v a t e d a c t i v i t y o f ALA-D. Doss e t a l . and San M a r t i n de V i a l e e t a l . C118,1433 a l s o o b s e r v e d i n c r e a s e d a c t i v i t y o f SYN i n t h e i r p o r p h y r i c r a t l i v e r p r e p a -r a t i o n s . Our e x p e r i m e n t s , on t h e o t h e r h a n d , showed t h a t t o -t a l p o r p h y r i n s y n t h e s i s , i n p o r p h y r i c a n d n o n - p o r p h y r i c r a t s , t o be t h e same a t 4 h r s . HPLC a n a l y s i s showed t h a t t h e b u l k o f t h e p o r p h y r i n f r a c t i o n was made up o f URO and HEPTA. From t h e r e s u l t s o f t h e e x p e r i m e n t s c o n d u c t e d w i t h t h e n o r m a l r a t l i v e r s , i t was s u s p e c t e d t h a t U r o ' g e n may h a v e a c c u m u l a t e d s u f f i c i e n t l y ( a f t e r 1 h r ) t o s t i m u l a t e p o r p h y r i n s y n t h e s i s . S i n c e t y p e I a n d I I I i s o m e r s were n o t r e s o l v e d by t h e HPLC, i t was n o t p o s s i b l e t o e s t a b l i s h t h e i s o m e r i d e n t i t i e s o f t h e p o r p h y r i n o g e n s t h a t were s y n t h e s i z e d . A c t i v a t i o n a n d i n d u c t i o n o f enzyme s y n t h e s i s a r e two p o s -s i b l e mechanisms t h a t c a n i n c r e a s e enzyme a c t i v i t y . HCB i s - 8 7 -m e t a b o l i z e d t o e l e c t r o p h i l i c i n t e r m e d i a t e s w h i c h s u b s e q u e n t l y r e a c t w i t h m a c r o m o l e c u l e s ( i . e . U r o ' g e n D) a n d r e n d e r them i n a c t i v e C 1 1 5 D . T h e r e f o r e , t h e p o s s i b i l i t y t h a t HCB ( o r m e t a b o l i t e s ) p l a y e d a r o l e i n i n c r e a s i n g enzyme a c t i v i t y , i n t h e s e l i v e r s , i s v e r y s m a l l . S t i m u l a t i o n o f enzyme a c t i v i t y t h r o u g h a c t i v a t i o n was a l s o somewhat c o n t r a i n d i c a t e d by t h e a b i l i t y o f exogenous U r o ' g e n I t o f u r t h e r s t i m u l a t e ALA-D. I n a d d i t i o n , any a c t i v a t i n g f a c t o r w o u l d be d i l u t e d t o c o n c e n -t r a t i o n s f a r b e l o w c e l l u l a r c o n c e n t r a t i o n ; i n t h e c o u r s e o f homogenate p r e p a r a t i o n . S i n c e U r o ' g e n I s t i m u l a t e d ALA-D and SYN i n v i t r o , i t i s p o s s i b l e t h a t a c c u m u l a t i o n o f p o r p h y r i n o g e n s i n d u c e d s y n t h e s i s o f t h e above enzymes. T h e r e f o r e , r e s u l t s o f t h e s e e x p e r i m e n t s s u g g e s t t h a t U r o ' g e n I may a l s o i n d u c e ALA-D and SYN ( s e e C 1 1 8 , 1 4 3 D ) i n p o r p h y r i a . S e v e r a l o t h e r s t u d i e s h a v e r e p o r t e d i n c r e a s e d p o r p h y r i n s y n t h e s i s t o o c c u r c o i n c i d e n t l y w i t h i n a c t i v a t i o n o f COSYN. K u s h n e r e t a l . C 3 5 3 , s t u d i e d t h e e f f e c t o f i r o n on t h e a c t i v -i t y of COSYN, and f o u n d e l e v a t e d s y n t h e s i s o f p o r p h y r i n o g e n s ( a r o u n d 70% more) f r o m e i t h e r ALA o r PBG; upon a d d i t i o n o f i r o n . I r o n i n h i b i t e d COSYN a c t i v i t y . S u b s e q u e n t p o r p h y r i n s y n t h e s i s showed p r e s e n c e o f m a i n l y URO I . I n t h e same s t u d y , d e n a t u r a t i o n o f COSYN w i t h m i l d h e a t a l s o r e s u l t e d i n s t i m u -l a t i o n o f U r o ' g e n I s y n t h e s i s . L e v i n C32D o b s e r v e d 70% i n -c r e m e n t i n SYN a c t i v i t y a t a pH where COSYN a c t i v i t y was i n -h i b i t e d a p p r o x i m a t e l y 8 0 % . A l l o f t h e s e e x p e r i m e n t s demon-s t r a t e d i n c r e a s e d p o r p h y r i n o g e n s y n t h e s i s ( i n d i c a t i v e o f g r e a t e r SYN a c t i v i t y ) i n t h e p r e s e n c e o f d e c r e a s e d COSYN a c -t i v i t y ( i n c r e a s e d U r o ' g e n I s y n t h e s i s ) . - 8 8 -S a n c o v i c h e t a l . E 3 3 3 , s t u d i e d t h e S Y N / C O S Y N c o m p l e x ( a l s o c o l l e c t i v e l y k n o w n a s p o r p h o b i l i n o g e n a s e ) a n d o b t a i n e d a s i g m o i d a l c u r v e w h e n t h e a c t i v i t y o f t h i s c o m p l e x w a s p l o t t e d a g a i n s t P B G c o n c e n t r a t i o n . M i l d h e a t c h a n g e d t h e c u r v e f r o m s i g m o i d t o h y p e r b o l i c a n d i n c r e a s e d t h e a f f i n i t y o f t h e e n z y m e f o r t h e s u b s t r a t e ( l o w e r Km v a l u e ) . T h e r e s u l t s a l s o s h o w e d a s l i g h t l y g r e a t e r c a p a c i t y t o s y n t h e s i z e p o r p h y r i n o g e n s . I t i s w e l l k n o w n t h a t a l l o s t e r i s m c a n l e a d t o s i g m o i d a l s a t u r a t i o n c u r v e s . S i n c e e x p e r i m e n t s h a v e s h o w n S Y N a n d C O S Y N t o p h y s i -c a l l y i n t e r a c t d u r i n g U r o ' g e n I I I s y n t h e s i s C 3 1 3 , i t i s p o s -s i b l e t h a t C O S Y N may m o d u l a t e S Y N a c t i v i t y t h r o u g h p r o t e i n - p r o t e i n i n t e r a c t i o n s . E x c e s s U r o ' g e n I may b e p e r c e i v e d a s d e c r e a s e d C O S Y N a c t i v i t y , a n d p r o b a b l y s e n s e d t h r o u g h b i n d i n g o f t h i s p o r p h y r i n o g e n t o A L A - D a n d S Y N / C O S Y N c o m p l e x . O u r r e s u l t s s u g g e s t t h a t U r o ' g e n I may f u n c t i o n a s a n e f f e c t o r t o s e q u e s t e r t h e m o d u l a t i o n b y C O S Y N . T h e r e -s u l t i n g i n c r e a s e i n S Y N a c t i v i t y may b e t h o u g h t o f a s a c o m p e n s a t o r y m e c h a n i s m w h i c h a t t e m p t s t o i n c r e a s e C O S Y N a c -t i v i t y ( t o s t i m u l a t e U r o ' g e n I I I s y n t h e s i s ) b y i n c r e a s i n g p r o d u c t i o n o f p r e - U r o ' g e n . S t i m u l a t i o n o f A L A - D i s p r o b a b l y a n a d d i t i o n a l s t e p t o s u p p l y a d e q u a t e P B G f o r U r o ' g e n s y n t h e -s i s . T h i s m e c h a n i s m i s h y p o t h e t i c a l a n d r e q u i r e s a d d i t i o n a l e x p e r i m e n t a l e v a l u a t i o n . T h e e f f e c t s o f U r o ' g e n I , o n t h e p a t h w a y , a r e s c h e m a t i c a l l y s u m m a r i z e d i n f i g . 2 3 . C l i n i c a l l y , i n c o n d i t i o n s s u c h a s E P a n d P C T , w h e r e t h e r e i s m a r k e d a c c u m u l a t i o n o f U r o ' g e n I , i n c r e a s e d a c t i v i t i e s o f A L A - D a n d / o r S Y N h a v e b e e n d e t e c t e d . S t u d i e s w i t h p e r i p h e r a l gure 23. Schematic representation of the ef f e c t s of Uro'gen I on the pathway. (+): stimulatory e f f e c t ; (-): i n h i b i t o r y e f f e c t . POSITIVE FEEDBACK MECHANISM BY URO'GEN 1 Heme «- Proto IX /(-) Glycine, SuccinylCoA B 6 ALA-S ALA J l Fe +2 Copro'gen oxidase Proto'gen oxidase Proto'gen \X_ Copro'gen HI Mitochondrion _JL_ R. o o I ALA Cytoplasm . . . T /„„.„ , Uro gen J_ • (+) ALA-D SYN x-'COSYN Uro'gen-D • PBG • Pre-uro'gen • Uro'gen J J J L • Copro'gen -91-b l o o d c e l l s and h e p a t i c t i s s u e f r o m t h e s e p a t i e n t s h a v e shown an i n c r e a s e d c a p a c i t y t o s y n t h e s i z e p o r p h y r i n o g e n s C2,119,120,127,1443. E x p e r i m e n t a l r e s u l t s and t h e a b o v e c l i n i c a l o b s e r v a t i o n s , s u g g e s t t h a t U r o ' g e n I , w h i c h d i r e c t l y s t i m u l a t e d ALA-D and SYN i n v i t r o , i s p r o b a b l y r e s p o n s i b l e f o r s t i m u l a t i n g t h e pathway i n t h e two p o r p h y r i a s . T h i s may come a b o u t f r o m a combined e f f e c t o f a c t i v a t i o n and i n d u c t i o n o f enzyme a c t i v i t i e s . The c o n c o m i t a n t i n c r e a s e i n t h e c o n v e r s i o n o f ALA a n d PBG, t o p o r p h y r i n o g e n s , w o u l d a c c o u n t f o r t h e l a c k o f e x c e s s p o r p h y r i n p r e c u r s o r a c c u m u l a t i o n and t h e a b s e n c e o f a c u t e symptoms, s e e n i n A I P , HC, and PV. -92-BIBLIQGRAPHY 1. G r a n i c k , S., and H. G i l d e r . 1947. D i s t r i b u t i o n , s t r u c -t u r e , and p r o p e r t i e s o f t h e t e t r a p y r r o l e s . 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