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Correlation between nortriptyline and debrisoquine hydroxylation in the human liver
Life Sciences, Vol. 33, pp. 631-636 Printed in the U.S.A.
CORRELATION
BETWEEN
Pergamon Press
NORTRIPTYLINE AND DEBRISOQUINE IN THE H U M A N L I V E R 1
1
HYDROXYLATION
Christer von B@hr , Carol Birger§son , Agneta Blanck G S r a n s s o n I, B r i t t M e l l s t r ~ m I and K l a s N i l s e l l 3
2'
, Monica
D e p a r t m e n t s of C l i n i c a l P h a r m a c o l o g y 1, M e d i c a l N u t r i t i o n 2 and S u r g e r y 3, K a r o l i n s k a I n s t i t u t e , H u d d i n g e U n i v e r s i t y H o s p i t a l , S-141 86 H u d d i n g e , S w e d e n (Received in final form J~ne i, 1983)
Summary The b e n z y l i c h y d r o x y l a t i o n of n o r t r i p t y l i n e (NT) and d e b r i s o q u i n e (D) by i s o l a t e d h u m a n liver m i c r o s o m e s f r o m e i g h t s u b j e c t s was studied. T h e r e was a s t r o n g c o r r e l a t i o n b e t w e e n the 1 0 - h y d r o x y l a t i o n of NT and the 4 - h y d r o x y l a t i o n of D (r = 0.96). The a b i l i t y to h y d r o x y l a t e D was a l s o m e a s u r e d in vivo as the r a t i o b e t w e e n D and 4-OH-D in u r i n e a f t e r oral a d m i n i s t r a t i o n of the drug to four subjects. This e s t i m a t e of h y d r o x y l a t i o n c a p a c i t y a g r e e d w i t h the in vitro measurements. Liver microsomes from a subject defined as a p o o r in v i v o o x i d i z e r of D h y d r o x y l a t e d NT and D u n u s u a l l y slowly. S e p a r a t i o n of m i c r o s o m a l p r o t e i n s by S D S - g e l e l e c t r o p h o r e s i s i n d i c a t e d a r e l a t i v e lack of a c y t o c h r o m e P-450 i s o z y m e w i t h a m o l e c u l a r w e i g h t of 54,500 in the liver f r o m the poor o x i d i z e r . The o x i d a t i o n of d e b r i s o q u i n e (D) and s p a r t e i n (S) is m o n o g e n i c a l l y c o n t r o l l e d and some 3-9 per cent of C a u c a s i a n s are poor o x i d i z e r s of b o t h d r u g s (1,2). S t u d i e s on NT s t e a d y state p l a s m a c o n c e n t r a t i o n s s u g g e s t that in m a n n o r t r i p t y l i n e (NT) k i n e t i c s are l i k e l y to be p o l y g e n i c a l l y c o n t r o l l e d (3). NT is m e t a b o l i z e d b o t h by 1 0 - h y d r o x y l a t i o n and N - d e m e t h y l a t i o n (4). The a b i l i t y to c a r r y o u t the 1 0 - h y d r o x y l a t i o n of NT a p p e a r s to be r e l a t e d to the D h y d r o x y l a t i o n p h e n o t y p e (5). It has r e c e n t l y b e e n shown that liver m i c r o s o m e s f r o m a s u b j e c t i d e n t i f i e d as a poor D h y d r o x y l a t o r in v i v o did n o t c a t a l y z e the 4 - h y d r o x y l a t i o n of the d r u g (6). T h i s s t u d y w a s u n d e r t a k e n to i n v e s t i g a t e the 1 0 - h y d r o x y l a tion of NT in v i t r o in r e l a t i o n to D h y d r o x y l a t i o n in v i v o and and in v i t r o in d i f f e r e n t subjects. Methods The in v i v o a b i l i t y to h y d r o x y l a t e D was m e a s u r e d in four p a t i e n t s . A single 10 mg oral dose of D (Declinax R tablets, H o f f m a n - L a Roche) was g i v e n and u r i n e w a s c o l l e c t e d for 6 hrs. The r a t i o b e t w e e n the p a r e n t d r u g and the 4 - h y d r o x y m e t a b o l i t e (4-OH-D) e x c r e t e d in the u r i n e w a s d e t e r m i n e d (I).
with
L i v e r t i s s u e f r o m these cholecystectomy because
p a t i e n t s w a s o b t a i n e d in c o n n e c t i o n of g a l l s t o n e s (GL). A n a e s t h e s i a w a s
0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
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i n i t i a t e d w i t h t h i o p e n t h a l f o l l o w e d by a d m i n i s t r a t i o n of n i t r o u s oxide, d i a z e p a m and fentanyl. D u r i n g the o p e r a t i o n a I to 3 g l i v e r b i o p s y was t a k e n f r o m the left lobe. This was a p p r o v e d by the E t h i c a l C o m m i t t e e of the h o s p i t a l . L i v e r tissue was also o b t a i n e d from k i d n e y t r a n s p l a n t d o n o r s w i t h c o m p l e t e c e r e b r a l i n f a r c t i o n as p r e v i o u s l y d e s c r i b e d (HL) (7). The m i c r o s o m a l f r a c t i o n of the livers was p r e p a r e d (7). P r o t e i n c o n c e n t r a t i o n (8), c y t o c h r o m e P-450 and b 5 c o n c e n t r a t i o n s (9) and N A D P H - c y t o c h r o m e c r e d u c t a s e a c t i v i t y (10) w e r e d e t e r mined. S D S - p O l y a c r y l a m i d e - - g e l e l e c t r o p h o r e s i s was p e r f o r m e d (11). Ten ~g of m i c r o s o m a l p r o t e i n was a p p l i e d to each well of the gel. The a c r y l a m i d e c o n c e n t r a t i o n was 9 % (w/v). The gel w a s s t a i n e d w i t h C o o m a s s i e B r i l l i a n t Blue. D e n s i t o m e t r i c t r a c i n g s w e r e p e r f o r m e d at 550 nm. The rates of NT h y d r o x y l a t i o n and a m i t r i p t y l i n e (AT) d e m e t h y l a t i o n w e r e m e a s u r e d in a s y s t e m p r e v i o u s l y d e s c r i b e d (12) cont a i n i n g I mg of m i c r o s o m a l protein. The i n c u b a t i o n time was 10 and 30 min for AT a n d NT, r e s p e c t i v e l y . The D h y d r o x y l a t i o n was p e r f o r m e d a c c o r d i n g to K a h n et al (13) w i t h 0.5 mg of m i c r o s o m a l p r o t e i n for 30 min. NT f o r m e d from AT w a s q u a n t i t a t e d by m a s s f r a g m e n t o g r a p h y (12) and 1 0 - O H - N T f o r m e d f r o m NT by H P L C (14). The 4 - h y d r o x y l a t i o n rate of D was d e t e r m i n e d by q u a n t i t a t i o n of f o r m e d 4-OH-D a c c o r d i n g to K a h n et al (13). This m e t h o d u s e s a s o l v e n t e x t r a c tion p r o c e d u r e to s e l e c t i v e l y e l i m i n a t e the s u b s t r a t e , w h i c h i n t e r f e r s w i t h the q u a n t i t a t i o n of the m e t a b o l i t e by gas c h r o m a t o g r a p h y - m a s s s p e c t r o m e t r y . In our hands, however, the e x t r a c t i o n p r o c e d u r e was not s u f f i c i e n t and we h a d to r e p l a c e it by a m o r e e f f i c i e n t s e p a r a t i o n t e c h n i q u e . To the i n c u b a t i o n sample was a d d e d the i n t e r n a l s t a n d a r d (13) and the m i x t u r e was f i l t e r e d t h r o u g h a M i l l i p o r e f i l t e r (type H A W P 0.45 ~m) w i t h i n I min. The f i l t e r was r i n s e d w i t h I ml of s o d i u m p h o s p h a t e b u f f e r pH 2.0 (I = 0.05) and the c o m b i n e d f i l t r a t e s w e r e s u b j e c t e d to H P L C i m m e d i a t e l y or s t o r e d at -20 ° over night. The c h r o m a t o g r a p h i c s y s t e m c o n s i s t e d of a p u m p (LDC, C o n s t a m e t r i c III), a d e t e c t o r (LDC, S p e c t r o m o n i t o r III) o p e r a t e d at 262 nm, an i n j e c t o r (Altex, P h e o d y n e 7021) and a g u a r d c o l u m n (Brownlee Labs, RP 18 S p h e r i 5). The a n a l y t i c a l c o l u m n (4.6 m m i.d x 150 m m length) was p a c k e d w i t h 5 ~m o c t a d e c y l s i l a n (Altex, U l t r a s p h e r e ) and e l u t e d w i t h s o d i u m p h o s p h a t e b u f f e r pH 2.0 (I = 0.05) and m e t h a n o l (75:25) at a f l o w rate of 1.0 m l - m i n -I. A f r a c t i o n of the e l u a t e c o r r e s p o n d i n g to 4-OH-D was c o l l e c t e d . A f t e r a d d i t i o n of 0.1 ml 2.5 M N a O H f o l l o w e d by 0.2 ml a q u e o u s s o l u t i o n of s o d i u m b i c a r b o n a t e the samples w e r e d e r i v a t i z e d and p r o c e s s e d as d e s c r i b e d by Kahn et al (13). The r e t e n t i o n v o l u m e of 4-OH-D was e s t a b l i s h e d by i n j e c t i o n of ref e r e n c e s u b s t a n c e (obtained f r o m Roche). S a m p l e s for the c a l i b r a t i o n c u r v e (NADPH excluded) s p i k e d w i t h k n o w n a m o u n t s of 4 - O H - D w e r e t r e a t e d in the same way. Results P a t i e n t data and e n z y m e c o n c e n t r a t i o n s / a c t i v i t y o b t a i n e d are s u m m a r i z e d in T a b l e I. L i v e r s p e c i m e n GL4 was o b t a i n e d f r o m a p a t i e n t c l a s s i f i e d as a p o o r o x i d i z e r (I) a c c o r d i n g to the in vivo D h y d r o x y ~ a t i o n test (metabolic r a t i o 50). W i t h m i c r o s o m e s f r o m this l i v e r the rate of 4 - h y d r o x y l a t i o n of D w a s slower, 14
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p m o l . m g - 1 " m i n -I, c o m p a r e d to the o t h e r seven s p e c i m e n s r a n g i n g f r o m 25 to 95 p m o l ' m g - l . m i n -I. S i m i l a r l y , the rate of 1 0 - h y d r o x y l a t i o n of N T w a s s l o w e r w i t h GL4, 6 p m o l - m g - l . m i n -1 c o m p a r e d to the r a n g e of 12 to 55 a m o n g o t h e r s p e c i m e n s . AT d e m e t h y l a t i o n to N T w a s a ~ s o m e a s u r e d in GL4 (nonsmoker). It w a s 178 p m o l ' m g - -min -1 w h i c h is in t h e lower range e a r l i e r f o u n d (175 to 923; n = 12) (12). A c l o s e c o r r e l a t i o n b e t w e e n the 4 - h y d r o x y l a t i o n of D and the 1 0 - h y d r o x y l a t i o n of N T in v i t r o was found a m o n g the livers (r = 0.96 n = 8) (Fig I). 1 0 - h y d r o x y l a t i o n of N T c o r r e l a t e d w i t h c y t o c h r o m e b 5 c o n c e n t r a t i o n (r = 0.73; p < 0.05). T h e r e w a s n o s i g n i f i c a n t c o r r e l a t i o n b e t w e e n N T 1 0 - h y d r o x y l a t i o n and c y t o c h r o m e P-450 c o n c e n t r a t i o n (r = 0.33) or N A D P H - c y t o c h r o m e c r e d u c t a s e a c t i v i t y (r = 0.28). The h y d r o x y l a t i o n of D d i d not--correlate s i g n i f i c a n t l y w i t h any of these e n z y m e l e v e l s / a c t i v i t y (cyt P-450; r = 0.16, cyt b5; r = 0.59, N A D P H cyt ~ red.; r = 0.14). TABLE C h a r a c t e r i s t i c s of p a t i e n t s concentrations/activity Liver No
sex/ age
urinary ratio . D / 4 - O H - D a;
and
I liver m i c r o s o m a l
enzyme
cytochrome P-450 n m o l ' m g -I
cytochrome b5 n m o l - m g -1
NADPH cytochrome c reductase n m o l . m g - l . m i n -I
I
M/67
0.4
0.28
0.29
53
2
F/32
0.5
0.58
0.42
45
3
F/36
0.8
0.41
0.28
108
4
F/53
50
0.35
0.43
60
24
F/44
-
0.34
0.61
56
25
F/52
-
0.43
0.33
78
26 b)~
F/59
-
1.44
0.52
77
28
F/44
-
0.63
0.45
66
I-4 w e r e o b t a i n e d d u r i n g c h o l e c y s t e c t o m y and 24-28 d u r i n g n e p h r e c t o m y . The l a t t e r d o n o r s h a d c e r e b r a l a n e u r y s m s . a) D = debrisoquine; 4-OH-D = 4-hydroxydebrisoquine, D / 4 - O H - D r e p r e s e n t s the c o n c e n t r a t i o n r a t i o b e t w e e n D and 4 - O H - D in u r i n e c o l l e c t e d d u r i n g 6 h o u r s f o l l o w i n g an oral dose of 10 mg d e b r i s o q u i n e . b) P a t i e n t t r e a t e d w i t h p e n t o b a r b i t a l , at l e a s t 15 g d u r i n g 60 hours. The liver was o b t a i n e d 24 h o u r s a f t e r c e s s a t i o n . Fig 2 shows d e n s i t o m e t r i c t r a c i n g s of m i c r o s o m a l p r o t e i n s f r o m GL 4 and HL 24 run on the SDS-gel. The m o s t o b v i o u s d i f f e r e n c e b e t w e e n t h e s e l i v e r s was that liver GL 4 (poor h y d r o x y l a t o r ) h a d a r e l a t i v e lack of a p r o t e i n b a n d w i t h a m o l e c u l a r w e i g h t of a p p r o x i m a t e l y 54,500 c o m p a r e d to liver HL 24 exh i b i t i n g the h i g h e s t h y d r o x y l a t i o n a c t i v i t y in vitro. I n t e r e s t i n g ly liver HL 26 o r i g i n a t i n g f r o m a p a t i e n t t r e a t e d w i t h p e n t o b a r b i t a l e x h i b i t e d an u n u s u a l l y h i g h i n t e n s i t y of a p r o t e i n b a n d w i t h a m o l e c u l a r w e i g h t of a b o u t 52,000.
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Fig. 40H-D
I
pmol,mgl~mi~ 1
Relationship between 10-hydroxylation of n o r t r i p t y l i n e (10-OH-NT) and 4 - h y d r o x y l a t i o n of d e b r i s o q u i n e (4-OH-D) in m i c r o s o m e s from e i g h t d i f f e r e n t h u m a n livers (GL o, HL • , see table I).
80
60
o 40
o
o
Q
2 0-
o
20
10 040THN
60
80
pmo}. m~l~mi61
A
B a
.....
J
...............
Fig.
2
P h o t o (A) and d e n s i t o m e t r i c t r a c i n g s (B) of h u m a n liver m i c r o somes run on S D S - g e l e l e c t r o p h o r e s i s and s t a i n e d for p r o t e i n w i t h C o o m a s s i e B r i l l i a n t Blue. The d e p i c t e d m o l e c u l a r w e i g h t s are d e t e r m i n e d from the gel. The m o l e c u l a r w e i g h t s of the s t a n d a r d p r o t e i n s are 94,000, 68,000, 45,000 and 30,000. Ten ~g of m i c r o somal p r o t e i n w a s a p p l i e d to each w e l l of the gel. a) HL 24, e x t e n s i v e h y d r o x y l a t o r . A r r o w i n d i c a t e s the p r o t e i n b a n d w i t h a m o l e c u l a r w e i g h t of 54,500. b) GL4, p o o r h y d r o x y l a t o r . c) HL 26, p a t i e n t t r e a t e d w i t h p e n t o b a r b i t a l . A r r o w i n d i c a t e s the p r o t e i n b a n d w i t h a m o l e c u l a r w e i g h t of 52,000.
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Discussion The r e s u l t s show that a p o o r h y d r o x y l a t o r of D d e f i n e d in vivo, also h a d u n u s u a l l y low in v i t r o liver m i c r o s o m a l c a p a c i t y to h y d r o x y l a t e D and NT. In v i v o e x t e n s i v e D h y d r o x y l a t o r s (metabolic r a t i o c l o s e to unity) w e r e a l s o rapid h y d r o x y l a t o r s of D a n d NT in vitro. S t u d i e s w i t h liver tissue in v i t r o c o u l d thus be u s e f u l w h e ~ i n v e s t i g a t i n g m o l e c u l a r m e c h a n i s m s b e h i n d the g e n e t i c a l l y d e t e r m i n e d d e f i c i e n c y in drug oxidation. The strong c o r r e l a t i o n b e t w e e n the 4 - h y d r o x y l a t i o n of D and the 1 0 - h y d r o x y l a t i o n of NT s u g g e s t s that these two o x i d a t i o n s have rate l i m i t i n g factors in common. The same c y t o c h r o m e P-450 isozyme m a y h y d r o x y late b o t h drugs. This does not e x c l u d e the p o s s i b i l i t y that other e n z y m e s a l s o can c a t a l y z e NT 1 0 - h y d r o x y l a t i o n . Furthermore, a c o m m o n r e g u l a t i o n of d i f f e r e n t enzyme c o m p o n e n t s can not be excluded. I n t e r e s t i n g l y , the c y t o c h r o m e b 5 level c o r r e l a t e d w i t h the NT 1 0 - h y d r o x y l a t i o n , i n d i c a t i n g that this c y t o c h r o m e i n f l u e n c e s the o x i d a t i o n of NT. It has b e e n shown that O - d e m e t h y l a t i o n of p - n i t r o a n i s o l e is c a t a l y z e d by a c y t o c h r o m e P-450 form w i t h a b s o l u t e r e q u i r e m e n t of c y t o c h r o m e b 5 for a c t i v i t y (15). C y t o c h r o m e P-450 levels did not c o r r e l a t e w i t h the h y d r o x y l a t i o n s . This is in line w i t h our h y p o t h e s i s that only a f r a c t i o n of the total a m o u n t of c y t o c h r o m e P-450 c a t a l y z e s the reaction. The S D S - g e l e l e c t r o p h o r e s i s p a t t e r n i n d i c a t e d a r e l a t i v e lack of a p r o t e i n b a n d w i t h a m o l e c u l a r w e i g h t of a p p r o x i m a t e l y 54,500 in the p o o r h y d r o x y l a t o r . D e n s i t o m e t r i c t r a c i n g s of m i c r o somes f r o m a d d i t i o n a l livers (not shown) support this o b s e r v a t i o n . It is t e m p t i n g to s p e c u l a t e that this b a n d r e p r e s e n t s a cytoc h r o m e P-450 isozyme i n v o l v e d in the h y d r o x y l a t i o n of D and NT. It can not be e x c l u d e d that other m i n o r or only s l i g h t l y abn o r m a l e n z y m e c o m p o n e n t s not e a s i l y r e s o l v e d and d e t e c t e d are involved. Acknowledgement This study was s u p p o r t e d by the Swedish M e d i c a l R e s e a r c h C o u n c i l (14X-05667, 04X-03902), Funds from the K a r o l i n s k a I n s t i t u t e and N o r d i s k a S a m f u n d e t s S t i f t e l s e f6r v e t e n s k a p l i g f o r s k n i n g u t a n djurf~rs~k. References I .
2. 3. 4. 5. 6. 7.
A. MAHGOUB, J.R. IDLE, L.G. DRING, K. L A N C A S T E R and K.L SMITH, L a n c e t 2 584-586 (1977). M. EICHELBAUM, N. S P A N N B R U C K E R , B. S T E I N C K E and H.J. DENGLER, Eur. J. Clin. Pharmacol. 16 183-187 (1979). M. ASBERG, D.A. PRICE EVANS and F.--SJOQVIST, J. Med. Genet. 8 129-135 (1971). B. A L E X A N D E R S O N and O. BORGA, Eur. J. Clin. Pharmacol. 5 174-180 (1973). B. MELLSTROM, L. BERTILSSON, J. S~WE, H-U. SCHULZ and F. SJOQVIST, Clin. Pharmacol. Ther. 30 189-193 (1981). D.S. DAVIES, G.C. KAHN, S. MURRAY, M.J. B R O D I E and A.R. BOOBIS, Br. J. Clin. Pharmacol. 11 89-91 (1981). C. von BAHR, C-G. GROTH, H. JANSSON, G. LUNDGREN, M. LIND and H. GLAUMANN, Clin. Pharmacol. Ther. 27 711-725 (1980).
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8, 9. 10. 11. 12. 13. 14. 15.
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G.L. P E T E R S O N , A n a l . B i o c h e m . 83 3 4 6 - 3 5 6 (1977). T. O M U R A a n d R. SATO, J. B i o l . C h e m . 239 2 3 7 0 - 2 3 7 8 (1964). L. E R N S T E R , A c t a C h e m . S c a n d . 121 6 0 0 - 6 1 1 (1958). U.K. L A E M M L I , N a t u r e 227 6 8 0 - 6 8 5 (1970). B. M E L L S T R O M a n d C. v o n B A H R , D r u g M e t a b . D i s p o s . 9 5 6 5 - 5 6 8 (1981). G.C. K A H N , A.R. B O O B I S , S. M U R R A Y , M . J . B R O D I E a n d D.S. D A V I E S , Br. J. C l i n . P h a r m a c o l . 13 6 3 7 - 6 4 5 (1982). B. M E L L S T R O M , L. B E R T I L S S O N , C. B I R G E R S S O N , M. G O R A N S S O N a n d C. v o n B A H R , D r u g M e t a b . D i s p o s . , in p r e s s . T. S U G I Y A M A , N. M I K O a n d T. Y A M A N O , B i o p h y s . Res. C o m m . 90 7 1 5 - 7 2 0 (1979).
CORRELATION
BETWEEN
Pergamon Press
NORTRIPTYLINE AND DEBRISOQUINE IN THE H U M A N L I V E R 1
1
HYDROXYLATION
Christer von B@hr , Carol Birger§son , Agneta Blanck G S r a n s s o n I, B r i t t M e l l s t r ~ m I and K l a s N i l s e l l 3
2'
, Monica
D e p a r t m e n t s of C l i n i c a l P h a r m a c o l o g y 1, M e d i c a l N u t r i t i o n 2 and S u r g e r y 3, K a r o l i n s k a I n s t i t u t e , H u d d i n g e U n i v e r s i t y H o s p i t a l , S-141 86 H u d d i n g e , S w e d e n (Received in final form J~ne i, 1983)
Summary The b e n z y l i c h y d r o x y l a t i o n of n o r t r i p t y l i n e (NT) and d e b r i s o q u i n e (D) by i s o l a t e d h u m a n liver m i c r o s o m e s f r o m e i g h t s u b j e c t s was studied. T h e r e was a s t r o n g c o r r e l a t i o n b e t w e e n the 1 0 - h y d r o x y l a t i o n of NT and the 4 - h y d r o x y l a t i o n of D (r = 0.96). The a b i l i t y to h y d r o x y l a t e D was a l s o m e a s u r e d in vivo as the r a t i o b e t w e e n D and 4-OH-D in u r i n e a f t e r oral a d m i n i s t r a t i o n of the drug to four subjects. This e s t i m a t e of h y d r o x y l a t i o n c a p a c i t y a g r e e d w i t h the in vitro measurements. Liver microsomes from a subject defined as a p o o r in v i v o o x i d i z e r of D h y d r o x y l a t e d NT and D u n u s u a l l y slowly. S e p a r a t i o n of m i c r o s o m a l p r o t e i n s by S D S - g e l e l e c t r o p h o r e s i s i n d i c a t e d a r e l a t i v e lack of a c y t o c h r o m e P-450 i s o z y m e w i t h a m o l e c u l a r w e i g h t of 54,500 in the liver f r o m the poor o x i d i z e r . The o x i d a t i o n of d e b r i s o q u i n e (D) and s p a r t e i n (S) is m o n o g e n i c a l l y c o n t r o l l e d and some 3-9 per cent of C a u c a s i a n s are poor o x i d i z e r s of b o t h d r u g s (1,2). S t u d i e s on NT s t e a d y state p l a s m a c o n c e n t r a t i o n s s u g g e s t that in m a n n o r t r i p t y l i n e (NT) k i n e t i c s are l i k e l y to be p o l y g e n i c a l l y c o n t r o l l e d (3). NT is m e t a b o l i z e d b o t h by 1 0 - h y d r o x y l a t i o n and N - d e m e t h y l a t i o n (4). The a b i l i t y to c a r r y o u t the 1 0 - h y d r o x y l a t i o n of NT a p p e a r s to be r e l a t e d to the D h y d r o x y l a t i o n p h e n o t y p e (5). It has r e c e n t l y b e e n shown that liver m i c r o s o m e s f r o m a s u b j e c t i d e n t i f i e d as a poor D h y d r o x y l a t o r in v i v o did n o t c a t a l y z e the 4 - h y d r o x y l a t i o n of the d r u g (6). T h i s s t u d y w a s u n d e r t a k e n to i n v e s t i g a t e the 1 0 - h y d r o x y l a tion of NT in v i t r o in r e l a t i o n to D h y d r o x y l a t i o n in v i v o and and in v i t r o in d i f f e r e n t subjects. Methods The in v i v o a b i l i t y to h y d r o x y l a t e D was m e a s u r e d in four p a t i e n t s . A single 10 mg oral dose of D (Declinax R tablets, H o f f m a n - L a Roche) was g i v e n and u r i n e w a s c o l l e c t e d for 6 hrs. The r a t i o b e t w e e n the p a r e n t d r u g and the 4 - h y d r o x y m e t a b o l i t e (4-OH-D) e x c r e t e d in the u r i n e w a s d e t e r m i n e d (I).
with
L i v e r t i s s u e f r o m these cholecystectomy because
p a t i e n t s w a s o b t a i n e d in c o n n e c t i o n of g a l l s t o n e s (GL). A n a e s t h e s i a w a s
0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
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i n i t i a t e d w i t h t h i o p e n t h a l f o l l o w e d by a d m i n i s t r a t i o n of n i t r o u s oxide, d i a z e p a m and fentanyl. D u r i n g the o p e r a t i o n a I to 3 g l i v e r b i o p s y was t a k e n f r o m the left lobe. This was a p p r o v e d by the E t h i c a l C o m m i t t e e of the h o s p i t a l . L i v e r tissue was also o b t a i n e d from k i d n e y t r a n s p l a n t d o n o r s w i t h c o m p l e t e c e r e b r a l i n f a r c t i o n as p r e v i o u s l y d e s c r i b e d (HL) (7). The m i c r o s o m a l f r a c t i o n of the livers was p r e p a r e d (7). P r o t e i n c o n c e n t r a t i o n (8), c y t o c h r o m e P-450 and b 5 c o n c e n t r a t i o n s (9) and N A D P H - c y t o c h r o m e c r e d u c t a s e a c t i v i t y (10) w e r e d e t e r mined. S D S - p O l y a c r y l a m i d e - - g e l e l e c t r o p h o r e s i s was p e r f o r m e d (11). Ten ~g of m i c r o s o m a l p r o t e i n was a p p l i e d to each well of the gel. The a c r y l a m i d e c o n c e n t r a t i o n was 9 % (w/v). The gel w a s s t a i n e d w i t h C o o m a s s i e B r i l l i a n t Blue. D e n s i t o m e t r i c t r a c i n g s w e r e p e r f o r m e d at 550 nm. The rates of NT h y d r o x y l a t i o n and a m i t r i p t y l i n e (AT) d e m e t h y l a t i o n w e r e m e a s u r e d in a s y s t e m p r e v i o u s l y d e s c r i b e d (12) cont a i n i n g I mg of m i c r o s o m a l protein. The i n c u b a t i o n time was 10 and 30 min for AT a n d NT, r e s p e c t i v e l y . The D h y d r o x y l a t i o n was p e r f o r m e d a c c o r d i n g to K a h n et al (13) w i t h 0.5 mg of m i c r o s o m a l p r o t e i n for 30 min. NT f o r m e d from AT w a s q u a n t i t a t e d by m a s s f r a g m e n t o g r a p h y (12) and 1 0 - O H - N T f o r m e d f r o m NT by H P L C (14). The 4 - h y d r o x y l a t i o n rate of D was d e t e r m i n e d by q u a n t i t a t i o n of f o r m e d 4-OH-D a c c o r d i n g to K a h n et al (13). This m e t h o d u s e s a s o l v e n t e x t r a c tion p r o c e d u r e to s e l e c t i v e l y e l i m i n a t e the s u b s t r a t e , w h i c h i n t e r f e r s w i t h the q u a n t i t a t i o n of the m e t a b o l i t e by gas c h r o m a t o g r a p h y - m a s s s p e c t r o m e t r y . In our hands, however, the e x t r a c t i o n p r o c e d u r e was not s u f f i c i e n t and we h a d to r e p l a c e it by a m o r e e f f i c i e n t s e p a r a t i o n t e c h n i q u e . To the i n c u b a t i o n sample was a d d e d the i n t e r n a l s t a n d a r d (13) and the m i x t u r e was f i l t e r e d t h r o u g h a M i l l i p o r e f i l t e r (type H A W P 0.45 ~m) w i t h i n I min. The f i l t e r was r i n s e d w i t h I ml of s o d i u m p h o s p h a t e b u f f e r pH 2.0 (I = 0.05) and the c o m b i n e d f i l t r a t e s w e r e s u b j e c t e d to H P L C i m m e d i a t e l y or s t o r e d at -20 ° over night. The c h r o m a t o g r a p h i c s y s t e m c o n s i s t e d of a p u m p (LDC, C o n s t a m e t r i c III), a d e t e c t o r (LDC, S p e c t r o m o n i t o r III) o p e r a t e d at 262 nm, an i n j e c t o r (Altex, P h e o d y n e 7021) and a g u a r d c o l u m n (Brownlee Labs, RP 18 S p h e r i 5). The a n a l y t i c a l c o l u m n (4.6 m m i.d x 150 m m length) was p a c k e d w i t h 5 ~m o c t a d e c y l s i l a n (Altex, U l t r a s p h e r e ) and e l u t e d w i t h s o d i u m p h o s p h a t e b u f f e r pH 2.0 (I = 0.05) and m e t h a n o l (75:25) at a f l o w rate of 1.0 m l - m i n -I. A f r a c t i o n of the e l u a t e c o r r e s p o n d i n g to 4-OH-D was c o l l e c t e d . A f t e r a d d i t i o n of 0.1 ml 2.5 M N a O H f o l l o w e d by 0.2 ml a q u e o u s s o l u t i o n of s o d i u m b i c a r b o n a t e the samples w e r e d e r i v a t i z e d and p r o c e s s e d as d e s c r i b e d by Kahn et al (13). The r e t e n t i o n v o l u m e of 4-OH-D was e s t a b l i s h e d by i n j e c t i o n of ref e r e n c e s u b s t a n c e (obtained f r o m Roche). S a m p l e s for the c a l i b r a t i o n c u r v e (NADPH excluded) s p i k e d w i t h k n o w n a m o u n t s of 4 - O H - D w e r e t r e a t e d in the same way. Results P a t i e n t data and e n z y m e c o n c e n t r a t i o n s / a c t i v i t y o b t a i n e d are s u m m a r i z e d in T a b l e I. L i v e r s p e c i m e n GL4 was o b t a i n e d f r o m a p a t i e n t c l a s s i f i e d as a p o o r o x i d i z e r (I) a c c o r d i n g to the in vivo D h y d r o x y ~ a t i o n test (metabolic r a t i o 50). W i t h m i c r o s o m e s f r o m this l i v e r the rate of 4 - h y d r o x y l a t i o n of D w a s slower, 14
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p m o l . m g - 1 " m i n -I, c o m p a r e d to the o t h e r seven s p e c i m e n s r a n g i n g f r o m 25 to 95 p m o l ' m g - l . m i n -I. S i m i l a r l y , the rate of 1 0 - h y d r o x y l a t i o n of N T w a s s l o w e r w i t h GL4, 6 p m o l - m g - l . m i n -1 c o m p a r e d to the r a n g e of 12 to 55 a m o n g o t h e r s p e c i m e n s . AT d e m e t h y l a t i o n to N T w a s a ~ s o m e a s u r e d in GL4 (nonsmoker). It w a s 178 p m o l ' m g - -min -1 w h i c h is in t h e lower range e a r l i e r f o u n d (175 to 923; n = 12) (12). A c l o s e c o r r e l a t i o n b e t w e e n the 4 - h y d r o x y l a t i o n of D and the 1 0 - h y d r o x y l a t i o n of N T in v i t r o was found a m o n g the livers (r = 0.96 n = 8) (Fig I). 1 0 - h y d r o x y l a t i o n of N T c o r r e l a t e d w i t h c y t o c h r o m e b 5 c o n c e n t r a t i o n (r = 0.73; p < 0.05). T h e r e w a s n o s i g n i f i c a n t c o r r e l a t i o n b e t w e e n N T 1 0 - h y d r o x y l a t i o n and c y t o c h r o m e P-450 c o n c e n t r a t i o n (r = 0.33) or N A D P H - c y t o c h r o m e c r e d u c t a s e a c t i v i t y (r = 0.28). The h y d r o x y l a t i o n of D d i d not--correlate s i g n i f i c a n t l y w i t h any of these e n z y m e l e v e l s / a c t i v i t y (cyt P-450; r = 0.16, cyt b5; r = 0.59, N A D P H cyt ~ red.; r = 0.14). TABLE C h a r a c t e r i s t i c s of p a t i e n t s concentrations/activity Liver No
sex/ age
urinary ratio . D / 4 - O H - D a;
and
I liver m i c r o s o m a l
enzyme
cytochrome P-450 n m o l ' m g -I
cytochrome b5 n m o l - m g -1
NADPH cytochrome c reductase n m o l . m g - l . m i n -I
I
M/67
0.4
0.28
0.29
53
2
F/32
0.5
0.58
0.42
45
3
F/36
0.8
0.41
0.28
108
4
F/53
50
0.35
0.43
60
24
F/44
-
0.34
0.61
56
25
F/52
-
0.43
0.33
78
26 b)~
F/59
-
1.44
0.52
77
28
F/44
-
0.63
0.45
66
I-4 w e r e o b t a i n e d d u r i n g c h o l e c y s t e c t o m y and 24-28 d u r i n g n e p h r e c t o m y . The l a t t e r d o n o r s h a d c e r e b r a l a n e u r y s m s . a) D = debrisoquine; 4-OH-D = 4-hydroxydebrisoquine, D / 4 - O H - D r e p r e s e n t s the c o n c e n t r a t i o n r a t i o b e t w e e n D and 4 - O H - D in u r i n e c o l l e c t e d d u r i n g 6 h o u r s f o l l o w i n g an oral dose of 10 mg d e b r i s o q u i n e . b) P a t i e n t t r e a t e d w i t h p e n t o b a r b i t a l , at l e a s t 15 g d u r i n g 60 hours. The liver was o b t a i n e d 24 h o u r s a f t e r c e s s a t i o n . Fig 2 shows d e n s i t o m e t r i c t r a c i n g s of m i c r o s o m a l p r o t e i n s f r o m GL 4 and HL 24 run on the SDS-gel. The m o s t o b v i o u s d i f f e r e n c e b e t w e e n t h e s e l i v e r s was that liver GL 4 (poor h y d r o x y l a t o r ) h a d a r e l a t i v e lack of a p r o t e i n b a n d w i t h a m o l e c u l a r w e i g h t of a p p r o x i m a t e l y 54,500 c o m p a r e d to liver HL 24 exh i b i t i n g the h i g h e s t h y d r o x y l a t i o n a c t i v i t y in vitro. I n t e r e s t i n g ly liver HL 26 o r i g i n a t i n g f r o m a p a t i e n t t r e a t e d w i t h p e n t o b a r b i t a l e x h i b i t e d an u n u s u a l l y h i g h i n t e n s i t y of a p r o t e i n b a n d w i t h a m o l e c u l a r w e i g h t of a b o u t 52,000.
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Fig. 40H-D
I
pmol,mgl~mi~ 1
Relationship between 10-hydroxylation of n o r t r i p t y l i n e (10-OH-NT) and 4 - h y d r o x y l a t i o n of d e b r i s o q u i n e (4-OH-D) in m i c r o s o m e s from e i g h t d i f f e r e n t h u m a n livers (GL o, HL • , see table I).
80
60
o 40
o
o
Q
2 0-
o
20
10 040THN
60
80
pmo}. m~l~mi61
A
B a
.....
J
...............
Fig.
2
P h o t o (A) and d e n s i t o m e t r i c t r a c i n g s (B) of h u m a n liver m i c r o somes run on S D S - g e l e l e c t r o p h o r e s i s and s t a i n e d for p r o t e i n w i t h C o o m a s s i e B r i l l i a n t Blue. The d e p i c t e d m o l e c u l a r w e i g h t s are d e t e r m i n e d from the gel. The m o l e c u l a r w e i g h t s of the s t a n d a r d p r o t e i n s are 94,000, 68,000, 45,000 and 30,000. Ten ~g of m i c r o somal p r o t e i n w a s a p p l i e d to each w e l l of the gel. a) HL 24, e x t e n s i v e h y d r o x y l a t o r . A r r o w i n d i c a t e s the p r o t e i n b a n d w i t h a m o l e c u l a r w e i g h t of 54,500. b) GL4, p o o r h y d r o x y l a t o r . c) HL 26, p a t i e n t t r e a t e d w i t h p e n t o b a r b i t a l . A r r o w i n d i c a t e s the p r o t e i n b a n d w i t h a m o l e c u l a r w e i g h t of 52,000.
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Discussion The r e s u l t s show that a p o o r h y d r o x y l a t o r of D d e f i n e d in vivo, also h a d u n u s u a l l y low in v i t r o liver m i c r o s o m a l c a p a c i t y to h y d r o x y l a t e D and NT. In v i v o e x t e n s i v e D h y d r o x y l a t o r s (metabolic r a t i o c l o s e to unity) w e r e a l s o rapid h y d r o x y l a t o r s of D a n d NT in vitro. S t u d i e s w i t h liver tissue in v i t r o c o u l d thus be u s e f u l w h e ~ i n v e s t i g a t i n g m o l e c u l a r m e c h a n i s m s b e h i n d the g e n e t i c a l l y d e t e r m i n e d d e f i c i e n c y in drug oxidation. The strong c o r r e l a t i o n b e t w e e n the 4 - h y d r o x y l a t i o n of D and the 1 0 - h y d r o x y l a t i o n of NT s u g g e s t s that these two o x i d a t i o n s have rate l i m i t i n g factors in common. The same c y t o c h r o m e P-450 isozyme m a y h y d r o x y late b o t h drugs. This does not e x c l u d e the p o s s i b i l i t y that other e n z y m e s a l s o can c a t a l y z e NT 1 0 - h y d r o x y l a t i o n . Furthermore, a c o m m o n r e g u l a t i o n of d i f f e r e n t enzyme c o m p o n e n t s can not be excluded. I n t e r e s t i n g l y , the c y t o c h r o m e b 5 level c o r r e l a t e d w i t h the NT 1 0 - h y d r o x y l a t i o n , i n d i c a t i n g that this c y t o c h r o m e i n f l u e n c e s the o x i d a t i o n of NT. It has b e e n shown that O - d e m e t h y l a t i o n of p - n i t r o a n i s o l e is c a t a l y z e d by a c y t o c h r o m e P-450 form w i t h a b s o l u t e r e q u i r e m e n t of c y t o c h r o m e b 5 for a c t i v i t y (15). C y t o c h r o m e P-450 levels did not c o r r e l a t e w i t h the h y d r o x y l a t i o n s . This is in line w i t h our h y p o t h e s i s that only a f r a c t i o n of the total a m o u n t of c y t o c h r o m e P-450 c a t a l y z e s the reaction. The S D S - g e l e l e c t r o p h o r e s i s p a t t e r n i n d i c a t e d a r e l a t i v e lack of a p r o t e i n b a n d w i t h a m o l e c u l a r w e i g h t of a p p r o x i m a t e l y 54,500 in the p o o r h y d r o x y l a t o r . D e n s i t o m e t r i c t r a c i n g s of m i c r o somes f r o m a d d i t i o n a l livers (not shown) support this o b s e r v a t i o n . It is t e m p t i n g to s p e c u l a t e that this b a n d r e p r e s e n t s a cytoc h r o m e P-450 isozyme i n v o l v e d in the h y d r o x y l a t i o n of D and NT. It can not be e x c l u d e d that other m i n o r or only s l i g h t l y abn o r m a l e n z y m e c o m p o n e n t s not e a s i l y r e s o l v e d and d e t e c t e d are involved. Acknowledgement This study was s u p p o r t e d by the Swedish M e d i c a l R e s e a r c h C o u n c i l (14X-05667, 04X-03902), Funds from the K a r o l i n s k a I n s t i t u t e and N o r d i s k a S a m f u n d e t s S t i f t e l s e f6r v e t e n s k a p l i g f o r s k n i n g u t a n djurf~rs~k. References I .
2. 3. 4. 5. 6. 7.
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