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Bile Acid Metabolism in Mammals: IX. Conversion of Chenodeoxycholic Acid to Cholic Acid by Isolated Perfused Rat Liver ABSTRACT

Current dogma of bile acid synthesis in mammals insists that hydroxylation of the ring structure at C-12 precedes side chain oxidation, and that chenodeoxycholic acid is not converted to chotic acid under normal conditions. This report concerns the conversion of chenodeoxycholic acid to cholic acid by isolated, perfused rat liver. Results indicate that isolated perfused rat liver has a definite, but limited, capacity for synthesis of cholic acid from chenodeoxycholic acid. INTRODUCTION

Current concepts of bile acid formation in mammals suggest that hydroxylation at C-12 precedes side-chain oxidation (1) and that chenodeoxycholic acid (CDCA) is not hydroxylated to cholic acid (CA) in viva (2). However, this is not the case in nonmammalian species, and the python (3), eel (4), chicken (5) and trout (6) have been found to form CA from CDCA. Recently, Samuels and Palmer reported the conversion of CDCA to CA in humans with o b s t r u c t i v e jaundice; they suggested that 12a-hydroxylation of CDCA can be induced in man during biliary stasis (7). In studies on the metabolism of CDCA by the isolated perfused rat liver, we detected appreciable amounts of /~-murichohc acid in the bile produced after the addition of CDCA to the perfusion medium (8). Although we did not detect a-muricholic acid in this bile, we did detect a small amount of another trihydroxy bile acid which had the same specific activity as added CDCA, and which appeared to be CA on the basis of thin layer chromatography (TLC) and gas liquid chromatography (GLC). We have now confirmed by mass spectroscopy (MS) and radio gas chromatography that this latter trihydroxy bile acid synthesized from CDCA by the isolated perfused rat liver is CA. MATERIALS AND METHODS

Eighteen experiments involving perfusion of isolated rat liver were performed by methods reported previously (9-11). Wistar strain rats of both sexes, weighing 250-300 g, were maintained on Purina Rat Chow diet. After 2 hr of base line perfusion during which the bile acid

pool of the system apparently was depleted (8), e x o g e n o u s CDCA (Ikapharm, Ramat-Gan, Israel) was added to 100 ml perfusion medium. No contaminants were seen by GLC when large amounts of CDCA were analyzed. CDCA labeled with C l a in the C-24 position (Tracer Labs, Waltham, Massachusetts; specific activity, 35.8 mCi/mmole) contained a maximum impurity of 3%, which was a m o n o h y d r o x y bile acid. In 12 experiments on 6 animals of each sex, a mixture of 1 /~mole CDCA and 5/aCi radiolabeled CDCA was purified by TLC before addition to the perfusion medium. The other 6 experiments involved male rats and the addition of 10, 20, or 30/2moles of CDCA to the perfusion medium. Each perfusion was continued for 3 hr following the addition of CDCA to the perfusion medium. Bile was collected in hourly aliquots and its bile acid composition analyzed

FIG. 1. Radio gas chromatography of bile acids secreted in 1 ~tmole chenodeoxycholic acid -C-24-14C perfusion. Upper tracing, radioactivity; lower tracing, mass. Peaks 1, 2, and 3 represent 13-muricholic, cholic, and chenodeoxycholic acids, respectively. IS =internal standard, 5-~-cholanic acid: Instrument: Packard Model 1894 proportional radioactivity monitor and a copper oxide furnace. Column 120 cm x 2 mm inside diameter glass tube packed with 19:1 (w/w) mixture of 3% HI-EFF-8BP and 3% OV 210 on 100-120 mesh Gas Chrom Q, respectively. Carrier gas, helium 55 ml/min. Temperatures: Column, 225 C; injector, 255 C; detector, 240 C. Proportional Counter Conditions: Range, 3000 CPM; time constant, 10see; high voltage, 1650 v; quench gas, propane 5 ml/min. Combustion furnace, 750 C. Flame, ionization mass delector with 10:1 stream splitter. Sample 2 M of 1% solution of bile acid methyl ester trimethylsilylether in silylation mixture. Total radioactivity: 12,000 dpm. LIPIDS, VOL. 10, NO. 9

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TABLE I Biliary Bile Acid Secretion After Addition of Chenodeoxycholic Acid to Perfusion Medium Rat liver : Chenodeoxycholic acid added (/~mole) Liver wt (g) Added bile acid secreted into bile (%) Bile acid secretion (nmole/g liver) Total

Female a

Male a

1

1

7.5 -+ 0.9 89

+ 1.1

10

8.3 + 1,3 91

Male b

+ 2.3

119

_+ 17

109

-+ 8

Chenodeoxycholic acid

71

-+ 12 c

34

_+6 c

~3-Muricholic acid

12

-+ 5 d

41

-+ 7 d

Cholic acid

35

+ 9

34

_+4

9.9 8.4

20 10.2 10.8

30 9.8 9,2

90 91

89 91

90 89

960 1080 700 750 220 284 40 46

1769 1679 1222 1152 480 469 47 58

2750 2880 2090 2202 605 619 55 59

aAverage of 6 perfusions (mean +_standard error). bTwo perfusions. CSignificantly (P < 0.05) different. dSignificantly (P < 0.001) different. as d e s c r i b e d previously (12). A p p r o p r i a t e aliq u o t s o f t h e bile acid m e t h y l esters of t h e various p e r f u s i o n s were p o o l e d , c o n v e r t e d to trim e t h y l s i l y l e t h e r s (13) a n d a n a l y z e d b y a c o m b i n e d GLC-MS s y s t e m a n d b y radiogas c h r o m a t o g r a p h y (6). RESULTS AND DISCUSSION

I n c o n t r o l e x p e r i m e n t s in w h i c h n o exogen o u s bile acid was a d d e d t o t h e p e r f u s i o n m e d i u m , biliary s e c r e t i o n o f bile acids in 3 hr. was negligible (8). I n t h o s e e x p e r i m e n t s i n w h i c h 1 p m o l e C D C A was a d d e d t o t h e perf u s i o n m e d i u m , 91% o f t h e bile acid was s e c r e t e d in t h e bile p r o d u c e d b y t h e livers of male rats a n d 89% in t h o s e o f f e m a l e rats. /3M u f i c h o l i c acid a c c o u n t e d f o r 10% o f t h e biliary bile acids in females, 38% in males (P 0 . 0 0 1 ) ( T a b l e I). CDCA a c c o u n t e d for 60% o f biliary bile acids in females, a n d 31% in males (P ~ 0 . 0 0 1 ) ( T a b l e I). In b o t h male a n d f e m a l e rats, t r i h y d r o x y bile acid p r e v i o u s l y i d e n t i f i e d as CA oil t h e basis o f T L C a n d G L C a c c o u n t e d for ca. 30% o f t h e biliary bile acids secreted. T h e r e was n o significant d i f f e r e n c e in specific activity o f t h e s e 3 bile acids as m e a s u r e d b y direct c o u n t , G L C q u a n t i t a t i o n o f various T L C b a n d s of m e t h y l esters o b t a i n e d b y t h e s y s t e m o f S u b b i a h , et al., (14), or b y radio gas c h r o m a t o g r a p h y (6) (Fig. 1). W h e n stanclards o f a - m u f i c h o l i c acid, m u r i c h o l i c acid, a n d C A were s u b j e c t e d to LIPIDS, VOL. 10, NO. 9

GLC-MS as t h e t r i m e t h y l s i l y l e t h e r o f t h e i r m e t h y l ester, a - m u r i c h o l i c acid gave a base p e a k at m / e 4 5 8 , /3-muricholic acid at m / e 2 8 5 , and C A at m / e 253. T r i h y d r o x y bile acid in t h e a b o v e e x p e r i m e n t s , i d e n t i f i e d t e n t a t i v e l y as CA, gave a base p e a k at m / e 253, a n d its mass spect r u m was i d e n t i c a l t o t h a t o f s t a n d a r d C A obt a i n e d in o u r l a b o r a t o r y a n d b y o t h e r investigators (15). C D C A in greater c o n c e n t r a t i o n s is t o x i c t o i s o l a t e d female rat liver, b u t n o t to i s o l a t e d male rat liver (8). In studies using t h e livers of male rats a n d 1 0 - 3 0 p m o l e s CDCA, it was f o u n d t h a t t h e a m o u n t o f fl-muricholic acid s e c r e t e d in t h e bile i n c r e a s e d progressively as the amount of CDCA added to the perfnsion m e d i u m was i n c r e a s e d ( T a b l e I). This was n o t t h e case f o r CA; biliary s e c r e t i o n of CA increased very little w i t h t h e a d d i t i o n o f larger a m o u n t s of C D C A t o the p e r f u s i o n m e d i u m (Table I). T h e s e e x p e r i m e n t s have d e m o n s t r a t e d t h a t m a m m a l i a n liver c a n 1 2 a - h y d r o x y l a t e t h e ring s t r u c t u r e of bile acids a f t e r o x i d a t i o n o f t h e i r side chain. These e x p e r i m e n t s also have d e m o n s t r a t e d t h a t , at low p e r f u s i o n m e d i u m c o n c e n t r a t i o n s (0.01 m m o l a r ) C D C A , ca. 30% C D C A c a n be c o n v e r t e d t o CA. It a p p e a r s t h a t t h e c a p a c i t y o f i s o l a t e d p e r f u s e d liver of t h e male rat t o c o n v e r t C D C A t o CA is l i m i t e d .

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I.M. YOUSEF M.M. FISHER

Departments of Pathology and Medicine University of Toronto Toronto, Ontario, Canada REFERENCES 1. Boyd, G.S., and I.W. Percy-Robb, Amer. J. Med. 51:580 (1970). 2. Danielsson, H., Advan. Lipid Res. 1:335 (1963). 3. Bergstrorn, S., H. Danielsson, and T. Kazuno, J. Biol. Cherm 235:983 (1960). 4. Masui, T., F. Ueyarna, H. Yashima, and T. Kazuno, J. Biocherru 62:650 (1967). 5. Ahlberg, J.W., V.A. Ziboh, R.C. Sonders, and S.L. Hsia, Fed. Proc. 20:283 (1961). 6. Denton, J.E., M.K. Yousef, I.M. Yousef, and A. Kuksis, Lipids 9:945 (1974). 7. Samuels, A.D., and R.H. Palmer, 23rd Ann. Mtg. Amer. Assoc. for Study of Liver Dis., 1972, p. 17.

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8. Yousef, I.M., R. Magnusson, V.M. Price, and M.M. Fisher, Can J. Physiol. Pharmacol. 51:418 (1973). 9. Fisher, M.M., and M. Kerly, J. Physiol. 174:273 (1964). 10. Fisher, M.M., R. Magnusson, M.J. Phillips and K. Miyai, Lab. Invest. 27:254 (1972). 11. Fisher, M.M., R. Magnusson, and K. Miyai, Lab. Invest. 25:88 (1971). 12. Yousef, I.M., G. Kakis, and M.M. Fisher, Can. J. Biochem. 50:402 (1972). 13. Kuksis, A., in "Methods of Biochemical Analysis," Vol. XIV, Edited by D. Glick, Interscience Publishers, New York, NY, 1966, p. 325. 14. Subbiah, M.T., A. Kuksis, and S. Mookerjea, Can. J. Biochem. 47:847 (1969). 15. Sjovall, J., P. Eneroth, and R. Ryhage, in "The Bile Acids, Chemistry, Physiology, and Metabolism," Vol. 1: Chemistry. Edited by P.P. Nair and D. Kritchevsky, Plenum Press, New York, NY, 1971, p. 209.

[Received May 12, 1975]

A Guide for Authors is Located in Lipids 10(January):60 (1975) LIPIDS, VOL. 10, NO. 9

Bile acid metabolism in mammals: IX. Conversion of chenodeoxycholic acid to cholic acid by isolated perfused rat liver.

Current dogma of bile acid synthesis in mammals insists that hydroxylation of the ring structure at C-12 precedes side chain oxidation, and that cheno...
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