Conversion of Cholesterol Into 5Β-Cholestane-3Α,7Α,12Α-Triol in Liver Homogenates

Formation of bile acids in man: conversion of cholesterol into 5β-cholestane-3α,7α,12α-triol in liver homogenates

Ingemar Björkhem, … , Kurt Einarsson, Gunnar Johansson

J Clin Invest. 1968;47(7):1573-1582. https://doi.org/10.1172/JCI105849.

Research Article

The mechanisms of the conversion of cholesterol into bile acids in man were studied by examining the metabolism of cholesterol-1,2-3H, cholest-5-ene-3β,7α-diol-7β-3H, tritiumlabeled 7α-hydroxycholest-4-en-3-one, 7α,12α- dihydroxycholest-4-en-3-one, and cholest-5-ene-3β,7α,12α-triol in fractions of liver homogenates. The 20,000 g supernatant fluid catalyzed the conversion of cholesterol into cholest-5-ene-3β,7α-diol, 7α-hydroxycholest-4-en-3-one, 7α- 12α-dihydroxycholest-4-en-3-one, and 5β-cholestane-3α,7α,12α-triol. In the presence of microsomal fraction fortified with NAD+, cholest-5-ene-3β,7α-diol was converted into 7α-hydroxycholest-4-en-3-one, and when this fraction was fortified with NADPH small amounts of cholest-5-ene-3β-7α,12α-triol were formed. 7α-Hydroxycholest-4-en-3-one was metabolized into 7α-12α-dihydroxycholest-4-en-3-one in the presence of microsomal fraction fortified with NADPH and into 5β-cholestane-3α,7α-diol in the presence of 100,000 g supernatant fluid. Cholest-5-ene-3β,7α,12α-triol was converted into 7α,12α-dihydroxycholest-4-en-3-one in the presence of microsomal fraction fortified with NAD+. The 100,000 g supernatant fluid catalyzed the conversion of 7α,12α-dihydroxycholest-4-en-3-one into 5β-cholestane- 3α,7α,12α-triol. The sequence of reactions in the conversion of cholesterol into 5β-cholestane-3α,7α-diol and 5β- cholestane-3α,7α,12α-triol, the subcellular localization of the enzymes, and the cofactor requirements were found to be the same as those described for rat liver.

Find the latest version: https://jci.me/105849/pdf Fonnation of Bile Acids in Man: Conversion of Cholesterol into 53-Cholestane-3a,7a, 12a-triol in Liver Homogenates *

INGEMAR BJORKHEM, HENRY DANIELSSON, KuRT EINARSSON, and GUNNAR JOHANSSON From the Department of Chemistry, Karolinska Institutet, Stockholm, Sweden

A B S T R A C T The mechanisms of the conversion 5,1-cholestane-3a,7a-diol and 5,8-cholestane-3a,7a, of cholesterol into bile acids in man were studied 12a-triol, the subcellular localization of the en- by examining the metabolism of cholesterol- zymes, and the cofactor requirements were found 1,2-3H, cholest-5-ene-3,8,7a-diol-7,8-3H, tritium- to be the same as those described for rat liver. labeled 7a-hydroxycholest-4-en-3-one, 7a,12a-dihydroxycholest-4-en-3-one, and cholest-5-ene-3,8, INTRODUCTION 7a,12a-triol in fractions of liver homogenates. The 20,000 g supernatant fluid catalyzed the conversion Bile acids are major end products of cholesterol of cholesterol into cholest-5-ene-3,8,7a-diol, 7a- metabolism in vertebrates. (For a review, see hydroxycholest4-en-3-one, 7a-12a-dihydroxycho- reference 1.) The conversion of cholesterol into lest4-en-3-one, and 5,8-cholestane-3a,7al2a-triol. bile acids takes place in the liver, and the main In the presence of microsomal fraction fortified bile acids formed are, in most mammalian species, with NAD+, cholest-5-ene-3,3,7a-diol was con- cholic acid 1 and chenodeoxycholic acid (1). In verted into 7a-hydroxycholest-4-en-3-one, and bile, bile acids are present as conjugates with when this fraction was fortified with NADPH taurine and glycine. Bile acids are excreted with small amounts of cholest-5-ene-3f8-7a,12a-triol bile into the intestine, reabsorbed in the ileum, were formed. 7a-Hydroxycholest-4-en-3-one was and again excreted with bile. A small amount of metabolized into 7a-12a-dihydroxycholest-4-en-3- bile acids escapes reabsorption in each cycle of one in the presence of microsomal fraction fortified enterohepatic circulation, and these bile acids are with NADPH and into 5,8-cholestane-3a,7a-diol in excreted with the feces. During their enterohepatic the presence of 100,000 g supernatant fluid. circulation bile acids are subjected to the action Cholest-5-ene-3,/,7cv,12a-triol was converted into of intestinal microorganisms. Major reactions in- 7a,12a-dihydroxycholest-4-en-3-one in the pres- volve the hydrolysis of the conjugates and the ence of microsomal fraction fortified with NAD+. removal of the 7a-hydroxyl group yielding deoxy- The 100,000 g supernatant fluid catalyzed the cholic acid from cholic acid and lithocholic acid conversion of 7a,12a-dihydroxycholest-4-en-3-one from chenodeoxycholic acid (1). Further struc- into 5f3-cholestane-3a,7a,12a-triol. The sequence 'The following systematic names are given to bile of reactions in the conversion of cholesterol into acids referred to by trivial names: cholic acid, 3a,7a, 12a- trihydroxy-5f-cholanoic acid; chenodeoxycholic acid, 3a,- * Bile acids and steroids 197. 7a-dihydroxy-5#-cholanoic acid; deoxycholic acid, 3a,12a- Received for publication 5 February 1968 and in re- dihydroxy-5,B-cholanoic acid; lithocholic acid, 3a-hy- vised form 7 March 1968. droxy-5fi-cholanoic acid. The Journal of Clinical Investigation Volume 47 1968 1573 tural modifications of the bile acids in the intestine of atropine and 1 ml of Ketogin.2 The general anaesthesia lead to the complex mixture of bile acids excreted consisted of sodium hexobarbital, nitrous oxide, succinyl with the feces. The bile acids formed by microbial choline, and fluothane and was initiated 15 min or less action on cholic acid and chenodeoxycholic acid before operation. can be reabsorbed, but of all Preparation of homogenates and analysis of incuba- the different metabo- tions. Liver biopsies (3-5 g) were taken within 15 min lites formed only deoxycholic acid appears to be after the abdomen had been opened and were immediately efficiently reabsorbed, and deoxycholic acid is a put in cold homogenizing medium. Homogenates were major bile acid in many species. Thus, the main prepared within 30 min after the biopsy. The homogenates, bile acids in human bile are cholic acid, cheno- 20% (liver wet weight per volume), were prepared in a modified Bucher medium (5), pH 7.4, using a Potter- deoxycholic acid, and deoxycholic acid. Elvehjem homogenizer equipped with a loosely fitting The mechanisms of the conversion of cholesterol pestle. The homogenate was centrifuged at 800 g for 10 into cholic acid and chenodeoxycholic acid have min and then at 20,000 g for 10 min. The microsomal and been extensively studied in recent years (1). The soluble fractions were obtained by centrifuging the 20,- results of 000 g supernatant fluid at 100,000 g for 2 hr. The microso- investigations on the metabolism of mal fraction was suspended in the same volume of homog- cholesterol and other oxygenated C27 steroids in enizing medium as the volume of the 20,000 g supernatant bile fistula rats and in homogenates of rat liver fluid from which it had been isolated. The labeled com- have led to the elucidation of the major pathways pounds were added to the incubation mixtures dissolved for the formation of bile acids in the rat (1). The in 50 ,ul of acetone, and incubations were run at 37°C. In- cubations with labeled cholesterol were run for 60 min, conversion of cholesterol into bile acids in man has and those with the other substrates for 10 or 20 min. not been studied in any detail. Staple and Rabino- (See legends to figures.) Incubations were terminated witz (2) and Carey (3) have isolated labeled by the addition of 20 volumes of chloroform-methanol 3a,7a,12ca-trihydroxy-5,8-cholestanoic acid from (2:1, v/v). The precipitate was filtered off, and the bile of patients treated with injection of chloroform-methanol solution was washed with 0.2 vol- labeled umes of a 0.9%o (w/v) solution of sodium chloride. cholesterol. Herman, Weinstein, Staple, and Ra- The chloroform phase was collected, and the solvent was binowitz (4) have recently reported the isolation evaporated under reduced pressure. The methanol-saline from human bile of small amounts of 5,8-choles- phase, which in all experiments contained less than 10% tane-3a,7a-diol, another probable intermediate in of the total radioactivity, was discarded. The residue of the conversion of cholesterol into bile acids. the chloroform extract together with appropriate reference These compounds was subjected to thin-layer chromatography results indicate that the pathways for the forma- with Kieselgel G 3 as adsorbent. The solvent systems used tion of bile acids in man are analogous to those in to develop the chromatoplates are given in the legends rat. However, it appeared of interest to attempt to the figures. The internal standards were visualized by to define more closely the sequence of reactions exposing the chromatoplates to iodine vapor. The appropriate zones were scraped into test tubes and were in the conversion of cholesterol into bile acids in extracted with methanol by vigorous stirring. The silica man by studying the metabolism of cholesterol and gel was allowed to settle by gravity, and an aliquot of the cholesterol metabolites in homogenates of human methanol solution was assayed for radioactivity. Further liver. identification of the metabolites formed was done by crys- tallizing representative samples together with authentic METHODS material to constant specific radioactivity. The reference compounds were material synthesized in this laboratory Subjects. The patients in this investigation were: and used in earlier studies in this series of investigations. A.R., female, aged 60; I.-L.J., female, aged 41; and H.S., Labeled compounds. Cholesterol-1,2-3H (specific ra- male, aged 57. One patient (I.-L.J.) was moderately dioactivity, 33 mc/mg) was obtained commercially.4 Be- jaundiced (serum bilirubin, 6.0 mg/100 ml; alkaline phos- fore use, the labeled cholesterol was purified by chroma- phatase, 17 Buch U; thymol turbidity test, 1 Maclagan U; tography on a column of aluminum oxide,5 grade III glutamic oxaloacetic transaminase (GOT), 180 U; glu- activity. Cholest-5-ene-3j9,7a-diol-7,f-3H (specific radioac- tamic pyruvic transaminase (GPT), 480 U). The jaun- tivity, 10 ,c/mg), tritium-labeled 7a-hydroxycholest-4-en- dice disappeared after operation. The results obtained with 3-one (specific radioactivity, 10 ,c/mg), tritium-labeled liver tissue from this patient were not different from those 7a,12a-dihydroxycholest-4-en-3-one (specific radioactivity, obtained with liver tissue from the other two patients. The patients suffered from cholelithiasis. Except for pa- 2 Ketobemidon chloride, 5 mg/ml, and N,N-dimethyl- tient I.-L.J., there was no clinical or laboratory evidence 3,3-diphenyl-1-methylallylamine chloride, 25 mg/ml. for any complication or any other disease, and the re- 3 Merck, Darmstadt, Germany. covery after operation was uneventful. 1 hr before cho- 4 New England Nuclear Corp. Boston, Mass. lecystectomy the patients were premedicated with 0.5 mg 5 Woelm, Eschwege, Germany. 1574 I. Bjorkhem, H. Danielsson, K. Einarsson, and G. Johansson 6 /sc/mg), and tritium-labeled cholest-5-ene-3p,7a,12a-triol the conversion of about 2% of added substrate into (specific radioactivity, 7 ,uc/mg) were prepared as de- more polar products. Experiments were conducted scribed previously (6-8). Radioactivity assay. Radioactivity was assayed with a with liver tissue from each of the three patients, methane gas flow counter. Aliquots of the extracts were and the extent of conversion of added cholesterol plated on aluminum planchettes. Under the conditions of as well as the pattern of products formed were the assay 1 juc of 'H corresponded to 6 X 108 cpm. about the same in all the experiments. Fig. 1 A RESULTS shows a thin-layer chromatogram of the extract of an incubation of cholesterol-1,2-3H with the 20,000 Metabolism of cholesterol-1,2-3H. Incubation g supernatant fluid. Rechromatography of the of cholesterol-1,2-3H with the 20,000 g supernatant material in the most polar zone (1 and 2, Fig. 1 A) fluid and with 800 g supernatant fluid resulted in suggested that the main part of this labeled material (70%) was identical with cholestane- A B C D 3/3,5a,613-triol (2, Fig. 1 B). About 10 % of the material had chromatographic properties of cholest-5-ene-3,8,7a,12a-triol (1, Fig. 1 B). The iden- 3520 tity of the main part of the labeled material with cholestane-3,f,5a,6,3-triol was established by crys- 3320 9C 6440 tallization together with authentic material to con- 1480 so 18.A0 70 6000 TABLE I 1720 1480 160 Identification of Products Formed from Cholesterol-i, 2-3H 50 13,960 20 19.240 No. of 40 4840 crystal- Specific 10 3000 400 lizations Solvent Weight activity 3520 mg cpm/mg 1240880 _ x1o-3 Cholestane-3f3, 5a, 0 18.3 0.58 6,B-triol 1 Methanol-water 11.2 0.72 2 Acetone-water 7.0 0.63 3 Methanol-water 3.7 0.57 FIGURE 1 A, thin-layer chromatogram of chloroform 4 Acetone-water 3.0 0.58 extract of incubation of cholesterol-1,2-3H with 20,000 g supernatant fluid. 4 ml of 20,000 g supernatant fluid, di- 5,B-Cholestane-3a, 0 7.4 0.72 luted with 2 ml of homogenizing medium, were incubated 7a, 12a-triol 1 Methanol-water 7.3 0.62 for 1 hr with lug of cholesterol-1,2-'H. The numbers on 2 Acetone-water 6.3 0.20 the chromatogram represent counts per minute. Reference 3 Methanol-water 4.9 0.13 compounds were: 1, cholest-5-ene-3f8,7a,12a-triol; 2, cho- 4 Acetone-water 4.1 0.12 lestane-3fi,5a,6fi-triol; 3, 5fi-cholestane-3a,7a,12a-triol; 4, 7a, 12a-Dihydroxy- 0 7.7 0.36 7a,12a-dihydroxycholest-4-en-3-one; 5, cholest-5-ene-3#,- cholest-4-en-3-one 1 Methanol-water 7.5 0.35 7a-diol; 6, cholest-5-ene-3#3,7,p-diol; 7, 5,-cholestane-3a,- 2 Acetone-water 6.8 0.30 7a-diol; 8, 3,B-hydroxycholest-5-en-7-one; 9, cholest-5- 3 Methanol-water 4.6 0.32 ene-3fi,26-diol; 10, 7a-hydroxycholest-4-en-3-one; 11, cholesterol. Solvent, benzene-ethyl acetate (3: 7, v/v). B, Cholest-5-ene-305, 0 10.3 1.30 rechromatography of material in the zone corresponding 7a-diol 1 Methanol-water 9.7 1.33 to reference compounds 1 and 2 of the chromatogram 2 Acetone-water 9.4 1.27 shown in A. Reference compounds were the same as in 3 Methanol-water 3.9 1.25 A. Solvent, ethyl acetate. C, rechromatography of material in the zone corresponding to reference compounds 3t3-Hydroxycholest- 0 15.2 0.58 4 and 5 of the chromatogram shown in A. Reference 5-en-7-one 1 Acetone-water 10.5 0.50 compounds were the same as in A. Solvent, benzene-ethyl 2 Methanol-water 8.2 0.42 acetate-trimethylpentane (3: 7: 3, v/v/v). The chromato- 3 Acetone-water 6.2 0.43 gram was run twice in the same solvent with drying of 7a-Hydroxycholest- 0 7.6 2.77 the plate between the runs. D, rechromatography of 4-en-3-one 1 Methanol-water 7.3 2.48 material in the zone corresponding to reference compounds 2 Acetone-water 6.9 1.63 7, 8, and 9 of the chromatogram shown in A. Reference 3 Methanol-water 4.6 1.66 compounds and solvent were the same as in A.

Metabolism of Cholesterol in Homogenates of Human Liver 1575 A B C and cholest-5-ene-3,,7a-diol (4, 5, Fig. 1 A) con- FRONT tained about 0.5 %o of the total radioactivity put 70 S50 61.50 on the chromatoplate. Rechromatography of this labeled material (Fig. 1 C) showed that about 2150 30 143950 2 350 20 %7 of the material had chromatographic proper- 60 42.500 1150 ties of 7a,12a-dihydroxycholest-4-en-3-one (4, Fig. 1550 1 C) and the remainder the properties of cholest- 1500 1300 5-ene-3/3,7a-diol (5, Fig. 1 C). Identification was 50 1500 established by crystallization to constant specific 40 950 30 26.400 4350 radioactivity with authentic material (Table I). 300 80 The labeled material appearing in the zone corre- 3Q 20 22350 sponding to cholest-5-ene-3,3,26-diol, 3,8-hydroxy- 2) 53.550 650 cholest-5-en-7-one, and 5,8-cholestane-3a,7a-diol 650 (7, 8, and 9, Fig. 1 A) was rechromatographed 700 (Fig. 1 D), and about 75 of the labeled material 10 2150 800 % 3900 could be identified as 3,8-hydroxycholest-5-en-7- START 1 750 1500o 650 one (8, Fig. 1 D and Table I). The material in the zone just below that corresponding to cholesterol in the chromatogram shown in Fig. 1 A was FIGURE 2 A, thin-layer chromatogram of chloroform rechromatographed with benzene-ethyl acetate extract of incubation of cholest-5-ene-3#,7a-diol-75--'H (1: 1, v/v) as solvent and with 7a-hydroxycho- with 20,000 g supernatant fluid. 3 ml of 20,000 g superna- as internal stan- tant fluid was incubated for 20 min with 40 ,ug of cholest- lest-4-en-3-one and cholesterol 5-ene-3fi,7a-diol-7fi-'H. The numbers on the chromato- dards. Part of this labeled material could be iden- gram represent counts per minute. Reference compounds tified as c7a-hydroxycholest-4-en-3-one (Table I). were: 1, 5#-cholestane-3ca,7a,12a-triol; 2, 7a 12a-dihy- From these data is was calculated that about 0.1 % droxycholest-4-en-3-one; 3, cholest-5-ene-3#,7a-diol; 4, of added cholesterol had been converted into 5,6-cholestane-3a,7a-diol; 5, 7a,12a-dihydroxy-5,B-cholestan-3-one; 6, 7a-hydroxycholest-4-en-3-one; 7, 7a-hy- 7a-hydroxycholest-4-en-3-one. droxy-53-cholestan-3-one. Solvent, benzene-ethyl acetate Metabolism of cholest-5-ene-33,Z7a-diol-7,/-3H. (1: 1, v/v). B, rechromatography of material in the Cholest-5-ene-3,8,7a-diol-7,8-3H was metabolized zone corresponding to reference compounds 2 and 3 of efficiently in the presence of 20,000 g supernatant the chromatogram shown in A. Reference compounds were the same as in A. Solvent, benzene-ethyl acetate- fluid into compounds with chromatographic prop- trimethylpentane (3: 7: 3, v/v/v). The chromatogram erties of 7a-hydroxycholest-4-en-3-one (6, Fig. was run twice in the same solvent with drying of the plate 2A) and 7a,12a-dihydroxycholest-4-en-3-one (2, between the runs. C, thin-layer chromatogram of chloroform extract of incubation of cholest-5-ene-3p,7a-diol- 7p-'H with microsomal fraction fortified with NADPH. TABLE I I 1 ml of microsomal fraction was diluted with 2 ml of ho- Identification of 7a-Hydroxycholest-4-en-3-one Formed mogenizing medium, fortified with 3 ,umoles of NADPH, from Cholest-5-ene-3f3, 7a-diol-7,3-3H and was incubated for 10 min with 40,ug of cholest-5-ene- 3j8,7a-diol-7p8-'H. The numbers on the chromatogram No. of Source of labeled crystal- Specific represent counts per minute. Reference compounds were: compound lizations Solvent Weight activity 3, cholest-5-ene-3,8,7a-diol; 8, cholest-5-ene-3#,7a,12a- triol. Solvent, ethyl acetate. mg cpm/mg X10-3 20,000 g supernatant 0 8.3 1.28 stant specific radioactivity (Table I). The labeled 1 Acetone-water 6.5 1.50 material in the zone corresponding to 5,8-choles- 2 Methanol-water 4.6 1.43 tane-3a,7a,12a-triol (3, Fig. 1 A) was crystallized 3 Acetone-water 4.2 1.57 together with authentic material (Table I). About Microsomes+NAD+ 0 9.6 1.67 15 % of the labeled material could be accounted 1 Acetone-water 8.1 1.83 for as The zone cor- 2 Methanol-water 7.0 1.73 5,8-cholestane-3a,7a,12a-triol. 3 Acetone-water 6.2 1.77 responding to 7a,12a-dihydroxycholest-4-en-3-one 1576 I. Bjorkhem, H. Danielsson, K. Einarsson, and G. Johansson A B C 7a-diol into 7a-hydroxycholest-4-en-3-one (2, Fig. FRONT;- i I 3 C and Table II). In one experiment, the microsomal fraction was fortified by the addition of I 1 oso 1100 950 NADPH resulting in the formation of small amounts of labeled material with the chromato- 20 2135 3450 21,800 A B C D FRONT ISoo 950 3900 3550 2300 60 1450 60 5600 6800 3 950 4350 41050

50 51.300 50 70,100 so 117,250 62,100 10 132.250 131.250 93.0 2 200 1900 10 1750 550 5125 6300 2400 30 850 30 7800 1100 400 900 1450 1800 1000 400 20 41.700 20 600 30000 14.75 150 500 _ START 950 2100 10 600 10 3050 1550 FIGURE 3 A, thin-layer chromatogram of chloroform extract of incubation of cholest-5-ene-3fi,7a-diol-7fi-3H 31450 with microsomal fraction. 1 ml of microsomal fraction was diluted with 2 ml of homogenizing medium and was START 2,950 21450 1 300 incubated with 40 14g of cholest-5-ene-3#,7a-diol-7fi-8H for 20 min. The numbers on the chromatogram represent counts 'per minute. Reference compounds were: 1, cholest- FIGURE 4 A, thin-layer chromatogram of chloroform 5-ene-3fi,7a-diol; 2, 7a-hydroxycholest-4-en-3-one. Solvent, extract of incubation of tritium-labeled 7a-hydroxycho- benzene-ethyl acetate (1: 1, v/v). B, thin-layer chro- lest-4-en-3-one with 20,000 g supernatant fluid. 3 ml of matogram of chloroform extract of incubation of cholest- 20,000 g supernatant fluid was incubated for 20 min with 5-ene-3,8,7a-diol-7#-3H with microsomal fraction fortified 40 gg of tritium-labeled 7a-hydroxycholest-4-en-3-one. with NADP'. 1 ml of microsomal fraction was diluted The numbers on the chromatogram represent counts per with 2 ml of homogenizing medium, fortified with 0.3 minute. Reference compounds were: 1, 5,8-cholestane- Mtmoles of NADP+ and was incubated for 20 min with 40 3a,7a,12a-triol; 2, 7a,12a-dibydroxycholest-4-en-3-one; 3, ,ug of cholest-5-ene-3,8,7a-diol-7f8-8H. Reference compounds 5,B-cholestane-3a,7a-diol; 4, 7a,12a-dihydroxy-5fi-choles- and chromatographic conditions were the same as in A. tan-3-one; 5, 7a-hydroxycholest-4-en-3-one; 6, 7a-hy- C, thin-layer chromatogram of chloroform extract of droxy-5,-cholestane-3-one. Solvent, benzene-ethyl acetate incubation of cholest-5-ene-3p,7a-diol-7p-3H with micro- (3: 7, v/v). B, thin-layer chromatogram of chloroform somal fraction fortified with NAD+. 1 ml of microsomal extract of incubation of tritium-labeled 7a-hydroxycho- fraction was diluted with 2 ml of homogenizing medium, lest4-en-3-one with 100,000 g supernatant fluid. 3 ml of fortified with 0.3 ,umoles of NAD+ and was incubated for 100,000 g supernatant fluid were incubated for 20 min 20 min with 40 jg of cholest-5-ene-3,3,7a-diol-7,8-8H. with 40 ,ug of tritium-labeled 7a-hydroxycholest-4-en-3- Reference compounds and chromatographic conditions one. Reference compounds and chromatographic condi- were the same as in A. tions were the same as in A. C, thin-layer chromatogram of chloroform extract of incubation of tritium-la- Fig. 2 B). The identity of the labeled 7a-hydroxy- beled 7a-hydroxycholest4-en-3-one with microsomal fraction. 3 ml of microsomal fraction was incubated for cholest-4-en-3-one formed was established by crys- 10 min with 40 ,ug of tritium-labeled 7a-hydroxycholest- tallization to constant specific radioactivity with 4-en-3-one. Reference compounds and chromatographic authentic material (Table II). In the presence of conditions were the same as in A. D, thin-layer chro- microsomal fraction, or microsomal fraction forti- matogram of chloroform extract of incubation of tritium- fied by the addition of NADP+, cholest-5-ene- labeled 7a-hydroxycholest-4-en-3-one with microsomal fraction fortified with NADPH. 3 ml of microsomal was metabolized only to a small 3,3,7a-diol-7j8-3H fraction was fortified with 3 of NADPH and was 3 A ,umoles extent (Fig. and B). Addition of NAD+ to incubated for 10 min with 40 Mg of tritium-labeled 7a-hy- the microsomal fraction resulted in a marked droxycholest4-en-3-one. Reference compounds and chro- stimulation of the conversion of cholest-5-ene-3,8, matographic conditions were the same as in A.

Metabolism of Cholesterol in Homogenates of Human Liver 1577 TABLE I I I Identification of Products formed from Tritium-Labeled 7a-Hydroxycholest-4-en-3-one

No. of Source of labeled crystalli- Specific compound Compound zations Solvent Weight activity

mg cpmllmg X 10-3 20,000 g supernatant 7a, 1 2a-Dihydroxycholest-4-en-3-one 0 11.7 2.94 1 Acetone-water 9.5 2.76 2 Methanol-water 7.7 2.73 3 Acetone-water 5.3 2.81

Microsomes 7a, 12a- Dihydroxycholest-4-en-3-one 0 11.2 1.22 + NADPH 1 Acetone-water 10.0 1.06 2 Methanol-water 7.0 1.07 3 Acetone-water 3.3 1.05

100,000 g supernatant 7a-Hydroxy-5i3-cholestan-3-one 0 9.9 0.21 1 Acetone-water 8.5 0.21 2 Methanol-water 6.9 0.24 3 Acetone-water 5.4 0.21

100,000 g supernatant 5,3-Cholestane-3a,7a-diol 0 12.6 0.41 1 Acetone-water 11.8 0.39 2 Acetone-water 4.7 0.36 3 Acetone-water 3.2 0.35

FRONT graphic properties of cholest-5-ene-3,8,7a,12a-triol (8, Fig. 2 C). Metabolism of tritium-labeled 7a-hydroxycho- 900 lest-4-en-3-one. As shown in Fig. 4 A, the main metabolite formed from tritium-labeled 7a-hy- 30 13,750 droxycholest-4-en-3-one in the presence of 20,000 g supernatant fluid was 7a-12a-dihydroxycholest- 2050 4-en-3-one (compound 2). The formation of this compound was also catalyzed efficiently by the 20 11 1.750 microsomal fraction fortified by the addition of NADPH (2, Fig. 4C and D, and Table III). 450 After incubation of tritium-labeled 7a-hydroxy- 10 13,100 TABLE IV Identification of Products Formed from Tritium-Labeled 600 7a, 12a-Dihydroxycholest-4-en-3-one

850 No. of START - crystal- Specific lizations Solvent Weight activity mg cpm/mg FIGURE 5 Thin-layer chromatogram of chloroform ex- X10- tract of incubation of tritium-labeled 7a,12a-dihydroxy- 7a, 12a-Dihydroxy- 0 11.5 0.53 1 Acetone-water 8.7 0.43 cholest-4-en-3-one with 100,000 g supernatant fluid. 3 ml 5#3-cholestan-3-one 2 Methanol-water 7.6 0.43 of 100,000 g supernatant fluid were incubated for 20 min 3 Acetone-water 6.0 0.45 with 40 lsg of tritium-labeled 7a,12a-dihydroxycholest-4- en-3-one. The numbers on the chromatogram represent 503-Cholestane-3a, 0 13.1 0.74 counts per minute. Reference compounds were: 1, 5,8-cho- 7a, 12a-triol 1 Acetone-water 11.3 0.75 lestane-3a,7a,12a-triol; 2, 7a,12a-dihydroxycholest-4-en- 2 Methanol-water 9.1 0.74 3-one; 3, 7a,12a-dihydroxy-5,8-cholestan-3-one. Solvent, 3 Acetone-water 8.3 0.80 benzene-ethyl acetate (3: 7, v/v).

1578 I. Bjirkhem, H. Danielsson, K. Einarsson, and G. Johansson A B C D TABLE V Identification of 7a, 12a-Dihydroxycholest-4-en-3-one Formed 7a, 12a-triol so 150 0 0 from Tritium-Labeled Cholest-5-ene-3f,

4 o 500 350 1000 750 No. of 150 0 450 350 Source of labeled crystal- Specific 3 O8800 250 1850 17,200 compound lizations Solvent Weight activity

800 200 650 900 mg cpm/mg X103 450 300 2'Q 500 650 20,000 g supernatant 0 13.7 0.55 1000 550 1050 400 1 Methanol-water 10.7 0.52 2 Acetone-water 8.6 0.51 0o 46850 56,900 61,550 45,900 3 Methanol-water 6.5 0.47 14.2 0.68 1800 2450 2100 650 Microsomes+NAD+ 0 1 Acetone-water 8.5 0.75 2 Methanol-water 7.2 0.65 1950 700 850 900 3 Acetone-water 6.5 0.77

200 700 850_ STA ,RT 200 Metabolism of tritium-labeled 7a,12a-dihydroxycholest-4-en-3-one. Incubation of tritium-labeled FIGURE 6 A, thin-layer chromatogram of chloroform 7a,12a-dihydroxycholest4-en-3-one with the cholest-5-ene-3,B,- extract of incubation of tritium-labeled 100,000 g supernatant fluid yielded two main fluid. 1 ml of 7a,12a-triol with 20,000 g supernatant 20,000 products, 7a,12a-dihydroxy-5,8-cholestan-3-one and fluid was diluted with 2 ml of homogeniz- g supernatant 1, Fig. 5 ing medium and was incubated for 20 min with 40 Ag of 5,8-cholestane-3a,7a,12a-triol (3 and tritium-labeled cholest-5-ene-3,B,7a,12a-triol. The numbers and Table IV). on the chromatogram represent counts per minute. Refer- Metabolism of tritium-labeled cholest-5-ene-3,8, ence compounds were: 1, cholest-5-ene-3#,7a,12a-triol; 2, 7a,12a-triol. Upon incubation of tritium-labeled 5p-cholestane-3a,7a,12a-triol; 3, 7a,12a-dihydroxycholest- with the 20,000 g 4-en-3-one; 4, 7a,12a-dihydroxy-5P-cholestan-3-one. Sol- cholest-5-ene-3,8,7a,12a-triol vent, ethyl acetate. B, thin-layer chromatogram of supernatant fluid, the added substrate was con- chloroform extract of incubation of tritium-labeled cho- verted efficiently into 7a,12a-dihydroxycholest-4- lest-5-ene-3,B,7a,12a-triol with microsomal fraction. 1 ml en-3-one (3, Fig. 6 A and Table V). The substrate was diluted with 2 ml of homog- of microsomal fraction was also incubated with the microsomal fraction enizing medium and was incubated for 20 min with 40 ,ug fraction fortified of tritium-labeled cholest-5-ene-3p,7a,12a-triol. Reference alone and with the microsomal compounds and chromatographic conditions were the by the addition of NADP+ or NAD+. Without any same as in A. C, thin-layer chromatogram of chloro- addition to the microsomal fraction, no conversion form extract of incubation of tritium-labeled cholest-5- of the substrate was observed (Fig. 6 B), and the with microsomal fraction fortified with ene-3,B,7a,12a-triol addition of NADP+ had only a slight stimulatory was diluted with 2 NADP+. 1 ml of microsomal fraction of NAD+ ml of homogenizing medium, fortified with 0.3 ,umoles effect (Fig. 6 C), whereas the addition of NADP+ and was incubated for 20 min with 40 ,ug of resulted in an efficient conversion of the substrate tritium-labeled cholest-5-ene-3,,7a,12a-triol. Reference into 7a,12a-dihydroxycholest-4-en-3-one (3, Fig compounds and chromatographic conditions were the 6D and Table V). same as in A. D, thin-layer chromatogram of chloroform extract of incubation of tritium-labeled cholest-5- DISCUSSION ene-3,B,7a,12a-triol with microsomal fraction fortified with NAD+. 1 ml of microsomal fraction was diluted with 2 ml The major pathways for the conversion of choles- of homogenizing medium, fortified with 0.3 ,umoles of terol into cholic acid and chenodeoxycholic acid in NAD+ and incubated for 20 min with 40 /Ag of tritium- the rat have now been elucidated (1). Fig. 7 sum- Reference compounds labeled cholest-5-ene-3,,7a,12a-triol. marizes the main sequences of reactions in the were the same as in A. and chromatographic conditions biosynthesis of cholic acid and chenodeoxycholic indicates the existence of cholest-4-en-3-one with the 100,000 g supernatant acid. Present information fluid the main products formed were 7a-hydroxy- several pathways for the formation of cholic acid 5,8-cholestan-3-one and 5/3-cholestane-3a,7a-diol as well as chenodeoxycholic acid (1), but the (6 and 3, Fig. 4B and Table III). quantitative importance of the various pathways Metabolism of Cholesterol in Homogenates of Human Liver 1579 20000 9 (2) HO 4~~~~~~~~~ HO 'OH

MICJNAD

HO-C HO 'OH

MIC NAD HO a ~~~100,000 9jNADPH ON~ ~~ O o(S~~~() (6) 0 OH

100.000 g NADPH 100.000 9JNADPH +HO

o ( 7~~~() HO' ~ OH

100.000 g NADPH HO'J COH H +HO

FIGURE 7 The biological formation of cho- (9) (10) HO, -OH HO' 'OH lic acid, chenodeoxycholic acid, and deoxycholic acid from cholesterol. Abbreviations: 20,000 g, 20,000 g supernatant fluid; mic, microsomal fraction. Compounds: 1, cholesterol; 2, cholest-5-ene-3j3,7a-diol; 3, cholest- 5-ene-3,8,7a,12a-triol; 4, 7a-hydroxycholest- 4-en-3-one; 5, 7a,12a-dihydroxycholest-4-en- 3-one; 6, 7a-hydroxy-5#-cholestan-3-one; 7, 7ca,12a-dihydroxy-5,8-cholestane-3-one; 8, 5X8- cholestane-3a,7a-diol; 9, 5j3-cholestane-3a,7a,- HO HO 12a-triol; 10, 3a,7a-dihydroxy-5,8-cholesta- rN4OOH rLAPi~OOH noic acid; 11, 3a,7a,12a-trihydroxy-5fi-cho- INTESTINE l 1 (4) lestanoic acid; 12, chenodeoxycholic acid; 13, HO' "'OH AO^>9 HO' cholic acid; 14, deoxycholic acid. has not been finally established. Many of the indi- that the sequence of reactions in the conversion vidual reactions have been studied in detail in the of cholesterol into 5,3-cholestane-3a,7a,12a-triol rat, and some of the enzymes catalyzing these reac- and 5,/-cholestane-3a,7a-diol is the same in ho- tions have been partially characterized. The aim mogenates of human liver as in rat liver homoge- of the present investigation was to study in detail nates (6, 7, 9-14). The subcellular distribution as the conversion of cholesterol into 5,8-cholestane- well as the cofactor requirements of the enzymes 3a,7a,12a-triol in man in order to provide a basis catalyzing these reactions were also found to be for further studies of factors influencing the forma- the same. (See Fig. 7.) tion of bile acids in man. It was thus considered of In the presence of 20,000 g supernatant fluid of importance to identify as many of the intermediary homogenates of human liver, cholesterol (1, Fig. products as possible and to obtain information on 7) was converted into cholest-5-ene-3,8,7a-diol (2, the subcellular localization of the enzymes cata- Fig. 7), 7a-hydroxycholest-4-en-3-one (4, Fig. 7), lyzing the reactions. The results obtained show 7a,12a-dihydroxycholest-4-en-3-one (5, Fig. 7), 1580 I. Bjorkhem, H. Danielsson, K. Einarsson, and G. Johansson and 5/8-cholestane-3a,7a,12a-triol (9, Fig. 7). In tion by the microsomal fraction of the A4-3-keto- addition, small amounts of cholestane-3,8,5a,6,8- steroid 5,8-reductase(s) catalyzing the saturation triol and 3,8-hydroxycholest-5-en-7-one were of the z4 double bond in 7a-hydroxycholest-4-en-3- formed. These compounds are well known autoxi- one and 7a,12a-dihydroxycholest-4-en-3-one (17). dation products of cholesterol (15), but at least It is possible that a similar inhibition occurs also 3,8-hydroxvcholest-5-en-7-one can be formed also in homogenates of human liver. by enzymatic action on cholesterol (16). The No studies of the further metabolism of 5,8- experiments do not permit an evaluation of the cholestane-3ac,7a-diol (8, Fig. 7) and 5,8-choles- extent of enzymatic formation of 3ft-hydroxycho- tane-3a,7a,12a-triol (9, Fig. 7) were performed lest-5-en-7-one. This compound is not an inter- in the present investigation. As shown in Fig. 7, mediate in the biosynthesis of cholic acid or cheno- these compounds are in the rat the substrates for deoxycholic acid (16), and its metabolic role has the enzyme(s) catalyzing the 26-hydroxylation, not been established. Evidence has been obtained the initial reaction in the degradation of the C27 to indicate that the enzymatic formation of 3,3- side chain in bile acid formation (1). It is probable hydroxycholest-5-en-7-one increases concomitant that 5,8-cholestane-3a,7a-diol and 5,8-cholestane- with a decrease in the formation of cholest-5-ene- 3a,7a,12a-triol are metabolized in the same way 3,8,7a-diol (16). The enzyme catalyzing the 7a- in man. hydroxylation of cholesterol has been found to be easily inactivated (16), and it is possible that the ACKNOWLEDGMENTS anaesthesia of the patients as well as the time lag The authors wish to express their sincere gratitude to in the preparation of the homogenates might have Dr. Olof Johansson, Department of Surgery I, Soder- an on this enzyme system. sjukhuset, Stockholm, for generous help in supplying the had influence liver specimens. The skillful technical assistance of Miss The further metabolism of cholest-5- ene-3,8,7a- Madelaine Ekebrand and Miss Britt-Marie Gravander is diol, 7a-hydroxycholest-4-en-3-one and 7a,12a-di- gratefully acknowledged. hydroxycholest-4-en-3-one in homogenates of This work is part of investigations supported by the human liver was completely analogous to the me- Swedish Medical Research Council (Project 13X-218) tabolism of these compounds in rat liver homoge- and by the Loo and Hans Ostermans Foundation. nates (6. 7, 12-14). Cholest-5-ene-3,8,7a-diol (2, REFERENCES converted into Fig. 7) was readily 7a-hydroxycho- 1. Danielsson, H., and T. T. Tchen. 1968. Steroid lest-4-en-3-one (4, Fig. 7) in the presence of metabolism. In Metabolic Pathways. D. M. Green- microsomal fraction fortified with NAD+. In the berg, editor. Academic Press Inc., New York, 3rd presence of microsomal fraction fortified with edition. 2:117. NADPH cholest-5-ene-3,8,7a-diol (2, Fig. 7) was 2. Staple, E., and J. L. Rabinowitz. 1962. Formation of converted only to a limited extent into cholest-5- trihydroxycoprostanic acid from cholesterol in man. Biochim. Biophys. Acta. 59: 735. This contrasts with ene-3,8,7a,12a-triol (3, Fig. 7). 3. Carey, J. B., Jr. 1964. Conversion of cholesterol to the efficient 12a-hydroxylation of 7a-hydroxycho- trihydroxycoprostanic acid and cholic acid in man. lest-4-en-3-one (4, Fig. 7) in the same system J. Clin. Invest. 43: 1443. indicating that this compound is the major sub- 4. Rabinowitz, J. L., R. H. Herman, D. Weinstein, and strate for the 12a-hydroxylase in cholic acid for- E. Staple. 1966. Isolation of 3a,7a-dihydroxycopro- man well as in the rat Soluble stane derived from cholesterol in human bile. Arch. mation in as (6). Biochem. Biophys. 114: 233. enzymes wAere found to catalyze the conversion 5. Bergstrom, S., and U. Gloor. 1955. Studies on the of 7a-hydroxycholest-4-en-3-one (4, Fig. 7) into 7a-hydroxylation of taurodeoxycholic acid in rat liver 5,3-cholestane-3a,7a-diol (8, Fig. 7) and of 7a, homogenates. Acta Chem. Scand. 9: 34. 12a-dihydroxycholest-4-en-3-one (5, Fig. 7) into 6. Danielsson, H., and K. Einarsson. 1966. On the con- 5,8-cholestane-3a,7a,12a-triol (9, Fig. 7) by means version of cholesterol to 7a,12a-dihydroxycholest-4- of the en-3-one. Bile acids and steroids 168. J. Biol. Chem. of the intermediary formation corresponding 241: 1449. 3-keto-5,8-steroids. It might be added that these 7. Berseus, O., H. Danielsson, and A. Kallner. 1965. reactions occurred only to a limited extent in Synthesis and metabolism of cholest-4-ene-7a,12a- incubations with the 20,000 g supernatant fluid. diol-3-one and 5,B-cholestane-7a,12a-diol-3-one. J. Biol. Evidence has been obtained to indicate an inhibi- Chem. 240: 2396. Metabolism of Cholesterol in Homogenates of Human Liver 1581 8. Bjbrkhem, I., H. Danielsson, C. Issidorides, and A. 13. Berseus, O., and K. Einarsson. 1967. On the conver- Kallner. 1965. On the synthesis and metabolism of sion of cholest-5-ene-3fi,7a-diol to 7a-hydroxycholest- cholest-4-en-7a-ol-3-one. Bile acids and steroids 4-en-3-one in rat liver homogenates. Bile acids and 156. Acta Chem. Scand. 19: 2151. steroids 186. Acta Chem. Scand. 21: 1105. 9. Mendelsohn, D., and E. Staple. 1963. The in vitro 14. Berseus, O., H. Danielsson, and K. Einarsson. 1967. catabolism of cholesterol: formation of 3a,7a,12a- Synthesis and metabolism of cholest-5-ene-3#,7a,12a- trihydroxycoprostane from cholesterol in rat liver. triol. Bile acids and steroids 169. J. Biol. Chem. 242: Biochemistry. 2: 577. 1211. 10. Mendelsohn, D., L. Mendelsohn, and E. Staple. 1965. 15. Danielsson, H. 1960. On the oxidation of cholesterol The in vitro catabolism of cholesterol: formation of in liver mitochondrial preparations. Acta Chem. Scand. 5#-cholestane-3a,7a-diol and 5fi-cholestane-3a,12a-diol 14: 846. from cholesterol in rat liver. Biochemistry. 4: 441. 16. Bj6rkhem, I., K. Einarsson, and G. Johansson. For- 11. Mendelsohn, D., L. Mendelsohn, and E. Staple. 1966. mation and metabolism of of cholesterol: a 3#-hydroxycholest-5-en-7- The in vitro catabolism comparison Acta Chem. Scand. of the formation of cholest-4-en-7a-ol-3-one and one and cholest-5-ene-3fi-7,8-diol. 5j#-cholestan-7a-ol-3-one from cholesterol in rat liver. In press. Biochemistry. 5: 1286. 17. Bjbrkhem, I., H. Danielsson, and K. Einarsson. 1967. 12. Hutton, H. R. B., and G. S. Boyd. 1966. The metabo- On the conversion of cholesterol to 5#-cholestane- lism of cholest-5-en-3fi,7a-diol by rat-liver cell frac- 3a,7a-diol in guinea pig liver homogenates. European tions. Biochim. Biophys. Acta. 116: 336. J. Biochem. 2: 294.

1582 1. Bkirkhem, H. Danielsson, K. Einarsson, and G. Johansson