On the Conversion of Cholestanol into Allocholic Acid in Rat Liver

Abstract

The early steps in the conversion of cholestanol into allocholic acid were studied in homogenates of rat liver. The 2 0 0 0 0~g supernatant fluid was found to catalyze 7a-hydroxylation of cholestanol. Under the same conditions 7a-hydroxylation of 56-cholestan-3a-01 and 5a-cholestan%one was insignificant. The microsomal fraction fortified with NAD catalyzed oxidation of 5a-cholestane-3/l,7a-diol into 7a-hydroxy-5a-cholestan-3-one. The 100000 x g supernatant fluid catalyzed reduction of 7a-hydroxy-5a-cholestan-3-one into 5a-cholestane-3a,7a-diol. The same reaction was effected by a partially purified 3a-hydroxysteroid dehydrogenase from rat liver in the presence of NADPH. The microsomal fraction fortified with NADPH catalyzed 12a-hydroxylation of 5a-cholestane-3a,7a-diol at a rate about six times faster than that with either 5a-cholestane-3@,7a-diol or 7a-hydroxy-5a-cholestan-3-one. In rats with a biliary fistula 5a-cholestane-3@,7a-diol and 5a-cholestane-3a,7a-diol were converted into allocholic acid. The results indicate that the main pathway for the conversion of cholestanol into allocholic acid involves the following steps : cholestanol +-5a-cholestane-3@,7a-diol -+ 7a-hydroxy-5a-cholestan-3-one +-5a-cholestane-3n,7a-diol -+ 5a-cholestane-3a,7a,12a-triol+ allocholic acid.5or-Bile acids are major bile acids in some lower species and may occur in small amounts in mammals [1, 21. 5P-Bile acids, cholester61 and cholestanol have been shown to be precursors of 5cc-bile acids in mammals. The conversion of 5p-bile acids into 5or-bile acids is effected by intestinal microorganisms in reactions probably involving A4-3-oxosteroids as intermediates [3]. The conversion of cholesterol into 5@-bile acids in the rat involves the intermediary formation of A4-3-oxosteroids. These Ad-3-oxosteroids, i.e. 7a-hydroxy-4-cholesten-3-one and 7a,12a-dihydroxy-4-cholesten-3-one have been shown to be converted to some extent into the corresponding 3-0x0-5a-steroids in rat liver microsomes fortified with NADPH [4]. These 3-0x0-5a-steroids are converted into allocholic acid in rats with a biliary fistula [4]. A similar pathway from cholesterol to allocholic acid has been shown in the lizard, Iguana iguana [2], a species having allocholic acid as the main bile acid. Karavolas et al. [5] and Ziller, Doisy and Elliot [6] have shown that cholestanol is converted into allochenodeoxycholic acid and allocholic acid in rats with a biliary fistula. Hofmann and Mosbach [7] and Hofmann et al. [8] have found that also in the rabbit cholestanol is converted into alloNomenclature. The following systematic names are given to compounds referred to by trivial names: cholestanol, 5a-cholestan-3~-01; cholic acid, 3a,7a,12a-trihydroxy-5p-cholanoic acid; allocholic acid, 3a,7a,lZa-trihydroxy-5a-cholanoic acid; chenodeoxycholic acid, 3a,7a-dihydroxy-5p-cholanoic acid; allochenodeoxycholic acid, 3a,7a-dihydroxy5a-cholanoic acid.cholic acid. The sequence of reactions in the conversion of cholestanol into allocholic acid is largely unknown. Shefer, Hauser and Mosb...

Mechanism of Microbial Transformation of Cholesterol into Coprostanol

“…Unchanged cholesterol was isolated from the incubation mixture and was found to have a 3H/14C ratio which was 1.4 times that of the original cholesterol ( Table 1). The extent of conversion of cholesterol into coprostanol was about 50°/, and the increase in 3H/14C ratio of unchanged cholesterol was calculated to correspond to a kinetic isotope effect of about 2 in the over-all reaction, provided the reaction follows zero-or first-order kinetics [15]. The presence of an isotope effect of this order of magnitude indicates that breaking of the C-H bond a t C-3 plays a role in determining the rate of the conversion of cholesterol into coprostanol.…”

On the Rate-Limiting Step in Microsomal Hydroxylation of Steroids

“…The general technique used in the present work to show possible isotope effects in the hydroxylations of the specifically deuterium-labeled steroids was the same as that used previously [5,6] were not accompanied by significant isotope effect, thus showing that breaking of the C-H bond in the substrate is not rate-limiting in these hydroxylations.…”

“…I n recent work from this laboratory [5,6], it was suggested that breaking of the C-H bond in the substrate might be a rate-limiting step in the microsomal hydroxylation of some steroids and fatty acids. It was shown that 7a-hydroxylation of [7a-3H]-plus [24-14C]-labeled taurodeoxycholic acid and 9-hydroxylation of [9-2H]decanoic acid were accompanied by a kinetic isotope effect of such magnitude that it was likely that splitting of the C-3H and C 2 H bonds repectively was rate-limiting.…”

“…'la-Hydroxylation of taurodeoxycholic acid is relatively insensitive towards carbon monoxide which could indicate that 'In-hydroxylation of taurodeoxycholic acid is independent of cytochrome P-450 (cf. [ 5 ] ) . However, there are spectral data that suggest that taurodeoxycholic acid is a substrate for a cytochrome-P-450-containing system [35].…”

“…Liver homogenate, ZOO/, (w/v) was prepared in a modified Bucher medium and the isolation of the 20 000 x g supernatant fluid and the microsomal fraction was performed as described previously [5]. I n the case of experiments with deuterated water, the microsomes were suspended in a modified Bucher medium prepared in deuterated water (99.8O/, pure, Norsk Hydro).…”