24-Hydroxycholesterol is a substrate for hepatic cholesterol 7α-hydroxylase (CYP7A)

Maria Norlin,Anders Toll,Ingemar Björkhem,Kjell Wikvall

doi:10.1016/s0022-2275(20)31996-9

Abstract

(24S)-Hydroxycholesterol is formed from cholesterol in the brain and is important for cholesterol homeostasis in this organ. Elimination of (24S)-hydroxycholesterol has been suggested to occur in the liver but little is known about the metabolism of this oxysterol. In the present investigation, we report formation of 7α,24-dihydroxycholesterol in pig and human liver. 7α-hydroxylase activity toward both isomers of 24-hydroxycholesterol [(24S) and (24R)] was found in a partially purified and reconstituted cholesterol 7α-hydroxylase (CYP7A) enzyme fraction from pig liver microsomes. In contrast, a purified enzyme fraction of pig liver oxysterol 7α-hydroxylase with high activity toward 27-hydroxycholesterol did not show any detectable activity toward 24-hydroxycholesterol. 7α-Hydroxylation of 24-hydroxycholesterol was strongly inhibited by 7-oxocholesterol, a known inhibitor of CYP7A. Human CYP7A, recombinantly expressed in Escherichia coli and in simian COS cells, showed 7α-hydroxylase activity toward both cholesterol and the two isomers of 24-hydroxycholesterol, with a preference for the (24S)-isomer. Our results show that 24-hydroxycholesterol is metabolized by CYP7A, an enzyme previously considered to be specific for cholesterol and cholestanol and not active toward oxysterols. Because CYP7A is the rate-limiting enzyme in the major pathway of bile acid biosynthesis, the possibility is discussed that at least part of the 24-hydroxycholesterol is converted into 7α-hydroxylated bile acids by the enzymes involved in the normal biosynthesis of bile acids.—Norlin, M., A. Toll, I. Björkhem, and K. Wikvall. 24-Hydroxycholesterol is a substrate for hepatic cholesterol 7α-hydroxylase (CYP7A). J. Lipid Res. 2000. 41: 1629–1639.

Highlights

Abstract (24S)-Hydroxycholesterol is formed from cholesterol in the brain and is important for cholesterol homeostasis in this organ
Previous work has demonstrated the existence of two cytochrome P-450 enzymes catalyzing 7␣hydroxylation in the different pathways of bile acid biosynthesis, one that is active toward cholesterol called the cholesterol 7␣-hydroxylase (CYP7A) and another that is active toward 27-hydroxycholesterol, referred to as the oxysterol 7␣-hydroxylase (5 –11)
Because the studies with purified enzyme fractions from pig liver indicated that the enzyme responsible for 7␣hydroxylation of 24-hydroxycholesterol might be the CYP7A, we found it of interest to test this hypothesis in experiments with recombinantly expressed human CYP7A

Summary

EXPERIMENTAL PROCEDURES

DEAE-Sepharose CL6B, S-Sepharose Fast Flow, and Q-Sepharose Fast Flow were purchased from Pharmacia (Uppsala, Sweden) and hydroxylapatite (Macroprep ceramic HTP) was purchased from Bio-Rad (Hercules, CA). Cytochrome P-450 catalyzing the 7␣-hydroxylation of 27hydroxycholesterol was prepared from pig liver microsomes as previously described by Norlin and Wikvall [11]. Cytochrome P-450 catalyzing the 7␣-hydroxylation of cholesterol was prepared from pig liver microsomes according to the methods described by Toll et al [7]. This cytochrome P-450 fraction showed several bands on SDS-PAGE and contained 1 –2 nmol of cytochrome P-450 per mg of protein. Eluted enzyme fractions were pooled and dialyzed against 100 mm phosphate buffer, pH 7.4, containing 0.2% POEL, 20% glycerol, 0.1 mm EDTA, 0.1 mm DTT, and 0.25 mm PMSF prior to assay of catalytic activity. Microsomes prepared from cells transfected with pSVL vector containing CYP7A cDNA and cells transfected with vector alone were incubated as described below

Incubation procedures

RESULTS

Findings

DISCUSSION