Journal Article Biochimica et Biophysica Acta Get access Sune BergstrÖM, Sune BergstrÖM Department of Chemistry, Karolinska Institutet, Stockholm (Sweden) Search for other works by this author on: Oxford Academic PubMed Google Scholar Henry Danielsson, Henry Danielsson Department of Chemistry, Karolinska Institutet, Stockholm (Sweden) Search for other works by this author on: Oxford Academic PubMed Google Scholar Bengt Samuelsson Bengt Samuelsson Department of Chemistry, Karolinska Institutet, Stockholm (Sweden) Search for other works by this author on: Oxford Academic PubMed Google Scholar Nutrition Reviews, Volume 39, Issue 8, August 1981, Pages 313–315, https://doi.org/10.1111/j.1753-4887.1981.tb06800.x Published: 01 August 1981

Modulation of cholesterol 7 alpha-hydroxylase activity was studied in a purified, reconstituted system from rat liver microsomes. Cysteine, dithiothreitol, reduced glutathione, and thioredoxin activated the system whereas glutathione disulfide inactivated it. A protein, which stimulated cholesterol 7 alpha-hydroxylase activity in the presence of glutathione or thioredoxin, was purified to apparent homogeneity from rat liver cytosol. It has a minimum Mr of 25,000. The protein had no effect on 12 alpha-hydroxylation of 7 alpha-hydroxy-4-cholesten-3-one or 25-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol. The cholesterol 7 alpha-hydroxylase stimulatory protein could not be replaced by the thioltransferase-dependent disulfide-reducing system nor by glutathione S-transferase A, B, or C. Neither ATP and MgCl2 nor sodium fluoride had any effect on the activity of the cholesterol 7 alpha-hydroxylase stimulatory protein. The results show that purified cholesterol 7 alpha-hydroxylase can be regulated by a mechanism involving disulfide bonds in the cytochrome P-450 molecule.

The activity of purified 12 alpha-hydroxylase from rabbit liver microsomes was modulated by including protein fractions from rabbit liver microsomes and cytosol into the system. The microsomal protein fraction stimulated 12 alpha-hydroxylation two times. The cytosolic fraction inhibited the reaction markedly. The microsomal 12 alpha-hydroxylase stimulatory activity was labile and the cytosolic 12 alpha-hydroxylase inhibitory activity was stable to mild heat treatment. Addition of ATP and MgCl2 or NaF had no effect on the activities of the two protein fractions. The activity of the microsomal stimulatory fraction decreased upon storage but could be reactivated by addition of reduced glutathione to the system.

Newly synthesized cytochrome P-450 was labeled by administration of radioactive δ-aminolevulinic acid to the rats prior to killing. Cytochrome P-450 fractions were isolated by solubilization of microsomes with sodium cholate followed by chromatography on octylamine-Sepharose and hydroxylapatite. The cholesterol 7α-hydroxylase activity was separated from the 5β-cholestane-3α,7α-diol, 12α, 25- and 26-hydroxylase activities. Cholesterol 7α-hydroxylase activity was found in a minor cytochrome P-450 fraction with low specific radioactivity. On the other hand, the 12α, 25- and 26-hydroxylase activities were found in a major cytochrome P-450 fraction with higher specific radioactivity.

The activity of purified, reconstituted cholesterol 7α-hydroxylase from rat liver microsomes was modulated by including protein fractions from rat liver cytosol in the system. One fraction stimulated cholesterol 7α-hydroxylation two to three times. Another fraction inhibited the reaction markedly. The stimulatory activity was labile and the inhibitory activity was stable to mild heat treatment. The stimulatory and inhibitory effects of the cytosolic protein fractions were dependent on the amount of protein and time of preincubation with the cholesterol 7α-hydroxylase system. Addition of ATP and MgCl2 or NaF had no effect on the activities of the cytosolic protein fractions.

FEBS LettersVolume 66, Issue 2 p. 299-302 Full-length articleFree Access On the ability of cumene hydroperoxide and NaIO4 to support microsomal hydroxylations in biosynthesis and metabolism of bile acids Henry Danielsson, Henry Danielsson Department of Pharmaceutical Biochemistry, University of Uppsala, Box 578, S-751 23 Uppsala, SwedenSearch for more papers by this authorKjell Wikvall, Kjell Wikvall Department of Pharmaceutical Biochemistry, University of Uppsala, Box 578, S-751 23 Uppsala, SwedenSearch for more papers by this author Henry Danielsson, Henry Danielsson Department of Pharmaceutical Biochemistry, University of Uppsala, Box 578, S-751 23 Uppsala, SwedenSearch for more papers by this authorKjell Wikvall, Kjell Wikvall Department of Pharmaceutical Biochemistry, University of Uppsala, Box 578, S-751 23 Uppsala, SwedenSearch for more papers by this author First published: July 15, 1976 https://doi.org/10.1016/0014-5793(76)80526-1Citations: 18AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume66, Issue2July 15, 1976Pages 299-302 ReferencesRelatedInformation

7α-Hydroxylation of exogenous [4-14C]cholesterol and endogenous cholesterol by rat liver microsomes was studied with a mass fragmentographic technique which allows determination of weight of 7α-hydroxycholesterol with high accuracy. The technique could be used for determination of the ratio between 14C-labeled molecules and unlabeled molecules in cholesterol and 7α-hydroxy cholesterol. In agreement with a previous investigation by Balasubramaniam, Mitroupoulos and Myant [Eur. J. Biochem. 34. (1973) 77] it was found that the degree of equilibration of exogenous cholesterol with endogenous cholesterol depended on the mode of addition of the cholesterol. In addition, it was found that the degree of equilibration depended on the amount of cholesterol added. With a small amount of cholesterol, less than 5 μg, the degree of equilibration was similar with acetone and with Tween 80 in standard incubations with microsomal fraction from 0.6 g of liver. With increasing amounts of cholesterol in acetone the degree of equilibration decreased and endogenous cholesterol was utilized more efficiently than exogenous cholesterol, probably due to precipitations of exogenous cholesterol in the incubation mixture. With increasing amounts of cholesterol in Tween 80, there was a preferential utilization of exogenous cholesterol with all concentrations of cholesterol tested (up to 160 μg). The total amount of 7α-hydroxycholesterol formed increased with increasing amounts of exogenous cholesterol in Tween 80, indicating that the cholesterol 7α-hydroxylase system is not saturated with substrate under normal conditions. From the specific radioactivity of cholesterol and 7α-hydroxycholesterol isolated after incubation of [4-14C]cholesterol in Tween 80, it was calculated that the “substrate pool” of cholesterol 7α-hydroxylase maximally corresponds to about one third of the total amount of cholesterol normally present in the microsomal fraction. By analysis of specific radioactivity of 7α-hydroxycholesterol and cholesterol formed from [5-3H]mevalonate in incubations of the 10000 ×g supernatant fluid of rat liver homogenate, it was concluded that 7α-hydroxycholesterol was synthesized preferentially from newly synthesized cholesterol. Cholesterol 7α-hydroxylase activity was assayed under different conditions using the different types of assay previously used in this and other laboratories. Similar results were obtained with all assays with respect to degree of influence on cholesterol 7α-hydroxylase activity of biliary drainage and cholesterol feeding. However, it was concluded that measurement of weight of 7α-hydroxycholesterol is the preferred method.

The 7α-hydroxylation of taurodeoxycholic acid and the 6β-hydroxylation of taurochenodeoxycholic acid and lithocholic acid were studied with a reconstituted system from rat liver microsomes consisting of partially purified cytochrome P-450, NADPH-cytochrome P-450 reductase, a synthetic phosphatidylcholine and NADPH or an NADPH-generating system. 7α-Hydroxylase activity was observed with cytochrome P-450 from either male or female rats whereas 6β-hydroxylase activity was observed only with cytochrome P-450 from male rats. With male rats, the ratio between 7α-hydroxylase activity and 6β-hydroxylase activity was considerably higher in the reconstituted system than in the original microsomal fraction. The 6β-hydroxylase activity of the reconstituted system was more stable than the 7α-hydroxylase activity during prolonged storage at -20°. The rate of the hydroxylations in the reconstituted system was linear with the concentration of cytochrome P-450 and increased with the concentration of NADPH-cytochrome P-450 reductase up to a certain level and then remained constant. The lipid dependence of the system could be correlated to some extent with the amount of phospholipids in the cytochrome P-450 fraction. Preparations of cytochrome P-450 that had been recentrifuged at 100,000 x g just prior to incubation showed a greater requirement for lipid and had a lower ratio between phospholipid and cytochrome P-450 than before centrifugation. Addition of superoxide dismutase to the reconstituted system did not inhibit 7α or 6β hydroxylation. Treatment with phenobarbital, known to increase 7α- as well as 6β-hydroxylase activity in rat liver microsomes 3- to 4-fold, increased the specific catalytic activity (hydroxylase activity per nanomole of cytochrome P-450) of cytochrome P-450 2- to 5-fold. The catalytic activity of NADPH-cytochrome P-450 reductase per unit of NADPH-cytochrome c reductase activity was about the same regardless of the source of the preparation. The possibility was discussed that different catalytic types of phenobarbitalinducible cytochrome P-450 are involved in 7α- and 6β-hydroxylation of bile acids.

Recent development in research concerning hydroxylations in biosynthesis and metabolism of bile acids is reviewed. In the conversion of cholesterol into bile acids, hydroxyl groups may be introduced into the following positions: C-6α, C-6β, C-7α, C-12α, C-16α, C-23, C-24, C-25, and C-26. With the exception of the 26-hydroxylation and probably the 24-hydroxylation, which are also catalyzed by the mitochondrial fraction, the different hydroxylations are catalyzed by the microsomal fraction of liver homogenate and require NADPH and molecular oxygen. With the possible exception of the 12α-hydroxylase, all the microsomal hydroxylations appear to involve participation of cytochrome P-450 and NADPH-cytochrome P-450 reductase. Several of the hydroxylations have been demonstrated in reconstituted systems consisting of partially purified cytochrome P-450, NADPH-cytochrome P-450 reductase and a phospholipid. The specificity of the hydroxylation is determined mainly by the cytochrome P-450 fraction. The cholesterol 7α-hydroxylase catalyzes the major rate-limiting step in the overall conversion of cholesterol into bile acids. The combined activities of the microsomal 26-hydroxylase and the 12α-hydroxylase play a major role in determining the ratio between cholic acid and chenodeoxycholic acid formed from cholesterol. There is a close relationship between the cholesterol 7α-hydroxylase activity and the rate of cholesterol biosynthesis. The regulatory role of the cholesterol 7α-hydroxylase, the 12α-hydroxylase and the microsomal 26-hydroxylase may be correlated with some specific properties of the enzyme systems which differ markedly from the properties of the other hydroxylases involved in biosynthesis and metabolism of bile acids. The influence of different factors on the hydroxylations has been studied and the physiological implications of the different effects are discussed.

A preparation of partially purified cytochrome P-450 from rat liver microsomes was found to catalyze 12α-hydroxylation of 7α-hydroxy-4-cholesten-3-one in the presence of NADPH and phosphatidyl choline. The reaction was stimulated two- to four-fold by addition of a preparation of cytochrome P-450 reductase. The reaction was inhibited by carbon monoxide to a considerably less extent than other hydroxylations catalyzed by the reconstituted system. In the presence of optimal concentrations of cytochrome P-450 reductase, cytochrome P-450 prepared from livers of starved rats catalyzed the 12α-hydroxylation more efficiently than cytochrome P-450 prepared from livers of normal rats or rats treated with phenobarbital.

The rate of incorporation of acetate into cholesterol by rat liver slices and the rates of 7α-hydroxylation of cholesterol, of 12α-hydroxylation of 7α-hydroxy-4-cholesten-3-one and of 7α-hydroxylation of taurodeoxycholic acid by rat liver microsomes were assayed at six different times of the day. The diurnal variations in rate of incorporation of acetate into cholesterol (maximum between 8 p.m. and midnight and minimum at noon) and in rate of 7α-hydroxylation of cholesterol (maximum at 8 p.m. and minimum at noon) followed each other closely. 12α-Hydroxylation of 7α-hydroxy-4-cholesten-3-one and 7α-hydroxylation of taurodeoxycholic acid remained at about the same level during the day. The pattern of diurnal variations in incorporation of acetate into cholesterol and in 7α-hydroxylation of cholesterol was changed by reversing the daily rhythm of the rats. The results indicate that changes in rates of biosynthesis of cholesterol and bile acids occur practically concomitantly.

The synthesis of tritium‐labeled 14‐methylpentadecanoic acid, 15‐methylhexadecanoic acid and 16‐methylheptadecanoic acid is described. These acids were found to be oxidized into a number of water‐soluble and ether‐soluble products by the 800×g supernatant fluid of rat liver homogenate. In the presence of mitochondrial or microsomal fraction fortified with NADPH, the iso‐fatty acids were converted mainly into ω‐oxidized products. Addition of boiled 100 000×g supernatant fluid or NAD increased the yield of ω‐oxidized products. Gas‐chromatographic and mass‐spectrometric analyses of the products formed showed that the main metabolites were the ω‐hydroxy iso‐fatty acid and the corresponding dicarboxylic acid.

The metabolism of endogenous cholesterol was studied in fractions of liver homogenates obtained from rats that had received a single injection of [1,2‐3H]cholesterol 48 hours before preparation of the homogenates. The pattern of labeled products formed in incubations of the 800×g supernatant fluid, of the mitochondrial fraction, and of the 20,000×g supernatant fluid was essentially the same as that described in earlier investigations, in which the labeled cholesterol had been added to the homogenates in acetone solution or as an emulsion. The results are consistent with the view that a major pathway for the conversion of cholesterol into 5β‐cholestane‐3α,7α,12α‐triol involves the intermediate formation of cholest‐5‐ene‐3β,7α‐diol, 7α‐hydroxycholest‐4‐en‐3‐one, and 7α,12α‐dihydroxycholest‐4‐en‐3‐one.

The synthesis of unlabeled and tritium-labeled cholest-5-ene-3β,7α,12α-triol is described. The formation of cholest-5-ene-3β,7α,12α-triol was shown in incubations of cholesterol and cholest-5-ene-3β,7α-diol with the 20,000 x g supernatant fluid of rat liver homogenates and of cholest-5-ene-3β,7α-diol with the microsomal fraction fortified by addition of reduced nicotinamide adenine dinucleotide phosphate. Cholest-5-ene-3β,7α,12α-triol was found to be converted to 7α,12α-dihydroxycholest-4-en-3-one in the presence of mitochondrial or microsomal fraction fortified with NAD or NADP. The microsomal fraction was more active than the mitochondrial fraction and NAD was several times more active than NADP.

The synthesis of unlabeled and tritium‐labeled cholest‐4‐ene‐3α, 7α‐diol and cholest‐4‐ene 3β, 7α‐diol is described. Both compounds were found to be efficiently converted into cheodeoxycholic acid and cholic acid in the bile fistula rat. The presence in liver of microsomal and soluble Δ 4 ‐3‐hydroxysteroid dehydrogenases active on Δ 4 ‐3‐hydroxysteroids of the C 27 series was demonstrated. The soluble dehydrogenase(s) was more active on the Δ 4 ‐3α‐hydroxysteroids than on the Δ 4 ‐3β‐hydroxysteroids. The microsomal fraction contained dehydrogenase(s) capable of oxidizing both the Δ 4 ‐3α‐hydroxysteroids and the Δ 4 ‐3α‐hydroxysteroids in the presence of NAD or NADP, NAD being preferred. No significant conversion of cholest‐5‐ene‐3β, 7α‐diol to cholest‐4‐ene‐3β, 7α‐diol could be shown nor could any direct saturation of the double bond in cholest‐4‐ene‐3α, 7α‐diol or that in cholest‐4‐ene‐3α, 7α,12α‐triol be shown. Cholest‐4‐ene‐3α, 7α‐diol was hydroxylated in the 12α‐position more efficiently than cholest‐4‐ene‐3β, 7α‐diol. It is suggested that the Δ 4 ‐cholestenols are converted to bile acids by first being oxidized to the corresponding Δ 4 ‐3‐ketosteroids.