Identification of CYP3A4 as the major enzyme responsible for 25-hydroxylation of 5beta-cholestane-3alpha,7alpha,12alpha-triol in human liver microsomes.

Human liver microsomes catalyze an efficient 25-hydroxylation of 5beta-cholestane-3alpha,7alpha,12alpha-triol. The hydroxylation is involved in a minor, alternative pathway for side-chain degradation in the biosynthesis of cholic acid. The enzyme responsible for the microsomal 25-hydroxylation has been unidentified. In the present study, recombinant expressed human P-450 enzymes have been used to screen for 25-hydroxylase activity towards 5beta-cholestane-3alpha, 7alpha,12alpha-triol. High activity was found with CYP3A4, but also with CYP3A5 and to a minor extent with CYP2C19 and CYP2B6. Small amounts of 23- and 24-hydroxylated products were also formed by CYP3A4. The Vmax for 25-hydroxylation by CYP3A4 and CYP3A5 was 16 and 4.5 nmol/(nmolxmin), respectively. The Km was 6 microM for CYP3A4 and 32 microM for CYP3A5. Cytochrome b5 increased the hydroxylase activities. Human liver microsomes from ten different donors, in which different P-450 marker activities had been determined, were incubated with 5beta-cholestane-3alpha,7alpha, 12alpha-triol. A strong correlation was observed between formation of 25-hydroxylated 5beta-cholestane-3alpha,7alpha,12alpha-triol and CYP3A levels (r2=0.96). No correlation was observed with the levels of CYP2C19. Troleandomycin, a specific inhibitor of CYP3A4 and 3A5, inhibited the 25-hydroxylase activity of pooled human liver microsomes by more than 90% at 50 microM. Tranylcypromine, an inhibitor of CYP2C19, had very little effect on the conversion. From these results, it can be concluded that CYP3A4 is the predominant enzyme responsible for 25-hydroxylation of 5beta-cholestane-3alpha, 7alpha,12alpha-triol in human liver microsomes.

Biochemical characterization of the 7alpha-hydroxylase activities towards 27-hydroxycholesterol and dehydroepiandrosterone in pig liver microsomes.

Microsomal cytochrome P-450 catalyzing the 7alpha-hydroxylation of 27-hydroxycholesterol and dehydroepiandrosterone was partially purified from pig liver. This enzyme fraction also catalyzed 7alpha-hydroxylation of 25-hydroxycholesterol and pregnenolone but did not 7alpha-hydroxylate cholesterol or testosterone. Studies with extrahepatic tissues have suggested the possibility of one common enzyme responsible for the 7alpha-hydroxylation of 27-hydroxycholesterol and dehydroepiandrosterone. A series of experiments was performed to study if there are one or several enzymes 7alpha-hydroxylating these steroids in the liver. The activities towards the two substrates copurified but the ratio between 27-hydroxycholesterol and dehydroepiandrosterone 7alpha-hydroxylation varied considerably in different purification steps and between different preparations. The enzyme inhibitors disulfiram, N-bromosuccinimide, ketoconazole, metyrapone and alpha-naphthoflavone affected the activities in a similar way. Dehydroepiandrosterone inhibited 27-hydroxycholesterol 7alpha-hydroxylation whereas 27-hydroxycholesterol had almost no inhibitory effect on dehydroepiandrosterone 7alpha-hydroxylation. Experiments to examine the nature of inhibition by dehydroepiandrosterone indicated that the two steroids did not compete for the same active site. The results of this study do not rule out the possibility of one single enzyme catalyzing 7alpha-hydroxylation of the two steroids. However, taken together the data suggest that hepatic microsomal 7alpha-hydroxylation of 27-hydroxycholesterol and dehydroepiandrosterone involves at least two, probably closely related, enzymes. (c) 1998 Elsevier Science B. V.

6alpha-hydroxylation of taurochenodeoxycholic acid and lithocholic acid by CYP3A4 in human liver microsomes.

The aim of the present study was to identify the enzymes in human liver catalyzing hydroxylations of bile acids. Fourteen recombinant expressed cytochrome P450 (CYP) enzymes, human liver microsomes from different donors, and selective cytochrome P450 inhibitors were used to study the hydroxylation of taurochenodeoxycholic acid and lithocholic acid. Recombinant expressed CYP3A4 was the only enzyme that was active towards these bile acids and the enzyme catalyzed an efficient 6alpha-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid. The Vmax for 6alpha-hydroxylation of taurochenodeoxycholic acid by CYP3A4 was 18.2 nmol/nmol P450/min and the apparent Km was 90 microM. Cytochrome b5 was required for maximal activity. Human liver microsomes from 10 different donors, in which different P450 marker activities had been determined, were separately incubated with taurochenodeoxycholic acid and lithocholic acid. A strong correlation was found between 6alpha-hydroxylation of taurochenodeoxycholic acid, CYP3A levels (r2=0.97) and testosterone 6beta-hydroxylation (r2=0.9). There was also a strong correlation between 6alpha-hydroxylation of lithocholic acid, CYP3A levels and testosterone 6beta-hydroxylation (r2=0.7). Troleandomycin, a selective inhibitor of CYP3A enzymes, inhibited 6alpha-hydroxylation of taurochenodeoxycholic acid almost completely at a 10 microM concentration. Other inhibitors, such as alpha-naphthoflavone, sulfaphenazole and tranylcypromine had very little or no effect on the activity. The apparent Km for 6alpha-hydroxylation of taurochenodeoxycholic by human liver microsomes was high (716 microM). This might give an explanation for the limited formation of 6alpha-hydroxylated bile acids in healthy humans. From the present results, it can be concluded that CYP3A4 is active in the 6alpha-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid in human liver.

Renal and hepatic 1 alpha-hydroxylation of 25-hydroxyvitamin D3 in piglets suffering from pseudo vitamin D-deficiency rickets, type I.

The piglets examined suffer from rickets and have symptoms similar to those of classic pseudo vitamin D-deficiency rickets, type I (PVDRI), including plasma concentrations of 1 alpha, 25-dihydroxyvitamin D3 considerably lower than in healthy control piglets. It has been suggested that the rachitic piglets have a defective renal 1 alpha,25-dihydroxyvitamin D3 production. The present study shows that partially purified mitochondrial and microsomal cytochrome P450 from kidney and liver of both rachitic and control animals is able to catalyze 1 alpha-hydroxylation of 25-hydroxyvitamin D3. The renal mitochondrial 1 alpha-hydroxylase activity was higher in the rachitic piglets whereas the renal microsomal 1 alpha-hydroxylase activity was decreased. The immunodetectable levels in kidney of a mitochondrial 1 alpha-hydroxylase (CYP27) and a microsomal 1 alpha-hydroxylase (vitamin D3 25-hydroxylase) were correlated with the 1 alpha-hydroxylase activities. The results suggest that the renal microsomal 1 alpha-hydroxylase is affected by the rachitic condition. It is concluded that the primary genetic defect of systemic 1 alpha,25-dihydroxyvitamin D3 deficiency in the rachitic PVDRI piglets does not reside in a defective function or absence of renal mitochondrial 25-hydroxyvitamin D3 1 alpha-hydroxylase. From this, it may also be concluded that PVDRI in man and pig appear to be two different forms of the disease.

24-, 25- and 27-hydroxylation of cholesterol by a purified preparation of 27-hydroxylase from pig liver.

Pig liver mitochondria were found to catalyze 27-, 25- and 24-hydroxylation of cholesterol at relative rates of about 1:0.2:0.04. An apparently homogeneous preparation of pig liver mitochondrial cytochrome P-450-27 was found to catalyze the same three hydroxylations at about the same relative rates when reconstituted with adrenodoxin and adrenodoxin reductase. The 24-hydroxycholesterol formed was shown to consist of one of the two possible stereoisomers. When using specifically deuterium-labeled substrates a significant isotope effect was observed in the case of 24-hydroxylation (KH/KD > 10), but not 25-hydroxylation (KH/KD = 1.1), or 27-hydroxylation (KH/KD = 1.1). The difference between the 24-hydroxylation and the other two hydroxylations may be due to different interactions between cholesterol and the same enzyme, with a resulting difference with respect to the rate-limiting step in the reaction. The physiological significance of the mitochondrial 24-hydroxylation is discussed.

7 alpha-hydroxylation of 26-hydroxycholesterol, 3 beta-hydroxy-5-cholestenoic acid and 3 beta-hydroxy-5-cholenoic acid by cytochrome P-450 in pig liver microsomes.

Pig liver microsomes were found to catalyze the 7 alpha-hydroxylation of several potential bile acid precursors besides cholesterol. 26-Hydroxycholesterol, 3 beta-hydroxy-5-cholestenoic acid and 3 beta-hydroxy-5-cholenoic acid were all efficiently converted into the 7 alpha-hydroxylated products. Two cytochrome P-450 fractions showing 7 alpha-hydroxylase activity could be isolated. One fraction catalyzed 7 alpha-hydroxylation of 26-hydroxycholesterol, 3 beta-hydroxy-5-cholestenoic acid and 3 beta-hydroxy-5-cholenoic acid but was inactive towards cholesterol. The other fraction catalyzed 7 alpha-hydroxylation of cholesterol in addition to the other substrates. 26-Hydroxycholesterol in equimolar concentration did not inhibit the cholesterol 7 alpha-hydroxylase activity of this fraction. It is concluded that liver microsomes contain a cytochrome P-450 catalyzing 7 alpha-hydroxylation of 26-hydroxycholesterol, 3 beta-hydroxy-5-cholestenoic acid and 3 beta-hydroxy-5-cholenoic acid. The results indicate that this cytochrome P-450 is different from that catalyzing 7 alpha-hydroxylation of cholesterol.

Microsomal 25-hydroxylation of vitamin D2 and vitamin D3 in pig liver.

A microsomal cytochrome P-450 catalysing 25-hydroxylation of vitamin D2 was purified from both male and female pigs to apparent homogeneity and a specific cytochrome P-450 content of 13 and 15.4 nmol x mg of protein-1, respectively. The enzyme also catalysed 25-hydroxylation of vitamin D3. The ratio between the 25-hydroxylase activities towards vitamin D2 and D3 was essentially the same in the different purification steps as well as in the apparently homogeneous enzyme preparation. The two enzyme activities showed the same pH optimum and decreased in parallel upon partial denaturation of the enzyme. Cholecalciferol competitively inhibited 25-hydroxylation of vitamin D2 and vice versa. The non-steroidal cytochrome P-450 inhibitor ketoconazole inhibited both enzyme activities and the Ki values were the same. The cytochrome P-450 showed the same apparent M(r), substrate specificity and N-terminal amino acid sequence as the previously purified vitamin D3 25-hydroxylase from pig liver microsomes. A monoclonal antibody raised against the vitamin D3 25-hydroxylase also recognized the vitamin D2 25-hydroxylase. The antibody immunoprecipitated the 25-hydroxylase activity towards both vitamin D2 and D3 in the purified enzyme. Taken together, the results show that the 25-hydroxylation of vitamin D2 and D3 is catalysed by the same microsomal cytochrome P-450 in pig liver microsomes. The properties of this 25-hydroxylase are discussed in relation to present knowledge concerning previously well-characterized vitamin D3 25-hydroxylases that are not able to catalyse 25-hydroxylation of vitamin D2.

Cytochrome P450 CYP27-catalyzed oxidation of C27-steroid into C27-acid.

Rabbit liver cytochrome P450 CYP27 has been previously shown to catalyze the complete conversion of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol into 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid. This study compares some properties of the reactions involved, the 27-hydroxylation of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol and the further oxidation of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,27-tetrol. The Km was the same for the two substrates, whereas the Vmax was three times higher for 27-hydroxylation than for the oxidation of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,27-tetrol. Ketoconazole inhibited both reactions, whereas disulfiram did not. Carbon monoxide inhibited the 27-hydroxylation of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol but not the further oxidation of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,27-tetrol. There was no difference in sensitivity to varying oxygen concentrations between the two reactions. The present study shows that CYP27 also converts, although less efficiently, 5 beta-cholestane-3 alpha,7 alpha-diol into 3 alpha,7 alpha-dihydroxy-5 beta-cholestanoic acid and cholesterol into 3 beta-hydroxy-5-cholestanoic acid. The rate of oxidation of cholesterol into C27-acid was very low--less than 1% of that with the other C27-steroids.

Cytochrome P450 enzymes in the bioactivation of vitamin D to its hormonal form (review).

The formation of 1alpha,25-dihydroxyvitamin D3 requires a 25-hydroxylation followed by a 1alpha-hydroxylation catalyzed by cytochrome P450 (CYP) enzymes in liver and kidney. The aim of this review is to give a brief summary of our research on the cytochrome P450 enzymes catalyzing the 25-hydroxylation and 1alpha-hydroxylation and to discuss the results in relation to other published literature on these enzymes. Two hepatic P450 enzymes catalyzing 25-hydroxylation of vitamin D3 exist in mammalian liver - one mitochondrial and one microsomal. The mitochondrial vitamin D3 25-hydroxylase is apparently identical with CYP27A, an obligatory enzyme in bile acid biosynthesis in liver. The microsomal 25-hydroxylase has been purified to apparent homogeneity from pig liver. The enzyme catalyzed 25-hydroxylation of vitamin D3, 1alpha-hydroxyvitamin D3, vitamin D2 and 1alpha-hydroxyvitamin D2. A cDNA encoding pig liver microsomal vitamin D3 25-hydroxylase has been isolated in this laboratory. The primary structure of vitamin D3 25-hydroxylase shows 70-80% identity with members of the CYP2D subfamily and has been designated CYP2D25. Three different 1alpha-hydroxylating cytochromes P450 in kidney, i.e. CYP27A, CYP27B and a microsomal 1alpha-hydroxylase, have been described. Mitochondrial cytochrome P450, catalyzing 1alpha-hydroxylation and 27-hydroxylation but not 24-hydroxylation of 25-hydroxyvitamin D3, was partially purified from pig kidney. Purification and inhibition experiments as well as experiments with a monoclonal antibody against CYP27A indicated that one single enzyme catalyzes both 1alpha- and 27-hydroxylation. Treatment of rats with a single i.v. dose of 1alpha,25-dihydroxyvitamin D3 resulted in a marked suppression of CYP27A mRNA levels in kidney. The results suggest a role for CYP27A as a renal mitochondrial 1alpha-hydroxylase. Subsequently, several research groups reported the isolation of cDNA encoding mouse, rat and human kidney 25-hydroxyvitamin D3 1alpha-hydroxylase. The amino acid sequences deduced from these cDNA clones were similar but differed from that of CYP27A. This 1alpha-hydroxylase constitutes a new CYP27 subfamily, CYP27B. The expression of CYP27B was found to be influenced by vitamin D status and parathyroid hormone. Mutations in the CYP27B gene have been identified in patients with pseudovitamin D-deficiency rickets. A microsomal P450 catalyzing 1alpha-hydroxylation of 25-hydroxyvitamin D3 has been purified to apparent homogeneity from pig kidney. This finding demonstrate the presence of a microsomal 1alpha-hydroxylase in addition to the mitochondrial 1alpha-hydroxylases in kidney. The relative importance and regulation of the different renal 1alpha-hydroxylases in the bioactivation of vitamin D3 under normal and pathological conditions will be subject for future studies.

Purification and properties of a 3 beta-hydroxy-delta 5-C27-steroid oxidoreductase from rabbit liver microsomes.

The enzyme converting 5-cholestene-3 beta, 7 alpha-diol to 7 alpha-hydroxy-4-cholesten-3-one has been solubilized from rabbit liver microsomes by treatment with a mixture of sodium cholate and the nonionic detergent Renex 690. The enzyme was purified about 200-fold, with a recovery of more than 50%, by chromatography on DEAE-cellulose, hydroxylapetite, 2',5',ADP-Sepharose 4B and 5'-AMP-Sepharose 4B. The purified enzyme showed only one protein band, with an apparent molecular weight of 46,000, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was eluted as a single peak on gel filtration on Ultrogel AcA 34. The elution volume corresponded to that observed for globular proteins with molecular weights in the 45,000 to 50,000 region. The substrate specificity of the microsomal fraction and of the purified oxidoreductase in oxidation and reduction of various 3-oxygenated C19-, C21-, and C27-steroids was studied in the presence of NAD and NADH. Whereas the microsomal fraction had a broad substrate specificity, NAD-supported oxidation with the purified oxidoreductase only occurred with 5-cholestene-3 beta, 7 alpha-diol as substrate. NADP could not replace NAD in the reaction. NADH-supported reduction with the purified oxidoreductase only occurred with 7 alpha-hydroxy-5 alpha-cholestan-3-one as substrate. The results suggest that conversion of 5-cholestene-3 beta, 7 alpha-diol to 7 alpha-hydroxy-4-cholesten-3-one is catalyzed by a single enzyme specific for certain C27-steroids.

Hydroxylations in biosynthesis of bile acids. Isolation of a cytochrome P-450 from rabbit liver mitochondria catalyzing 26-hydroxylation of C27-steroids.

A cytochrome P-450 catalyzing 26-hydroxylation of C27-steroids was purified from liver mitochondria of untreated rabbits. The enzyme fraction contained 10 nmol of cytochrome P-450/mg of protein and showed only one protein band with a minimum Mr = 53,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified mitochondrial cytochrome P-450 showed apparent molecular weight similar to microsomal cytochromes P-450LM4 but differed in spectral and catalytic properties from these microsomal isozymes. The purified cytochrome P-450 catalyzed 26-hydroxylation of cholesterol, 5-cholestene-3 beta,7 alpha-diol, 7 alpha-hydroxy-4-cholesten-3-one, 5 beta-cholestane-3 alpha,7 alpha-diol, and 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol up to 1000 times more efficiently than the mitochondria. The cytochrome P-450 required both ferredoxin and ferredoxin reductase for catalytic activity. Microsomal NADPH-cytochrome P-450 reductase could not replace ferredoxin and ferredoxin reductase. The cytochrome P-450 was inactive in 7 alpha-, 12 alpha- and 25-hydroxylations of C27-steroids. The results suggest that mitochondrial 26-hydroxylation of various C27-steroids is catalyzed by the same species of cytochrome P-450.

Hydroxylations in biosynthesis of bile acids. Cytochrome P-450 LM4 and 12alpha-hydroxylation of 5beta-cholestane-3alpha, 7alpha-diol.

12alpha-Hydroxylation of 5beta-cholestane-3alpha, 7alpha-diol was studied in reconstituted systems consisting of electrophoretically homogeneous cytochrome P-450 LM4 fractions and NADPH-cytochrome P-450 reductase from rabbit liver microsomes. Cytochrome P-450 LM4 fractions were prepared from untreated, phenobarbital-treated, beta-naphthoflavone-treated and starved rabbits. The purified cytochromes catalyzed 12alpha-hydroxylation more efficiently than the corresponding microsomes. In the reconstituted systems, carbon monoxide inhibited 12alpha-hydroxylation by 50-80%. The rate of 12alpha-hydroxylation was three to four times higher with cytochrome P-450 LM4 fractions from starved rabbits than with cytochrome P-450 LM4 fractions from untreated, phenobarbital-treated and beta-naphthoflavone-treated animals. Amino acid analyses, peptide mapping experiments as well as absorption spectral and circular dichroism spectral analyses revealed physical differences between cytochrome P-450 LM4 fractions from starved animals and preparations from phenobarbital-treated animals. The results indicate the presence of a cytochrome P-450 species in the cytochrome P-450 LM4 fraction specific for 12alpha-hydroxylation.

Hydroxylations in biosynthesis of bile acids. Isolation of subfractions with different substrate specificity from cytochrome P-450LM4.

Chromatography of electrophoretically homogeneous cytochrome P-450LM4 from cholestyramine-treated rabbits on octylamine-Sepharose resulted in the isolation of two subfractions, cytochrome P-450LM4 I and cytochrome P-450LM4 II, with different catalytic properties. The original cytochrome P-450LM4 fraction catalyzed 7 alpha-hydroxylation of cholesterol, 12 alpha-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha-diol, 25-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha-diol and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol, 6 beta-hydroxylation of testosterone, and demethylation of ethylmorphine. Cytochrome P-450LM4 I was inactive in cholesterol 7 alpha-hydroxylation, but catalyzed the other hydroxylations. Cytochrome P-450LM4 II catalyzed efficient cholesterol 7 alpha-hydroxylation. It also catalyzed the other hydroxylations, although at lower rates than cytochrome P-450LM4 I. Emulgen inhibited all steroid hydroxylase activities in cytochrome P-450LM4 II except the cholesterol 7 alpha-hydroxylase activity. Cytochrome P-450LM4 I and cytochrome P-450LM4 II showed the same apparent molecular weight and spectral properties as the original cytochrome P-450LM4 fraction. The two subfractions differed in amino acid composition. They produced similar but not identical one-dimensional peptide maps upon limited proteolysis with papain, chymotrypsin, and trypsin. The results show that cytochrome P-450LM4 from cholestyramine-treated rabbits contains at least two species with different amino acid compositions and different substrate specificities toward C27-steroids involved in biosynthesis of bile acids.

Purification of a cytochrome P-450 from pig kidney microsomes catalysing the 25-hydroxylation of vitamin D3.

Cytochrome P-450 catalysing 25-hydroxylation of vitamin D3 was purified from pig kidney microsomes. The enzyme fraction contained 7 nmol of cytochrome P-450/mg of protein and showed only one protein band with an apparent Mr of 50,500 upon SDS/polyacrylamide-gel electrophoresis. The purified cytochrome P-450 catalysed 25-hydroxylation of vitamin D3 up to 1,000 times more efficiently, and 25-hydroxylation of 1 alpha-hydroxyvitamin D3 up to 4000 times more efficiently, than the microsomes. The cytochrome P-450 required microsomal NADPH-cytochrome P-450 reductase for catalytic activity. Mitochondrial ferredoxin and ferredoxin reductase could not replace microsomal NADPH-cytochrome P-450 reductase. The enzyme preparation showed no detectable 25-hydroxylase activity towards vitamin D2 or 1 alpha-hydroxylase activity towards 25-hydroxyvitamin D3. CO inhibited the 25-hydroxylation by more than 85%. Mannitol, hydroquinone, catalase and superoxide dismutase did not affect the 25-hydroxylation. The possible role of the kidney microsomal cytochrome P-450 in the metabolism of vitamin D3 is discussed.

25-hydroxylation of vitamin D3 in rat liver: roles of mitochondrial and microsomal cytochrome P-450.

A mitochondrial cytochrome P-450 fraction, which catalyzed 25-hydroxylation of vitamin D3 much more efficiently than intact mitochondria was isolated from livers of male and female rats. For comparison, a microsomal cytochrome P-450 fraction was isolated by the same procedures. The mitochondrial cytochrome P-450 from female rats catalyzed 25-hydroxylation as efficiently as the same material from male rats. The microsomal 25-hydroxylation was male specific. The 25-hydroxylase activity in intact mitochondria and the 25-hydroxyvitamin D3 concentration in serum were similar in male and female rats. There was no correlation between the 25-hydroxylase activity in microsomal cytochrome P-450 and the 25-hydroxyvitamin D3 concentration in serum.

Evidence for the formation of 26-hydroxycholesterol by cytochrome P-450 in pig kidney mitochondria.

Pig kidney mitochondria were found to catalyze the formation of 26-hydroxycholesterol, an inhibitor of cholesterol biosynthesis. The cholesterol 26-hydroxylase was purified 600-fold. It was present in a mitochondrial enzyme fraction enriched in cytochrome P-450. The cytochrome P-450 fraction required NADPH, mitochondrial ferredoxin and ferredoxin reductase for 26-hydroxylase activity. The mitochondria and the purified 26-hydroxylase preparation also catalyzed 26-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol, and intermediate in cholic acid biosynthesis, and of 25-hydroxyvitamin D3. The role of extra-hepatic formation of 26-hydroxycholesterol is discussed.

25-Hydroxylation of vitamin D3 by a cytochrome P-450 from rabbit liver mitochondria.

A cytochrome P-450 catalysing 25-hydroxylation of vitamin D3 was purified from liver mitochondria of untreated rabbits. The enzyme fraction contained 9 nmol of cytochrome P-450/mg of protein and showed only one protein band with an apparent Mr of 52,000 upon SDS/polyacrylamide-gel electrophoresis. The preparation showed a single protein spot with an apparent isoelectric point of 7.8 and an Mr of approx. 52,000 upon two-dimensional isoelectric-focusing-polyacrylamide-gel electrophoresis. The purified cytochrome P-450 catalysed 25-hydroxylation of vitamin D3 up to 5000 times more efficiently than did the mitochondria. The cytochrome P-450 required both ferredoxin and ferredoxin reductase for catalytic activity. Microsomal NADPH-cytochrome P-450 reductase could not replace ferredoxin and ferredoxin reductase. The cytochrome P-450 catalysed, in addition to 25-hydroxylation of vitamin D3, the 25-hydroxylation of 1 alpha-hydroxyvitamin D3 and the 26-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol. The enzyme did not catalyse side-chain cleavage of cholesterol, 11 beta-hydroxylation of deoxycorticosterone, 1 alpha-hydroxylation of 25-hydroxyvitamin D3, hydroxylations of lauric acid and testosterone or demethylation of benzphetamine. The results raise the possibility that the 25-hydroxylation of vitamin D3 and the 26-hydroxylation of C27 steroids are catalysed by the same species of cytochrome P-450 in liver mitochondria. The possible role of the liver mitochondrial cytochrome P-450 in the metabolism of vitamin D3 is discussed.

Porcine microsomal vitamin D(3) 25-hydroxylase (CYP2D25). Catalytic properties, tissue distribution, and comparison with human CYP2D6.

The metabolic activation of the prohormone vitamin D(3) requires a 25-hydroxylation that has been reported to be catalyzed by both mitochondrial CYP27A and a microsomal vitamin D(3) 25-hydroxylase in the liver. CYP27A has been extensively studied, but its role as a physiologically important vitamin D(3) 25-hydroxylase has been questioned. The present paper reports that the microsomal vitamin D(3) 25-hydroxylase, purified from pig liver, converted vitamin D(3) into 25-hydroxyvitamin D(3) in substrate concentrations which are within the physiological range (apparent K(m) = 0.1 microm). The enzyme 25-hydroxylated vitamin D(3), 1 alpha-hydroxyvitamin D(3) and vitamin D(2) and also converted tolterodine, a substrate for human CYP2D6, into its 5-hydroxymethyl metabolite. Tolterodine inhibited the microsomal 25-hydroxylation, whereas quinidine, an inhibitor of CYP2D6, did not markedly inhibit the reaction. The primary structure of the microsomal vitamin D(3) 25-hydroxylase, designated CYP2D25, shows 77% identity with that of human CYP2D6. Northern blot and reverse transcription-polymerase chain reaction experiments revealed that CYP2D25 mRNA is expressed in higher levels in liver than in kidney and in small amounts in adrenals, brain, heart, intestine, lung, muscle, spleen, and thymus. Experiments with human liver microsomes and recombinantly expressed CYP2D6 strongly indicate that the microsomal 25-hydroxylation of vitamin D(3) in human liver is catalyzed by an enzyme different from CYP2D6.