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Results for “Steroid 12-alpha-Hydroxylase”

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At least 19 recordsLinked to original sources
PubMed1983

Effect of feeding cholesterol and sitosterol on hepatic steroid 12 alpha-hydroxylase activity in female hamsters.

The effects of dietary cholesterol and sitosterol on the activity of the hepatic steroid 12 alpha-hydroxylase, gallbladder bile acid composition, and serum and liver cholesterol concentrations were studied in female hamsters. The 12 alpha-hydroxylase activity was inhibited by 63% in cholesterol-fed animals and by 30% in sitosterol-fed animals. Cholesterol feeding increased percentages of chenodeoxycholic and lithocholic acids in gallbladder bile with reciprocal decrease of cholic acid, while sitosterol feeding had no significant effect on bile acid composition. Cholesterol feeding increased levels of serum and liver cholesterol, on the contrary, sitosterol decreased both concentrations. A positive correlation between the 12 alpha-hydroxylase activity and the ratio of cholic acid plus its metabolites to chenodeoxycholic acid plus its metabolite was also observed. These results support the proposal that steroid 12 alpha-hydroxylase is a major factor in determining the relative proportion of cholic acid and chenodeoxycholic acid synthesized in the liver and indicate that cholesterol feeding increased percentage of chenodeoxycholic acid by inhibiting the activity of steroid 12 alpha-hydroxylase.

Animals↗
PubMed1983

Effect of bile acid feeding on hepatic steroid 12 alpha-hydroxylase activity in hamsters.

The effects of feeding cholic acid, chenodeoxycholic acid and ursodeoxycholic acid on the activity of the hepatic steroid 12 alpha-hydroxylase, gallbladder bile acid composition, fecal neutral sterol output, cholesterol synthesis and bile acid synthesis were determined in female hamsters. The 12 alpha-hydroxylase activity was inhibited to 56% by cholic acid, to 62% by chenodeoxycholic acid, and to 78% by ursodeoxycholic acid compared with the control. Bile acid composition was altered by feeding of cholic acid and chenodeoxycholic acid to be rich in the given bile acids. Fecal neutral sterol output increased about twice by feeding chenodeoxycholic acid and ursodeoxycholic acid, whereas cholic acid had no significant effect. Body cholesterol synthesis increased to 217% by chenodeoxycholic acid and to 274% by ursodeoxycholic acid, whereas effect of cholic acid was not significant. Bile acid synthesis was suppressed to 48% of control only by chenodeoxycholic acid. A positive correlation between the 12 alpha-hydroxylase activity and the bile acid synthesis was observed in the control, chenodeoxycholate-fed and ursodeoxycholate-fed animals. In conclusion, ursodeoxycholic acid might have less inhibitory effect on the steroid 12 alpha-hydroxylase and the bile acid synthesis than chenodeoxycholic acid.

Animals↗
PubMed1983

Sex differences in gallbladder bile acid composition and hepatic steroid 12 alpha-hydroxylase activity in hamsters.

The gallbladder bile acid composition and the activity of the hepatic steroid 12 alpha-hydroxylase were determined in male and female hamsters. Cholic acid, chenodeoxycholic acid, and deoxycholic acid were the major bile acids in both sexes; in addition, 7-ketodeoxycholic acid and lithocholic acid were present. A sex-linked difference in the ratio of cholic acid (plus its metabolites) to chenodeoxycholic acid (plus its metabolite) was observed. The ratio was 1.93 +/- 0.39 in males and 2.74 +/- 0.54 in females. Another sex-linked difference was found in the activity of the 12 alpha-hydroxylase. The extent of the 12 alpha-hydroxylation of 7 alpha-hydroxycholest-4-en-3-one to yield 7 alpha, 12 alpha-dihydroxycholest-4-en-3-one was about two times greater in the microsomal suspension obtained from the liver of female hamsters than in that of male hamsters. A positive correlation between the 12 alpha-hydroxylase activity and the ratio of cholic acid/chenodeoxycholic acid was also observed. These results strongly support the proposal that the activity of the 12 alpha-hydroxylase is the major factor in determining the relative proportion of cholic acid and chenodeoxycholic acid formed from cholesterol in the liver.

Animals↗
PubMed1986

Competitive inhibitors of rabbit hepatic microsomal steroid 12 alpha-hydroxylase.

The sterols 7 alpha-hydroxycholest-4-en-3-one (I) and 5 alpha-cholestane-3 alpha,7 alpha-diol (II) are competitive inhibitors for rabbit hepatic microsomal preparations of steroid 12 alpha-hydroxylase with apparent Ki values of 56 and 93 microM, respectively. To ascertain the optimum structure for a substrate with maximal enzymic activity, nine sterols or steroidal acids containing the 7 alpha-hydroxy-4-en-3-one or 3 alpha,7 alpha-dihydroxy-5 alpha configuration were prepared and studied as inhibitors with enzyme preparations in the presence of NADPH, oxygen and appropriate cofactors. Although each of these compounds exhibited competitive inhibition, the best inhibitor for sterol (I) was 7 alpha,25-dihydroxycholest-4-en-3-one (IV) (Ki 36 microM). Steroidal acids (3-oxo-7 alpha-hydroxychol-4-enoic acid and 3-oxo-7 alpha-hydroxy-4-cholene-24-carboxylic acid) were poor inhibitors (Ki 1080 and 654 microM, respectively). For sterol (II) the best inhibitors were sterol (IV) (Ki 35 microM) and 5 alpha-cholestane-3 alpha,7 alpha,25-triol (VIII) (Ki 45 microM). The 12 alpha-hydroxylated products of sterols (I) and (IV) were less tightly bound to the enzyme (Ki 88 and 98 microM, respectively) in the presence of sterol (II). Allochenodeoxycholic acid (Ki 495 microM) was not a good inhibitor for sterol (II). 12 alpha-Hydroxylated products of sterols (IV) and (VIII) were isolated from larger scale incubations, separated by HPLC and identified by mass spectrometry.

Animals↗
PubMed1979

Hepatic cholesterol metabolism in normo- and hyperlipidemic patients with cholesterol gallstones.

In vivo studies have shown abnormalities in cholesterol and bile acid metabolism in primary hyperlipoproteinemia (HLP). The aim of the present investigation was to determine if the increased production of cholesterol in HLP type IV can be attributed to a correspondingly high level of the hepatic 3-hydroxy-3-methylglutaryl (HMG) CoA reductase activity and if the low cholic acid: chenodeoxycholic acid synthesis ratio in HLP type II is due to some hydroxylase deficiency. Liver biopsies from 26 normolipidemic and 25 hyperlipidemic (10 type IIa, 6 type IIb, and 9 type IV) patients undergoing elective cholecystectomy were assayed for HMG CoA reductase activity, 12 alpha-hydroxylase activity, and 25-hydroxylase activity. The HMG CoA reductase activity was normal in HLP type IIa and type IIb and was increased about twice HLP type IV (P less than 0.001). The 12 alpha- and 25-hydroxylase activities were normal in all groups of patients. The results are compatible with a normal cholesterol synthesis in the liver in HLP type II. A reduced 12 alpha- or 25-hydroxylase activity cannot explain the low production of cholic acid relative to chenodeoxycholic acid in this type of HLP. The elevated HMG CoA reductase activity found in the liver of type IV patients may, however, be part of the explanation for the elevated synthesis of cholesterol often seen in these patients.

Adult↗
PubMed1979

Competitive inhibitors of rabbit hepatic microsomal 12 alpha-steroid hydroxylase.

Rabbit hepatic microsomal 12 alpha-steroid hydroxylase which is stable to storage at -70 degrees C in the pellet form was assayed for activity with [5 alpha,6 alpha-3H2]cholestane-3 alpha,7 alpha-diol solubilized with Tween 80 since methanol was incapable of maintaining the sterol in aqueous solution. Under optimized conditions in phosphate buffer, pH 7.4, containing nicotinamide, magnesium chloride, and NADPH, the enzyme conversion appeared linear for the initial 10 min. The rate of hydroxylation was proportional to protein concentration up to 4 mg/ml. Apparent Km and Vmax were 71 microM and 323 pmol of product/mg of protein/min. Based on the known structural requirements of the enzyme system, competitive inhibitors were prepared with the C-12 position derivatized as an alkene, hydroxyl, or oxo functional group. A Dixon plot revealed that 5 alpha-cholest-11-ene-3 alpha,7 alpha,26-triol was the best inhibitor with an apparent Ki of 26 microM.

Animals↗
PubMed1985

Assays for cholesterol 7 alpha-hydroxylase and 12 alpha-hydroxylase using high performance liquid chromatography.

Rapid and accurate assay methods for cholesterol:NADPH oxidoreductase (EC 1.14.13.17, 7 alpha-hydroxylating) and 7 alpha-hydroxy-4-cholesten-3-one 12 alpha-hydroxylase (enzyme not yet registered) are described. 7 alpha-Hydroxylase utilizes the endogenous cholesterol of liver microsomes as substrate. The reaction products were separated by high performance liquid chromatography monitored at 214 nm. Much higher activity was obtained with the method compared to literature values, which were obtained using externally added radioactive cholesterol as the substrate. The 12 alpha-hydroxylase activity was measured using non-radioactive steroid as the substrate. The reaction products were separated by the chromatography and detected at 240 nm. Comparable activities were obtained by this method compared to those that were obtained using radioactive substrate.

Animals↗
PubMed1988

Effect of streptozotocin-induced diabetes on the activity of 7 alpha-hydroxy-4-cholesten-3-one-specific 12 alpha-hydroxylase in rats.

The activity of 12 alpha-hydroxylase in hepatic microsomes from normal, streptozotocin-induced diabetic, and insulin-treated diabetic rats was studied with 7 alpha-hydroxy-4-cholesten-3-one as a substrate. In the diabetic rats, the 12 alpha-hydroxylase activity was about 50% lower than in the normal rats. Treatment of the diabetics with insulin cancelled the reduction of the activity. These results show that an insulin-deficient state causes a paradoxical decrease in the activity of the key enzyme for cholic acid formation.

Animals↗
PubMed1983

Regulation of hydroxylations in biosynthesis of bile acids: modulation of reconstituted 12 alpha-hydroxylase activity by protein fractions from rabbit liver cytosol and microsomes.

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.

Adenosine Triphosphate↗
PubMed1992

Purification and characterization of 7 alpha-hydroxy-4-cholesten-3-one 12 alpha-hydroxylase.

The isoform of cytochrome P450 that catalyzes the 12 alpha-hydroxylation of 7 alpha-hydroxy-4-cholesten-3-one, an intermediate in the conversion of cholesterol to cholic acid, was purified to homogeneity from rabbit liver microsomes. The extent of purification in the various steps was judged by an assay involving high performance liquid chromatography. The purified enzyme showed a single band on SDS-polyacrylamide gel electrophoresis (M(r) = 50,000). The NH2-terminal amino acid sequence is as follows: Val-Leu-Trp-Gly-Leu-Leu-Gly-Ala-Leu-Leu-Met-Val-Met-Val-Gly-, which is different from that of any other P450s so far reported. The specific content of the enzyme was 13.3 nmol of cytochrome P450/mg of protein. Upon reconstitution with NADPH-cytochrome P450 reductase and cytochrome b5, the P450 enzyme showed a high activity of 12 alpha-hydroxylation with a turnover number of 36.6 min-1 at 37 degrees C. The omission of either cytochrome P450 or NADPH-cytochrome P450 reductase resulted in complete loss of activity, and the omission of cytochrome b5 resulted in 40% loss of activity. Antibodies prepared from mouse inhibited the 12 alpha-hydroxylase activity of rabbit liver microsomes about 90% and that of the rat liver microsomes 50%. The enzyme activity was not inhibited by other antibodies raised against other forms of P450 that catalyze different monooxygenation reactions toward xenobiotics or endogenous substrates. Anti-cytochrome b5 antibody inhibited the activity 40%, suggesting the functional role of this protein, and anti-reductase inhibited the activity almost completely. The microsomal enzyme activity was markedly elevated by starvation or streptozotocin administration to the animals. However, an immunoblotting experiment showed no correlation between the enzyme activity and the amount of protein, suggesting that post-translational modification may occur.

Amino Acid Sequence↗
PubMed1991

Characteristics of cholesterol 7 alpha-hydroxylase and 7 alpha-hydroxycholesterol hydroxylase activities of rodent liver.

A second cholesterol-derived metabolite in addition to 7 alpha-hydroxycholesterol was observed to be produced from endogenous microsomal cholesterol in the presence of hamster liver microsomal fractions and NADPH, when analyzed by HPLC using the method of Ogishima and Okuda (Anal Biochem 158: 228-232, 1986). However, only 7 alpha-hydroxycholesterol was produced in the presence of rat hepatic microsomal protein fractions and NADPH. The second metabolite was facilely produced when endogenous 7 alpha-hydroxycholesterol was incubated with hamster liver microsomes and NADPH, but not with rat liver microsomes. The second metabolite derived from either endogenous cholesterol or exogenous 7 alpha-hydroxycholesterol contained three hydroxyl groups as shown by mass spectrometric analysis. After oxidation of the 3 beta-ol group by cholesterol oxidase, the metabolite comigrated with 7 beta-hydroxycholest-3-one on normal phase HPLC, but was resolved from both 7 alpha- and 7 beta-hydroxycholest-3-one on reverse phase HPLC. The data indicate that the second metabolite is a hydroxylated product of 7 alpha-hydroxycholesterol, possibly cholest-5-ene-3 beta,7 alpha, 12 alpha-triol. Cholestyramine feeding increased production of both 7 alpha-hydroxycholesterol and its metabolite from endogenous cholesterol by 3-fold in hamster liver microsomes in vitro. However, the direct conversion of 7 alpha-hydroxycholesterol to the metabolite by hamster liver microsomes was not increased appreciably after cholestyramine feeding (20-30%). The hydroxylation of 7 alpha-hydroxycholesterol was similar in characteristics to cholesterol 7 alpha-hydroxylase activity in that it was dependent on NADPH, was inhibited by several known P450 inhibitors, and was affected by an inhibitory autobody elicited against rat hepatic NADPH: cytochrome P450 oxidoreductase. 5,6- and 7,8-Benzoflavone were poor inhibitors (IC50 approximately 1 mM) of cholesterol 7 alpha-hydroxylase activity in liver microsomes from cholestyramine-fed rats, but caused a striking enhancement of the 7 alpha-hydroxylase activity of liver microsomes from untreated rats in vitro. In contrast, 7,8-benzoflavone inhibited cholesterol 7 alpha-hydroxylase and 7 alpha-hydroxycholesterol hydroxylase activities of microsomes from normal and cholestyramine-fed hamsters. However, 5,6-benzoflavone stimulated cholesterol 7 alpha-hydroxylase activity in liver microsomes from normal and cholestyramine-fed hamsters, but inhibited 7 alpha-hydroxycholesterol hydroxylase activity by approximately 50%. These results suggest that hepatic cholesterol 7 alpha-hydroxylase and 7 alpha-hydroxycholesterol hydroxylase activities apparently involve multiple forms of cytochrome P450 in untreated and cholestyramine-treated hamsters.

Animals↗
PubMed1985

Bile acid synthesis during development. Mitochondrial 12 alpha-hydroxylation in human fetal liver.

Hydroxylation of 5 beta-[7 beta-3H]cholestane-3 alpha, 7 alpha-diol was studied in mitochondrial preparations from human fetal livers. The livers were obtained at legal abortions between weeks 14 and 24. In addition to hydroxylation in the 26-position, 5 beta-cholestane-3 alpha, 7 alpha-diol was hydroxylated in the 12 alpha-position. In one experiment, mitochondrial protein was solubilized and partially purified. Material with such chromatographic properties as those of cytochrome P450 showed 12 alpha-hydroxylase activity when combined with adrenodoxin and adrenodoxin reductase from bovine adrenal mitochondria. Because adrenodoxin and adrenodoxin reductase are components specific for mitochondrial hydroxylase systems, the results exclude microsomal contamination as the origin of this 12 alpha-hydroxylase activity. Further, there was no hydroxylase activity when NADPH-cytochrome P450 reductase from rat liver microsomes was added instead of adrenodoxin and adrenodoxin reductase. The microsomal fraction of fetal liver was also shown to possess 12 alpha-hydroxylase activity. Microsomal and mitochondrial hydroxylase activities per milligram of protein towards 5 beta-cholestane-3 alpha, 7 alpha-diol were of the same order of magnitude. The occurrence of an efficient sterol nucleus hydroxylase activity in liver mitochondria appears to be unique for fetal liver.

Adrenodoxin↗
PubMed1992

12 alpha-hydroxylase activity in human liver and its relation to cholesterol 7 alpha-hydroxylase activity.

Interruption of the enterohepatic circulation by cholestyramine causes a several-fold increase in bile acid synthesis, reflected in a stimulation of cholesterol 7 alpha-hydroxylase activity; the synthesis of cholic acid being stimulated to a greater extent than chenodeoxycholic acid. It is not known if this preferential increase in cholic acid is due to an increase of the 12 alpha-hydroxylase activity. The present study aimed at investigating the 12 alpha-hydroxylase activity and its relation to cholesterol 7 alpha-hydroxylase activity in liver microsomes of patients with different levels of cholesterol 7 alpha-hydroxylase activity. Liver biopsies were obtained from four gallstone-free patients, and seven untreated and two cholestyramine-treated gallstone patients undergoing cholecystectomy, and four patients with Crohn's disease undergoing intestinal resection. The combined group of cholestyramine-treated and ileum-resected patients had four times higher cholesterol 7 alpha-hydroxylase activity and two times higher 12 alpha-hydroxylase activity than the other patients. A positive correlation was obtained between cholesterol 7 alpha-hydroxylase activity and 12 alpha-hydroxylase activity (r = +0.69; n = 16). These results indicate that the increased ratio between the synthesis of cholic acid and chenodeoxycholic acid during cholestyramine treatment is due to a compensatory increase of the 12 alpha-hydroxylase activity.

Adult↗
PubMedDate not supplied

12alpha- and 7alpha-hydroxysteroid dehydrogenase activities from Fusobacterium spp.

On screening fecal organisms for hydroxysteroid dehydrogenase activities applicable to bile acid metabolism studies, we have isolated a gram negative "Bacteroides-like" anaerobe which yields both 12alpha- and 7alpha-hydroxysteroid dehydrogenase (HSDH) activities in cell-free preparation. At the optimal harvest time of 36 hours, approximately 4500 units 12alpha-HSDH and 360 units 7alpha-HSDH were produced per 10(10) viable cells. The two enzymes appear to be separate entities in the basis of their stabilities on freezing, and prolonged storage at room temperature and elution volumes on Sephadex G 200. Thin layer chromatography studies on oxidation products confirmed the respective sites of oxidation to be the 12alpha-OH and 7alpha-OH position. No 3alpha-OH oriented activity was measurable. Preliminary kinetic studies of the 12alpha-HSDH revealed a broad pH curve with optimal activity at pH 9.5. Michaelis constants for glycodeoxycholate and NADP were estimated at 1.5 x 10(-4)M and 3.3 x 10(-5)M respectively.

Animals↗
PubMed1983

Evidence for a lack of regulatory importance of the 12 alpha-hydroxylase in formation of bile acids in man: an in vivo study.

The possibility that the 12 alpha-hydroxylase involved in formation of bile acids is of regulatory importance for the ratio between cholic acid and chenodeoxycholic acid in bile was studied with an in vivo technique. [4-14C]7 alpha-Hydroxy-4-cholesten-3-one and [6 beta-3H]7 alpha, 12 alpha-dihydroxy-4-cholesten-3-one were synthesized, and a mixture of these two bile acid intermediates was administered intravenously in five healthy subjects and in one patient with severe liver cirrhosis. The patient with liver cirrhosis was included in the study because of a considerable reduction in biosynthesis of cholic acid. Since the [4-14C]-labeled steroid is an intermediate just proximal to and since the [6 beta-3H]-labeled steroid is an intermediate just distal to the 12 alpha-hydroxylase step, the 3H/14C ratio in the cholic acid formed should reflect the relative 12 alpha-hydroxylase activity. The 3H/14C ratio varied between 1.8 and 3.9 in the cholic acid isolated from the healthy subjects and was 3.6 in the cholic acid isolated from the patient with liver cirrhosis. The ratio between cholic acid and chenodeoxycholic acid varied between 0.6 and 3.9 in the bile from the control subjects and was only 0.4 in the bile from patients with liver cirrhosis. There was no correlation between the 3H/14C ratios and the ratios between cholic acid and chenodeoxycholic acid in bile.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗