A comparative study of the conversion of 7-hydroxycholesterol in rabbit,guinea pig,rat, hamster,and chicken

The metabolism of epimeric 7-hydroxycholesterol was studied in vitro. 7Alpha-hydroxycholesterol or 7beta-hydroxycholesterol were incubated with rabbit, guinea pig, rat, hamster, and chicken microsomal suspensions and then extracted and analyzed using high-performance liquid chromatography (HPLC). 7Alpha-hydroxy-4-cholesten-3-one was the main product from 7alpha-hydroxycholesterol in the rabbit, guinea pig, and rat. A considerable amount of 7-ketocholesterol was also produced in the hamster and chicken. In all vertebrates, 7beta-hydroxycholesterol was converted only to 7-ketocholesterol in all vertebrates. 7Beta-hydroxy-4-cholesten-3-one was not detected. Reduction of 7-ketocholesterol was also studied in the rat and hamster. Whereas 7-ketocholesterol was converted to 7beta-hydroxycholesterol in the rat, it was converted to both 7alpha- and 7beta-hydroxycholesterol in the hamster. These results suggest that 7alpha-hydroxycholesterol is converted not only to 7alpha-hydroxy-4-cholesten-3-one but also to 7-ketocholesterol in the hamster and chicken. 7Beta-hydroxycholesterol was converted to 7-ketocholesterol in all vertebrates tested. The interconversion between 7alpha- and 7beta-hydroxycholesterol via 7-ketocholesterol was observed in the hamster in this in vitro study.     

Biosynthesis of cholestanol from bile acid intermediates in the rabbit and the rat

Biliary 7 alpha-hydroxy-4-cholesten-3-one (an intermediate in bile acid biosynthesis) may be 7 alpha-dehydroxylated in the gut and further metabolized to cholestanol (Skrede, S., and Bj?rkhem, I. (1982) J. Biol. Chem. 257, 8363-8367). We have now evaluated the quantitative importance of pathway(s) to cholestanol with 7 alpha-hydroxylated C27 steroids as intermediates. After feeding conventionally fed rabbits or rats or germ-free rats with [7 alpha-3H]cholesterol and [4-14C]cholesterol, tissue cholestanol could be isolated with about a 20% lower 3H/14C ratio than present in cholesterol. We conclude that there is a pathway to cholestanol involving 7 alpha-hydroxylated intermediates. Intestinal microorganisms are not essential for this pathway, which accounts for at most 20% of the cholestanol formed in these species. In bile fistula rats, there was also a significant conversion of intraperitoneally injected [7 beta-3H]7 alpha-hydroxycholesterol and [4-14C]7 alpha-hydroxy-4-cholesten-3-one into cholestanol. The enzymes involved in the 7 alpha-hydroxylation/dehydroxylation pathway for the biosynthesis of cholestanol are probably located in the liver. Both 7 alpha-hydroxycholesterol and 7 alpha-hydroxy-4-cholesten-3-one may be intermediates.     

Conversion of 7alpha-hydroxycholesterol and 7alpha-hydroxy-beta-sitosterol to 3alpha, 7alpha-dihydroxy- and 3alpha, 7alpha, 12alpha-trihydroxy-5beta-steroids in vitro.

The metabolism of 7alpha-hydroxycholesterol and 7alpha-hydroxy-beta-sitosterol (24alpha-ethyl-5-cholestene-3beta,7alpha-diol) has been compared in rat liver subcellular fractions. 7alpha-Hydroxy-beta-sitosterol was shown to be metabolized in the same manner as 7alpha-hydroxycholesterol. Thus, the following C29 metabolites have been identified: 24alpha-ethyl-7alpha-hydroxy-4-cholesten-3-one, 24alpha-ethyl-7alpha,12alpha-dihydroxy-4-cholesten-3-one, 24alpha-ethyl-7alpha-hydroxy-5beta-cholestan-3-one, 24alpha-ethyl-5beta-cholestane-3alpha,7alpha-diol, 24alpha-ethyl-7alpha,12alpha-dihydrozy-5beta-cholestan-3-one, and 24alpha-ethyl-5beta-cholestane-3alha,7alpha,12alpha-triol. The C29 compounds were generally less efficient substrates. The most pronounced difference was noted for the delta4-3-oxosteroid 5beta-reductase. Thus, 7alpha-hydroxy-4-cholesten-3-one was three to four times as efficiently reduced as the C29 analog. The oxidation of the 3beta,7alpha-dihydroxy-delta5-steroid to the 7alpha-hydroxy-delta4-3-oxosteroid, the 12alpha-hydroxylation of the 7alpha-hydroxy-delta4-3-oxosteroid, and the reduction of the 7alpha-hydroxy-5beta-3-oxosteroid to the 3alpha,7alpha-dihydroxy-5beta-steroid occurred in up to two times better yields for the C27 steroids.     

Novel LC-MS/MS method for assay of 7alpha-hydroxy-4-cholesten-3-one in human plasma. Evidence for a significant extrahepatic metabolism

A new isotope dilution LC-MS/MS method for assay of 7alpha-hydroxy-4-cholesten-3-one without need for derivatization is described. This method was used in catheterization experiments on healthy fasting volunteers. The levels of this generally used marker for bile acid synthesis were slightly but significantly higher in the hepatic vein than in the brachial artery. In contrast, the levels of the precursor to 7alpha-hydroxy-4 cholesten-3-one, 7alpha-hydroxycholesterol, were the same in the two vessels. It is concluded that there is a net extrahepatic metabolism of 7alpha-hydroxy-4-cholesten-3-one. The similarity and very high correlation between the levels in the two vessels (r=0.97) are consistent with the contention that 7alpha-hydroxy-4-cholesten-3-one is a suitable marker for the activity of the hepatic cholesterol 7alpha-hydroxylase and thus bile acid synthesis.     

On the origin of the cholestenoic acids in human circulation

3 Beta-hydroxy-5-cholestenoic acid, 3 beta,7 alpha-dihydroxy-5-cholestenoic acid, and 7 alpha-hydroxy-3-oxo-4-cholestenoic acid are metabolites of cholesterol present at significant concentrations (40-80 ng/ml) in human circulation. The 7 alpha-hydroxylated acids may be formed from cholesterol via two major pathways initiated by oxidations at either the 7 alpha- or 27-positions. In an attempt to clarify the origin and possible precursor-product relationships between these cholestenoic acids, we measured their deuterium enrichment in a unique experiment, after infusion of 10 g of [2H(6)]-cholesterol to a healthy volunteer. The observed extent and time-course of deuterium enrichment of circulating 3 beta-hydroxy-5-cholestenoic and 3 beta,7 alpha-dihydroxy-5-cholestenoic acid were almost identical, while different from that of cholesterol and 7 alpha-hydroxycholesterol. Notably, the deuterium enrichment of 7 alpha-hydroxy-3-oxo-4-cholestenoic acid was similar to that of 7 alpha-hydroxycholesterol (and its metabolite 7 alpha-hydroxy-4-cholesten-3-one), though distinct from the other cholestenoic acids. Finally, the enrichment of unesterified 27-hydroxycholesterol followed a similar, though less pronounced, time curve to the delta(5)-cholestenoic acids. In conclusion, these results suggest that plasma 3 beta-hydroxy-5-cholestenoic acid is formed from a pool of cholesterol distinct from that used for the formation of the bulk of 27-hydroxycholesterol. The results are also in accordance with a formation of 3 beta,7 alpha-dihydroxy-5-cholestenoic acid directly from 3 beta-hydroxy-5-cholestenoic acid, and a formation of most of the circulating 7 alpha-hydroxy-4-cholesten-3-one from 7 alpha-hydroxycholesterol. These results are consistent with a flux of 7 alpha-hydroxycholesterol from the liver into the circulation, and an extrahepatic metabolism of this steroid into 7 alpha-hydroxy-3-oxo-4-cholestenoic acid.     

Hepatic 7 alpha-dehydroxylation of bile acid intermediates, and its significance for the pathogenesis of cerebrotendinous xanthomatosis

The bile acid precursor 7 alpha-hydroxy-4-cholesten-3-one was found to be enzymatically dehydroxylated at a slow rate by liver tissues from the rat, human, and guinea pig. The rat liver enzyme is localized in the microsomal fraction, has a pH optimum of about 8.5, an apparent Km of 0.03-0.04 mM, and a Vmax of 10-15 nmoles.mg protein-1.hr-1. The product from 7 alpha-hydroxy-4-cholesten-3-one was identified as cholesta-4,6-dien-3-one by its chromatographic properties and by mass spectrometry. The reaction proceeded both in air and N2, and pyridine nucleotides were not required as cofactors. In addition to the enzymatic reaction, there was a significant nonenzymatic dehydroxylation of 7 alpha-hydroxy-4-cholesten-3-one, in particular at high pH and with high concentrations of protein. No 7 alpha-dehydroxylation occurred with various 7 alpha-hydroxylated 3 beta-hydroxy-delta 5-steroids. We have previously shown that at least part of the accumulation of cholestanol in cerebrotendinous xanthomatosis (CTX) is due to accelerated 7 alpha-dehydroxylation of bile acid intermediate(s), which are further converted into cholestanol. The capacity to dehydroxylate 7 alpha-hydroxy-4-cholesten-3-one was found to be about the same in homogenates of liver biopsies from two patients with CTX as in preparations from control subjects. It is suggested that increased levels of substrate (7 alpha-hydroxy-4-cholesten-3-one) in the liver, rather than increased amounts of 7 alpha-dehydroxylase is the explanation for the accelerated 7 alpha-dehydroxylation in CTX that leads to increased biosynthesis of cholestanol.     

An improved method for assay of cholesterol 7 alpha-hydroxylase activity

An improved assay method for cholesterol 7 alpha-hydroxylase which is accurate, sensitive, and yet still simple is described. The method consists of two parts: the first part is cholesterol 7 alpha-hydroxylation in liver microsomes utilizing cholesterol in situ as the substrate, and the second part is conversion of the product, 7 alpha-hydroxycholesterol, into 7 alpha-hydroxy-4-cholesten-3-one having an intense absorption at 240 nm by the action of cholesterol oxidase. The converted sterol is then analyzed by high-performance liquid chromatography. During the second enzyme reaction, the first enzyme reaction is halted and further metabolism of the product is prevented. In consequence, the method had more than 10-fold increase in the sensitivity than the previous one.     

Correlation between plasma levels of 7alpha-hydroxy-4-cholesten-3-one and cholesterol 7alpha-hydroxylation rates in vivo in hyperlipidemic patients

BACKGROUND/AIM: Hepatic bile acid synthesis is the main mechanism whereby the organism can degrade cholesterol. Plasma levels of 7alpha-hydroxy-4-cholesten-3-one have been reported to reflect bile acid synthesis and the expression or activity of the limiting enzyme of the main biosynthetic pathway, cholesterol 7alpha-hydroxylase. Aim of this study was to correlate the levels of this metabolite with the rates of cholesterol 7alpha-hydroxylation in vivo, a direct measurement of bile acid synthesis, in hyperlipidemic patients. DESIGN: Concentrations of 7alpha-hydroxy-4-cholesten-3-one were assayed by gas-liquid chromatography: mass spectrometry in plasma samples obtained in 18 patients with primary hyperlipoproteinemia who previously underwent determination of cholesterol 7alpha-hydroxylation rates in vivo by tritium release analysis. Both determinations were performed in basal conditions and after treatment with hypolipidemic drugs (the fibric acid derivatives gemfibrozil and bezafibrate, cholestyramine alone or associated with simvastatin). RESULTS: Changes in plasma 7alpha-hydroxy-4-cholesten-3-one profile closely reflected in vivo cholesterol 7alpha-hydroxylation rates during treatment with fibrates, cholestyramine and cholestyramine plus simvastatin. When plotting determinations from all studies (n=40), a very strict correlation was disclosed between plasma 7alpha-hydroxy-4-cholesten-3-one and cholesterol 7alpha-hydroxylation rates (r=0.81, P<0.001). CONCLUSIONS: Plasma 7alpha-hydroxy-4-cholesten-3-one closely mirrors measurements of cholesterol 7alpha-hydroxylation rates in vivo in hyperlipidemic subjects and therefore stands as a reliable marker of global bile acid synthesis. In view of the correlation observed, these data may help to interpret changes of plasma levels of this metabolite in terms of cholesterol balance quantification.     

Potential bile acid precursors in plasma--possible indicators of biosynthetic pathways to cholic and chenodeoxycholic acids in man

The plasma concentrations of 3 beta-hydroxy-5-cholestenoic acid, 3 beta,7 alpha-dihydroxy-5-cholestenoic acid and 7 alpha-hydroxy-3-oxo-4-cholestenoic acid have been compared with that of 7 alpha-hydroxy-4-cholesten-3-one in healthy subjects and in patients with an expected decrease or increase of the bile acid production. In controls and patients with liver disease, the level of 7 alpha-hydroxy-3-oxo-4-cholestenoic acid was positively correlated to that of 3 beta,7 alpha-dihydroxy-5-cholestenoic acid and not to that of 7 alpha-hydroxy-4-cholesten-3-one. In patients with stimulated bile acid formation the levels of the acids were not correlated to each other but there was a significant positive correlation between the levels of 7 alpha-hydroxy-3-oxo-4-cholestenoic acid and 7 alpha-hydroxy-4-cholesten-3-one. These findings indicate that the precursor of 7 alpha-hydroxy-3-oxo-4-cholestenoic acid differs depending on the activity of cholesterol 7 alpha-hydroxylase. Since the activity of this enzyme is reflected by the level of 7 alpha-hydroxy-4-cholesten-3-one in plasma the findings are compatible with a formation of 7 alpha-hydroxy-3-oxo-4-cholestenoic acid from 3 beta,7 alpha-dihydroxy-5-cholestenoic acid when the rate of bile acid formation is normal or reduced and from 7 alpha-hydroxy-4-cholesten-3-one under conditions of increased bile acid synthesis. In support of this interpretation, 7 alpha,26-dihydroxy-4-cholesten-3-one was identified at elevated levels in plasma from patients with ileal resection or treated with cholestyramine. The levels of 7 alpha,12 alpha-dihydroxy-4-cholesten-3-one were also higher than normal in these patients. Based on these findings and previous knowledge, a model is proposed for the biosynthesis of bile acids in man. Under normal conditions, two major pathways, one "neutral" and one "acidic" or "26-oxygenated", lead to the formation of cholic acid and chenodeoxycholic acid, respectively. These pathways are separately regulated. When the activity of cholesterol 7 alpha-hydroxylase is high, the "neutral" pathway is most important whereas the reverse is true when cholesterol 7 alpha-hydroxylase activity is low. In cases with enhanced activity of cholesterol 7 alpha-hydroxylase, the "neutral" pathway is connected to the "acidic" pathway via 7 alpha,26-dihydroxy-4-cholesten-3-one, whereas a flow from the acidic pathway to cholic acid appears to be of minor importance.     

Bile acids. LXXIII. Synthesis of analogs of 7 alpha-hydroxy-4-cholesten-3-one as substrates for hepatic steroid 12 alpha-hydroxylase

Analogs of 7 alpha-hydroxy-4-cholesten-3-one were prepared to ascertain structural features necessary for maximal activity of hepatic microsomal 12 alpha-steroid hydroxylase. Methyl 3 alpha,7 alpha-dihydroxy-5 beta-cholane-24-carboxylate derived from chenodeoxycholic acid was oxidized at C-3 with silver carbonate/Celite. The product was hydrolyzed and dehydrogenated with SeO2 to provide 3-oxo-7 alpha-hydroxy-4-cholene-24-carboxylic acid. 5 beta-Cholestane-3 alpha,7 alpha,25-triol and 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25-tetrol were similarly oxidized at C-3 and dehydrogenated to provide 7 alpha,25-dihydroxy-4-cholesten-3-one and 7 alpha,12 alpha,25-trihydroxy-4-cholesten-3-one, respectively. The products were characterized by thin-layer and gas chromatography, ultraviolet, infrared, proton resonance and mass spectrometry.     

Purification and characterization of delta 4-3-ketosteroid 5 beta-reductase

delta 4-3-Ketosteroid 5 beta-reductase was purified about 230-fold from 100,000 X g supernatant of rat liver homogenate using 7 alpha-hydroxy-4-cholesten-3-one as substrate throughout. The purified enzyme was electrophoretically homogeneous, and its molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 37,000 and that determined by gel filtration chromatography on calibrated Sephadex G-100 column was 37,200. The absorption spectrum of the purified enzyme showed only a peak at 276 nm due to aromatic amino acids, precluding the presence of a prosthetic group such as flavine in the molecule. The enzyme is highly labile in a low buffer concentration, but is markedly stabilized in the presence of 20% glycerol in 10 mM phosphate buffer. Higher buffer concentration such as 300 mM potassium phosphate buffer was also effective to prevent deterioration in the absence of glycerol, but the effect was somewhat lower compared to glycerol. The purified enzyme showed the activity toward a variety of substrates including testosterone, cortisol, cortisone, progesterone, 4-androstene-3,17-dione, 7 alpha-hydroxy-4-cholesten-3-one, and 7 alpha,12 alpha-dihydroxy-4-cholesten-3-one. The optimal pH for the 5 beta-reduction of 7 alpha-hydroxy-4-cholesten-3-one was 7.4, and the cofactor required for the reaction was NADPH, while NADH revealed no effect. The enzyme activity was inhibited by p-chloromercuribenzoate, but its inhibition was prevented by the presence of a reduced form of glutathione.     

Dehydroxylation of a 7 beta-hydroxy-C27 plant sterol in rat liver

(22R)-Cholest-5-ene-3 beta,7 alpha,22-triol and the isomeric (22R)-cholest-5-ene-3 beta,7 beta,2-triol were 7-dehydroxylated by rat liver microsomes, after addition of NAD+ to the incubations. The product from both sterols was identified as (22R)-22-hydroxycholesta-4,6-dien-3-one by gas chromatography-mass spectrometry. The overall conversion of the 7 alpha-compound had an apparent Vmax of 5 nmol/mg protein per h, about 3-times higher than that of the 7 beta-isomer. Km was about 0.018 mmol/l in both reactions. NAD+ was required for the 7-dehydroxylation to proceed, conceivably by serving as cofactor in formation of the intermediate 7 beta,2-dihydroxy-4-cholesten-3-one. EDTA had a stimulatory effect upon the product formation. 7 alpha-Dehydroxylation of the bile acid precursor 7 alpha-hydroxy-4-cholesten-3-one has been demonstrated in liver tissue, but a 7 beta-hydroxy-C27-steroid dehydroxylating enzyme has previously not been identified. The results are discussed in relation to the marked differences in effect on neoplastic growth by the two isomeric hydroxysterols.     

Differences in hepatic levels of intermediates in bile acid biosynthesis between Cyp27(-/-) mice and CTX

Cerebrotendinous xanthomatosis (CTX) is a rare, recessively inherited lipid storage disease characterized by a markedly reduced production of chenodeoxycholic acid and an increased formation of 25-hydroxylated bile alcohols and cholestanol. Patients with this disease are known to have mutations in the sterol 27-hydroxylase (Cyp27) gene. However, one study showed that mice with a disrupted Cyp27 gene did not have any CTX-related clinical or biochemical abnormalities. To explore the reason, hepatic cholesterol, cholestanol, and 12 intermediates in bile acid biosynthetic pathways were quantified in 10 Cyp27(-/-) and 7 Cyp27(+/+) mice, two CTX patients (untreated and treated with chenodeoxycholic acid), and four human control subjects by high resolution gas chromatography-mass spectrometry. Mitochondrial 27-hydroxycholesterol and 5beta-cholestane-3alpha,7alpha,12alpha,27-tetrol were virtually absent in both Cyp27(-/-) mice and CTX patients. In Cyp27(-/-) mice, microsomal concentrations of intermediates in the early bile acid biosynthetic pathway (7alpha-hydroxycholesterol, 7alpha-hydroxy-4-cholesten-3-one, 7alpha,12alpha-dihydroxy-4-cholesten-3-one, and 5beta-cholestane-3alpha,7alpha,12alpha-triol), 25-hydroxylated bile alcohols (5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol, 5beta-cholestane-3alpha,7alpha,12alpha,23R,25-pentol, and 5beta-cholestane-3alpha,7alpha,12alpha,24R, 25-pentol), and cholestanol were all significantly elevated compared with those in Cyp27(+/+) mice, although the levels were lower than those in untreated CTX patients. The intermediate levels in early bile acid biosynthesis were more elevated in male (16;-86% of CTX) than in female Cyp27(-/-) mice (7-30% of CTX). In contrast, 25-hydroxylated bile alcohol concentrations were not significantly different between male and female Cyp27(-/-) mice and were considerably lower (less than 14%) than those in CTX patients.These results suggest that 1) in Cyp27(-/-) mice, especially in females, classic bile acid biosynthesis via 7alpha-hydroxycholesterol is not stimulated as much as in CTX patients; and 2) formed 25-hydroxylated bile alcohols are more efficiently metabolized in Cyp27(-/-) mice than in CTX patients.     

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.     

On the mechanism of cerebral accumulation of cholestanol in patients with cerebrotendinous xanthomatosis

The most serious consequence of sterol 27-hydroxylase deficiency in humans [cerebrotendinous xanthomatosis (CTX)] is the development of cholestanol-containing brain xanthomas. The cholestanol in the brain may be derived from the circulation or from 7alpha-hydroxylated intermediates in bile acid synthesis, present at 50- to 250-fold increased levels in plasma. Here, we demonstrate a transfer of 7alpha-hydroxy-4-cholesten-3-one across cultured porcine brain endothelial cells (a model for the blood-brain barrier) that is approximately 100-fold more efficient than the transfer of cholestanol. Furthermore, there was an efficient conversion of 7alpha-hydroxy-4-cholesten-3-one to cholestanol in cultured neuronal and glial cells as well as in monocyte-derived macrophages of human origin. It is concluded that the continuous intracellular production of cholestanol from a bile acid precursor capable of rapidly passing biomembranes, including the blood-brain barrier, is likely to be of major importance for the accumulation of cholestanol in patients with CTX. Such a mechanism also fits well with the observation that treatment with chenodeoxycholic acid, which normalizes the level of the bile acid precursor, results in a reduction of cholestanol-containing xanthomas even in the brain.     

Levels of 7 alpha-hydroxy-4-cholesten-3-one in plasma reflect rates of bile acid synthesis in man

A method for analysis of 7 alpha-hydroxy-4-cholesten-3-one in plasma is described. Following solid-phase extraction/purification the compound is determined by high-performance liquid chromatography using a UV detector. The median concentration in healthy subjects was 12 ng/ml (range 3-40). The levels were lower in diseases associated with a low bile acid production: extrahepatic cholestasis, less than 1.5 ng/ml (range less than 0.9-3); liver cirrhosis less than 1.5 ng/ml (range less than 0.9-38), and higher in diseases associated with a high bile acid production: cholestyramine treatment, 188 ng/ml (range 54-477); ileal resection 397 ng/ml (range 128-750). The levels were essentially normal in patients with colon resection. The results are consistent with a strong positive correlation between the levels of 7 alpha-hydroxy-4-cholesten-3-one in plasma and the rate of bile acid synthesis.     

Isolation of 5 alpha-cholestane-3 beta, 7 alpha-diol from bile of patients with cerebrotendinous xanthomatosis. Inefficiency of this steroid as a precursor to cholestanol

Cerebrotendinous xanthomatosis is a rare, inherited disease characterized by defective bile acid biosynthesis as well as by accumulation of cholesterol and cholestanol. The mechanism behind the accumulation of cholestanol is unknown. Using combined gas chromatography-mass spectrometry, 5 alpha-cholestane-3 beta, 7 alpha-diol could be identified as a minor component in bile from two such patients. There were no significant amounts of this steroid in bile from control subjects. Most probably, the 5 alpha-cholestan-3 beta, 7 alpha-diol found is formed from 7 alpha-hydroxy-4-cholesten-3-one in the liver. 7 alpha-Hydroxy-1-cholesten-3-one, being a normal intermediate in bile acid biosynthesis, is known to accumulate in the liver and bile of patients with cerebrotendinous xanthomatosis, due to a defect of the mitochondrial 26-hydroxylase. The possibility was tested that (7 beta-3H)-labeled 5 alpha-cholestane-3 beta, 7 alpha-diol could be converted into cholestanol by a direct 7 alpha-dehydroxylation in the intestine. This conversion did not occur in rabbits, however, regardless of whether the labelled steroid was administered orally or intracoecally. It is concluded that 5 alpha-cholestane-3 beta, 7 alpha-diol is of little or no importance as a precursor to cholestanol in rabbits. Most probably, this is also the case in patients with cerebrotendinous xanthomatosis.     

Purification and characterization of cholesterol 7 alpha-hydroxylase from rat liver microsomes

Cholesterol 7 alpha-hydroxylase (cholesterol, NADPH: oxygen oxidoreductase, 7 alpha-hydroxylating, EC 1.14.13.17) was purified from liver microsomes of cholestryramine-fed male rats by using high-performance ion-exchange chromatography. The purified enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 52,000), and its dithionite-reduced CO complex exhibited an absorption maximum at 450 nm. The specific content of the enzyme was 9 nmol of cytochrome P-450/mg of protein. Upon reconstitution with NADPH-cytochrome P-450 reductase, the enzyme showed a high activity of cholesterol 7 alpha-hydroxylation with the turnover number of 50 min-1 at 37 degrees C. The reaction was inhibited neither by aminoglutethimide nor by metyrapone, but inhibited markedly by iodoacetamide and disulfiram. The reaction was also inhibited significantly by CO. The enzyme catalyzed hydroxylation of cholesterol with strict regio- and stereoselectivity and was inert toward other sterols which are intermediates in the conversion of cholesterol to bile acids, i.e. 7 alpha-hydroxy-4-cholesten-3-one (12 alpha-hydroxylation), 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol (25-hydroxylation), and taurodeoxycholate (7 alpha-hydroxylation). Unlike other cytochromes P-450 isolated from rat liver microsomes, the enzyme showed no activity toward testosterone and xenobiotics such as 7-ethoxycoumarin and benzo[a] pyrene. The NH2-terminal amino acid sequence of the enzyme was Met-Phe-Glu-Val(Ile)-Ser-Leu-, which was distinct from those of any other cytochromes P-450 of rat liver microsomes hitherto reported. These results indicate that the enzyme is a novel species of cytochrome P-450 so far not isolated from liver microsomes.     

Membrane properties of oxysterols. Interfacial orientation, influence on membrane permeability and redistribution between membranes

The membrane properties of cholesterol auto-oxidation products, 7-ketocholesterol, 7 beta-hydroxycholesterol, 7 alpha-hydroxycholesterol and 25-hydroxycholesterol were examined. Monolayer studies show that these oxysterols are perpendicularly orientated at the interphase. Only 7 beta-hydroxycholesterol and 7 alpha-hydroxycholesterol are tilted at low surface pressures. In mixed monolayers with dioleoylphosphatidylcholine, 7-ketocholesterol, 7 beta-hydroxycholesterol and 7 alpha-hydroxycholesterol show a condensing effect in this order, although to a lesser extent that that observed for cholesterol. In liposomes these oxysterols also reduce glucose permeability and in the same order as their condensing effect. On the other hand 25-hydroxycholesterol shows no condensing effect in monomolecular layers whereas glucose permeability in liposomes is enormously increased. The permeability increase is already maximal at 2.5 mol% 25-hydroxycholesterol. Differential scanning calorimetry experiments reveal that all four oxysterols tested reduce the heat content of the gel----liquid-crystalline phase transition. It is concluded that 7-ketocholesterol, 7 beta-hydroxycholesterol and 7 alpha-hydroxycholesterol have a cholesterol like effect, although less efficient than cholesterol, whereas 25-hydroxycholesterol showing no condensing effect acts as a spacer molecule. Packing defects in the hydrophobic core of the bilayer due to the presence of the C-25 hydroxyl group are believed to cause the permeability increase. The transfer of radiolabelled (oxy)sterols from the monolayer to lipoproteins or vesicles in the subphase was studied. The transfer rate increases in the following order 7-ketocholesterol, 7 beta-hydroxycholesterol, 7 alpha-hydroxycholesterol, 25-hydroxycholesterol. The difference in rate between 7-ketocholesterol and 25-hydroxycholesterol is 20-fold. A higher rate of transfer is observed in the presence of high density lipoproteins and small unilamellar vesicles. A transfer rate for cholesterol is hardly measurable under these conditions. The transfer measured is consistent with the involvement of a water-soluble intermediate.     

Assay method for mitochondrial sterol 27-hydroxylase with 7alpha-hydroxy-4-cholesten-3-one as a substrate in the rat liver

Mitochondrial sterol 27-hydroxylase (EC 1.14.13.15) is an important enzyme, not only in the formation of bile acids from cholesterol intermediates in the liver but also in the removal of cholesterol by side chain hydroxylation in extrahepatic tissues. The enzyme has been assayed by complicated methods using radiolabeled substrates or deuterium-labeled tracers. These methods may be inaccurate for measuring enzyme activity, because the amount of electron-transferring proteins may be insufficient for maximal velocity. To solve this problem, after solubilization of the enzyme from rat liver mitochondria with n-octyl-beta-d-glucopyranoside (OGP), we measured the enzyme activity by incubating the solubilized enzyme with saturated amounts of electron-transferring proteins. In our assay system, using 7alpha-hydroxy-4-cholesten-3-one (HCO) as a substrate, we could easily measure the product, 7alpha,27-dihydroxy-4-cholesten-3-one, with HPLC monitoring absorbance at 240 nm. The product formation was proportionate to the time up to 5 min and the protein concentration up to 0.5 mg of protein/ml. The maximal velocity of the enzyme was 1.1 nmol/min/mg of protein, which was 4- to 16-fold higher than previously reported values. A simple and accurate assay method for sterol 27-hydroxylase in rat liver mitochondria is herein described.