Metabolism of the cholestanol precursor cholesta-4,6-dien-3-one in different tissues

Cerebrotendinous xanthomatosis (CTX) is a lipid storage disease where the basic defect is a lack of the mitochondrial C27-steroid 26-hydroxylase involved in bile acid synthesis (EC 1.14.13.15). Cholestanol and cholesterol accumulate in all tissues. At least part of the accumulation of cholestanol is due to a 7 alpha-dehydroxylation of early bile acid intermediates. Cholesta-4,6-dien-3-one, a proposed intermediate in this pathway, is found in increased concentrations in serum of the patients. This study shows that cholesta-4,6-dien-3-one may be metabolized to 4-cholesten-3-one and cholestanol by liver, adrenals and brain. No conversion was found in intestinal mucosa or in kidneys. The capacity to convert cholesta-4,6-dien-3-one into 4-cholesten-3-one and cholestanol varied in different tissues as well as in different species. The results are discussed in relation to the cholestanol accumulation in CTX.     

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.     

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.     

On the substrate specificity of human CYP27A1: implications for bile acid and cholestanol formation

The mitochondrial sterol 27-hydroxylase (CYP27A1) is required for degradation of the C27-sterol side chain in bile acid biosynthesis. CYP27A1 seems, however, to have roles beyond this, as illustrated by patients with a deficient sterol 27-hydroxylase due to mutations of the CYP27A1 gene [cerebrotendinous xanthomatosis (CTX)]. These subjects have symptoms ranging from accumulation of bile alcohols and cholestanol to accelerated atherosclerosis and progressive neurologic impairment. The present work describes a detailed investigation on the substrate specificity of recombinant human CYP27A1. In accordance with some previous work with rat liver mitochondria, the activity in general increased with the polarity of the substrate. An obvious example was the finding that cholesterol was 27-hydroxylated more efficiently than cholesterol oleate but less efficiently than cholesterol sulfate. The oxysterols 24S-hydroxycholesterol and 25-hydroxycholesterol were 27-hydroxylated less efficiently than cholesterol, possibly due to steric hindrance. Surprisingly, sterols with a 3-oxo-Delta4 structure were found to be hydroxylated at a much higher rate than the corresponding sterols with a 3beta-hydroxy-Delta5 structure. The rates of hydroxylation of the sterols were: 7alpha-hydroxy-4-cholesten-3-one>4-cholesten-3-one>7alpha-hydroxycholesterol>24-hydroxy-4-cholesten-3-one> cholesterol>25-hydroxy-4-cholesten-3-one>24-hydroxycholesterol>or=25-hydroxycholesterol. The possibility is discussed that the findings may have implications for oxysterol-mediated regulation of gene expression. The very high activity of CYP27A1 towards the cholestanol precursor 4-cholesten-3-one may be of importance in connection with the accumulation of cholestanol in patients with CTX.     

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.     

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.     

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.     

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.     

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.     

Cerebrotendinous xanthomatosis: An inborn error in bile acid synthesis with defined mutations but still a challenge

Cerebrotendinous xanthomatosis [CTX] is a rare disease characterized by the accumulation of cholesterol and cholestanol in brain and tendons caused by a mutation in the sterol 27-hydroxylase gene [CYP27A1] involved in bile acid synthesis. Disruption of this gene in mice does not give rise to xanthomas. The gene defect leads to reduced bile acid synthesis with a compensatory increase in the activity of the rate-limiting enzyme in bile acid synthesis, cholesterol 7α-hydroxylase. This leads to a marked accumulation of 7α-hydroxylated bile acid precursors, in particular 7α-hydroxy-4-cholesten-3-one. The latter oxysterol passes the blood-brain barrier and is an efficient precursor to cholestanol. The activity of cholesterol 7α-hydroxylase is normalized by treatment with bile acids. Such treatment reduces the xanthomas in CTX patients in parallel with decreased cholestanol levels. The relationship between the accumulation of cholestanol and the development of cholesterol-rich xanthomas has however not been clarified and a suitable animal model is still lacking.     

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.     

On the mechanism of accumulation of cholestanol in the brain of mice with a disruption of sterol 27-hydroxylase

The rare disease cerebrotendinous xanthomatosis (CTX) is due to a lack of sterol 27-hydroxylase (CYP27A1) and is characterized by cholestanol-containing xanthomas in brain and tendons. Mice with the same defect do not develop xanthomas. The driving force in the development of the xanthomas is likely to be conversion of a bile acid precursor into cholestanol. The mechanism behind the xanthomas in the brain has not been clarified. We demonstrate here that female cyp27a1^−/− mice have an increase of cholestanol of about 2.5- fold in plasma, 6-fold in tendons, and 12-fold in brain. Treatment of cyp27a1^−/− mice with 0.05% cholic acid normalized the cholestanol levels in tendons and plasma and reduced the content in the brain. The above changes occurred in parallel with changes in plasma levels of 7α-hydroxy-4-cholesten-3-one, a precursor both to bile acids and cholestanol. Injection of a cyp27a1^−/− mouse with ²H₇-labeled 7α-hydroxy-4-cholesten-3-one resulted in a significant incorporation of ²H₇-cholestanol in the brain. The results are consistent with a concentration-dependent flux of 7α-hydroxy-4-cholesten-3-one across the blood-brain barrier in cyp27a1^−/− mice and subsequent formation of cholestanol. It is suggested that the same mechanism is responsible for accumulation of cholestanol in the brain of patients with CTX.     

Metabolism of potential precursors of chenodeoxycholic acid in cerebrotendinous xanthomatosis (CTX).

In patients with cerebrotendinous xanthomatosis (CTX), diminished cholic acid production is associated with incomplete oxidation of the cholesterol side chain and the excretion of C(25)-hydroxy bile alcohols. The aims of this investigation were 1) to provide quantitative information on the pool size and production rate of chenodeoxycholic acid by the isotope dilution technique; and 2) to investigate the possible existence of a block in chenodeoxycholic acid synthesis and explain the absence of chenodeoxycholic acid precursors in CTX. After the injection of [24-(14)C]chenodeoxycholic acid, measurements of chenodeoxycholic acid pool size and production rate in a CTX subject were, respectively, 1/20 and 1/6 as great as controls. Further, three potential precursors of chenodeoxycholic acid, namely [G-(3)H]7alpha-hydroxy-4-cholesten-3-one, [G-(3)H]5beta-cholestane-3alpha,7alpha,25-triol, and [G-(3)H]5beta-cholestane-3alpha,7alpha,26-triol, were administered to the CTX and control subjects and the specific activity curves of [G-(3)H]cholic acid and [G-(3)H]chenodeoxycholic acid were constructed and compared. In the control subjects, the two bile acids decayed exponentially, but in the CTX patient maximum specific activities were abnormally delayed, indicating the hindered transformation of precursor into bile acid. These results show that chenodeoxycholic acid synthesis is small in CTX and that the conversion of 7alpha-hydroxy-4-cholesten-3-one, 5beta-cholestane-3alpha,7alpha,25-triol, and 5beta-cholestane-3alpha,7alpha,26-triol to both chenodeoxycholic acid and cholic acid were similarly impaired.     

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.     

Disrupted coordinate regulation of farnesoid X receptor target genes in a patient with cerebrotendinous xanthomatosis

Cerebrotendinous xanthomatosis (CTX), sterol 27-hydroxylase (CYP27A1) deficiency, is associated with markedly reduced chenodeoxycholic acid (CDCA), the most powerful activating ligand for farnesoid X receptor (FXR). We investigated the effects of reduced CDCA on FXR target genes in humans. Liver specimens from an untreated CTX patient and 10 control subjects were studied. In the patient, hepatic CDCA concentration was markedly reduced but the bile alcohol level exceeded CDCA levels in control subjects (73.5 vs. 37.8 +/- 6.2 nmol/g liver). Cholesterol 7alpha-hydroxylase (CYP7A1) and Na+/taurocholate-cotransporting polypeptide (NTCP) were upregulated 84- and 8-fold, respectively. However, small heterodimer partner (SHP) and bile salt export pump were normally expressed. Marked CYP7A1 induction with normal SHP expression was not explained by the regulation of liver X receptor alpha (LXRalpha) or pregnane X receptor. However, another nuclear receptor, hepatocyte nuclear factor 4alpha (HNF4alpha), was induced 2.9-fold in CTX, which was associated with enhanced mRNA levels of HNF4alpha target genes, CYP7A1, 7alpha-hydroxy-4-cholesten-3-one 12alpha-hydroxylase, CYP27A1, and NTCP. In conclusion, the coordinate regulation of FXR target genes was lost in CTX. The mechanism of the disruption may be explained by a normally stimulated FXR pathway attributable to markedly increased bile alcohols with activation of HNF4alpha caused by reduced bile acids in CTX liver.     

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.     

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.     

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.     

The effect of some analogues of disulfiram on the aldehyde dehydrogenases of sheep liver.

1. The liver microsomal metabolism of [4-14C]cholesterol, endogenous cholesterol, 7 alpha-hydroxy-4-[6 beta-3H]cholesten-3-one, 5-beta-[7 beta-3H]cholestane-3 alpha, 7 alpha-diol and [3H]lithocholic acid was studdied in control and clofibrate (ethyl p-chlorophenoxyisobutyrate)-treated rats. 2. The extent of 7 alpha-hydroxylation of exogenous [414C]cholesterol and endogenous cholesterol, the latter determined with a mass fragmentographic technique, was the same in the two groups of rats. The extent of 12 alpha-hydroxylation of 7 alpha-hydroxy-4-cholesten-3-one and 5 beta-cholestane-3 alpha, 7 alpha-diol was increased by about 60 and 120% respectively by clofibrate treatment. The 26-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha-diol was not significantly affected by clofibrate. The 6 beta-hydroxylation of lithocholic acid was about 80% higher in the clofibrate-treated animals than in the controls. 3. The results are discussed in the context of present knowledge about the liver microsomal hydroxylating system and bile acid formation in patients with hypercholesterolaemia, treated with clofibrate.     

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.