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At least 19 recordsLinked to original sources
PubMed1993

Oxidation/isomerization of 5-cholesten-3 beta-ol and 5-cholesten-3-one to 4-cholesten-3-one in pure sterol and mixed phospholipid-containing monolayers by cholesterol oxidase.

In this study we have examined the cholesterol oxidase (Streptomyces cinnamomeus) catalyzed conversion of either 5-cholesten-3 beta-ol or 5-cholesten-3-one into 4-cholesten-3-one in pure sterol or mixed phospholipid-containing monolayers at the air/buffer interface. The mean molecular area requirement of 5-cholesten-3-one in a pure monolayer was slightly smaller than the comparable area required by 5-cholesten-3 beta-ol (although the collapse pressure was markedly lower for 5-cholesten-3-one), and both sterols were about equally capable of condensing the lateral packing density of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine at a lateral surface pressure of 20 mN/m. Both sterols were converted by cholesterol oxidase to 4-cholesten-3-one, the reaction being faster with 5-cholesten-3-one as compared to 5-cholesten-3-beta-ol. When the temperature-dependency of the cholesterol oxidase catalyzed conversion of the sterols to 4-cholesten-3-one was examined, the Arrhenius activation energy was calculated to +30 kJ/mol and +27 kJ/mol for 5-cholesten-3 beta-ol and 5-cholesten-3-one, respectively, when the sterols were presented to the enzyme as pure sterol monolayers at a lateral surface pressure of 20 mN/m. With a mixed monolayer containing 40 mol% sterol and 60 mol% EPC, the corresponding activation energies were +107 kJ/mol and +96 kJ/mol for 5-cholesten-3 beta-ol and 5-cholesten-3-one, respectively. With the monolayer system used, it appeared that the over all rate-limiting step in the enzyme-catalyzed conversion of 5-en-sterols to 4-en-3-one was the desorption of the sterol molecules from the monolayer into the active site of the enzyme at the interface. This appeared to be true both with pure sterol monolayers as well as with mixed monolayers containing phosphatidylcholine.

Cholestenones↗
PubMed1984

Transformation of 4-cholesten-3-one and 7 alpha-hydroxy-4-cholesten-3-one into cholestanol and bile acids in cerebrotendinous xanthomatosis.

In order to determine whether cholestanol and bile acids are derived from the same precursor, key intermediates of both biosynthetic pathways beyond cholesterol were administered intravenously to a patient with cerebrotendinous xanthomatosis and to a control subject. After pulse-labeling with [4-14C]4-cholesten-3-one and [G-3H]7 alpha-hydroxy-4-cholesten-3-one, cholestanol, cholesterol, and the two primary bile acids, cholic acid and chenodeoxycholic acid were isolated from specimens of bile. Each compound was purified by thin-layer chromatography and conclusively identified by gas-liquid chromatography-mass spectrometry. In other studies, the in vitro formation of 4-cholesten-3-one from cholesterol was measured in hepatic microsomal fractions prepared from a subject with cerebrotendinous xanthomatosis and from 3 control individuals. In all subjects, cholic acid and chenodeoxycholic acid were labeled with tritium, but neither cholesterol nor cholestanol contained this isotope. In contrast, 14C was detected in the cholestanol fraction with trace amounts in chenodeoxycholic acid, cholic acid, and cholesterol. Hepatic microsomes prepared from liver biopsy specimens obtained from a subject with cerebrotendinous xanthomatosis produced three times more 4-cholesten-3-one than the controls. The results indicate that 4-cholesten-3-one was converted primarily into cholestanol and 7 alpha-hydroxy-4-cholesten-3-one into cholic acid and chenodeoxycholic acid. Neither ketonic steroid was transformed into cholesterol. The increased production of cholestanol in cerebrotendinous xanthomatosis may be accounted for by enhanced hepatic formation of 4-cholesten-3-one. 7 alpha-Hydroxy-4-cholesten-3-one is a precursor of bile acids, but not of cholestanol.

Adult↗
PubMed1986

Metabolism of 24-ethyl-4-cholesten-3-one and 24-ethyl-5-cholesten-3 beta-ol (sitosterol) after intraperitoneal injection in the rat.

14C-labeled C29- and C27-steroids were injected in rats, which were killed after 14 days. Phytosterols (C29) were excreted mainly as such, whereas C27-steroids were recovered essentially as water soluble metabolites. The total 14C-excretion was lower from 3-oxo, delta 4-steroids (5 alpha-stanol precursors) than from 3 beta-hydroxy,delta 5-steroids. 14C-Phytosterols were accumulated more than C27-steroids in liver, serum (mainly in HDL) and especially in adrenal glands and ovaries. In relation to serum, particularly the 5 alpha-stanols were enriched in the adrenal glands and ovaries. No striking lysosomal accumulation of any of the steroids was found.

Animals↗
PubMed1966

Biosynthesis of delta-7-cholesten-3-beta-ol, delta-5,7-cholestadien-3-beta-ol, and delta-5-cholesten-3-beta-ol by guinea pig intestinal mucosa in vitro.

Methods were developed for the separation and determination of the various 27-carbon sterols of intestinal mucosa by means of thin-layer chromatography. Scrapings of the mucosa of the small intestine of guinea pig and rat were shown to incorporate isotope from (14)C-labeled acetate and mevalonate into sterols in vitro. For each substrate this activity was lowest in mucosa from the proximal third of the small intestine and greatest in mucosa from the more distal regions of the small intestine. The total 27-carbon sterol content of guinea pig mucosa varied only slightly along the length of the small intestine, but the concentration of cholesterol was highest distally. More than 95% of the radioactivity incorporated from acetate-2-(14)C into 27-carbon sterols by guinea pig mucosa in 4 hr was recovered as lathosterol and 7-dehydrocholesterol; less than 5% was in cholesterol. The specific activities of the 27-carbon sterols were consistent with the concept that synthesis proceeds from lathosterol to 7-dehydrocholesterol to cholesterol.

Acetates↗
PubMed1982

Biosynthesis of cholestanol from intestinal 7 alpha-hydroxy-4-cholesten-3-one.

Using isotope dilution-mass spectrometry, it was shown that human bile contains significant amount of 7 alpha-hydroxy-4-cholesten-3-one, an intermediate in the major pathway for bile acid biosynthesis. In bile from 14 healthy subjects, the concentration was 0.14 +/- 0.01 micrograms/ml (mean +/- S.E.). Four bile samples collected from two patients with cerebrotendinous xanthomatosis contained considerably higher amounts of this steroid, 0.47-1.32 micrograms/ml. After oral administration of [4-14C]7 alpha-hydroxy-4-cholesten-3-one to rabbits, 14C-labeled cholestanol could be isolated from the intestinal wall, liver, and blood after 24 h. The label incorporated into the intestinal wall was about 10% of that obtained with [4-14C]cholesta-4,6-dien-3-one or [4-14C]4-cholesten-3-one as precursors. Labeled cholesta-4,6-dien-3-one and 4-cholesten-3-one could be isolated from the intestinal contents 12 h after feeding [4-14C]7 alpha-hydroxy-4-cholesten-3-one to rabbits. It is proposed that cholesta-4,6-dien-3-one and 4-cholesten-3-one are formed from 7 alpha-hydroxy-4-cholesten-3-one by the same mechanism as that involved in 7 alpha-dehydroxylation of primary bile acids. We suggest that biliary 7 alpha-hydroxy-4-cholesten-3-one may be a physiological precursor to cholestanol. The possibility is discussed that part of the increased formation of cholestanol in patients with cerebrotendinous xanthomatosis is due to excess biliary 7 alpha-hydroxy-4-cholesten-3-one or some metabolite of this steroid.

Animals↗
PubMed2002

Safety evaluation of phytosterol esters. Part 7. Assessment of mutagenic activity of phytosterols, phytosterol esters and the cholesterol derivative, 4-cholesten-3-one.

Phytosterol esters are phytosterols derived from vegetable oils following esterification to fatty acids. When phytosterols are added to foods, they inhibit the absorption of dietary and endogenous cholesterol and thereby reduce blood cholesterol concentrations. As part of a comprehensive programme of safety assessment, the mutagenic potential of phytosterols and phytosterol esters has been assessed in a bacterial mutation assay and an in vitro chromosome aberration assay. In addition, an in vitro mammalian cell gene mutation assay and two in vivo mutagenicity studies, namely rat bone marrow micronucleus and liver unscheduled DNA synthesis (UDS) assays, were conducted on phytosterol esters only. Phytosterols and phytosterol esters did not show any evidence of mutagenic activity in any of these assays. A breakdown product of cholesterol is 4-cholesten-3-one and thus the amount of 4-cholesten-3-one in the gut may increase following supplementation of foods with phytosterol-esters. 4-cholesten-3-one had been previously reported as mutagenic but, due to various shortcomings, these data could not be used to assess the mutagenic activity of 4-cholesten-3-one. The mutagenic activity of 4-cholesten-3-one and its major faecal by-products, 5beta-cholestan-3-one, was assessed in two in vitro assays, a bacterial mutation assay and an in vitro chromosome aberration assay. Neither 4-cholesten-3-one nor 5beta-cholestan-3-one showed evidence of mutagenic activity in these assays.

Animals↗
PubMed1996

27-hydroxylated low density lipoprotein (LDL) cholesterol can be converted to 7alpha,27-dihydroxy-4-cholesten-3-one (cytosterone) before suppressing cholesterol production in normal human fibroblasts. Evidence that an altered metabolism of ldl cholesterol can underlie a defective feedback control in malignant cells.

The formation of oxysterols in cultured human fibroblasts and their physiological roles as intracellular regulators of cholesterol production have been investigated. In the presence of low density lipoproteins (LDL), normal fibroblasts converted LDL cholesterol to 27hydroxycholesterol, which was further metabolized to 7alpha, 27-dihydroxycholesterol, 7alpha,27-dihydroxy-4-cholesten-3-one, and 7alpha-hydroxy-3-oxo-4-cholestenoic acid. Autooxidation products of cholesterol contaminating the lipoproteins were also metabolized in the cells. 7alpha-Hydroxycholesterol was converted to 7alpha-hydroxy-4-cholesten-3-one prior to 27-hydroxylation and further oxidation to 7alpha-hydroxy-3-oxo-4-cholestenoic acid. 7beta-Hydroxycholesterol and 7-oxocholesterol were 27-hydroxylated and then oxidized to C27-acids. Oxidation of the 7beta-hydroxy group also occurred. 25-Hydroxycholesterol was 7alpha-hydroxylated and further oxidized to 7alpha,25-dihydroxy-4-cholesten-3-one. 25-Hydroxylation of sterols was observed only under specific conditions. In contrast, only small amounts of oxysterols were formed in virus-transformed human fibroblasts when incubated with lipoproteins. This was due to very low activities of the 27- and 7alpha-hydroxylating enzymes. The rate of oxidation at C-3 was also decreased moderately. A defective suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase by LDL and autooxidation products of cholesterol observed in the transformed fibroblasts could be caused by the deficiencies of the sterol-metabolizing enzymes, since these cells responded normally to the sterol metabolites 7alpha,27-dihydroxy-4-cholesten-3-one, 7alpha, 25-dihydroxy-4-cholesten-3-one, and 27-hydroxy-7-oxo-cholesterol. These metabolites, which all possessed an oxo group with a conjugated double bond in the steroid nucleus and a hydroxyl group in the side chain, did not seem to require further metabolism in order to be active. An impaired response to LDL was also seen in other human tumor cells, including breast carcinoma, colonic carcinoma, and malignant melanoma cells. Common to all the malignant cells was an intracellular shortage of 7alpha, 27-dihydroxy-4-cholesten-3-one caused by a decreased formation or an increased metabolism.

Cell Line, Transformed↗
PubMedDate not supplied

Accumulation of 7 alpha-hydroxy-4-cholesten-3-one and cholesta-4,6-dien-3-one in patients with cerebrotendinous xanthomatosis: effect of treatment with chenodeoxycholic acid.

Evidence was recently presented that an essential part of the accumulation of cholestanol in patients with cerebrotendinous xanthomatosis is due to acceleration of a novel pathway, involving 7 alpha-hydroxylated intermediates in bile acid biosynthesis as precursors (J. Clin. Invest. 1985; 75:448-456). Such intermediates accumulate in patients with cerebrotendinous xanthomatosis due to lack of the mitochondrial 26-hydroxylase involved in the major pathway for bile acid biosynthesis. The new pathway may involve the following steps: 7 alpha-hydroxycholesterol----7 alpha-hydroxy-4-cholesten-3-one----cholesta-4,6- dien-3-one----4-cholesten-3-one----cholestanol. Accurate methods have been developed for assay of 7 alpha-hydroxy-4-cholesten-3-one and cholesta-4,6-dien-3-one in serum, based on isotope dilution-mass spectrometry. The serum levels of 7 alpha-hydroxy-4-cholesten-3-one as well as those of cholesta-4,6-dien-3-one were found to be markedly elevated in the three patients with cerebrotendinous xanthomatosis. Treatment of two of the patients with chenodeoxycholic acid reduced the serum levels of the two steroids by more than 80%. The concentration of cholestanol was reduced by 72% in one patient and by 48% in the other. The possibility is discussed that accumulation of cholestanol in patients with cerebrotendinous xanthomatosis is secondary to accumulation of 7 alpha-hydroxy-4-cholesten-3-one and cholesta-4,6-dien-3-one.

Brain Diseases↗
PubMed1994

Determination of 7 alpha-hydroxy-4-cholesten-3-one level in plasma using isotope-dilution mass spectrometry and monitoring its circadian rhythm in human as an index of bile acid biosynthesis.

A highly sensitive and specific method has been developed for determination of the level of 7 alpha-hydroxy-4-cholesten-3-one in plasma. This method is based on a stable isotope-dilution technique by gas chromatography-selected-ion monitoring mass spectrometry. 7 alpha-Hydroxy-4-cholesten-3-one was extracted from plasma by saltingout extraction, and then purified by serial solid-phase extractions. The extract was treated with O-methylhydroxyl-amine hydrochloride and then dimethylethylsilylated. The resulting methyloxime-dimethylethylsilyl ether derivative was quantified by gas chromatography-selected-ion monitoring mass spectrometry with a high-resolution mode. The plasma levels of 7 alpha-hydroxy-4-cholesten-3-one were correlated with the cholesterol 7 alpha-hydroxylase activity to a higher degree than those of any other form of 7 alpha-hydroxycholesterol (r = 0.84, n = 16, p < 0.0001). The present method was applied to monitor the circadian rhythm of 7 alpha-hydroxy-4-cholesten-3-one levels in human plasma. It was concluded that the plasma level of 7 alpha-hydroxy-4-cholesten-3-one is a useful index for the monitoring of bile acid biosynthesis in the human liver.

Adult↗
PubMed1993

Bacterial cholesterol oxidases are able to act as flavoprotein-linked ketosteroid monooxygenases that catalyse the hydroxylation of cholesterol to 4-cholesten-6-ol-3-one.

A new metabolite of cholesterol was found in reaction mixtures containing cholesterol or 4-cholesten-3-one as a substrate and extra- or intracellular protein extracts from recombinant Streptomyces lividans and Escherichia coli strains carrying cloned DNA fragments of Streptomyces sp. SA-COO, the producer of Streptomyces cholesterol oxidase. The new metabolite was identified as 4-cholesten-6-ol-3-one based on comparisons of its high-performance liquid chromatography, gas chromatography/mass spectrometry, infrared and proton-nuclear magnetic resonance spectra with those of an authentic standard. Genetic analyses showed that the enzyme responsible for the production of 4-cholesten-6-ol-3-one is cholesterol oxidase encoded by the choA gene. Commercially purified cholesterol oxidase (EC 1.1.3.6.) of a Streptomyces sp., as well as of Brevibacterium sterolicum and a Pseudomonas sp., and a highly purified recombinant Streptomyces cholesterol oxidase were also able to catalyse the 6-hydroxylation reaction. Hydrogen peroxide accumulating in the reaction mixtures as a consequence of the 3 beta-hydroxysteroid oxidase activity of the enzyme was shown to have no role in the formation of the 6-hydroxylated derivative. We propose a possible scheme of a branched reaction pathway for the concurrent formation of 4-cholesten-3-one and 4-cholesten-6-ol-3-one by cholesterol oxidase, and the observed differences in the rate of formation of the 6-hydroxy-ketosteroid by the enzymes of different bacterial sources are also discussed.

Biodegradation, Environmental↗
PubMed1976

Formation of bile acids in man. Metabolism of 7alpha-hydroxy-4-cholesten-3-one in normal subjects with an intact enterohepatic circulation.

The formation of bile acids in man is thought to involve a series of reactions in which the initial steps are the same for both cholic acid and chenodeoxycholic acid. The point of bifurcation of the pathway is postulated to occur after the formation of 7alpha-hydroxy-4-cholesten-3-one. To test the hypothesis that the entire synthesis of both bile acids proceeds through this intermediate we studied the metabolism of labeled 7alpha-hydroxy-4-cholesten-3-one in eight normal subjects with an intact enterohepatic circulation. If all the production of cholic acid and chenodeoxycholic acid takes place via 7alpha-hydroxy-4-cholesten-3-one, the areas under the specific decay curves of cholic acid and chenodeoxycholic acid should be identical following a single injection of this labeled intermediate. However, in 6 of the 8 subjects studied the area under the cholic acid specific activity decay curve was significantly less than the area under the chenodeoxycholic acid specific activity decay curve. These results that the production of cholic acid in man may not always involve the intermediate 7alpha-hydroxy-4-cholesten-3-one.

Adult↗
PubMed1984

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.

Animals↗
PubMed1990

Effect of 4-cholesten-3-one on lecithin-cholesterol acyltransferase activity and the lipid concentration in the serum of normocholesterolaemic and hypercholesterolaemic rats.

Male Wistar strain rats and PHHC (Prague hereditary hypercholesterolaemic) rats received an intraperitoneal injection of 4-cholesten-3-one for five days. Lecithin-cholesterol acyltransferase activity and total cholesterol, triglyceride and phospholipid levels were determined in their serum. A significant drop in the total cholesterol level was found in normocholesterolaemic Wistar rats after the administration of cholestenone. The serum triglyceride content remained unaltered and the phospholipid concentration showed a downward trend. Lecithin-cholesterol acyltransferase activity was also significantly reduced. In PHHC rats, no significant changes occurred in total cholesterol, triglyceride and phospholipid levels, or in lecithin-cholesterol acyltransferase activity, after the administration of 4-cholesten-3-one. A comparison of serum 4-cholesten-3-one concentrations in the two groups of experimental animals shows that the turnover time for this substance in hypercholesterolaemic rats is only half as long as in normocholesterolaemic rats.

Animals↗
PubMed1981

Effect of 4-cholesten-3-one on steroidogenesis and morphology of rat adrenal cortex.

The production of corticosteroids from endogenous precursors was decreased in adrenal glands of male rats fed a diet containing one per cent of 4-cholesten-3-one for 3 and 7 days. In contrast, the conversion of labelled progesterone into corticosteroids was only slightly affected as shown by the increased aldosterone to 18-hydroxydeoxycorticosterone ratio. Morphological method revealed lipoid adrenal hyperplasia developing progressively during the observation period. In the adrenal gland, the content of 4-cholesten-3-one rose slightly and, as shown in the experiment with labelled 4-cholesten-3-one, it can serve only as a weakly effective precursor of corticosteroid biosynthesis. Cholesterol content in the adrenals decreased and lipoid hyperplasia could not be explained therefore by cholesterol accumulation.

Adrenal Cortex↗
PubMed1991

The plasma level of 7 alpha-hydroxy-4-cholesten-3-one reflects the activity of hepatic cholesterol 7 alpha-hydroxylase in man.

Circulating levels of 7 alpha-hydroxy-4-cholesten-3-one have been compared with activities of the rate-limiting enzyme in bile acid synthesis, microsomal cholesterol 7 alpha-hydroxylase, measured in liver biopsies obtained from patients undergoing surgery for gallstone disease. Some patients were treated with cholestyramine or bile acids prior to operation in order to alter the feed-back inhibition of the enzyme. The levels of the sterol were similar in untreated patients and in patients treated with ursodeoxycholic acid (median concentration 17 and 13 ng/ml, respectively), and so were the activities of the enzyme (median activity 7.0 and 5.5 pmol/min/mg protein, respectively). The sterol levels and enzyme activities were significantly increased in patients treated with cholestyramine (91 ng/ml and 45 pmol/min/mg protein) and decreased in patients treated with chenodeoxycholic acid (less than 2.0 ng/ml and 0.7 pmol/min/mg protein). There was a strong positive correlation (r = 0.90, P less than 0.00001) between levels of 7 alpha-hydroxy-4-cholesten-3-one in plasma and the activities of cholesterol 7 alpha-hydroxylase in the whole patient group. The results show that analysis of 7 alpha-hydroxy-4-cholesten-3-one in plasma is a sensitive and convenient method to determine relative rates of bile acid production in man.

Adult↗