Effect of cholesta-3,5-dien-7-one on human liver aldehyde dehydrogenase

A recently isolated cholesterol oxidation product, cholesta-3,5-dien-7-one, which was present at high concentrations in fatty/cirrhotic alcoholic liver was identified as a potent endogenous inhibitor of the cytosolic, E1, isozyme of aldehyde dehydrogenase (EC 1.2.1.3). The oxysterol was a less potent inhibitor of mitochondrial, E2, isozyme. The inhibition of the E1 isozyme was irreversible on the IEF gels, upon dilution and with 33 microM 2-mercaptoethanol during activity assay. The calculated 1-50% values from the inhibition curves for the E1 isozyme were 5-10 microM and approx. 180 microM for the E2 isozyme. The E3 isozyme was not sensitive to the oxysterol. Judging from the Lineweaver-Burk plot, the inhibition of the E1 isozyme with a constant concentration of cholesta-3,5-dien-7-one (52 microM) appeared to be noncompetitive.     

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.     

Synthesis of 5alpha-pregna-1,20-dien-3-one

5ALPHA-Pregna-1,20-dien-3-one has been synthesized from 3beta-hydroxy-5alpha-pregnan-20-one by reaction of the tosylhydrazone with butyllithium to introduce the vinyl moiety. Oxidation of the alcohol and treatment of the anion of the resulting ketone with benzeneselenenyl chloride gave the 2-phenylseleno derivative. This afforded the conjugated enone on oxidative elimination.     

Mechanism and stereochemistry in the sequential enzymatic saturation of the two double bonds in cholesta-4,6-dien-3-one.

The mechanism and stereochemistry in connection with enzymatic conversion of cholesta-4,6-dien-3-one into cholestanol was studied. Rat and mouse liver microsomes are able to catalyze NADPH-dependent sequential saturation of the two double bonds. Evidence was obtained that the saturation of the delta 6-double bond includes transfer of a hydride ion from the B-side of the cofactor to the 7-position of the steroid (mainly 7 beta-position), followed by addition of a proton to the 6 alpha-position (mainly trans addition). The saturation of the delta 4-double bond includes transfer of a hydride ion from the B-side of the cofactor to the 5 alpha-position of the steroid followed by addition of a proton to the 4 beta-position (trans addition). The reduction of the 3-oxo group was found to involve transfer of a hydride ion from the B-side of the cofactor NADPH to the 3 alpha-position of the steroid. The results are in accord with the contention that the enzymatic saturation of the two double bonds involves a polarization of the 3-oxo group making C-7 electrophilic and C-6 nucleophilic in connection with the saturation of the delta 6-double bond and C-5 electrophilic and C-4 nucleophilic in connection with the saturation of the delta 4-double bond.     

The role of a cholesta-8,14-dien-3β-ol system in cholesterol biosynthesis

The biosynthesis of cholesterol from squalene and tritiated water is described. Degradation of the cholesterol indicated that C-15 may be involved in cholesterol biosynthesis. In accordance with this view it is shown that in the conversion of [2RS-(3)H(2)]mevalonic acid into cholesterol one of the hydrogen atoms at C-15 is removed. A mechanism for the removal of the 14alpha-methyl group in steroid biosynthesis that involves the labilization of a C-15 hydrogen atom is outlined. In accordance with the requirement of this scheme it is shown that 4,4'-dimethyl-cholesta-8,14-dien-3beta-ol is converted into cholesterol.     

5alpha-Pregna-1,20-dien-3-one and related compounds from a soft coral.

Four unusual pregnane derivatives, 5alpha-pregna-1,20-dien-3-one (1), 1,4,20-pregnatrien-3-one (3), 5alpha-pregn-20-en-3alpha-o1 o-acetate (4), and 5alpha-pregn-20-en-1alpha,3alpha-diol 3-acetate (6) have been isolated from an unidentified soft coral from Canton Island.     

Synthesis and testing of 17a beta-hydroxy-7 alpha-methyl-D-homoestra-4,16-dien-3-one: a highly potent orally active androgen

The title compound, 17a beta-hydroxy-7 alpha-methyl-D-homoestra-4,16-dien-3-one (3), was synthesized in five steps (17% overall yield) from 7 alpha-methylestrone methyl ether (5) and was found to possess oral androgenic activity, in excess of other known androgens, without using 17 alpha-alkyl substitution.     

Towards an ecdysone synthesis. 20 -acetoxy-5 -pregna-2,7-dien-6-one

Pregnenolone (3β-hydroxy-5-pregnen-20-one) was converted to 20β-acetoxy-3, 5-cyclo-5α-pregnan-6-one by existing procedures. This compound was ring opened with bromine, and the resultant 3, 5-dibromide was subsequently rearranged to the 3, 7-dibromide. Dehydrohalogenation of the latter gave 20β-acetoxy-5α-pregna-2, 7-dien-6-one. This substance is of interest as intermediate for the synthesis of ecdysone.     

Synthesis of 3 beta-hydroxy-5 alpha-cholest-8-en-7-one and 3 beta-hydroxy-5 alpha-cholest-8-en-11-one: evaluation as potential hypocholesterolemic agents

An efficient procedure for the chemical synthesis of 3 beta-hydroxy-5 alpha-cholest-8-en-7-one and 3 beta-hydroxy-5 alpha-cholest-8-en-11-one is described. These ketosterols have been shown to have possible significant hypocholesterolemic effects when fed to normal rats at a level of 0.15% in a laboratory chow diet. The diets containing the steroids caused significant decreases in food consumption which were associated with decreases in the rate of gain in body weight.     

Synthesis of [3alpha-3H]cholesta-5,8-dien-3beta-ol and tritium-labeled forms of other sterols of potential importance in the Smith-Lemli-Optiz syndrome

Five unsaturated sterols relevant to the Smith-Lemli-Opitz syndrome have been prepared in high radiochemical purity with a tritium label at the 3alpha position. Swern oxidation of cholesta-5,8-dien-3beta-ol and other unlabeled C27 sterols afforded the corresponding 3-ketosteroids, and reduction with tritiated NaBH4 gave the desired 3alpha-3H sterols, with double bonds at the delta(5,8), delta(5,8(14)), delta(6,8), delta(6,8(14)), and delta8 positions. High radiochemical purity of the tritiated sterols was demonstrated by normal phase, reversed phase, and silver-ion (Ag+) high-performance liquid chromatography (HPLC). In the course of this work, we developed a medium-pressure variant of Ag+-HPLC for purifying radiolabeled samples, documented significant isotopic fractionation of the 3alpha-tritiated sterols and their acetates on Ag+-HPLC, and discovered unexpected effects of a delta(8(14)) bond on the conformation of 3-keto-delta5-steroids. The synthetic and analytical methodologies described herein should provide a sound basis for investigating the origin and metabolism of sterols involved in the Smith-Lemli-Opitz syndrome and in late stages of cholesterol biosynthesis.