Implications of cholesterol autoxidation products in the pathogenesis of inflammatory diseases.

There is rising interest in non-enzymatic cholesterol oxidation because the resulting oxysterols have biological activity and can be used as non-invasive markers of oxidative stress in vivo. The preferential site of oxidation of cholesterol by highly reactive species is at C7 having a relatively weak carbon-hydrogen bond. Cholesterol autoxidation is known to proceed via two distinct pathways, a free radical pathway driven by a chain reaction mechanism (type I autoxidation) and a non-free radical pathway (type II autoxidation). Oxysterols arising from type II autoxidation of cholesterol have no enzymatic correlates, and singlet oxygen ((1)ΔgO2) and ozone (O3) are the non-radical molecules involved in the mechanism. Four primary derivatives are possible in the reaction of cholesterol with singlet oxygen via ene addition and the formation of 5α-, 5ß-, 6α- and 6ß-hydroxycholesterol preceded by their respective hydroperoxyde intermediates. The reaction of ozone with cholesterol is very fast and gives rise to a complex array of oxysterols. The site of the initial ozone reaction is at the Δ5,6 -double bond and yields 1,2,3-trioxolane, a compound that rapidly decomposes into a series of unstable intermediates and end products. The downstream product 3ß-hydroxy-5-oxo-5,6-secocholestan-6-al (sec-A, also called 5,6-secosterol), resulting from cleavage of the B ring, and its aldolization product (sec-B) have been proposed as a specific marker of ozone-associated tissue damage and ozone production in vivo. The relevance of specific ozone-modified cholesterol products is, however, hampered by the fact sec-A and sec-B can also arise from singlet oxygen via Hock cleavage of 5α-hydroperoxycholesterol or via a dioxietane intermediate. Whatever the mechanism may be, sec-A and sec-B have no enzymatic route of production in vivo and are reportedly bioactive, rendering them attractive biomarkers to elucidate oxidative stress-associated pathophysiological pathways and to develop pharmacological agents.

Faecal steroid excretion in humans is affected by calcium supplementation and shows gender-specific differences.

BACKGROUND: Previous human studies on the effect of dietary calcium supplementation on faecal excretion of bile acids (BA) and faecal water concentrations of animal neutral sterols (NSt, cholesterol and its metabolites) lack detailed information about single BA and NSt. AIM OF THE STUDY: We investigated whether single BA and NSt in faeces and especially in faecal water are affected by calcium supplementation and whether this affects genotoxicity of faecal water. In addition, we differentiated between men and women with regard to the concentrations of BA and NSt in faecal water. METHODS: Thirty-one healthy volunteers consumed a calcium supplemented bread (1.0 g/day) and a placebo bread, respectively, for 4 weeks in a double-blind, randomised cross-over trial. Faeces were collected quantitatively for 5 days in the last week of each period. NSt and BA were analysed by GC-MS. RESULTS: Due to calcium supplementation faecal concentrations of lithocholic acid (LCA, 14%, P = 0.008), deoxycholic acid (DCA, 19%, P < 0.001) and 12 keto-deoxycholic acid (12 keto DCA, 29%, P = 0.049) significantly increased whereas BA concentrations in faecal water were only marginally affected. In contrast, concentrations of cholesterol (30%, P = 0.020) and its metabolites coprostanol (43%, P = 0.004), coprostanone (36%, P = 0.003), cholestanol (44%, P = 0.001) and cholestenone (32%, P = 0.038) in faecal water significantly decreased. Total NSt concentration in faecal water was found to be significantly higher in women compared to men (P = 0.018). The genotoxicity of faecal water was neither affected by calcium supplementation nor were there gender-specific differences. CONCLUSIONS: Dietary calcium supplementation diversely affects BA and NSt in faeces and in faecal water but does not influence the genotoxicity of faecal water in healthy adults.

[Analysis of steroids in crude drugs by capillary gas chromatography].

The steroids, cholest-4-ene-one and cholestane-3, 6-dion, were determined in 8 species of crude drugs of Haima and Hailong by capillary gas chromatography.

Identification of 27-nor-3 alpha, 7 alpha, 12 alpha-trihydroxycoprostan-24-one apparently derived from 3 alpha, 7 alpha, 12 alpha-trihydroxy-24-oxocoprostanoic acid, a postulated intermediate of bile acid biosynthesis.

Incubation of [27-13C]-3 alpha, 7 alpha, 12 alpha-trihydroxycoprost-24-en-26-oic acid with rat liver homogenate followed by 13C-NMR analysis of the incubation product has resulted in the identification of [26-13C]-27-nor-3 alpha, 7 alpha, 12 alpha-trihydroxycoprostan-24-one, supporting the idea that the substrate has been metabolized into 3 alpha, 7 alpha, 12 alpha-trihydroxy-24-oxocoprostan-26-oic acid CoA derivative.

Synthesis and 13C-NMR analysis of 5 alpha- and 5 beta-cholestane-3,6-dione.

Starting from cholesterol a simple and efficient synthesis of 5 alpha-cholestane-3,6-dione and 5 beta-cholestane-3,6-dione is described. The 13C shielding data of C-7, C-9, and C-19 in both isomers can be used in the determination of the stereochemistry at C-5 of these compounds. The combination of 13C NMR spectroscopy and the simple synthesis of both isomers offers good opportunities for the determination of the stereochemistry at C-5 of 3,6-dioxosteroids.

Gas-liquid chromatography of fecal neutral steriods.

A method is described for the analysis of fecal neutral steriods with a dual-column gas-liquid chromatography (GLC) system. After saponification of the fecal slurry, the neutral steroids were extracted with hexane. The GLC separation of the compounds and quantitation were achieved by simultaneous injection of the derivatized and derivatized aliquots of the extract onto dual colmuns under identical conditions. The neutral steroids of interest were than identified by matching the retention times with those of known standards, and identification was confirmed by use of an interfaced GLC high-resolution mass spectrometry system. The detection limit was 0.003 mg of steroid/g of fecal slurry. The pricision of the method is illustrated by a relative standard diviation of 2-10% and a recovery of neutral steroids from 73-96%. The method was applied to the determination of fecal neutral steroids in a "High protein diet in colon cancer study". A considerably larger level of coprostanone than of coprostanol was observed. Data on neutral steroids in fecal samples from subjects on different diets are the subject of a separate publication.