Protective effect of ebselen on cytotoxicity induced by cholestane-3 beta, 5 alpha, 6 beta-triol in ECV-304 cells

The protective effect of ebselen, with documented glutathione peroxidase-like activity and antioxidative and anti-inflammatory properties, on the cytotoxicity induced by oxysterol was investigated in ECV-304 cells with cholestane-3beta, 5alpha, 6beta-triol (3-triol), one of the most toxic oxysterols. 3-triol exhibited significant cytotoxicity to ECV-304 cells in dose- and time-dependent manners. Pre-incubations with ebselen at different concentrations for 4 h effectively inhibited the decreases of the cell viability and the intracellular thiols level induced by 3-triol; suppressed the 3-triol-caused increases of the GPx and NOS activities, the LDH leakage and MDA formation. The inhibition of ebselen to the generation of intracellular ROS induced by 3-triol was monitored by luminol-, lucigenin-derived chemiluminescence and DCFH-DA-derived fluorescence assays. Our results suggest that ebselen inhibited 3-triol-induced enhancement of intracellular ROS level and the cytotoxicity of 3-triol is contributed to, for the most part, an enhanced formation of intracellular O2.-; nevertheless, the mitochondria were not the main source of intercellular O2.- contributed to the cytotoxicity of 3-triol. Ebselen lost its high protection against 3-triol-induced injuries in the presence of GSH probably due to the formation of the ebselen-GSH adduct. In conclusion, our investigations provide new utility for ebselen as a prospective antiatherosclerotic in both clinical and non-clinical situations.     

Metabolism of bile alcohols in the perfused rabbit liver.

The mechanism and sequence of side chain hydroxylation of cholesterol in bile acid synthesis was studied in the isolated perfused rabbit liver. A comparison was made between the importance of 26- and 25-hydroxylation in cholic acid biosynthesis in the rabbit. The formation of [G-3H]cholic acid was observed when the liver was perfused with 5beta-[G-3H]cholestane-3alpha, 7alpha-diol, 5beta-[G-3H]cholestane-3alpha, 7alpha-12alpha-triol, and 5beta-[G-3H]cholestane-3alpha, 7alpha, 26-triol. No [G-3H]chenodeoxycholic acid was detected in the bile. These findings indicate that potential precursors of chenodeoxycholic acid were hydroxylated at position 12alpha either subsequent to or before hydroxylation of the cholesterol side chain. In addition, no other intermediates (tetrahydroxy or pentahydroxy bile alcohols) were found in the bile when these compounds were perfused in the liver. Bile acid precursors were detected in bile when the rabbit liver was perfused with 5beta-[24-14C]cholestane-3alpha, 7alpha, 25-triol. The 5beta-[24-14C]cholestane-3alpha, 7alpha, 25-triol was hydroxylated in the liver at the 12alpha position to yield the corresponding 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol. The tetrol was further metabolized to a series of pentols (5beta-cholestane-3alpha, 7alpha, 12alpha, 22, 25-pentol; 5beta-cholestane-3alpha, 7alpha, 12alpha, 23, 25-pentol; 5beta-cholestane-3alpha, 7alpha, 12alpha, 24, 25-pentol; and 5beta-cholestane-3alpha, 7alpha, 12alpha, 25, 26-pentol). The major bile acid obtained from the perfusion of the 5beta-cholestane-3alpha, 7alpha, 25-triol was cholic acid. The experiments indicated that in the rabbit liver 12alpha-hydroxylation can occur after hydroxylation of the cholesterol side chain at either C-25 (5 beta-cholestane-3alpha, 7alpha, 25-triol) or C-26 (5beta-cholestane-3alpha, 7alpha-26-triol). Apparently, the rabbit can form cholic acid via the classical 26-hydroxylation pathway as well as via 25-hydroxylated intermediates.     

Cholestane-3beta, 5alpha, 6beta-triol promotes vascular smooth muscle cells calcification

Oxysterols found in atherosclerotic plaque may be associated with vascular calcification. We investigated the effect of oxysterol cholestane-3beta, 5alpha, 6beta-triol (Triol) on in vitro calcification of rat vascular smooth muscle cells (VSMCs). In vitro calcification was induced by incubation of VSMCs with beta-glycerophosphate. Calcifying nodule formation, calcium deposition in extracellular matrix, and alkaline phosphatase (ALP) activity were measured as indices of calcification. Because apoptotic bodies can serve as nucleation sites for calcification, apoptosis of calcifying VSMCs was determined by Hoechst 33258 staining, TUNEL, and FITC-labeled annexin V/PI double staining. The calcium deposition and ALP activity in calcifying VSMCs were much higher than those in non-calcifying VSMCs. Triol increased calcifying nodule formation, calcium deposition, ALP activity, and apoptosis of nodular cells in calcifying VSMCs. As determined by 2,7-dichlorofluorescein fluorescence, Triol induced the generation of reactive oxygen species (ROS) in calcifying VSMCs dose- and time-dependently. Triol-induced increases in calcium deposition, ALP activity, apoptosis, and ROS generation were all attenuated by antioxidant vitamin C plus vitamin E (VC + VE). The results demonstrated that Triol promoted VSMCs calcification through direct increase of ALP activity and apoptosis, probably by ROS-related mechanism.     

Conversion of a pentane-1,3,5-triol derivative using lipases as chiral catalysts and possible function of the lid for the regulation of substrate selectivity and enantioselectivity

Abstract

The enantioselective desymmetrization of a prochiral 3-O-silyl protected pentane-1,3,5-triol derivative was achieved by lipase-catalysed hydrolysis. The lipase from B. cepacia led to 95.4% enantiomeric excess (ee)of the (R)-configured compound (R)-4 at a theoretical yield of 79% and was isolated with 98.2% ee and 27% yield. Furthermore, it was found that the ee switched from an excess of (R)-4 to an excess of (S)-4 during the course of the reaction using crude lipase from C. rugosa under the same conditions. This finding was investigated in detail and compared to the change of substrate selectivity known for the lipase from M. miehei. It is supposed that both phenomena may result from a movement of the lid.     

Metabolism of cholestane-3 beta,5 alpha,6 beta-triol. II. Identification of two major neutral metabolites in the rat

Rats were given a single oral dose of cholestane-3beta,5alpha,6beta-triol-4-(14)C, and their feces were collected. The two major neutral metabolites were separated and isolated by use of solvent fractionation and chromatographic methods. The metabolites were identified as cholestane-3beta,5alpha-diol-6-one and a mixture of long-chain fatty acid esters of cholestane-3beta,5alpha,-6beta-triol. Cholestane-3beta,5alpha-diol-6-one was identified using thin-layer and gas-liquid chromatography, infrared spectroscopy, and the spectrum produced by reaction with 65% sulfuric acid. The mixed esters of cholestane-3beta,5alpha,6beta-triol were subjected to basic hydrolysis, and the steroid moiety was identified using the same techniques employed for cholestane-3beta,5alpha-diol-6-one. The fatty acids were analyzed by gas-liquid chromatography of their methyl esters.     

Inhibition of lymphatic absorption of cholesterol by cholestane-3 beta, 5 alpha, 6 beta-triol

The effect of cholestane-3,5alpha,6-triol (CT) on the intestinal absorption of cholesterol and oleic acid, as well as the absorption of labeled CT, was studied in lymph ductcannulated rats. Intragastric administration of 50 mg of CT in an emulsion with cholesterol-7alpha-(3)H and oleic acid-1-(14)C resulted in 50% inhibition of sterol transfer into lymph but only 8% depression of fatty acid absorption over an 8 hr period. The absorption of labeled CT into lymph was only 2-3% compared with 50% absorption of cholesterol when each was fed alone. 10% of the fed CT was recovered in the intestinal mucosa, and of this, one-half was associated with the brush border fraction. In rats fed CT 6 days prior to cholesterol and fatty acid administration, there was no effect on fatty acid absorption, while cholesterol absorption was reduced by almost 30%. When the intestinal mucosa from these animals were investigated by electron microscopy, it appeared that CT feeding resulted in numerous enlarged mitochondria and a marked increase in length of the microvilli. If animals were allowed to recover for 6 days from the CT prefeeding regime, the intestinal mucosa appeared normal, and the absorption of cholesterol approached that in controls. A possible mechanism for CT inhibition of cholesterol absorption was shown to be competition for the enzyme cholesterol esterase which esterifies cholesterol prior to entrance into the lymphatic system. CT itself is poorly esterified and poorly absorbed, but it is effective in inhibiting esterification of cholesterol in vitro.     

Quantitative aspects of the conversion of 5 beta-cholestane intermediates to bile acids in man.

The in vivo conversion of several 5 beta-cholestane intermediates to primary bile acids was investigated in three patients with total biliary diversion. The following compounds were administered intravenously: 5 beta-[G-3H]-cholestane-3 alpha, 7 alpha-diol, 5 beta-[G-3H]cholestane-3 alpha, 7alpha, 26-triol, and 5 beta-[24-14C]cholestane-3 alpha, 7 alpha-25-triol. Bile was then collected quantitatively at frequent intervals for the next 21 to 28 h. The administered 5 beta-[G-3H]cholestane-3alpha, 7alpha, 26-triol was found to be efficiently converted to cholic and chenodeoxycholic acids in two patients; 61 and 75% of the administered label was found in primary bile acids. The proportion of labeled cholic to chenodeoxycholic acid was 1.20 and 1.02 in the bile of these patients, indicating that the C-26 triol was efficiently converted to cholic acid. The ratio of cholic to chenodeoxycholic acid (mass) in the bile of these patients was 1.23 and 2.32. The 5 beta-cholestane-3alpha, 7alpha-diol intermediate was also efficiently converted (71%) to both primary bile acids. The cholic to chenodeoxycholic acid ratios by mass and label were similar (2.97 versus 2.23). By contrast, the 5beta-cholestane-3alpha, 7alpha, 25-triol was poorly converted to bile acids in three patients. Following the administration of this compound almost all of the administered radioactivity found in the bile acid fraction was in cholic acid (5 to 19%) and very little (less than 5%) was found in chenodeoxycholic acid. These findings indicate that ring hydroxylation at position 12 is not materially hindered by the presence of a hydroxyl group on the side chain at C-26 in patients with biliary diversion. The labeled C-26-triol which was efficiently converted to both primary bile acids in a proportion similar to that which was observed for the bile acids synthesized by the liver suggests that this 5beta-cholestane derivative may be a major intermediate in the synthesis of both cholic and chenodeoxycholic acids.     

A sensitive and specific LC-MS/MS method for rapid diagnosis of Niemann-Pick C1 disease from human plasma

Niemann-Pick type C1 (NPC1) disease is a rare, progressively fatal neurodegenerative disease for which there are no FDA-approved therapies. A major barrier to developing new therapies for this disorder has been the lack of a sensitive and noninvasive diagnostic test. Recently, we demonstrated that two cholesterol oxidation products, specifically cholestane-3β,5α,6β-triol (3β,5α,6β-triol) and 7-ketocholesterol (7-KC), were markedly increased in the plasma of human NPC1 subjects, suggesting a role for these oxysterols in diagnosis of NPC1 disease and evaluation of therapeutics in clinical trials. In the present study, we describe the development of a sensitive and specific LC-MS/MS method for quantifying 3β,5α,6β-triol and 7-KC human plasma after derivatization with N,N-dimethylglycine. We show that dimethylglycine derivatization successfully enhanced the ionization and fragmentation of 3β,5α,6β-triol and 7-KC for mass spectrometric detection of the oxysterol species in human plasma. The oxysterol dimethylglycinates were resolved with high sensitivity and selectivity, and enabled accurate quantification of 3β,5α,6β-triol and 7-KC concentrations in human plasma. The LC-MS/MS assay was able to discriminate with high sensitivity and specificity between control and NPC1 subjects, and offers for the first time a noninvasive, rapid, and highly sensitive method for diagnosis of NPC1 disease.     

Preanalytical standardization for reactive oxygen species derived oxysterol analysis in human plasma by liquid chromatography–tandem mass spectrometry

The analysis of the oxysterols 7-keto-, 7-α/β-hydroxy-, 5α,6α-epoxy-, 5β,6β-epoxycholesterol and cholestane-3β,5α,6β-triol derived from reactive oxygen species (ROS) is of interest as biomarkers in the field of atherosclerosis. Preanalytical validation is a crucial point to minimize the susceptibility of oxysterols to in vitro autoxidation. The aim of this study was to standardize a preanalytical protocol for ROS-derived oxysterol analysis by liquid chromatography–tandem mass spectrometry in human plasma.     

Glycosides of ergosterol derivatives from Hericum erinacens

A new glycoside of ergosta-7,22-diene-3,5,6-triol was isolated from the dry fruiting body of Hericum erinacens and the structure was established by chemical and spectroscopic means. Six ergosterol derivatives were also isolated from H. erinacens.     

The response of the antioxidant defense system in rat hepatocytes challenged with oxysterols is modified by Covi-ox

Suspension cultures of isolated rat hepatocytes were used to investigate whether 7-ketocholesterol and cholestane-3,5,6-triol exert oxidative stress in cells as manifested by increased lipid peroxidation and the induction of the antioxidant enzymes catalase, glutathione peroxidase and superoxide dismutase. The oxysterols were found to increase the levels of both superoxide dismutase and catalase and to have variable effects on glutathione peroxidase activity. Increased lipid peroxidation was not observed, indicating that the endogenous antioxidant defense system was capable of protecting against any oxidative stress that might otherwise by exerted by 7-ketocholesterol or cholestane-3,5,6-triol. Covi-ox, a natural tocopherol blend reduced the effects of both oxysterols on the antioxidant enzymes. A concurrent reduction in the production of thiobarbituric acid-reactive substances in Covi-ox-treated cells is indirect evidence that reactive oxygen species were produced by oxysterols in hepatocyte suspension cultures.     

Bile acids. 39. Metabolism of 5 -cholestane-3 ,26-diol and 5 -cholestane-3 ,7 ,26-triol in the rat with a bile fistula

25r-5alpha-[5alpha,6alpha-(3)H(2)]Cholestane-3beta,7alpha,26-triol was prepared from 3beta,26-diacetoxy-5alpha[5alpha,6alpha-(3)H(2)]cholestan-7-one that was obtained from kryptogenin. Huang-Minlon reduction of the ketone provided 25r-5alpha-[5alpha-(3)H]cholestane-3beta,26-diol. Results from mass spectrometry, molecular rotation, and several types of chromatography are consonant with the assigned structures. Bile was collected for 8 days from adult male rats, with cannulated bile ducts, that had received approximately 0.8 mg of the triol or diol intraperitoneally. Bile from the first 12 hr was hydrolyzed, and the bile acids were separated by partition chromatography. The chromatographic pattern of separated bile acids was much simpler for the triol than the diol. Approximately 50% of the bile acids derived from the triol were trihydroxy allo acids (allocholic acid, 44%, and its 3beta isomer, 5.3%); only 16.4% allocholic acid was obtained from the diol. Comparable amounts of allochenodeoxycholic acid were derived from the diol and triol (21.2% and 28.2%, respectively). Unidentified metabolites in the dihydroxy acid fraction derived from the diol constitute 15.8% of chromatographed material.     

Metabolism of cholesteryl palmitate by rat brain in vitro; formation of cholesterol epoxides and cholestane-3beta,5alpha,6beta-triol

Incubation of [4-(14)C]cholesteryl palmitate with the 12,000 g supernatant fraction of adult rat brain fortified with an NADPH-generating system and beta-mercaptoethylamine resulted in formation (2-5%) of more polar metabolites characterized as a mixture of cholesterol-5,6-epoxides. Under extended incubation conditions, cholestane-3beta-5alpha-6beta-triol was isolated as the major end product of the incubations. Free [4-(14)C]cholesterol incubated under similar conditions was not oxidized, whereas oxidation of [4-(14)C]cholesteryl palmitate appeared to be dependent upon hydrolysis of the ester by the rat brain microsomal subcellular fraction. Elimination of the NADPH-generating system or the addition of EDTA to the incubation mixture inhibited epoxide formation, suggesting that the products are derived from an NADPH-dependent enzymatic lipoperoxidation mechanism. The in vitro conversion of [4-(14)C]cholesterol-5alpha,6alpha-epoxide to cholestane-3beta,5alpha,6beta-triol was also demonstrated in rat brain subcellular fractions in the absence of added cofactors.     

New bile alcohols--synthesis of 5beta-cholestane-3alpha, 7alpha, 25-triol and 5beta-cholestane-3alpha, 7alpha, 25-24 (14C)-triol.

5beta-Cholestane-3alpha, 7alpha, 25-triol and 5beta-cholestane-3alpha, 7alpha, 25-24(14-C)-triol were synthesized from 3alpha, 7alpha-dihydroxy-5beta-cholanoic acid (chenodeoxycholic acid). Chenodeoxycholic acid was converted to the diformoxy derivative (II) using formic acid. Reaction of II with thionyl chloride yielded the acid chloride which was treated with diazomethane (CH-2-N-2 or 14-CH-2-N-2) to produce 3alpha, 7alpha-diformoxy-24-oxo-25-diazo-25-homocholane (III, A or B). 25-Homochenodeoxycholic acid (IV, A or B) was formed from III by means of the Wolff rearrangement of the Arndt-Eistert synthesis. The methyl ester of V (A or B) was treated with methyl magnesium iodidi in ether to provide the desired triol, VI (A and B). The triol was identified by mass spectrometry and elemental analysis and was characterized by thin-layer and gas-liquid chromatography. The 3alpha, 7alpha, 25-triol is of possible significance as an intermediate in the pathway of bile acid formation from cholesterol.     

The structure of the benzeneboronate of pentane-1,3,5-triol

The relative ease of formation of five-, six-, seven-, and eight-membered cyclic benzeneboronates is discussed. Pentane-1,3,5-triol forms, by interaction with benzene-boronic anhydride, exclusively DL-4-(2-hydroxyethyl)-2-phenyl-1,3,2-dioxaborinane. It is suggested that, in this compound, the oxygen atom of the hydroxyl group is intramolecularly co-ordinated with the boron atom.     

Effect of selenium compounds on the damage induced by oxysterol on rat arterial walls

Wistar rats were fed Se-deficient (0.017±0.002 mg Se/kg) and Seadequate (0.32±0.045 Se mg/kg) diets for 12 mo and then were given 5 mg/kg of cholestane-3β,tα,6β-triol (3-triol), intravenously. Se compounds (Na₂SeO₃ and ebselen) were supplemented in different doses and times to the Se-deficient rats. Twenty-four hours after 3-triol infusion, the changes in ultrastructures of rat aorta were examined by scanning electron micrography (SEM) and transmission electron micrography (TEM). SEM examinations showed that 3-triol induced diffused injuries on arterial endothelial urfaces of long-term Se-deficient rat, and a large number of holes or craterlike defects were observed. TEM examinations further showed that 3-triol induced swelling, necrosis, and shedding of endothelial cells, which resulted in the destruction of endothelial integrity. Mean-while, smooth muscle cells proliferated and migrated toward intimae; the breakage of internal elastic lamina benefited the migration of smooth muscle cells. Supplemented with Na₂SeO₃ (40 μg/kg, 10 d per continuum) and ebselen (20 mg/kg), respectively, exhibited significant protection from damages induced by 3-triol. It seems that protecting mechanisms were different between Na₂SeO₃ and ebselen. The present investigation gave visual evidence that both injuries induced by cholesterol oxides and the Se nutritional status contributed to the development of atherosclerosis.     

Urinary excretion of 5beta-pregnane-3alpha,20alpha,21-triol in human gestation

The steroids in urine from normal pregnant women have been studied. After extraction of conjugate steroids, solvolysis and enzymatic hydrolysis, the liberated steroids were separated by chromatography on Sephadex LH-20, and were analysed by gas-liquid chromatography and gas chromatography mass spectrometry. The following steroids were isolated and completely identified in the LH-20 fraction 7: 5beta-pregnane-3alpha,20alpha-diol, 5beta-pregnane-3alpha,17,20alpha-triol, 5beta-pregnane-3alpha,20alpha,21-triol and 5alpha-pregnane-3beta,16alpha,20alpha-triol. In addition, two metabolites tentatively identified as 5xi-pregnane-2xi,3xi,20xi-triol and 2xi,3xi,16xi-trihydroxy-5xi-pregnan-20-one, have not been reported as occcurring in urine from pregnant women. The 5beta-pregnane-3alpha,20alpha,21-triol was detected only in the third trimester of pregnancy and the urinary excretion values are between 320 and 650 microgram per 24 h. With the present data, it is not possible to establish the precursor(s) of this steroid. However, these results tentatively suggest that 5beta-pregnane-3alpha,20alpha,21-triol arises from foeto-placental unit.     

Chemical synthesis of 5beta-cholestane-3alpha, 7alpha, 24, 25-tetrol and its metabolism in the perfused rabbit liver

5beta-[G-3H]Cholestane-3alpha, 7alpha, 24xi, 25-tetrol (IV) was synthesized via dehydration and peroxidation of 5beta-[G-3H]cholestane-3alpha, 7alpha, 25-triol. Following perfusion of the labeled compound in the isolated rabbit liver, the bile alcohol and bile acid metabolites secreted into the bile were identified by a combination of thin layer chromatography, gas-liquid chromatography, and gas-liquid chromatography/mass spectrometry. The following bile alcohols were tentatively identified: 5beta-cholest-23-ene-3alpha, 7alpha, 25-triol, 5beta-cholest-25-ene-3alpha, 7alpha, 12alpha, 24xi-tetrol, and 5beta-cholestane-3alpha, 7alpha, 12alpha, 24xi, 25-pentol. The amount of administered tetrol recovered unchanged ranged from 1 to 88%. Cholic acid was the major product, but limited amounts of chemodeoxycholic acid were also formed. The 24-hydroxyl group in the steroid side chain did not prevent 12alpha-hydroxylation.     

One step synthesis of 6-oxo-cholestan-3β,5α-diol

Cholesterol metabolism has been recently linked to cancer, highlighting the importance of the characterization of new metabolic pathways in the sterol series. One of these pathways is centered on cholesterol-5,6-epoxides (5,6-ECs). 5,6-ECs can either generate dendrogenin A, a tumor suppressor present in healthy mammalian tissues, or the carcinogenic cholestane-3β,5α,6β-triol (CT) and its putative metabolite 6-oxo-cholestan-3β,5α-diol (OCDO) in tumor cells. We are currently investigating the identification of the enzyme involved in OCDO biosynthesis, which would be highly facilitated by the use of commercially unavailable [¹⁴C]-cholestane-3β,5α,6β-triol and [¹⁴C]-6-oxo-cholestan-3β,5α-diol. In the present study we report the one-step synthesis of [¹⁴C]-cholestane-3β,5α,6β-triol and [¹⁴C]-6-oxo-cholestan-3β,5α-diol by oxidation of [¹⁴C]-cholesterol with iodide metaperiodate (HIO₄).