cholesterol-alpha-oxide and 25-hydroxycholesterol

Oxidative stress plays an important role in the pathophysiology of diabetes mellitus. The aim of this study was to evaluate the formation of cholesterol oxides (ChOx) as biomarkers of oxidative stress in subjects with impaired glucose tolerance (IGT) and diabetes.. Blood plasma levels of cholesterol oxidation products were determined in the following groups: type 1 diabetes mellitus (DM1), type 2 diabetes (DM2), impaired glucose tolerance (IGT), children without diabetes (C1) and adults without diabetes (C2). The serum levels of cholest-5-ene-3alpha,7alpha-diol (7alpha-hydroxycholesterol, 7alpha-OH), cholest-5-ene-3beta,7beta-diol (7beta-hydroxycholesterol, 7beta-OH), 3beta-hydroxycholest-5-7-one (7-ketocholesterol, 7-K), 5alpha-cholestane-3beta,5,6beta-triol (cholestanetriol), 5,6alpha-epoxy-5alpha-cholestan-3alpha-ol (cholesterol-5alpha,6alpha-epoxide,), 5,6beta-epoxy-5beta-cholestan-3beta-ol (cholesterol-5beta,6beta-epoxide) and cholest-5-eno-3beta,25-diol (25-hydroxycholesterol, 25-OH) (trivial name and abbreviations indicated in parentheses) were quantified by gas chromatography using flame ionization detection.. The levels of total ChOx were elevated in the DM1 and DM2 groups compared to age-matched subjects without diabetes (p < 0.05). The concentrations of 7beta-hydroxycholesterol, cholesterol-alpha-epoxide and cholesterol-beta-epoxide were higher in the blood plasma of subjects in the DM2 group than in the blood plasma of subjects in the C2 and IGT groups (p < 0.05). Treatment of type 2 diabetic patients with oral hypoglycemic drugs associated with insulin resulted in lower concentrations of nitrotyrosine in the blood plasma without significant changes in the concentrations of glucose and glycated hemoglobin. Moreover, combination with statins in both treatments decreased the concentrations of ChOx.. ChOx are suitable biomarkers of oxidative stress and may be useful in clinical studies to follow drug effects on lipid oxidative modifications in diabetic patients.

Topics: Adolescent; Adult; Aged; Angiotensin-Converting Enzyme Inhibitors; Biomarkers; Child; Cholestanols; Cholesterol; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Female; Glucose Intolerance; Humans; Hydroxycholesterols; Hypoglycemic Agents; Insulin; Ketocholesterols; Male; Middle Aged; Oxidative Stress

Increasing the energy value of diets with dietary fat, particularly fats rich in saturated fatty acids, can result in the elevation of plasma total and lipoprotein cholesterol. In the present study, experimental diets were designed to examine the effects of increasing the energy content of diets with a saturated fat source and cholesterol in a non-purified diet on hyperlipoproteinaemia and aortic plaque composition in the atherosclerosis-susceptible Japanese quail (Coturnix japonica) model of human atherosclerosis. Commercial poultry diets containing two levels (i.e. 60 or 120 g/kg) of beef tallow as the primary source of saturated fat were balanced for endogenous cholesterol or supplemented with cholesterol (i.e. 0.5 or 5.0 g/kg) and fed to quail for 9 weeks to examine the effects on whole plasma, lipoprotein and aortic plaque lipid composition in relation to aortic plaque formation. Hypercholesterolaemia (P < 0.001) was confirmed in birds fed on high-cholesterol (HC) diets only. An interaction (P = 0.05) between dietary cholesterol and fat intake level was observed for plasma triacylglycerols (TG) and was specific to changes observed in VLDL composition. Diet-induced changes in lipoprotein total cholesterol, TG and phospholipid composition were greatest in the portomicron and VLDL fractions in birds fed on atherogenic diets. Hyperlipoproteinaemia induced by the 60 g/kg added beef tallow-HC diet resulted in significant (P < 0.001) aortic plaque deposition, which was further enhanced in birds fed on the 120 g/kg beef tallow-HC diet. Quail fed on 120 g/kg beef tallow-HC diets exhibited the most severe aortic plaque formation, with marked increases in aortic tissue cholesterol content and quantifiable amounts of several cholesterol oxides (5,6 alpha-epoxy-5 alpha-cholesterol, 7 beta-hydroxycholesterol, cholestanetriol, 7-ketocholesterol and 25-hydroxycholesterol). In summary, hyperlipoproteinaemia associated with HC diets with a greater proportion of energy from saturated fat produced a combined effect in altering plasma and lipoprotein lipid composition as well as aortic tissue cholesterol and cholesterol oxide content in the Japanese quail.

Topics: Analysis of Variance; Animals; Aorta; Arteriosclerosis; Bird Diseases; Cholestanols; Cholesterol; Cholesterol, Dietary; Coturnix; Dietary Fats; Disease Models, Animal; Energy Intake; Hydroxycholesterols; Hypolipidemic Agents; Ketocholesterols; Lipids; Lipoproteins; Male; Regression Analysis

Two novel populations of spontaneous PC12 cell mutants resistant to a toxic concentration of 25-OH-cholesterol (5 microg/ml, 12.5 microM) were isolated and designated as R25R and F25R based on cell morphology. R25R consisted of round cells that were morphologically similar to the parent PC12 cells, and responded to nerve growth factor by extending neurites. F25R was a group of process-bearing flat cells that did not assume a neuronal morphology in the presence of nerve growth factor. These two cell lines also acquired some cross-resistance toward other cholesterol oxides. Nerve growth factor induced prominent voltage-dependent calcium currents in parent PC12 cells and in R25R, but not in F25R. Further experiments indicated that the parent PC12 cells, R25R and F25R exhibited different properties when challenged with a variety of toxic insults, including amphotericin B, serum withdrawal and beta-amyloid protein treatment.

Topics: Amyloid beta-Peptides; Animals; Calcium Channels; Cell Culture Techniques; Cell Death; Cell Differentiation; Cholesterol; Culture Media, Serum-Free; Drug Resistance, Neoplasm; Hydroxycholesterols; Ion Channel Gating; Membrane Lipids; Nerve Growth Factors; PC12 Cells; Rats

The effects of the cholesterol oxides on low density lipoprotein receptor (LDLR) gene expression were investigated. Cultured rabbit aortic smooth muscle cells were incubated with 1, 2, and 5 micrograms/ml culture medium concentrations of pure cholesterol, 25-hydroxycholesterol (25-OH), 7-ketocholesterol (7-keto), cholestane-3 beta, 5 alpha, 6 beta-triol (triol) and cholesterol-5 alpha, 6 alpha-epoxide (epoxide) for 12 hours and with vehicle only as control. Total mRNAs were extracted and electrophoresed. Northern blot hybridization analyses were performed. The results showed mRNA expressions of LDLR gene were inhibited to 16.1 +/- 4.4%, 33.8 +/- 0.6%, 42.8 +/- 1.8% and 46.9 +/- 3.9% of control by 25-OH, 7-keto, epoxide and triol respectively. Pure cholesterol showed only minimal inhibition. The inhibitions were time dependent. Although cholesterol oxides have been shown to alter many membrane-related functions and the LDLR domain are located in the cell membrane. The findings of this study suggested that the cholesterol oxides exerted their repressive actions on LDLR function primarily by down-regulating LDLR gene expression rather than directly upon cell membrane.

Topics: Animals; Blotting, Northern; Cells, Cultured; Cholestanols; Cholesterol; Gene Expression Regulation; Hydroxycholesterols; Ketocholesterols; Muscle, Smooth, Vascular; Rabbits; Receptors, Lipoprotein; RNA, Messenger

A method for isolation, detection and quantification of cholesterol oxidation products based on solid phase extraction in combination with preparative HPLC and gas chromatography-mass spectrometry selected ion monitoring has been developed for dairy products. The isolation procedure had a high recovery and artifact formation was minimal, as shown by isotope labelling. The limits of detection ranged from 0.3 to 35 pg/microliters of the isomeric forms of 7-hydroxycholesterol, 20 alpha-hydroxycholesterol, the isomeric forms of cholesterol-5,6-epoxides, cholestanetriol, 25-hydroxycholesterol and 7-ketocholesterol corresponding to a limit of quantification of 2-6 ng oxysterol/g lipid in the dairy product, depending on the nature of the cholesterol oxidation product.

Topics: Cholestanols; Cholesterol; Cholesterol Esters; Chromatography, High Pressure Liquid; Dairy Products; Gas Chromatography-Mass Spectrometry; Hydroxycholesterols; Ketocholesterols; Mass Spectrometry; Oxidation-Reduction; Oxides; Triglycerides

The homogenizing effect of cholesterol and its oxidative derivatives, 7-ketocholesterol, cholesterol 5 alpha, 6 alpha-epoxide and 25-hydroxycholesterol, in liquid-crystalline 1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC) bilayer vesicles was studied using the fluorescence lifetimes of 2-(3-(diphenylhexatrienyl)propanoyl)-1-hexadecanoyl-sn-glycero-3-p hosphocholine (DPH-PC). The phase and modulation data were fitted either to discrete exponential models or to models characterized by continuous distributional lifetimes. Among all the models tested, it was found that the best one to account for the experimental data was the unimodal Lorentzian distribution. Thus, the DPH-PC lifetime was adequately described by a distributional center and a full width at half-maximum, for DOPC vesicles these values being 6.23 and 0.48 ns, respectively. Increasing the concentration of cholesterol, 7-ketocholesterol, or cholesterol 5 alpha, 6 alpha-epoxide from 0 to 30 mol% resulted in an increase of the lifetime center (e.g., 7.16 ns at 30 mol% cholesterol) and a decrease of the distributional width (e.g., 0.05 ns at 30 mol% cholesterol). On the other hand, up to 30 mol% of 25-hydroxycholesterol incorporated into the bilayer vesicles showed little influence on both lifetime parameters. Our results support the use of lifetime distributional width to evaluate membrane heterogeneity and suggest that oxysterols, depending on their molecular structural particulars, may exert cholesterol-like homogenizing effect in membranes.

Topics: Cholesterol; Diphenylhexatriene; Hydroxycholesterols; Ketocholesterols; Lipid Bilayers; Membrane Fluidity; Membrane Lipids; Phosphatidylcholines; Spectrometry, Fluorescence

A spectrum of cholesterol oxidation derivatives (oxysterols) is generated in food products exposed to heat or radiation in the presence of oxygen. One of these derivatives (cholestan-3 beta,5 alpha,6 beta-triol) was shown to compromise the selective barrier function of cultured vascular endothelial cell monolayers, an action that may initiate atherosclerotic lesion formation. This study sought to investigate the relationship of cholesterol synthesis inhibition by several naturally occurring oxysterols to depression of vascular endothelial cell monolayer barrier function, determined as an increase in albumin transfer across cultured endothelial monolayers. All oxysterols tested caused a variable time- and dose-dependent elevation in trans-endothelial albumin transfer, and they were also able to inhibit cholesterol biosynthesis to varying degrees. Pure cholesterol was without effect on both counts. The correlation between the increase in albumin transfer related to oxysterol exposure and the ability of oxysterols to suppress cholesterol biosynthesis was, however, poor. Moreover, mevinolin, a water-soluble competitive inhibitor of cholesterol synthesis, reduced the rate of cholesterol synthesis to 0.9% of control but did not significantly increase albumin transfer. Cholestan-3 beta,5 alpha,6 beta-triol caused a 660% elevation in albumin transfer while cholesterol synthesis remained at 11% of control. We conclude that changes in endothelial barrier function caused by exposure to the oxysterols examined, but not pure cholesterol, are probably related to factors other than the well-known action of cholesterol biosynthesis inhibition. These findings may have implications in the development of atherosclerosis.

Topics: Animals; Biological Transport; Cell Membrane Permeability; Cholestanes; Cholestanols; Cholesterol; Dehydrocholesterols; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelium; Hydroxycholesterols; Hypolipidemic Agents; In Vitro Techniques; Ketocholesterols; Lovastatin; Serum Albumin; Swine; Time Factors

Human umbilical vein endothelial cells and rabbit aortic smooth muscle cells in culture were incubated for intervals up to 24 hrs with varying concentrations of cholesterol, 7-ketocholesterol, 25-hydroxycholesterol, cholestane-3 beta,5 alpha,6 beta-triol or cholesterol-5 alpha, 6 beta-epoxide. Endocytosis, as measured by uptake of horseradish peroxidase (HRP), was inhibited in a dose and time dependent manner in both endothelial and smooth muscle cell cultures by cholestane-3 beta,5 alpha,6 beta-triol and 25-hydroxycholesterol. Inhibition by 7-ketocholesterol in endothelial cells occurred only at higher concentrations, and cholesterol and cholesterol epoxide showed no significant inhibitory effects. The viability of the cells exposed to the cholesterol oxides at the concentrations that inhibited the uptake of HRP was not changed. Cholesterol oxides induce functional endothelial injury, not morphologically apparent, which may be involved in atherogenesis.

Topics: Animals; Aorta; Cells, Cultured; Cholestanols; Cholesterol; Endocytosis; Endothelium, Vascular; Horseradish Peroxidase; Humans; Hydroxycholesterols; Hypolipidemic Agents; Ketocholesterols; Kinetics; Muscle, Smooth, Vascular; Rabbits; Umbilical Veins