Cholestane Triol Research Articles

Circulating oxysterol metabolites as potential new surrogate markers in patients with hormone receptor-positive breast cancer: Results of the OXYTAM study

Accumulating evidence indicates that cholesterol oxygenation products, also known as oxysterols (OS), are involved in breast cancer (BC) promotion. The impact of Tam, as well as aromatase inhibitors (AI), an alternative BC endocrine therapy (ET), on OS metabolism in patients is currently unknown. We conducted a prospective clinical study in BC patients receiving Tam (n=15) or AI (n=14) in adjuvant or in metastatic settings. The primary end point was the feasibility of detecting and quantifying 11 different OS in the circulation of patients before and after 28days of treatment with Tam or AI. Key secondary end points were the measurements of variations in the concentrations of OS according to differences between patients and treatments. OS profiling in the serum of patients was determined by gas chromatography coupled to mass spectrometry. OS profiling was conducted in all patients both at baseline and during treatment regimens. An important inter-individual variability was observed for each OS. Interestingly 5,6β-epoxycholesterol relative concentrations significantly increased in the entire population (p=0.0109), while no increase in Cholestane-triol (CT) levels was measured. Interestingly, we found that, in contrast to AI, Tam therapy significantly decreased blood levels of 24-hydroxycholesterol (24-HC), 7α-HC and 25-HC (a tumor promoter) (p=0.0007, p=0.0231 and p=0.0231, respectively), whereas 4β-HC levels increased (p=0.0010). Interestingly, levels of 27-HC (a tumor promoter) significantly increased in response to AI (p=0.0342), but not Tam treatment. According to these results, specific OS are promising candidate markers of Tam and AI efficacy. Thus, further clinical investigations are needed to confirm the use of oxysterols as biomarkers of both prognosis and/or the efficacy of ET.

Consumption of cholesterol oxides from fast foods fried in beef fat in New Zealand

AbstractLevels of cholesterol oxides were determined in samples of beef dripping used for deep frying in retail fast‐food outlets in Christchurch, New Zealand. The average levels (n = 8) of cholesterol oxides were: β‐epoxy cholesterol (15 mg/kg), α‐epoxy cholesterol, 7β‐hydroxy cholesterol, 7‐keto cholesterol (10 mg/kg each), 7α‐hydroxy cholesterol (5 mg/kg), and cholestane triol, 25‐hydroxy cholesterol (1 mg/kg each). Based on the amount of fat in a typical deep‐fried “fish and chips” meal, the amounts of individual cholesterol oxides consumed would then range from 0.05–0.7 mg. This is three to nine times less than reported in a test meal experiment with humans but could still be expected to cause detectable increases in plasma cholesterol oxide levels.

Effects of Antioxidants and Packing on Cholesterol Oxidation in Processed Anchovy during Storage

Effects of added butylated hydroxyanisole (BHA) and tocopherols on oxidation of cholesterol during processing and subsequent storage of boiled and dried anchovy were evaluated by quantitative determination of constituent fatty acids and cholesterol oxides. Boiling and air-drying used for preparation of the sample yielded certain cholesterol oxides, including 7β-hydroxycholesterol, 7-ketocholesterol, cholesterol-5,6-α- and β-epoxides, cholestane triol and 25-hydroxycholesterol, even though BHA or tochopherols were added. As the levels of polyunsaturated fatty acids of the total lipids decreased, the levels of cholesterol oxides increased in all samples regardless of the presence of BHA or tocopherol. Formation of cholesterol oxides was controlled to some extent in the sample with added BHA. Tocopherols were also effective in controlling cholesterol oxidation, however they were less effective than BHA. Addition of BHA and tocopherols did not affect the rate of formation of cholesterol oxides in the early stage, up to 28 d of storage in air. The most effective method in controlling cholesterol oxidation was vacuum-packing of the sample with a metal-based oxygen absorber. For this sample, levels of cholesterol oxides after 156 d were similar to those of the control sample without added antioxidant stored in air for 14 d.

Plasma oxysterols and angiographically determined coronary atherosclerosis: a case control study

Several in vitro and in vivo experiments have implicated oxysterols in the aetiology and progression of atherosclerosis. Oxysterols may be formed endogenously by oxidation of cholesterol and thus may form a marker of LDL oxidation. They may also be obtained exogenously through dietary intake. We investigated the association of oxysterols with the degree of coronary stenosis in patients undergoing coronary angiography. Cases with severe coronary atherosclerosis 80 stenosis in one of the major coronary vessels, n =80 were compared with controls with no or minor stenosis 50 stenosis in all three major coronary vessels, n =79 . Cases and controls were prestratified on age, gender and smoking habits. Evaluated were plasma levels of unesterified 7 hydroxycholesterol, 7 hydroxycholesterol, 25 hydroxycholesterol, 7 ketocholesterol, cholestane triol and 5,6 epoxycholestanol. 7 Hydroxycholesterol made up 67 of the total amount of plasma oxysterol concentration and was the only one significantly higher in cases 1.53 mu g per 100 ml vs 1.27 mu g per 100 ml, p 0.05 . Further, cases had somewhat higher LDL cholesterol levels and significantly lower HDL cholesterol levels than controls. After multivariate adjustment to account for this difference in lipid levels and for the prestratification factors the mean difference between cases and controls for 7 hydroxycholesterol 0.14 mu g per 100 ml was no longer significant. Also the other oxysterols showed no significant association with the degree of coronary stenosis. Multiple logistic regression analyses showed an adjusted odds ratio of 1.07 95 CI, 0.45-2.59 in the highest tertile of total plasma oxysterol level. We conclude, that this study does not support the hypothesis that plasma oxysterols form an additional risk factor for coronary atherosclerosis.

Effects of the degree of unsaturation of coexisting triacylglycerols on cholesterol oxidation

AbstractIn order to evaluate the effects of the degree of unsaturation of triacylglycerols on cholesterol oxidation, mixtures of purified sardine oil triacylglycerols (iodine value, IV=182.6) and cholesterol; of partially hydrogenated sardine oil triacylglycerols (IV=174.5) and cholesterol; and of fully hydrogenated sardine oil triacylglycerols (IV=92.0) and cholesterol were incubated at 25°C in the dark. The oxidative stability of the samples decreased with increasing degree of unsaturation of the triacylglycerols in the sample mixtures; the induction period for peroxide values (PV) of the sardine oil triacylglycerols and cholesterol was shorter than that of the partially hydrogenated sardine oil triacylglycerols and cholesterol. Certain polyunsaturated fatty acids (PUFAs) in the constituent fatty acids of sardine oil triacylglycerols started to decrease after a shorter induction period compared with that of the partially hydrogenated triacylglycerols. The prominent cholesterol oxides accumulated in the samples were 7β‐hydroxycholesterol, 7‐ketocholesterol, β‐epoxide and cholestane triol. The tendency for accumulation of cholesterol oxides in the time course coincided with the changes in PV as well as the decrease in PUFAs. Cholesterol was oxidized in conjunction with autoxidation of coexisting fish oil triacylglycerols. Although lowering the degree of unsaturation of fish oil triacylglycerols was effective in prolonging the induction period of cholesterol oxidation, the rate of cholesterol oxidation in the cholesterol oxides' formation phase after the induction period was not affected by the difference in the proportion of highly unsaturated fatty acids in the natural and partially hydrogenated triacylglycerols of fish oils.

Oxidative decomposition of cholesterol in fish products

Cholesterol oxides in fish products popular in Japan, including salted and dried, boiled and dried and smoked products, were qualitatively and quantitatively determined as trimethylsilyl ether derivatives by gas‐liquid chromatography and mass spectrometry. The level of total cholesterol oxides ranged widely between 8.3 ppm in boiled and dried shrimp and 188.0 ppm in boiled and dried anchovy. 7β‐Hydroxycholesterol and 7‐ketocholesterol were the most prominent oxidative decomposition products of cholesterol. The levels of epimeric epoxides, cholestane triol and 25‐hydroxycholesterol were relatively low. To elucidate a mechanism of cholesterol oxidation proceeding during fish processing and subsequent preservation, four model systems, consisting of a mixture of purified cod liver triglycerides plus cholesterol, of a mixture of authentic triolein plus cholesterol, of triolein alone and of cholesterol alone, were stored separately at 25°C in dry air for up to 104 d. The residual fatty acids of the triglycerides, and the cholesterol oxides produced, were recovered and determined. Oxygen uptake remained almost unchanged for the mixture of triolein plus cholesterol. No detectable amount of cholesterol oxides was produced, and the fatty acid content of the residual oleic acid, measured by an internal standard, remained almost unchanged. For the mixture of cod liver triglycerides plus cholesterol, a remarkable increase in oxygen uptake was observed. A continuous increase in the amount of cholesterol oxides was observed, accompanied by a remarkable concurrent decrease in polyunsaturated fatty acid residues, as well as of the oleic acid naturally present. These results strongly suggest that cholesterol oxidation in fish products proceeds in conjunction with oxidative decomposition of the coexisting polyunsaturated fatty acids of fish oils.

Qualitative and comparative studies of cholesterol oxides in commercial and home-made Indian ghees

Cholesterol oxides are reported to be cytotoxic, mutagenic and many be atherogenic. Cholestane triol and epoxide are particularly important and are studied qualitatively in ghee samples obtained from different sources. In home-made ghee, both cholesterol oxides are identified whereas in commercial ghees they are absent. It is also observed that subjecting butter to high temperatures causes cholesterol to be converted into a high content of cholesterol oxide in ghee. Thus, dietary exposure of these cholesterol oxides from home-made ghee might lead to adverse effects and be a risk factor for atherosclerosis.

Uptake, metabolism, and cytotoxicity of isomeric cholesterol-5,6-epoxides in rabbit aortic endothelial cells.

The isomeric cholesterol-5,6-epoxides represent two common cholesterol autoxidation products and along with their principal metabolic product, 3 beta,5 alpha,6 beta-cholestane triol, are purportedly angiotoxic. The uptake and cytotoxic action of these compounds was examined in cultured rabbit aortic endothelial cells emphasizing mechanisms of uptake and metabolic fate. The isomeric cholesterol epoxides are incorporated with equal facility and in a dose-dependent manner. The pattern of uptake, which is markedly influenced by media serum concentration and by temperature, suggests that these compounds are partly incorporated through association with serum lipoproteins. After incorporation, both epoxide isomers are rapidly converted to cholestane triol which readily exits the cells. Cholestane triol is further metabolized to an ester-type product representing up to 10% of the added cholesterol epoxides by 24 h of incubation. The order of cytotoxic potency of these cholesterol oxides is: cholestane triol greater than cholesterol-beta-epoxide greater than cholesterol-alpha-epoxide, with LD50 concentrations ranging from 23 to greater than 150 microM in confluent cells. Cholestane triol and cholesterol-beta-epoxide are twice as cytotoxic to subconfluent cells as compared to confluent cells, whereas cholesterol-alpha-epoxide is essentially equitoxic to confluent and subconfluent cells. Cholesterol epoxide cytotoxicity is significantly reduced by treatments in the absence of serum in accord with substantial reduction in uptake when incubations are performed in serum-free media. Our findings show that these cytotoxic cholesterol oxides are incorporated by endothelial cells through a combination of receptor-mediated and nonspecific or passive mechanisms; however, the efficacy of uptake and resulting toxicity is substantially influenced by serum lipoproteins.

Increased cholesterol epoxide hydrolase activity in clofibrate-fed animals

Cholesterol epoxide hydrolase (mCE) is a microsomal enzyme that hydrolyzes cholesterol-5,6-epoxides (CE) to cholestanetriol (CT). In the present study, hepatic mCE activity was measured in mice pretreated with several different xenobiotics known to induce a variety of hepatic drug-metabolizing enzymes. Only the phenoxyacetate hypolipidemics (clofibrate and ciprofibrate, included in the diet for 14 days) were found to increase hepatic mCE activity (1.8-fold increase). Clofibrate administration also increased rabbit hepatic (2.9-fold increase) and rat and rabbit renal mCE activity (1.5- and 2.3-fold increase respectively). On a subcellular level, mCE activities in rabbit nuclear and light mitochondrial fractions were increased (3.3- and 1.8-fold increase respectively), whereas activities in the cytosolic and heavy mitochondrial fractions were unchanged. In rabbits, clofibrate administration enhanced the hepatic microsomal hydrolysis of CE without increasing the hydrolysis of arene epoxides (benzopyrene-4,5-oxide) or fatty acid epoxides (methyl cis-9,10-epoxystearate). Increase of tissue mCE activity may significantly enhance tissue CT levels. In this light, it is worth noting similarities in the mechanisms of hypocholesterolemic action caused by cloflbrate or CT administration.

Generation of complement anaphylatoxins and C5b-9 by crystalline cholesterol oxidation derivatives depends on hydroxyl group number and position

Cholesterol crystals activate the human alternative complement pathway. Loss of Factor B hemolytic activity in C2-deficient serum was comparable to that in normal human serum after incubation with cholesterol crystals. Consumption of Factor B hemolytic activity in normal serum incubated with cholesterol occurred in a time- and dose-dependent manner. The reduced capacity of crystals-absorbed serum to activate C2, but not Factor B, on fresh crystals, indicated that cholesterol mediates antibody-dependent classical pathway activation in addition to alternative pathway activation in whole serum. Cholestane triol, an oxidation derivative of cholesterol which bears three hydroxyl groups, cleaved 5-fold more C3 than cholesterol in normal human serum. Three cholesterol derivatives, each bearing two hydroxyl groups, were intermediate activators between cholesterol and cholestane triol. The compounds differed, however, in their complement-activating ability, indicating that hydroxyl position as well as number exerts an influence on complement activation. Measurements of C3adesArg and C5adesArg antigens in cholesterol crystal treated serum revealed that approx. 10% of total serum C3 was cleaved and that, on a molar basis, only 3% C5 cleavage occurred relative to C3 cleavage. For 1 mole of C5a generated, 0.1 moles of fluid-phase C5b-9 was detected. Although the extent of C3 cleavage varied with each cholesterol derivative depending on the position and number of hydroxyl groups, the relative coupling efficiency of C3 and C5 cleavage and C5a and C5b-9 generation was similar for all compounds. The ability of cholesterol and its oxidation products to generate anaphylatoxins and C5b-9 complexes may be of importance in mediating inflammatory processes involved in atherogenesis.

The cytotoxic and mutagenic properties of cholesterol oxidation products

The oxidation of cholesterol proceeds as part of the lipid peroxidation process in membranes. Several oxidation products characteristic of a free-radical mechanism are formed and some can serve as indices of the nature and extent of cholesterol oxidation and of lipid peroxidation in general. Among the most typical oxidation products of lipid peroxide-dependent propagation reactions are the enantiomeric 5,6-epoxides and 7-ketocholestanol. Small amounts of these compounds may persist in tissues experiencing lipid peroxidation at a low but steady flux of free-radical reactions. Supporting evidence includes the routine detection of small quantities of cholesterol epoxides in tissues of normal animals, and the increase of these epoxides under conditions of oxidant stress or antioxidant deficiency. Conversion of cholesterol epoxides to cholestane triol is expected in cells possessing cholesterol epoxide hydrolase. All of these oxidation products possess remarkable cytotoxicity (at least part of which may be due to effects on the cell membrane) causing an increase in intracellular calcium. The cholesterol epoxides are also weakly mutagenic, although the mechanism for this mutagenicity remains to be clarified. In contrast, the other lipid epoxides normally encountered in tissues (chiefly fatty acid epoxides) are not mutagenic, and are much less toxic than the oxysterols described. The cytotoxicity of several oxysterols may be due to a number of mechanisms. That only the epoxides are mutagenic suggests that genotoxicity is a function of their electrophilic reactivity. This is not consistently apparent with the other compounds examined.

Cholesterol epoxide is a direct-acting mutagen.

A 24-hr treatment of V79 Chinese hamster lung fibroblasts with 12.4 microM cholesterol 5 alpha, 6 alpha-epoxide induced 8-azaguanine-resistant mutants at frequencies 4.6- to 11.8-fold higher than the spontaneous mutation rate. We show that cholesterol epoxide, which is produced by in vivo cholesterol oxidation, is a weak direct-acting mutagen. Cholesterol epoxide was found to be accumulated by cells and transformed to cholestane-3 beta, 5 alpha, 6 beta-triol, which was more toxic and a more potent inhibitor of DNA synthesis than the epoxide but, at concentrations less than 17.8 microM, was not significantly mutagenic. Consideration of the rates of cholesterol epoxide conversion to cholestane triol shows that this conversion can result in abolition of the mutagenicity of the epoxide. Conditions under which conversion of the epoxide to the triol is low, as in the case of low epoxide hydrolase activity, favor mutagenicity whereas rapid conversion to triol favors cytotoxicity.

The bile acid composition of gastric contents from neonates with high intestinal obstruction.

The study was designed to identify 'atypical' bile acids in gastric contents from three neonates with high intestinal obstruction on the basis that this was likely to represent a rich source of primary bile acids. Cholic acid was the major component, and related 'atypical' bile acids included its C-3 and C-7 oxidation products, its 3 beta-epimer and 2 beta- and 6 alpha-hydroxylation products. Allocholic acid was the only 5 alpha-cholanic acid derivative identified. 7 alpha, 12 alpha-Dihydroxy-3-oxochol-4-en-24-oic acid was found in all three specimens and might be an intermediate in a biosynthetic pathway from cholesterol to cholic acid in which side-chain oxidation precedes at least some of the nuclear changes. Side-chain-hydroxylated derivatives of trihydroxycoprostanic acid were also detected and these may represent intermediates in biosynthetic pathways from cholesterol to cholic acid via 5 beta-cholestan-3 alpha, 7 alpha, 12 alpha-triol. The most abundant bile acid of this type was (25 epsilon)-3 alpha, 7 alpha, 12 alpha, 25-tetrahydroxy-5 beta-cholestan-26-oic acid, which suggested that C-25 hydroxylation may be an important step in the shortening of the C8 side chain of the cholestane triol to the C5 side chain of cholic acid in the neonatal period. Bile acids lacking a substituent at C-12 included chenodeoxycholic acid, its C-3 and C-7 oxidation products, its 3 beta-epimer and its 6 alpha-hydroxylation product (hyocholic acid).