Patients with atherosclerosis may have increased circulating levels of 27-hydroxycholesterol and cholestenoic acid.

Extrahepatic sterol 27-hydroxylase (CYP27) appears to have a role in the elimination of excess cholesterol from various cells, particularly macrophages, and there is a net flux of 27-hydroxyycholesterol and its metabolites from different extrahepatic sources to the liver. In this study we tested the hypothesis that patients with advanced atherosclerosis may have higher levels of 27-oxygenated products in the circulation than control subjects. Concordant with previous studies, a strong correlation was observed between circulating levels of 27-hydroxycholesterol and cholesterol, in both healthy subjects and subjects with hypercholesterolemia and documented atherosclerosis. A group of male subjects with normal or only slightly elevated serum cholesterol and rapidly progressing carotid atherosclerosis (n = 20) had serum levels of 27-oxygenated cholesterol not statistically different from those of a matched group of subjects with little or no development of atherosclerosis (n = 20). The situation was similar in a group of patients (n = 20) with advanced general atherosclerosis associated with severe clinical symptoms. Among the two groups of patients with atherosclerosis, a few patients had relatively high levels of 27-oxygenated products. Among the healthy controls, two healthy volunteers (brother and sister) were found to have high levels of 27-hydroxycholesterol, most probably due to genetic reasons. The possibility is discussed that the high levels of 27-oxygenated products in the circulation of a few patients with atherosclerosis may be related to high amounts of active macrophages present in atherosclerotic lesions. In view of the number of factors that could affect the levels in the circulation, other explanations cannot be ruled out. At the present state of knowledge, measurements of circulating levels of 27-oxygenated metabolites do not seem to add useful information about the atherosclerotic process.

Preparation and gas chromatographic-mass spectrometric analysis of N-acetyl-O-trimethylsilyl derivatives of long-chain base residues of natural sphingomyelin.

A procedure for the preparation of long-chain base residues of egg yolk, bovine milk and bovine brain sphingomyelin was developed. The bases were converted to N-acetyl-O-trimethylsilyl (TMS) derivatives before being submitted to gas chromatography and mass spectrometry. The chromatographic profile of the milk sample was complex with thirteen peaks, whereas the profiles of brain and egg yolk long-chain bases were simple and remarkably similar.

Formation of oxysterols during oxidation of low density lipoprotein by peroxynitrite, myoglobin, and copper.

Oxidation of low density lipoprotein (LDL) in the artery wall leads to the formation of cholesterol oxidation products that may result in cytotoxicity. Different mechanisms could contribute to LDL oxidation in vivo resulting in characteristic and specific modification of the cholesterol molecule. Alternatively, attack on cholesterol by chain propagating peroxyl radicals could result in the same distribution of oxidation products irrespective of the initial pro-oxidant mechanism. To distinguish between these possibilities we have monitored the formation of nine oxysterols during LDL oxidation, promoted by copper, myoglobin, peroxynitrite, or azo bis amidino propane. Regardless of the oxidant used, the pattern of oxysterol formation was essentially the same. The yields of products identified decreased in the order 7-oxocholesterol > 7 beta-hydroxycholesterol > 7 alpha-hydroxycholesterol > 5,6 beta-epoxycholesterol > 5,6 alpha-epoxycholesterol except in the case of peroxynitrite in which case a higher yield of 5, 6 beta-epoxycholesterol relative to 7-oxocholesterol was found. No formation of cholestane 3 beta, 5 alpha, 6 beta-triol, or the 24-,25-,27-hydroxycholesterols was seen. Concentration of 7-oxocholesterol levels in LDL was positively correlated with the degree of protein modification. Endogenous alpha-tocopherol in LDL or supplementation with butylated hydroxytoluene prevented oxysterol formation. Taken together these data indicate that the oxidation of cholesterol and protein in LDL occur as secondary oxidation events consequent on the attack of fatty acid peroxyl/alkoxyl radicals on the 7-position of cholesterol, and with amino acids on apoB. Furthermore, oxidant processes with atherogenic potential, such as peroxynitrite, copper, and myoglobin are capable of producing oxidized LDL containing cytotoxic mediators.

The oxysterols cholest-5-ene-3 beta,4 alpha-diol, cholest-5-ene-3 beta,4 beta-diol and cholestane-3 beta,5 alpha,6 alpha-triol are formed during in vitro oxidation of low density lipoprotein, and are present in human atherosclerotic plaques.

Isolated human low density lipoprotein (LDL) was oxidized with either cupric ions or soybean lipoxygenase and linoleic acid. Cholesterol oxidation products (oxysterols) were determined by isotope dilution gas chromatography-mass spectrometry. A new cholestane-3,5,6-triol isomer, cholestane-3 beta,5 alpha,6 alpha-triol, which has not previously been recognized as a cholesterol autoxidation product, was found at similar concentrations as the well-known cytotoxic cholestane-3 beta,5 alpha,6 beta-triol during both copper- and lipoxygenase-mediated LDL oxidation. Furthermore, two epimeric cholest-5-ene-3 beta,4-diols were identified in the oxidized LDL at similar concentrations. These two isomers were also identified in human atherosclerotic tissue in a ratio of 1:1 at a concentration more than 10-times higher than in non-atherosclerotic vessels. In vitro oxidation of LDL under an 18O2 atmosphere revealed that molecular oxygen was the only source of the oxygen functions at C-4 in the cholest-5-ene-3 beta,4-diols. Taken together, these findings suggest that the cholest-5-ene-3 beta,4-diols in atherosclerotic plaques are formed by autoxidation.

Time course of oxysterol formation during in vitro oxidation of low density lipoprotein.

Cholesterol oxidation products (oxysterols) have been implicated in several aspects of atherogenesis; they affect key enzymes in cholesterol homeostasis, induce calcification in vascular cells and possess cytotoxic properties. Oxysterols are formed during oxidative modification of low density lipoprotein (LDL). Using a recently developed method based on isotope dilution-mass spectrometry, the kinetics of formation of oxysterols during oxidation of LDL by cupric ions or soybean lipoxygenase was studied. The same products, mainly 7- and 5-oxygenated cholesterol, were formed by the two oxidation methods. Virtually no side-chain oxidized oxysterols were formed. During the oxidations, preferentially esterified cholesterol was consumed and consumption of polyunsaturated fatty acids and formation of conjugated dienes preceded the appearance of oxysterols. Cholesterol 7-hydroperoxides potential cytotoxins, were present in LDL oxidized by copper or lipoxygenase.

Determination of cholesterol oxidation products in human plasma by isotope dilution-mass spectrometry.

A method based on isotope dilution-mass spectrometry was developed for the determination of nine cholesterol oxidation products in human plasma. The cholesterol oxidation products determined were cholest-5-ene-3 beta,7 alpha-diol, cholest-5-ene-3 beta,7 beta-diol (7 alpha- and 7 beta-hydroxycholesterol, respectively), 3 beta-hydroxycholest-5-en-7-one(7-oxocholesterol),5,6 alpha-epoxy-5 alpha- cholestan-3 beta-ol (cholesterol-5 alpha,6 alpha-epoxide),5,6 beta-epoxy-5 beta-cholestan-3 beta-ol (cholesterol-5 beta,6 beta-epoxide), (cholesterol-5 beta,6 beta-epoxide), cholestane-3 beta,5 alpha,6 beta-triol, cholest-5-ene-3 beta,24-diol (24-hydroxycholesterol), cholest-5-ene-3 beta,25-diol (25-hydroxycholesterol), and cholest-5-ene-3 beta,27-diol (27-hydroxycholesterol). A corresponding deuterium-labeled internal standard, containing 3 to 6 deuterium atoms, was synthesized for each cholesterol oxidation product except 5 beta,6 beta-epoxycholesterol which was determined using the internal standard for 5 alpha,6 alpha-epoxycholesterol. Plasma from 31 healthy volunteers was analyzed by the new method and 27-, 24-, and 7 alpha-hydroxycholesterol were the most abundant cholesterol oxidation products (mean values 154, 64, and 43 ng/ml, respectively). The other oxysterols determined were present in concentrations lower than 30 ng/ml. Males had higher 27-hydroxycholesterol concentrations in plasma than females. The 5,6-oxygenated products were present mainly unesterified while the other oxidation products were mostly in esterified form.