Jon I. Teng

Sterol metabolism—XLVII. oxidized cholesterol esters in human tissues☆

Abstract Human aorta, liver, and plasma have been examined for the presence of fatty acylesters of oxidized cholesterol derivatives using high performance liquid chromatography and chemical ionization mass spectrometry. Many such sterol esters have been detected, including eight individual diesters and six individual monoesters of six sterols and six C 14 -C 18 fatty acids. Mono- and di-esters of the epimeric 5-cholestene-3β,7-diols, 5-cholestene-3β,25-diol, and a 5-cholestene-3β,26-diol were found at μ/g levels in aorta; palmitate esters of 5-cholestene-3β,25-diol and 3β-hydroxy-5-cholesten-7-one were detected at ng/g levels in liver; and esterified 5-cholestene-3β,7α-diol,5-cholestene-3β,7β-diol, 3β-hydroxy-5-choles ten-7-one, a 5-cholestene-3β.24-diol, 5-cholestene-3β,25-diol, and a 5-cholestene-3β,26-diol were present in plasma at ng/ml levels. The presence of esterified 5-cholestene-3β,7α-diol, 5-cholestene-3β,7β-diol, and 3β-hydroxy-5-cholesten-7-one together may be interpreted as evidence of in vivo lipid peroxidation of cholesterol.

High-performance liquid chromatography of linoleic acid hydroperoxides and their corresponding alcohol derivatives

The four major hydroperoxides derived from autoxidation or lipoxygenase action on linoleic acid or on methyl linoleate and their corresponding alcohol derivatives are resolved by high-performance liquid chromatography on Zorbax SIL 3 micron particulate columns irrigated with hexane-diethyl ether-acetic acid mixtures. The four major linoleic acid hydroperoxides are interconverted at different rates in benzene or carbon tetrachloride solutions but are stable to storage under nitrogen at -70 degrees C for several months.

BIOSYNTHESIS OF CHOLEST-5-ENE-3β, 24-DIOL (CEREBROSTEROL) BY BOVINE CEREBRAL CORTICAL MICROSOMES1

The presence of cholest‐5‐ene‐3β, 24‐diol (cerebrosterol) in samples of human, bovine, and rabbit brains has been established by isolation of the sterol therefrom and identification by comparison of physical properties. Cholest‐5‐ene‐3β, 24‐diol was present at the level of 66.5 sg/g of dried tissue in human brain, 42.9 μg/g in cattle brain, and 89.5 pg/g in rabbit brain. Cholest‐5‐ene‐3β, 24‐diol was the only readily detectable hydroxycholesterol derivative in these brain tissues and was concentrated in the 105,000 g pellet (microsomal fraction) of both human and bovine cerebral cortex, with no demonstrable amounts of the sterol present in nuclear or mitochondrial fractions. Incubation of [1,2‐3H]‐ or of [4‐14C]‐cholesterol with the 105,000 g microsomal pellet from bovine cerebral cortical homogenates demonstrated 0.1‐0.38 per cent conversions to radioactive cholest‐5‐ene‐3β, 24‐diol, isolated and purified as the 3β, 24‐dibenzoate. The bioconversion required oxygen, and a stimulation of hydroxylation by added NADPH2 was demonstrated. Our observations establish that a sterol 24‐hydroxylase system is present in bovine cerebral cortex.

High-performance liquid chromatography of cardiolipin.

Resolution of freshly prepared and of commercially available (degraded) samples of cardiolipin into 15-30 components has been accomplished by reversed-phase high-performance liquid chromatography using a 3-micron particulate Microsorb C18 column irrigated with linear gradients of acetonitrile--methanol--10 mM phosphate buffer pH 7.4. Selected resolved components were crystallized and characterized by infrared absorption spectra. Saponification of other components and identification of component fatty acids by reversed-phase high-performance liquid chromatography demonstrated the presence of ten fatty acids (14:0, 14:1, 16:0, 16:1, 18:0, 18:1, 18:2, 18:3, 20:0, 20:4), with linoleic acid (18:2) identified in all resolved components. From fatty acid composition data it appears that several resolved fractions consist of single cardiolipin molecular species.

Sterol metabolism. XXVI. Pyrolysis of some sterol allylic alcohols and hydroperoxides

Abstract The thermal decomposition of the allylic alcohols 5α-cholest-6-ene-3β,5-diol, cholest-5-ene-3β,7α-diol, and cholest-5-ene-3β,7β-diol and of the allylic hydroperoxides 3β-hydroxy-5α-cholest-6-ene-5-hydroperoxide, 3β-hydroxycho lest-5-ene-7α-hydroperoxide, and 3β-hydroxycholest-5ene-7β-hydroperoxide to six common major pyrolysis products cholest-5-ene-3β,7α-diol, cholest-5-ene-3β,7β-diol, 3β-hydroxycholest-5-en-7-one, cholesta-3,5-dien-7-one, cholesta-4,6-dien-3-one, and cholesta2,4,6-triene was established.

High-performance liquid chromatographic analysis of human erythrocyte oxyterols as Δ4-3-ketone derivatives

Abstract Following oxidation by cholesterol oxidase human erythrocyte oxysterols were analyzed as the corresponding hydroxy- Δ 4 -3-ketones by HPLC with effluent monitoring at 235 nm. Variable amounts of epimeric cholest-5-ene-3s,7-diols, 3s-hydroxycholest-5-en-7-one, cholest-5-ene-3s,19-diol, cholest-5-ene-3s,20-diol, cholest-5-ene-3s,25-diol and cholest-5-ene-3s,26-diol were detected in erythrocyte membranes of patients with sickle cell anemia or sickle cell trait. Only 3s-hydroxycholest-5-en-7-one was detected in erythrocyte membranes of healthy donors.

Sterol metabolism: XXXVII. On the oxidation of cholesterol by dioxygenases☆

Abstract The oxidation of cholesterol by plant and mammalian dioxygenases yielding cholesterol 7α- and 7β-hydroperoxides has been demonstrated. Cholesterol oxidation is coupled to the oxygenation of polyunsaturated fatty acid esters by soybean lipoxygenase, to the reduction of hydrogen peroxide catalyzed by horseradish peroxidase, and to the oxidation of NADPH by the NADPH-dependent microsomal lipid peroxidation system of rat liver. The initially formed epimeric cholesterol 7-hydroperoxides are transformed in each case to the commonly encountered corresponding 7-alcohol and 7-ketone derivatives. These dioxygenase transformations thus mimic in detail the radiation-induced free radical oxidation of cholesterol by molecular oxygen. Electronically excited (singlet) molecular oxygen is not implicated in these transformations.

Sterol metabolism. XL. On the failure of superoxide radical anion to react with cholesterol

Superoxide radical anion (O-2) failed to react with cholesterol under a variety of conditions. In some instances products indicative of free radical oxidation by molecular oxygen (O2) were found, but no products of electronically excited (singlet) molecular oxygen (1O2) attack on cholesterol were detected. These results do not support a direct role of O-2 in lipid peroxidation of cholesterol-rich membranes or of the formation of 1O2 from O-2 dismutation.

Sterol peroxidation by Pseudomonas fluorescens cholesterol oxidase

Cholesterol is oxidized by commercially available Pseudomonas fluorescens cholesterol oxidase to 6 beta-hydroperoxycholest-4-en-3-one as the initial product, with none of the expected produce, cholest-4-en-3-one, formed. The transformation indicates that P. fluorescens cholesterol oxidase also acts as a flavoprotein dioxygenase.

Sterol hydroperoxide metabolism by Salmonella typhimurium

In order to rationalize multiphasic dose-response data evincing mutagenicity towards Salmonella typhimurium TA1537 for sterol hydroperoxides 3 beta-hydroxy-5 alpha-cholest-6-ene-5-hydroperoxide and 3 beta-hydroxycholest-5-ene-7 alpha-hydroperoxide their metabolism by the bacterial test strain was investigated. The 5 alpha-hydroperoxide was isomerized to the 7 alpha-hydroperoxide and reduced to 5 alpha-cholest-6-ene-3 beta,5-diol; the 7 alpha-hydroperoxide was reduced to cholest-5-ene-3 beta,7 alpha-diol and transformed to 3 beta-hydroxycholest-5-en-7-one. The 3 beta,5 alpha-diol and 3 beta,7 alpha-diol were not interconverted nor was either transformed to the 7-ketone.