Johan E. van Lier

Sterol metabolism: Part 9. 26-hydroxycholesterol levels in the human aorta☆

Abstract The concentration of 26-hydroxycholesterol in 36 samples of human intimal and 19 samples of human medial tissue of varying extent of atherosclerosis was estimated using thin-layer and gas chromatographic procedures. Levels of 26-hydroxycholesterol were compared with total lipid and cholesterol levels and all three entities were shown to increase in about the same manner with increasing severity of atherosclerosis both in intimal tissue samples and in medial tissue samples. These results are discussed in terms of their possible significance to the origin and progression of atherosclerosis.

Sterol metabolism: II. Gas chromatographic recognition of cholesterol metabolites and artifacts

Abstract The gas chromatographic characterization of common sterols related to cholesterol or implicated in cholesterol autoxidation is deseribed. Relative retention data are given for sixty cholesterol derivatives, including free sterols, steryl acetates, and steryl trimethylsilyl ethers, on four gas chromatographic systems.

Sterol metabolism VIII. Conversion of cholesterol 20α-hydroperoxide to 20α, 21- and 20α,22R-dihydroxycholesterol by adrenal cortex mitochondria

Abstract 1. 1. Cholesterol 20α-hydroperoxide incubated with bovine and murine adrenal cortex mitochondrial enzyme preparations yields 20α, 22 R -dihydroxycholesterol and 20α, 21-dihydroxycholesterol, isolated as crystalline material, both sterols being identified by direct comparison of physical properties with authentic reference samples. 2. 2. A chemical synthesis of the previously undescribed 20α, 21-dihydroxycholesterol from 3β, 21-diacetoxypregn-5-en-20-one is described. 3. 3. The enzyme involved could be solubilized from acetone-dried mitochondria by sonic treatment, giving a specific actifity of 2–5 nmoles/min per mg protein (at 30°). Preparations of partially purified cytochrome P-450 derived from these mitochondrial extracts were also enriched in the sought enzyme. 4. 4. The transformations of cholesterol 20α-hydroperoxide to 20α, 21- and 20α, 22 R -dihydroxycholesterols do not occur with heat inactivated preparations nor with preparations subjected to prolonged sonication. 5. 5. The transformations do not require molecular oxygen or added NADPH. 6. 6. Decomposition of cholesterol 20α-hydroperoxide to pregnenolone, androst-5-ene-3β, 17β-diol, and androst-5-en-3β-ol is catalyzed slightly by protein, but at a much lower reaction velocity than the enzymic transformation, thus becoming noticeable only after prolonged incubations. 7. 7. A speculative proposal is made that the adrenal cortical cleavage of the isohexyl moiety of the cholesterol side-chain may proceed via 20α-hydroperoxide formation followed by enzymic rearrangement to 20α, 22 R -dihydroxycholesterol, which then undergoes bond cleavage between C-20 and C-22 to yield pregnenolone.

Sterol metabolism. XVI. Cholesterol 20α-hydroperoxide as an intermediate in pregnenolone biosynthesis from cholesterol

Abstract Incubation of adrenal cortex mitochondrial enzymes with cholesterol 20α-hydroperoxide, 20α, 22R-dihydroxycholesterol, 20α-hydroxycholesterol, or pregnenolone prior to incubation with cholesterol-1, 2- 3 H inhibits incorporation of tritium into pregnenolone and other steroids. By means of added carrier sterols it was demonstrated that both cholesterol 20α-hydroperoxide and 20α, 22R-dihydroxycholesterol contained radioactivity after incubation. The combination of reaction inhibition and of incorporation of tritium into cholesterol 20α-hydroperoxide and 20α, 22R-dihydroxycholesterol suggest that both be important intermediates in the biosynthesis of pregnenolone from cholesterol.

Chromatography of some cholesterol autoxidation products on sephadex LH-20

Abstract The resolution on Sephadex LH-20 lipophylic gel of several C 27 , C 24 , C 21 , and C 19 steroids of interest in our studies of cholesterol autoxidation has been achieved. Relative mobilities of twenty steroids on adsorption thin-layer chromatograms are compared with relative mobilities on Sephadex LH-20, and a marked retardation effect on Sephadex LH-20 of four sterol hydroperoxides was noted.

Sterol metabolism. XI. Thermal decomposition of some cholesterol hydroperoxides

Summary The decomposition of cholesterol 20α- and 25-hydroperoxides was studied by gas chromatography. Cholesterol 20α-hydroperoxide decomposed to 3β-hydroxypregn-5-en-20-one, androst-5-en-3β-ol, cholest-5-ene-3β, 20α-diol, androst-5-ene-3β, 17β-diol, and androsta-5,16-dien-3β-ol. Cholesterol 25-hydroperoxide decomposed to cholest-5-ene-3β, 25-diol, chol-5-en-3β-ol, pregn-5-en-3β-ol, and 3β-hydroxy-27-norcholest-5-en-25-one. These thermal decomposition patterns, similar to but different from that of autoxidation products in air-aged cholesterol, account suitably for the presence of degraded steroids in air-aged cholesterol. Analogies are drawn between the autoxidation of cholesterol via hydroperoxide intermediates with subsequent spontaneous decomposition and the biological oxidation of cholesterol to give similar types of products.

24‐Hydroxycholesterol levels in human brain

24‐Hydroxycholesterol (cerebrosterol, cholest‐5‐ene‐3β;, 24ξ‐diol) occurs at low levels in human (Di Frisco, De Ruggieri and Ercoli, 1953; Ercoli and De Ruggieri, 1953a, 19536; Schubert, Rose and Burger, 1961; Van Lier and Smith, 1969, 1970), equine (Ercoli, Di Frisco and De Ruggieri, 1953a; Ercoli and De Ruggieri, 1953a, 1953b; Fieser, Huang and Bhattacharyya, 1957) and bovine (Richter and Dannenberg, 1969) brain tissue. Only one of two possible C‐24 epimeric alcohols appears to occur in human brain (Van Lier and Smith, 1970) and the sterol may be regarded as a true endogenous trace‐level sterol and not as an artifact of autoxidation derived during isolation and analysis. As a phase of our continuing interests in the presence of trace‐level polar sterols in human tissues (Van Lier and Smith, 1967, 1969, 1970, 1971a; Smith and Van Lier, 1970), we sought to measure levels of 24‐hydroxycholesterol in different parts of human brain by gas chromatographic means. The present report deals with our measurements of 24‐hydroxycholesterol in human cortex, subcortical white matter, midbrain, pons, and cerebellum.

Cholesterol autoxidation: Identification of the volatile fragments

The volatile fragments of air-aged cholesterol were analysed by means of gas chromatography-mass spectrometry; The following fourteen compounds were identified: ethanol, acetic acid, acetone, 2-methylpropene, 2-methyl-1-propanol, 2-methyl-2-propanol, 2-butanone, 2-methylpropionic acid, 2-methyl-2-butanol, 2-pentanone, 3-methyl-2-butanone, 2-methyl-1-pentene, 2-methyl-2-pentanol, and 2-methyl-4-penten-2-ol. Their formation via decomposition of initially formed sterol hydroperoxides is discussed.

Sterol metabolism XV. Rearrangement of cholesterol 20α-hydroperoxide by bovine adrenal cortex mitochondrial cytochrome P-450 preparations

Abstract Cholesterol 20α-hydroperoxide was converted by bovine adrenal cortex mitochondrial cytochrome P-450 preparations at pH 8 to 20α, 21-dihydroxycholesterol and 20α, 22R-dihydroxycholesterol. The rearrangement reaction previously followed by gas chromatography was followed spectrally by means of induced difference spectra. Cholesterol-20α-hydroperoxide did not give an induced difference spectrum with cytochrome P-450 nor did 20α, 22R-dihydroxycholesterol, but the product 20α, 21-dihydroxycholesterol gave a strong Type II induced difference spectrum, thus permitting direct observation of the rearrangement of cholesterol 20α-hydroperoxide to 20α, 21-dihydroxycholesterol. A spectral dissociation constant Ks could be derived from the spectral binding data for 20α, 21-dihydroxycholesterol. These results are discussed in light of our suggestion that cholesterol 20α-hydroperoxide may be an intermediate in the biosynthesis of pregnenolone from cholesterol.

Crystalline sterols obtained by gas chromatography

Abstract A gas chromatographic method utilizing capillary tube collection of eluted sterols has been examined for preparative use. A variety of sterols may be collected in a state of high purity after gas chromatography, their high purity being demonstrated by melting point and thin-layer and other gas chromatographic evidences.