Yong Yeng Lin

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.

Sterol metabolism XXVIII. Biosynthesis and accumulation of cholest-5-ene-3β, 24-diol (cerebrosterol) in developing rat brain

Abstract Cholest-5-ene-3β,24-diol (cerebrosterol) was identified as a minor sterol of rat brain. The sterol occurs at levels comparable to those of human and other mammalian brain and appears to accumulate during maturation. Immature rat brain had 12.5–13.8 μg/g fresh brain; adult rat brain had 27.5–34.4 μg/g. The sterol was detected in nuclear, mitochondrial, microsomal and soluble fractions but was greatest in myelin and nerve endings fractions. Rat brain homogenates and microsomal (105000 × g pellet) fractions therefrom transformed [1,2-3H]cholesterol to [3H]cholest-5-ene-3β-diol in 0.15–0.18% yields.

Microbial hydroxylations. VII. Kinetic studies on the hydroxylation of 19-norsteroids by Curvularia lunata.

Abstract 19-Nortestosterone induces hydroxylases for its own metabolism by Curvularia lunata NRRL 2380, optimum induction times of 1–3 h being indicated. Comparison of product spate and hydroxylation rates for vegetative cell culture incubations and for cell-free enzyme systems suggests that the vegetative cell system may be used for study of the initial kinetics of hydroxylation of steroid substrates without interference of endogenous cellular metabolism. Double reciprocal plots of kinetic data establish that the three major modes of transformation of 19-nortestosterone (to the 10β-, the 11β-, and the 14α-monohydroxylated derivatives) have identical apparent Michaelis constants (Km 0.34 mM). Mathematical treatment of a model system supports identical Km values as found experimentally. Arguments based on these kinetics are presented which suggest that the 19-norsteroid hydroxylase system of C. lunata NRRL 2380 not be three individual hydroxylases acting independently but rather be a single hydroxylase of more generalized function.

Sterol metabolism--XLV. Differentiation among monohydroxylated cholesterol derivatives by chromatography and mass spectrometry.

Abstract Twenty monohydroxylated cholesterol derivatives have been characterized by high performance liquid column chromatography and by chemical ionization (CI) mass spectrometry. With ammonia as reagent gas four ions are formed: an ammonium adduct [M + NH 4 ] + , a substitution ion [M + NH 3 -OH] + and elimination ions [M-OH] + and [M-H 2 O-OH] + ]. Thermally stable stenediols exhibit [M + NH 4 ] + as principal ion, less stable allylic or tertiary alcohol derivatives the elimination ions [M-OH] + or [M-H 2 O-OH] + . With methane or isobutane as reagent gas monohydroxylated cholesterols yield five ions: the protonated molecular ion [M + H] + , molecular ion[M] + , hydride abstraction ion [M-H] + , and elimination ions [M-H 2 O + H] + and [M-2H 2 O + H] + , the principal ion uniformly being the [M-H 2 + H] + ion. Methane CI mass spectra of trimethylsilyl ether derivatives of the side-chain hydroxycholesterols exhibit unique fragmentation ions [C n H 2n OSi(CH 3 ) 3 ] + derived from α-cleavages diagnostic of the site of side-chain hydroxylation.

Microbial hydroxylations: VIII. Induction of steroid hydroxylases of Curvularia lunata by 19-nortestosterone analogs

Abstract 19-Nortestosterone, rac -19-nortestosterone, and testosterone induce hydroxylases of Curvularia lunata NRRL 2380 for their own metabolism and for the metabolism of rac -13β-ethyl-17β-hydroxygon-4-en-3-one. Products formed include derivatives monohydroxylated at the 10β-, ent -10β-, 11β-, ent -11β, ent -12α-, and 14α-sites, depending on substrate. Formation of specific ent -10β-, ent -11β-, and ent -12α-monohydroxylated products from racemic substrates in incubations induced by 19-nortestosterone or by testosterone implies that steroids of natural optical configuration induce hydroxylase activities which metabolize enantiomeric substrates of unnatural optical configuration. These points are interpreted in terms of our previously suggested possibility that a generalized hydroxylase be that agent causing formation of the complex spate of monohydroxylated products of both natural and unnatural optical configurations from racemic 19-norsteroid substrates in these systems.

Microbial hydroxylations: VI. Hydroxylation of rac-17β-hydroxyestra-4,8(14)-dien-3-one by Curvularia lunata

Abstract Aerobic incubations of rac-17β-hydroxyestra-4,8(14)-dien-3-one with Curvularia lunata NRRL 2380 gave rac-10β,17β-dihydroxyestra-4,8(14)-dien-3-one, rac-11β, 17β-dihydroxyestra-4,8(14)-dien-3-one, and rac-7α,17β-dihydroxyestra-4, 8(14)-dien-3-one. Some resolved 17β-hydroxyestra-4,8(14)-dien-3-one of natural configuration was also recovered. Structures of the hydroxylated products were determined from their spectral behavior. These results differ from our prior work where resolved hydroxylation products were obtained from rac-17β-hydroxyestr-4-en-3-one with C. lunata. These and other related points are discussed in speculation regarding microbial hydroxylase substrate specificity.