James C. Orr

Preliminary observations on the occurrence of cholesterol sulfate in man

Abstract In the course of studies on the incorporation of sodium [ 35 S]sulfate into sulfatide in patients with neurological disorders, a metabolically active substance was found which resembles sulfatide both in solubility and behavior on column chromatography but is separable from it on thin-layer chromatography, A method was devised for the isolation of larger quantities of this material. It was found to have the same melting point and Chromatographie properties as authentic cholesterol sulfate and strongly resembles it in its infrared spectrum, Mild acid hydrolysis yielded a substance identical to cholesterol in its melting point, and infrared and nuclear magnetic spectra. A survey of human tissues revealed the presence of small amounts of cholesterol sulfate in plasma, bile, urine, liver, kidney, and larger quantities in feces. It was also found in the brain of a 4-day-old infant. Intravenously administered sodium [ 35 S]sulfate was rapidly incorporated into cholesterol sulfate. Until recently, cholesterol sulfate had not been isolated from any biological material. This study demonstrates that it is metabolically active and that it is present in a variety of human tissues and body fluids.

The twin ion technique for detection of metabolites by gas chromatography-mass spectrometry: Intermediates in estrogen biosynthesis☆

Abstract [4- 14 C + 7-D 0.44 ]Androstenedione and [4- 14 C + 7β-D 0.42 ]testosterone were prepared. When they were examined by mass spectrometry, the above proportion of deuterium and protium forms resulted in mass spectra in which the molecular ion (M + ) and (M + + 1) were of equal intensity. Fragment ions that contained deuterium were also twins. When doubly-labeled androstenedione and testosterone were used as substrates for the aromatizing enzymes of human placenta, the mass spectra of metabolites were characteristically labeled and thus readily distinguished from unlabeled material. Metabolites were quantitated by counting 14 C. 17β,19-Dihydroxyandrost-4-en-3-one, 19-hydroxyandrost-4-ene-3,17-dione, 17β-hydroxy-3-oxoandrost-4-en-19-al, 3,17-dioxoandrost-4-en-19-al, estradiol-17β, and estrone were isolated, identified by their mass spectra, and quantitated following incubation of doubly-labeled androstenedione and testosterone with human placental microsomes.

Methylation of 2-acetamido-2-deoxy-d-hexoses

Abstract Methyl iodide in the presence of the dimethylsulfinyl carbanion leads to complete O -methylation and N -methylation of 2-acetamido-2-deoxyglycosides. Thus, methylation of methyl 2-acetamido-2-deoxy-α- d -glucopyranoside and -galactopyranoside gave, respectively, methyl 2-deoxy-3,4,6-tri- O -methyl-2-( N -methylacetamido)-α- d -glucopyranoside and -galactopyranoside. Methylation, under these conditions, with methyl- 14 C iodide showed that fully methylated 2-acetamido-2-deoxyglycosides, detectable in small amounts by radioautography, are obtained in quantitative yield. Methylation of methyl 2-acetamido-2-deoxy-6- O -trityl-α- d -hexopyranosides, followed by removal of the trityl group and acetylation gave methyl 6- O -acetyl-2-deoxy-3,4-di- O -methyl-2-( N -methylacetamido)-α- d -hexopyranosides. Separation and identification of these N -methylated 2-acetamido-2-deoxyhexosides was accomplished by g.l.c. and m.s. Acid hydrolysis of these derivatives gave the corresponding hydrochlorides.

Quantitative gas chromatography of steroid methoxime-trimethylsilyl ethers

Abstract A method of preparing the 0-methyloxime trimethylsilyl ethers of nanomole quantitites of C 19 -and C 21 -steroids is described. The derivatives, identified by their mass spectra, can be separated and quantitated by gas chromatography using either isothermal or temperature programmed conditions. Thermal instability renders many free steroids unsuitable for gas-liquid chromatography. Derivative formation, such as the conversion of hydroxyl groups to trimethylsilyl ethers and of ketones to methoximes (0-methyloximes) overcomes this problem (3–9). In earlier investigations, large amounts of steroids were employed and quantitation was not investigated. We describe a gas chromatographic method of separation and quantitation of small amounts of C 19 — and C 21 -steroids as their methoxime-trimethylsilyl ethers. The derivatives were identified by gas chromatography-mass spectrometry. This technique is rapid and has been applied to the analysis of steroids in tissues and biological fluids (9).

Identification by gas chromatography-mass spectrometry of intermediates in the aromatization of modified C19 steroids by human placental microsomes☆

Abstract Analogs of 4-androstene-3,17-dione and testosterone were tested as substrates for the aromatizing enzyme complex of human placenta. Compounds modified in rings B, C and D were found to be aromatized via a pathway similar to that postulated for 4-androstene-3,17-dione and testosterone, in which oxidation to the 19-hydroxy and 19-oxo (or corresponding gem-diol) intermediates occurs. No evidence of additional intermediates was obtained.

The stereochemistry of hydrogen transfer to NADP+ by enzymes acting upon stereoisomeric substrates☆

Abstract The stereochemistry of hydrogen transfer from estradiol-17α and estradiol-17β to NADP + in the presence of chicken liver estradiol-17α and estradiol-17β dehydrogenases was found in both cases to involve the 4-pro-S proton of the pyridine nucleotide. One of these enzymes must therefore use the stereochemically less favorable mode of interaction of steroid with coenzyme.

A time study of the in vitro Metabolism of 3β-hydroxyandrost-5-en-17-one by human adrenocortical tissue

Abstract A kinetic study of the in vitro metabolism of 3β-hydroxyan-drost-5-en-17-one-7 2H by a normal human adrenal gland and an adrenocortical adenoma causing Cushings syndrome is presented. The principal product, androst-4-ene-3, 17-dione-7 2H, was separated, identified and quantitated by combined gas chromatography-mass spectrometry,, The results are compared with those obtained with 3β-hydroxyandrost-5-en-17-one-7 3H as substrate and analysis of the products by isotope dilution.

THE STEREOCHEMISTRY OF ENZYMIC REDUCTION OF 21-DEHYDROCORTISOL TO CORTISOL

A 21-hydroxysteroid dehydrogenase of sheep liver that causes reduction of 21-dehydrocortisol to cortisol does so with transfer of hydrogen from the 4-pro-S-position of NADH to the 21-pro-S-position of cortisol.

The stereochemistry of enzymic reduction of 21-dehydrocortisol to cortisol.

Abstract A 21-hydroxysteroid dehydrogenase of sheep liver that causes reduction of 21-dehydrocortisol to cortisol does so with transfer of hydrogen from the 4-pro-S-position of NADH to the 21-pro-S-position of cortisol.