Enzymatic estimation of steroids in subpicomole quantities by hydroxysteroid dehydrogenases and nicotinamide nucleotide cycling

Extremely sensitive methods are described for the measurement of 3 alpha- and 3 beta-hydroxysteroids, as well as 3-ketosteroids, based on their reaction with highly purified bacterial hydroxysteroid dehydrogenases and the amplification of the accompanying changes in nicotinamide nucleotides by enzym...

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Bibliographic Details
Published in:The Journal of biological chemistry 1982-01, Vol.257 (2), p.633-642
Main Authors: PAYNE, D. W, SHIKITA, M, TALALAY, P
Format: Article
Language:English
Subjects:
Analytical, structural and metabolic biochemistry
Animals
Biological and medical sciences
Chromatography, High Pressure Liquid - methods
Female
Fundamental and applied biological sciences. Psychology
Humans
Hydroxysteroid Dehydrogenases - metabolism
Kinetics
Male
Microchemistry
NAD
Other biological molecules
Oxidation-Reduction
Pregnancy
Prostate - analysis
Rats
Rats, Inbred Strains
Spectrophotometry, Ultraviolet - methods
Steroids - analysis
Steroids - urine
Terpenes, steroids. Hormones
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Summary:Extremely sensitive methods are described for the measurement of 3 alpha- and 3 beta-hydroxysteroids, as well as 3-ketosteroids, based on their reaction with highly purified bacterial hydroxysteroid dehydrogenases and the amplification of the accompanying changes in nicotinamide nucleotides by enzymatic cycling procedures. Conditions have been devised under which the steroid oxidation and reduction reactions lead to the formation of stoichiometric quantities of NADH or NAD+, respectively, even in the presence of large excesses of reaction products. The scope of these methods is illustrated by application to the analysis of minute volumes of human pregnancy urine, high pressure liquid chromatography fractions derived from such urine samples, and human serum. The steroid contents of milligram quantities of rat prostate have been determined. The methods have been applied also to the measurement of the activities of steroid-transforming enzymes, such as the 3 alpha-hydroxysteroid dehydrogenase of prostate microsomes. At the present time the sensitivity of the described methods allows the accurate determination of 0.2-0.4 pmol of steroids.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(19)68240-2