A FLUORESCENCE ASSAY OF PROGESTERONE

Abstract

SUMMARY A fluorescence assay for the quantitative estimation of progesterone has been developed. The method consists of ether extraction of progesterone from alkali-treated tissue or plasma, isolation by descending paper chromatography, conversion to 20β-hydroxypregn-4-en-3-one using the enzyme 20β-hydroxysteroid dehydrogenase, purification by paper chromatography and fluorimetric estimation of the 20β-hydroxy isomer in 2:1 conc. H2SO2: 80% ethanol reagent. The addition of a trace amount of [4-14C]progesterone to each sample was used to correct for losses incurred during the above procedure. The method was more sensitive and more specific than available methods of chemical estimation. Increased specificity was achieved by a number of modifying stages including enzymic conversion and chromatographic purification, the fluorescence reaction being carefully controlled to overcome its lack of specificity. The fluorescence of eluted paper blanks had a consistently low value of 4·5 ± 0·1 nanogram (ng.) Recoveries of known amounts of steroid from male guinea-pig plasma were 38·3 ± 7·3% and 35·4 ± 3·3%, measured by fluorescence assay and 14C counting respectively. Progesterone was estimated in rabbit ovarian tissue by fluorescence assay and u.v. spectrophotometry when the concentrations were sufficiently high. The fluorescence assay gave values which were slightly lower than by spectrophotometry. The technique has been applied routinely to blood plasma and other tissues of several mammalian species including laboratory animals.