A rapid measurement of delta 5-3 beta-hydroxysteroid dehydrogenase activity coupled with delta 4-delta 5 isomerase by a high pressure liquid chromatography.

ABSTRACT

By a high pressure liquid chromatography, progesterone (lambda max = 240 nm, epsilon 240 = 17,000) was separable from the other delta 4-3-oxosteroids. Relationship between the amount of progesterone and the area of the peak corresponding to progesterone was established as linear at least between 0-2.0 g. Progesterone is formed from pregnenolone by delta 5-3 beta-hydroxysteroid dehydrogenase coupled with delta r-delta 5 isomerase. Because pregnenolone is devoid of absorption oat 240 nm, the enzyme activity was measurable by the high pressure liquid chromatography from the amount of progesterone produced from pregnenolone in vitro in the presence of nicotinamide adenine dinucleotide. As an example of its application, the dehydrogenase activities in the ovaries of the immature rats treated with gonadotropins were measured by the above method. The estimates obtained by this method were agreeable with those by thin layer chromatography, employing radioactive pregnenolone as the substrate.