A Double Isotope Procedure for the Determination of Progestins in Rat Ovarian Vein Blood

Publisher Website
Google Scholar
Abstract
A double isotope dilution method is described for the quantitative determination of progestins in rat ovarian vein blood. Pure samples and extracts from blood (which include trace quantities of carbon-14 labeled indicators) are incubated with alumina-3H2O to form tritium-labeled steroids by the exchange of enolic hydrogens for tritium. The tritiated progestins are easily purified by thin-layer chromatography to a constant tritium-carbon-14 ratio. The incorporation of tritium into progesterone and into 20[alpha]-hydroxypregn-4-en-3-one (20[alpha]-OHP) is directly proportional to the quantity of steroid. It was calculated that the amount of steroid that can be confidently distinguished from the tracers is 0.02 fig for progesterone and 0.05 [mu]g for 20[alpha]-OHP, and that the error of a measurement of progesterone at a level of 0.18 [mu]g is [plus or minus] 9.2% and the error of a measurement of 20[alpha]-OHP at a level of 0.5 [mu]g is [plus or minus] 8.2%. Progesterone is quantitatively recovered from 1 ml of blood from hypophysectomized rats. Results are presented to show that the method is adequate for the determination of progesterone and 20[alpha]-OHP in a single 5-min. collection of ovarian venous blood. It was found that ovarian secretion of progesterone remained quite constant throughout a 40-min. collection period and that the mean rate of secretion of 20[alpha]-OHP in the estrous rat is 3-fold greater than that of progesterone.