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P. K. SIITERI, P. TIPPIT, C. YATES, J. C. PORTER, A Double Isotope Procedure for the Determination of Progestins in Rat Ovarian Vein Blood, Endocrinology, Volume 82, Issue 4, 1 April 1968, Pages 837–843, https://doi.org/10.1210/endo-82-4-837
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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 tritiumlabeled 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 20α-hydroxypregn-4-en-3-one (20α-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 μg for progesterone and 0.05 μg for 20α-OHP, and that the error of a measurementc of progesterone at a level of 0.18 μg is ± 9.2 % and the error of a measurement of 20α-OHP at a level of 0.5 μg is ±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 20α-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 20α-OHP in the estrous rat is 3-fold greater than that of progesterone. (Endocrinology82: 837, 1968)