A double-isotope derivative method for plasma progesterone assay

Abstract

Studies of the metabolism of progesterone have been hampered by the lack of a specific and sensitive method for the measurement of this steroid in plasma. Application of the double-isotope derivative method offered a solution to this problem. The usual derivative that is used in methods of this type is a tritiated acetate. Since progesterone cannot be acetylated, an alternative was devised by reducing the steroid with sodium borotritide followed by partial re-oxidation with manganese dioxide. The resultant product is isolated and purified by paper chromatography prior to counting. The accuracy of the method was demonstrated by assay of solvent samples and analysis of two pools of pregnancy plasma, results of which are given in this paper. Another study showed that as little as one milliliter of pregnancy plasma was sufficient to give an accurate result. Authentic progesterone was added to male plasma in amounts of 0.1 μg/5 cm 3 of plasma for recovery studies. The measured values of 0.140 ± 0.008 μg/5 ml and 0.032 ± 0.04 μg/5 ml, when compared to values of 0.033 ± 0.009 μg/5 ml, led us to believe that the latter value represents progesterone endogenous to male plasma. The specificity of the method is shown by a comparison of partition coefficients. Derivatives were made from authentic progesterone-4C 14 and from a plasma extract. Aliquots of each were partitioned in two systems. The resultant coefficients were 2.86 vs. 2.87, and 0.225 vs. 0.219. The final derivative has been identified as 20-hydroxy-pregn-4-ene-3-one by R f values on several chromatogram systems and by crystallization to constant specific activity.