Estimation of Progesterone in Human Peripheral Blood Using³⁵S-Thiosemicarbazide1

Abstract

A double isotope derivative method for the estimation of progesterone in 10 ml peripheral plasma, using 35S-thiosemicarbazide (100 mc/mM) as reagent and 1,2-3H-progesterone (0.8 mμg, 87 μc/μg) a s indicator, is described. Purification is achieved by chromatography of the first derivative formed, progesterone bisthiosemicarbazone, by a silica gel thin layer and a paper system, hydrolysis to progesterone 3-thiosemicarbazone and chromatography of this derivative by a thin layer system, and finally acetylation to the 3-thiosemicarbazone 2′,4′-diacetate, and subsequent chromatography of this derivative on one thin layer and one paper system. The values given by 10 ml water samples and the same volume of plasma from ovariectomized, adrenalectomized subjects were 0.0020 ±0.0009 (sd) ±0.0002 (se) μg (23 samples) and 0.0029 ±0.0013 (sd) ±0.006 (se) μg (5 samples), respectively. These mean values were therefore the nonspecific blank of the method. No known naturally occurring steroids were found to interfere with the estimations. The precision and accuracy of the analysis of quantities of progesterone present in the plasma of normal women was found to be 7.5%. The standard error of the estimate was 0.0009 μg/10 ml in the lower range of values without subtraction of the nonspecific blank, which also had a standard deviation of 0.0009 μg/10 ml. Values (corrected for the appropriate water blank) for plasma taken from normal young women in the luteal and follicular phase of the cycle were 1.04 ±0.32 (sd) ±0.13 (se) (7 samples) and 0.113 ±0.049 (sd) 0.021 (se) ±/100 ml (5 samples). Plasma from ovariectomized females gave 0.039 ±0.010 (sd) ±0.004 (se) μg/100 ml (6 samples) (corrected for blank), indicating that the ovaries of normal women secrete progesterone or a precursor throughout the cycle. Plasma from males gave 0.028 ±0.013 (sd) ±0.004 (se) μg/100 ml (13 samples) (corrected for blank), which is similar to the values for ovariectomized females, although the excretion of urinary pregnanediol is much higher in males than in ovariectomized females. This indicates that urinary pregnanediol is not a unique metabolite of plasma progesterone in young males.