A Method for the Measurement of Estrone and Estradiol-17β in Peripheral Human Blood and Other Biological Fluids Using35S Pipsyl Chloride

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Abstract
A double-isotope derivative method suitable for the estimation of estrone and estradiol–17β in human peripheral blood and ovarian or adrenal venous blood of sheep is described. 3H-estrone or estradiol-17β (42.4 c/ mmol) was added as indicator to the biological sample before extraction to allow for the calculation of losses. The phenolic extract of blood was reacted with 35S p.-iodobenzene sulfonyl chloride (150–200 me/mmol), and the derivatives purified by 4 chromatography and the 2 additional derivative steps. About 10 % of both steroids was present in the final sample which was used for counting. Specificity was indicated by the fact that the nonspecific blank of the method, 0.26 ±0.10 (sd) ng for estrone and 0.24±0.16 (sd) ng for estradiol, was the same for water and plasma from adrenalectomized-oophorectomized sheep or women. There was no statistically significant difference in the value calculated from aliquots taken before the acetylation and chromatography, the final stage and following an additional reverse-phase chromatography step. The coefficient of variation of replicate samples when 0.80 ng was added to 10 ml aliquots of a pool of plasma from an adrenalectomized-oophorectomized sheep was 20% for estrone and 18% for estradiol-17β. Regression analysis of estimates of known quantities of estrone and estradiol-17β in the lower range (0.80–8.00 ng) added to a pool of sheep plasma indicated no systematic error in the method. No estrone could be detected in sheep ovarian venous blood in the presence of large amounts of estradiol-17β; adrenal-venous blood of sheep contained no estradiol-17β and small amounts of estrone. The mean values in ng/100 ml (corrected for the method blank) of peripheral plasma obtained from 6 normal women in the follicular and luteal phase of the menstrual cycle were 5.7±1.8 (sd) and 11.1 ± 1.40 (sd) for estrone, and 6.4 ±3.62 (sd) and 19.6 ±1.55 (sd) for estradiol-17β, respectively. The values in 4 normal men were 6.40 ± 1.50 (sd) for estrone and 2.1 ±0.52 (sd) for estradiol-17β.

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