Increased Estrogenic Potency of Human Urine after Hydrogenation

Abstract

A method described by us1 for the hydrolysis and extraction of urine for estrogens involves boiling with 15 vol. % of HC1 for 10 minutes, followed by continuous extraction with benzene (Benzol, Merck's Reagent) for 24 hours. It has been shown that the benzene extraction recovers all estrogens present after hydrolysis. It has also been demonstrated that this short hydrolysis with a high concentration of acid gives values which check with those obtained after the Cohen and Marrian 2 technique,∗ and does not affect the potency of estrone or estriol added to urine of known “total” estrogenic content.

Although the application of this method of quantitation has been found to yield consistent physiological curves of estrogen excretion, 3 , 4 there is no proof that all bound estrogens are freed by the acid hydrolysis or that destruction of combined estrogens may not occur. With the idea of possibly increasing hydrolysis as well as preventing any destruction of combined estrogens through oxidation, zinc (Zinc Dust, Merck's Reagent) has been added to urines prior to the acid treatment. A marked rise in the estrogenic potency of urines from both pregnant and non-pregnant women has resulted (Table 1).

“To” signifies the addition of 15 vol. % HC1 and 10-minute boiling under a reflux condenser; T_zn the addition of 15 vol. % HC1 and 4% Zn and 3-hour boiling under a reflux. Four percent zinc constitutes an excess with 15 vol. % HC1. Maximum increase in potency occurs after 2 hours and is not changed after 5 hours of boiling with acid and zinc. Evolution of hydrogen continues both during boiling and extraction. The titratable acidity is reduced from around 1.5 N to around 1.3 N in 3 hours of boiling. It is to be noted that hydrogenation does not affect a uniform increase in potency, since the ratios of T_zn to To, when assayed in olive oil, vary between 2.0 and 6.6.

The processes involved in this augmentation of urinary estrogen by zinc hydrolysis have not as yet been identified. The results thus far, howrever, are in accord with the hypothesis that the explanation lies in increased hydrolysis, and also conversion of estrone (but not estriol) into a reduced form of greater estrogenic activity, possibly dihydro-estrin (dihydro-theelin, estradiol). It is apparent for the present that hydrolysis with the addition of zinc may not be employed in physiological studies of estrogen excretion, although there is some indication that the ratio of T_zn to To may provide an index of the relative estrone content of specimens analyzed.