The hydrolysis of the combined forms of oestrone and oestriol present in human pregnancy urine

Abstract

FOR several years it has been recognised that the yields of oestrogenic material which can be obtained from human pregnancy urine by extraction with immiscible fat-solvents may be considerably increased by preliminary acidification of the urine [Marrian, 1930;1933;Doisy et al., 1930]. Zondek [1934, 1] did, indeed, state that such preliminary treatment was unnecessary to ensure complete extraction of the hormones, but later [1934, 2] revised his opinions as a result of work carried out by Borchardt et al. [1934]. The latter authors were responsible for the first systematic attempt to determine the optimum conditions for the liberation of oestrin from its combined state in human pregnancy urine. They showed that the amount of hormone that could be extracted by prolonged boiling with benzene was greatly increased by acidification of the urine to PH 3 0, and still further increased by the addition of hydrochloric acid to a concentration of 3-6 %.The present authors [1934] developed a colorimetric method, based on the Kober test [1931] for oestrin, for the separate quantitative estimation of oestrone and oestriol in ethereal extracts of human pregnancy urine. This method enabled a more complete study of the factors influencing the hydrolysis by acids of the " oestrin-esters " in urine to be made than would have been possible if only the biological test for oestrin had been available. It was shown that when pregnancy urine was acidified to PH 1-2 and then heated at 1000, there occurred a slow liberation of ether-soluble oestrin from the combined form, which reached a maximum after 16 hours. Heating for longer periods resulted in a decrease in the amount of ether-extractable oestrin, indicating that destruction of the liberated hormone had occurred. By heating the urine acidified to PH 1-2 in an autoclave at 15 lbs. pressure for 2-4 hours, a larger amount of ether-soluble oestrin was liberated than when the urine was heated at 1000. Autoclaving for longer periods destroyed the liberated oestrin. This work clearly showed that, as might be expected, the rate of hydrolysis of the "oestrin esters " in urine is considerably increased by an increase in temperature, and that destruction of the hormone may occur on prolonged heating in an acid aqueous medium.Few attempts have been made to effect liberation of the combined oestrin in urine by means other than that of hydrolysis with acids. Marrian [1933] found that keeping the urine until considerable bacterial decomposition had occurred was extremely effective as a means of liberating the combined oestrin, but that heating to 1000 at an alkaline reaction for short periods was ineffective. Both these findings were confirmed by Cohen and Marrian [1934].For two reasons it was clearly necessary to conduct a more detailed investigation of the factors involved in the hydrolysis of the " oestrin esters " in human pregnancy urine. In the first place, estimation of the oestrin content of urine ( 1577 )

The determination of oestrogens in human pregnancy urine. A new method of correcting for the brown colour developed in the Kober reaction by non-oestrogenic substances

“…On the basis of these observations a simple procedure for correcting for the non-oestrogen brown colour given by human pregnancy urine oestrogen concentrates in the Kober reaction has been worked out. This correction procedure which, in principle, has much in common with the one suggested by Jayle et al (1943), is undoubtedly superior to the H202 method of Cohen & Marrian (1934) and because of its simplicity would seem to have certain advantages over the methods employed by others for the avoidance of, or correction for, the interfering brown colour. EXPERIMENTAL Hydrolysis and extraction of urine A method of hydrolysis of urine based on that suggested by Smith & Smith (1935) has been employed.…”

“…The oestrogen was added to the urine after hydrolysis rather than before in order to avoid complicating the issue by the losses due to destruction which are known to occur during the hydrolysis (cf. Cohen & Marrian, 1935 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 Fraction of 24 hr. urine specimen used (%) Vol.…”

“…The phenolsulphonic acid reagent was prepared according to the method of Cohen & Marrian (1934). The technique of colour development adopted was essentially similar to that described by Venning et at.…”

“…HCI to the urine, heated to boiling and continued the boiling for 10 min. It was shown by Smith, Smith & Pincus (1938) that the yields of free oestrogen extractable from human pregnancy urine after such a hydrolysis were as high as those obtained after the less convenient method of Cohen & Marrian (1935 Urine specimens were collected with toluene as preservative. In order to standardize the procedure as far as possible, 24 hr.…”

“…The problem of correcting for this interfering brown colour, or of eliminating without loss of oestrogen the substances which give rise to it has been approached by a number of workers in different ways. Cohen & Marrian (1934) measured the red colour of the final reaction mixture in Lovibond units and then subtracted the red component of the residual brown colour after fading the oestrogen pink colour with H202. However, as was pointed out by Venning, Evelyn, Harkness & Browne (1937) and by Bachman & Pettit (1941), this method of correction is not satisfactory since treatment with H202 causes some fading of the non-oestrogen brown colour.…”