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SUMMARY
A purification step involving separation of the ketonic fraction through the Girard complex has been incorporated in an earlier method for estimating urinary oestrogens. The new method, which measures oestrone only, involves acid or enzymic hydrolysis of the urine, extraction with ether, removal of the acidic fraction with alkaline carbonate solution, evaporation of the ether, separation of the ketonic fraction through the Girard complex, saponification, methylation, and chromatography on alumina columns. The oestrone methyl ether present in the final extract is measured by a semi-micro modification of the Kober reaction, which, with the larger volume of urine processed, increases the sensitivity of the estimation ninefold. This increase in sensitivity is made possible by the purification achieved in the new method, and fractions of 1 μg oestrone/24 hr urine can be measured. The reliability of the new method has been investigated and data concerning its accuracy, precision, sensitivity and specificity are presented. By comparison with the new method, the earlier method gives overestimates of approx. 0.9 μg oestrone/24 hr urine.
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SUMMARY
The hydrolysis of the conjugates of oestrone, oestradiol-17β and oestriol present in human urine has been investigated using acids and the enzymes β-glucuronidase and phenol sulphatase derived from Patella vulgata.
Using acid hydrolysis, maximum yields were obtained from the majority of urines by boiling 60 min with 15 vol. % concentrated hydrochloric acid. Under these conditions losses amounting to approx. 20% of the oestrogens present occur in concentrated urine specimens. These losses can be diminished by diluting the urine with water before hydrolysis.
Using enzymic hydrolysis, maximum yields were obtained by incubating the urine for 96 hr at 37° C and pH 4·7 with approx. 600 u./ml. β-glucuronidase.
When allowance was made for the losses which occur during acid hydrolysis, the yields from pregnancy and non-pregnancy urine were approximately the same by the two hydrolytic procedures. From this, it is inferred that no unknown major error exists in either procedure.
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At low oestrogen concentrations the assay methods of Brown (1955) and of Bauld (1956) for measuring oestriol, oestrone, and oestradiol-17β in urine are limited by impurities remaining in the final fractions. This interference has been diminished in Brown's method by an additional step of purification taken from Bauld's method, thus enabling the three oestrogens to be measured in most urines from men and pre-and postmenopausal women (Brown, Bulbrook & Greenwood, 1957 a). However, more complete purification is necessary for the measurement of still smaller amounts of oestrogens present, for example, in the urine of children, or of patients who have been subjected to gonadectomy, adrenalectomy or hypophysectomy. A procedure giving highly purified oestrogen fractions in quantitative yield would be useful in the study of oestrogen excretion after the administration of small doses of tritium-labelled precursors. Such a procedure would obviate the necessity of purifying to constant specific activity with its