Colorimetric determination of substances containing the grouping —CH2.CO— in urine extracts as an indication of androgen content

Abstract

IN the expectation that a measure of endocrine activity in the human patient might be given by the level of excretion in the urine of compounds related to the male hormones, attention has recently been directed to the extraction of urinary constituents possessing androgenic activity and their measurement.As an alternative to biological assay, chemical methods have been applied.Zimmermann [1935] first suggested that steroid sex hormones could be deter- mined quantitatively by the use of m-dinitrobenzene, which gives a red colour in presence of alkali with compounds containing an active methylene group.A year later [Zimmermann, 1936] he published a modified method which was applied to pure compounds (androsterone, testosterone, oestrone and equilin), and to urine extracts.Wu & Chou [1937] described a modification of the method, and used it on urine extracts with androsterone as a reference substance.Oesting & Webster [1938; cf.Oesting, 1937] used the Zimmermann technique, and roughly correlated their figures with the comb-growth produced in capons by inunction of the same extracts on the comb.None of these investigators has considered simultaneously both capon assay and colorimetric assay in terms of pure hormones, and there was thus an obvious gap to be filled before it would be possible to test the colorimetric assay as an indicator of the androgenic activity of urine extract in terms of international units.,The correlation of the figures obtained in this way would be expected to give data which, in addition to testing the value of the colorimetric assay on the assumption that excretion of androgenic activity was diagnostically sig- nificant (as has been done by Oesting & Webster), would also throw light on the chemical nature of the androgenic substances excreted in the urine, providing clues to the metabolism of steroid hormones, and giving a tangible chemical property which would serve as a guide in the analytical investigation of urine extracts.As part of a scheme of work on the determination of hormones in blood and, urine, undertaken under the auspices of the Hormones Committee of the Medical Research Council, investigations of methods of extraction of androgens from urine and their assay on capons have been in progress at this Institute for some time.These have been extended to a comparison of colorimetric and capon assays, and, on the chemical side in particular, to attempts to improve the sensitivity of the colorimetric method and to investigate its specificity.The account of experimental work which follows deals in turn with the description of the modified colorimetric method finally adopted, a study of some of the many factors influencing the reaction, an investigation of the behaviour ( 1312 )1313'