The Colorimetric Determination of Urinary Estrin

Kober (1931) was the first to suggest a calorimetric test for urinary estrin. He compared the degree of red color developed after treatment with phenolsulfonic acid in a test sample with a standard cresol red solution. Cohen and Marrian (1934) modified the Kober procedure and determined the hormone content of estrone and estriol fractions by comparison with a standard curve from a set of determinations with pure hormone, using a Lovibond tintometer for color standards. Cartland, Meyer, Miller, and Rutz (1935) have also employed the phenolsulfonic acid reagent, using both the Lovibond tintometer and a colorimeter for color comparisons of their extracts against a crystalline hormone standard. These methods have been employed for the quantitative determination of the estrins. Sala (1935) has employed the greenish fluorescence that develops after the hormones are treated with concentrated H&$04 as a qualitative test for pregnancy in the mare. Chevallier, Cornil, and Verdollin (1935) have used the characteristic ultraviolet absorpt,ion at 2800 A. to detect estrogenic substances in human pregnancy urine extracted by alkali hydrolysis. Zimmerman (1935) has suggested that the reaction of estrone with m-dinitrobenzene in alkaline alcoholic solution should afford a good test. The violet color that develops occurs also with a number of other ketones. Schmulovitz and Wylie (1935) have used the orange color developed when the estrins are coupled with diazotized p-nitroaniline as a means of detecting these substances in human pregnancy urine, but no separat.ion of estrone and estriol is made in their extractions. David (1934) has noted a typical blue color when estriol crystals are treated with sulfuric acid followed by arsenic acid.