The application of the Kober test to the quantitative estimation of oestrone and oestriol in human pregnancy urine

Abstract

IT is well known that both oestrone and oestriol when warmed with concentrated sulphuric acid give a green-fluorescing orange-coloured solution, thus resembling sterols and the bile acids. The colours given by the latter compounds are discharged by dilution with water, but as Kober [1931] showed, the oestrin colour on dilution with water is changed to a clear red, the green fluorescence being retained. His evidence suggested that this test was highly specific for oestrin. Kober also showed that the addition of phenol to the sulphuric acid eliminated the green fluorescence from, and intensified the red colour of, the finally diluted solution. He stated that under the conditions of his procedure, the intensity of the red colour bore a direct but not linear relationship to the amount of oestrin, and he claimed that by the use of this test it was possible to assay the oestrin content of impure extracts. The obvious desirability of a rapid chemical method for the quantitative determination of the oestrin content of human urines led the authors to investigate the value of the Kober test for such a purpose. EXPERIMENTAL. Modification8 introduced into the Kober technique. Kober suggested the following technique. About 5y of crystalline hormone (presumably oestrone) are heated to 100' for 2 minutes with 0-2 ml. of a mixture of equal parts2 of concentrated sulphuric acid and phenolsulphonic acid (o-+p-). The mixture is then cooled, and after the addition of 02 ml. of water is heated momentarily to boiling. Finally the solution is made up to 1 ml. with water and immediately cooled. After standing in the cold for 5-10 minutes, the colour is compared with that of a standard solution of acid cresol red (10 mg. in 100 ml.) in a colorimeter. Kober states that 5y of the hormone give a colour of the same intensitv as that of 25y of cresol red. Since the greater part of the oestrogenic material in human pregnancy urine is oestriol, most of the earlier experiments described here were carried out on l The names oestrone and oestriol are here used to designate the two oestrogenic substances present in human pregnancy urine which have been known previously as ketohydroxyoestrin and trihydroxyoestrin, theelin and theelol, etc. This new system of nomenclature has been approved by the majority of the interested English workers [Nature, 1933, 132, 205]. The name oestrin is used where it is desired to refer to the oestrogenic material in urine without specifying its exact chemical nature. 2 It is assumed here that Kober used equimolecular parts of sulphuric and phenolsulphonic acids.

Analytical Methods for the Determination of Neuroactive Steroids

“…Efforts to quantify steroid hormones using colorimetry-based methods have existed since the 1930s [13][14][15]. One of the first methods used to define and quantify hormonal activity was also a bioassay [16].…”

“…Quantitative MS analyses are mainly based on the use of internal standards, which are compounds of either similar structure, analyte analogues, or stable isotope-labeled (SIL) standards bearing, for example, 13 C, 15 N, 17 O, or 2 H [69,70]. The use of SIL standards has become increasingly dominant in recent years.…”

The isolation of 16-epioestriol from the urine of pregnant women

“…Oestriol fractions containing KC-4 were prepared from ether extracts of the acid-hydrolysed urine both by the usual benzene-water partition procedure and by the original procedure of Cohen & Marrian (1934) involving extraction of a 'strong phenolic' fraction by 0-1 N-NaOH from ether. The latter procedure, being more economical in solvents, was clearly preferable for large-scale use.…”

Methods of Extracting Compounds Related to the Steroid Hormones From Human Urine

“…Marrian [1933], and Cohen and Marrian [1934] observed that allowing urine of pregnancy to putrefy was a more effective method of liberating oestrogens than hydrolysis by acid for 1 hour at 100°C, and Patterson [1937] based a routine method of chemical diagnosis of pregnancy on liberation of oestriol by incubation of urine with Bact. coli.…”