Some Observations on the Kober Colour and Fluorescence Reactions of the Natural Oestrogens

Abstract

The two-stage Kober reaction with oestriol, oestrone and oestradiol-17\g=b\ has been investigated. The factors involved in the maximum production of colour are summarized.Reducing agents were important in both the first and second stages of the Kober reaction. Oestriol in small amounts did not form the red Kober colour when reducing agents were not present in the first stage.In the absence of water, sulphuric acid did not give optimal results in the first stage. Oestradiol-17\g=b\ failed to give the Kober colour reaction in the presence of high concentrations of sulphuric acid. Optimal sulphuric acid-water ratios differed for the three oestrogens and were 76 % sulphuric acid for oestriol, 66 % acid for oestrone and 60 % acid for oestradiol-17\g=b\. The development of fluorescence was similarly affected by sulphuric acid concentration.The phenol-sulphuric acid reagent used by many workers appears to owe its efficacy to the action of the phenol, partly as a reducing agent and partly as a diluent of the sulphuric acid.Oestriol reacted in the first stage at a slower rate than oestrone and oestradiol.Further water and heating was usually required after the first stage (i.e. for the second stage of the Kober reaction) for complete formation of the red colour. Maximum intensity and stability of the red colour in the second stage was obtained in the presence of 50-60 % sulphuric acid and water. Under these optimum conditions the second heating time was not critical.In the second stage, concentrations of sulphuric acid lower than 50 % caused instability of the colour and fading during heating.In the second stage of the Kober reaction, reducing agents with oxidation-reduction potentials of the order of the hydroquinone-quinhydrone couple were also required for the production of maximum density and stability of the red colour.A colour method based on these findings is presented.Marrian [1930] reported that when oestriol was warmed with concentrated sulphuric acid, an orange colour with a green fluorescence was produced. Kober [1931] noted that when this orange fluorescent solution was diluted with water and warmed, the colour changed to a clear red, also with a greenish fluorescence, and that this change appeared to be almost specific for the natural oestrogens. Phenol added to the reaction mixture quenched the final fluorescence making the red colour both more intense and suitable for visual colorimetry. This colour reaction is known generally as the Kober reaction, although many modifications have since been proposed. Kober's original method was thus carried out in two stages, the first being the formation of the orange (or under certain conditions, yellow) intensely fluorescing colour and the second the conversion of this to the red colour. The intense greenish fluorescence obtained in the first stage of the Kober colour reaction has been used by a number of workers [Jailer, 1947;, for the fluorimetrie estima¬ tion of the natural oestrogens. These fluorimetrie methods, though not highly specific, are very sensitive and...

An Investigation into the Automated Measurement of Urinary Oestrogens in Pregnancy

“…The fluoacid and quinol concentrations in Kober's reagent, rescence of free oestriol, oestradiol, oestrone, and heating coil temperature, and final acid con centra-their monoglucuronides was measured relative to tions. Brown (1952) has shown that these factors this standard under differing conditions of acid affect both the colour and fluorescence produced and quinol concentration in Kober's reagent, heating by all the oestrogens and also that different oestro-coil temperature, and final acid concentration. A gens react in different ways to changes in these number of urines, including a pooled urine, with conditions.…”

Urinary Inhibitors of ß-Glucuronidase

“…An example of such an inhibitor is saccharolactone. (6) It is possible to separate saccharolactone from the estrogen glucuronosides in urine by gel-filtration. (7) It is likely that competitive substrates of either normal dietary or of therapeutic origin and saccharolactone account for much of the inhibition ob served on adding urine to ß-glucuronidase.…”

“…The extent of hy drolysis of phenolphthalein glucuronide and p-nitrophenyl glucuronide was determined by adding glycine buffer (pH 10-5) to an appropri ately diluted aliquot of the incubation medium and comparing the optical density at 545 and 395 mji,, respectively with previously determined standard curves for phenolphthalein and p-nitrophenol. The extent of hydrolysis of estradiol glucuronide (17 ß-monoglucuronide) and estriol glucuronide (principally the 16 a-glucuronide, but probably a mixture, of mono-and di-glucuronides, [7]) was deter mined by extracting the incubation media with ethyl acetate, evapo rating the solvent and applying the Kober reaction [6]. Pregnanediol was extracted with a mixture of benzene and ethyl acetate (6:4) and was determined by the procedure of Ronan et al [23].…”

“…should be a linear function of the concentration of sac-V V1 (pH 6) charolactone or saccharic acid and should be equal to zero in the absence of saccharolactone or saccharic acid. That this plot is linear may be seen from Figure 4, where the appropriate plot has been made after determination of the extent of inhibition with several concen trations of inhibitor pretreated at pH 4 and 6.…”

Urinary Estrogens as a Measure of Fetoplacental Well-Being

“…Brown 15 substituted hydroquinone for phenol to lessen the oxidation of the estriol fraction by the sulfuric acid. With this modification, the Kober method lacked maximum sensitivity because numerous impunttes in urine produce a yellow color in the Kober…”