Kober Color Research Articles

Proton Acid‐Catalysed Transformations of Estrogen Derivatives: New Results and Some Mechanistic Aspects of the Kober Colour Reaction

AbstractThe 3‐O‐methylated estrogen derivatives 1a‐d and α‐estradiol (1e) underwent sulfuric acid‐catalysed transformations to furnish the steroids 2–6. The processes involved in the reaction sequence are regioselective sulfonation and, above all, the Wagner‐Meerwein rearrangement of the methyl group at C‐13. With the objective of obtaining further information on the course of the Kober colour reaction of estrogens, some UV/VIS and ESR spectroscopic investigations were also carried out.

Pregnancy Urinary Estrogen Determination Using a Quality Control Material

Urinary estrogen determinations are now commonplace for monitoring high-risk pregnancies. In this study, a simple spectrophotometric modification of the Kober color reaction is evaluated, using a stable urine control material. It is essential that a procedure of this importance include a quality control material with each batch of assays. The preparation procedure for this stable control material and the long-term data obtained on its use are described in detail. The method used in this study is simple and can be performed in any clinical chemistry laboratory.

Chromogenic reactions of steroids with strong acids. IX. Behavior of estrone methyl ether in concentrated sulfuric acid.

The behavior of estrone methyl ether (V) in strong acid was studied, in order to elucidate the mechanism of the Kober color reaction. When V was dissolved into concentrated sulfuric acid, a maximum absorption gradually appeared at 465 nm which was then transferred to 452 nm with elapse of time. On the other hand, 3-methoxy-17α-methylestra-1, 3, 5 (10), 9 (11)-tetraen-17β-ol (XIIIb) immediately gave the chromophoric cation χ-465 (VIIb) in concentrated sulfuric acid. Similarly, 3-methoxyestra-1, 3, 5 (10), 9 (11)-tetraen-17β-ol (XIIIa) gave the cation χ-364 (XIVa) which changed in turn to χ-465 (VIIa). Sulfonation gradually occurred at C (2)s of VIIa and VIIb in the same acid to give the corresponding cations XIXa and XIXb ; the maximum absorption at 465 nm altered to 452 nm. The mechanism of the conversion of V into the C (2)-sulfonated χ-465 (XIXa) was elucidated from these behavior of XIIIa and XIIIb in concentrated sulfuric acid.

A semi-automated fluorometric method for total estrogens in pregnancy urine.

We report a continuous-flow fluorometric method for total urinary estrogens that involves the Kober reaction, with extraction of the reaction product into dichloroethane containing trichloroacetic acid as described by Hahnel and Jones [Clin. Chim. Acta 16, 185 (1967)]. The dichloroethane extraction gives greater stability to the Kober color and sharper separation of aqueous and organic phases. The analytical system is adjusted to give maximum response to estriol 16alpha,beta-D-glucuronide, the principal estrogen conjugate in urine from late pregnancy, when calibrated with estriol standards. An initial 20-fold dilution of the sample with water increased analytical recovery of estriol conjugates from urine while maintaining adequate fluorescent response. Glucose interference was reduced by dilution and eliminated by treatment with sodium borohydride. Urinary protein up to 20 g/liter did not interfere. Total estriol, as determined by gas-liquid chromatography, comprises about 70% of total urinary estrogens in late pregnancy as measured by our continuous-flow fluorometric method. A reference range is presented based on 209 randomly collected urine specimens in which total urinary estrogens are expressed as a ratio to creatinine.

Chromogenic Reactions of Steroids with Strong Acids. V. Products in the Kober Reaction of Steroidal Estrogens

Formation and chemical structures of some products were studied in the Kober color reactions of estradiol 3-methyl ether (I) and of estrone methyl ether (II). After heating I or II with 78% (w/w) sulfuric acid at 100°for about half-hour, extraction of the reaction-mixture with benzene left the Kober-pigment in the acidic layer, which was collected in a reddish purple regin adn was instable in a neutral or alkaline aqueous solution. In this reaction, were produced 3-methyoxy-17ξ-methyl-8ξ, 9ξ, 13ξ, 14ξ-18 norestra-1, 3, 5(10)-triene (III), 3'-methyl-7-methoxy-1, 2-cyclopenteno-9, 10-dihydrophenanthrene (IV), 3'-methyl-7-methoxy-1, 2-cyclopentenophenanthrene (V), 3-methoxy-17ξ-methyl-13ξ, 14ξ-18-norestra-1, 3, 5(10), 8-tetraene (VI), and 3-methoxy-17ξ-methyl-13ξ, 14ξ-18-norestra-1, 3, 5(10), 6, 8-pentaene (VII) from I. The same reaction of II gave these compounds (III-VII) and the new products, methyl 3'-methyl-7-methoxy-1, 2-cyclopenteno-9, 10-dihydrophenanthrene-6-sulfonate (VIII) and 3'-methyl-7-methoxy-1, 2-cyclopentadieno-9, 10-dihydrophenanthrene (IX).

Oestrogen determination in pregnancy urine

Modifications have been made at the acid hydrolysis and the Kober colour reaction stages of the Oakey et al. 1 procedure for assaying total oestrogens in pregnancy urine. These modifications have resulted in a method whereby a single determination of urine oestriol can be carried out in 1 1 2 hours.

A Note on Reported Levels of Estrone in Laying Hens’ Blood

Brown’s (1955) method for the measurement of human urinary estrogens by the Kober colour reaction has been applied successfully to the determination of estrone in the urine of the domestic hen (Common et al., 1965). Similar techniques, but based on the fluorescence of the Ittrich-Kober reaction product, have been used to measure estrone in human blood (Roy, 1962) and in the blood of pregnant cows (Robinson et al., 1970). When we applied Roy’s procedure (1962) to the blood of laying and non-laying hens, we obtained consistently negative results, even though recoveries of reference estrone (equivalent to 20 ng./ml. blood) added after the acid hydrolysis (64.3±8.2%) were comparable to those reported for the recovery from human blood, viz., 61.0 ± 5.0% (Roy, 1962).Accordingly, the initial extraction step was modified by varying the acid concentration and reflux time during hydrolysis and also by replacing the acidic hydrolysis by enzymatic hydrolysis. In…

Further observations on the Kober colour and Ittrich fluorescence reactions in the measurement of oestriol, oestrone and oestradiol.

SUMMARY Higher temperatures allow lower sulphuric acid concentrations and shorter heating times to be used in the Kober colour reaction for oestrogens. A one-stage reaction which is completed in 5 min. at 120° is described for oestrone and oestradiol, and a two-stage reaction which requires two periods of heating for 5 min. at 120° is described for oestriol. The conditions were applied to the Ittrich fluorescence procedure. A spectrophotofluorimetric correction was developed in which fluorescence was measured at wavelengths for excitation and emitted light near the optima for the oestrogens and at another combination at which the oestrogens produced virtually no fluorescence whereas that of impurities was not diminished. Extraction, centrifugation and fluorimetry were performed in specially designed cells. The sensitivity is 0·05–0·1 ng./sample with a linear response up to 300 ng. and a precision better than 4% in the range 1·0–100 ng.

Observations on the Mechanism of Action of Clomiphene (MRL-41)

Serial follicle stimulaint hormone (FSH) and luteinizing hormone (LH) determinations (by the methods of Steelman and Pohley and Parlow respectively) in 4 23-53 year old anovulatory patients given short-term clomiphene treatment (100-400 mg orally/day for 6 days) were employed in studying the mechanism of clomiphene action. Serial determinations of 3 estrogens (estrone estradiol and estriol) were also determined using thin-layer absorption chromatography before Kober color development. FSH levels rose in all cases during or immediately following clomiphene therapy. LH did not rise prior to a rise in estrogens. The estradiol fraction was found to contain at least 3 different components. During clomiphene therapy the estradiol fraction fell. Since the estradiol fraction was composed of 2 compounds in addition to estradiol and since it fell during treatment it was postulated that it was the unidentified estrogens whose excretion had decreased. It was suggested that this fall in unidentified estrogens may be responsible for the increase in FSH release observed during treatment.

Two dimensional thin layer chromatography of estradiol and estriol glucosiduronic acids extracted from buffered solution

Estriol and estradiol glucosiduronic acids are selectively extracted from acid solution with a chloroform-isopropyl alcohol solvent mixture. They are recovered from the organic phase into acetate buffer pH 4.5 and returned to ethyl acetate upon acidification and extraction. On evaporation of ethyl acetate, the estrogen glucosiduronic acid residue is dissolved in ammoniated isopropyl alcohol. These conjugates are separated on thin layers of silica gel H on glass plates in three consecutively-applied solvent mixtures (I) chloroform-isopropyl alcohol; (II) chloroform-isopropyl alcohol-formate and (III) ethyl acetate-pyridine-acetic acid-water. Migrations in solvents I and II are conducted in one direction and in solvent III in a second dimension. The estradiol and estriol glucosiduronic acids are located from the position of standards used as markers in this two-dimensional migration and are determined directly by the Kober color reaction and by glucosiduronic acid analysis. A technique is also described for the quantitative elution of the estrogen conjugates from chromatoplates and for their complete hydrolysis by beta-glucuronidase. The free steroids so obtained are in a purified state and these should be readily measurable with the sensitive physical techniques now available.

SPECTROFLUORIMETRY OF OESTROGENS: APPLICATION OF CORRECTION METHODS WHEN THE UNFILTERED WOLFRAM CONTINUUM IS USED AS ACTIVATING LIGHT.

ABSTRACT The application of the wolfram continuum as activating light in spectrofluorimetry of oestrogens has been examined using oestrone as a model substance. When primary filters were omitted, the scatter, deriving from the incident beam, formed a continuous background over which the fluorescence spectra could be recorded in their full extension. In quantitative determinations, fluorescent light could be differentiated by application of correction equations based on recordings of the intensities of narrow spectral bands at the wave lengths of maximal specific emission and at one or two additional wave lengths. When correction methods were to be applied under the conditions examined, the procedure of Ittrich (1958) for colour development and extraction was found to offer the highest sensitivity. The correction equations used in fluorimetry of the Kober colour complex (in methylene chloride) were Fcorr = 2 F 548 mμ − (F 518 mμ + F 578 mμ) according to Allen (1950) or Fcorr = F 548 mμ − F 518 mμ where F represents light intensity units. The standard deviation of the corrected blank value in these methods corresponded to less than ± 0.2 ng oestrone.

Urinary Excretion of Estrogenic Compounds During Estrus and Gestation by the Bovine as Determined by Three Assay Methods

Summary Aliquots of 24-hr urine collections from cows during estrus and at the 50th, 100th, 200th, and 275th days of gestation were extracted and assayed for the estrogenic content by the Kober color reaction, fluorometrically, and with bioassay. The bioassay procedure gave results for estradiol-17 α that averaged 1.19 times higher than those obtained by the Kober color reaction and 1.74 times higher than the fluorometric results. However, in measuring estradiol-17 β , the bioassay results were 0.7 times greater than the Kober results and 0.54 times greater than, the fluorometric results. The greatest differences between the methods were revealed in measuring urinary estrone, in that the bioassay results averaged only 0.21 times greater than the Kober results and 0.42 times higher than the fluorometric results. Excretion of the three estrogens was lowest at 50 days, but after 100 days of gestation an increase in urinary estrogenic excretion occurred and it was at an accelerated rate during the last stages of pregnancy. The most pronounced increase was in the excretion of estradiol-17 α , although estrone excretion was also considerably higher during the latter part of gestation. The urinary excretion of estradiol-17 β increased at a much slower rate than did that of two other estrogens.

APPLICATION OF THE ITTRICH EXTRACTION PROCEDURE TO THE FLUORIMETRIC MEASUREMENT OF OESTROGENS IN BLOOD

SUMMARY The fluorescence reaction described by Ittrich (1958) for the quantitative estimation of oestrogens has been investigated, and applied to the measurement of oestriol, oestrone and oestradiol in fractions from blood prepared by the procedure of Roy & Brown (1960). The results obtained by this method from blood taken during pregnancy agreed closely with those given by the less sensitive Kober colour reaction. Fluorescing impurities derived from solvents and reagents gave 'base-line' values equivalent to approximately 0·02 μg. of each oestrogen per 100 ml. blood. These background values masked the oestriol and the majority of the oestradiol levels when the procedure was applied to peripheral venous blood obtained during the menstrual cycle. However, positive values for oestrone were encountered reaching 0·14 μg./100 ml. at the peak; these values correlated closely with the urinary oestrone output. The ratio of blood:urinary concentration of oestrone was the same during the menstrual cycle as during pregnancy.

Some observations on the Kober colour and fluorescence reactions of the natural oestrogens.

The two-stage Kober reaction with oestriol, oestrone and oestradiol-17β 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-17β 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-17β. 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.