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J. B. BROWN

Interest in methoxylated oestrogens has been stimulated by the discovery of 2-methoxyoestrone and 2-methoxyoestriol in human urine (Kraychy & Gallagher, 1957; Fishman & Gallagher, 1958; Loke & Marrian, 1958). Furthermore, the 3-methyl ether of 17α-ethinyloestradiol is widely used as a potent oral oestrogen. The question arises whether the oestrogen methyl ethers can be demethylated in vivo to the parent phenols. To test this possibility, the 3-methyl ethers of oestrone, oestradiol and oestriol have been administered to human subjects, and the resulting outputs of oestrone, oestradiol and oestriol and of their methyl ethers in the urine have been measured.

EXPERIMENTAL PROCEDURES

The methyl ethers were purified by chromatography on alumina and recrystallization from ethanol. Their melting points (uncorrected) were: oestrone methyl ether 172° c; oestradiol methyl ether 97–98° c, 117–118° C; oestriol methyl ether 148–154° c. The pure compounds were dissolved in olive oil (2·5 mg./ml. for oestrone and oestradiol

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J. B. BROWN

SUMMARY

Oestrone, oestradiol-17β and oestriol were administered by intramuscular injection to two men and four women. The amounts of oestrone, oestradiol-17β and oestriol excreted in the urine as a result of the injections were measured. The figures derived from these injection experiments have been used to speculate on the nature and the amounts of the oestrogens secreted by the ovaries during the menstrual cycle and by the placenta during pregnancy.

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J. B. BROWN

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.

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K. FOTHERBY and J. B. BROWN

SUMMARY

The urinary excretion of pregnanetriol, pregnanediol and oestrogens was measured throughout five anovulatory cycles in three subjects, and throughout an anovulatory cycle and an ovulatory cycle in an adrenalectomized subject. Cyclical changes in pregnanetriol excretion, which paralleled the changes in oestrogen output, were observed during some of the anovulatory cycles and also during the ovulatory cycle in the adrenalectomized subject.

These findings suggest that one factor contributing to the increase in pregnanetriol excretion during the normal ovulatory menstrual cycle is the secretion by the ovary, at times of maximal oestrogen secretion, of a precursor (possibly 17α-hydroxyprogesterone) common to both oestrogens and pregnanetriol. However, that other factors are probably also involved is suggested by the particular pattern of pregnanetriol excretion during the normal ovulatory cycle and by the finding that the increase in pregnanetriol excretion at about the time of ovulation is usually greater than the increase found in the anovulatory cycle.

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J. B. BROWN and J. A. STRONG

SUMMARY

The urinary excretion of endogenous and exogenous oestrogens was studied in patients with disordered thyroid function, and in obese and non-obese patients without evident thyroid disease. Hypothyroid patients and obese patients converted a larger percentage of administered oestradiol-17β to urinary oestriol and a lower percentage to urinary oestrone than hyperthyroid patients and non-obese patients. The recovery of administered oestradiol as oestriol, oestrone and oestradiol in urine was the same in the hypothyroid, obese and non-obese patients but considerably lower in the hyperthyroid patients. Similar differences were found in the urinary excretion of endogenous oestrogens. The excretion of endogenous oestrogens varies with the weight of the patient; it was greater in the obese than in the non-obese. The findings indicate that the pathways of oestrogen metabolism are influenced by a number of the factors, including thyroid function, associated with the weight of the patient.

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EVELINE J. ROY and J. B. BROWN

SUMMARY

A chemical method for the estimation of oestriol, oestrone and oestradiol-17β in the blood of the pregnant woman and of the foetus is described. It is based on a method previously developed for urine and involves acid hydrolysis of whole blood, extraction of the phenolic fraction, methylation, chromatography on alumina columns and colorimetric measurement using a micro-modification of the Kober reaction. Kober-chromogenic impurities in the final extracts are derived from the procedure, not from the blood, and are eliminated by the spectrophotometric correction of Allen. The accuracy, precision, sensitivity and specificity of the method have been determined.

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J. A. LORAINE and J. B. BROWN

SUMMARY

A method for the quantitative determination of gonadotrophins in the urine of non-pregnant human subjects is described.

The reliability criteria for the method have been studied and the technique has been found to be satisfactory in terms of accuracy, precision, sensitivity and specificity.

The importance of expressing results of urinary gonadotrophin assays in terms of a reference preparation such as HMG 20A is emphasized.

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SUSAN A. BARRETT and J. B. BROWN

SUMMARY

Cox's method for the analysis of pregnanediol in human urine by gas—liquid chromatography (GLC) of the free steroid on neopentyl glycol adipate in conjunction with a semi-automatic extractor for processing the urine was evaluated. Without automation of the GLC, one worker could complete 24 assays/day. The reliability of Cox's method was confirmed. Results obtained throughout the menstrual cycle in 15 women, during the pregnancies of 480 patients, in 18 postmenopausal women and in 20 men are presented.

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J. B. BROWN and H. A. F. BLAIR

SUMMARY

A purification step involving separation of the ketonic fraction through the Girard complex has been incorporated in an earlier method for estimating urinary oestrogens. The new method, which measures oestrone only, involves acid or enzymic hydrolysis of the urine, extraction with ether, removal of the acidic fraction with alkaline carbonate solution, evaporation of the ether, separation of the ketonic fraction through the Girard complex, saponification, methylation, and chromatography on alumina columns. The oestrone methyl ether present in the final extract is measured by a semi-micro modification of the Kober reaction, which, with the larger volume of urine processed, increases the sensitivity of the estimation ninefold. This increase in sensitivity is made possible by the purification achieved in the new method, and fractions of 1 μg oestrone/24 hr urine can be measured. The reliability of the new method has been investigated and data concerning its accuracy, precision, sensitivity and specificity are presented. By comparison with the new method, the earlier method gives overestimates of approx. 0.9 μg oestrone/24 hr urine.

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J. B. BROWN and H. A. F. BLAIR

SUMMARY

The hydrolysis of the conjugates of oestrone, oestradiol-17β and oestriol present in human urine has been investigated using acids and the enzymes β-glucuronidase and phenol sulphatase derived from Patella vulgata.

Using acid hydrolysis, maximum yields were obtained from the majority of urines by boiling 60 min with 15 vol. % concentrated hydrochloric acid. Under these conditions losses amounting to approx. 20% of the oestrogens present occur in concentrated urine specimens. These losses can be diminished by diluting the urine with water before hydrolysis.

Using enzymic hydrolysis, maximum yields were obtained by incubating the urine for 96 hr at 37° C and pH 4·7 with approx. 600 u./ml. β-glucuronidase.

When allowance was made for the losses which occur during acid hydrolysis, the yields from pregnancy and non-pregnancy urine were approximately the same by the two hydrolytic procedures. From this, it is inferred that no unknown major error exists in either procedure.