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SUMMARY
During the normal menstrual cycle the excretion of pregnanetriol increased on the day that the excretion of oestrone and oestradiol reached a maximum during the follicular phase. Pregnanediol excretion did not begin to increase until 2 days later. The excretion of pregnanetriol reached a peak and began to decrease before that of pregnanediol. Thus the pattern of pregnanetriol excretion was different from that of the excretion of oestrogens or pregnanediol.
Progestational steroids in a dose which inhibited ovulation prevented the rise in both pregnanetriol and pregnanediol excretion during the second half of the menstrual cycle.
The administration of oestradiol dibenzoate to two subjects did not affect pregnanetriol excretion.
It is suggested that the increased amounts of pregnanetriol excreted during the second half of the menstrual cycle arise from a precursor of pregnanetriol, most probably 17α-hydroxyprogesterone, secreted by the ovary.
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The metabolism in human subjects of ethynyloestradiol (EE) or its 3-methylether (mestranol) labelled with 14C or 3H has been studied by numerous investigators (see Fotherby & James, 1972; Goldzieher & Kraemer, 1972). The rate of excretion of metabolites of the two compounds appears to be similar. Oestrogen methylethers are hydrolysed in vivo. After administration of the 3-methylethers of oestrone, oestradiol or oestriol to human subjects only small amounts of the ethers were detected in urine and excretion of the free oestrogens was slower by about 24 h than after administration of the free compounds (Brown, 1962). Williams (1969) identified EE in the urine of women receiving mestranol. However the rate of hydrolysis of mestranol and the blood level of EE attained, have not been studied.
In the present investigation plasma levels of EE were measured in normal, healthy volunteers (3 male and 1 female). Plasma samples were obtained
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SUMMARY
After injection of [4-14C]norethisterone or norgestrel labelled with 14C in the ethynyl group into rabbits, 45% and 57·4% of the radioactivity was excreted in the urine, mostly within 2 days. Although small amounts of radioactivity were excreted in the expired air of rabbits receiving norgestrel, little metabolism of the ethynyl group occurred. Radioactivity in plasma declined quickly after injection and 24 hr. later less than 0·5% of the dose remained in the circulation. Up to 5 hr. after injection large amounts of radioactivity were found in liver, kidney, intestine and bile; a large proportion of the dose appeared to undergo an entero-hepatic circulation. Although the concentration in fat and muscle was low the total amount in these tissues may approach 5%. The uterus contained a high concentration of radioactivity suggesting that this tissue may bind the progestins or their metabolites. By 24 hr. the amount of radioactivity in the tissues had markedly decreased. Small amounts of radioactivity crossed the placenta of pregnant animals near term and small amounts were found in the foetus and amniotic fluid. Within 3 hr. of the injection at least half of the radioactivity in the tissues was in a conjugated form and considerable metabolism of the injected progestins had occurred. Little, if any, norethisterone or norgestrel was present in the tissues. The metabolites of norethisterone were mainly polar compounds showing that hydroxylation of the steroid had occurred, whereas the metabolites of norgestrel appeared to be similar to the compounds produced by partial or complete reduction of the α,β-unsaturated ketone grouping in ring A.
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SUMMARY
Antibodies to norethisterone and norgestrel were raised in rabbits by injecting the steroid 3-carboxymethyloxime coupled to bovine serum albumin. Within 6 months of the first injection, antisera were obtained which gave 50% binding of the labelled steroid at dilutions of 1:100000; after treatment with Rivanol the antisera had titres of 1:20000 to 1:30000. Labelled ligands were prepared by iodination of the tyrosine methyl esters of the steroid oximes; minimum specific activities of 100 to 150 Ci/mmol were obtained. Dextran-charcoal suspension was used for the separation of bound and free labelled steroid. The effect of changes in the incubation conditions on the sensitivity of the assay were investigated. The reliability criteria for the assays were satisfactory, blank values were low and the mean recoveries were greater than 90%. The sensitivity of the method corresponded to a concentration of the steroids in plasma of 30 pg/ml. The coefficient of variation for replicate analyses on the same sample varied from 13·5 to 16%. Apart from the parent steroid, the 3-oxime and the tyrosine methyl ester of the oxime, significant cross-reaction with the antiserum was also obtained with the dihydro and tetrahydro metabolites of the progestogens. After administration orally of 1 mg norgestrel or 1 mg norethisterone to human subjects, peak levels of the steroids in plasma were obtained within 1 to 2 h. Norgestrel was metabolized more slowly than norethisterone. For the periods 2–8 h and 8–24 h after administration of norethisterone the half-lives were approximately 3 h and 5 h respectively; for norgestrel the half-lives were 2·8 h and 10 h. Twenty-four hours after administration, the mean levels of norgestrel and norethisterone in plasma were 1·5 and 0·2 ng/ml respectively corresponding approximately to 0·4% and 0·05% of the dose in the total plasma volume.
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SUMMARY
A simple method modified from those developed by Stern [1957] and Bongiovanni & Eberlein [1958] for the estimation of pregnanetriol in urine is described.
The accuracy, sensitivity, precision and specificity of the method are discussed.
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SUMMARY
The conversion of three possible precursors of pregnanetriol, namely 11-deoxycortisol, 3β,17α-dihydroxypregn-5-en-20-one and 17α-hydroxyprogesterone, to pregnanetriol has been studied quantitatively. After the intravenous injection of pregnanetriol, 66% of the dose was recovered as pregnanetriol in urine. The amounts of the three possible precursors converted to pregnanetriol were 0, 8 and 35% of the injected dose, respectively.
Evidence is presented to show that the pregnanetriol excreted after the injection of 3β,17α-dihydroxypregn-5-en-20-one was 5β-pregnance-3α,17α,20α-triol. The excretion of dehydroepiandrosterone was not increased by the injection of 3β,17α-dihydroxypregn-5-en-20-one.
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SUMMARY
[14C]Norgestrel was administered to seven human subjects and 43% of dose was excreted in the urine within 5 days; the biological half-life of the radioactivity was 24 hr. Enzymic hydrolysis released only 32% of the the urinary radioactivity and a further 25% was excreted as sulphate conjugates. The metabolites excreted in the urine were much less polar than those following the administration of the related compounds, norethisterone or lynestrenol. The 3αOH,5β and 3βOH,5β isomers of the tetrahydronorgestrel (13β-ethyl-17α-ethynyl-5β-gonane-3α,17β-diol) were isolated from urine and identified by mass spectrometry and thin-layer and gas—liquid chromatography. Plasma radioactivity decreased more rapidly than after the administration of norethisterone and lynestrenol. About 2% of the administered dose was converted to acidic compounds. There was no apparent difference in the rate of excretion of radioactivity or in the metabolites after either oral or intravenous administration of norgestrel.
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SUMMARY
After the administration of norethisterone to seven human subjects 37·4% to 80·6% of the dose was excreted in the urine within 5 days. About half of the urinary radioactivity was extractable after acid or enzymatic hydrolysis and about 80% was extracted by using a serial hydrolytic and extraction procedure. About 15% of the urinary radioactivity was present as sulphate conjugates. About 5% of the urinary radioactivity was present in acidic or phenolic compounds. No metabolism of the ethynyl group seemed to occur and the Girard reaction showed that about half of the urinary metabolites were ketonic. The metabolites in the glucuronide fraction of urine were predominantly more polar on paper chromatography than tetrahydronorethisterone, whereas in the sulphate fraction most of the metabolites had a polarity similar to the simple reduction products of norethisterone. Two days after injection the plasma still contained about 5% of the administered dose.
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SUMMARY
After the administration of [4-14C]lynestrenol (17α-ethynyl-19-nor-androst-4-en-17β-ol) to 7 human subjects 31·–57·6% of the dose, whether administered orally or i.v., was excreted in the urine within 5 days. The biological half-life of radioactivity was 26·5 hr. After acid and enzymatic hydrolysis, 58·7 and 45·6% respectively of the urinary radioactivity was extractable. About 10% of the urinary metabolites were excreted as sulphate conjugates. A mean value of 1·75% of the administered dose was converted to phenolic compounds. The metabolites in the free fraction and enzymehydrolysed extract of urine were almost entirely polar compounds, whereas 70% of the metabolites in the sulphate fraction were much less polar. The chromatographic evidence showed that hydroxylation of lynestrenol must have occurred at two points in the molecule. Plasma radioactivity decreased more rapidly than after administration of norethisterone.
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Kamyab, Fotherby & Klopper (1968) showed that there were similarities between the in-vivo metabolism in man of 14C-labelled norethisterone (17α-ethynyl-19-nortestosterone) and lynestrenol (3-deoxynorethisterone) and suggested that some of these similarities might be due to the conversion of the latter to the former. The present communication describes such a conversion in vitro.
Rabbit liver was homogenized and incubated by the procedure described by Davidson & Fotherby (1965) except that: (1) each flask contained in addition 0·0012 m-NADPH, (2) the incubations proceeded for 16 hr., (3) ethyl acetate was used for extraction instead of benzene and (4) the residue obtained, following evaporation of the ethyl acetate, was submitted to a hexane-methanol partition (Fotherby, Colas, Atherden & Marrian, 1957). For the large-scale incubations, 20 mg. steroid in 0·2 ml. ethanol were added to an homogenate of 20 g. liver. Thin-layer chromatography was carried out using either silica gel G (Merck and
