ABSTRACT
The metabolism, in vitro, of [6,7-3H]-oestrone-3-sulfate and [6,7-3H]-oestradiol-17beta-3-sulfate by the uteri of pregnant female Guinea-Pigs has been studied using 900 g supernatants. A hydrolysis of the sulfate moiety was observed: 35% of the radioactivity was recovered in the unconjugated fractions after 1 hr. of incubation. Gluruconide oestrogens were also recovered but the rate of synthesis was very low (3.6% after 1 hr.). OEstrone and oestradiol-17beta have been identified in unconjugated, glucuro and sulfo-conjugated fractions after incubation with the tritiated oestrogen sulfates.
Subject(s)
Estradiol/analogs & derivatives , Estrogens, Conjugated (USP)/metabolism , Estrone/analogs & derivatives , Pregnancy, Animal , Uterus/metabolism , Animals , Estradiol/metabolism , Estrone/metabolism , Female , Guinea Pigs , Hydrolysis , Kinetics , Pregnancy , TritiumABSTRACT
We describe a mechanized method for determining total estrogens in plasma during the last trimester of pregnancy. This method involves rapid enzymic hydrolysis, automatic extraction (in a tube) with cyclohexane/ethyl acetate, purification of the extracted material by anion-exchange chromatography on a disposable "mini-column" of Dowex AG1 X 2 (acetate form), and continuous-flow fluorometric quantification. Conditions of hydrolysis and extraction were studied. The mean within-day imprecision (CV) was 7% and between-day imprecision was 8.1%. Sensitivity was 75 nmol/L of plasma. Results are obtained in 4 h. The technique is quite suitable for routine determinations: 90 assays can be done by a team of three technicians in one working day.
Subject(s)
Estrogens/blood , Fluorometry/methods , Female , Humans , Hydrolysis , Pregnancy , Pregnancy Trimester, Third , Time FactorsABSTRACT
We describe a new method of extraction and purification of estrogens in low concentration in urine, involving a rapid enzymic hydrolysis of a 10-mL sample, automatic extraction (in a tube) with CHCl3/ethyl acetate, purification by chromatography on a disposable "mini-column" of AG 1-X2, and fluorometry by continuous flow according to Itrich's procedure [Acta Endocrinol. (Copenhagen) 35, 34 (1960)]. Conditions of hydrolysis, extraction, and purification were studied. Within-day precision (CV) was 7.5%, the mean between-day precision 8.5%. The sensitivity was 6 microgram of total estrogens per liter of urine. The specificity was assessed particularly by comparison with the results obtained by Schöller et al. [Acta Endocrinol. (Copenhagen) 57, suppl. 107 (1966)] and by gas-chromatography-mass-spectrometry. The normal limits as calculated by this technique were identical to values reported by others. The technique is rapid; results are obtained in 3 h. It is quite suitable for routine determinations: 100 assays can be done by a team of three technicians in one working day.
Subject(s)
Estrogens/urine , Chloroform , Chromatography, Ion Exchange , Estrogens/isolation & purification , Estrogens, Conjugated (USP)/urine , Female , Fluorometry , Humans , Hydrolysis , MethodsABSTRACT
An automated method has been developed for the measurement of estrogens in the urine of pregnant and non-pregnant subjects. Using the Kober-Ittrich fluorescence procedure, this automated method was applied to the assay of estrogens in the purified urinary extracts of non-pregnant women, men or children. For pregnant-women it had been possible to simplify the purification of urinary extracts, and the reaction was performed directly on the sodium hydroxide fraction. The precision, accuracy, and sensitivity of the method had been analysed.
Subject(s)
Estrogens/urine , Autoanalysis/methods , Child , Estriol/urine , Estrone/urine , Female , Humans , Male , Pregnancy , Sodium Hydroxide , Spectrometry, Fluorescence/methodsABSTRACT
Different cellular fractions of guinea-pig placenta were incubated in the presence of (7n-3H) testosterone. Microsomal aromatization of 3H-testosterone into estrone and estradiol-17beta was demonstrated in the presence of NADPH. The predominance of estrone after incubation with 17beta-hydroxylated precursors, (7n-3H) testosterone and (6,7-3H) estradiol-17beta, indicate that there is a microsomal 17beta-hydroxysteroid dehydrogenase activity. In this report, cytosolic sulfurylation of estrogens is demonstrated. This latter activity represents a quite original characteristic of the placental metabolism of estrogens in guinea-pigs. In contrast with the human placenta where there is considerable sulfatase activity, the guinea-pig placenta can sulfurylate estrogens.
Subject(s)
Estradiol/biosynthesis , Estrone/biosynthesis , Guinea Pigs/metabolism , Placenta/metabolism , Testosterone/metabolism , 17-Hydroxysteroid Dehydrogenases/metabolism , Animals , Chromatography, Gas , Chromatography, Thin Layer , Female , In Vitro Techniques , Pregnancy , Sulfates/metabolismABSTRACT
The metabolism of [6,7-3H] estrone and of [6,7(3)H] estrone-3-sulfate have been comparatively studied in the maternal and fetal guinea-pig livers. The appearance of estradiol-17 beta resulting from the activity of the 17 beta-hydroxysteroid-dehydrogenase is more important in the fetal than in the maternal hepatic tissue. This suggests the direct transformation of estrone-3-sulfate into estradio-3-sulfate in the fetus. After incubation of the [3H] estrone, there is an abundant hepatic conjugation. The glycuroconjugated components are predominant, as well in the maternal as in the fetal hepatic tissue. For the latter-one the sulfoconjugation is inexistant. The sulfatasic activity shown after the incubation of [3H] estrone-3-sulfate is very low in the fetal hepatic tissue; in contrast, this activity is higher in the maternal tissue.
Subject(s)
Estrone/analogs & derivatives , Estrone/metabolism , Fetus/metabolism , Liver/metabolism , Animals , Estradiol/biosynthesis , Estrogens, Conjugated (USP)/metabolism , Female , Guinea Pigs , In Vitro Techniques , Liver/embryologyABSTRACT
Slices of pregnant guinea pig liver were incubated with (6,7-3H)estrone and with (6,7-3H)estradiol. Free, glucuro- and sulfo-conjugated fractions were isolated by specific extraction and hydrolysis. The radioactivity distribution in these 3 fractions demonstrated a predominance of conjugated compounds (95% of isolated estrogens) with slightly more glucuro-conjugated than sulfo-conjugated compounds. After isolating estrogens by TLC, we were able to determine estrone and estradiol in these 3 fractions from incubations with 3H-estrone or with 3H-estradiol by means of specific activity recrystallisation. Estriol was determined in glucuro-and sulfo-conjugated fractions after incubation with 3H-estrone as well as in sulfo-conjugated fraction after incubation with 3H-estradiol. Glucuro- or sulfo-conjugated estrone was the predominant estrogen after incubation with 3H-estrone just as after incubation with 3H-estradiol. This led us to conclude to an important 17beta-hydroxysteroid-dehydrogenase activity. The 16alpha-hydroxylastic-activity is weaker since estriol represented only 1,43 % of estrogens isolated after incubation with 3H-estrone and 0.82% after incubation with 3H-estradiol.
Subject(s)
Estradiol/metabolism , Estrone/metabolism , Liver/metabolism , Pregnancy, Animal , 17-Hydroxysteroid Dehydrogenases/metabolism , Animals , Female , Guinea Pigs , In Vitro Techniques , Liver/enzymology , Mixed Function Oxygenases/metabolism , PregnancyABSTRACT
The metabolism of (6-7-3H) estradiol-17 beta was studied in vitro in different cellular fractions of Guinea Pig placenta. The substrate was transformed into free as well as conjugated (hydrosoluble) estrone. It was thus possible to conclude, on one hand the existence of a very active microsomial 17 beta-hydroxy steroid-dehydrogenase and on the other the presence of a cytosolic system of estrogen conjugation. No estradiol-17 alpha or estriol were determined.
Subject(s)
Estradiol/metabolism , Estrone/biosynthesis , Placenta/metabolism , Animals , Cytosol/metabolism , Estradiol Dehydrogenases/metabolism , Female , Guinea Pigs , In Vitro Techniques , Microsomes/metabolism , Placenta/enzymology , Placenta/ultrastructure , PregnancyABSTRACT
The excretion of seven urinary estrogens was studied during the last two or three weeks of five normal pregnant women. A study of the urinary excretion curves demonstrated that 16 alpha-hydroxyestrone and 16-oxo-estradiol followed the same pattern with the particularity of presenting two excretion peaks which were persistently observed on the 8th or 7th day prior to delivery for the first one, and around the 4th or 3rd day prior to delivery for the second one. It is possible that there is a relation between this phenomenon and the onset of delivery.
