[Evidence, in vitro, of estrone and estradiol-17 beta 3-sulfoconjugate hydrolysis in the uterus of pregnant guinea pigs]. / Mise en évidence, in vitro, de l'hydrolyse de l'oestrone et de l'oestradiol-17beta 3-sulfoconjugue's par l'utérus gravide de Cobaye.

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.

[3 alpha-hydroxy-5 alpha-androstane-17-one-3-sulphate in hirsute women's plasma (author's transl)]. / Sulfate de 3 alpha-hydroxy-5 alpha-androstane-17-one plasmatique chez la femme hirsute.

The determination of serum androsterone-3-sulphate was carried out by radioimmunoassay with an antiserum specific against 15 alpha-carboxymethyl-androsterone-BSA. The values obtained (mean +/- SD) in ng/ml were 739 +/- 190 (n = 9) for Men; 285 +/- 157 (n = 13) for Women; 677 +/- 464 (n = 131) for hirsute women respectively and were compared to the values of urinary sulphate of dehydroepiandrosterone in the same subjects.

The preparation of three fluorescence-labeled derivatives of testosterone.

Three fluorescence-labelled derivatives of testosterone were prepared consisting of the steroid separated from the fluorochrome by a hydrocarbon "bridge". "Bridges" of different lengths (C2 to C7) were used as the length required to avoid steric hindrance effects by the fluorochrome in studies on steroid-protein binding was unknown. The three derivatives prepared were: 17 beta-hydroxy--4-androsten-3-one 3-(O-(N-(2'-mercapto)ethyl)carbamoylmethyl)oxime, 17 beta-hydroxy-4-androsten-3-one 3-(O-(N-(3'-amino)propyl)carbamoylmethyl)oxime and 17 beta-hydroxy-4-androsten-3-one 3-(O(N-(7'-amino)heptyl)carbamoylmethyl)oxime. These were then coupled with either a dansyl or a fluorescein molecule. Overall yields were sufficient and the products immunoreactive with anti-testosterone antiserum.

Kinetic study of the interaction between rat haptoglobin and rat liver cathepsin B.

One of the roles of the acute phase reactants (APR), according to Koj, is to regulate the action of tissue proteinases released during the inflammatory reaction. To study this phenomenon in vitro, rat liver cathepsin B (EC 3.4.22.1), a lysosomal proteinase, and a typical APR, rat serum haptoglobin, were purified. By kinetic study, haptoglobin was found to inhibit cathepsin B and the inhibitory activity of a competitive type was increased with the molecular weight of the substrate. When 125I-labelled denatured bovine serum albumin (BSA) was used as a substrate, the apparent Michaelis constant (Km app.) for cathepsin B was 5 +/- 0.4 X 10(-6) M. When haptoglobin was added, the apparent inhibition constant (Ki app.) was 3.5 +/- 1.5 X 10(-8) M. Native haptoglobin was not catabolized by cathepsin B while under the same conditions denatured haptoglobin was degraded by the enzyme.

Semi-automated fluorometry of total estrogens in plasma during late pregnancy.

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.

[In vitro aromatization of androgens in the presence of testosterone binding proteins from human placenta or serum]. / Aromatisation in vitro des androgènes en présence de protéines humaines, placentaire ou sérique, liant la testostérone.

Two related 17 beta hydroxy steroid binding proteins of serum and placental origin respectively are shown to inhibit significantly the aromatization of testosterone and androstenedione by isolated human placenta microsomes. Quantitative correlations suggest that this inhibitory effect is due to the fixation of the testosterone substrate (or androstenedione previously converted to testosterone) on the binding proteins. A possible role for these macromolecules in the steroidogenic activity of the placenta is discussed.

Rapid fluorometry of estrogens in nonpregnancy urine, with use of chloroform extraction and purification by anion-exchange chromatography.

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.

Identification of haptoglobin in chicken serum and specificity of the chicken haptoglobin-hemoglobin complex formation.

1. The presence of haptoglobin in chicken serum has been demonstrated by three different techniques: gel filtration, cellulose acetate electrophoresis and fluorescence quenching. 2. Chicken haptoglobin shows a narrow species specificity; it binds only avian and reptilian but not mammalian hemoglobins. 3. Haptoglobin seems to have been subjected to profound changes during the course of evolution.

[An automated spectrofluorometry method for urinary estrogens determination in pregnant and non-pregnant subjects (author's transl)]. / Méthode de dosage automatique par spectrofluorimétrie des estrogènes urinaires au cours et en dehors de la grossesse.

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.

Conversion, in vitro, of (7n-3H) testosterone to estrone and estradiol-17beta and their 3-sulfate conjugate by the guinea-pig placenta.

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.

Fluorescence studies of the interactions of serum albumin and rat alpha1-fetoprotein with aflatoxin B1. Specificity and binding parameters.

The interaction of bovine serum albumin and rat alpha1-fetoprotein with aflatoxin B1 has been followed by the fluorescence quenching of the protein in presence of the ligand. The binding parameters (n, number of sites and Kd, dissociation constant) have been determined for the bovine serum albumin-alflatoxin B1 system: n = 3.5 and Kd = 3.1 +/- 0.5 . 10(-5) M and for the alpha-fetoprotein-aflatoxin system: n = 4 and Kd = 3.7 +/-0.5 . 10(-5) M. The competition of anilino-naphthalene-sulfonate and aflatoxin B1 for the same hydrophobic sites on bovine serum albumin has been demonstrated. The fluorescence quenching of various proteins (lysozymes, egg-albumin, gamma-globulin) by aflatoxin B1 have shown that there is not a strict specificity of aflatoxin towards proteins.

[In vitro comparative metabolism of 6,7-3H estrone and 6,7-3H estrone-3-sulfate in maternal and fetal guinea-pig liver]. / Etude comparée du métabolisme, in vitro, de la [6, 7-3H-A1 estrone et du [6, 7-3H] estrone-3-sulfate par le foie maternel et foetal du cobaye.

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.

Rat and human embryo and post-natal sera contain a potent endogenous competitor of estrogen-rat alpha-fetoprotein interactions.

A highly active inhibitor of the binding of estrone and estradiol-17beta to rat alpha-fetoprotein is demonstrated for the first time in embryo, immature and adult rat sera as well as in fetal and adult human sera. The competitive character and the narrow specificity of this inhibition effect is shown. The major compound responsible for this activity is isolated by successive column Sephadex LH20 and thin layer chromatography: it is characterized as a nonpolar, nonphenolic, dialysable and thermostable substance, unreactive towards anti-estrone and anti-estradiol-17beta antibodies. The possible biological role of an endogenous non-estrogen ligand of rodent fetoproteins is discussed.

[Kinetics of transcutaneous penetration and fixation of 3-propoxy-17-methoxyestradiol and estradiol in various rat tissues]. / Cinétiques de pénétration transcutanée et de fixation du 3-propyl éther, 17-méthyl éther oestradiol et de l'oestradiol au niveau de différents tissus chez la ratte.

The transcutaneous penetration of 3-propyl ether, 17-methyl ether oestradiol (POM) occurs by a diffusion phenomenon and does not seem to be modulated by a cutaneous receptor as it is the case for oestradiol. After transcutaneous administration of POM and oestradiol, a comparison of the kinetics of uptake on the uterus and of uterotrophic effects, as well as an analysis of radioactivity taken up by a partition method between petroleum ether and sodium hydroxide, indicates that cleavage of both ether groups of POM occurs leading to estradiol. It is likely that this de-etherification takes place in the liver after a period of quiescence. The lipophilic nature of POM allows an obvious uptake by the aorta and a very significant uptake by the adipose tissue. The etherification of the alcohol functions of oestradiol allows an adequate protection of the hormone against hepatic catabolism. This may explain, along with the release of metabolites taken up by the adipose tissue, that POM is bound to a greater extent than oestradiol by various tissues.