Max-Fernand Jayle

Comparative study on the binding of estrogens by human and rat serum proteins in development

Summary The binding of estrone, 17β-estradiol and testosterone on rat and human sera as well as on pure serum proteins has been studied comparatively. It is known that the rat embryo serum contains an α 1 -fetoprotein with high binding affinity for estrogens. By contrast, no estrogen fixation has been found with human sera containing α 1 -fetoprotein (fetus, pregnant women, cord blood and hepatoma). However, the adult human and particularly the female at the end of pregnancy, display a serum sex binding globulin with high affinity for both 17β-estradiol and testosterone. The amount of this protein is extremely low in fetuses up to the 5th month of pregnancy and in the cord blood.

One step preparation of both human C-reactive protein and CIt.

C-reactive protein (CRP) is a trace constituent of normal human sera, which increases as much as 1000fold in many inflammatory reactions [ 1 ]. Comparison of amino acid sequences of many different proteins has shown the closest relationship between CRP and CIt. Moreover, examination of these proteins by negative stain electron microscopy revealed a similar cyclic structure of five subunits. The term pentraxin has been therefore proposed to describe these related proteins [2]. These data have been confirmed by complete amino acid sequence of human CRP [3 ]. Otherwise CIt appears to be identical to serum P-component and to 9.5 S ~-glycoprotein [4,5]. CRP preparation using affinity for the pneumococcal C-polysaccharide, which is at the origin of the discovery and the name of CRP [6], is still widely employed, particularly for sequence determinations and biological interactions studies. This practice is maintained, although the preparation of pneumococcal C-polysaccharide is unanimously recognized as tedious. In fact, the other proposed methods are not any more convenient [7-10]. This paper describes a fast method to prepare CRP by affinity chromatography with a high yield. This preparation takes advantage of the calcium dependent affinity of CRP for numerous ligands. The chosen system allows successive elution of CRP and CIt, both obtained as pure constituents, according to immunological and electrophoretic criteria. 2. Materials and methods

The conjugation of testosterone with horseradish peroxidase and a sensitive enzyme assay for the conjugate.

The formation of a horseradish peroxidase-testosterone conjugate for the enzyme-linked immunoassay of testosterone was investigated, using tritiated testosterone to follow the reaction. The formation of testosterone-3-(carboxymethyl) oxime-peroxidase by the mixed anhydride method was found to give a conjugate of high enzymatic activity and with three molecules of testosterone per molecule of peroxidase. The optimum conditions for the assay of peroxidase activity were studied and an assay capable of measuring 1 to 5 ng of the conjugate developed; the standard curve being virtually linear. The stability of the conjugate in solution and the effect of lyophilisation on enzymatic activity are also described. The peroxidase-testosterone conjugate was suitable for enzyme-linked immunoassay and the quantities measurable with the peroxidase assay covered the range necessary for a plasma testosterone assay. The stability of the conjugate was such that no particular precautions were necessary for its storage.

Etude des constantes de liaison entre les oestrogenes et 1'α1-foetoproteine de rat

The binding constants of the α1‐foetoprotein of the rat embryo serum for oestrone and oestradiol‐17β have similar values, i.e. 1 × 108 M−1 in average at 25°. There is probably one binding site per mole of binding protein. The high α1‐foetoprotein concentration in the rat embryo serum at 17–19 days of pregnancy explains the exceptionally high levels of fixation of the phenolsteroids by this serum.

A comparative study of the metabolic fate of testosterone, 17α-methyl-testosterone, 19-nor-testos-terone, 7α-methyl-19-nor-testosterone and 17α-methyl-estr-5(10)-ene-17β-ol-3-one in normal males

Abstract Single doses of 150 mg each of five different testosterone and nor-testosterone derivatives were given to four normal male subjects and urinary excretions of 17-ketosteroids, 17-hydroxycorticoids, hydroxy steroids and phenolsteroids by Browns method in basal urine and two 24 h post-treatment urines were measured. The conversions to Zimmerman chromogens, phenolsteroids, and hydroxysteroids were compared from the point of view of total percentage recovery of metabolites and of the rapidity of excretion. The three 17α-methylated steroids studied were metabolised to Zimmerman positive chromogens in a small but constant percentage. Testosterone and nor-testosterone had equivalent conversions to 17-ketosteroids, but the metabolites of the nor-derivatives were excreted more rapidly. The percentage recovery of metabolites appeared distinctly greater if the steroid could be easily metabolized to 17-ketosteroids. There was no phenolsteroid increase found after methyl-testosterone administration and only a small amount in one of the four subjects after testosterone. The 19-nor-steroids, on the contrary, gave rise to significant amounts. Nor-testosterone gave a strong increase in the estrone fraction. 17α-methyl-estr-5(10) ene-17β-ol-3-one and 17α-methyl-19-nor-testosterone gave increases in the estriol fraction, the former being the more potent precursor of the two.

A new approach for quantitative evaluation of cross-reactivity of steroids with an antiserum by radioimmunoassay: Application to a highly specific antiestriol

Abstract The study of several antiestriol antisera in the presence of a series of analogues of estriol has led to a critical discussion of the classical cross-reaction test. A more accurate test (CR 1 ng) which permits a more precise determination of specificity of antisera is described.

Gas chromatography profile of estrogens: Application to pregnancy urine

A method for the simultaneous quantitation of 7 estrogens in pregnancy urine is described. It involves enzymatic hydrolysis extraction of free steroids, ion-exchange column chromatography and quantitation of the trimethylsilyl derivatives by gas chromatography on OV 1. Data obtained from normal and twin pregnancies and from women with anencephalic fetus or intra uterine fetal death are analysed. The sensibility of the method is about 40 mug of each estrogen by liter of urine.

Préparation et propriétés physiqes des haptoglobines de lapin et de rat

Abstract Rabit haptoglobin is obtained by chromatography of DEAE-cellulose. The purification of rat haptoglobin required three chromatographies on DEAE, CM- and TEAE-cellulose. The principal physical constants of these two proteins are given and they are compared with those of human haptoglopin HP 1-a.

Purification and estrogen binding properties of mouse alpha1-fetoprotein and of two forms of the protein with different affinities for concanavalin-A.

Summary Mouse alpha1-fetoprotein (AFP) has been purified to electrophoretic and immunological homogeneity by preparative polyacrylamide gel electrophoresis and its binding parameters with estrone (E1) and estradiol (E2) have been measured by an equilibrium dialysis technique. Association constants are slightly more elevated for E2 than for E1 (KaE1 ∼ 0.4 108 M−1 and KaE2 ∼ 0.7 108 M−1 at 25°C), whereas the number of binding sites per mole of AFP displays similar fractional values (about 0.3) with both ligands. Purified mouse AFP has been submitted to affinity chromatography on Concanavalin-A-Sepharose, and two fractions have been obtained and purified in ponderal amounts. Binding parameters have been measured comparatively with E1 and E2 for the readily excluded fraction, as well as for the initially retained one. Significant differences have been demonstrated between the two forms; the excluded variant exhibits unique binding features, such as the presence of two classes of E2 saturable binding sites (Ka2 ∼ 1 109 M−1 and Ka1 ∼ 0.6 108 M−1) and a nearly 1:1 stoichiometry for the binding of E1. The results are discussed in relation to the nature and the significance of the heterogeneity of mouse AFP in the estrogen binding process. They are briefly compared to similar studies with the estrophilic rat AFP.

Determination of progesterone and of free and conjugated estrogens in pregnant and pseudo-pregnant rats ☆

Abstract Rat progesterone and estrogen levels have been determined in peripheral, ovarian, foetal plasma and in amniotic fluid, during estrous cycle, pseudo-pregnancy and pregnancy. From the progesterone levels, it was concluded that the predominant source of this hormone is the ovary. Though an ovarian contribution could not be excluded, placental origin of estrogens during pregnancy appeared evident. The estrogen levels showed the preponderance of the free form in a ratio E 2 /E 1