Abstract1. A radioimmunoassay has been developed for the measurement of 2-methoxyoestrogens in samples of human serum without chromatography after solvent extraction. Antibodies raised against 2-methoxyoestradiol-17\g=b\-hemi-succinyl-BSA served as binding protein and 2-methoxyoestrone -17cmo -[125I]iodohistamine served as 125I-labelled ligand. A specific (little or no cross-reactivities to other oestrogens), sensitive (2.4 \ m=+-\0.9 pg/tube) and precise (6.5% intra-assay; 9.1% inter-assay) assay was obtained by employing this heterologous bridge system. 1 The following trivial names and abbreviations were used 2-methoxyoestrone: 2-methoxy-3-hydroxy-1,3,5(10)oestratrien-17-one, 2-methoxyoestradiol: 2-methoxy-l,3,5(10)-oestratriene-3,l7ß-diol, 2-methoxyoestriol: 2-methoxy-l,3,5(10)-oestratriene-3,16ct,17ß-triol, 2-hydroxyoestrone: 2,3-dihydroxy-l,3,5(10)-oestratrien-17-one, 2-hydroxyoestradiol: l,3,5(10)-oestratriene-2,3,17ß-triol, 2-hydroxyoestriol: 1,3,5( 10)-oestratriene-2,3,16 , 17ßtetrol, oestrone: 3-hydroxy-l,3,5(10)-oestratrien-17-one, oestradiol-17ß: l,3,5(10)-oestratriene-3,17ß-diol, oestriol: l,3,5(10)-oestratriene-3,16a, 17ß-triol, 2-methoxyestrone-17-cmo : 2-methoxyoestrone-17carboxymethoxime : 17-(0-carboxymethyl)-oximino-2methoxy-3-hydroxy-1,3,5( 10)-oestratriene, 2-methoxy-oestradiol-17ß-succinyl-BSA : 2-methoxyl,3,5(10)-oestratriene-3,17ß-diol-17ß-ylhemisuccinate-bovine serum albumin. Dedicated to Professor Dr. med. J. Zander on the occasion ofhb 65th birthday. The following concentrations were found in serum sam¬ ples of healthy subjects (median, range in parentheses): men (19-58 years): < 10.3 (< 10.3-35.5) pg/ml (n = 22); women follicular phase: 46 (18-63) pg/ml (n = 8); luteal phase: 70 (31 -138) pg/ml (n = 8); post-menopausal women: 33 (21 -76) pg/ml (n = 10); pregnant women 11th-16th week: 674 (216-1678) pg/ml (n = 46); 37th-40th week: 3768 (2035-10691) pg/ml (n = 34); newborn cord serum 1608 (575-3095) pg/ml (n = 41).2-Hydroxyoestrogens and their monomethyl ethers are a major group of oestrogen metabolites in both laboratory animals and humans. Their role in endocrine physiology is still poorly understood. Investigations into the physiological significance of this type of oestrogens require sensitive and speci¬ fic methods for the quantitative determination of these compounds in body fluids and tissues. 2-Hydroxyoestrogens are rapidly metabolized, ini¬ tially in the vascular space (Bates et al. 1977) and from the quantitative point of view predominantly in the liver (Knuppen et al. 1970), by the catechol-O-methyltransferase. Therefore in addition to the measurement of 2-hydroxyoestrogens (Berg et al. 1982) the 2-methoxyoestrogens should be meas¬ ured for a sufficient description of the production and excretion rates of 2-substituted oestrogens.The radioimmunological method described hitherto utilizes tritium labelled radioligands, with high specific radioactivities not yet available com¬ mercially (Emons et al. 1979).
Catecholestrogens, 2-methoxyestrogens and "classical" estrogens (estrone, estradiol, estriol) were measured simultaneously in serum and urine samples of 220 pregnant women from the 8th week of pregnancy until to delivery. From these data we established the central 0.80 centile intervals as time specified reference intervals for each substance analyzed. Serum and urinary estradiol rise steadily during the progress of pregnancy, whereas estrone, catecholestrogens and 2-methoxyestrogens reach a plateau during the last trimester. These observations support the hypothesis, that the amount of the latter compounds may be regulated by separate mechanisms. The values of concentration and excretion of 2- and 4-substituted estrogens varied widely throughout pregnancy. Even very high or very low concentrations of these substances had no recognizable relation to the outcome of pregnancy. This supports the assumption that catecholestrogens and their methylethers are metabolites without any regulatory function in pregnancy.
The enzymatic properties of a homogeneous Sterylsulfatase preparation isolated from human term placenta were studied. The enzyme exhibited both arylsulfatase and Sterylsulfatase activity: it catalysed the hydrolysis of sulfuric acid esters of (in the order of decreasing specific activity) non-steroidal phenols, of a phenolic steroid, and of neutral 3/3-, 21-and (though at a very low rate) 17/3-hydroxysteroids. However, among all the substrates tested only the 3-sulfates of phenolic and neutral steroids exhibited high affinity towards the sulfatase. Vitamin D 3 sulfate was not hydrolysed by the Sterylsulfatase but strongly inhibited its activity. The products of the catalytic reaction, free steroids or phenols as well as the sulfate anion or analogues thereof, likewise interfered with the enzyme's activity. K\ values of unconjugated steroids were ten-to hundredfold higher than K m values of the respective sulfoconjugates. Inorganic sulfate only slightly inhibited the sulfatase activity; its inhibitory potency, however, increased in a time-dependent manner when it was preincubated with the enzyme prior to assay. In contrast to sulfate, the hypothetical transition-state analogues sulfite and vanadate acted as strong inhibitors of the sulfatase activity.According to the results of an analysis of the effect of pH on Sterylsulfatase kinetics, enzyme constituents with pK values of approximately 5.8 and 8.0 are involved in a general acid-base catalysed reaction. Treatment of the sulfatase with amino-acid side chain modifying reagents directed against arginine, cysteine, cystine, serine or tyrosine residues did not result in significant alteration of its activity. Diethylpyrocarbonate known to react primarily with histidyl groups, however, rapidly inactivated the enzyme; this inactivation reaction was markedly retarded in the presence of substrate. Histidine thus appears to be essential for the catalytic activity of the sulfatase.Taken together, the present results reveal a considerable similarity between the catalytic mechanism of human placental Sterylsulfatase and the ones already proposed for the lysosomal arylsulfatases A and B.Taurocholate, salicylate, ouabain, and 4,4'-substituted stilbene-2,2'-disulfonates are well known inhibitors of carrier-mediated transport of anions across Enzyme: Sterylsulfatase, steryl-sulfate sulfohydrolase (EC 3.1.6.2).
(1 -e-C'(t)/kT)rstdrdsdt (3) (r, s und t = Abstande zwischen den drei Molekulen). Fur das Potential U ( r ) werden in diese Beziehungen geeignete Funktionen eingefulirt, die jedoch zu sehr kompli-* Horrn Dip1.-Ing. E . RGmw, Darnistadt, zum 65. Geburtstay gewiclmet.
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