Biomedical subjects
Publications and source records attributed to E Kuss.
The N-terminus of the recently isolated sterylsulfatase of human placental cellular membranes was sequenced. According to our results, the enzyme preparation proved to be homogeneous at least with respect to this part of the polypeptide chain; the n-terminal sequence of the sulfatase previously proposed by others, however, had to be revised partially.
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Glutathione transferases exhibit both isomerase and transferase activity. The acceptance of steroids as substrates for or inhibitors of these activities was studied using a 350-fold enriched preparation of the enzyme from human placenta. As an isomerase, the enzyme preparation catalyzed the conversion of pregn-5-ene-3,20-dione (Km 0.03 mmol/l) and androst-5-ene-3,17-dione (Km 0.05 mmol/l) to the respective 4-ene-3-oxosteroids (specific activity 0.8 U/mg protein). This isomerase activity strictly depended on the presence of glutathione (Km 0.04 mmol/l). As a transferase, the enzyme preparation catalyzed the conjugation of glutathione (Km 0.5 mmol/l) with 1-chloro-2,4-dinitrobenzene (Km 1.0 mmol/l) (specific activity 100 U/mg protein). This transferase activity was inhibited by all phenolic (KI values 0.2-1.5 mmol/l) and some of the neutral steroids (KI values 1.4-3.5 mmol/l) tested. Phenolic steroids inhibited the enzyme activity competitively to 1-chloro-2,4-dinitrobenzene and non-competitively to both substrates. The results indicate that steroids can interact with the placental glutathione transferase in vitro both as substrates and as inhibitors. Since, however, the observed Km and KI values of the steroids are far above the values of their concentrations in the placenta, these interactions are of only minor physiological relevance.
The serum concentration of the tumour marker CA 125 was determined during an inflammatory adnexitis of a young woman of 22 years of age, the measured value being 478 kU/l. After the inflammation had subsided, a malignoma was largely excluded by laparoscopy; the CA 125 value had dropped to 70 kU/l. In a woman of 34 years of age who had an endometriosis that had been identified for the last ten years, as well as an acute suppurative fibrinous peritonitis, the CA 125 value was found to be 205 kU/l. On the fourth postoperative day the value was around 165 kU/l, on the 13th and 26th postoperative day 273 kU/l and 168 kU/l, respectively. There were no signs of an inflammation.
The concept of "placental insufficiency" appears to offer a plausible explanation for foetal emergency situations; in reality, however, it leads to unproven and possibly wrong ideas on the genesis of these phenomena. It is applied in a semantically inconsistent manner, and that is one of the reasons why it is unsuitable as a term that can be used in scientific language. It is nevertheless being continually used in present-day "medicalese" and even in recent textbooks of obstetrics as a definition of nosological entities. The claim that the term "placental insufficiency" is necessary for mutual communication in obstetrics, is not substantiated or, if at all, with the self-confirming claim that it cannot be eliminated because it is already firmly rooted in clinical thinking. The conditions in the foetus and the newborn labelled "placental insufficiency syndrome" could be defined without additional linguistic effort by means of a number of sufficiently precise and far more important terms, namely "underweight", "dystrophy", "asphyxia/hypoxia". These terms stand for results of direct observation without hypothetical connotations. The conditions they describe are--at least in principle--objectifiable and measurable. The expressions allow and induce further deliberations and studies on actual causes of chronic or acute deficient supply of the foetus, the nascent and newborn infant, and how to avoid these causes. The criticism of the concept "placental insufficiency" presented and reviewed here, is not new.(ABSTRACT TRUNCATED AT 250 WORDS)
Concentrations of unconjugated and conjugated 2-hydroxyoestrogens and their 2-methylethers were determined by radioimmunoassay in serum obtained from the umbilical artery, umbilical vein, retroplacental space, and maternal peripheral vein at the time of delivery. The concentrations of unconjugated 2-hydroxyoestrogens and 2-methoxyoestrogens were found to be significantly higher in the post-placental vessels than in the pre-placental vessels. On the basis of the concentrations measured it was estimated that the placenta secretes about 1.4 nmol/min 2-hydroxyoestrogens and about 1.7 nmol/min of 2-methoxyoestrogens. The present in vivo findings confirm the placental 2-hydroxylase- and catechol-O-methyl-transferase activities previously demonstrated in vitro.
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The steryl-sulfatase of normal human placental microsomes was solubilized and enriched about 350-fold. Chromatography on Sepharose 6B of the purified enzyme preparation revealed a single protein peak which eluted according to an apparent molecular mass of 270 +/- 30 kDa; when electrophorized on sodium dodecyl sulfate polyacrylamide gel the sulfatase migrated according to a molecular mass of 64 +/- 4 kDa. Estrogensulfatase activity was co-purified with the steryl-sulfatase activity; obviously, both activities belong to the same enzyme species. The purified sulfatase was injected into three rabbits. Antisera produced by the rabbits yielded a single sharp immunoprecipitation line in Ouchterlony double diffusion experiments when tested with the isolated sulfatase or with a solubilized microsomal fraction of normal placentas. The activity of sulfatase preparations incubated with antiserum was precipitated by addition of polyethylene glycol followed by centrifugation; none of the antibodies reacting with the sulfatase therefore appeared to interfere with its enzymatic activity. Using these antisera, steryl-sulfatase protein could be detected by immunoblotting analysis in solubilized microsomal fractions of normal placentas but not in solubilized microsomal fractions of three steryl-sulfatase activity-deficient placentas. This finding argues in favour of human placental steryl-sulfatase deficiency being due to extremely diminished or absent enzyme protein in the placenta.
Human placental steroid-sulfatase was extracted nearly quantitatively from microsomes as well as from acetone dry powder of placenta homogenates using CHAPS as detergent. The solubilized enzyme was enriched 10-fold by ammonium sulfate precipitation and gel chromatography. The sulfatase extracted from both microsomes and acetone dry powder eluted as a single fraction on Sepharose 6B, but with different apparent molecular masses (390 and 270 kDa, respectively). Kinetic experiments with the sulfate esters of dehydroepiandrosterone, 16 alpha-hydroxydehydroepiandrosterone, estrone, and estriol as substrates or inhibitors indicated that the solubilized sulfatase was fully active. Both the particulate and the extracted enzyme showed higher affinities for the 16-unsubstituted than for the 16 alpha-hydroxylated substrates. Whereas a competitive inhibition was observed in mixed substrate incubations with dehydroepiandrosterone sulfate/16 alpha-hydroxydehydroepiandrosterone sulfate and estrone sulfate/estriol sulfate, diverse patterns of inhibition were obtained with dehydroepiandrosterone sulfate/estrone sulfate, depending on the sulfatase preparation used. However, evidence for the distinct nature of the steroid-sulfatase and the estrogen-sulfatase was not obtained. The membrane-bound, but not the solubilized enzyme was to a certain degree sensitive to lipase and acetone. The solubilized sulfatase strongly bound to ConA-Sepharose. This observation together with the elution by alpha-methyl mannoside were indicative of the presence of carbohydrates on the sulfatase. Since its enzymatic activity was markedly decreased by the effects of alpha-mannosidase and N-acetylglucosaminidase, a possible involvement of the carbohydrate moiety in the catalytic activity of the sulfatase might be considered.
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The standard concepts, symbols, and formulae of reversible association reactions are comprehensively illustrated with the aid of the immune reaction, and presented in the context of their use in analysis (section 1). The enthalpy and entropy values of activation and reaction are interpreted in the light of the results of recent investigations (section 2). Common methods that can be performed in the ordinary clinical chemical laboratory are discussed for the determination of physical chemical constants, including those of complicated association reactions (section 3); Processing of experimental data was recently reviewed in this journal (E. Kuss, J. Clin. Chem. Clin. Biochem. 20, 227-234 (1982]. The use of physical chemical constants in analysis is illustrated with examples (section 4).
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The new method of solid-liquid extraction of oestrogens from pregnancy urine with Sep-Pak C18 cartridges was compared to the approved method of extraction with Amberlite XAD-2 columns. At about equal costs the extraction by Sep-Pak cartridges turned out to be more economical with respect to time and work. The yield was higher by about 35% and the precision was slightly improved. On account of these results the hitherto applied method of extraction by Amberlite XAD-2 was replaced by the Sep-Pak C18 method within our routine analysis for oestrogen determination in pregnancy monitoring. The method proved to be good over one year; therefore it can be recommended generally.
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 beta-hemi-succinyl-BSA served as binding protein and 2-methoxyoestrone-17-cmo-[125I]iodohistamine served as 125I-labelled ligand. A specific (little or no cross-reactivities to other oestrogens), sensitive (2.4 +/- 0.9 pg/tube) and precise (6.5% intra-assay; 9.1% inter-assay) assay was obtained by employing the heterologous bridge system. The following concentrations were found in serum samples of healthy subjects (median, range in parentheses): men (19-58 years): less than 10.3 (less than 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).
35S-labeled sulfate esters of dehydroepiandrosterone and 16 alpha-hydroxydehydroepiandrosterone were synthesized and used as substrates for the in vitro kinetic assay of human placental steroid-sulfatase. Both steroid sulfates were hydrolysed by placenta homogenates and microsomal fractions with V values comparable to each other. The Km value of the 16 alpha-hydroxy compound, however, was found to be about tenfold higher than that of dehydroepiandrosterone sulfate. Both sulfate esters competitively inhibited each other's hydrolysis. The results suggest that dehydroepiandrosterone sulfate, as compared to its 16 alpha-hydroxy derivative, is the preferred substrate of the sulfatase. As far as conclusions can be drawn from experiments in vitro, this finding excludes the possibility that the preponderance of placental estriol production over that of estradiol and estrone in human late pregnancy is due to a preferential binding and cleavage of the estriol precursor 16 alpha-hydroxydehydroepiandrosterone sulfate by the placental steroid-sulfatase.