Studies on ouabain-like endogenous natriuretic factors in human urine. Inhibition of Na-K-ATPase and 3H-ouabain binding.

An endogenous inhibitor of sodium transport and of the Na-K-ATPase enzyme was previously detected in the small molecular weight postsalt fraction SIV of serum from saline-loaded rats after gel filtration on Sephadex G-25. In addition, a natriuretic factor present in this fraction of urine from salt-loaded subjects was found to bind to a specific digoxin antibody. Therefore, in the present study the small molecular weight natriuretic and digoxin antibody-binding activities present in the urine of salt-loaded healthy volunteers were purified by reverse-phase chromatography and by immunoprecipitation with the digoxin antibody and were studied for their in vitro effects on Na-K-ATPase derived from hog cerebral cortex. Na-K-ATPase inhibitory activities were found to roughly parallel the natriuretic activities at the various stages of purification. After reverse-phase chromatography, the material of fraction SIV which was bound to the digoxin antibody revealed the highest specific natriuretic activity of 3.95 +/- 0.29 mumol/min X mg injected material and showed strongest inhibition of the enzyme with I50 at a concentration of 0.08 microgram/ml, as compared with 2.4 micrograms/ml of the original postsalt urine fraction SIV. Fraction SIV revealed a noncompetitive inhibition of the enzyme with respect to potassium, but also significantly inhibited 3H-ouabain binding to the enzyme. Thus, the natriuretic substance(s) present in the urine of salt-loaded healthy subjects exhibit a potent inhibitory effect on the Na-K-ATPase enzyme which is similar, but not identical to that of ouabain.

Further characterization of the endogenous natriuretic and digoxin-like immunoreacting activities in human urine: effects of changes in sodium intake.

In the present study natriuretic activity and digoxin-like immunoreacting activity (DLIA) were determined in small molecular weight (MW) fractions of urine from healthy subjects during low (35 mmol/day) and high (greater than 400 mmol/day) sodium intake by bioassay and by a radioimmunoassay for digoxin, respectively. After gel filtration of urine on a Sephadex G-25 column the natriuretic activity appeared in the post-salt fraction SIV, whereas DLIA was present in small amounts in the salt fraction SIII and, with consistently higher activity, in the post-salt fraction SIV. Natriuretic activity significantly increased and DLIA decreased in fraction SIV with high sodium intake, but total urinary excretion of DLIA remained unaltered during changes in sodium intake. In addition, anion-exchange and reverse-phase chromatography revealed that DLIA is not specifically related to the natriuretic activity but also reflects unspecific binding of various urine constituents to this digoxin antibody. Although the antibody binds a natriuretic material, this radioimmunoassay is thus unsuitable to determine the endogenous natriuretic activity in urine fractions. Whereas they elute differently on reverse-phase chromatography, amino acid analyses revealed that both the natriuretic factor directly purified from the post-salt fraction SIV and the natriuretic material bound to the digoxin antibody have in common four amino acids at similar molar ratios. The physicochemical properties as evidenced by chromatographic and electrophoretic studies as well as enzymatic inactivation suggest that the low MW natriuretic factor(s) in human urine may be associated with a small peptide(s) of weak acidic nature.

An improved method for the preparation of human fetal and adult hepatocytes.

A method to prepare isolated hepatocytes from the small amounts of liver or liver fragments obtained from the human fetus following therapeutic abortion in early pregnancy is described. The hepatocytes have been characterized by electron microscopy and by their ability to dealkylate the benzodiazepine drug prazepam. The preparation of hepatocytes from a liver sample obtained from a kidney transplant donor is described and discussed.

Quantitative analysis of prazepam and its metabolites by electron capture gas chromatography and selected ion monitoring. Application to diaplacetal passage and fetal hepatic metabolism in early human pregnancy.

Methods have been developed for the determination of the benzodiazepine tranquilizer prazepam and its metabolites desmethyl diazepam, 3-hydroxy-prazepam and oxazepam by electron capture gas chromatography and selected ion monitoring with diazepam as the internal standard. The benzodiazepines were isolated from blood serum or homogenized tissue samples, either by extraction with ethyl acetate or on small Extrelut columns packed with porous silica. The concentrated extracts were directly injected into the gas chromatograph equipped with an electron capture detector. Following trimethylsilylation, analysis on a gas chromatography--mass spectrometry--computer system operated in the selected ion-monitoring mode was performed. Using 50--200 mg (microliter) biological material, concentrations of prazepam and metabolites of 5 ng/g(ml) could be determined with signal-to-noise ratios of greater than 10. Using 1 g(ml) samples, the same signal-to-noise ratios were obtained with 1 ng/g(ml) concentrations. The methods developed were applied to the analysis of the diaplacental transfer of prazepam and desmethyl diazepam in early human pregnancy. Furthermore, prazepam metabolism in human fetal liver and cell cultures was studied.