Fractionation of the Diuretic Factor Present in Pregnant Mare Urine

Abstract

Urine preparations obtained by dialysis and lyophilization were fractionated and the various fractions assayed for diuretic activity in the dog. The assay procedure employed yields good reproducibility of results when different samples of the same urine preparation are injected intravenously in doses which are similar. Refluxing of the urine sample at pH 1.0 or pH 10.0 for 30 minutes does not alter the diuretic activity of the samples. A significant diuretic response was found in fractions obtained by extraction of the urine samples with chloroform and petroleum ether but not with diethyl ether or ethyl acetate. In no instance was the extraction of activity by these organic solvents complete. Fractional precipitation with ammonium sulphate did not yield a complete separation of the activity. There is evidence that the fraction obtained with half-saturation with ammonium sulphate has a greater activity per milligram of dry weight than the fraction obtained with subsequent complete saturation. Acetone, in a sufficiently high concentration, precipitates the activity completely. When the urine sample is refluxed for 30 minutes at pH 1.0. and subjected to fractional acetone precipitation at pH 10.0, the greatest activity is found in the precipitate obtained at an acetone concentration between 63% and 75%.