Progress in isolation and purification of an inhibitor of sodium transport obtained from dog urine.

Among the potential modulators of transtubular sodium transport is the putative natriuretic hormone. Widespread efforts are underway to isolate this substance in pure form. The present studies describe a series of experiments directed to this goal. Urine samples in the amount of 150 liters were taken from normal, mineralocorticoid hormone "escape" dogs and were chromatographed through Sephadex G-25. The active fraction of the eluate (that is, the fraction containing the inhibitor of sodium transport) was then subjected to high pressure liquid chromatography (HPLC) in four consecutive steps using three different resins. Approximately 5% of the each column eluate was diverted by use of a stream-splitting apparatus, and the fluorescent pattern was measured and recorded, in most instances following the addition of fluorescamine. Based on the respective fluorescent patterns obtained from the eluates of the successive chromatographic steps, the residual portion (95%) of each eluate was divided into fractions, and each fraction was bioassayed. In each instance only the biologically active fraction was subjected to further purification. In the first step involving HPLC and a cation exchange resin, six fractions were obtained. Only one was active. When this fraction was subjected to reverse-phase chromatography, seven new fractions emerged. Again only one was active. When it was chromatographed using a second cation exchange resin, two fluorometrically detectable peaks, termed N ad H, were identifiable. N exhibited spontaneous fluorescence; H exhibited fluorescamine-dependent fluorescence. In the final step, N and H were separated and bio-assayed individually. N was inactive; H proved to be a potent inhibitor of sodium transport. Accumulation of H in sufficient quantity will determine whether it is a single compound and will permit analysis of its chemical nature.