ENDOGENOUS SODIUM-PUMP INHIBITORS IN HUMAN URINE - FURTHER IDENTIFICATION OF INHIBITORS OF NA-K-ATPASE

We investigated the presence of endogenous Na-K-ATPase inhibitor(s), i e, ouabain-like factors (OLFs), in the urine of salt-loaded healthy su bjects. For this purpose 24-h urine was collected on days 3, 4, and 5 of high sodium intake (>30 g NaCl/day). The samples then were lyophili zed. Redissolved urine concentrates were acidified (pH 3.5) and subjec ted to gelchromatography on a Sephadex G-25 column where the OLFs elut ed in the post-salt fraction IV. When lyophilized fraction IV was rech romatographed on Sephadex G-10, OLFs with molecular mass (M(r)) of app roximately 400 eluted in a late fraction IV/8 separate from added ouab ain, ouabagenin (or digoxin), which eluted shortly after void volume. With the subsequent reverse-phase HPLC of fraction IV/8 a polar OLF-1 eluted in fraction IV/8a after the void volume in the water phase and a more apolar OLF-2 eluted at 20% acetonitrile in fraction IV/8d. Only the more apolar OLF-2 cross-reacted with a digoxin antibody. By prepa rative thin-layer chromatography OLF-1 and OLF-2 were purified as sing le compounds with potent dose-dependent Na-K-ATPase inhibition and K-i -values approximating 1.5 x 10(-5) mol/L and 1.5 x 10(-4) mol/L, respe ctively. Mass-spectroscopy (MS) showed M(r) of 391 and H-1-NMR charact erized the endogenous urinary apolar OLF-2 as a compound that is struc turally totally unrelated to ouabain; infrared (IR) spectroscopy of OL F-1 and OLF-2 also revealed no similarity with ouabain. In contrast, d ata from MS, NMR-, and IR-spectroscopy show structural resemblance wit h ascorbic acid derivatives, some of these having strong inhibitory ef fects on Na-K-ATPase.