Abstract
We investigated endogenous Na-K-ATPase inhibitors, i.e. ouabain-like factors(OLFs), in the urine of salt-loaded healthy subjects. During an intake of > 30g NaCVday 24h-urines were collected, lyophilized, redissolved and acidified to pH 3.5. With gelchromatography the inhibitory activity eluted in a post-salt fraction FIV from Sephadex G-25. When this fraction was again passed through Se hadex G-10, one of three OLFs eluted in the early subfractions FIV/1-2 close to H-ouabain and cross-reacted strongly with a ouabain antibody (NEN). Two additional OLFs with M, around 400 eluted in a late subfraction FIV/8 which resolved after reverse-phase HPLC into a more polar OLF-1 (water phase) and a more apolar OLF-2 (20% acetonitrile). Only the more apolar OLF-2 crossreacted with digoxin and ouabain antibodies. OLF-1 and OLF-2 purified to single compounds by preparative thin layer chromatography inhibited Na-KATPase with ICso of around 1.5 x 10-5M and 1.5 x 104M, respectively. Identification of OLF-2 was first attempted because most material was available for further processing. Data from mass-spectroscopy, nuclear magnetic resonance ('H-NMR) and infrared spectroscopy characterized OLF-2 as structurally unrelated to ouabain but resembling ascorbic acid derivatives, i.e. vanadium 07) diascorbates (M, 403) with similar elution times from RP-HPLC I:
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