Ouabain-Like Factors in Human Urine: Identification of a Na-K-ATPase Inhibitor as Vanadium-Diascorbate Adduct

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 NaCl/day 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 Sephadex G-10, one of threc OLFs eluted in the early subfractions FIV/1–2 close to ³H-ouabain and cross-reacted strongly with a ouabain antibody (NEN). Two additional OLFs with M_raround 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 cross-reacted with digoxin and ouabain antibodies. OLF-1 and OLF-2 purified to single compounds by preparative thin layer chromatography inhibited Na-K-ATPase with IC₅₀of around 1.5 × 10^-5M and 1.5 × 10^-4M, respectively. Identification of OLF-2 was first attempted because most material was available for further processing. Data from mass-spectroscopy, nuclear magnetic resonance (¹H-NMR) and infrared spectroscopy characterized OLF-2 as structurally unrelated to ouabain but resembling ascorbic acid derivatives, i.e. vanadium (V) diascorbates (M^r403) with similar elution times from RP-HPLC as OLF-2. They inhibited the enzyme in its E₂-configuration with IC₅₀of 9 × 10^-5M and 2 × 10^-6M for V^iv- and V^v-diascorbate, respectively. OLF-1, OLF-2 and V-diascorbate raise intracellular free calcium in inner medullary collecting duct and vascular smooth muscle cells which also contract in vitro. V-diascorbate was also natriuretic in a bioassay. We suggest that V-diascorbates represent one of several OLFs excreted in human urine

Key Words:

• Na-K-ATPase inhibitor
• ouabain
• vanadium-diascorbate
• human urine