The endogenous ouabainlike factor in human plasma and MHS rat tissues: comparison of two independent assays.

In view of the controversy regarding the detectability and nature of an endogenous ouabainlike factor (OLF) in mammals, tissues and plasma from MHS and MNS rat strains and plasma from healthy volunteers were purified and tested for the presence of OLF using two independent types of assay. Plasma and tissues were obtained from two pools of 150 adult MHS and MNS rats and 50 ml of plasma was obtained from untreated normotensive subjects. After HPLC fractionation, OLF was in parallel quantified by a radioimmunoassay (RIA) using an anti-ouabain polyclonal antibody (OU Ab) and by enzymatic inhibition of dog kidney Na-K ATPase (ATPase). 1) One fraction with HPLC retention time similar to ouabain was identified by both assays; 2) OLF showed a parallel dilution curve and similar Kd to ouabain both in RIA (Kd=3nM) and ATPase (Kd=14nM); 3) comparable OLF yield was obtained from the tested tissues of both rat strains by both methods: a linear regression analysis of RIA and ATPase OLF tissue measurements showed a highly significant correlation coefficient (r=0.88, N=16, p<0.001);4) OLF was present in greater amount in hypothalamus, hypophysis and plasma of MHS than those of MNS, while OLF was similar in adrenals;5) HPLC fractionation of human plasma showed a major fraction with the same retention time of ouabain. Similarly to rat plasma, the measurements of OLF levels both with RIA and ATPase gave comparable results also in the human plasma samples, (range between 20-60 pM). In conclusion these data clearly demonstrate that rat plasma and tissue extracts contain measurable amounts of OLF which is selectively recognized by Ou Ab and similarly quantified by RIA and ATPase assays. The higher OLF levels measured in plasma and central tissues of MHS rats further confirms previous findings on the pathogenetic role of OLF in MHS hypertension. Furthermore, OLF levels are similarly detected by both methods also in human plasma, even after HPLC fractionation.