Ouabain-like factor quantification in mammalian tissues and plasma: comparison of two independent assays.
The resolution of controversies that concern the detectability of an endogenous ouabain-like factor (OLF) in mammalian tissues and plasma was approached by the application of a standardized method for its extraction and quantification. Two independent assays were used to quantify the OLF: (1) a radioimmunoassay, which used a polyclonal anti-ouabain antiserum, and (2) a radioenzymatic assay based on the inhibition of dog kidney Na+,K+-ATPase. Plasma and tissues were obtained from the Milan hypertensive strain (MHS) and the Milan normotensive strain (MNS) of rats and from healthy human volunteers. Results indicate that (1) a single high-performance liquid chromatography (HPLC) fraction identical to that of ouabain was identified by both assay methods in the rat hypothalamus and hypophysis and in both rat and human plasma; (2) dilution curves of OLF and standard ouabain were parallel and with a similar Kd, both in radioimmunoassay (3 nmol/L) and ATPase assay (14 nmol/L); (3) after HPLC, OLF was similarly quantified by the two methods in the hypothalamus, hypophysis, adrenals, and plasma of rats and in human plasma; (4) OLF was present in larger amounts in the hypothalamus, hypophysis, and plasma of MHS rats than that of MNS rats; (5) the HPLC fraction of human plasma was quantified similarly by both assays (range, 60 to 150 pmol/L); (6) recovery of standard ouabain in pre-HPLC plasma extracts was approximately 90%; and (7) pre-HPLC OLF concentrations in human plasma ranged between 0.05 and 0.75 nmol/L. Rat cerebral tissues and both rat and human plasma contained measurable amounts of OLF, which were quantified similarly by radioimmunoassay and ATPase assay, both before and after HPLC fractionation. The increased MHS tissue and plasma levels of OLF are in keeping with the pathogenetic role of this factor in MHS hypertension.
Ferrandi
M
M
Prassis-Sigma Tau, Research Institute, Milan, Italy.
Manunta
P
P
Balzan
S
S
Hamlyn
J M
JM
Bianchi
G
G
Ferrari
P
P
D000311:Adrenal Glands / Q000737:chemistry; D000818:Animals; D001685:Biological Factors / Q000032:analysis* / Q000097:blood*; D002298:Cardenolides; D002851:Chromatography, High Pressure Liquid; D004077:Digoxin; D004285:Dogs; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000097:blood*; D006801:Humans; D007031:Hypothalamus / Q000737:chemistry; D007106:Immune Sera / Q000276:immunology; D008297:Male; D008722:Methods; D009994:Osmolar Concentration; D010042:Ouabain / Q000032:analysis / Q000276:immunology; D010902:Pituitary Gland / Q000737:chemistry; D011863:Radioimmunoassay; D051381:Rats; D011919:Rats, Inbred Strains; D012503:Saponins; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis; D014020:Tissue Extracts / Q000737:chemistry
D001685:Biological Factors; D002298:Cardenolides; D004791:Enzyme Inhibitors; D007106:Immune Sera; D012503:Saponins; D014020:Tissue Extracts; C034504:digoxin-like factors; D010042:Ouabain; D004077:Digoxin; D000254:Sodium-Potassium-Exchanging ATPase