SIMULTANEOUS ISOLATION OF ENDOGENOUS DIGOXIN-LIKE IMMUNOREACTIVE FACTOR, OUABAIN-LIKE FACTOR, AND DEGLYCOSYLATED CONGENERS FROM MAMMALIAN-TISSUES

DLIF (digolrin-like immunoreactive factor) and OLF (ouabain-Like facto r) are endogenous steroid-like ligands (approximately 781 and 595 Da, respectively) with molecular and structural properties similar to the plant-derived cardiac glycosides, digoxin and ouabain. We developed a purification method with a sufficiently wide range of extraction solub ility to separate compounds with polarities spanning those of ouabain and digoxin. This technique provides a rapid, reliable, and efficient method for simultaneously isolating DLIF, OLF, and several naturally e xisting deglycosylated congeners, including three deglycosylated speci es of DLIF (DLIF-genin, DLIF-mono, and DLIF-bis) and one deglycosylate d species of OLF (OLF-genin). Separation is achieved using acid extrac tion, C-18 reverse-phase HPLC chromatography, and signal detection usi ng two antibodies, one specific for digoxin and one for ouabain. The a verage-extraction efficiency is 400 pmol digoxin equivalent (range 300 -500) and 42 pmol ouabain equivalent (range 37-50) per gram of adrenal cortex for DLIF and OLF, respectively. The relative molar immunoreact ivity of DLIF is 10(3)-fold less than that of digoxin, whereas that of OLF is unity compared to ouabain, suggesting that OLF is structurally more similar to ouabain than DLIF is to digoxin. Of interest is the p resence of a compound reacting with both digoxin and ouabain antibodie s, This unique immunoreactive species is likely to have structural sim ilarity to both digoxin and ouabain and thus may represent a metabolic link between DLIF and OLF. (C) 1996 Academic Press, Inc.