Simultaneous Isolation of Endogenous Digoxin-like Immunoreactive Factor, Ouabain-like Factor, and Deglycosylated Congeners from Mammalian Tissues

Abstract

DLIF (digoxin-like immunoreactive factor) and OLF (ouabain-like factor) 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 solubility 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 existing deglycosylated congeners, including three deglycosylated species of DLIF (DLIF-genin, DLIF-mono, and DLIF-bis) and one deglycosylated species of OLF (OLF-genin). Separation is achieved using acid extraction, C-18 reverse-phase HPLC chromatography, and signal detection using two antibodies, one specific for digoxin and one for ouabain. The average 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 immunoreactivity of DLIF is 103-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 presence of a compound reacting with both digoxin and ouabain antibodies. This unique immunoreactive species is likely to have structural similarity to both digoxin and ouabain and thus may represent a metabolic link between DLIF and OLF.