Isolation of digoxin-like immunoreactive factors from mammalian adrenal cortex

Endogenous digoxin-like immunoreactive factors (DLIF) are present in serum and tissues of humans and animals. To date, a tissue source for these factors has not been rigorously defined nor have these factors been isolated to identifiable homogeneity. In this study, we define the distribution of DLIF in mammalian tissues, demonstrate the adrenal cortex to be the principal source of this factor in bovine, and isolate DLIF to chromatographic homogeneity using high performance liquid chromatography (HPLC). DLIF concentrations in tissue extracts from rats measured as follows: adrenal glands, 44.3; serum, 6.3; liver, 5.2; kidney, 1.2; heart, brain, or lungs, less than 1.4 ng of digoxin-equivalent per g of protein. Human tissues showed similar results. In dogs, the ratio of the DLIF concentration in lumbar vein serum to that in infrarenal inferior vena cava serum was 3.3 +/- 0.4 (mean +/- S.E., n = 4). Bovine adrenal cortex contained 7 times more DLIF per g of tissue than the adrenal medulla. 70 +/- 4% (n = 7) of the total bovine cortical DLIF activity (6,159 pg of digoxin-equivalent) applied to a reverse phase HPLC column eluted as one definitive fraction. 60% of the digoxin-like immunoreactivity extracted from bovine serum also co-eluted with DLIF from adrenal. None of the 14 steroid molecules or 7 cardiac glycoside congeners co-eluted with the major DLIF activity. Our data indicate that 947 pmol of DLIF is equivalent to 1 pmol of digoxin-equivalent immunoreactivity. Preliminary mass spectral analysis suggests that purified DLIF has a molecular mass of 780 daltons comprised of one 390-dalton aglycone component plus several sugar moieties. This study establishes a definitive link between DLIF in serum and the adrenal cortex as a source tissue. We also demonstrate a method for purifying DLIF to chromatographic homogeneity with an extraction capacity of 1.2 nmol of DLIF per g of adrenal cortex.