Abstract
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.
Highlights
(DLIF) are present in serum and tissuoefshumans and 3)
A definitive total bovine cortical digoxin-likeimmunoreactive factors (DLIF) activity (6,159 pg of di- correlation has notbeen established between DLIF molecules goxin-equivalent)appliedto a reverse phase HPLC andthepresence of high affinity ligands for the digitalis column eluted as one definitive fraction. 60%of the glycoside binding sites on cell-membrane Na,K-ATPase
We demonstrate that the adrenal cortex is the principal source of digoxin-like immunoreactive factors (DLIF) found in bovine plasma
Summary
To determine whether the adrenal gland was a source of DLIF, serum DLIF concentrationins the left lumbar vein and infrarenalinferiorvena cava were measured in four dogs. These results are shown in TabI1leand demonstrate that the lumbar veins consistently had higher DLIF values (on averfrom polyclonal digoxin-specific antibodies relative to digoxin stand- age, 3.3-fold higher) than the infrarenalinferior vena cava in Isolation ofMfDraoLmmIFmAadliCraeonnratel x. DLIF and cortisol measured in blood samples drawn from infrarenal inferior vena cava and the left lumbar vein of four 15- to 20-kg mongrel dogs (seetext for details).Hydrocortisonemeasurements demonstrate that the lumbavrein blood represents adrenal drainage. This was demonstrated when the water-trichloroacetic acid mixture alone was chromatographed and eluted digoxin-like immunoactivity at onemajor peak
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