IS OUABAIN AN AUTHENTIC ENDOGENOUS MAMMALIAN SUBSTANCE DERIVED FROM THE ADRENAL

Ouabain has recently been reported to be an endogenous mammalian substance released by the adrenal cortex and present in normal plasma. We have attempted to confirm and extend this observation. Using a ouabain radioimmunoassay developed in this laboratory, we fractionated by high-performance liquid chromatography (HPLC) normal human plasma from healthy volunteers to determine the presence of ouabain immunoreactivity and compare this immunoreactivity with authentic ouabain. In most subjects no ouabain immunoreactivity that coeluted with authentic ouabain was observed. Some subjects had ouabain-immunoreactive material present at low levels, but it was largely attributable to cross-reactivity with diverse substances found not to be ouabain. Similar results were obtained after analysis of plasma collected from 10 patients entering a medical intensive care unit. Studies of serum-free medium conditioned by bovine adrenocortical cells showed some ouabain immunoreactivity. To determine whether this material might be a steroid product of cholesterol side-chain cleavage, we performed chemical blockade of steroidogenesis, which effectively suppressed progesterone production by these cells but had no consistent effect on ouabain immunoreactivity in this medium. Stimulation of steroidogenesis with 22-R-OH-cholesterol in bovine adrenocortical cells did not produce any increase in the ouabain immunoreactivity present in conditioned medium. Subsequent HPLC studies of ouabain immunoreactivity in bovine adrenocortical cell-conditioned medium indicated that authentic ouabain did not account for most of the ouabain immunoreactivity in serum-free medium. Studies with bovine adrenocortical cells incubated in a minimal salt and glucose medium indicated a small peak of immunoreactivity that may correspond to authentic ouabain. Examination of ouabain immunoreactivity in serum-supplemented medium conditioned by the murine adrenocortical tumor cell line Y1 indicated much higher levels of immunoreactivity; however, the major portion of this immunoreactivity eluted during reversed-phase HPLC with much lower polarity than authentic ouabain. These studies indicate only a weak likelihood that ouabain is a biologically relevant product of the mammalian adrenal cortex.