Application of Supercritical Fluid Chromatography to Characterize a Labile Digitalis-Like Factor

Abstract

Abstract —A sodium pump inhibitor (digitalis-like factor), isolated from the peritoneal dialysate of volume-expanded, hypertensive patients with kidney failure who were treated with this dialysis modality, was further purified and characterized by means of supercritical fluid chromatography, a separation technique whose application to very-low-concentration biomolecules is new. Previous studies suggested that after high-performance liquid chromatography (HPLC) purification, this inhibitor was the only factor correlated with volume status and blood pressure in these patients. When this same HPLC fraction was furthered purified on 2-dimensional supercritical fluid chromatography, a single peak coeluted with [Na,K]ATPase inhibitory activity. When split specimens were used, there was a strict correlation between the peak area, measured by flame ionization detection, and activity (n=10, R =0.98, P =0.00001). Inhibitory activity after supercritical fluid chromatography was still correlated with the degree of volume expansion of donor patients ( P =0.01). After HPLC purification, this volume-sensitive inhibitor was chemically labile. With further purification on supercritical fluid chromatography, the active peak was still labile with comparable half-life. Supercritical fluid chromatography coupled with flame ionization detection provided an estimate of the amount of the inhibitor present. Again using split specimens, we determined that the labile digitalis-like factor was ≈30-fold more effective than ouabain in inhibiting renal [Na,K]ATPase activity and ≥500 times more effective than ouabain in causing vascular smooth muscle contraction. The data suggest that we have purified to homogeneity a labile digitalis-like factor that is readily distinguished from ouabain or bufalin, based on chromatographic characteristics, chemical lability, and a much lower effective concentration for its biological activity.