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(Hypertension. 2000;36:1059.)
© 2000 American Heart Association, Inc.

Scientific Contributions

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

Steven W. Graves; Karen E. Markides; Norman K. Hollenberg

From the Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah (S.W.G.); the Department of Analytical Chemistry, Uppsala University, Uppsala, Sweden (K.E.M.); and the Department of Radiology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Mass (N.K.H.).

Correspondence to Dr Steven W. Graves, Department of Chemistry and Biochemistry, Brigham Young University, BNSN C-212, Provo, UT 84602. E-mail swgraves{at}chemdept.byu.edu

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.

Key Words: sodium pump • sodium • hypertension, sodium-dependent • ouabain