This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Graves, S. W. Articles by Hollenberg, N. K. Search for Related Content PubMed PubMed Citation Articles by Graves, S. W. Articles by Hollenberg, N. K. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB DIGOXIN OUABAIN Medline Plus Health Information High Blood Pressure Related Collections Hypertension - basic studies Ion channels/membrane transport

(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