Application of supercritical fluid chromatography to characterize a labile digitalis-like factor.
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 approximately 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.
Graves
S W
SW
Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah, USA. swgraves@chemdept.byu.edu
Markides
K E
KE
Hollenberg
N K
NK
D002298:Cardenolides; D002849:Chromatography, Gas / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004077:Digoxin; D004791:Enzyme Inhibitors / Q000302:isolation & purification / Q000494:pharmacology; D006801:Humans; D006973:Hypertension / Q000150:complications / Q000378:metabolism*; D007676:Kidney Failure, Chronic / Q000209:etiology / Q000378:metabolism; D010042:Ouabain / Q000494:pharmacology; D012503:Saponins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors
D002298:Cardenolides; D004791:Enzyme Inhibitors; D012503:Saponins; C034504:digoxin-like factors; D010042:Ouabain; D004077:Digoxin; D000254:Sodium-Potassium-Exchanging ATPase