Application of supercritical fluid chromatography to characterize a labiledigitalis-like factor

A sodium pump inhibitor (digitalis-like factor), isolated from the peritone al dialysate of volume-expanded, hypertensive patients with kidney failure who were treated with this; dialysis modality, was further purified and cha racterized by means of supercritical fluid chromatography, a separation tec hnique whose application to very-low-concentration biomolecules is new. Pre vious studies suggested that after high-performance liquid chromatography ( HPLC) purification, this inhibitor was the only factor correlated with volu me status and blood pressure in these patients. When this same HPLC fractio n was furthered purified on 2-dimensional supercritical fluid chromatograph y, 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 wa s still correlated with the degree of volume expansion of donor patients (P = 0.01). After HPLC purification, this volume-sensitive inhibitor was chem ically labile. With further purification on supercritical fluid chromatogra phy, the active peak was still labile with comparable half-life. Supercriti cal fluid chromatography coupled with flame ionization detection provided a n estimate of the amount of the inhibitor present, Again using split specim ens, we determined that the labile digitalis-like factor was approximate to 30-fold more effective than ouabain in inhibiting renal [Na,K]ATPase activ ity and greater than or equal to 500 times more effective than ouabain in c ausing vascular smooth muscle contraction. The data suggest that we have pu rified to homogeneity a labile digitalis-like factor that is readily distin guished from ouabain or bufalin, based on chromatographic characteristics, chemical lability, and a much lower effective concentration for its biologi cal activity.