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UBC Theses and Dissertations

Digoxin-like immunoreactive substances in the neonate Matthewson, Beryl Ellen

Abstract

Digoxin, a steroidal glycoside that inhibits Na⁺/K⁺-ATPase, is the most commonly prescribed cardiac medication in North America. Blood levels of this drug are routinely measured to reduce the risks of toxicity. Reports questioning the specificity of antisera used in radioimmunoassays for serum digoxin measurements began to appear after 1975¹ when plasma from patients with renal failure, not on glycoside therapy, showed false-positive digoxin levels. Since then, digoxin-like immunoreactive substances (DLIS) have been found in sera from patients with hepatic failure, hypertension, pre-eclampsia, in amniotic fluid and cord blood. Some of the highest values for DLIS have been detected in premature infants, where levels have often exceeded the therapeutic range (0.2-2.0 µg/L) for digoxin. Cord blood has been identified as a rich source of DLIS. Dahl et al² were the first to suggest that a circulating saluretic substance "endoxin", may cause hypertension in salt sensitive rats. Gruber et al³ reported on the existence of digoxin-like factor(s) in the plasma of volume-expanded dogs. Plasma from these dogs inhibited Na⁺/K⁺ATPase activity. A number of other studies have supported the concept that such digoxin-like factors may be of etiological significance in hypertension⁴. In view of these observations, a study was undertaken to isolate and fractionate DLIS from mixed cord blood and determine whether or not any of this digoxin-like material possessed Na⁺/K⁺-ATPase inhibitory properties. Cord blood collected in the Grace Hospital Maternity Unit (Vancouver, BC), was pooled and DLIS extracted using C₁₈,R-Sep Paks. Extracts were resolved by high performance liquid chromatography (HPLC) into several fractions containing digoxin equivalent immunoactivity as measured by radioimmunoassay (RIA). A number of steroids and bile acids (dehydroepi-androsterone-sulfate, cortisone, Cortisol, deoxycortisone, ∆⁴androstene-dione, progesterone and glycochenodeoxycholic acid) cross-reacted with digoxin antisera and had HPLC retention times similar to DLIS-containing fractions. The ability of HPLC generated DLIS positive cord blood fractions to inhibit Na⁺/K⁺-ATPase activity was determined in three different assay systems; red cell ⁸⁶Rb uptake canine kidney-Na⁺/K⁺-ATPase and red cell membrane-Na⁺/K⁺-ATPase. At least six fractions contained DLIS and inhibited Na⁺/K⁺-ATPase activity. Inhibition varied with the assay system used but none of the fractions inhibited ⁸⁶Rb uptake by erythocytes. One fraction (which eluted at 29 minutes) contained progesterone; 72% of the inhibitory activity present in this fraction was attributable to this steroid. Another inhibitory fraction co-eluted with dehydroepiandrosterone-sulfate (DHEAS-S). The only fractions found to inhibit both the red cell membrane and canine kidney Na⁺ /K⁺-ATPase enzymes eluted at 7 and 29 minutes. In summary, a number of digoxin-like immunoreactive substances were isolated from cord blood by HPLC fractionation and found to inhibit Na⁺/K⁺-ATPase activity. Inhibition varied with the assay system used. There was no apparent correlation between inhibition and digoxin immunoreactivity. Very large quantities (500 mL) of cord blood were extracted to demonstrate these properties. It remains to be determined whether or not DLIS isolated during the perinatal period is of physiological significance.

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