Digifab and immunoneutralization of cardiotonic steroids in preeclampsia

Abstract

Background. Elevated levels of Na + /K + -ATPase(NKA)-inhibitory cardiotonic steroids (CTS) including marinobufagenin (MBG) contribute to pathogenesis of preeclampsia (PE) via induction of vasoconstriction and of vascular stiffness. In PE, Digibind (affi nity-purifi ed digoxin antibody) exhibits benefi cial effects primarily due to immunoneutralization of CTS including MBG. Because Digibind is not available and the only other «clinically-usable» digoxin antibody is DigiFab (BTG International Ltd), we compared DigiFab and Digibind with respect to their ability to interact with CTS in PE plasma and to ex-vivo reverse PE-induced NKA inhibition. Objective. We compared effects of Digibind and Digifab on erythrocyte NKA from patients with PE and normotensive pregnant subjects. Using competitive fl uoroimmunoassays based on Eu-labeled DigiFab and Digibind we compared profi le of elution of digoxin-immunoreactive material following fractionation of PE plasma on reverse-phase HPLC column. Design and methods. 7 patients with PE (28 ± 2 years; gestational age, 39,0 ± 0,5 weeks, urinary protein excretion, 2,12 ± 0,46 g/24 hours, blood pressure 157 ± 5/94 ± 2 mm Hg) and 6 normotensive pregnant subjects (26 ± 1 years; gestational age, 37 ± 1 weeks, blood pressure 112 ± 2/75 ± 3 mm Hg) were studied. Results. PE was associated with substantial inhibition of erythrocyte NKA (1,47 ± 0,17 vs. 2,65 ± 0,16 umol Pi/mL/hr in control group, p < 0,001). At concentration 10 ug/mL (which mimics dose of Digibind administered in PE) both Digibind and DigiFab partially, but signifi cantly restored NKA activity (2,1 ± 0,2 and 2,05 ± 0,3 umol Pi/mL/hr, respectively). In competitive immunoassay Digibind and Digifab exhibited comparable cross-reactivity with MBG and endogenous ouabain. Following HPLC fractionation of plasma, both Digibind and Digifab detected PE-associated increase in CTS material in fractions 16–18; 176 vs. 75 pmoles for Digibind, control and preeclampsia, respectively; 221 vs. 70 pmoles for Digifab, control and PE, respectively. 4G4 anti-MBG monoclonal antibody detected preeclampsia-induced CTS increase (1056 vs. 421 pmoles) mainly in fraction 16. Conclusions. We conclude that: (i) Digifab and Digibind exhibit comparable cross-immunoreactivity with CTS; (ii) in patients with PE plasma levels of digoxin-like immunoreactivity are elevated proportionally when measured by both Digifab and Digibind and both antibodies reverse preeclampsia-induced NKA inhibition, and (iii) following HPLC fractionation of PE plasma, Digifab and Digibind interact with endogenous bufadienolides including MBG, rather than endogenous ouabain.