Magnesium sulfate potentiates effect of DigiFab on marinobufagenin-induced Na/K-ATPase inhibition.

BACKGROUND: Immunoneutralization of elevated circulating levels of endogenous digitalis-like Na/K-ATPase inhibitors (i.e. cardiotonic steroids (CTS)) represents a novel approach in the treatment of preeclampsia (PE). Recently we demonstrated that DigiFab (Fab fragments of affinity-purified ovine digoxin antibody) restores PE-induced inhibition of Na/K-ATPase in erythrocytes ex vivo. Previously magnesium ions were shown to antagonize digitalis-induced toxicity, which is mediated by Na/K-ATPase inhibition. We hypothesized that magnesium sulfate would potentiate the effect of DigiFab in the reversal of CTS-induced Na/K-ATPase inhibition. METHODS: To test this hypothesis, we studied the ex vivo effect of DigiFab on Na/K-ATPase activity in erythrocytes from patients with PE in the absence and in the presence of 3 mmol/L magnesium sulfate. RESULTS: Compared with 11 normotensive pregnant subjects (29 ± 1 years; gestational age = 39.0 ± 0.2 weeks; blood pressure = 111 ± 2/73 ± 2 mm Hg), the 12 patients with PE (30 ± 1 years; gestational age = 37.9 ± 0.3 weeks; blood pressure = 159 ± 5/99 ± 3 mm Hg) had plasma levels of marino-bufagenin increased 3-fold (1.38 ± 0.40 vs. 0.38 ± 0.10 nmol/L; P < 0.01) and activity of Na/K-ATPase in erythrocytes was inhibited (1.16 ± 0.11 vs. 2.80 ± 0.20 µmol Pi/ml/h; P < 0.01). Ex vivo, DigiFab (1 µg/ml) restored erythrocyte Na/K-ATPase activity (1.72 ± 0.13 µmol Pi/ml/h; P < 0.01), and 3 mmol magnesium sulfate potentiated the effect of DigiFab (2.30 ± 0.20 µmol Pi/ml/h; P < 0.01). CONCLUSIONS: Magnesium is capable of increasing the efficacy of immunoneutralization of marinobufagenin-induced Na/K-ATPase inhibition.

DigiFab interacts with endogenous cardiotonic steroids and reverses preeclampsia-induced Na/K-ATPase inhibition.

Elevated levels of endogenous Na/K-ATPase (NKA) inhibitors, cardiotonic steroids (CTSs) including marinobufagenin (MBG), contribute to pathogenesis of preeclampsia (PE) and represent a target for immunoneutralization by Digibind (Ovine Digoxin Immune Antibody, Glaxo-Smith Kline). Because Digibind is no longer commercially available, we studied whether DigiFab (BTG International Ltd, UK) can substitute Digibind for immunoneutralization of CTS in patients with PE. We compared DigiFab, Digibind, and anti-MBG monoclonal antibody (mAb) with respect to their ability to interact with CTS in PE plasma and to restore NKA activity in erythrocytes from patients with PE. Using immunoassays based on DigiFab, Digibind, and anti-MBG mAb, we studied the elution profile of CTS following high-performance liquid chromatography (HPLC) fractionation of PE plasma. Totally, 7 patients with mild PE (28 ± 2 years; gestational age, 39 ± 0.5 weeks; blood pressure 156 ± 5/94 ± 2 mm Hg) and 6 normotensive pregnant participants (28 ± 1 years; gestational age, 39 ± 0.4 weeks; blood pressure 111 ± 2/73 ± 2 mm Hg) were enrolled. Preeclampsia was associated with a substantial inhibition of erythrocyte NKA (1.47 ± 0.17 vs 2.65 ± 0.16 µmol Pi/mL per h in control group, P < .001). Ex vivo, at 10 µg/mL concentration, which is consistent with the clinical dosing of Digibind administered previously in PE, DigiFab and Digibind as well as anti-MBG mAb (0.5 µg/mL) restored erythrocyte NKA activity. Following HPLC fractionation of pooled PE and control plasma, PE-associated increase in CTS material was detected by Digibind (176 vs 75 pmoles), DigiFab (221 vs 70 pmoles), and anti-MBG mAb (1056 vs 421 pmoles). Therefore, because DigiFab interacts with CTS from PE plasma and reverses PE-induced NKA inhibition, it can substitute Digibind for immunoneutralization of CTS in patients with PE.