Rapid detection of convallatoxin using five digoxin immunoassays

Abstract

Context: Cardiac glycosides of plant origin are implicated in toxic ingestions that may result in hospitalization and are potentially lethal. The utility of commonly available digoxin serum assays for detecting foxglove and oleander ingestion has been demonstrated, but no studies have evaluated the structurally similar convallatoxin found in Convallaria majalis (lily of the valley) for rapid laboratory screening, nor has digoxin immune Fab been tested as an antidote for this ingestion.

Objective: We aimed to (1) evaluate multiple digoxin assays for cross-reactivity to convallatoxin, (2) identify whether convallatoxin could be detected in vivo at clinically significant doses, and (3) determine whether digoxin immune Fab could be an effective antidote to convallatoxin.

Materials and methods: Cross-reactivities of purified convallatoxin and oleandrin with five common digoxin immunoassays were determined. Serum from mice challenged with convallatoxin was tested for apparent digoxin levels. Binding of convallatoxin to digoxin immune Fab was determined in vitro.

Results: Both convallatoxin and oleandrin were detectable by a panel of commonly used digoxin immunoassays, but cross-reactivity was variable between individual assays. We observed measurable apparent digoxin levels in serum of convallatoxin intoxicated mice at sublethal doses. Convallatoxin demonstrated no binding by digoxin immune Fab.

Conclusion: Multiple digoxin immunoassays detect botanical cardiac glycosides including convallatoxin and thus may be useful for rapid determination of severe exposures, but neutralization of convallatoxin by digoxin immune Fab is unlikely to provide therapeutic benefit.

Keywords: Convallotoxin; Digoxin; Digoxin immunoassays; Lily of the valley; Oleandrin.

Figure 1. Structural similarity of botanic cardiac glycosides with digoxin

Common backbone highlighted with variable glycosides digitoxose (digoxin and digitoxin), oleandrose (oleandrin) and rhamnose (convallatoxin).

Figure 2. Cross-reactivity of digoxin immunoassays for purified convallatoxin

Digoxin-free serum was supplemented with varying concentrations of convallatoxin and apparent digoxin was determined using the indicated immunoassays. Means of two measurements are shown.

Figure 3. Cross-reactivity of digoxin immunoassays for purified oleandrin

Digoxin-free serum was supplemented with varying concentrations of oleandrin and apparent digoxin was determined using the indicated immunoassays. Means of two measurements are shown.

Figure 4. Detection of convallatoxin in the serum of intoxicated mice

Convallatoxin was administered by intraperitoneal injection at the LD50 for mice (10mg/kg; n=3) or 10% of the LD50 (1mg/kg; n=5). One mouse received a sham saline injection. Apparent digoxin was measured in the serum after 10 minutes using the Abbott Architect assay. Convallatoxin levels were calculated based on a standard curve. N.D. not detected.

Figure 5. Digoxin immune FAB binds digoxin

Serum supplemented with digoxin was incubated with digoxin immune Fab as indicated. Digoxin was measured in an unmanipulated sample (total). Protein bound drug was removed by ultrafiltration and remaining unbound (free) digoxin was measured. Note that effective binding of digoxin by DigiFab necessitated a broken y-axis to accommodate the range of measured values. Means of two measurements are shown.

Figure 6. Digoxin immune FAB does not bind convallatoxin

Serum supplemented with convallatoxin was incubated with digoxin immune Fab as indicated. Apparent digoxin was measured in an unmanipulated sample (total). Protein bound toxin was removed by ultrafiltration and apparent digoxin was measured as a surrogate for remaining unbound (free) convallatoxin. Means of two measurements are shown.