Mechanism of Action of Divicine in a Cell-free System and in Glucose-6-phosphate Dehydrogenase-deficient Red Cells

Abstract

Favism is an acute hemolysis occurring in glucose-6-phosphate dehydrogenase (G6PD)-deficient (Mediterranean variant) individuals after intake of fava beans. Divicine (D), 2,6-diamino-4,5-dihydroxypyrimidine, is present in high amounts in the beans, and is suspected to play a role in hemolysis. Its mechanism of action was studied in a cell-free system and in G6PD (Mediterranean variant)-deficient red cells (RBC). Upon hydrolysis of the inactive β-glucoside vicine, reduced divicine is formed. Oxygen acts as a one- or two-electron acceptor; superoxide anion and hydrogen peroxide are formed, respectivley, together with the semiquinoid free-radical form of D. This free radical gives an electron spin resonance (ESR) signal, which is similar to that of the alloxan free radical. Added reduced glutathione (GSH) is rapidly oxidized with a stoichiometry of one to one, and the ESR signal is abolished. Additional GSH is oxidized by hydrogen peroxide and by a slow redox cycle which continuously regenerates oxidized D. The fast-direct and the slow-indirect oxidation result in nonstoichiometric oxidation of GSH. D added to G6PD-deficient RBC rapidly oxidizes GSH with an end point kinetics and a stoichiometry of one to one. Hydrogen peroxide and superoxide anion are scavenged in the RBC and no redox cycling is taking place. No GSH is regenerated even after long incubation periods. After the primary event, i.e., oxidation of GSH and —SH groups, a number of metabolic, rheologic, and membrane modifications, together with increased erythrophagocytosis take place in G6PD-deficient, D-treated RBC only. There is almost complete correspondence between D effects and RBC changes during the favic crisis. Among these effects, increased erythrophagocylosis, membrane cross-bonding, and formation of Heinz bodies seem to be causally related with the hemolytic process.