pyrimidinones and Glucosephosphate-Dehydrogenase-Deficiency

Topics: Erythrocytes; Favism; Free Radicals; Glucosephosphate Dehydrogenase Deficiency; Glucosides; Humans; Pyrimidinones; Vicia faba

Topics: Adult; Child; Child, Preschool; Erythrocytes; Female; Glucosephosphate Dehydrogenase Deficiency; Glucosides; Hemolysis; Humans; Male; Pyrimidinones; Seeds; Vicia faba

The balanced polymorphism of glucose-6-phosphate dehydrogenase deficiency (G6PD-) is believed to have evolved through the selective pressure of malarial combined with consumption of fava beans. The implicated fava bean constituents are the hydroxypyrimidine glucosides vicine and convicine, which upon hydrolysis of their beta-O-glucosidic bond, became protein pro-oxidants. In this work we show that the glucosides inhibit the growth of Plasmodium falciparum, increase the hexose-monophosphate shunt activity and the phagocytosis of malaria-infected erythrocytes. These activities are exacerbated in the presence of beta-glucosidase, implicating their pro-oxidant aglycones in the toxic effect, and are more pronounced in infected G6PD- erythrocytes. These results suggest that G6PD- infected erythrocytes are more susceptible to phagocytic cells, and that fava bean pro-oxidants are more efficiently suppressing parasite propagation in G6PD- erythrocytes, either by directly affecting parasite growth, or by means of enhanced phagocytic elimination of infected cells. The present findings could account for the relative resistance of G6PD- bearers to falciparum malaria, and establish a link between dietary habits and malaria in the selection of the G6PD- genotype.

Topics: Animals; Erythrocytes; Fabaceae; Female; Glucosephosphate Dehydrogenase; Glucosephosphate Dehydrogenase Deficiency; Glucosides; Humans; Hydrogen-Ion Concentration; Hydrolysis; Malaria, Falciparum; Male; Pentose Phosphate Pathway; Phagocytosis; Plants, Medicinal; Plasmodium falciparum; Pyrimidinones; Uridine

In the present study we have assayed the effect of divicine in G6PD-deficient red blood cells in the presence of deferoxamine (iron-chelating drug) and NaN3 (inhibitor of catalase). The effect of divicine has been compared to oxidative stress by H2O2; haemolysis is regarded as an index of cellular toxicity. In addition, we have tested antioxidant enzymatic systems. No significant change in antioxidant enzymatic systems was found in RBCs from subjects with G6PD deficiency when compared to the control group, either in oxidative haemolysis by divicine or by H2O2; a significant decrease in oxidative haemolysis by H2O2 was observed in the presence of deferoxamine, whereas no change was found in oxidative haemolysis by divicine. The replacement of incubation medium by homologous plasma or the supplementation with bovine serum albumin resulted in a marked decrease of percentage of haemolysis by divicine.

Topics: Adult; Catalase; Deferoxamine; Erythrocytes; Glucosephosphate Dehydrogenase Deficiency; Glutathione Peroxidase; Glutathione Reductase; Hemolysis; Humans; Hydrogen Peroxide; In Vitro Techniques; Male; Oxidation-Reduction; Pyrimidinones

The effects of primaquine (PQ), its enantiomers [(+)PQ,(-)PQ] and hydroxy metabolites [5-hydroxyprimaquine (5HPQ) and 6-desmethyl-5-hydroxyprimaquine (6D5HPQ)] on cell membranes of glucose-6-phosphate dehydrogenase (G-6-PD) deficient red cells were studied in vitro. There was no significant effect of PQ on the malonyldialdehyde (MDA) content of normal and heterozygous red cells, but it caused a significant increase in MDA in G-6-PD deficient red cells (P less than 0.05). There was no noticeable difference between the effects of the two enantiomers on this variable (P greater than 0.05). Compared to PQ, the hydroxy metabolites produced a significantly greater increase in MDA in all the groups studied (P less than 0.001). Of the two hydroxy metabolites, 6D5HPQ was more toxic than 5HPQ. Staining with MC540 showed that exposure to PQ, its enantiomers and two putative metabolites produced significant fluorescence, indicating that the drug produces marked alterations in membrane fluidity. Although the fluorescence was seen both in normal and heterozygous cells, the effect was marked in hemizygous deficient red cells (P less than 0.001). Scanning electron microscopic (SEM) studies revealed that PQ enantiomers had a stomatocytic effect on red cells of normal, heterozygous and hemizygous G-6-PD deficient red cells, whereas the putative metabolites had an echinocytic effect. The effects were most pronounced in G-6-PD deficient red cells.

Topics: Erythrocyte Membrane; Erythrocytes; Female; Glucosephosphate Dehydrogenase Deficiency; Humans; Lipid Peroxidation; Membrane Fluidity; Microscopy, Electron, Scanning; Primaquine; Pyrimidinones; Stereoisomerism

Damaged RBC drawn from favic patients during acute hemolysis showed marked alterations in their two major proteolytic systems. Cytosolic procalpain (i.e., the proenzyme species of Ca2+-activated neutral proteinase, or calpain) had considerably lower activity than in matched RBC from asymptomatic G6PD-deficient subjects. The total RBC activity of the three acid endopeptidases that are normally membrane-bound was not reduced in favism, but its subcellular distribution was mostly cytosolic, suggesting quantitative release from membranes. Changes in procalpain activity are the result of both autoxidation of divicine and of the intracellular elevation of Ca2+ that is found in favism. Changes in acid endopeptidase activity are the consequence of perturbed Ca2+ homeostasis. Overall, the picture shows a marked impairment of the RBC proteolytic machinery that in turn may worsen cellular damage.

Topics: Calcium; Calpain; Endopeptidases; Enzyme Precursors; Erythrocytes; Favism; Glucosephosphate Dehydrogenase Deficiency; Hemolysis; Humans; Male; Peptide Hydrolases; Pyrimidinones

Topics: Ascorbic Acid; Carbon Monoxide; Erythrocytes; Glucosephosphate Dehydrogenase Deficiency; Hemolysis; Humans; Oxidation-Reduction; Peroxides; Plasma; Pyrimidinones; tert-Butylhydroperoxide

The influence of O2-, H2O2 and metal ions on the auto-oxidation of divicine, a pyrimidine aglycone, was studied. In air at pH 7.4, the hydroquinonic form oxidized within a few minutes. Superoxide dismutase (SOD) markedly decreased the initial rate, giving a lag phase followed by rapid oxidation. Although catalase or diethylenetriamine-penta-acetic acid (DTPA) alone had little effect, each in the presence of SOD further slowed the initial rate and increased the lag. H2O2 decreased the lag time, as did Cu2+, Fe2+ or haemoglobin. GSH substantially increased the lag phase, but it eventually reacted with the divicine to form a 305 nm-absorbing adduct. These results indicate that an O2(-)-dependent mechanism of divicine auto-oxidation normally predominates. Auto-oxidation can also occur by a mechanism involving H2O2 and transition metal ions or haemoglobin, and if both these reactions are prevented by SOD and DTPA or catalase, a third mechanism, requiring build-up of an autocatalytic intermediate, becomes operative. Oxyhaemoglobin did not react directly with divicine, but reacted with the H2O2 produced by divicine auto-oxidation to give mainly an oxidized derivative presumed to be ferrylhaemoglobin. Divicine was shown to reduce ferylhaemoglobin to methaemoglobin, and this reaction was probably responsible for the acceleratory effect of haemoglobin on divicine oxidation. These results indicate that O2 rather than oxyhaemoglobin is likely to initiate divicine oxidation in the erythrocyte. Haemolytic crises, which are thought to result from this oxidation, occur only sporadically in glucose-6-phosphate dehydrogenase deficient individuals following ingestion of fava beans. A characteristic of the crises is acute depletion of erythrocyte GSH, and the vulnerability of these cells could relate to the ability of GSH, in combination with SOD, to protect against the autocatalytic mechanism of divicine auto-oxidation. Our demonstration of a variety of auto-oxidation pathways also suggests possible areas of individual variation.

Topics: Catalase; Erythrocytes; Glucosephosphate Dehydrogenase Deficiency; Glutathione; Hemoglobins; Humans; Hydrogen Peroxide; Metals; Methemoglobin; Oxidation-Reduction; Oxyhemoglobins; Pentetic Acid; Pyrimidinones; Superoxide Dismutase; Superoxides

Calpain, the micromolar Ca2+-requiring form of Ca2+-stimulated neutral proteinase purified from human red cells, is remarkably inactivated during autoxidation of divicine (2,6-diamino-4,5-dihydroxypyrimidine), an aglycone implicated in the pathogenesis of favism. Inactivation of purified calpain is produced, in decreasing order of efficiency, by transient, probably semiquinonic species arising from autoxidation of divicine, by the H2O2 that is formed upon autoxidation itself, and by quinonic divicine, respectively. Purified procalpain, the millimolar Ca2+-requiring form that can be converted to the fully active calpain form by a variety of mechanisms, is less susceptible than calpain itself to inactivation by the same by-products of divicine autoxidation. When intact red cells are exposed to autoxidizing divicine, procalpain undergoes a significant loss of activity. At 1 mM divicine, intracellular inactivation is observed with procalpain only, while the activity of a number of red cell enzymes is unaffected. Inactivation of procalpain is consistently greater in red cells from glucose-6-phosphate dehydrogenase-deficient subjects than in normal cells. Restoration of normal levels of glucose-6-phosphate dehydrogenase activity by means of entrapment of homogeneous human glucose-6-phosphate dehydrogenase in the deficient red cells results in normal stability of intracellular reduced glutathione; decreased susceptibility of procalpain to inactivation by autoxidizing divicine. These findings suggest that in the glucose-6-phosphate dehydrogenase-deficient red cells the procalpain-calpain system is a major target of divicine cytotoxicity.

Topics: Calpain; Enzyme Precursors; Erythrocytes; Glucosephosphate Dehydrogenase Deficiency; Glutathione; Humans; Oxidation-Reduction; Pyrimidinones

Red cells of G6PD (D-glucose-6-phosphate:NADP+ 1-oxidoreductase; G6PD) deficient (Mediterranean variant) subjects were studied during a fava bean haemolytic crisis. Two representative cases are described. In Case 1, haemolysis was still going on. In more than 50% of the red cells the Hb was confined to one part of the cell, leaving the other part as transparent as a Hb-free ghost. In this part the membranes appeared tightly bonded because swelling did not peel apart the bonded membrane areas. This feature is defined as membrane cross bonding (MCB). In Case 2, haemolysis had terminated and MCB-cells were less than 1%. MCB was reproduced in vitro by incubating G6PD-deficient whole blood with 1 mM divicine for up to 10 h. Subsequent shrinkage of red cells in hypertonic plasma (400 mOsm) resulted in the rapid formation of MCB. Membrane modifications by divicine, contained in fava beans, followed by osmotic shrinkage in the kidney and/or squeezing in the microcirculation are proposed as the cause of MCB during the favic crisis. MCB reduces the effective surface area of red cells. This is a plausible cause for sequestration by the reticulo-endothelial system. Intravascular haemolysis observed in favic crisis cannot be explained by mechanical forces, but it is possible that the effective surface area is reduced by MCB to such an extent that red cells lyse osmotically.

Topics: Child; Child, Preschool; Erythrocyte Membrane; Erythrocytes; Favism; Glucosephosphate Dehydrogenase Deficiency; Heinz Bodies; Hemolysis; Humans; Male; Pyrimidinones

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 beta-glucoside vicine, reduced divicine is formed. Oxygen acts as a one- or two-electron acceptor; superoxide anion and hydrogen peroxide are formed, respectively, 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Ascorbic Acid; Cell-Free System; Cytochrome c Group; Erythrocytes; Favism; Glucosephosphate Dehydrogenase Deficiency; Glutathione; Hemolysis; Humans; Hydrogen Peroxide; In Vitro Techniques; Kinetics; Male; Oxidation-Reduction; Pyrimidinones; Superoxides

Complete removal of leucocytes and platelets from erythrocytes and the development of a sensitized procedure for the assay of G6PD activity allowed the biochemical mechanisms of the Mediterranean variety of G6PD deficiency to be re-evaluated. Activity in the young erythrocytes from 9 G6PD-deficient subjects averaged 0.1% of the levels observed in the corresponding erythrocyte fraction from normal individuals: moreover, the decline of activity during aging of the G6PD-deficient erythrocytes was comparable with that observed for the normal enzyme. Mutant G6PD purified from granulocytes of a G6PD-deficient subject and entrapped within the corresponding erythrocytes was remarkably stable. Exposure of native erythrocytes to an oxidative stress (divicine plus ascorbate) resulted in a decrease of G6PD activity that was significantly more rapid and extensive in control than in G6PD-deficient cells. These results seem to exclude enhanced intracellular breakdown of the mutant protein within the circulating erythrocytes.

Topics: Ascorbic Acid; Erythrocyte Aging; Erythrocytes; Glucosephosphate Dehydrogenase; Glucosephosphate Dehydrogenase Deficiency; Hemolysis; Humans; Male; Mediterranean Islands; Mutation; Pyrimidinones

Intravenous injection of divicine into mice infected with Plasmodium vinckei rapidly killed the parasites and caused haemolysis. Degenerating parasites were observed frequently inside intact circulating erythrocytes, implying that parasite death was not a passive consequence of haemolysis. Both parasite death and haemolysis were prevented by the iron chelator desferrioxamine. In vitro, divicine caused the accumulation of malonyldialdehyde and the depletion of reduced glutathione in normal mouse erythrocytes. Desferrioxamine inhibited the former event, but not the latter. These observations support the hypothesis advanced by Huheey & Martin (Experientia, 31, 1145, 1975) to explain the patchy geographical distribution of glucose-6-phosphate dehydrogenase deficiency in historic malarial areas and also suggest that desferrioxamine, a drug already in clinical use, is a potential treatment for favism and other examples of oxidative haemolysis.

Topics: Animals; Deferoxamine; Erythrocytes; Favism; Female; Glucosephosphate Dehydrogenase Deficiency; Glutathione; Hemolysis; Malaria; Male; Malondialdehyde; Mice; Mice, Inbred CBA; Microscopy, Electron; Pyrimidinones

A combination of divicine (an aglycone from the fava bean beta-glucoside vicine) and ascorbate results in a marked production of ethylene from methional, as a probable indication of OH radical formation. Addition of iron-EDTA to this oxidising system enhances the ethylene production significantly. The enhancing effect of iron-EDTA is also observed when both normal and Glucose 6-phosphate dehydrogenase (G6PD)-deficient red cells are exposed to the divicine-ascorbate system. Moreover, iron-EDTA magnifies other consequences of oxidant damage afforded by divicine-ascorbate or by ascorbate alone on the target red cells, such as depletion of reduced glutathione, formation of methemoglobin, stimulation of hexose monophosphate shunt activity and lipid peroxidation. Although the biochemical changes induced by this oxidative system are not remarkably different in normal and in G6PD-deficient red cells, the extra-damaging effect of chelated iron might be important in the mechanism of hemolysis.

Topics: Ascorbic Acid; Catalase; Edetic Acid; Erythrocytes; Ferric Compounds; Free Radicals; Glucosephosphate Dehydrogenase Deficiency; Glutathione; Humans; Hydroxides; Hydroxyl Radical; Iron; Male; Oxygen; Pyrimidinones

Fava beans contain high amounts (up to 6.7 g/100 g dry weight) vicine and convicine. Their active aglycones divicine and isouramil have equivalent metabolic effects. They rapidly oxidize GSH to GSSG in normal and G6PD-deficient red cells. No regeneration of GSH occurs in deficient cells. The stoichiometry of the divicine oxidation of GSH is 1:1. Ascorbic acid is quickly oxidized by isouramil in both normal and deficient cells but regenerates only in normal cells. Isouramil oxidizes NADH at a much lesser extent than NADPH. Glycolysis is activated at the glyceraldehyde 3-phosphate dehydrogenase step. Divicine strongly stimulates hexone monophosphate shunt only in normal red cells. Divicine alone or associated with ascorbic acid has almost no effect in deficient red cells. Malonyl dialdehyde production is slight and virtually the same in normal and deficient cells treated with 5 mM isouramil. Large polypeptide aggregates are formed after 12 and 24 hours incubation with 1 mM divicine in deficient cells only. Divicine (0.25 mM) markedly decreases the filterability of deficient cells. The results are consistent with a causal role of divicine/isouramil in the genesis of the hemolytic crisis occurring in G6PD-deficient subjects after fava bean ingestion.

Topics: Ascorbic Acid; Barbiturates; Erythrocytes; Favism; Glucosephosphate Dehydrogenase Deficiency; Glucosides; Glutathione; Humans; Kinetics; Pyrimidinones; Reference Values; Species Specificity; Toxins, Biological; Uracil; Uridine