Favic Patients Research Articles

Do favic patients resume fava beans ingestion later in their life, a study for this, and a new hypothesis for favism etiology

The etiology of favism remains unclear and the fate of favic patients has not previously been studied. Therefore, individuals who had experienced an episode of favism were studied regarding subsequent fava bean ingestion, including the reason for fava bean ingestion after the initial favic attack and any adverse reactions. In addition, a new hypothesis for the etiology of favism is proposed. From June 2005 to June 2012, a total of 38 patients with a history of favism were included in this study. Circumstances regarding the initial favic attack were obtained from medical records and patient interviews, and subsequent fava bean ingestion and recurrence of symptoms were investigated. Three of the 38 patients (7.9%) were female, and 35 (92.1%) were male. The mean age was 27.9 years (14-63 years). The first attack of favism occurred before 10 years of age for 31 patients (81.6%) and in the springtime for 35 patients (92.1%). Thirty-three patients (86.7%) regularly ate fava beans before the attack, and 35 (92.1%) resumed eating fava beans within 1-17 years after the attack without symptoms. Two patients (5.2%) experienced a single recurrence of symptoms. No evidence of hemolysis was found in the four patients checked after fava bean re-ingestion. Patients resumed eating fava bean for various reasons, and the recurrence of symptoms was uncommon. An infectious agent such as a virus may play a role in the development of favism.

Life and Death of Glucose-6-Phosphate Dehydrogenase (G6PD) Deficient Erythrocytes – Role of Redox Stress and Band 3 Modifications

G6PD catalyzes the first, pace-making reaction of pentosephosphate cycle (PPC) which produces NADPH. NADPH maintains glutathione and thiol groups of proteins and enzymes in the reduced state which is essential for protection against oxidative stress. Individuals affected by G6PD deficiency are unable to regenerate reduced glutathione (GSH) and are undefended against oxidative stress. G6PD deficiency accelerates normal senescence and enhances the precocious removal of chronologically young, yet biologically old cells. The term hemolytic anemia is misleading because RBCs do not lyse but are removed by phagocytosis. Acute hemolysis by fava bean ingestion in G6PD deficient individuals (favism) is described being the best-studied natural model of oxidant damage. It bears strong analogies to hemolysis by oxidant drugs or chemicals. Membrane alterations observed in vivo during favism are superimposable to changes in senescent RBCs. In summary, RBC membranes isolated from favic patients contained elevated amounts of complexes between IgG and the complement fragment C3b/C3c and were prone to vesiculation. Anti-band 3 IgG reacted to aggregated band 3-complement complexes. In favism extensive clustering of band 3 and membrane deposition of hemichromes were also observed. Severely damaged RBCs isolated from early crises had extensive membrane cross-bonding and very low GSH levels and were phagocytosed 10-fold more intensely compared to normal RBCs.

The high incidence of acute hemolysis due to favism in Ahvaz, Iran—clinical features and laboratory findings

Objective To collect comprehensive information about the features of favic patients in Ahvaz (Capital of Khouzestan, Iran) and analyze the extent of the differences with their corresponding in other regions. Methods A total of 103 patients with acute hemolysis admitted to pediatric division of Abouzar Hospital located in the city of Ahvaz, Iran during 21st of June 2008 to 20 th of June 2009 were analyzed retrospectively. Results 95.14% of the patients had favism while 4.86% of them underwent hemolysis due to other reasons. These patients were male (68.93%) and female children (31.06%) admitted mostly during the spring season. The three main symptoms were urine discoloration, jaundice and vomiting. At the admission time, the main hematologic findings were as follows: G6PD sufficient status (45.63%), G6PD deficient status (54.36%) and hemoglobin concentration: 2.5–11.8 (mean±SD: 6.45 ±2.12) g/dL. Conclusions In conclusion, Ahvaz was determined as a black zone for favism in which the disease can be considered a life threatening health problem. Moreover, slight differences were observed in the three main symptoms compared with favic patients in other regions.

Capillary electrohoretic determination of purine nucleoside phosphorylase activity in human erythrocytes

Favism is an acute hemolytic anemia triggered by ingestion of fave beans in genetically susceptible subjects with severe deficiency of glucose-6-phosphate dehydrogenase (G6PD) activity. Erythrocytes from 10 favic patients had constantly and markedly increased calcium levels, as compared with values detected in 4 asymptomatic G6PD-deficient controls. Correspondingly, the calcium permeability of erythrocytes, estimated as the fraction of intracellular calcium exchangeable with externally added 45Ca2+, was invariably enhanced in favism and returned to normal patterns after several months from the acute hemolytic crisis. In favic patients, the levels of erythrocyte calcium ATPase activities showed wide variability, ranging from 2.0–12.9 μmol Pi/ml RBC/h, while control values in asymptomatic G6PD-deficient subjects were 10.62 ± 2.03 μmol Pi/ml RBC/h. Analysis of the calcium ATPase in situ in erythrocyte membranes from favic patients showed the same molecular mass of 134 kD as observed in the control subjects. Exposure of G6PD-deficient erythrocytes in vitro to autoxidizing divicine, a pyrimidine aglycone strongly implicated in the pathogenesis of favism which leads to late accumulation of intracellular calcium, caused: (i) a marked inactivation of calcium ATPase, without changes in the molecular mass of 134 kD; and (ii) the concomitant loss of spectrin, band 3 and band 4.1, all known substrates of the calcium activated procalpain-calpain proteolytic system. Thus, the increased intraerythrocytic calcium apparently results in the degradation of calcium ATPase observed in some favic patients. It is proposed that both enhanced calcium permeability and a calcium-stimulated degradation of the calcium pump are the mechanisms responsible for the perturbation of erythrocyte calcium homeostasis in favism.

Mechanisms of perturbation of erythrocyte calcium homeostasis in favism

Favism is an acute hemolytic anemia triggered by ingestion of fave beans in genetically susceptible subjects with severe deficiency of glucose-6-phosphate dehydrogenase (G6PD) activity. Erythrocytes from 10 favic patients had constantly and markedly increased calcium levels, as compared with values detected in 4 asymptomatic G6PD-deficient controls. Correspondingly, the calcium permeability of erythrocytes, estimated as the fraction of intracellular calcium exchangeable with externally added 45Ca 2+, was invariably enhanced in favism and returned to normal patterns after several months from the acute hemolytic crisis. In favic patients, the levels of erythrocyte calcium ATPase activities showed wide variability, ranging from 2.0–12.9 μmol P i/ml RBC/h, while control values in asymptomatic G6PD-deficient subjects were 10.62 ± 2.03 μmol P i/ml RBC/h. Analysis of the calcium ATPase in situ in erythrocyte membranes from favic patients showed the same molecular mass of 134 kD as observed in the control subjects. Exposure of G6PD-deficient erythrocytes in vitro to autoxidizing divicine, a pyrimidine aglycone strongly implicated in the pathogenesis of favism which leads to late accumulation of intracellular calcium, caused: (i) a marked inactivation of calcium ATPase, without changes in the molecular mass of 134 kD; and (ii) the concomitant loss of spectrin, band 3 and band 4.1, all known substrates of the calcium activated procalpain-calpain proteolytic system. Thus, the increased intraerythrocytic calcium apparently results in the degradation of calcium ATPase observed in some favic patients. It is proposed that both enhanced calcium permeability and a calcium-stimulated degradation of the calcium pump are the mechanisms responsible for the perturbation of erythrocyte calcium homeostasis in favism.

Favism: impairment of proteolytic systems in red blood cells

Red blood cells (RBC) from favic patients are characterized by (a) severe oxidative damage (contributed by autoxidation of divicine and isouramil, two pyrimidine aglycones present in fava beans) and (b) greatly increased calcium levels. In vitro, both autoxidation of divicine and calcium loading produced marked alterations of proteolytic systems in intact RBC. Specifically, autoxidizing divicine inactivated procalpain, the proenzyme species of calcium-activated cytosolic neutral proteinase, or calpain. Inactivation was much greater with glucose-6-phosphate dehydrogenase (G6PD)-deficient RBC than with normal RBC. On the other hand, loading of normal and G6PD-deficient RBC with calcium resulted in conversion of procalpain to calpain and eventual autoproteolytic inactivation of calpain itself, and extensive release of acid endopeptidase activity from the membranes into the cytosol. Damaged RBC from favic patients had significantly lowered procalpain activity and an abnormal subcellular distribution of acid proteinase activity that was found mostly in the cytosol. When purified calpain was incubated with membranes from acetylphenylhydrazine (APH)-treated RBC, significant proteolysis was observed affecting mostly band 3 and hemoglobin chains, ie, the two proteins involved in the onset of aggregation of Heinz bodies. Moreover, exposure of intact RBC to 20 mmol/L APH induced depletion of procalpain activity for which the time course was inversely related to formation of Heinz bodies. These findings support the role of procalpain in protecting G6PD-deficient RBC from oxidant-induced Heinz body formation and imply that exhaustion of the procalpain-calpain system is an important step in the mechanisms of RBC damage and destruction in favism.

Favism: Impairment of Proteolytic Systems In Red Blood Cells

Abstract Red blood cells (RBC) from favic patients are characterized by (a) severe oxidative damage (contributed by autoxidation of divicine and isouramil, two pyrimidine aglycones present in fava beans) and (b) greatly increased calcium levels. In vitro, both autoxidation of divicine and calcium loading produced marked alterations of proteolytic systems in intact RBC. Specifically, autoxidizing divicine inactivated procalpain, the proenzyme species of calcium-activated cytosolic neutral proteinase, or calpain. Inactivation was much greater with glucose-6-phosphate dehydrogenase (G6PD)-deficient RBC than with normal RBC. On the other hand, loading of normal and G6PD-deficient RBC with calcium resulted in conversion of procalpain to calpain and eventual autoproteolytic inactivation of calpain itself, and extensive release of acid endopeptidase activity from the membranes into the cytosol. Damaged RBC from favic patients had significantly lowered procalpain activity and an abnormal subcellular distribution of acid proteinase activity that was found mostly in the cytosol. When purified calpain was incubated with membranes from acetylphenylhydrazine (APH)-treated RBC, significant proteolysis was observed affecting mostly band 3 and hemoglobin chains, ie, the two proteins involved in the onset of aggregation of Heinz bodies. Moreover, exposure of intact RBC to 20 mmol/L APH induced depletion of procalpain activity for which the time course was inversely related to formation of Heinz bodies. These findings support the role of procalpain in protecting G6PD-deficient RBC from oxidant-induced Heinz body formation and imply that exhaustion of the procalpain-calpain system is an important step in the mechanisms of RBC damage and destruction in favism.

Hexose monophosphate shunt-stimulated reduction of methemoglobin by divicine

Reduced divicine (2,6-diamino-4,5-dihydroxypyrimidine), an aglycone implicated in the pathogenesis of favism, reduces methemoglobin efficiently in intact erythrocytes and in hemolysates. Oxidized divicine produces the same effect when glucose or an NADPH-generating system is added to intact erythrocytes or to hemolysates. Although NADPH, NADH, and GSH have no direct methemoglobin-reducing activity in vitro, they convert oxidized divicine to the reduced hydroquinone species, which is responsible for the electron transfer to methemoglobin. Reduction of methemoglobin is optimally observed under nitrogen since, in the presence of oxygen, reduced divicine undergoes autoxidation. Several lines of evidence rule out the reduction of methemoglobin by divicine through an enzyme-catalyzed process, although it is certainly sustained by the hexose monophosphate shunt activity of erythrocytes through the generation of both NADPH and GSH. Thus, the strong enhancing effect that glucose produces on the divicine-dependent methemoglobin reduction within intact normal erythrocytes is completely absent in erythrocytes from glucose-6-phosphate dehydrogenase-deficient subjects. This distinctive behavior might account for the enhanced methemoglobin levels that are found both in vitro in glucose-6-phosphate dehydrogenase-deficient erythrocytes exposed to divicine and in vivo as a typical feature of the acute hemolytic crisis of favic patients.

Favism: disordered erythrocyte calcium homeostasis

The biochemical events that take place during acute hemolysis of G6PD-deficient subjects in favism are far from being elucidated. Evidence is here reported for a constantly and heavily disordered calcium homeostasis in the erythrocytes from seven favic patients. The abnormality, ie, a significantly impaired calcium ATPase activity and a parallel marked increase of intracellular calcium levels, was characteristic of the acute hemolytic crisis although unrelated to the attendant reticulocytosis. Concomitantly, a remarkable decrease of intracellular potassium was also observed. The mean +/- SD Ca2+-ATPase activity in the favic patients was 20.8 +/- 7.8 mumol Pi/g Hb/h compared with 37.2 +/- 8.5 in the matched controls represented by 12 healthy G6PD-deficient subjects (P less than .001). The mean +/- SD intraerythrocytic calcium content was 288 +/- 158 mumol/L of erythrocytes in the favic patients as compared with 22.0 +/- 8.2 in the G6PD-deficient controls (P less than .001). The intraerythrocytic potassium content was 76.6 +/- 19.3 mmol/L of erythrocytes in the favic patients and 106.6 +/- 8.2 in the G6PD-deficient controls (P less than .001). In vitro incubation of normal and G6PD-deficient erythrocytes with divicine, a pyrimidine aglycone present in fava beans and strongly implicated in the pathogenesis of favism, reproduces most of these events, including drop of calcium ATPase, increased intracellular calcium, and leakage of erythrocyte potassium.

Favism: Disordered Erythrocyte Calcium Homeostasis

Abstract The biochemical events that take place during acute hemolysis of G6PD- deficient subjects in favism are far from being elucidated. Evidence is here reported for a constantly and heavily disordered calcium homeostasis in the erythrocytes from seven favic patients. The abnormality, ie, a significantly impaired calcium ATPase activity and a parallel marked increase of intracellular calcium levels, was characteristic of the acute hemolytic crisis although unrelated to the attendant reticulocytosis. Concomitantly, a remarkable decrease of intracellular potassium was also observed. The mean +/- SD Ca2+-ATPase activity in the favic patients was 20.8 +/- 7.8 mumol Pi/g Hb/h compared with 37.2 +/- 8.5 in the matched controls represented by 12 healthy G6PD-deficient subjects (P less than .001). The mean +/- SD intraerythrocytic calcium content was 288 +/- 158 mumol/L of erythrocytes in the favic patients as compared with 22.0 +/- 8.2 in the G6PD-deficient controls (P less than .001). The intraerythrocytic potassium content was 76.6 +/- 19.3 mmol/L of erythrocytes in the favic patients and 106.6 +/- 8.2 in the G6PD-deficient controls (P less than .001). In vitro incubation of normal and G6PD-deficient erythrocytes with divicine, a pyrimidine aglycone present in fava beans and strongly implicated in the pathogenesis of favism, reproduces most of these events, including drop of calcium ATPase, increased intracellular calcium, and leakage of erythrocyte potassium.

Impairment of the calcium pump of human erythrocytes by divicine

Divicine (2,6-diamino-4,5-dihydroxypyrimidine), an aglycone implicated in the pathogenesis of favism, produces a remarkable and consistent inactivation of the Ca 2+-ATPase activity of the erythrocyte calcium pump. The patterns of inactivation are similar in normal and glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes. Inactivation of Ca 2+-ATPase is apparently unrelated to the cellular GSH system, to the proteolytic machinery of mature erythrocytes, and to calmodulin, and also occurs in hemoglobin-free, unsealed erythrocyte membranes at 50–100 μ m concentrations of divicine. Analysis of erythrocytes that have escaped destruction during the acute hemolytic crisis of a number of favic patients revealed a dramatic elevation of erythrocyte calcium and a significant decrease of Ca 2+-ATPase activity. These results support the view that divicine plays a toxic role in the pathogenesis of favism and suggest that acute electrolyte imbalances, mostly affecting calcium homeostasis, are involved in the mechanisms of erythrocyte damage and destruction in this hemolytic disease.

Comparative patterns of ‘in vitro’ oxidative hemolysis of normal and glucose 6-phosphate dehydrogenase (G6PD)-deficient erythrocytes

(ii) This enzyme disorder is characterized by a particularly severe deficiency of catalytic activity, being 9.1% of the G6PD activity found in erythrocytes from normal subjects [ 11; It is associated, although in a characteristically non-constant way, with episodes of acute intravascular hemolysis which are triggered by exogenous agents such as fava beans and (or) drugs resulting in a severe oxidative challenge to the affected erythrocytes [2,3]. Peroxidation of membrane lipids has long been considered as an obligatory step of oxidative hemolysis [4]. A preliminary study on the structure of membrane lipid components failed to show significant differences among erythrocytes from: (a) normal subjects; (b) healthy G6PD-deficient subjects; (c) favic patients after hemolytic crisis, excepting for a distinctive content of palmitic acid [ 51. This is an investigation exploring, on a comparative basis between control,and G6PDdeficient erythrocytes, the dynamic changes in these lipid components which underlie the oxidative hemolysis.

A Serum Defect in Favism

The inhibition of fava bean hemagglutination by normal serum is not observed with favic serum. The inhibitory principle resides in the IgA fraction of normal serum. Serum with a selective deficiency of IgA also fails to inhibit fava bean hemagglutination. The normal serum IgA values obtained in favic patients suggest a qualitative difference in their IgA to account for this failure to inhibit fava bean hemagglutination.