G6PD-deficient Red Blood Cells Research Articles

Glucose-6-phosphate dehydrogenase deficiency detection using fluorocytometric assay: Evaluation after 1 year of clinical implementation.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common enzymopathy that affects red blood cells (RBCs) and renders them susceptible to oxidative stress. G6PD deficiency can cause hemolytic anemia, especially after exposure to certain drugs or infections. The diagnosis of G6PD deficiency is usually based on spectrophotometric measurement of enzyme activity, but this method has limitations in heterozygous females and in patients with other hematological disorders. In this study, we evaluated the use of flow cytometry as an alternative method for detecting G6PD deficiency in 514 samples (265 females and 249 males) from a clinical laboratory. We compared the results of flow cytometry with those of spectrophotometry and molecular analysis, and assessed the performance of flow cytometry in different subgroups of patients. We found that flow cytometry was able to identify G6PD deficiency in most cases, with high sensitivity and specificity. Flow cytometry also allowed the quantification of the percentage of G6PD-deficient RBCs, which varied among heterozygous females due to X-chromosome inactivation. Moreover, flow cytometry detected several cases of G6PD deficiency that were missed by spectrophotometry, especially in heterozygous females with normal or subnormal enzyme activity. However, flow cytometry also showed some false negative results, mainly in patients with sickle cell disease. Therefore, flow cytometry is a reliable and efficient tool for screening G6PD deficiency, but some precautions should be taken in interpreting the results in patients with other hematological conditions.

Primaquine-5,6-Orthoquinone Is Directly Hemolytic to Older G6PD Deficient RBCs in a Humanized Mouse Model.

Primaquine and Tafenoquine are the only approved drugs that can achieve a radical cure for Plasmodium vivax malaria but are contraindicated in patients who are deficient in glucose 6-phosphate dehydrogenase (G6PDd) due to risk of severe hemolysis from reactive oxygen species generated by redox cycling of drug metabolites. 5-hydroxyprimaquine and its quinoneimine cause robust redox cycling in red blood cells (RBCs) but are so labile as to not be detected in blood or urine. Rather, the quinoneimine is rapidly converted into primaquine-5,6-orthoquinone (5,6-POQ) that is then excreted in the urine. The extent to which 5,6-POQ contributes to hemolysis remains unclear, although some have suggested that it is a minor toxin that should be used predominantly as a surrogate to infer levels of 5-hydroxyprimaquine. In this report, we describe a novel humanized mouse model of the G6PD Mediterranean variant (hG6PDMed-) that recapitulates the human biology of RBC age-dependent enzyme decay, as well as an isogenic matched control mouse with human nondeficient G6PD hG6PDND In vitro challenge of RBCs with 5,6-POQ causes increased generation of superoxide and methemoglobin. Infusion of treated RBCs shows that 5,6-POQ selectively causes in vivo clearance of older hG6PDMed- RBCs. These findings support the hypothesis that 5,6-POQ directly induces hemolysis and challenges the notion that 5,6-POQ is an inactive metabolic waste product. Indeed, given the extreme lability of 5-hydroxyprimaquine and the relative stability of 5,6-POQ, these data raise the possibility that 5,6-POQ is a major hemolytic primaquine metabolite in vivo. SIGNIFICANCE STATEMENT: These findings demonstrate that 5,6-POQ, which has been considered an inert waste product of primaquine metabolism, directly induces ROS that cause clearance of older G6PDd RBCs. As 5,6-POQ is relatively stable compared with other active primaquine metabolites, these data support the hypothesis that 5,6-POQ is a major toxin in primaquine induced hemolysis. The findings herein also establish a new model of G6PDd and provide the first direct evidence, to our knowledge, that young G6PDd RBCs are resistant to primaquine-induced hemolysis.

Glucose-6-Phosphate Dehydrogenase Deficiency: Difficulties in Diagnosis at the Souro Sanou University Hospital, Burkina Faso

Glucose-6-phosphate dehydrogenase deficiency is the most common enzymopathy worldwide. The precise prevalence of G6PD is unknown in Burkina Faso. The objective of the study was to describe the difficulties to diagnose this disease at the Souro Sanou University hospital (CHUSS) in Bobo-Dioulasso. It involved five patients comprising one child with homozygous SS sickle cell disease, one adolescent screened following a family investigation, and three adults including a man and two women. Blood smear stained with May Grunwald Giemsa was performed to look for specific signs of G6PD-deficient red blood cell and brilliant cresyl Blue for Heinz Bodies. A microscope Olympus BX53 equipped with a Camera (XC10) and connected to a computer was used to read blood smears and capture images. Genes sequencing by Sanger method were performed in a specialized laboratory in molecular genetics. For each analysis, the protocol and instructions of the equipment and reagent manufacturer were applied. Of the five patients, three had anemia and only one had hyperreticulocytosis. Two patients had biological signs of hemolysis and one patient had an elevated CRP. Blood smear stained with MGG and cresyl blue showed specific signs of G6PD-deficient red blood cells and Heinz bodies in all patients. Biochemical analysis and molecular typing confirmed G6PD deficiency. The presence of G6PD-deficient red blood cells in the blood smear guides the diagnosis of G6PD deficiency. The diagnosis is biochemical and is based on the combined measurement of G6PD plus pyruvate kinase and/or hexokinase.

Glucose 6-P Dehydrogenase-An Antioxidant Enzyme with Regulatory Functions in Skeletal Muscle during Exercise.

Hypomorphic Glucose 6-P dehydrogenase (G6PD) alleles, which cause G6PD deficiency, affect around one in twenty people worldwide. The high incidence of G6PD deficiency may reflect an evolutionary adaptation to the widespread prevalence of malaria, as G6PD-deficient red blood cells (RBCs) are hostile to the malaria parasites that infect humans. Although medical interest in this enzyme deficiency has been mainly focused on RBCs, more recent evidence suggests that there are broader implications for G6PD deficiency in health, including in skeletal muscle diseases. G6PD catalyzes the rate-limiting step in the pentose phosphate pathway (PPP), which provides the precursors of nucleotide synthesis for DNA replication as well as reduced nicotinamide adenine dinucleotide phosphate (NADPH). NADPH is involved in the detoxification of cellular reactive oxygen species (ROS) and de novo lipid synthesis. An association between increased PPP activity and the stimulation of cell growth has been reported in different tissues including the skeletal muscle, liver, and kidney. PPP activity is increased in skeletal muscle during embryogenesis, denervation, ischemia, mechanical overload, the injection of myonecrotic agents, and physical exercise. In fact, the highest relative increase in the activity of skeletal muscle enzymes after one bout of exhaustive exercise is that of G6PD, suggesting that the activation of the PPP occurs in skeletal muscle to provide substrates for muscle repair. The age-associated loss in muscle mass and strength leads to a decrease in G6PD activity and protein content in skeletal muscle. G6PD overexpression in Drosophila Melanogaster and mice protects against metabolic stress, oxidative damage, and age-associated functional decline, and results in an extended median lifespan. This review discusses whether the well-known positive effects of exercise training in skeletal muscle are mediated through an increase in G6PD.

Fyn specifically Regulates the activity of red cell glucose-6-phosphate-dehydrogenase

Fyn is a tyrosine kinase belonging to the Src family (Src-Family-Kinase, SFK), ubiquitously expressed. Previously, we report that Fyn is important in stress erythropoiesis. Here, we show that in red cells Fyn specifically stimulates G6PD activity, resulting in a 3-fold increase enzyme catalytic activity (kcat) by phosphorylating tyrosine (Tyr)-401. We found Tyr-401 on G6PD as functional target of Fyn in normal human red blood cells (RBC), being undetectable in G6PD deficient RBCs (G6PD-Mediterranean and G6PD-Genova). Indeed, Tyr-401 is located to a region of the G6PD molecule critical for the formation of the enzymatically active dimer. Amino acid replacements in this region are mostly associated with a chronic hemolysis phenotype. Using mutagenesis approach, we demonstrated that the phosphorylation status of Tyr401 modulates the interaction of G6PD with G6P and stabilizes G6PD in a catalytically more efficient conformation. RBCs from Fyn-/−mice are defective in G6PD activity, resulting in increased susceptibility to primaquine-induced intravascular hemolysis. This negatively affected the recycling of reduced Prx2 in response to oxidative stress, indicating that defective G6PD phosphorylation impairs defense against oxidation. In human RBCs, we confirm the involvement of the thioredoxin/Prx2 system in the increase vulnerability of G6PD deficient RBCs to oxidation. In conclusion, our data suggest that Fyn is an oxidative radical sensor, and that Fyn-mediated Tyr-401 phosphorylation, by increasing G6PD activity, plays an important role in the physiology of RBCs. Failure of G6PD activation by this mechanism may be a major limiting factor in the ability of G6PD deficient RBCs to withstand oxidative stress.

Fyn Specifically Regulates the Activity of Red Cell Glucose-6-Phosphate-Dehydrogenase

Fyn is a tyrosine kinase belonging to the Src family (Src-Family-Kinase, SFK), ubiquitously expressed. Previously, we reported that Fyn is important in stress erythropoiesis. Here, we show that in red cells Fyn specifically stimulates G6PD activity, resulting in a 3-fold increase of the enzyme catalytic activity (kcat) by phosphorylating tyrosine (Tyr)-401. We found Tyr-401 on G6PD as functional target of Fyn in normal human red blood cells (RBC), being undetectable in G6PD deficient RBCs (G6PD-Mediterranean and G6PD-Genova). Indeed, Tyr-401 is located to a region of the G6PD molecule critical for the formation of the enzymatically active dimer. Amino acid replacements in this region are mostly associated with a chronic hemolysis phenotype. Using mutagenesis approach, we demonstrated that the phosphorylation status of Tyr401 modulates the interaction of G6PD with G6P and stabilizes G6PD in a catalytically more efficient conformation. RBCs from Fyn-/-mice are defective in G6PD activity, resulting in increased susceptibility to primaquine-induced intravascular hemolysis. This negatively affected the recycling of reduced Prx2 in response to oxidative stress, indicating that defective G6PD phosphorylation impairs defense against oxidation. In human RBCs, we confirm the involvement of the thioredoxin/Prx2 system in the increase vulnerability of G6PD deficient RBCs to oxidation. In conclusion, our data suggest that Fyn is an oxidative radical sensor, and that Fyn-mediated Tyr-401 phosphorylation, by increasing G6PD activity, plays an important role in the physiology of RBCs. Failure of G6PD activation by this mechanism may be a major limiting factor in the ability of G6PD deficient RBCs to withstand oxidative stress. Disclosures Cappellini: Genzyme/Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Vifor Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Honoraria; CRISPR Therapeutics: Membership on an entity's Board of Directors or advisory committees. Forni:Novartis, Iron chelation: Research Funding; Celgene, Erithropoiesis Stimulation: Research Funding; Roche, Erithropoiesis Stimulation: Research Funding; BlueBirdBio: Consultancy.

Increased Methylation of Deamidated Asparagines and Aspartates in Stored Red Blood Cells from Glucose 6-Phosphate Dehydrogenase-Deficient Blood Donors

Blood transfusion is a life-saving intervention for millions of recipients worldwide every year. However, refrigerated storage of red blood cells (RBCs) for up to 42 days promotes impairments in energy and redox homeostasis, which impact RBC hemolytic propensity and post-transfusion performance of the storage-damaged RBC. Since mature RBCs are devoid of de novo protein synthesis - owing to the lack of organelles and nuclei - they evolved metabolic mechanisms to cope with oxidative stress. Two of these involve (i) activation of the pentose phosphate pathway (PPP), which generates reducing equivalents (NADPH) to preserve glutathione homeostasis and recharge NADPH-dependent antioxidant enzymes; and (ii) recycling of oxidatively damaged proteins via methylation of dehydrated and deamidated aspartate and asparagine residues, a process that consumes methionine as the main methyl group donor. Thus, we hypothesize that the latter mechanism is relevant to routine blood bank storage, especially in glucose-6-phosphate dehydrogenase (G6PD)-deficient donors. In this routinely accepted donor population (~10% of donors of African descent), mutations of G6PD, the rate-limiting enzyme of the PPP, result in instability and decreased enzymatic activity (e.g., <1% residual activity in the Mediterranean variant).Within the framework of the REDS-III RBC-Omics study, packed RBC units (n=599) were stored under blood bank conditions for 10, 23 and 42 days and profiled for oxidative hemolysis (following AAPH insult) and time-dependent metabolic dysregulation of the trans-sulfuration pathway. The rate of methionine consumption positively correlated with storage age and oxidative hemolysis. In a separate cohort, RBCs from G6PD-deficient and G6PD-normal donors were also collected and metabolically profiled during storage. The G6PD-deficient donors had higher pre-storage methionine levels as compared against G6PD-sufficient donors, although one week of storage was sufficient to significantly flip this ratio (p<0.01). To ensure that the phenomenon was driven by storage-induced oxidative insults, RBCs were also stored under normoxic, hyperoxic (SO2 >95%), or hypoxic conditions (SO2 = 20%, 10%, 5% of < 3%) for up to 42 days. Of note, hypoxia decreased methionine consumption rates and increased S-adenosylmethionine/S-adenosylhomocysteine ratios in comparison to normoxic control RBCs, while opposite trends were observed in response to oxidative stress. In parallel, RBCs were incubated under normoxic or hypoxic conditions for 16h, in presence or absence of a generic methyltransferase inhibitor, adenosine dialdehyde, or hydrogen peroxide (0.1 or 0.5%). 13C,15N-Methionine uptake and consumption rates were faster in response to pro-oxidant insults and were prevented by hypoxia or methyltransferase inhibition. Through a combination of proteomics approaches and 13C-methionine tracing, we observed oxidation-induced increases in both Asn deamidation to Asp and the formation of methyl-Asp on key structural proteins and enzymes, including band 3, hemoglobin, ankyrin, 4.1, spectrin beta, aldolase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), biphosphoglycerate mutase, lactate dehydrogenase and catalase. Methylated regions tended to map proximal to the active site (e.g., N316 of GAPDH) and/or residues interacting with the N-terminal cytosolic domain of band 3. Hypoxic incubation with hydrogen peroxide phenocopied normoxic conditions in the presence of pro-oxidant insults, suggesting that the beneficial role of hypoxia in preventing methionine consumption and protein methylation is attributable to decreased oxidative stress. G6PD-deficient donor RBCs had significantly (p<0.0001) higher levels of methylated-deamidated Asn residues in hemoglobin, band 3, and GAPDH, as compared to stored G6PD-normal donor RBCs.Although methylation of basic amino acid residues is an epigenetic modification in nucleated cells, protein methylation at carboxylate side chains and deamidated Asn residues is a non-epigenetic post-translational sensor of oxidative stress and refrigerated storage in anucleate human RBCs. This damage repair mechanism is exacerbated in RBCs from G6PD-deficient donors, as a compensatory mechanism for the lack of normal PPP function. DisclosuresD'Alessandro:Hemanext inc: Membership on an entity's Board of Directors or advisory committees; Omix Technologies inc: Equity Ownership. Nemkov:Omix Technologies inc: Equity Ownership. Yoshida:Hemanext inc: Employment. Dunham:Hemanext inc: Employment. DuMont:Hemanext inc: Membership on an entity's Board of Directors or advisory committees. Hansen:Omix Technologies inc: Equity Ownership. Spitalnik:Hemanext inc: Membership on an entity's Board of Directors or advisory committees. Zimring:Rubius Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Metabolic Linkage and Correlations to Storage Capacity in Erythrocytes from Glucose 6-Phosphate Dehydrogenase-Deficient Donors.

In glucose 6-phosphate dehydrogenase (G6PD) deficiency, decreased NADPH regeneration in the pentose phosphate pathway and subnormal levels of reduced glutathione result in insufficient antioxidant defense, increased susceptibility of red blood cells (RBCs) to oxidative stress, and acute hemolysis following exposure to pro-oxidant drugs and infections. Despite the fact that redox disequilibrium is a prominent feature of RBC storage lesion, it has been reported that the G6PD-deficient RBCs store well, at least in respect to energy metabolism, but their overall metabolic phenotypes and molecular linkages to the storability profile are scarcely investigated. We performed UHPLC-MS metabolomics analyses of weekly sampled RBC concentrates from G6PD sufficient and deficient donors, stored in citrate phosphate dextrose/saline adenine glucose mannitol from day 0 to storage day 42, followed by statistical and bioinformatics integration of the data. Other than previously reported alterations in glycolysis, metabolomics analyses revealed bioactive lipids, free fatty acids, bile acids, amino acids, and purines as top variables discriminating RBC concentrates for G6PD-deficient donors. Two-way ANOVA showed significant changes in the storage-dependent variation in fumarate, one-carbon, and sulfur metabolism, glutathione homeostasis, and antioxidant defense (including urate) components in G6PD-deficient vs. sufficient donors. The levels of free fatty acids and their oxidized derivatives, as well as those of membrane-associated plasticizers were significantly lower in G6PD-deficient units in comparison to controls. By using the strongest correlations between in vivo and ex vivo metabolic and physiological parameters, consecutively present throughout the storage period, several interactomes were produced that revealed an interesting interplay between redox, energy, and hemolysis variables, which may be further associated with donor-specific differences in the post-transfusion performance of G6PD-deficient RBCs. The metabolic phenotypes of G6PD-deficient donors recapitulate the basic storage lesion profile that leads to loss of metabolic linkage and rewiring. Donor-related issues affect the storability of RBCs even in the narrow context of this donor subgroup in a way likely relevant to transfusion medicine.

Prevalence and hematological indicators of G6PD deficiency in malaria-infected patients.

BackgroundThis study aimed to evaluate the prevalence and alteration of hematological parameters in malaria patients with a glucose-6-phosphate dehydrogenase (G6PD) deficiency, in the western region of Thailand, an endemic region for malaria.MethodsData about patients with malaria hospitalized between 2013 and 2015 were collected. Clinical and sociodemographic characteristics such as age and gender, diagnosis on admission, and parasitological results were mined from medical records of the laboratory unit of the Phop Phra Hospital in Tak Province, Thailand. Venous blood samples were collected at the time of admission to hospital to determine G6PD deficiency by fluorescence spot test and detect malaria parasites by thick and thin film examination. Other data such as complete blood count and parasite density were also collected and analyzed.ResultsAmong the 245 malaria cases, 28 (11.4 %) were diagnosed as Plasmodium falciparum infections and 217 cases (88.6 %) were diagnosed as P. vivax infections. Seventeen (6.9 %) patients had a G6PD deficiency and 228 (93.1 %) patients did not have a G6PD deficiency. Prevalence of male patients with G6PD deficiency was higher than that of female patients (P < 0.05, OR = 5.167). Among the patients with a G6PD deficiency, two (11.8 %) were infected with P. falciparum, while the remaining were infected with P. vivax. Malaria patients with a G6PD deficiency have higher monocyte counts (0.6 × 103/μL) than those without a G6PD deficiency (0.33 × 103/μL) (P < 0.05, OR = 5.167). Univariate and multivariate analyses also confirmed that malaria patients with a G6PD deficiency have high monocyte counts. The association between G6PD status and monocyte counts was independent of age, gender, nationality, Plasmodium species, and parasite density (P < 0.005).ConclusionThis study showed a prevalence of G6PD deficiency in a malaria-endemic area. This study also supported the assertion that patients with G6PD-deficient red blood cells had no protection against the P. falciparum infection. In addition, malaria patients with a G6PD deficiency had higher monocyte counts than those without a G6PD deficiency. These findings will help to recognize and diagnose malaria patients with a G6PD deficiency, as well as to identify the risks and protective factors against malaria in endemic areas.Electronic supplementary materialThe online version of this article (doi:10.1186/s40249-016-0130-0) contains supplementary material, which is available to authorized users.

Inability to maintain GSH pool in G6PD-deficient red cells causes futile AMPK activation and irreversible metabolic disturbance.

Glucose 6-phosphate dehydrogenase (G6PD) is essential for maintenance of nicotinamide dinucleotide hydrogen phosphate (NADPH) levels and redox homeostasis. A number of drugs, such as antimalarial drugs, act to induce reactive oxygen species and hemolytic crisis in G6PD-deficient patients. We used diamide (DIA) to mimic drug-induced oxidative stress and studied how these drugs affect cellular metabolism using a metabolomic approach. There are a few differences in metabolome between red blood cells (RBCs) from normal and G6PD-deficient individuals. DIA causes modest changes in normal RBC metabolism. In contrast, there are significant changes in various biochemical pathways, namely glutathione (GSH) metabolism, purine metabolism, and glycolysis, in G6PD-deficient cells. GSH depletion is concomitant with a shift in energy metabolism. Adenosine monophosphate (AMP) and adenosine diphosphate (ADP) accumulation activates AMP protein kinase (AMPK) and increases entry of glucose into glycolysis. However, inhibition of pyruvate kinase (PK) reduces the efficacy of energy production. Metabolic changes and protein oxidation occurs to a greater extent in G6PD-deficient RBCs than in normal cells, leading to severe irreversible loss of deformability of the former. Normal and G6PD-deficient RBCs differ in their responses to oxidants. Normal cells have adequate NADPH regeneration for maintenance of GSH pool. In contrast, G6PD-deficient cells are unable to regenerate enough NADPH under a stressful situation, and switch to biosynthetic pathway for GSH supply. Rapid GSH exhaustion causes energy crisis and futile AMPK activation. Our findings suggest that drug-induced oxidative stress differentially affects metabolism and metabolite signaling in normal and G6PD-deficient cells. It also provides an insight into the pathophysiology of acute hemolytic anemia in G6PD-deficient patients.

Evaluation of antimalarial activity and toxicity of a new primaquine prodrug.

Plasmodium vivax is the most prevalent of the five species causing malaria in humans. The current available treatment for P. vivax malaria is limited and unsatisfactory due to at least two drawbacks: the undesirable side effects of primaquine (PQ) and drug resistance to chloroquine. Phenylalanine-alanine-PQ (Phe-Ala-PQ) is a PQ prodrug with a more favorable pharmacokinetic profile compared to PQ. The toxicity of this prodrug was evaluated in in vitro assays using a human hepatoma cell line (HepG2), a monkey kidney cell line (BGM), and human red blood cells deficient in the enzyme glucose-6-phosphate-dehydrogenase (G6PD). In addition, in vivo toxicity assays were performed with rats that received multiple doses of Phe-Ala-PQ to evaluate biochemical, hematological, and histopathological parameters. The activity was assessed by the inhibition of the sporogonic cycle using a chicken malaria parasite. Phe-Ala-PQ blocked malaria transmission in Aedes mosquitoes. When compared with PQ, it was less cytotoxic to BGM and HepG2 cells and caused less hemolysis of G6PD-deficient red blood cells at similar concentrations. The prodrug caused less alteration in the biochemical parameters than did PQ. Histopathological analysis of the liver and kidney did show differences between the control and Phe-Ala-PQ-treated groups, but they were not statistically significant. Taken together, the results highlight the prodrug as a novel lead compound candidate for the treatment of P. vivax malaria and as a blocker of malaria transmission.

Development of a novel mouse model of severe glucose-6-phosphate dehydrogenase (G6PD)-deficiency for in vitro and in vivo assessment of hemolytic toxicity to red blood cells

Studies of hemolytic agents on G6PD-deficient subjects have been extensively performed on red blood cells obtained from donors, only using in vitro methods. However, there has been no adequate G6PD-deficient animal model for in vivo assessment of potentially hemolytic agents. The objective of this study is to establish a novel mouse model of severe G6PD-deficiency, with high susceptibility to hemolytic damage upon oxidative agents. To create this model, G6PD mutant Gpdx allele was introduced into the C57L/J mouse strain background by breeding program. The hemolytic toxicity of naphthalene and its metabolite α-naphthol on G6PD-deficient red blood cells was evaluated. Our data showed that the F2 homozygous Gpdx mutant with C57L/J background exhibiting the G6PD activity was 0.9 ± 0.1 U/g Hb, level similar to those of G6PD deficiency in human. A significantly negative correlation was demonstrated between GSH percentage reduction and G6PD activity (r = − 0.51, p < 0.001) upon challenge of the red blood cells with alpha-naphthol in vitro. Similar correlation was also found between GSSG elevation and G6PD activity. Our in vivo studies showed that the administration of naphthalene at 250 mg/kg inflicted significant oxidative damage to the G6PD-deficient mice, as illustrated by the decrease of the GSH-to-GSSG ratio (by 34.2%, p = 0.005) and the increase of the methemoglobin level (by 1.9 fold, p < 0.001). Hemolytic anemia was also found in G6PD-deficient mice at this dosage of naphthalene. In summary, this novel mouse model could be utilized as a screening platform to more accurately determine the hemolytic toxicity of pharmacological agents on G6PD-deficient subjects.

Hyperbilirubinemia in African American neonates: clinical issues and current challenges

African American neonates evidence a low incidence of hyperbilirubinemia yet account for more than 25% of the reported kernicterus cases in the USA. Glucose-6-phosphate dehydrogenase (G6PD) deficiency accounts for approximately 60%, and late preterm gestation and ABO hemolytic disease approximately 40% of these cases. Females heterozygous for G6PD A- harbor a population of G6PD-deficient red blood cells and are at risk for hyperbilirubinemia. Pre-discharge bilirubin measurement coupled with gestational age enhances the identification of neonates at hyperbilirubinemia risk. Parental education at the time of birth hospitalization discharge combined with timely follow-up may help to reduce the risk of developing hazardous hyperbilirubinemia.

Co-ordinated stage-dependent enhancement of Plasmodium falciparum antioxidant enzymes and heat shock protein expression in parasites growing in oxidatively stressed or G6PD-deficient red blood cells

BackgroundPlasmodium falciparum-parasitized red blood cells (RBCs) are equipped with protective antioxidant enzymes and heat shock proteins (HSPs). The latter are only considered to protect against thermal stress. Important issues are poorly explored: first, it is insufficiently known how both systems are expressed in relation to the parasite developmental stage; secondly, it is unknown whether P. falciparum HSPs are redox-responsive, in view of redox sensitivity of HSP in eukaryotic cells; thirdly, it is poorly known how the antioxidant defense machinery would respond to increased oxidative stress or inhibited antioxidant defense. Those issues are interesting as several antimalarials increase the oxidative stress or block antioxidant defense in the parasitized RBC. In addition, numerous inhibitors of HSPs are currently developed for cancer therapy and might be tested as anti-malarials. Thus, the joint disruption of the parasite antioxidant enzymes/HSP system would interfere with parasite growth and open new perspectives for anti-malaria therapy.MethodsStage-dependent mRNA expression of ten representative P. falciparum antioxidant enzymes and hsp60/70–2/70–3/75/90 was studied by quantitative real-time RT-PCR in parasites growing in normal RBCs, in RBCs oxidatively-stressed by moderate H2O2 generation and in G6PD-deficient RBCs. Protein expression of antioxidant enzymes was assayed by Western blotting. The pentosephosphate-pathway flux was measured in isolated parasites after Sendai-virus lysis of RBC membrane.ResultsIn parasites growing in normal RBCs, mRNA expression of antioxidant enzymes and HSPs displayed co-ordinated stage-dependent modulation, being low at ring, highest at early trophozoite and again very low at schizont stage. Additional exogenous oxidative stress or growth in antioxidant blunted G6PD-deficient RBCs indicated remarkable flexibility of both systems, manifested by enhanced, co-ordinated mRNA expression of antioxidant enzymes and HSPs. Protein expression of antioxidant enzymes was also increased in oxidatively-stressed trophozoites.ConclusionResults indicated that mRNA expression of parasite antioxidant enzymes and HSPs was co-ordinated and stage-dependent. Secondly, both systems were redox-responsive and showed remarkably increased and co-ordinated expression in oxidatively-stressed parasites and in parasites growing in antioxidant blunted G6PD-deficient RBCs. Lastly, as important anti-malarials either increase oxidant stress or impair antioxidant defense, results may encourage the inclusion of anti-HSP molecules in anti-malarial combined drugs.

Oxidized and poorly glycosylated band 3 is selectively phosphorylated by Syk kinase to form large membrane clusters in normal and G6PD-deficient red blood cells

Oxidative events involving band 3 (Anion Exchanger 1) have been associated with RBC (red blood cell) removal through binding of NAbs (naturally occurring antibodies); however, the underlying mechanism has been only partially characterized. In addition to inducing direct membrane protein oxidative modification, oxidative treatment specifically triggers the phosphorylation of band 3 tyrosine residues. The present study reports that diamide, a thiol group oxidant, induces disulfide cross-linking of poorly glycosylated band 3 and that the oligomerized band 3 fraction is selectively tyrosine phosphorylated both in G6PD (glucose-6-phosphate dehydrogenase)-deficient and control RBCs. This phenomenon is irreversible in G6PD-deficient RBCs, whereas it is temporarily limited in control RBCs. Diamide treatment caused p72 Syk phosphorylation and translocation to the membrane. Diamide also induced p72 Syk co-immunoprecipitation with aggregated band 3. Moreover, following size-exclusion separation of Triton X-100-extracted membrane proteins, Syk was found only in the high-molecular-mass fraction containing oligomerized/phosphorylated band 3. Src family inhibitors efficiently abrogated band 3 tyrosine phosphorylation, band 3 clustering and NAbs binding to the RBC surface, suggesting a causal relationship between these events. Experiments performed with the non-permeant cross-linker BS(3) (bis-sulfosuccinimidyl-suberate) showed that band 3 tyrosine phosphorylation enhances its capability to form large aggregates. The results of the present study suggest that selective tyrosine phosphorylation of oxidized band 3 by Syk may play a role in the recruitment of oxidized band 3 in large membrane aggregates that show a high affinity to NAbs, leading to RBC removal from the circulation.

Pro-oxidative effects of Chinese herbal medicine on G6PD-deficient erythrocytes in vitro

Glucose-6-phosphate dehydrogenase (G6PD)-deficient subjects are susceptible to chemical-induced oxidative haemolysis. Little is known concerning the haemolytic properties of Chinese herbal medicine on G6PD-deficient subjects. Our objective was to investigate the pro-oxidative effect of 18 commonly used Chinese herbal medicine (CHM) on human G6PD-deficient red blood cells. G6PD-deficient (n=10) and normal (n=10) whole blood samples were incubated with water extracts of CHM. The resulting levels of reduced glutathione (GSH) and methaemoglobin (MetHb) were determined by biochemical assays. Rhizoma Coptidis significantly reduced GSH level by 48.9+/-5.4% (at 1 mg/mL) in the G6PD-deficient erythrocytes (P<0.001) compared with the respective control group without challenge. Similar dose-dependent responses were observed at higher concentrations of Cortex Moutan, Radix Rehmanniae, Radix Bupleuri, Rhizoma Polygoni Cuspidati and Flos Chimonanthi (P<0.01, 5-10 mg/mL). In addition, the levels of MetHb were elevated significantly when challenged with Rhizoma Coptidis (2.8 fold at 5 mg/mL) and Cortex Moutan (3.4 fold at 10 mg/mL). This is the first report on the pro-oxidative action of CHM on G6PD-deficient blood samples in vitro as demonstrated by the decrease of GSH and increase of MetHb. G6PD-deficient subjects should restrain from excessive consumption of these pro-oxidative herbs.

H 2O 2 injury in β thalassemic erythrocytes: Protective role of catalase and the prooxidant effects of GSH

Redox-mediated injury is an important pathway in the destruction of β thalassemic red blood cells (RBC). Because of the autoxidation of the unstable hemoglobin chains and subsequent release of globin free heme and iron, significant amounts of superoxide (O 2 −) and, more importantly, hydrogen peroxide (H 2O 2) are generated intracellularly. Hence, catabolism of H 2O 2 is crucial in preventing cellular injury. Removal of H 2O 2 is mediated via two primary pathways: GSH-dependent glutathione peroxidase or catalase. Importantly, both pathways are ultimately dependent on NADPH. In the absence of any exogenous oxidants, model thalassemic RBC demonstrated significantly decreased GSH levels ( P < 0.001 at 20 h). Perhaps of greater pathophysiologic importance, however, was the finding that the model thalassemic RBC exhibited significantly ( P < 0.001) decreased catalase activity. Following 20 h incubation at 37°C only 61.5 ± 2.9% of the initial catalase activity remained in the α-hemoglobin chain-loaded cells versus 104.6 ± 4.5 and 108.2 ± 3.2% in the control and control-resealed cells, respectively. The mechanism underlying the loss of both catalase activity and GSH appears to be the same in that both catabolic pathways require adequate NADPH levels. As shown in this study, model β thalassemic cells are unable to maintain a normal (∼1.0) NADPH/NADP total ratio and, after 20 h, the model β thalassemic cells have a significantly ( P < 0.001) lower ratio (∼0.5) which is quite similar to a G6PD-deficient RBC. In support of these findings, direct inactivation of catalase gives rise to significantly increased oxidant damage. In contrast, GSH depletion is not closely associated with oxidant sensitivity. Indeed, the consumption of GSH noted in the thalassemic RBC may be via a prooxidant pathway as augmentation of cellular GSH levels actually enhances α-hemoglobin chain-mediated injury.

N-Acetyl cysteine, L-cysteine, and β-mercaptoethanol augment seleniumglutathione peroxidase activity in glucose-6-phosphate dehydrogenasedeficient human erythrocytes

In glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes, failure to maintain normal levels of reduced glutathione (GSH) due to decreased NADPH regeneration in the hexose monophosphate pathway results in acute hemolytic anemia following exposure to oxidative insults, such as ingestion of Vicia fava beans or use of certain drugs. GSH is a source of protection against oxidative attack, used by the selenium-dependent glutathione peroxidase (Se-GSH-Px)/reductase (GR) system to detoxify hydrogen peroxide and organic peroxides, provided that sufficient GSH is made available. In this study, Se-GSH-Px activity was analyzed in G6PD-deficient patients in the presence of reducing agents such as N-Acetyl cysteine, L-cysteine, and beta-mercaptoethanol. Se-GSH-Px activity was decreased in G6PD-deficient red blood cells (RBCs). N-Acetyl cysteine, L-cysteine, and beta-mercaptoethanol increased Se-GSH-Px activity in G6PD-deficient human erythrocytes, indicating that other reducing agents can be utilized to complement Se-GSH-Px activity in G6PD deficiency. Based on the increased susceptibility of G6PD-deficient patients to oxidative stress, the reported increase in Se-GSH-Px activity can facilitate the detoxification of reactive oxygen species.

Glutathione and Glucose-6-Phosphate Dehydrogenase Deficiency Can Increase Protein Glycosylation

Glycosylation (glycation) of proteins can cause cataractogenesis and cellular damage. Using a human red blood cells (RBC) model and two different methods, this study shows that the glycation of hemoglobin increases significantly in glutathione (GSH) deficient RBC exposed to high glucose levels in vitro. Hemoglobin glycation was assessed using both affinity and ion-exchange chromatography. GSH-deficiency was accomplished artificially by treating RBC with 1-chloro-2,4-dinitrobenzene (CDNB) as well as by using hereditary G6PD-deficient RBC. GSH replenishment by dithioerythritol (DTE) and dithiothreitol (DTT) significantly blocked the glycation of hemoglobin in GSH-deficient cells. There was a significant negative correlation between levels of GSH and glycated hemoglobin following treatment with DTE ( r = −0.98, n = 5, p < .001) and DTT ( r = −0.94, n = 5, p < .02). This demonstrates that GSH depletion can indeed increase glycation. Thus, GSH and G6PD-deficient cells are at a significantly greater risk for the glycation of proteins, which in turn can cause increased cellular damage.

Free Radical and Oxidative Damage in Human Blood Cells.

Free radicals and oxidative damage play important roles in aging and many degenerative disorders such as cancer, cardiovascular diseases, and Alzheimer disease. Antioxidants can alleviate some of the harmful effects of oxidative damage. In this report, we describe that we have been using human red blood cells (RBCs) as a model system to delineate the effects of oxidative damage on human cells, particularly on glucose-6-phosphate dehydrogenase (G6PD)-deficient human RBCs. By using a monolayer technique, we found that oxidative denaturation of hemoglobin leads to the release of hemin into the RBC membrane and the released hemin is capable of oxidizing membrane proteins via a thiyl radical intermediate as detected by the electron spin resonance technique. By using a Laser Viscodiffractometer (Vidometer) to measure RBC deformability, we found that the deformability of G6PD-deficient RBCs was drastically reduced by hydroxyl radicals. Perhaps as a consequence of enhanced susceptibility to oxidative stress, G6PD-deficient individuals have lower antioxidant levels, particularly vitamin C, than normal individuals. Interestingly, we have also found that RBC deformability could be affected by two environmental pollutants, namely, platinum and palladium, which can enhance hydroxyl radical formation in the presence of hydrogen peroxide and ferrous ion (Fenton reaction).