Glucose-6-phosphate Dehydrogenase Deficient Cells Research Articles

269 - G6PD knockdown-mediated impairment of inflammasome activation via dysregulation of p38/MAPK pathway

Glucose-6-phosphate dehydrogenase (G6PD) is a rate-limiting enzyme of pentose phosphate pathway to maintain cellular redox homeostasis via the production of NADPH. Through NADPH oxidase (NOX), NADPH modulates the generation of reactive oxygen species (ROS) against pathogen infection. G6PD deficient cells have low sensitivity to induce inflammation pathway for elimination of pathogen infection. Inflammasome activation is a kind of inflammatory response and plays a regulatory role in innate immunity by producing pro-inflammatory cytokines including interleukine-1β and 18 (IL-1β and IL-18). However, the role of G6PD in the activation of inflammasome remains elusive. To investigate whether G6PD modulates inflammasome activation, peripheral blood mononuclear cells (PBMCs) were isolated from well-known G6PD genetic variants in Taiwan and differentiated into macrophage-liked cells by phorbol 12-myristate 13-acetate (PMA) treatment. The signal 1 and 2 pathways of inflammasome were activated by lipopolysaccharides (LPS) and adenosine triphosphate (ATP) stimulation, respectively. During inflammasome activation, a decreased secretion of IL-1β was observed in PMA-primed G6PD deficient PBMCs as compared with their counterparts. We further used human monocytic cells (THP-1) as model to delineate whether the expression of molecules in inflammasome signal 1 and 2 pathway could be modulated by G6PD. Indeed, inflammasome activation-induced IL-1β secretion and p38 phosphorylation of signal 1 pathway were downregulated upon G6PD knockdown in THP-1 cells. In contrast, the effect of G6PD on signal 2 pathway was not significant in THP-1 cells. Taken together, these results suggest that G6PD deficiency down regulates inflammasome activation via MAPK pathway and the detail mechanism is currently under investigation.

The Role of Glucose‐6‐Phosphate Dehydrogenase of Inflammasome Activation

Glucose‐6‐phosphate dehydrogenase (G6PD) is a rate‐limiting enzyme of pentose phosphate pathway to maintain cellular redox homeostasis via the production of NADPH. Through NADPH oxidase (NOX), NADPH modulates the generation of reactive oxygen species (ROS) against pathogen infection. G6PD deficient cells have low sensitivity to induce inflammation pathway for elimination of pathogen infection. Inflammasome activiation is a kind of inflammatory response and plays a regulatory role in innate immunity by producing pro‐inflammatory cytokines including interleukine‐1β and 18 (IL‐1β and IL‐18). However, the role of G6PD in the activation of inflammasome is remains elusive. Since well‐known G6PD genetic variation in Taiwan is 1376 (G>T; 42.3 ~ 52.3%) and 1388 (G>A; 10.5 ~ 34%), we collected peripheral blood from patients with either of these two point mutations. To investigate whether G6PD modulates inflammasome activation, peripheral blood mononuclear cells (PBMCs) were isolated and differentiated into macrophage‐liked cells by phorbol 12‐myristate 13‐acetate (PMA) treatment. The signal 1 and 2 pathways of inflammasome were activated by lipopolysaccharides (LPS) and adenosine triphosphate (ATP) stimulation, respectively. During inflammasome activation, a decreased secretion of IL‐1β was observed in PMA‐primed G6PD deficient PBMCs as compared with their counterparts. We further used human monocytic cells (THP‐1) as model to delineate whether the expression of molecules in inflammasome signal 1 and 2 pathway could be modulated by G6PD. Indeed, inflammasome activation‐induced IL‐1β secretion and NF‐κB phosphorylation of signal 1 pathway were downregulated upon G6PD knockdown in THP‐1 cells. Nevertheless, the effect of G6PD on signal 2 pathway is still unclear. Taken together, these results suggest that G6PD deficiency down regulates inflammasome activation and the underlying mechanism is currently under investigation.

A comprehensive analysis of membrane and morphology of erythrocytes from patients with glucose-6-phosphate dehydrogenase deficiency

Acute hemolytic anemia could be triggered by oxidative stress in the patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. However, the underlying hemolytic mechanism is unknown. To make clear the hemolytic mechanisms, a systematic study on membrane ultrastructure had been undertaken. A comprehensive method was used including atomic force microscopy, scanning electron microscopy, flow cytometer and fluorescence microscopy to analyze the membrane ultrastructure, externalized phosphatidylserine (PS), intracellular Ca(2+) concentration, morphology and the distributions of band 3 protein in G6PD deficient red blood cells (RBCs) after tert-butyl-hydroperoxide (t-BHP) oxidation. The results showed that erythrocyte shrinkage, annexin-V binding to externalized PS on the membrane of early-stage apoptotic cells, the increased membrane roughness and intracellular Ca(2+) concentration, as well as the change of distributions of band 3 protein in RBCs. Compared with the control RBCs, as the concentration of t-BHP up to 0.1mM, the membrane roughness of G6PD deficient RBCs showed significant difference (p<0.05) and as the concentration of t-BHP up to 0.3mM, externalized PS showed significant difference (p<0.05). Furthermore, the population types of RBCs showed dramatic difference between control groups and G6PD deficient groups. Oxidative stress induced more serious erythrocyte apoptosis and resulted in increased roughness of erythrocyte membrane and abnormal distributed band 3 protein in G6PD deficient RBCs. Echinocytes are the predominant abnormal erythrocyte shape occurring in the peripheral blood from patients with G6PD deficiency, which may shorten the RBCs lifespan. The results in the present study will give an increased understanding for the hemolytic mechanism of G6PD deficiency.

Unexpected glucose‐6‐phosphate dehydrogenase deficiency

A 50-year-old man with refractory intestinal T-cell lymphoma was started on combination chemotherapy (ifosfamide, methotrexate, l-asparaginase, etoposide, dexamethasone). Cotrimoxazole was commenced for pneumocystis prophylaxis after a normal glucose-6-phosphate dehydrogenase (G6PD) spot fluorescence screen [>4·5 iu/g haemoglobin (Hb)]. Initial tumour shrinkage was accompanied by renal impairment (creatinine 393 μmol/l, normal 67–109) and hyperuricaemia (1259 μmol/l, normal 260–530), and rasburicase (4·5 mg × 1 dose) was given for tumour lysis. Despite a dramatic fall in uric acid level (55 μmol/l), anaemia suddenly occurred (Hb fell from 113 g/l to 59 g/l), followed by hypotension and oliguria. Biochemical screening showed raised unconjugated bilirubin (41 μmol/l, normal <6) and lactate dehydrogenase (798 iu/l, normal 118–211). Intravascular haemolysis was confirmed by raised methaemalbumin (14·3 mg/l, normal <01·0) levels and haemoglobinuria. A peripheral blood film showed bite cells and abundant hemi-ghost cells (left), classical for oxidative haemolysis. The patient survived with transfusion, haemodialysis and supportive treatment, with complete renal recovery. A repeat G6PD enzyme assay on his stored blood sample showed a marginal G6PD level of 5·5 iu/g Hb, which lies within the range expected for female heterozygotes. His four brothers were all G6PD deficient (0·21–1·24 iu/g Hb). Using allele-specific polymerase chain reaction, a G6PD Kaiping allele (nt 1388 G→A) was detected in the patient and all siblings. Surprisingly the patient also carried a second normal G6PD allele, suggesting extra X chromosome material. On further questioning, the patient volunteered a history of azoospermia and bilateral absent vas deferens. There were no other abnormal features suggestive of Klinefelter syndrome. Karyotype analysis on stimulated peripheral blood lymphocytes and fluorescence in situ hybridization with an X whole chromosome painting probe (Oncor Inc., Gaithersburg, MD, USA) confirmed the presence of extra X chromosome material on the Y-chromosome (right, red arrow) in all 20 metaphases analysed: 46, XY.ish add(Y)(p11)(wcpX+). Hence the patient was an occult carrier of extra X chromosome material and a heterozygous carrier of G6PD deficiency. Deficiency of G6PD is the commonest red cell enzymopathy world-wide. It affects 4·8% of all Southern Chinese males, and is screened for at birth and before the commencement of oxidative medications. In female heterozygotes, random lyonization of the X chromosomes results in a mixture of normal and G6PD deficient red cells. The resultant average enzyme levels usually lie within the normal range. However female heterozygotes for G6PD deficiency can still suffer haemolysis as a result of skewed lyonization because of age, haemopoietic transplantation or clonal haemopoiesis. Without mandatory screening, this can cause unexpected severe haemolysis. Our case showed that a simple fluorescent screen might still be insufficient. Severe oxidative stresses because of a combination of drugs (cotrimoxazole and rasburicase) could still cause en masse destruction of the G6PD deficient subpopulation, resulting in life-threatening intravascular haemolysis. Hence, a quantitative enzyme level assay is a better option in areas endemic for G6PD deficiency.

Mutation in G6PD gene leads to loss of cellular control of protein glutathionylation: Mechanism and implication

More than 400 million people are susceptible to oxidative stress due to glucose-6-phosphate dehydrogenase (G6PD) deficiency. Protein glutathionylation is believed to be responsible for loss of protein function and/or cellular signaling during oxidative stress. To elucidate the implications of G6PD deficiency specifically in cellular control of protein glutathionylation, we used hydroxyethyldisulfide (HEDS), an oxidant which undergoes disulfide exchange with existing thiols. G6PD deficient (E89) cells treated with HEDS showed a significant increase in protein glutathionylation compared to wild-type (K1) cells. In order to determine whether increase in global protein glutathionylation by HEDS leads to loss of function of an important protein, we compared the effect of HEDS on global protein glutathionylation with that of Ku protein function, a multifunctional DNA repair protein, using a novel ELISA. E89 cells treated with HEDS showed a significant loss of Ku protein binding to DNA. Cellular protein thiol and GSH, whose disulfide is involved in protein glutathionylation, were decreased by HEDS in E89 cells with no significant effect in K1 cells. E89 cells showed lower detoxification of HEDS, that is, conversion of disulfide HEDS to free sulfhydryl mercaptoethanol (ME), compared to K1 cells. K1 cells maintained their NADH level in the presence of HEDS but that of E89 cells decreased by tenfold following a similar exposure. NADPH, a cofactor required to maintain reduced form of the thiols, was decreased more in E89 than K1 cells. The specific role of G6PD in the control of such global protein glutathionylation and Ku function was further demonstrated by reintroducing the G6PD gene into E89 (A1A) cells, which showed a normal phenotype.

Band 3 tyr-phosphorylation in normal and glucose-6-phospate dehydrogenase-deficient human erythrocytes

Haemolysis is usually episodic in glucose-6-phosphate dehydrogenase (G6PD) deficiency, often triggered by a period of oxidative stress. In the present work, we investigate a possible biochemical mechanism underlying the enhanced susceptibility of G6PD deficient red blood cells (RBC) to oxidative stress. We analysed eight male subjects with Mediterranean glucose-6P-dehydrogenase deficiency (G6PDd), class II, for their ability in phosphorylating erythrocyte membrane band 3 following oxidative and osmotic stress. Our findings show that this sensitivity is connected to an early membrane band 3 Tyr-phosphorylation in the presence of diamide. However, since both Syk, and Lyn kinases, and SHP-2 phosphatase, mostly implicated in the band 3 P-Tyr level regulation, are alike in content and activity in normal and patient erythrocytes, an alteration in the membrane organization is likely the cause of the anomalous response to the oxidant. We report, in fact, that hypertonic-induced morphological change in G6PDd erythrocyte induces a higher membrane band 3 Tyr-phosphorylation, suggesting a pre-existing membrane alteration, likely due to the chronic lowering of the redox systems in patients. We also report that 1-chloro-2,4-dinitrobenzene-pre-treatment of normal red cells can alter the normal protein-protein and protein-membrane interaction under hypertonic rather than oxidative stress, thus partially resembling the response in patients, and that RBC may utilize a wider range of redox defence, under oxidative conditions, including, but not exclusively, NADPH and glutathione. On the whole, these results would encourage a different approach to the evaluation of the effects of pharmacological administration to patients, giving more attention to the possible drug-induced membrane alteration evidenced by the abnormal band 3 Tyr-phosphorylation.

G6PD Deficient Cells and the Bioreduction of Disulfides: Effects of DHEA, GSH Depletion and Phenylarsine Oxide

We used Glucose 6 phosphate dehydrogenase (G6PD) minus cells (89 cells) and G6PD containing cells (K1) to understand the mechanisms of bioreduction of disulfide and the redox regulation of protein and non protein thiols in mammalian cells. The 89 cells reduce hydroxyethyldisulfide (HEDS) to mercaptoethanol (ME) at a slower rate than K1 cells. HEDS reduction results in loss of nonprotein thiols (NPSH) and a decrease in protein thiols (PSH) in 89 cells. The effects are less dramatic with K1 cells. However, the loss of NPSH and PSH in K1 cells are increased in the absence of glucose. Glutathione-depletion with L-BSO partially blocks HEDS reduction in K1 and 89 cells. Treatment with the vicinal thiol reagent phenyl arsenic oxide (PAO) blocks reduction of HEDS in both cells. Surprisingly, dehydroepiandrosterone (DHEA), a known inhibitor of G6PD, inhibits the growth and blocks the reduction of HEDS both in 89 and K1 cells suggesting that its mechanism for inhibition of growth is not G6PD related.

Oxidation of cellular thiols by hydroxyethyldisulphide inhibits DNA double-strand-break rejoining in G6PD deficient mammalian cells

Purpose : We investigated the effect of protein- and non proteinthiol oxidation on DNA double-strand-break (DSB) rejoining after irradiation and its relevance in the survival of CHO cells. Materials and methods : We used mutant cells null for glucose 6 phosphate dehydrogenase (G6PD) activity since reducing equivalents, required for reduction of oxidized thiols, are typically generated through G6PD regulated production of NADPH. Cellular thiols were oxidized by pre-incubating the cells with hydroxyethyldisulphide (HEDS), the oxidized form of mercaptoethanol (ME). The concentrations of the intracellular and extracellular non-protein thiols (NPSH), glutathione, cysteine and mercaptoethanol were quantitated by HPLC. Protein thiols (PSH) were estimated using Ellman's reagent. Cell survival was determined by clonogenic assay. The induction and rejoining of DSB in cells was quantitated by Pulse Field Gel Electrophoresis after exposure to ionizing radiation. Results : Much lower bioreduction of HEDS was found in the G6PD deficient mutants (E89) than in the wild-type cells (K1). A 1 h treatment of E89 cells with HEDS produced almost complete depletion of non-protein thiol (NPSH) and a 26% decrease in protein thiols. Only minor changes were found under similar conditions with K1 cells. When exposed to gamma radiation in the presence of HEDS, the G6PD null mutants exhibited a higher cell killing and decreased rate and extent of rejoining of DSB than were observed in K1 cells. Moreover, when the G6PD deficient cells were transfected with the gene encoding wild-type G6PD (A1A), they recovered close to wild-type cellular thiol status, cell survival and DSB rejoining. Conclusions : These results suggest that a functioning oxidative pentose phosphate pathway is required for DSB rejoining in cells exposed to a mild thiol oxidant.

G6PD activity and gene expression in leukemic cells from G6PD-deficient subjects

In the present study we examined gene expression and glucose-6-phosphate dehydrogenase (G6PD) activity in leukemic cells isolated from G6PD normal and deficient subjects. The results have shown that G6PD activity strongly increases in G6PD normal leukemic cells as well as in G6PD deficient leukemic cells when compared to peripheral blood mononuclear cells (PBMC). Higher levels of G6PD gene expression were observed in leukemic cells from G6PD deficient patients compared to G6PD normal. A similar pattern of gene expression was also observed for 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase. These results support the hypothesis that G6PD deficient cell, in order to sustain their growth, must respond to the low activity of their mutant enzyme with an increase in quantity through an induction of gene expression.

Pathways for the Reduction of Oxidized Glutathione in the Plasmodium falciparum-Infected Erythrocyte: Can Parasite Enzymes Replace Host Red Cell Glucose-6-Phosphate Dehydrogenase?

Plasmodium falciparum-infected human red cells possess at least two pathways for the generation of reduced nicotinamide adenine dinucleotide phosphate (NADPH): (1) the glucose-6-phosphate dehydrogenase (G6PD) pathway and (2) the glutamate dehydrogenase (GD) pathway using glutamate as a substrate. Uninfected erythrocytes lack the GD pathway. The NADPH generated can be used to reduce oxidized glutathione (GSSG), which accumulates in the presence of an oxidative stress. In red cell G6PD deficiency, this pathway is reduced or absent, and the host cells as well as the parasites within them are vulnerable to oxidant stress. In view of the presence of the GD pathway in parasitized red cells and the recent description of a parasite-derived G6PD enzyme, we have asked whether the pathways for the reduction of GSSG provided by the parasite can substitute for the host G6PD in red cells deficient in G6PD activity. We have devised a functional assay in which the reduction rate of GSSG is monitored in the presence of buffered infected or control red cell lysates and substrates. Infected G6PD-deficient erythrocytes were obtained from in vitro cultures after a single prior growth cycle of the parasites in G6PD deficient cells to eliminate contaminating normal red cells. The results show that only parasitized red cells can reduce GSSG via the GD pathway. In parasitized G6PD Mediterranean red cells (completely G6PD-deficient), there is a detectable GSSG reduction via the G6PD pathway, not found in uninfected lysates from the same individual. In G6PD A- (African type, featuring partial deficiency), a small increment in the G6PD-dependent reduction of GSSG can also be detected. However, when compared to G6PD normal red cells, the activities from the parasite-derived pathways are small and could not be considered substitutes for normal host enzyme activity. It is concluded that while the plasmodium provides additional pathways for the generation of NADPH that may serve its own metabolic needs, the host red cells and hence the parasite itself remain vulnerable to oxidant stress.

Pathways for the reduction of oxidized glutathione in the Plasmodium falciparum-infected erythrocyte: can parasite enzymes replace host red cell glucose-6-phosphate dehydrogenase?

Plasmodium falciparum-infected human red cells possess at least two pathways for the generation of reduced nicotinamide adenine dinucleotide phosphate (NADPH): (1) the glucose-6-phosphate dehydrogenase (G6PD) pathway and (2) the glutamate dehydrogenase (GD) pathway using glutamate as a substrate. Uninfected erythrocytes lack the GD pathway. The NADPH generated can be used to reduce oxidized glutathione (GSSG), which accumulates in the presence of an oxidative stress. In red cell G6PD deficiency, this pathway is reduced or absent, and the host cells as well as the parasites within them are vulnerable to oxidant stress. In view of the presence of the GD pathway in parasitized red cells and the recent description of a parasite-derived G6PD enzyme, we have asked whether the pathways for the reduction of GSSG provided by the parasite can substitute for the host G6PD in red cells deficient in G6PD activity. We have devised a functional assay in which the reduction rate of GSSG is monitored in the presence of buffered infected or control red cell lysates and substrates. Infected G6PD-deficient erythrocytes were obtained from in vitro cultures after a single prior growth cycle of the parasites in G6PD deficient cells to eliminate contaminating normal red cells. The results show that only parasitized red cells can reduce GSSG via the GD pathway. In parasitized G6PD Mediterranean red cells (completely G6PD-deficient), there is a detectable GSSG reduction via the G6PD pathway, not found in uninfected lysates from the same individual. In G6PD A- (African type, featuring partial deficiency), a small increment in the G6PD-dependent reduction of GSSG can also be detected. However, when compared to G6PD normal red cells, the activities from the parasite-derived pathways are small and could not be considered substitutes for normal host enzyme activity. It is concluded that while the plasmodium provides additional pathways for the generation of NADPH that may serve its own metabolic needs, the host red cells and hence the parasite itself remain vulnerable to oxidant stress.

A contradiction between in vivo and in vitro activities of normal and variant glucose 6-phosphate dehydrogenase.

The presumed "physiologic activity" of normal glucose 6-phosphate dehydrogenase (G6PD), i.e. activity assayed in the presence of physiologic concentrations of ATP, 2,3-diphosphoglycerate, glucose 6-phosphate, NADP and NADPH in the normal red cells, is comparable to shunt pathway activity of intact normal red cells. In contrast, the presumed "physiologic activity" of G6PD variants associated with enzyme deficiency (Gd A- and Gd Mediterranean) is one order of magnitude higher than actual shunt pathway activity of the variant red cells. The difference of kinetic parameters of the enzyme in artificial buffer media and presumed physiologic medium in red cells cannot explain the discrepancy. The apparent discrepancy could be attributed either to (a) over-estimation of NADP and under-estimation of NADPH concentrations in the G6PD deficient red cells due to rapid oxidation of NADPH to NADP in preparation of hemolysate, or to (b) a large portion of NADP which accumulated in the G6PD deficient red cells binds with cellular components of intact red cells and is not available as substrate for G6PD.

In vitro correction of erythrocyte glucose 6-phosphate dehydrogenase (G6PD) deficiency

A high-yield procedure based on hypotonic lysis and isotonic resealing of erythrocytes was used in order to achieve entrapment of homogeneous human glucose 6-phosphate dehydrogenase and of other metabolically related enzyme proteins (hexokinase and 6-phosphogluconate dehydrogenase). Erythrocytes from normal subjects and from individuals affected by glucose 6-phosphate dehydrogenase deficiency (glucose 6-phosphate dehydrogenase Mediterranean) were used for such studies. The following properties of the variously loaded erythrocyte suspensions were investigated: (a) hexose monophosphate shunt activity, (b) glycolytic activity, (c) intracellular concentrations of glucose 6-phosphate, 6-phosphogluconate, NADP, and NADPH. These parameters were evaluated on the erythrocyte suspensions both in the resting state and under oxidative stimulation by methylene blue. In the glucose 6-phosphate dehydrogenase-deficient cells, complete normalization of hexose monophosphate shunt activity and of the other metabolic properties under study was achieved by restoring the intracellular glucose 6-phosphate dehydrogenase activity up to the levels of normal erythrocytes. Specifically, hexokinase became the rate-limiting step of the overall glucose oxidation pathway under methylene blue stimulation, this reproducing the situation found in normal erythrocytes. The extent of Heinz bodies formation upon incubation with acetylphenylhydrazine confirmed the full metabolic competence of the glucose 6-phosphate dehydrogenase-reconstituted Mediterranean erythrocytes compared with the unloaded ones.

Inherited Haemolytic States: Glucose-6-phosphate Dehydrogenase Deficiency

Acute hemolytic anemia could be triggered by oxidative stress in the patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. However, the underlying hemolytic mechanism is unknown. To make clear the hemolytic mechanisms, a systematic study on membrane ultrastructure had been undertaken. A comprehensive method was used including atomic force microscopy, scanning electron microscopy, flow cytometer and fluorescence microscopy to analyze the membrane ultrastructure, externalized phosphatidylserine (PS), intracellular Ca2+ concentration, morphology and the distributions of band 3 protein in G6PD deficient red blood cells (RBCs) after tert-butyl-hydroperoxide (t-BHP) oxidation. The results showed that erythrocyte shrinkage, annexin-V binding to externalized PS on the membrane of early-stage apoptotic cells, the increased membrane roughness and intracellular Ca2+ concentration, as well as the change of distributions of band 3 protein in RBCs. Compared with the control RBCs, as the concentration of t-BHP up to 0.1 mM, the membrane roughness of G6PD deficient RBCs showed significant difference (p < 0.05) and as the concentration of t-BHP up to 0.3 mM, externalized PS showed significant difference (p < 0.05). Furthermore, the population types of RBCs showed dramatic difference between control groups and G6PD deficient groups. Oxidative stress induced more serious erythrocyte apoptosis and resulted in increased roughness of erythrocyte membrane and abnormal distributed band 3 protein in G6PD deficient RBCs. Echinocytes are the predominant abnormal erythrocyte shape occurring in the peripheral blood from patients with G6PD deficiency, which may shorten the RBCs lifespan. The results in the present study will give an increased understanding for the hemolytic mechanism of G6PD deficiency.

Enzyme assays at the single cell level using a new type of microfluorimeter.

A direct method for the microfluorimetric determination of NADPH2 and 4-methylumbelliferon in the sub-picomole range is described. By means of an inverted fluorescence microscope with epi-illumination it has been possible to measure low concentrations of these fluorophores in volumes down to 17 nl. The aqueous microvolumes to be measured are suspended in oil to prevent evaporation. Since the droplets are sunk on thin teflon foil, to attain a spherical form, they can be measured in a reproducible way. The excitation of the microdroplet takes place from below through the teflon foil, while the fluorescence emission follows the same route back to a photomultiplier. The possibilities of this method are illustrated with the biochemical measurements of glucose-6-phosphate dehydrogenase (G6PD) activity in single fibroblasts. It was possible to distinguish between normal and G6PD deficient cells on a single cell level. The microfluorimetric measurement of lysosomal enzymes with 4-methylumbelliferyl substrates is discussed.

Glucose-dependent Protection by Dietary Selenium against Haemolysis of Rat Erythrocytes in vitro

THE occurrence in man of drug-induced haemolysis in glucose-6-phosphate dehydrogenase (G6PD) deficient erythrocytes1 suggested the possibility of an analogy to the haemolysis which occurs in vitamin E deficient red blood cells. Cohen and Hochstein2 have shown that haemolysis in G6PD deficient cells is associated with the inability of the cell to generate adequate reduced glutathione (GSH) through GSSG reductase because of the impaired generation of NADPH. Moreover, there is evidence that glucose protects red blood cells from haemolysis by its ability to provide NADPH through G6PD which subsequently generates GSH3. The G6PD deficient cell, however, cannot maintain an adequate concentration of GSH in the cell, even in the presence of glucose4, whereas the normal cell can maintain a normal concentration of GSH in the presence of glucose, preserving the integrity of the red blood cell. Vitamin E protects red blood cells from haemolysis whether supplied in vivo or in vitro and its effect has usually been demonstrated without glucose in the incubation medium. Although selenium prevents many of the same deficiency symptoms as vitamin E, it has not been uniformly effective in preventing the in vitro haemolysis of red blood cells. If a protective action of selenium against haemolysis were dependent on the presence of GSH, or if selenium were involved in the generation of GSH, selenium would not be expected to prevent haemolysis unless glucose was present in the incubation medium to provide a constant source of NADPH for the generation of GSH from GSSG through GSSG reductase (Fig. 1).

Enhanced binding of FAD to glutathione reductase in G6PD deficiency.

THE activity of glutathione reductase (GR) in erythrocytes deficient in glucose-6-phosphate dehydrogenase (G6PD) is approximately 1.5 to 2 times higher than in red cells with normal G6PD activity1. This may provide a compensatory mechanism for the defect in glutathione reduction in G6PD deficient cells. The cause of the increased activity of GR in G6PD deficiency is unknown and the possibility of increased production of GR in erythrocyte precursors of G6PD deficient individuals seems unlikely. The lower mean red cell age, resulting from a moderately shortened life span of G6PD deficient erythrocytes2, may play a part but is unlikely to account entirely for the marked increase in GR activity. Recently Beutler3 reported a stimulation of erythrocyte GR in normal subjects by flavin–adenine dinucleotide (FAD) in vitro and by supplementation of its precursor riboflavin in vivo. GR activity was shown to depend on dietary intake of riboflavin, but even in persons with a relatively high intake of this vitamin GR does not seem to be saturated with FAD4. These findings suggest that differences of FAD binding of GR may provide an explanation for the increased GR activity in G6PD deficient red cells.