Detoxification of xenobiotics by glutathione S-transferases in erythrocytes: the transport of the conjugate of glutathione and 1-chloro-2,4-dinitrobenzene

The incubation of human erythrocytes with 1-chloro-2,4-dinitrobenzene (CDNB) results in almost quantitative conjugation of glutathione (GSH) to form S-(2,4-dinitrophenyl) glutathione. The reaction is catalysed by erythrocyte glutathione S-transferase. During the present studies we have identified the conjugate in the incubation medium of CDNB-treated erythrocytes, indicating that the conjugate of GSH and CDNB is transported out by the erythrocytes. Quantitation of the conjugate in the incubation medium by amino acid analysis and thin layer chromatography indicates that the erythrocytes transport the conjugate at an approximate rate of 140 nmol/h/ml erythrocytes. The transport of the conjugate is inhibited by sodium fluoride. Exhaustion of ATP from the erythrocytes results in a significant decrease in the rate of transport which is restored with the regeneration of ATP by incubating the erythrocytes with adenine and inosine. This indicates that the transport of conjugate is an energy dependent process.     

Glutathione S-transferases of lung: purification and characterization of human lung glutathione S-transferases

Glutathione S-transferases play a major role in the protection of tissues from the toxic effects of exnobiotics and the products of lipid peroxidation. In the present studies we demonstrate that human lung has two forms of glutathione (GSH) S-transferase having isoelectric pH of 4.9 and 9.2. These anionic and cationic forms represent about 98% and 2% of the total GSH S-transferase activity towards 1-chloro-2,4-dinitrobenzene (CDNB). Although the cationic form accounts for only about 2% of the total GSH S-transferase activity of human lung, it comprises 28% of the total GSH S-transferase protein of lung. Both these enzymes express highest activity with CDNB and have similar K_m values with this substrate, but the anionic enzyme is more active towards a wider range of substrates. Only the cationic enzyme expresses glutathione peroxidase II activity. Anionic and cationic human lung GSH S-transferases are homodimers of about Mr 22,000 and 24,000 subunits, respectively. Antiserum raised against the anionic GSH S-transferase does not cross react with the cationic form, but does cross react with anionic GSH S-transferases of human placenta, liver, and erythrocytes. A close structural interrelationship between the lung anionic form and placental GSH S-transferases is indicated by similar immunological characteristics and the presence of the same N-terminals for both enzymes.     

NADPH, not glutathione, status modulates oxidant sensitivity in normal and glucose-6-phosphate dehydrogenase-deficient erythrocytes

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is characterized by the loss of NADPH and enhanced erythrocyte oxidant sensitivity. Historically, it has been theorized that the elevated oxidant sensitivity of G6PD-deficient erythrocytes arises as the direct consequence of decreased intracellular glutathione (GSH) concentrations. To directly investigate the basis of G6PD deficiency oxidant sensitivity, the effects of altered GSH and NADPH concentrations were examined in normal and G6PD-deficient erythrocytes. The results of this study demonstrated that GSH depletion, by 1-chloro-2,4-dinitrobenzene (CDNB), had no effect on hemoglobin oxidation in response to hydrogen peroxide (H2O2) generating systems (phenazine methosulfate and menadione bisulfite) in either normal or G6PD-deficient cells. Furthermore, a fourfold to sixfold increase in intracellular GSH concentration also did not protect against H2O2-generating systems in the normal or G6PD-deficient erythrocytes. Conversely, introduction of an NADPH-generating system (purified G6PD) into G6PD-deficient cells resulted in a significant decrease in oxidant sensitivity and an ability to cycle GSH. Further experiments demonstrated that the reduced oxidant sensitivity of the G6PD-reconstituted erythrocytes was not due to the maintenance of GSH levels because CDNB-mediated depletion of GSH did not alter this protective effect. Analysis of these results demonstrated a direct correlation between NADPH, but not GSH, concentration and hemoglobin oxidant sensitivity.     

谷胱甘肽抑制蒿甲醚的抗血吸虫作用

目的　观察还原型谷胱甘肽 (GSH)对蒿甲醚伍用氯化血红素抗日本血吸虫作用的影响。　方法　将蒿甲醚、氯化血红素、GSH及谷胱甘肽耗竭剂 2 ,4 -二硝基氯苯 (CDNB)单一或伍用加入含 5周龄血吸虫的培养液内 ,温育2 4 h后 ,测定虫体的丙二醛含量 ,并观察培养至 96 h虫体存活情况。感染小鼠经蒿甲醚 30 0 mg/kg治疗后 6、12或 2 4h,测定虫体的 GSH水平。　结果　体外 ,蒿甲醚伍用氯化血红素作用血吸虫 2 4 h后 ,虫丙二醛含量明显升高。培养时间延长 ,虫陆续死亡。GSH对蒿甲醚伍用氯化血红素诱导血吸虫脂质过氧化及杀虫作用具有拮抗作用 ,CDNB则有增强作用。体内 ,蒿甲醚作用血吸虫 6～ 2 4 h,虫体内 GSH水平先降低再明显升高。　结论　 GSH可能在血吸虫防御蒿甲醚衍生的毒性过氧化物和自由基攻击中起重要作用。     

The Gardos channel is responsible for CDNB-induced dense sickle cell formation

The red blood cells (RBCs) derived from blood taken from homozygous sickle cell (SS) patients demonstrate densities that are inversely proportional to the intracellular reduced glutathione (GSH) content. Addition of 1 mM 1-chloro-2,4-dinitrobenzene (CDNB) to low-density sickle cells (LDSS), at 4 degrees C, results in a shift of LDSS erythrocytes to high-density sickle cells (HDSS), with corresponding decreases in GSH. We have previously demonstrated that this CDNB effect was due to increased K(+) leakage and that dense cell formation could be inhibited by clotrimazole (specific for the Gardos channel) but not DIOA (specific for the K(+)-Cl(-) co-transport system) at pH 7.4 (Shartava et al. Am. J. Hematol. 1999;62:19-24). Here we demonstrate that clotrimazole (10 microM) inhibits dense cell formation at pH 7.1 and 6.8, while DIOA (1 mM) has no effect. As pH 6.8 is the optimal pH for the K(+)-Cl(-) co-transport system, we can now reasonably conclude that damage to the Gardos channel is responsible for CDNB-induced dense cell formation.     

Erythrocyte-oxidized glutathione transport in pyrimidine 5'-nucleotidase deficiency

The oxidized form of glutathione transport was studied in human erythrocytes in pyrimidine 5'-nucleotidase (P5N) deficiency, a disorder in which the amounts of CTP and UTP in the erythrocytes are elevated. The inhibition of ATP-requiring oxidized glutathione (GSSG) transport by CTP and UTP is believed to play a role in elevating the levels of the reduced form of glutathione (GSH) in the erythrocytes of patients with P5N deficiency. The current investigation was undertaken to determine if GSSG transport actually decreases in the erythrocytes of such patients. Erythrocytes from a 17-year-old patient and a 13-year-old patient with P5N deficiency hemolytic anemia and from ten normal subjects were used as materials for the experiment. Erythrocytes, which had been previously incubated with [3H]glycine, were incubated at 37 degrees C, and the rate of [3H]GSSG transported by the cells was estimated. The velocity of GSSG transport out of the erythrocytes was quite low in the patients, 3.17-3.65 nmol GSSG/ml erythrocytes/hr at 37 degrees C in one case, and 3.30 nmol GSSG/ml erythrocytes/hr in the other case, vs that in the normal controls (6.00 +/- 0.80 nmol GSSG/ml erythrocytes/hr; mean +/- SD). The activity of gamma-glutamylcysteine synthetase and glutathione synthetase did not decrease in the patients. Decreased transport activity of GSSG in addition to a normal synthesis rate for GSH may explain the increased concentration of erythrocyte GSH in P5N deficiency.     

Increased glutathione conjugate transport: a possible compensatory protection mechanism against oxidative stress in obesity?

OBJECTIVE: To compare glutathione S-conjugate transport in obese and nonobese persons, and how glutathione S-conjugates are involved in the antioxidant status in obesity. MATERIALS AND METHODS: The efflux of glutathione conjugates and malondialdehyde (MDA) levels were measured in erythrocytes of obese (N = 33) and nonobese (N = 28) persons at every 30 min during a 120 min incubation time in vitro. 2,4-dinitrophenyl-S-glutathione (DNP-SG) represented the glutathione S-conjugate. RESULTS: The efflux of conjugate in erythrocytes from obese subjects (708 +/- 147 DNP-SG efflux nmol/ml erythrocytes/h) was significantly higher than that of control group (490 +/- 105 DNP-SG efflux nmol/ml erythrocytes/h) (P < 0.05). At all time points measured (30-120 min), there was an increase in DNP-SG efflux in obese group (P < 0.05). This is manifested by a decrease in cellular DNP-SG levels. The susceptibility of erythrocytes to in vitro 1-chloro-2,4-dinitrobenzene (CDNB)-induced oxidative stress were greater for cells of control group (P < 0.05), although hemolysis sensitivity of these cells are not different between both groups (P > 0.05). Following CDNB pretreatment, incubation of erythrocyte with vanadate, a DNP-SG transport inhibitor, resulted in an increase of MDA in both groups. However, in this case, the difference in susceptibility was not related to obesity. On the other hand, while erythrocyte glutathione level was lower in obese subjects (79% of control) than in controls (P < 0.05), the adenosine 5'-triphosphate (ATP) levels, the enzyme activities of glutathione S-transferase (GST) and the conjugation capacities of the erythrocytes were not different between groups (P>0.05). CONCLUSION: Obesity may increase erythrocyte glutathione conjugate transport independent from ATP and GST activity that may protect against MDA formation in vitro.     

Erythrocyte glutathione S-transferase deficiency and hemolytic anemia

A patient with unexplained erythrocyte glutathione-S-transferase (GST) deficiency has been detected among 513 unrelated persons with hemolytic anemia. An otherwise healthy adult male, the deficient individual had a mild hemolytic anemia with splenomegaly, indirect hyperbilirubinemia, and cholelithiasis. Because he was adopted and childless, the hereditary nature of the defect could not be established. The residual enzyme activity was only about 15% of mean normal. Depletion of glutathione (GSH) from the cells by 1-chloro-2,4-dinitrobenzene (CDNB), a substrate for GST, was somewhat decreased in the red cells from the patient, suggesting that a functional defect existed. The kinetic properties of the residual enzyme and the ratio of activity to antigenicity were normal. Modest decreases in leukocyte and platelet GST activities were documented. Although a cause-and-effect relationship between the GST deficiency and hemolysis may exist, this cannot be proven in the absence of affected family members.     

N-acetylcysteine and clotrimazole inhibit sickle erythrocyte dehydration induced by 1-chloro-2,4-dinitrobenzene.

Clotrimazole, a specific inhibitor of the Ca(2+) activated potassium (Gardos) channel, and the antioxidant N-acetylcysteine were found to inhibit the in vitro formation of high-density sickle cells induced by treatment with 1-chloro-2,4-dinitrobenzene (CDNB). The CDNB induced leakage of K(+) can be inhibited by treatment of SS erythrocytes with 20 mM N-acetylcysteine. We conclude that the effect of N-acetylcysteine in preventing K(+) efflux and formation of high-density sickle cells is related to its ability to protect the Gardos channel from oxidative damage caused by diminished levels of reduced glutathione. This effect is due to the ability of N-acetylcysteine to maintain an appropriate level of reduced glutathione and its direct antioxidant activity.     

Normal glutathione content and some related enzyme activities in the fetal erythrocytes

Pure fetal blood was obtained by direct-vision fetoscopy from 66 fetuses at 17-24 weeks gestation. The concentration of GSH and the activities of the enzymes gamma-glutamylcysteine synthetase (GCS), glutathione synthetase (GS), glutathione reductase (GR) and glutathione peroxidase (GPx) were analysed by established techniques to find the normal ranges for this gestational age. The ranges were relatively narrow and could serve as reference values for the prenatal diagnosis of defects in the GSH metabolism of erythrocytes. The results were compared with those obtained from 38 normal adults and with published values on neonatal blood. In the case of GR a comparison was also made with maternal blood. In comparison with adults, fetal erythrocytes showed higher GSH concentration and GCS activity and lower GS and GPx activities. This pattern resembled that found in neonatal erythrocytes except for the GCS activity, which was higher in the fetal cells. Furthermore the differences between fetal and adult erythrocytes were more pronounced than those between neonatal and adult cells. The GR activity of fetal erythrocytes was also higher than that of either normal adult or maternal blood. This difference, however, was reduced to an insignificant level when the enzyme was activated in vitro by flavin adenine dinucleotide (FAD) because of a relatively low per cent activation of the GR in the fetal erythrocytes.     

Elevated glutathione accelerates oxidative damage to erythrocytes produced by aromatic disulfide

It has been shown that certain dogs have erythrocytes characterized by an inherited high concentration of reduced glutathione (GSH), five to seven times the normal level (high-GSH RBCs). We examined whether increased GSH in dog erythrocytes leads to increased protection against oxidative damage induced by acetylphenylhydrazine (APH) and/or 4-aminophenyl disulfide (4-AD). When erythrocytes were incubated with 30 mmol/L APH, the Heinz body count was appreciably higher in normal RBCs than in high-GSH RBCs, while there was no difference in the increase of the methemoglobin (metHb) concentration in both RBCs. In contrast, both the Heinz body count and metHb production were much higher in high-GSH RBCs than in normal RBCs when erythrocytes were incubated with 4-AD. Furthermore, the generation of the superoxide in erythrocytes treated with 4-AD, which was measured by spin trapping combined with electron spin resonance (ESR), was obviously higher in high-GSH RBCs than in normal RBCs. These results clearly indicate that erythrocyte GSH is an important defense against oxidative damage induced by certain compounds such as APH, but that, in contrast, elevated GSH appears to accelerate oxidative damage to erythrocytes produced by aromatic disulfides, such as 4-AD, which generated a superoxide in erythrocytes via its redox reaction with GSH.     

Red blood cells as a physiological source of glutathione for extracellular fluids

Plasma low molecular mass thiols are represented by glutathione, cysteine, cysteinylglycine and homocysteine. The physiological mechanisms responsible for maintaining the homeostasis of these compounds in the intracellular and extracellular spaces have not been fully clarified. Erythrocytes possess the enzymatic machinery to synthesize glutathione and an efflux of glutathione disulfide and glutathione conjugates from erythrocytes under various conditions occurs. In this study, the property of red blood cells (RBCs) to export low molecular mass thiols has been assessed. Plasma concentration of low molecular mass thiols has been measured in healthy volunteers by HPLC and a significant correlation with RBC number has been observed for glutathione and cysteinylglycine. A sustained export of reduced glutathione has been observed (about 21 nmol/h/ml RBCs) together with a lower, though significant, efflux of both cysteine and homocysteine. These results suggest that erythrocytes can contribute significantly to the extracellular pool of glutathione (GSH), thus cooperating with liver and other tissues to the dynamics of inter-organ GSH metabolism.     

Oxidant damage to erythrocyte membrane in glucose-6-phosphate dehydrogenase deficiency: correlation with in vivo reduced glutathione concentration and membrane protein oxidation

Chronic nonspherocytic hemolytic anemia has been observed in a recently described glucose-6-phosphate dehydrogenase (G6PD) variant, G6PDWayne. The mechanical properties of these erythrocytes and other G6PD variants were examined. The deformability of G6PD-deficient erythrocytes was normal, as determined by osmotic scan ektacytometry, and was not significantly affected by hemolytic crisis. In the common varieties of G6PD deficiency, the mechanical stability of the red blood cell (RBC) membrane was greater than normal, but G6PDWayne membranes were abnormally susceptible to shear-induced fragmentation. There was no evidence for a concurrent genetic defect in spectrin, because self- association constants and tryptic digests were normal. The fragility of G6PDWayne membranes appeared to be a consequence of oxidative damage to membrane thiol groups associated with a low glutathione (GSH) level in these RBCs. Associations among GSH level, thiol oxidation, and membrane instability were also found when a larger group of G6PD-deficient RBCs were examined. In normal erythrocytes, 1-chloro-2,4-dinitrobenzene was used to reduce GSH levels by 50%. Membrane thiol oxidation and membrane fragility both increased when these cells were kept at 4 degrees C for 3 to 5 days. Our findings suggest that chronic depletion of GSH leads to the destabilization of membrane skeleton through oxidation of membrane protein thiols.     

Hydroxylamine Treatment Increases Glutathione-Protein and Protein-Protein Binding in Human Erythrocytes

ABSTRACT: Hydroxylamine is a direct-acting hematotoxic agent leading to hemolytic anemia in animals and man. The effect of hydroxylamine on the morphology, sulfhydryl status and membrane skeletal proteins of human erythrocytes were studied. Loss of reduced glutathione (GSH) from the red blood cells was directly proportional to the hydroxylamine concentration used. This loss of GSH was larger than the sum of the increase in the amounts of extracellular glutathione and intracellular oxidized glutathione (GSSG). The extracellular glutathione is mainly present as GSSG, which is in agreement with the fact that only GSSG is exported from the erythrocytes by membrane bound ATPases. Lack of GSSG export was not limited by decreased ATP levels in the erythrocytes and we concluded that the GSH that disappeared did not become available as intracellular GSSG. After reduction of the erythrocyte incubates the lost GSH was almost completely recovered indicating that the lost GSH is present in the cell as protein-glutathione mixed disulfides. Glutathione thus stored within the cell can be quickly recovered by combined thioltransferase and glutathione reductase activity when conditions become more favorable again. SDS-polyacrylamide gel electrophoresis of membrane ghosts from human red cells revealed changes in skeletal proteins with a smearing of bands 1, 2 and 3 to the higher molecular weight end of the gel and the appearance of new monomeric and dimeric hemoglobin bands at about 16 and 30 kD. The observed alterations are probably a consequence of disulfide bridge formation between cellular proteins (mainly hemoglobin) and skeletal proteins as well as between hemoglobin monomers. Exposure of hydroxylamine to erythrocytes caused severe Heinz body formation but the outside morphology of the cells was only marginally altered. The described changes in sulfhydryl status of the red blood cells are likely to play a major role in the premature splenic sequestration of hydroxylamine-damaged erythrocytes.     

Biochemical analysis of a recombinant glutathione transferase from the cestode Echinococcus granulosus

Glutathione transferases (GSTs) are believed to be a major detoxification system in helminths. We describe the expression and functional analysis of EgGST, a cytosolic GST from Echinococcus granulosus, related to the Mu-class of mammalian enzymes. EgGST was produced as an enzymatically active dimeric protein (rEgGST), with highest specific activity towards the standard substrate 1-chloro-2,4-dinitrobenzene (CDNB; 2.5 μmol min⁻¹ mg⁻¹), followed by ethacrynic acid. Interestingly, rEgGST displayed glutathione peroxidase activity (towards cumene hydroperoxide), and conjugated reactive carbonyls (trans-2-nonenal and trans,trans-2,4-decadienal), indicating that it may intercept damaging products of lipid peroxidation. In addition, classical GST inhibitors (cybacron blue, triphenylthin chloride and ellagic acid) and a number of anthelmintic drugs (mainly, hexachlorophene and rafoxanide) were found to interfere with glutathione-conjugation to CDNB; suggesting that they may bind to EgGST. Considered globally, the functional properties of rEgGST are similar to those of putative orthologs from Echinococcus multilcularis and Taenia solium, the other medically important cestodes. Interestingly, our results also indicate that differences exist between these closely related cestode GSTs, which probably reflect specific biological functions of the molecules in each parasitic organism.     

Efflux of glutathione and glutathione complexes from human erythrocytes in response to vanadate

The main objective of the present study was to investigate if vanadate is extruded from the cells in a glutathione dependent manner resulting in the appearance of extracellular glutathione and complexes of glutathione with vanadium. Vanadate significantly depleted intracellular non-protein sulfhydryl (NPSH) levels in a time- and concentration-dependent manner. The intracellular NPSH level was decreased to 0.0 ± 0.0 μmol/ml erythrocyte when exposed to 10 mM of vanadate for 4 h. Extracellular NPSH level was increased concomitantly with the intracellular decrease and reached to 0.1410 ± 0.005 μmol/ml erythrocyte in 4 h. Intracellular decrease and extracellular increase in NPSH levels were significantly inhibited in the presence of DIDS, a chloride-bicarbonate exchanger which also mediates phosphate and arsenate transport in erythrocytes. In parallel with the increase in extracellular NPSH levels, significant increases in extracellular glutathione levels were detected following exposure to vanadate. Extracellular glutathione levels reached to 0.0150 ± 0.0.001, 0.0330 ± 0.001, and 0.0576 ± 0.002 μmol/ml erythrocyte with 1, 5, and 10 mM of vanadate respectively. Dimercaptosuccinic acid treatment of supernatants significantly increased the glutathione levels measured in the extracellular media. Utilization of MK571 an MRP inhibitor decreased the rate of glutathione efflux from erythrocytes suggesting a role for this membrane transporter in the process. A known methylation inhibitor periodate oxidized adenosine decreased the rate of glutathione efflux from erythrocytes. This observed decrease in extracellular GSH levels suggests that GSH release partly requires a proper cellular methylation process and that part of GSH detected in the extracellular media may arise from GSH–vandium complexes. The results of the present study indicate that human erythrocyte efflux glutathione in reduced free form and in conjugated form/s that can be recovered with dimercaptosuccinic acid when exposed to vanadate.     

The effects of exogenous glutathione on reduced glutathione level, glutathione peroxidase and glutathione reductase activities of rats with different ages and gender after whole-body Γ-irradiation

Age-and gender-related changes on reduced glutathione (GSH) level, glutathione peroxidase (GPx) and glutathione reductase (GR) activities in the liver of rat exposed to different dose of whole-body g-ray irradiation were determined. In addition, the effect of administration of exogenous GSH on endogenous GSH levels, GPx and GR activities was investigated. For this aim, male and female rats aged 1 and 5 moths were divided into two groups as g-ray and g-ray+GSH. Both groups were again divided into four groups as irradiated with 2, 4, 6 and 8 Gy doses. GSH level and GPx activity did not change with age while GR activity was decreased with age. Gender-dependent changes in GPx and GR activities were observed, but GSH values were not affect by sex. GSH levels, GPx and GR activities were not observed dose-associated changes of g-irradiation. GSH level and GPx activity in the 8Gy group were increased by GSH. GR activities of old male rats were found to be increased by glutathione in the 6 and 8Gy groups. These results indicate that radiation and administration of exogenous GSH affect gender-and age-dependent GSH level, GPx and GR activities in the rats.     

Activation of rat hepatic stellate cells leads to loss of glutathione S-transferases and their enzymatic activity against products of oxidative stress.

Oxidative stress, mediated partly by lipid peroxidation products, may lead to increased collagen synthesis by hepatic stellate cells (HSC). Stellate cells are protected from oxidative stress by enzymes of detoxication such as the glutathione S-transferases (GSTs), which form glutathione conjugates with lipid peroxidation products (e.g., 4-hydroxy-2-nonenal [HNE]). To better understand the role of GSTs in stellate cell biology, we examined the expression and enzymatic activity of GSTs in normal and activated (both culture- and in vivo-activated) stellate cells. Normal stellate cells contained numerous isoforms of GST including those that detoxify HNE. High levels of enzymatic activity toward 1-chloro-2,4-dinitrobenzene (CDNB) and HNE were present in normal stellate cells and were similar to levels present in whole liver. Following activation by growth in culture, the expression of several GSTs (rGSTA1/A2, A3, and M1) was lost. Also, enzymatic activities toward CDNB and HNE fell approximately 90%. However, expression of rGSTP1 was maintained. A similar loss of rGSTA1/A2, A3, and M1 with persistent expression of rGSTP1 was present after activation in vivo. Furthermore, we identified 2 subpopulations of activated stellate cells with different GST phenotypes from injured livers. In summary, activated stellate cells lose most forms of GST and associated enzymatic activities that are present in normal stellate cells. The findings raise the possibility that activated stellate cells have less ability to detoxify lipid peroxidation products and may be susceptible to oxidative stress. Additionally, we propose that the phenotypic change in GSTs is a sensitive marker of stellate cell activation.     

Oxidative state of glutathione in arterial and venous red blood cells and plasma of rabbit

Reduced (GSH) and oxidized (GSSG) glutathione levels in arterial and venous rabbit red blood cells (RBC) and plasma are reported. No significant differences were noted between arterial and venous erythrocytes GSH and GSSG content. Plasma GSH levels were found to be the same in both arterial and venous samples. Contrarily, the concentrations of GSSG were higher in venous than in arterial plasma. These findings cannot be attributed solely to the increased RBC oxidative activity that occurs in the venous system. The phenomenon may reflect the oxidative state present in other tissues and may thus be a harbinger of differences in GSH content.     

Selective occurrence of glutathione instability in red blood corpuscles of the various Jewish tribes

1. Experience with GSH stability test performed on blood samples to whichglucose has been added is summarized and the procedure is recommendedfor routine use for the detection of sensitive subjects.

2. Glutathione stability of erythrocytes has been studied in various population groups of Israel. No case with instable GSH has been found among Jewishsubjects originating from Eastern, Central or Western Europe. Instabilityof GSH was however found in about 20 per cent of subjects originating fromIraq and about 5 per cent of subjects originating from Yemen or North Africa.Isolated cases of this abnormality were also discovered among a small numberof persons from other Oriental or Mediterranean countries as well as amongthe Arab inhabitants of Israel.

3. The genetic analysis points to a transmission of glutathione instabilityby a sex-linked, incompletely dominant gene with variable expressivity.

4. Variable expressivity of glucose-6-phosphate dehydrogenase activity hasalso been detected in the defective erythrocytes.

Submitted on December 5, 1957 Accepted on April 12, 1958