Pentose Phosphate Shunt Activity Research Articles

Maintenance of cellular NADPH is critical to intestinal cell survival during oxidative challenge: Role of glucose and pentose phosphate shunt activity

Cellular redox balance is maintained by the status of reduced glutathiona (GSH) and glutathione disulfide (GSSG) through the function of GSH pemxidase and GSSG reductase coupled to the NADP+/NADPH redox pair. DUring oxidative challenge enhanced glucose supply and pentose phosphate shunt activity to support NADPH production for GSSG reduction is critical to restonng redox homeostasis and maintaining cell survival. The current objective was to test the hypothesis using two intestinal cell lines with distinct cellular redox status, viz., the normal human colon epithelial NCM 460 and the human coIon adenocarcinoma G'~Co2 cell lines Cells were treated with the thiol oxidam, diamide, to induce cellular GSH/ GSSG imbalance without oxyradical generation. Cellular redox, NADPH, redox cycle enzymes and cell apopmsis were determined. Cells were cultured in normal (25 raM) or reduced (5 raM) glucose DMEM to evaluate the role of glucose in diamideqnduced apoptosis. NCM 460 cells exhibited 50% more cellular GSH and a 10-fold higher glucose 6-phosphate dehydrogenase (~PD) activity. Diamide at 200, 500 or 750 ~M significantly increase cell apoptosis at 24h in CaCo-2 (15%. 25% & 55%, versus 5%, 10% & 20% in NCM460, respectively). Diamide initiated an early ( i 5 rain) GSH loss with GSSG increase in both cell lines, but NCM460 ceils recovered more rapidly fi'om this imbalance. Basehne NADPH levels were similar in tbe two cell lines, but fell markedly after diamide treatment in CaCo-2 cells, indicating a decreased capacity for NADPH production, consistent with higher GSSG and reduced G6PD activity in these cells. CaCo-2 cells grown in reduced glucose DMEM (5 raM), were more vulnerable to diamide challenge (45% at t.500 M diamide), confirnung the importance ot glucose supply in NADPH generation to sustain GSSG reduction and cell survival These results show that intestinal cells like NCM460, which possess higher redox and antioxidant potentials, are more resistant to diamide-induced apoptosis. This resistance is related to the cell's ability to maintain intracdlular GSH redox balance during diantide stress through enhanced NADPH produetinn by the pentose phosphate pathway-. The results underscore tim rate limiting role of G6PD and glucose availability in the function of the shunt Supported hy NIH grant DK 44510,

Pyrimidine 5' nucleotidase deficiency.

Pyrimidine 5' nucleotidase deficiency.

Increased islet DNA synthesis and glucose-derived lipid and amino acid production in association with beta-cell hyperproliferation in normoglycaemic 60 % pancreatectomy rats.

Glycaemia does not change following a 60 % pancreatectomy in rats because of enhanced beta-cell function and proliferation (so-called beta-cell adaptation). We previously studied these rats 4 weeks after surgery and showed hypersensitization of glucose-induced insulin secretion because of increased glucokinase activity. In this study of 60 % pancreatectomy rats 5 days after surgery, when beta-cell proliferation increased threefold, we investigated whether increases in glucose metabolism enhance the production of glucose-derived lipid, amino acids and DNA. Isolated islets from 60 % pancreatectomy and sham-operated control rats 5 days or 4 weeks after surgery were studied. Five days after 60 % pancreatectomy surgery, islet glucose phosphorylation increased threefold, but overall glucose usage increased only twofold. The glucose-6-phosphate (G6P) concentration thus doubled, resulting in a sixfold increase in G6P metabolism through the pentose phosphate shunt (PPS). The pentose phosphate shunt generates ribose-5-phosphate for nucleotide synthesis, and DNA synthesis doubled in the partial pancreatectomy islets. In contrast, partial pancreatectomy rats 4 weeks after surgery had a smaller increase in glucokinase activity and their islet glucose-6-phosphate concentration and pentose phosphate shunt activity were equal to that of the control rats. DNA synthesis and beta-cell proliferation, based on BrdU incorporation were close to normal. Another consequence of the heightened glucose metabolism in the 5-day partial pancreatectomy islets was twofold increase in production of glucose-derived lipid and the amino acids, alanine and glutamate. The enhanced glucokinase activity in 60 % pancreatectomy rats supports the compensatory beta-cell hyperproliferation by increasing production of glucose-derived DNA, lipids and amino acids.

Fatty acid-induced beta cell hypersensitivity to glucose. Increased phosphofructokinase activity and lowered glucose-6-phosphate content.

Diabetic states are characterized by a raised serum/islet level of long chain fatty acids and a lowered ED50 for glucose-induced insulin secretion. Prolonged culture (> 6 h) of islets with long chain fatty acids replicates the basal insulin hypersecretion. We examined this effect in rat islets cultured for 24 h with 0.25 mM oleate. Insulin secretion at 2.8 mM glucose was doubled in combination with a 60% lowered islet content of glucose-6-phosphate (G6P). Investigation of the lowered G6P showed: (a) increased glucose usage from 0.5 to 100 mM glucose with identical values measured by [2-3H]glucose and [5-3H]glucose, (c) indicating little glucose- 6-phosphatase activity, (b) unchanged low pentose phosphate shunt activity, (c) 50% increased phosphofructokinase (PFK) Vmax, (d) a normal ATP/ADP ratio, and (e) unchanged fructose 2,6 bisphosphate content. Triacsin C, an inhibitor of fatty acyl-CoA synthetase, prevented the increase in PFK activity and the lowered G6P content. These results suggest that long chain acyl-CoA mediates the rise in PFK activity, which in turn lowers the G6P level. We speculate that the inhibition of hexokinase by G6P is thus attenuated, thereby causing the basal insulin hypersecretion.

The Regulatory Role for Magnesium in Glycolytic Flux of the Human Erythrocyte

31P NMR was used to measure the intracellular free magnesium concentration ([Mg2+]i) in human erythrocytes while [Mg2+]i was changed between 0.01 and 1.2 mM using the divalent cationophore A23187. 13C NMR and [2-13C]glucose were used to determine the kinetic effects of [Mg2+]i by measuring the flux through several parts of the glucose pathway. Glucose utilization was strongly dependent on [Mg2+]i, with half-maximal flux occurring at 0.03 mM. The rate-limiting step was most likely at phosphofructokinase, which has a Km(Mg2+) of 0.025 mM in the purified enzyme. Phosphorylated glycolytic intermediate concentration was also strongly dependent on [Mg2+]i and [MgATP], and glucose transport plus hexokinase may have been partially rate-determining at [Mg2+]i below approximately 0.1 mM. The pentose phosphate shunt activity was too low to determine the dependence on [Mg2+]i. Phosphoglycerate kinase and 2, 3-diphosphoglycerate mutase fluxes were also measured, but were not rate-limiting for glycolysis and showed no Mg2+ dependence. Human erythrocyte [Mg2+]i varies between 0.2 mM (oxygenated) and 0.6 mM (deoxygenated), well above the measured [Mg2+]i(1/2). It is unlikely, then, that [Mg2+]i plays a regulatory role in normal erythrocyte glycolysis.

Oxidative damage to RBC membranes and pentose phosphate shunt activity in hemodialysis patients after suspension of erythropoietin treatment.

During follow-up of anemic hemodialysis patients (HDP) treated with recombinant human erythropoietin (rHuEpo), it was noticed that in five HDP, some time after suspension of rHuEpo, hemoglobin (Hb) levels remained at acceptable levels. A metabolic block of the pentose phosphate shunt (PPS) has been described in HDP, which leads to increased oxidative damage of red blood cell (RBC) membranes and increased susceptibility to hemolysis. The increased production of short-chain fatty aldehydes, including malonyldialdehyde (MDA), is an appropriate index of oxidative damage. This study aimed to verify whether the maintenance of acceptable levels of Hb was related to a change in RBC membrane oxidative damage and pentose phosphate shunt activity. In the five HDP in question who required rHuEpo (150 U/kg/week) for severe anemia (Hb = 7.48 +/- 0.95 g/dl), after a stable level of Hb > 10 g/dl was reached for at least 1 month, rHuEpo treatment was stopped. Hb levels remained adequate (Hb = 10.68 +/- 0.77 g/dl) after 14.6 +/- 7.64 months. The oxidative damage was evaluated by measuring RBC MDA (microgram/ml packed RBC) basal levels, and PPS activity by measuring MDA levels after incubation with ascorbate and cyanide (delta % RBC MDA production). Ten anemic HDP not treated with rHuEpo were used as controls (Hb = 8.12 +/- 1.32 g/dl). It was found that the maintenance of adequate levels of serum Hb after suspension of rHuEpo therapy is related to a decrease in RBC membrane oxidative damage (RBC MDA HDP = 2.40 +/- 0.41 vs. RBC MDA controls = 18.23 +/- 6.56; P < 0.005) in consequence of the normalization of pentose phosphate shunt activity.

Cytophotometric assay of cytochrome oxidase, lactate dehydrogenase and glucose-6-phosphate dehydrogenase activities in human peroxidized spermatozoa

Human spermatozoa contain appreciable amounts of intracellular glutathione, which has a protective function against peroxidative degradation of spermatozoal polyunsaturated fatty acids by the NADPH-dependent glutathione peroxidase/reductase enzymatic system. The glutathione system provides a basic defense against peroxidative damage, without which the superoxide dismutase system would dominate. Since oxidative damage is said to include enzyme leakage and changes in metabolism, cytochrome oxidase and lactate dehydrogenase activities were used as indicators of the energy metabolism in unwashed and washed human spermatozoa during lipid peroxidation. Lipid peroxidation was induced by aerobic incubation of sperms in the presence of sodium ascorbate and ferrous sulphate. In addition, since NADPH concentrations influence the concentration of reduced glutathione, we studied glucose-6-phosphate dehydrogenase activity as an indicator of pentose phosphate shunt activity, the main source of NADPH. Microdensitometric measurements of the three enzymes were made by a Vickers M85a scanning microdensitometer. We found that the lipid peroxidation process greatly affects the 3 enzymatic activities examined and that seminal plasma protects against the extensive deleterious effects of lipid peroxidation.

Characterization of Red Blood Cell Metabolism in Rainbow Trout

Red blood cell metabolism was studied in vitro using whole blood obtained by catheter from resting rainbow trout (Oncorhynchus mykiss). Preparations were viable as shown by stable NTP, metabolite and catecholamine levels and acid-base status, all of which remained at in vivo levels over the 2 h incubation period. Enzymes diagnostic of glycolysis, the tricarboxylic acid (TCA) cycle and phosphagen metabolism were all present in significant amounts in red blood cells. In direct comparisons of 14C-labelled substrates at normal resting plasma concentrations, rates of CO2 production were in the order: glucose greater than lactate greater than alanine greater than oleate. Total CO2 production rates from these four oxidative substrates did not equal directly measured O2 consumption rates, indicating that other substrates may also be important in vivo. Oxidative pathway Km values for glucose (8.4 mmol l-1), lactate (3.3 mmol l-1) and alanine (0.8 mmol l-1) were well within the normal physiological ranges of plasma concentrations. Glucose concentration did not affect lactate oxidation rates, but there was some inhibition (27%) of glucose oxidation by high lactate concentrations (20 mmol l-1). The observed Km values and competitive interactions suggest that changes in plasma concentrations associated with environmental stresses can considerably alter the relative rates of oxidation of glucose and lactate in vivo. Considerable pentose-phosphate shunt activity was detected in red cells, as indicated by high activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and high CO2 production rates from (1-14C)-labelled glucose. Even in the presence of normal O2 levels, a significant percentage (28%) of glucose metabolism was directed to lactate production. Taken together, these results demonstrate that rainbow trout whole blood incubated in vitro constitutes a dynamic and viable system for metabolic studies at the pathway level.

Integration of lipid metabolism in the mammary gland and adipose tissue by prolactin during lactation

Prolactin deficiency, induced by bromocryptine treatment, brought about reciprocal changes in the ability of adipocytes and acini isolated from lactating rats to synthesize lipids. The capacity to synthesize fatty acids and phospholipids decreased in the mammary gland and increased in adipocytes by bromocryptine treatment. In the mammary gland, the maximum potential activity of the pentose shunt as well as the specific activities of the pathway dehydrogenases were significantly reduced by bromocryptine treatment. Simultaneously, adipose tissue increased its lipogenic capacity but neither the maximum potential of the shunt nor the specific activities of the pentose phosphate shunt dehydrogenases were significantly changed with respect to the control lactating rats. Thus, a differential regulatory mechanism(s) of the pentose phosphate shunt activity appears to operate in these two tissues. Adipocytes from lactating rats showed a poor responsiveness to insulin in terms of lipid synthesis from glucose. In contrast, in adipocytes from bromocryptine treated rats insulin was able to increase lipid synthesis (105%). Sheep prolactin administration 'in vivo' partially reversed the effects of bromocryptine. These data suggest that prolactin mediates adipocytes resistance to insulin during lactation. Phospholipid synthesis, as occurred in fatty acid synthesis, is increased in adipose tissue and decreased in mammary gland by bromocryptine treatment. However, alpha 1-adrenergic stimulation increases phosphatidylinositol turnover to about the same percentages in both mammary gland acini and adipocytes from lactating rats independently of bromocryptine treatment.

Prostaglandin E 2 and α2 adrenoceptor agonists inhibit the pentose phosphate shunt in pancreatic islets

Glucose utilization in isolated pancreatic islets of the rat was inhibited by prostaglandin (PG) E 2 and the α 2 adrenoceptor agonist, clonidine, to a similar extent; other prostaglandins did not affect glucose utilization. Islet oxidation of [1- 14C]glucose and [6- 14C]glucose demonstrated that the pentose phosphate shunt was inhibited by PGE 2 and clonidine. Pertussis toxin antagonizes the effects of clonidine and PGE 2 on total glucose utilization and pentose phosphate shunt activity. The results suggest that PGE 2 and α 2 adrenoceptor agonists may regulate glucose metabolism through similar transduction mechanisms, and that a guanine nucleotide binding regulatory (G) protein modulates certain metabolic effects of prostaglandins and adrenergic agonists.

Effect of CCK-8 on pentose phosphate shunt activity, pyridine nucleotides, and glucokinase of rat islets.

In rat pancreatic islets the effects of cholecystokinin octapeptide (CCK-8) on pentose phosphate shunt (PPS) activity, glucokinase and hexokinase activity, and NADPH, NADP+, NADH, and NAD+ were studied. By elevating the glucose concentration from 3.0 to 8.3 and 16.7 mM the oxidation of [1-14C]- and [6-14C]glucose and the calculated PPS activity were increased in a concentration-dependent manner; 10 nM CCK-8 enhanced selectively the effect on [1-14C]glucose oxidation thereby increasing the PPS activity but only at an intermediate glucose concentration (8.3 mM). CCK-8 had no effect on glucokinase or hexokinase activity and CCK-8 did not influence glucose utilization. By elevating the glucose concentration, total NADPH and NADH were increased and total NADP+ and NAD+ were decreased. CCK-8 (10 nM) increased selectively NADPH and decreased NADP+ but did not change NADH or NAD+; the effect of CCK-8 on NADPH and NADH was only observed in the presence of an intermediate stimulatory glucose concentration (8.3 mM) but not at either a substimulatory glucose concentration or a maximally stimulatory glucose concentration for insulin release (3.0 or 16.7 mM). The data indicate first that CCK-8 does not act on glucose phosphorylation or glucose utilization and second that CCK-8 increases PPS activity and NADPH levels in rat pancreatic islets. Since the concentrations of glucose necessary for these CCK-8 effects are in the range of 8.3 mM and parallel with those necessary for insulin release as shown in earlier observations, glucose oxidation via pentose phosphate shunt and NADPH are suggested to be related to the CCK-8-modulated insulin release.

Pentose phosphate shunt metabolism by cells of the chick growth cartilage

We have measured the activity of the pentose shunt pathway in the chick growth cartilage. Measurement of D-[1- 14C] glucose and D-[6- 14C] glucose metabolism by chondrocytes indicated that pentose phosphate shunt activity was low. However, when the cells were stimulated with phenazine methosulfate (PMS) and t-butyl hydroperoxide, a significant elevation in shunt activity was observed. This activity was further increased by dithiothreitol. Enzymatic and substrate requirements of the shunt pathway were examined and related to morphology of the tissue. It was found that as chondrocytes mature, there is increased glucose-6-phosphate dehydrogenase activity, and decreased quantities of glucose-6-phosphate and NADPH. While these investigations indicated that shunt activity was maximum in hypertrophic cartilage, the results of cytochemical studies suggested that the activity was greatest in those cells that were most removed from the O 2 supply. Experiments were performed to examine O 2 requirements of chondrocytes in relationship to the pentose phosphate shunt. First, using a phosphorescence quenching technique, total O 2 uptake by these cells was found to be constant over a large part of the physiological range of O 2 tensions. Over the same range, when stimulated by PMS, O 2 uptake by CN − treated cells was increased. In the 1–5 μM O 2 range, non-mitochondrial O 2 consumption decreased more slowly than total respiration. Finally, the observation that NADPH directly stimulated chondrocyte O 2 consumption suggest that cartilage cells may be able to form O 2 metabolites.

Impaired erythrocyte phosphoribosylpyrophosphate formation in hemolytic anemia due to pyruvate kinase deficiency

RBCs from patients with hemolytic anemia due to pyruvate kinase (PK) deficiency are characterized by a decreased total adenine and pyridine nucleotide content. Because phosphoribosylpyrophosphate (PRPP) is a precursor of both adenine and pyridine nucleotides, we investigated the ability of intact PK-deficient RBCs to accumulate PRPP. The rate of PRPP formation in normal RBCs (n = 11) was 2.89 +/- 0.80 nmol/min.mL RBCs. In contrast, the rate of PRPP formation in PK-deficient RBCs (n = 4) was markedly impaired at 1.03 +/- 0.39 nmol/min.mL RBCs. Impaired PRPP formation in these cells was not due to the higher proportion of reticulocytes. To study the mechanism of impaired PRPP formation, PK deficiency was simulated by incubating normal RBCs with fluoride. In normal RBCs, fluoride inhibited PRPP formation, caused adenosine triphosphate (ATP) depletion, prevented 2,3-diphosphoglycerate (DPG) depletion, and inhibited pentose phosphate shunt (PPS) activity. These results together with other data suggest that impaired PRPP formation is mediated by changes in ATP and DPG concentration, which lead to decreased PPS and perhaps decreased hexokinase and PRPP synthetase activities. Impaired PRPP formation may be a mechanism for the decreased adenine and pyridine nucleotide content in PK-deficient RBCs.

Impaired Erythrocyte Phosphoribosylpyrophosphate Formation in Hemolytic Anemia due to Pyruvate Kinase Deficiency

RBCs from patients with hemolytic anemia due to pyruvate kinase (PK) deficiency are characterized by a decreased total adenine and pyridine nucleotide content. Because phosphoribosylpyrophosphate (PRPP) is a precursor of both adenine and pyridine nucleotides, we investigated the ability of intact PK-deficient RBCs to accumulate PRPP. The rate of PRPP formation in normal RBCs (n = 11) was 2.89 +/- 0.80 nmol/min.mL RBCs. In contrast, the rate of PRPP formation in PK-deficient RBCs (n = 4) was markedly impaired at 1.03 +/- 0.39 nmol/min.mL RBCs. Impaired PRPP formation in these cells was not due to the higher proportion of reticulocytes. To study the mechanism of impaired PRPP formation, PK deficiency was simulated by incubating normal RBCs with fluoride. In normal RBCs, fluoride inhibited PRPP formation, caused adenosine triphosphate (ATP) depletion, prevented 2,3-diphosphoglycerate (DPG) depletion, and inhibited pentose phosphate shunt (PPS) activity. These results together with other data suggest that impaired PRPP formation is mediated by changes in ATP and DPG concentration, which lead to decreased PPS and perhaps decreased hexokinase and PRPP synthetase activities. Impaired PRPP formation may be a mechanism for the decreased adenine and pyridine nucleotide content in PK-deficient RBCs.

Topography of basal glucose utilization in the hippocampus determined with [1- 14C]glucose and [6- 14C]glucose

The activity of the pentose phosphate shunt was assessed under basal conditions in subregions of the hippocampus by measuring the uptake and retention of [1- 14C]glucose and [6- 14C]glucose and their 14C-labelled metabolites. The relative and absolute retention of carbon-14 from each of the two compounds was nearly identical in all regions examined. For each compound, the highest accumulation of 14C occurred in the granule cell layer of the dentate gyrus and in the pyramidal cell layer. Relatively high retention of radioactivity was also found in the molecular layer of dentate gyrus and in the stratum lacunosum-moleculare. The stratum radiatum and stratum oriens contained the lowest levels of radioactivity among hippocampal regions. The equal retention of radioactivity from [1- 14C]glucose and [6- 14C]glucose implies that pentose phosphate shunt activity is very low throughout the hippocampus under the conditions of this study. The uptake and retention of radioactivity was evaluated in different hippocampal regions 10 or 30 min following intravenous injection of [1- 14C]glucose. Although there was significantly more radioactivity at 30 min than at 10 min, the same topographic pattern of radioactivity within the hippocampus was observed in rats after both survival periods, indicating that an equal fraction of the [1- 14C]glucose utilized in different hippocampal regions is oxidized to 14CO 2 under these conditions. Most regions of high glucose utilization in the hippocampus determined with [1- 14C]glucose and [6- 14C]glucose correspond to regions of intense histochemical staining for cytochrome oxidase reported in the literature. In regions that exhibit low cytochrome oxidase activity and high [1- 14C]glucose utilization, high nicotinamide adenine dinucleotide phosphate-diaphorase activity is present and high amino acid uptake occurs. These observations suggest that measurement of the uptake and retention of radioactivity from [1- 14C]glucose and [6- 14C]glucose provides an index of glucose utilized for biosynthetic mechanisms as well as for energy transducing processes.

Impaired antioxidant defense in hemoglobin E-containing erythrocytes: a mechanism protective against malaria?

Red blood cell (RBC) antioxidant defense was investigated in eight individuals with hemoglobin E (Six EE and two E-B(+) thalassemia) and compared to that in six individuals with thalassemia and ten normal subjects. Individuals with hemoglobin E had increased incubated Heinz body formation (68% +/- 18%; p less than 0.001) compared to normal and thalassemic RBC (10% +/- 2% and 11% +/- 5%, respectively). Stimulated pentose phosphate shunt activity was increased in the thalassemic and decreased in the hemoglobin E RBC as compared to normal. The 2,3-diphosphoglycerate (DPG) content of the EE RBC was increased to 5.59 +/- 0.69 mumol/ml RBC as compared to normal (4.51 +/- 0.77; p less than 0.001). In the EE RBC, there was a direct correlation between Heinz body formation and DPG content (r = 0.73). Ascorbic and dehydroascorbic acid (0.1 and 1.0 mM) were able to decrease the degree of Heinz body formation in the hemoglobin E RBC. Ascorbic acid (0.1 mM) prolonged the response of the pentose shunt. Thus impaired antioxidant defense may account for the persistence of the hemoglobin E gene in areas where malaria is endemic. Oxidant medications should be used with caution in individuals of Southeast Asian origin.

Increased Heinz body formation and impaired erythrocyte pentose phosphate shunt function during pregnancy.

The erythrocytes of 90 pregnant women were evaluated for the presence of in vivo or in vitro oxidant damage. The reduced glutathione (P less than 0.005) and the membrane reduced sulfhydryl (P less than 0.001) concentrations were decreased in fresh erythrocytes. Following incubation with acetylphenylhydrazine, Heinz body formation was significantly increased (P less than 0.001). Both the increase in Heinz body formation and the reduction in membrane reduced sulfhydryl content correlated strongly with duration of pregnancy. Glucose consumption was significantly decreased before, but not after, new methylene blue stimulation. Pentose phosphate shunt activity was impaired both before (P less than 0.05) and after (P less than 0.001) stimulation. No changes were observed in pentose phosphate recycling. The only alteration observed in the activity of the enzymes of the pentose shunt was an elevation of 6-phosphogluconate dehydrogenase activity. Although the clinical significance of these findings remains to be determined, medications with an oxidant potential should be used judiciously during gestation.

Antioxidant metabolism during blood storage and its relationship to posttransfusion red cell survival.

The status of the erythrocyte antioxidant defense system and its relationship to posttransfusion red cell survival were determined in erythrocytes stored for 35 or 42 days in CPD-A1 anticoagulant with a saline-adenine-glucose additive. As storage progressed, there was a significant increase in incubated Heinz body formation (P less than .001) and a significant decrease in reduced glutathione (GSH) stability (P less than .001). Stimulated pentose phosphate shunt activity also declined during storage (P less than .06), while unstimulated shunt activity remained unchanged. The increase in Heinz body formation was associated with decreased GSH stability (r = -.77, P less than .001), which in turn was associated with the decline in stimulated pentose shunt activity (r = .67, P less than .001). The changes in Heinz body formation (r = -.85), GSH stability (r = .83), and stimulated pentose shunt activity (r = .54) were all significantly (P less than .001) related to the decline in adenosine triphosphate (ATP) content of the erythrocyte. Red cell survival 24 hours after transfusion was significantly related to the GSH stability (r = .80, P less than .001) and to the ATP concentration (r = .76, P less than .005) on the day of transfusion. Thus, dysfunction of the erythrocyte antioxidant defense system occurs during blood storage and appears to be related, in part, to ATP depletion. The ability to maintain a normal reduced glutathione concentration during oxidant stress appears to be an important determinant of red cell survival in the peritransfusion period.

Inhibition of the pentose phosphate shunt by lead: a potential mechanism for hemolysis in lead poisoning

Recent investigations have disclosed a decrease in pentose phosphate shunt activity in hereditary pyrimidine 5′-nucleotidase deficiency. Clinical lead poisoning is associated with an acquired decrease in pyrimidine 5′-nucleotidase activity. The current investigations were undertaken (1) to determine if pentose shunt activity was decreased in erythrocytes exposed to lead, and (2) to compare the mechanism of inhibition to that seen in hereditary pyrimidine 5′-nucleotidase deficiency. Normal erythrocytes incubated with lead acetate in vitro demonstrated increased Heinz body formation, decreased reduced glutathione, a positive ascorbate cyanide test, and a reversible suppression of pentose shunt activity in the intact erythrocyte. Lead acetate added to normal red cell hemolysates markedly inhibited the activities of glucose-6-phosphate dehydrogenase (G6PD) and phosphofructokinase. The mean Kis of lead for glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (NADP) for G6PD were 1.5 microM and 2.1 microM, respectively, which is within the range of intraerythrocytic lead concentrations found in clinical lead poisoning. Magnesium enhanced the ability of lead to inhibit G6PD. Thus, the shortened erythrocyte survival in lead poisoning appears to be due, in part, to increased oxidant sensitivity secondary to inhibition of G6PD and the pentose shunt. The mechanism of shunt inhibition is, in part, similar to that seen in hereditary pyrimidine 5′-nucleotidase deficiency.

Inhibition of the Pentose Phosphate Shunt by Lead: A Potential Mechanism for Hemolysis in Lead Poisoning

Recent investigations have disclosed a decrease in pentose phosphate shunt activity in hereditary pyrimidine 5'-nucleotidase deficiency. Clinical lead poisoning is associated with an acquired decrease in pyrimidine 5'-nucleotidase activity. The current investigations were undertaken (1) to determine if pentose shunt activity was decreased in erythrocytes exposed to lead, and (2) to compare the mechanism of inhibition to that seen in hereditary pyrimidine 5'-nucleotidase deficiency. Normal erythrocytes incubated with lead acetate in vitro demonstrated increased Heinz body formation, decreased reduced glutathione, a positive ascorbate cyanide test, and a reversible suppression of pentose shunt activity in the intact erythrocyte. Lead acetate added to normal red cell hemolysates markedly inhibited the activities of glucose-6-phosphate dehydrogenase (G6PD) and phosphofructokinase. The mean Kis of lead for glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (NADP) for G6PD were 1.5 microM and 2.1 microM, respectively, which is within the range of intraerythrocytic lead concentrations found in clinical lead poisoning. Magnesium enhanced the ability of lead to inhibit G6PD. Thus, the shortened erythrocyte survival in lead poisoning appears to be due, in part, to increased oxidant sensitivity secondary to inhibition of G6PD and the pentose shunt. The mechanism of shunt inhibition is, in part, similar to that seen in hereditary pyrimidine 5'-nucleotidase deficiency.