Substances Stimulating Glucose Catabolism by the Oxidative Reactions of the Pentose Phosphate Pathway in Human Erythrocytes*

Szeinberg, A.; Marks, Paul A.

doi:10.1172/jci104330

Cited by 118 publications

(41 citation statements)

References 46 publications

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“…Incomplete recovery of added carbon (owing to CO2 evolution) and higher concentrations of some metabolites (like pentose phosphate and heptulose phosphate) are consistent with this interpretation, which is in agreement with findings of other authors (19,25).…”

Section: Resultssupporting

confidence: 93%

Glucose Metabolism in Human Erythrocytes From Normal and Fava Bean-Sensitive Subjects*

Fornaini¹,

Leoncini²,

Luzzatto³

et al. 1962

J. Clin. Invest.

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Many studies during the last decade have been concerned with the biochemical properties underlying various states of the human red blood cell, including its maturation, aging, and some abnormal conditions.A considerable amount of work has been devoted particularly to the study of erythrocyte metabolism in the fava bean-and drug-induced hemolytic anemias. Observations were first made by Beutler and his co-workers (1, 2) concerning the instability and decreased content of reduced glutathione (GSH) in red cells in drug-induced anemia. Carson (3) and Gross (4) and their colleagues, and Sansone and Segni (5, 6) demonstrated glucose-6-phosphate dehydrogenase (G6PD) deficiency in these cells. Further work has since been done in an attempt to find a link between the genetically determined metabolic defect and the hemolytic phenomenon.Such studies have been concerned mainly with the activity and properties of various enzymes, and some work in this field has been done also in our laboratory (7-10). In the present paper a somewhat different approach is described. Glucose metabolism in fava bean-sensitive versus normal erythrocyte suspensions has been followed by quantitatively estimating the different metabolites that arise from its consumption in vitro. On these transformations, as well as on triose phosphate oxidation by hemolyzed red cells, the action of methylene blue (Mb) has also been studied. METHODSSubjects. Blood was obtained from seven men and two women from Sardinia, five of whom belong to the same *This work was supported by Grant H 4586 of the United States Public Health Service. Preliminary communications concerning some of the results reported in the present paper have appeared in Boll. Soc. ital. Biol. sper. 1961, 37, 1046, 1048 family. Two of them had experienced acute hemolytic anemia (24 and 16 months ago, respectively) after fava bean ingestion. In all of them red cell G6PD was lacking, reduced glutathione was decreased, the Beutler test (2) was positive, and the reticulocyte count was normal. Normal subjects of both sexes and of approximately corresponding ages were used as controls.Preparation of red blood cell suspensions. The blood was collected by venipuncture, and heparin added. After centrifugation the plasma and buffy coat were removed by suction, the red blood cells (RBC) were washed thrice with buffered isotonic KCl, pH 7.4, and then made up to volume. The packed cell volume was determined on this suspension by centrifuging in a hematocrit tube for 60 minutes at 1,200 X G. RBC suspensions were never completely free from leukocytes. The contamination, however, was always less than 0.05 per cent.Hemolyzed preparations. These were prepared by treating the washed RBC with 2 vol of ice-cold water. The ghosts were removed by centrifuging for 30 minutes at 30,000 X G. The supernatant fluid was then diluted as required. All manipulations were performed at + 20 C.Incubation and extraction. In the experiments concerning glucose metabolism in intact RBC, each reaction vessel contained, per milliliter: 1...

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Section: Resultssupporting

confidence: 93%

Glucose Metabolism in Human Erythrocytes From Normal and Fava Bean-Sensitive Subjects*

Fornaini¹,

Leoncini²,

Luzzatto³

et al. 1962

J. Clin. Invest.

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“…although not by others (20), to function in human erythrocytes as well. Unlike catalase, GSH peroxidase efficiently detoxifies low levels of H202, doing so by reversibly oxidizing reduced glutathione (17).…”

mentioning

confidence: 68%

Oxidative Hemolysis and Erythrocyte Metabolism in Hereditary Acatalasia*

Jacob

Ingbar²,

Jandl³

1965

J. Clin. Invest.

141

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“…Finally, it has been estimated that about 30% of the flux through the oxidatively stimulated HMS is from recycled glucose-6-P rather than glucose-6-P formed directly via the H K reaction [45,461. This flux does not occur in the reconstituted metabolic system and was not incorporated into the computer simulation scheme.…”

Section: Discussionmentioning

confidence: 99%

Regulation of the human‐erythrocyte hexose‐monophosphate shunt under conditions of oxidative stress

Thorburn

Kuchel

1985

European Journal of Biochemistry

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The regulation of the hexose monophosphate shunt of human erythrocytes under conditions of oxidative stress has been investigated by monitoring the reduction of oxidised glutathione (GSSG) to reduced glutathione (GSH) in erythrocytes containing high levels of GSSG; 'H NMR and a biochemical assay were used to measure the changes.A reconstituted metabolic system prepared with the purified erythrocyte enzymes was used in conjunction with studies of intact cells and haemolysates to determine the dependence of the rate of GSH production on the activities of hexokinase and glucose-6-phosphate dehydrogenase. Both of these enzymes have previously been claimed to be the rate-limiting step of oxidatively stimulated flux through the hexose monophosphate shunt.The absence of a kinetic isotope effect on the rate of GSH production in these systems, when [l-ZH]glucose replaced glucose as the source of reducing equivalents, showed that glucose-6-phosphate dehydrogenase activity was not a strong determinant of the flux. The dependence of the rate of GSH production on the concentration of the hexokinase inhibitors glucose 1,6-bisphosphate and glycerate 2,3-bisphosphate showed that, under conditions of oxidative stress, hexokinase was the principal determinant of flux through the shunt. Glucose 1,6-bisphosphate at the concentration present in vivo appears to be more important in limiting hexokinase activity, and thus the rate of glucose utilisation, than was previously assumed.A detailed computer model of the system was developed based on the reported kinetic parameters of the enzymes involved. A sensitivity analysis of this model predicted that the hexokinase reaction would have a sensitivity coefficient of 0.995 with respect to the maximal rate of GSH production.The hexose monophosphate shunt (HMS) provides reducing equivalents in the form of NADPH and pentose sugars for nucleotide metabolism. In human erythrocytes, flux through the HMS maintains glutathione in the reduced form as one of the main cellular anti-oxidative mechanisms [l]. Red cells are regularly subjected to high oxygen tension and are among the first body cells exposed to exogenous oxidative substances that are ingested, injected or inhaled. The importance of this anti-oxidative function is shown by the massive oxidant-induced haemolysis seen in subjects with a marked deficiency of glucose-6-phosphate dehydrogenase (G6PD), the first enzyme unique to the HMS [l].The regulation of the erythrocyte HMS under oxidatively stressed or unstressed conditions has been extensively studied.Correspondence to P. W. Kuchel,

show abstract

Substances Stimulating Glucose Catabolism by the Oxidative Reactions of the Pentose Phosphate Pathway in Human Erythrocytes*

Cited by 118 publications

References 46 publications

Glucose Metabolism in Human Erythrocytes From Normal and Fava Bean-Sensitive Subjects*

Glucose Metabolism in Human Erythrocytes From Normal and Fava Bean-Sensitive Subjects*

Oxidative Hemolysis and Erythrocyte Metabolism in Hereditary Acatalasia*

Regulation of the human‐erythrocyte hexose‐monophosphate shunt under conditions of oxidative stress

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