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

Abstract

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...

Agglutination of Stored Erythrocytes by a Human Serum. Characterization of the Serum Factor and Erythrocyte Changes*

“…It is known to stimulate the hexose monophosphate shunt and to increase glucose utilization and CO2 formation; in the presence of MB, erythrocyte oxygen uptake may increase as much as twentyfold (52)(53)(54). Even in the absence of glucose, increased uptake of oxygen is observed that presumably results from utilization of intracellular phosphate esters (51); the rate of breakdown of triose phosphate is increased, and lactate is oxidized to pyruvate (53,55). Washed red cells contain almost no glucose and limited amounts of other substrates (56).…”

“…Not only does the hexose monophosphate shunt fail, but pentose phosphate is not returned to the glycolytic pathway from the pentose shunt (57). Fructose 6-phosphate and triose phosphate become undetectable, and glucose accumulates (53). These differences may at least partly account for the more pronounced effect of MB in inducing agglutinability of G6PD-deficient cells and the lack of a protective or ameliorative effect of added glucose.…”

Pyridine nucleotides in erythrocyte metabolism.

“…In recent years investigations of erythrocyte metabolism have been concerned with the interrelationships of enzymes, coenzymes, substrates, cofactors, and pH, inter alia (1)(2)(3)(4)(5)(6)(7)(8). Although data have accumulated on these various aspects of the metabolism of normal and pathological erythrocytes, little direct information has been available heretofore on pyridine nucleotide coenzymes, despite their importance in both the glycolytic and the hexose monophosphate shunt pathways.…”

Neonatal Hemolysis Due to a Transient Severity of Inherited Pyruvate Kinase Deficiency

“…The glucose utilisation by intact erythrocytes was determined according to previously described procedure [2], and the reduced glutathione stability according to Beutler [3] in presence of 5 x 10-3 M glucose.…”