Abstract
Hemolytic episodes in patients with erythrocyte glucose-6-phosphate dehydrogenase (G6PD) deficiency are known to occur during diabetic acidosis (1). The mechanism for this hemolysis is not understood. Whether hyperglycemia has a role in diabetic acidosis-induced hemolysis of G6PD deficient red blood cells is not known. Travis and coworkers have reported that elevated glucose concentrations stimulated the hexose monophosphate shunt (HMP) of normal erythrocytes, presumably due to activation of the sorbitol pathway and the associated oxidation of dihydronicotinamide adenine dinucleotide diphosphate (NADPH) (2). The experiments described in this report were designed to answer whether glucose-induced NADPH-oxidation increased the vulnerability of G6PD deficient erythrocytes to oxidant injury. Specifically, the effects of normal and elevated glucose concentrations on NADPH-dependent reactions integrally involved with hemoglobin protection were measured.
Methods. Red blood cells (RBC's) from normal and Black G6PD deficient males were collected in EDTA and washed thrice in a pH 7.4 glycylglycine-buffered salt solution (GBS) with the following composition: 140 mM NaCl, 5 mM KC1, 1 tnM Na2HPO4, 1 mM MgCl2 and 20 mM glycylglycine. Following the last wash, erythrocytes were resuspended in GBS to a hematocrit of 25-30%. Glucose was added to the incubation medium such that the final concentration was 5 or 50 mM. As the glucose concentration was varied, equiosmolal amounts of sucrose were added to maintain a constant osmolality in all experimenta suspensions. The HMP shunt was assessed by measurement of glucose-1-14C oxidation to 14CO2 under resting conditions and in the presence of 10 mM sodium ascorbate (3). Glutathione (GSH) stability and hemoglobin oxidation were determined in normal and G6PD deficient RBC's incubated with 5 mM NaCN, 10 mM ascorbate and 5 or 50 mM glucose. Glutathione was measured by the method of Beutler (4).
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