MATERIALS AND METHODS

Subjects studied. All subjects were adult males. The G6PD-deficient subjects wereall Negroes who had normal hemoglobin by electrophoretic analysis performed on starch blocks (8). In all subjects the leukocyte count was normal-, the hematocrit above 40 per cent, and the reticulocyte count below 3.3 per cent. The. hematologic studies were performed by standard methods (9). G6PD was assayed by following the appearance of reduced triphosphopyridine nucleotide (TPNH) spectrophotometrically under the conditions described by Motulsky (10): The reaction mixture contained 3, umoles disodium ethylenediamine tetraacetic acid;. 20, umoles MgCl,; 39.5, umoles nicotinamide; 222 Amoles Tris-(hydroxymethyl) aminomethane; 5, umoles glucose-6-phosphate; 0.4 mg triphosphopyridine nucleotide (TPN);-and 2 ml of a 1: 250 aqueous hemolysate of whole blood in a final volume of 3 ml. The reaction was carried out at room temperature incuvets of 1 cm light path in a Beckman model DU spectrophotometer. Units of activity are defined as change in optical density at 340 mA per minute per ml of erythrocytes. Preparation of blood. Whole blood was collected in heparin (0.1 mg per ml of blood) under sterile conditions from fasting subjects. Whole blood was used for most experiments to keep manipulation of the erythrocytes to a minimum. The data of Bishop, Rankine, and Talbott (11) and of Whittam (12) indicate that essen-tially all the ATP of whole blood is in the erythrocytes, and in the present study no significant difference was found between the ATP content of whole blood and an equivalent amount of washed erythrocytes from a sus-pension which had a hematocrit of 35 per cent and contained 2,000 platelets and 1,850 leukocytes per mm3. Washed erythrocytes were prepared at 40 C as de-scribed by Whittam (12) except that balanced salt solution buffered with bicarbonate was used throughout. The erythrocytes were finally suspended in sufficient balanced salt solution to restore the volume to that of the original blood.