Kinetic analysis in single, intact cells by microspectrophotometry: Evidence for two populations of erythrocytes in an individual heterozygous for glucose‐6‐phosphate dehydrogenase deficiency

Abstract

A microspectrophotometric technique was used to measure the kinetics of methemoglobin reduction in intact, unaltered human erythrocytes. Reduction was catalyzed by endogenous NADPH‐methemoglobin reductase in the presence of Nile Blue. The technique was applied to the study of erythrocytes from a female donor with decreased glucose‐6‐phosphate dehydrogenase (G‐6‐PD) activity. The individual was shown to be heterozygous for deficiency of G‐6‐PD. The kinetic study revealed two distinct populations of erythrocytes that were nearly equal in number. One cell population showed reduction rates between 0 and 25% of normal, whereas the second cell population displayed rates within the range seen for normal cells. Single‐cell indices of cell size, cell hemoglobin content, and ratio of cell hemoglobin to cell size did not correlate with single‐cell reduction rates and were not significantly different between the two populations. These results provide quantitative support for the X‐inactivation hypothesis in G‐6‐PD deficiency.