Characterization of S-glutathionyl Hemoglobin in Homozygous Sickle Cell Disease

Abstract

S-glutathionyl hemoglobin is a proposed biomarker of oxidative stress but has not been measured in sickle cell disease patients. Unlike the S-glutathionyl adduct of normal adult hemoglobin, S-glutathionyl sickle hemoglobin (HbSSG) cannot be directly measured by capillary isoelectric focusing, because it coelutes with fetal hemoglobin (HbF). This suggests that HbF, measured in sickle cell patients with or without hydroxyurea therapy, might contain endogenous HbSSG. As S-glutathionyl hemoglobin can form during sample storage, HbSSG could falsely elevate HbF levels in stored samples. We measured HbSSG based on the quantitative difference in the heterogeneous HbF/HbSSG peak before and after hemolysates were treated with dithiothreitol. Paired t tests showed that dithiothreitol reduced HbF/HbSSG in blood from pediatric sickle cell patients (n=25, mean decrease+/-SD=1.0%+/-0.6, P<0.05) but not in normal infants (n=25). Higher HbF levels in hydroxyurea-treated patients (n=5) were not attributable to HbSSG. HbSSG increased significantly within 1 day in samples stored at -20 degrees C but was unchanged in samples stored 60 days at-70 degrees C. We conclude that blood from sickle cell patients contained up to 2.2% HbSSG, and that endogenous HbSSG is a minor interferent in the measurement of HbF in fresh blood but a major interferent in improperly stored samples.