Characterization of a novel hemoglobin-glutathione adduct that is elevated in diabetic patients

Background: Typically, a diagnosis of diabetes mellitus is based on elevated circulating blood glucose levels. In an attempt to discover additional markers for the disease and predictors of prognosis, we undertook the characterization of HbA(1d3) in diabetic and normal patients. Material and Methods: PolyCAT A cation exchange chromatography and liquid chromatography-mass spectroscopy was utilized to separate the alpha- and beta -globin chains of HbA(1d3) and characterize their presence in normal and diabetic patients. Results: We report the characterization of HbA(1d3) as a glutathionylated, minor hemoglobin subfraction that occurs in higher levels in diabetic patients (2.26 +/- 0.29%) than in normal individuals (1.21 +/- 0.14%, p < 0.001). The alpha -chain spectrum displayed a molecular ion of m/z 15126 Da, which is consistent with the predicted native mass of the HbA(0) alpha -globin chain. By contrast, the mass spectrum of the beta -chain showed a mass excess of 307 Da (m/z = 16173 Da) versus that of the native HbA(0) beta -globin chain (m/z = 15866 Da). The native molecular weight of the modified beta -globin chain HbA(0) was regenerated by treatment of HbA(1d3) with dithiothreitol, consistent with a glutathionylated adduct. Conclusions: We propose that HbA(1d3) (HbSSG) forms normally in vivo, and may provide a useful marker of oxidative stress in diabetes mellitus and potentially other pathologic situations.