Characterization of a Novel Hemoglobin-Glutathione Adduct That Is Elevated in Diabetic Patients

Yousef Al-Abed, Sonya VanPatten, Hongwei Li, John A. Lawson, Garret A. FitzGerald, Kirk R. Manogue, Richard Bucala

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 HbA1d3 in diabetic and normal patients. PolyCAT A cation exchange chromatography and liquid chromatography-mass spectroscopy was utilized to separate the alpha- and beta-globin chains of HbA1d3 and characterize their presence in normal and diabetic patients. We report the characterization of HbA1d3 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 HbA0 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 HbA0 beta-globin chain (m/z = 15866 Da). The native molecular weight of the modified beta-globin chain HbA0 was regenerated by treatment of HbA1d3 with dithiothreitol, consistent with a glutathionylated adduct. We propose that HbA1d3 (HbSSG) forms normally in vivo, and may provide a useful marker of oxidative stress in diabetes mellitus and potentially other pathologic situations.