Impact of glutathione-HbA1c on HbA1c measurement in diabetes diagnosis via array isoelectric focusing, liquid chromatography, mass spectrometry and ELISA

Hemoglobin A 1c (HbA 1c ) has been proven to be a key biomarker for diabetes screening, and glutathiolation of HbA 1c (viz., GSS-HbA 1c ) has been identified. However, the impact of GSS-HbA 1c on the measurement of HbA 1c for diabetes screening has not been quantitatively assessed yet. To address the issue, the micropreparative capillary isoelectric focusing (cIEF) developed in our previous work was used for the high resolution separation and purification of hemoglobin (Hb) species. The main fractions of HbA 0 , HbA 3 and HbA 1c extracted from the developed cIEF were identified by validated Mono S method. The proposed GSS-HbA 1c fractions in the cIEF were pooled and identified by electrospray ionization mass spectrometry (ESI-MS). The HbA 1c enzyme-linked immunosorbent assay (ELISA) kit was employed for further quantitative analysis of GSS-HbA 1c . A total of 34 blood samples with HbA 1c levels from 4.2% to 13.4% were assessed via the above comprehensive strategy of IEF-HPLC-MS-ELISA. It was demonstrated that the HbA 1c levels detected by cation exchange LC were considerably influenced by the glutathiolation of Hb and the range of detected GSS-HbA 1c values was between 0.23% and 0.74%. The results and developed cIEF methods have considerable significances for investigation of diabetes and clinical diagnosis.