Candidate reference methods for hemoglobin A1c based on peptide mapping

Abstract

A reference method that specifically measures hemoglobin (Hb) A1c is an essential part of the reference system for the international standardization of Hb A1c/glycohemoglobin. We have developed a new method for quantification, based on the specific N-terminal residue of the hemoglobin β-chains. Enzymatic cleavage of the intact hemoglobin molecule with endoproteinase Glu-C has been optimized to obtain the β-N-terminal hexapeptides of Hb A1c and Hb A0. These peptides have been separated by reversed-phase HPLC and quantitated by electrospray ionization-mass spectrometry (method A) or by capillary electrophoresis (method B). With these peptides and hyphenated separation techniques, it has been possible to overcome the insufficient resolution of currently used protein separation systems for Hb A1c.

Elevated plasma semicarbazide‐sensitive amine oxidase (SSAO) activity in Type 2 diabetes mellitus complicated by retinopathy

“…(S Eckerbom, personal communication). Results obtained using this method closely agree with those from a proposed world standard method [20], giving about 1.2% U lower values than the Bio-Rex method in use during the Diabetes Control and Complications Trial [21].…”

ISPAD Clinical Practice Consensus Guidelines 2018: Glycemic control targets and glucose monitoring for children, adolescents, and young adults with diabetes

“…31,33 The International Federation of Clinical Chemistry (IFCC) has developed a reference method that precisely measures glycated HbA1c only. 92,93 The reference measurement procedure has been defined as bN1deoxyfructosyl-hemoglobin, and the recommended SI measurement units are mmol/mol. 94 The IFCC, American Diabetes Association, European Association for the Study of Diabetes, and the International Diabetes Federation have issued a consensus statement regarding this standardization process.…”

Enzymes used for the determination of HbA1C

“…cleavage of the intact glycated hemoglobin molecule with the endoproteinase Glu-C, using HPLC-ESI/MS or HPLC-CE ( [3,4], Fig. 1(left)).…”

“…At each substrate concentration, the absorbance for Fru-hexapeptide was 80% of that for Fru-ValHis, indicating that about 80% of Fru-hexapeptide was digested by the protease releasing Fru-ValHis. These results indicated that FPOX is applicable for determining the amount of Fru-hexapeptide that is supposed to be liberated from HbA 1C by Glu-C endoproteinase [3,4].…”

“…Fig. 1(left) shows the IFCC reference method for the measurement of HbA 1C [3,4]. In the first step, HbA 1C is cleaved into peptides by the endopeptidase Glu-C and in the second step, N -(1-deoxyfructosyl)-Val-His-Leu-Thr-Pro-Glu (Fru-hexapeptide) released from the b-chain of HbA 1C is quantified using HPLC and electospray ionization mass spectrometry or in a two-dimensional approach using HPLC and capillary electrophoresis with UV-detection.…”