Higher degree of glycation of hemoglobin S compared to hemoglobin A measured by mass spectrometry: Potential impact on HbA1c testing

Abstract

BACKGROUND Glycated hemoglobin (GHb), reported as HbA1c, is used as marker of long-term glycemia for diabetic patients. HbA1c results from boronate affinity methods are generally considered to be unaffected by most hemoglobin variants; this assumes comparable glycation of variant and non-variant (HbAA) hemoglobins. In this report, glycation of HbA beta chain (βA) and HbS beta chain (βS) for the most common Hb variant trait (HbAS) are examined. METHODS We analyzed 41 blood samples from subjects with HbAS, both with and without diabetes. Using LC-MS, ratios of glycated HbS to glycated HbA were determined by comparison of areas under the curves from extracted ion chromatograms. RESULTS Glycation of βS chains was significantly higher (p<0.001) than βA chains; this difference was consistent across subjects. Total (α+β) glycated HbAS was theoretically estimated to be ~5% higher than glycated HbAA. CONCLUSION This novel mass-spectrometric approach described allows for relative quantification of glycated forms of βS and βA. Although βS glycation was significantly higher than that of βA, the difference in total glycation of HbAS versus HbAA was smaller and unlikely to impact clinical interpretation of boronate affinity HbA1c results. These data support the continued use of boronate affinity to measure HbA1c in patients with HbAS.

Capillary Zone Electrophoresis-Top-Down Tandem Mass Spectrometry for In-Depth Characterization of Hemoglobin Proteoforms in Clinical and Veterinary Samples

“…Determination of HbA1c is performed using various analytical methods such as cation-exchange chromatography and capillary electrophoresis with optical detection as well as immunological methods . However, numerous studies have demonstrated that Hb sequence variants or other proteoforms (term used according to Smith and Kelleher) can lead to interferences in identification and quantification of HbA1c. − Therefore, mass spectrometry (MS) has been considered as an alternative for unambiguous identification and quantification, not only for glycated Hb, but also for Hb sequence variants. Research has been focused on matrix-assisted laser desorption/ionization-MS and direct-infusion electrospray ionization-MS. , These studies demonstrated good correlation between clinically determined HbA1c values and individually glycated α- and β-chains.…”

Interpretation of HbA1c lies at the intersection of analytical methodology, clinical biochemistry and hematology (Review)

“…Therefore, the glycosylation rate of β S appears to be higher than that of β A . However, Kabytaev et al (40) concluded that the clinical interpretation was relatively unaffected by the tiny net difference between HbAS (sickle phenotype) and uncharacterized total glycosylation. A summary of the altered glycosylation rates presented in mutants located in the first 10 amino acids of the β chain and at amino acid positions 139-140 is presented in Table II (41)(42)(43)(44).…”

The use of LC tandem mass spectrometry as part of a workflow for the screening and identification of hemoglobin variants. Characterization of Hb Ullevaal as an example

“…Antibody based HbA 1c assays are principally not able to detect variants. Even variants which are prevalent in several populations have a direct or indirect influence on the measured HbA 1c what makes it desirable to gain knowledge about their presence [6,7].…”