Hemoglobin Raleigh as the cause of a falsely increased hemoglobin A1C in an automated ion-exchange HPLC method

Abstract

Irreversible glycation of the hemoglobin A0 (HbA0) beta chain leads to the production of HbA1C, which can be used to monitor long-term blood glucose control in patients with diabetes mellitus. HbA1C is less positively charged than nonglycated HbA0, and this decrease in charge is the basis of ion-exchange and electrophoretic methods that measure HbA1C. We recently identified a sample that appeared to contain 46% HbA1C by an automated ion-exchange HPLC method (Bio-Rad Variant) but only 3.8% by an immunoinhibition latex agglutination method. A combination of traditional and mass spectrometric protein analysis and genomic DNA analysis of the Hb beta chain and genes revealed that the patient was heterozygotic for Hb-Raleigh, a variant containing a valine-->alanine substitution at position 1 of the beta chain. The amino-terminal alanine in this variant Hb is posttranslationally modified by acetylation, leading to a charge difference similar to glycation and making the behavior of HbA1C and Hb Raleigh virtually identical in the ion-exchange HPLC method. This observation suggests that it is important to confirm HbA1C values in excess of 15%, especially if they are not consistent with the clinical picture, by an independent HbA1C method such as immunoassay or boronic acid affinity chromatography. However, for this particular variant Hb, even these latter methods might be misleading, because the acetylated N-terminal amino acid of the Hb-Raleigh beta chain cannot be glycated.