HEMOGLOBIN-RALEIGH AS THE CAUSE OF A FALSELY INCREASED HEMOGLOBIN-A(1C) IN AN AUTOMATED ION-EXCHANGE HPLC METHOD

Irreversible glycation of the hemoglobin A(0) (HbA(0)) beta chain lead s to the production of HbA(1C), which can be used to monitor long-term blood glucose control in patients with diabetes mellitus. HbA(1C) is less positively charged than nonglycated HbA(0), and this decrease in charge is the basis of ion-exchange and electrophoretic methods that m easure HbA(1C). We recently identified a sample that appeared to conta in 46% HbA(1C) by an automated ion-exchange HPLC method (Bio-Rad Varia nt(TM)) but only 3.8% by an immunoinhibition latex agglutination metho d. A combination of traditional and mass spectrometric protein analysi s and genomic DNA analysis of the Hb beta chain and genes revealed tha t the patient was heterozygotic for Hb-Raleigh, a variant containing a valine-->alanine substitution at position 1 of the beta chain. The am ino-terminal alanine in this variant Hb is posttranslationally modifie d by acetylation, leading to a charge difference similar to glycation and making the behavior of HbA(1C) and Hb Raleigh virtually identical in the ion-exchange HPLC method. This observation suggests that it is important to confirm HbA(1C) values in excess of 15%, especially if th ey are not consistent with the clinical picture, by an independent HbA (1C) method such as immunoassay or boronic acid affinity chromatograph y. However, for this particular variant Hb, even these latter methods might be misleading, because the acetylated N-terminal amino acid of t he Hb-Raleigh beta chain cannot be glycated.