Measurement of hemoglobin-acetaldehyde adduct in alcoholic patients

A sensitive and specific test for chronic alcohol abuse is useful in the diagnosis and management of alcoholic patients. Herein, we report the measurement of hemoglobin-acetaldehyde adducts (Hb-AA) in alcoholic patients by a sandwich ELISA using different antibodies. Keyhole limpet hemocyanin (KLH), a peptide consists of eight amino acid residues (8-pep, V1 to K8) at the N-terminus of beta-chain of human sickle-cell Hb and a segment of HbA beta-chain consists of 11 amino acids rich in lysine or K (11-pep, G56-K66) were incubated with acetaldehyde and NaCNBH3 to form protein-AAs. 8-Pep-AA and 11-pep-AA were individually conjugated to unmodified KLH as the carrier. Anti-protein-AA IgGs were raised in rabbits using these three protein-AA immunogens. When anti-KLH-AA IgG was used in ELISA, optical densities for alcoholic patients and controls were 0.311 +/- 0.124 and 0.147 +/- 0.042 (means +/- SD, n = 40/group, p < 0.001), respectively. Using mean value +/- 2 SD of controls as the cut-off, sensitivities to detect alcoholic patients were 78, 75, and 43%, respectively, when anti-KLH-AA, anti-11-pep-AA, and anti-8-pep-AA were used. Correlation among optical densities obtained from the first two IgGs was excellent (R2 = 0.905). We conclude that: (1) Hb-AA has the potential of being a good marker for alcohol abuse, and (2) the site of Hb that is modified by acetaldehyde in vivo is primarily located in a surface-accessible domain near the center of the beta-chain of HbA where several lysine residues are clustered.