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Carbohydrate-deficient transferrin (CDT) is one of the best indicators for chronic alcohol abuse and detection of relapse. In this study, we explore the microheterogeneity of β-hexosaminidase (β-HEX) in chronic alcohol abusers in the framework of a driver's license regranting program. Studies have shown that increased serum activity of β-HEX B (isoforms P, I, and B) may be a sensitive marker for chronic alcohol abuse. Here, we describe methodology, limitations, and correlation of β-HEX isoforms with CDT.CDT was assayed at the central laboratory of the Ghent University Hospital by capillary zone electrophoresis, measured on the Capillarys 2™ system and was expressed as a percentage of total serum transferrin (%CDT). Serum of chronic alcohol abusers was compared to nonheavy drinkers using agarose gel isoelectric focusing (IEF). Total β-HEX activity was assayed fluorimetrically following preparative IEF in 81 subjects. β-HEX isoforms were investigated and compared between nonheavy drinkers and heavy drinkers.Agarose gel IEF shows additional cathodal bands in serum of chronic alcohol abusers. Mean total β-HEX activity between pH 6.8 and 7.7, designated as HEX-7, showed the highest correlation with %CDT (r = 0.70, p < 0.0001, n = 68). In a selected subgroup, where CDT could not be quantified (n = 13) because of an atypical electropherogram, HEX-7 was in concordance with either estimated %CDT value or liver enzyme activities.In this proof-of-concept study, we introduce a novel approach to quantify β-HEX isoforms using preparative IEF and fluorimetry. A highly significant correlation of HEX-7 and %CDT has been found. Because of exclusion of the P isoform, HEX-7 could be a useful supplementary marker for detecting chronic alcohol abuse.