Improved method for carbohydrate-deficient transferrin determination in human serum by capillary zone electrophoresis

Carbohydrate-deficient transferrin (CDT) is a reliable marker of chronic or repeated alcohol abuse. It indicates a group of isoforms of human transferrin (Tf), the main iron transport serum protein, deficient in sialic acid residues (asialo-, monosialo- and disialo-Tf) in comparison to the main isotransferrin which contains four sialic acid groups (tetrasialo-Tf). The aim of the present work was to develop a capillary electrophoretic method suitable for rapid determination of CDT components in serum. Serum samples (0.1 ml) were saturated with iron by incubation with 10 mM FeCl3 (2 microl) and 500 mM NaHCO3 (3 microl) for 30 min, then diluted 1:10 in water and injected by positive pressure (0.5 p.s.i. for 10 s). Separation was performed with a capillary zone electrophoretic method using bare fused-silica capillaries (57 cm x 20 microm I.D.) and a buffer composed of 100 mM sodium tetraborate adjusted with 6 M HCl to pH 8.3 added with 1.5 mM diaminobutane. Applied voltage was 20 kV and temperature 25 degrees C. Detection was by UV absorption at 200 nm wavelength. Under the described conditions, asialo-, monosialo-, disialo-, trisialo- and tetrasialo-transferrin were baseline separated. The limit of detection (signal-to-noise ratio of 2) was about 0.3% for disialo-Tf, and 0.5% of trisialo-Tf, expressed as percentages of the terasialo-Tf peak area. Day-to-day RSDs of relative migration times were < or = 0.2%. Quantitation showed day-to-day RDSs < or = 6.9% and < or = 10.9% for disialo- and trisialo-Tf, respectively. The results from 79 control subjects, including social drinkers, and 23 alcoholics showed disialo- and trisialo-Tf significantly increased in patients (P<0.0001 and <0.01, respectively). A clear interference from trisialo-Tf in an immunoassay for CDT was demonstrated. The present method is suitable for confirmation of CDT immunoassays by independent technique.