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

• Published in: Journal of chromatography. B, Biomedical sciences and applications Vol. 739; no. 1; pp. 81 - 93
• Main Authors: Crivellente, Federica, Fracasso, Giulio, Valentini, Roberta, Manetto, Giulia, Riviera, Anna Pia, Tagliaro, Franco
• Format: Journal Article Conference Proceeding
• Language: English
• Published: Amsterdam Elsevier B.V 28.02.2000; Elsevier Science
• Subjects: Analysis of complex biological substances; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Blood and biological fluids; Carbohydrate-deficient transferrin; Electrophoresis, Capillary - methods; Fundamental and applied biological sciences. Psychology; Humans; Immunoassay - methods; Quality Control; Reproducibility of Results; Transferrin - analogs & derivatives; Transferrin - analysis; Transferrins; Carbohydrate-deficient transferrin; Transferrins; Human; Ferroprotein; Biological fluid; Transferrin; Separation; Capillary electrophoresis; Deficiency; Biological marker; Alcohol; Analysis method; Serum; Carbohydrate; Quantitative analysis
• ISSN: 0378-4347; 1387-2273
• DOI: 10.1016/S0378-4347(99)00309-6

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 m M FeCl 3 (2 μl) and 500 m M NaHCO 3 (3 μl) 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×20 μm I.D.) and a buffer composed of 100 m M sodium tetraborate adjusted with 6 M HCl to pH 8.3 added with 1.5 m M diaminobutane. Applied voltage was 20 kV and temperature 25°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 ≤0.2%. Quantitation showed day-to-day RDSs ≤6.9% and ≤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.