Determination of Carbohydrate-deficient Transferrin Using Capillary Zone Electrophoresis

Abstract

Current methods for carbohydrate-deficient transferrin (CDT) often suffer from low precision, complexity, or risk of false positives attributable to genetic variants. In this study, a new capillary zone electrophoresis (CZE) method for CDT was developed. CZE was performed on a P/ACE 5000 using fused-silica capillaries [50 microm (i.d.) x 47 cm] and the CEOFIX CDT buffer system with addition of 50 microL of anti-C3c and 10 microL of anti-hemoglobin. Native sera were loaded by high-pressure injection for 3 s, separated at 28 kV over 12 min, and monitored at 214 nm. CDT was completely resolved by differences in migration times (di-trisialotransferrin, 9.86 +/- 0.05 min; monosialotransferrin, 9.72 +/- 0.05 min; asialotransferrin, 9.52 +/- 0.04 min), with a CV of 0.15%. The number of theoretical plates was 312,000 +/- 21,000 for the mono- and 199 000 +/- 6500 for the di-trisialylated transferrin. Genetic CB and CD variants showed prominent peaks with migration times of 10.12 +/- 0.06 and 9.89 +/- 0.03 min, respectively, and the carbohydrate-deficient glycoprotein syndrome could be detected, excluding false-positive results. CZE results (as a percentage; y) correlated with the Axis %CDT TIA (x) values by Deming regression analysis: y = 1.92x - 7.29; r = 0.89. CDT values in 130 healthy nonalcoholics were determined. The 2.5th and 97.5th percentiles were 1.84% and 6.79%. CZE without sample pretreatment can determine CDT with good precision, allows detection of variants, and correlates with ion-exchange chromatography.