Evaluation and optimization of capillary zone electrophoresis with different dynamic capillary coatings for the determination of carbohydrate-deficient transferrin in human serum

Abstract

Serum transferrin (Tf) comprises several isoforms with up to two complex oligosaccharide chains containing zero to eight sialic acid residues and neutral sugars. The major glycoform, known as tetrasialo-Tf, contains four sialic acid residues and accounts for about 80% of whole Tf in human serum. Carbohydrate-deficient transferrin (CDT) encompasses isoforms that are deficient in carbohydrate chains and consequently in sialic acid residues (including asialo-, monosialo- and disialo-Tf) and is a well known marker for chronic alcohol abuse. Recently capillary zone electrophoresis (CZE) has been reported as a tool extremely effective for the simultaneous, individual, quantitative determination of CDT isoforms. Three CZE methods that feature different dynamic capillary coatings were evaluated and optimized for CDT determination in human serum of alcohol abusers and control subjects. CZE separation was performed in alkaline borate buffers after serum sample saturation with iron, electropherograms were detected at 200 nm, data were evaluated as % area of disialo-Tf in relation to tetrasialo-Tf and peak identification was accomplished via relative migration times to tetrasialo-Tf, immunosubtraction and enzymatic sequential cleavage of sialic acid residues. Dynamic capillary coatings with diaminobutane, spermine and a double coating produced by commercially available proprietary agents were investigated and found to be suitable for determination of CDT in human serum. For all three approaches, best results were obtained in 50 microm I.D. fused-silica capillaries of 50 cm effective length and a capillary cartridge temperature of 20-25 degrees C. Using 3 mM 1,4-diaminobutane or 0.02 mM spermine in a borate-based running buffer of pH 8.3 provided data of remarkable similarity with resolution of di-, tri-, tetra- and pentasialo-Tf within 15-18 min. With the double coating, asialo-Tf and Tf isoforms with two to six sialic acid residues were baseline separated. Compared to the two amine-based procedures, the run times were found to be somewhat shorter, the detector signals higher, the applied power level significantly lower and the reproducibility better.