Determination of Carbohydrate-Deficient Transferrin in Human Serum by Capillary Zone Electrophoresis

Abstract

Abstract Serum transferrin (Tf) comprises several isoforms with up to two complexoligosaccharide chains containing 0 to 8 sialic acid residues and neutral sugars. Themajor glycoform, known as tetrasialo-Tf, contains 4 sialic acid residues and accounts forabout 80 % of whole Tf in human serum. Carbohydrate-deficient transferrin (CDT)encompasses isoforms that are deficient in carbohydrate chains and consequently insialic acid residues (including asialo-, monosialo- and disialo-Tf) and is a well knownmarker for chronic alcohol abuse. Recently capillary zone electrophoresis (CZE) hasbeen reported as a tool extremely effective for the simultaneous, individual, quantitativedetermination of CDT isoforms. Three CZE methods that feature different dynamiccapillary coatings were evaluated and optimized for CDT determination in human serumof alcohol abusers and control subjects. CZE separation was performed in alkalineborate buffers after serum sample saturation with iron, electropherograms were detectedat 200 nm, data were evaluated as area % of disialo-Tf in relation to tetrasialo-Tf andpeak identification was accomplished via relative migration times to tetrasialo-Tf,immunosubtraction and enzymatic sequential cleavage of sialic acid residues. Dynamiccapillary coatings with diaminobutane, spermine and a double coating produced bycommercially available proprietary agents were investigated and found to be suitable fordetermination of CDT in human serum. For all three approaches, best results wereobtained in 50 µm I.D. fused-silica capillaries of 50 cm effective length and a capillarycartridge temperature of 20 - 25 °C. Using 3 mM 1,4-diaminobutane (DAB) or 0.02 mMspermine in a pH 8.3 borate-based running buffer provided data of remarkable similaritywith resolution of di-, tri-, tetra- and pentasialo-Tf within 15 to 18 min. With the doublecoating, asialo-Tf and Tf isoforms with 2 to 6 sialic acid residues were baselineseparated. Compared to the two amine-based procedures, the run times were found to besomewhat shorter, the detector signals higher, the applied power level significantlylower and the reproducibility better.