Improved capillary electrophoresis method for the determination of carbohydrate-deficient transferrin in patient sera.

Abstract

Capillary zone electrophoresis (CZE) with a dynamic double coating formed by charged polymeric reagents represents an effective tool for the separation of iron-saturated transferrin (Tf) isoforms and thus the determination of carbohydrate-deficient transferrin (CDT, sum of asialo-, monosialo- and disialo-Tf in relation to total Tf) in human serum. Using the CEofix-CDT reagents, a 50 microm inner diameter (ID) capillary of 60 cm total length and the P/ACE MDQ under optimized instrumental conditions (20 kV and 30 degrees C) is demonstrated to provide outstanding assay precision for the determination of CDT in human serum. For CDT levels of 1.0% and 4.5%, precision relative standard deviation (RSD) values (n = 8) were determined to be < 3.0% and < 1.5%, respectively. During the first year of operation under routine conditions, more than 600 patient samples were analyzed in a total of 62 sets of runs. Except for selected samples of patients with severe liver diseases, interference-free Tf patterns were detected. Asialo-Tf was not detected in control sera and in patient sera with a CDT level < 1.70%, but became detectable in 89.6% of sera with > 2.3% disialo-Tf. Monosialo-Tf was only detected in two sera containing > 13.3% CDT. The optimized CZE assay was applied to confirm positive CDT results produced by an immunoassay during long-term monitoring of a patient which led to the determination of the elimination kinetics of asialo-Tf, disialo-Tf, and CDT after an episode of high alcohol consumption (estimated apparent half lifes of 4.86, 7.24, and 6.74 days, respectively). The optimized CZE assay with an upper reference limit for CDT of 1.70% represents an attractive alternative to high-performance liquid chromatography (HPLC). It features simpler sample preparation, faster analysis time, and higher isoform resolution compared to the most recent HPLC approach and can thus be regarded as a new candidate of a reference method for CDT.