Apllication of capillary electrophoresis to analyze human transferrin focusing on the detection and quantification of the asialo-Tf isoform

In forensic sciences, the analysis of Carbohydrate-deficient transferrin (CDT) is used in the diagnostics of alcohol abuse, because it is known that the concentration of CDT increases after alcohol consumption. The human Transferrin (Tf) is the iron transport serum glycoprotein characterized by oligosaccharide chains that can be n-“antennary” (from zero to four) each one with a sialic-acid residue. Consequently, there are different isoforms with different numbers of sialic-acid residues and CDT includes the monosialo-, the asialo-, and the disialo-Tf isoforms characterized by absence, one or two sialic-acid residues. Currently, CDT analyses are based on the relative quantification of disialo-Tf isoform in respect to the total Tf isoforms identified (up to pentasialo-Tf). At present, the available analytical methods to perform CDT analysis based on immunochemical assay, liquid chromatography (LC) and capillary electrophoresis (CE) do not allow for an accurate identification of asialo-Tf because of its low concentration. Taking into account these premises, this project, starting from an existing capillary electrophoretic method performing “routine” CDT analyses, was aimed at developing an improved procedure suitable for asialo-Tf determination. The “new” CE method was developed by optimizing different experimental parameters such as temperature, electrical potentials, injection time and types, stacking procedures and the running buffer composition. The selected conditions have been 28 °C of temperature and 30 kV of applied voltage combined with the increase of the amount of the loaded material by increasing the injection time up to 50 s and with the use of an optimized running buffer adjusted to the 1,4-diaminobutane (DAB) 9 mM concentration. In addition, a sample pre-treatment procedure with polyethylene glycol 8000 (PEG 8000) at final concentration of 20% was introduced to further improve the asialo-Tf detection through the precipitation of interfering proteins present in the serum sample. The analysis of results showed an additional peak, in both disialo-Tf “positive” and “negative” samples, with the relative migration time (T_mr) ascribable to asialo-Tf and quantifiable up to minimal concentration of 0.2%. The resolution (R) between other isoforms was maintained (R=1.5 between disialo- and trisialo-Tf) with an increase of signal intensity of two times in respected to the results obtained using the “traditional” method. The intra-day reproducibility study of asialo-Tf showed a coefficient of variation (CV) of 23%, for concentrations of asialo-Tf ≤ 0.2% and a CV of 7%, for concentrations ≥ 0.7%. The inter-days reproducibility was characterized by a CV range from 22.5% (for an asialo-Tf concentration of 0.21%) to 5% (for an asialo-Tf concentration of 1.23%). These results can be considered acceptable taking into account the instrumental sensitivity of CE. Although a final validation is still lacking (e.g. by applying immunoextraction and/or immunosubtraction test), at the present stage of the work, we can state that for the first time in the literature a method suitable for the detection and quantification of asialo-Tf in human serum has been developed and preliminarily validated.