Isabella Fermo 1 Luca Germagnoli 2 Armando Soldarini 2 Fernanda Dorigatti 2 Rita Paroni 1, 3 1 Laboratory of Separative Techniques, IRCCS H San Raffaele, 2 Diagnostica e Ricerca San Raffaele S.p.a, 3 Department of Medicine, Surgery and Dental Sciences, University of Milan, H San Paolo, Milan, Italy Capillary zone electrophoresis for determination of carbohydrate-deficient transferrin in human serum Carbohydrate-deficient transferrin (CDT) is the most specific marker for diagnosis of chronic excessive alcohol consumption and includes the serum transferrin (T f ) iso- forms with two or less sialic acid residues (di-, mono-, and asialo-T f ). To monitor serum CDT, we developed a capillary zone electrophoresis (CZE) method based on the dy- namic capillary coating with diethylenetriamine (DETA). The separation was performed in a bare fused-silica capillary (50 mm ID, 57 cm in length), applying a voltage of 25 kV and a temperature of 407C. Using a 100 mmol/L borate buffer, pH 8.4 with 3 mmol/L DETA, the T f isoforms (asialo- to pentasialo-T f ) were resolved within 16 min. Enzymatic cleavage of sialic acid residues with neuraminidase and immunosubtraction were used to identify CDT isoforms. The relative amount of CDT expressed as area % of disialo-T f isoform related to the area of tetrasialo-T f in 50 healthy donors (24 males and 26 females; aged 25–50 years) was 3.15 6 0.76% (mean 6 SD). The comparison between CDT values obtained by this CZE procedure and the “Axis-Shield %CDT” kit gave r = 0.644, p , 0.001 (n = 290). This easy to use and inexpensive CZE procedure could be an ideal tool to investigate CDT proteins for clinical or forensic purposes. Keywords: Alcohol abuse / Capillary zone electrophoresis / Carbohydrate-deficient transferrin / Diethylenetriamine DOI 10.1002/elps.200305694 1 Introduction Transferrin (T f ), the most important iron-transport protein in the blood, is characterized by two independent metal ion-binding sites and two N-linked branched carbohy- drate chains terminating with a sialic acid molecule [1]. Serum T f shows a microheterogenity in isoelectric points due to differences in the iron load or in the number of sialic acid residues that may vary from 0 to 8. The predominant serum isoform (,75% of total T f ) is the tetrasialo-T f that contains two biantennary N-glycans with four terminal sialic acid residues (pI 5.4). Normal serum also contains low amounts of pentasialo- (pI 5.3), trisialo- (pI 5.6), and disialo-T f (pI 5.7). An increase in the isoforms with pI . 5.7 (mono-, di-, and asialo-T f ), collectively referred as carbo- hydrate-deficient transferrine (CDT), was observed for the first time in the cerebrospinal fluid of patients with alcoholic cerebellar degeneration [2], and, later on, in the serum of subjects consuming large quantities of ethanol [3]. In the last years, CDT has gained attention as a sensitive and specific marker of chronic alcohol abuse and for monitoring abstinence, being characterized by a diagnos- tic specificity higher than the traditional laboratory tests for detecting consumption of ethanol (mean corpuscular volume, g-glutamyltransferase, aspartate, and alanine aminotransferase) [4, 5]. Early procedures for CDT determination were based on concanavalin A crossed affinity immunoelectrophoresis [6] and on IEF coupled with laser densitometry to evaluate T f band patterns after immunoblotting [7]. The latter highly selective technique, in spite of its complexity for routine clinical use, is considered the reference method for the diagnosis of congenital disorders of glycosylation. Later, methods based on enzyme (CDTect-EIA; Pharma- cia & UpJohn, Uppsala, Sweden) or turbidimetric (Axis- Shield %CDT; Axis-Shield ASA, Oslo, Norway) immu- noassay [8–10] have been proposed. Due to the lack of CDT-specific reactions or antibodies, however, CDT determination by immunoassay requires as a first step the separation from non-CDT isoforms and from serum matrix by electrophoresis (e.g., IEF) or by chromatog- raphy (e.g., anion exchange). The immunoassays are unable to differentiate between proteins with different pI, so the genetic T f variants could potentially impair the accuracy of the analysis. Thus, when CDT determination Correspondence: Dr. Rita Paroni, Department of Medicine, Sur- gery, and Dental Sciences, University of Milano, H San Paolo via Di Rudini’ 8, I-20142 Milan, Italy E-mail: rita.paroni@unimi.it Fax: 139-02-50316040 Abbreviations: CDT , carbohydrate-deficient transferrin; DAB, 1,4-diaminobutane; DETA, diethylenetriamine; T f , transferrin Electrophoresis 2004, 25, 469–475 469 CE and CEC  2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim