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Drug Monitoring and Toxicology |
1 Departments of Clinical Neuroscience and Clinical Chemistry, Karolinska Institutet and Hospital, SE-171 76 Stockholm, Sweden.
2 Axis-Shield ASA, N-0510 Oslo, Norway.
3 Department of Clinical Chemistry, Malmo University Hospital, SE-205 02 Malmo, Sweden.
aAddress correspondence to this author at: Alcohol Laboratory, L7:03, Karolinska Hospital, SE-171 76 Stockholm, Sweden. Fax 46-8-5177-1532; e-mail anders.helander{at}cns.ki.se.
Background: There is need for a reference method for transferrin glycoforms in serum to which routine immunologic methods for the alcohol marker carbohydrate-deficient transferrin (CDT) can be traceable. We describe an improved HPLC method for transferrin glycoforms.
Methods: Transferrin was iron-saturated by mixing the serum with ferric nitrilotriacetic acid, and lipoproteins were precipitated with dextran sulfate and calcium chloride. Separation of glycoforms was performed on a SOURCE 15Q anion-exchange column using salt gradient elution. Quantification relied on selective absorbance of the iron–transferrin complex at 470 nm. The relative amount of each glycoform was calculated as a percentage of the area under the curve, using baseline integration.
Results: The HPLC system provided reproducible separation and quantification of the asialo-, monosialo-, disialo-, trisialo-, tetrasialo-, pentasialo-, and hexasialotransferrin glycoforms. Most importantly, disialo- and trisialotransferrin were almost baseline separated. The intra- and interassay CV for disialotransferrin were <5%. Serum and the pretreated samples were stable for at least 2 days at 22 or 4 °C. Sera from 132 healthy controls contained [mean (SD)] 1.16 (0.25)% disialotransferrin, 4.77 (1.36)% trisialotransferrin, 80.18 (2.01)% tetrasialotransferrin, and 13.88 (1.69)% pentasialo- + hexasialotransferrin. In some cases of a high (>6%) trisialotransferrin, monosialotransferrin was detected at <0.25%. Asialotransferrin was not detected in control sera, but was detected in 57% of chronic heavy drinkers and in 62% of sera with 
2% disialotransferrin.
Conclusions: The HPLC method fulfills the requirements of a preliminary reference method for CDT and should work for any combination of serum transferrin glycoforms. This method could also be useful for confirming positive CDT results by immunoassays in medico-legal cases.
The following articles in journals at HighWire Press have cited this article:
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  V. Bianchi, A. Ivaldi, A. Raspagni, C. Arfini, and M. Vidali Use of Carbohydrate-Deficient Transferrin (CDT) and a Combination of GGT and CDT (GGT-CDT) to Assess Heavy Alcohol Consumption in Traffic Medicine Alcohol Alcohol., January 28, 2010; (2010): agq006v1 - agq006. [Abstract] [Full Text] [PDF]
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F. Schellenberg, L. Mennetrey, C. Girre, B. Nalpas, and J. C. Pages Automated Measurement of Carbohydrate-Deficient Transferrin Using the Bio-Rad %CDT by the HPLC Test on a VariantTM HPLC System: Evaluation and Comparison with Other Routine Procedures Alcohol Alcohol., September 1, 2008; 43(5): 569 - 576. [Abstract] [Full Text] [PDF]
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J. P. Bergstrom and A. Helander Clinical Characteristics of Carbohydrate-Deficient Transferrin (%Disialotransferrin) Measured by HPLC: Sensitivity, Specificity, Gender Effects, and Relationship with other Alcohol Biomarkers Alcohol Alcohol., July 1, 2008; 43(4): 436 - 441. [Abstract] [Full Text] [PDF]
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 J. B. Whitfield, V. Dy, P. A.F. Madden, A. C. Heath, N. G. Martin, and G. W. Montgomery Measuring Carbohydrate-Deficient Transferrin by Direct Immunoassay: Factors Affecting Diagnostic Sensitivity for Excessive Alcohol Intake Clin. Chem., July 1, 2008; 54(7): 1158 - 1165. [Abstract] [Full Text] [PDF]
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A. Helander and G. Nordin Insufficient Standardization of a Direct Carbohydrate-Deficient Transferrin Immunoassay Clin. Chem., June 1, 2008; 54(6): 1090 - 1092. [Full Text] [PDF]
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J. R. Delanghe, A. Helander, J. P.M. Wielders, J. M. Pekelharing, H. J. Roth, F. Schellenberg, C. Born, E. Yagmur, W. Gentzer, and H. Althaus Development and Multicenter Evaluation of the N Latex CDT Direct Immunonephelometric Assay for Serum Carbohydrate-Deficient Transferrin Clin. Chem., June 1, 2007; 53(6): 1115 - 1121. [Abstract] [Full Text] [PDF]
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S. ARADOTTIR, G. ASANOVSKA, S. GJERSS, P. HANSSON, and C. ALLING PHOSPHATIDYLETHANOL (PEth) CONCENTRATIONS IN BLOOD ARE CORRELATED TO REPORTED ALCOHOL INTAKE IN ALCOHOL-DEPENDENT PATIENTS Alcohol Alcohol., July 1, 2006; 41(4): 431 - 437. [Abstract] [Full Text] [PDF]
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F. Bortolotti, G. De Paoli, J. P. Pascali, M. T. Trevisan, M. Floreani, and F. Tagliaro Analysis of Carbohydrate-Deficient Transferrin: Comparative Evaluation of Turbidimetric Immunoassay, Capillary Zone Electrophoresis, and HPLC Clin. Chem., December 1, 2005; 51(12): 2368 - 2371. [Full Text] [PDF]
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A. Helander, J. P.M. Wielders, R. te Stroet, and J. P. Bergstrom Comparison of HPLC and Capillary Electrophoresis for Confirmatory Testing of the Alcohol Misuse Marker Carbohydrate-Deficient Transferrin Clin. Chem., August 1, 2005; 51(8): 1528 - 1531. [Full Text] [PDF]
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C. Papadea and R. J. Schlosser Rapid Method for {beta}2-Transferrin in Cerebrospinal Fluid Leakage Using an Automated Immunofixation Electrophoresis System Clin. Chem., February 1, 2005; 51(2): 464 - 470. [Abstract] [Full Text] [PDF]
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A. Helander, J. Bergstrom, and H. H. Freeze Testing for Congenital Disorders of Glycosylation by HPLC Measurement of Serum Transferrin Glycoforms Clin. Chem., May 1, 2004; 50(5): 954 - 958. [Full Text] [PDF]
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