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Fluorescent adduct formation with terbium: a novel strategy for transferrin glycoform identification in human body fluids and carbohydrate-deficient transferrin HPLC method validation

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Abstract

This paper puts forward a new method for the transferrin (Tf) glycoform analysis in body fluids that involves the formation of a transferrin-terbium fluorescent adduct (TfFluo). The key idea is to validate the analytical procedure for carbohydrate-deficient transferrin (CDT), a traditional biochemical serum marker to identify chronic alcohol abuse. Terbium added to a human body-fluid sample produced TfFluo. Anion exchange HPLC technique, with fluorescence detection (λ exc 298 nm and λ em 550 nm), permitted clear separation and identification of Tf glycoform peaks without any interfering signals, allowing selective Tf sialoforms analysis in human serum and body fluids (cadaveric blood, cerebrospinal fluid, and dried blood spots) hampered for routine test. Serum samples (n = 78) were analyzed by both traditional absorbance (Abs) and fluorescence (Fl) HPLC methods and CDT% levels demonstrated a significant correlation (p < 0.001 Pearson). Intra- and inter-runs CV% was 3.1 and 4.6%, respectively. The cut-off of 1.9 CDT%, related to the HPLC Abs proposed as the reference method, by interpolation in the correlation curve with the present method demonstrated a 1.3 CDT% cut-off. Method comparison by Passing-Bablok and Bland-Altman tests demonstrated Fl versus Abs agreement. In conclusion, the novel method is a reliable test for CDT% analysis and provides a substantial analytical improvement offering important advantages in terms of types of body fluid analysis. Its sensitivity and absence of interferences extend clinical applications being reliable for CDT assay on body fluids usually not suitable for routine test.

The formation of a transferrin-terbium fluorescent adduct can be used to analyze the transferrin glycoforms. The HPLC method for carbohydrate-deficient transferrin (CDT%) measurement was validated and employed to determine the levels in different body fluids.

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Acknowledgements

The authors wish to thank Veronica Paterlini for her excellent work measuring the fluorescence spectra and Andrew Bailey for his invaluable language revision. This work was supported in part by a grant from the Department of Diagnostics and Public Health, Unit of Forensic Medicine, University of Verona, Italy.

University of Verona Patent No. MI2014A000395 and No. PCT/EP2015/054896

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Authors and Affiliations

  1. Department of Diagnostics and Public Health, Unit of Forensic Medicine, University of Verona, Policlinico “G.B. Rossi”, Piazzale L.A. Scuro 10, 37134, Verona, Italy

    Daniela Sorio, Elio Franco De Palo, Anna Bertaso, Federica Bortolotti & Franco Tagliaro

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  1. Daniela Sorio
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  2. Elio Franco De Palo
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  3. Anna Bertaso
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Correspondence to Franco Tagliaro.

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The study protocol was approved by the local ethics committee. Informed consent was obtained before sample collection.

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University of Verona Patent No. MI2014A000395 and No. PCT/EP2015/054896.

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Sorio, D., De Palo, E.F., Bertaso, A. et al. Fluorescent adduct formation with terbium: a novel strategy for transferrin glycoform identification in human body fluids and carbohydrate-deficient transferrin HPLC method validation. Anal Bioanal Chem 409, 1369–1378 (2017). https://doi.org/10.1007/s00216-016-0069-9

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