title: Fluorescent adduct formation with terbium: a novel strategy for transferrin glycoform identification in human body fluids and carbohydrate-deficient transferrin HPLC method validation.
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 (λexc298 nm and λem550 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. Graphical Abstract 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.
authors.affiliation: 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.
authors.forename: Daniela
authors.initials: D
authors.lastname: Sorio
authors.affiliation: 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.
authors.forename: Elio Franco
authors.initials: EF
authors.lastname: De Palo
authors.affiliation: 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.
authors.forename: Anna
authors.initials: A
authors.lastname: Bertaso
authors.affiliation: 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.
authors.forename: Federica
authors.initials: F
authors.lastname: Bortolotti
authors.affiliation: 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. franco.tagliaro@univr.it.
authors.forename: Franco
authors.initials: F
authors.lastname: Tagliaro
mesh_terms: D001826:Body Fluids / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D013050:Spectrometry, Fluorescence; D013725:Terbium / Q000737:chemistry*; D014168:Transferrin / Q000031:analogs & derivatives* / Q000032:analysis
chemical_list: D005456:Fluorescent Dyes; D014168:Transferrin; C056759:carbohydrate-deficient transferrin; D013725:Terbium
keywords: Alcohol abuse; CDT; Dried blood spots; Fluorescence; Terbium; Transferrin