title: Development of a new HPLC method using fluorescence detection without derivatization for determining purine nucleoside phosphorylase activity in human plasma.
abstract: Purine nucleoside phosphorylase (PNP) activity is involved in cell survival and function, since PNP is a key enzyme in the purine metabolic pathway where it catalyzes the phosphorolysis of the nucleosides to the corresponding nucleobases. Its dysfunction has been found in relevant pathological conditions (such as inflammation and cancer), so the detection of PNP activity in plasma could represent an attractive marker for early diagnosis or assessment of disease progression. Thus the aim of this study was to develop a simple, fast and sensitive HPLC method for the determination of PNP activity in plasma. The separation was achieved on a Phenomenex Kinetex PFP column using 0.1% formic acid in water and methanol as mobile phases in gradient elution mode at a flow rate of 1ml/min and purine compounds were detected using UV absorption and fluorescence. The analysis was fast since the run was achieved within 13min. This method improved the separation of the different purines, allowing the UV-based quantification of the natural PNP substrates (inosine and guanosine) or products (hypoxanthine and guanine) and its subsequent metabolic products (xanthine and uric acid) with a good precision and accuracy. The most interesting innovation is the simultaneous use of a fluorescence detector (excitation/emission wavelength of 260/375nm) that allowed the quantification of guanosine and guanine without derivatization. Compared with UV, the fluorescence detection improved the sensitivity for guanine detection by about 10-fold and abolished almost completely the baseline noise due to the presence of plasma in the enzymatic reaction mixture. Thus, the validated method allowed an excellent evaluation of PNP activity in plasma which could be useful as an indicator of several pathological conditions.
authors.affiliation: Department of Medical, Oral and Biotechnological Sciences, Laboratory of Pharmacology and Toxicology, University of Chieti-Pescara, Via dei Vestini 29, 66013 Chieti, Italy. Electronic address: p.giuliani@unich.it.
authors.forename: Patricia
authors.initials: P
authors.lastname: Giuliani
authors.affiliation: Department of Medical, Oral and Biotechnological Sciences, Laboratory of Pharmacology and Toxicology, University of Chieti-Pescara, Via dei Vestini 29, 66013 Chieti, Italy. Electronic address: mariachiarazuccarini@libero.it.
authors.forename: Mariachiara
authors.initials: M
authors.lastname: Zuccarini
authors.affiliation: Department of Medical, Oral and Biotechnological Sciences, Laboratory of Pharmacology and Toxicology, University of Chieti-Pescara, Via dei Vestini 29, 66013 Chieti, Italy. Electronic address: sbuccella@unich.it.
authors.forename: Silvana
authors.initials: S
authors.lastname: Buccella
authors.affiliation: Department of Medical, Oral and Biotechnological Sciences, Laboratory of Pharmacology and Toxicology, University of Chieti-Pescara, Via dei Vestini 29, 66013 Chieti, Italy. Electronic address: margherita.rossini@unich.it.
authors.forename: Margherita
authors.initials: M
authors.lastname: Rossini
authors.affiliation: Department of Medical, Oral and Biotechnological Sciences, Laboratory of Pharmacology and Toxicology, University of Chieti-Pescara, Via dei Vestini 29, 66013 Chieti, Italy. Electronic address: i.dalimonte@unich.it.
authors.forename: Iolanda
authors.initials: I
authors.lastname: D'Alimonte
authors.affiliation: Department of Medical, Oral and Biotechnological Sciences, Laboratory of Pharmacology and Toxicology, University of Chieti-Pescara, Via dei Vestini 29, 66013 Chieti, Italy. Electronic address: r.ciccarelli@unich.it.
authors.forename: Renata
authors.initials: R
authors.lastname: Ciccarelli
authors.affiliation: Department of Medical, Oral and Biotechnological Sciences, Laboratory of Pharmacology and Toxicology, University of Chieti-Pescara, Via dei Vestini 29, 66013 Chieti, Italy. Electronic address: matteo.marzo@hotmail.com.
authors.forename: Matteo
authors.initials: M
authors.lastname: Marzo
authors.affiliation: Department of Medical, Oral and Biotechnological Sciences, Laboratory of Pharmacology and Toxicology, University of Chieti-Pescara, Via dei Vestini 29, 66013 Chieti, Italy. Electronic address: antoniop.marzo@libero.it.
authors.forename: Antonio
authors.initials: A
authors.lastname: Marzo
authors.affiliation: Department of Medical, Oral and Biotechnological Sciences, Laboratory of Pharmacology and Toxicology, University of Chieti-Pescara, Via dei Vestini 29, 66013 Chieti, Italy. Electronic address: pat.diiorio@unich.it.
authors.forename: Patrizia
authors.initials: P
authors.lastname: Di Iorio
authors.affiliation: Department of Medical, Oral and Biotechnological Sciences, Laboratory of Pharmacology and Toxicology, University of Chieti-Pescara, Via dei Vestini 29, 66013 Chieti, Italy. Electronic address: f.caciagli@unich.it.
authors.forename: Francesco
authors.initials: F
authors.lastname: Caciagli
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000191:economics / Q000379:methods*; D057075:Enzyme Assays / Q000191:economics / Q000379:methods; D005453:Fluorescence; D006147:Guanine / Q000097:blood / Q000378:metabolism; D006151:Guanosine / Q000097:blood / Q000378:metabolism; D006801:Humans; D057230:Limit of Detection; D011683:Purine-Nucleoside Phosphorylase / Q000097:blood* / Q000378:metabolism
chemical_list: D006151:Guanosine; D006147:Guanine; D011683:Purine-Nucleoside Phosphorylase
keywords: Adenine- and guanine-derivatives; Enzyme kinetics; HPLC; Human plasma; Purine nucleoside phosphorylase; UV-Fluorescence detection