Development and validation of a quantitative method for thymidine phosphorylase activity in peripheral blood mononuclear cells

Autor:	Anna Tzani, Dick Pluim, Bart A. W. Jacobs, Jan H.M. Schellens, Pia van der Laan, Jos H. Beijnen
Rok vydání:	2018
Předmět:	0301 basic medicine Antimetabolites Antineoplastic Thymidine phosphorylase activity fluoropyrimidines Biochemistry Peripheral blood mononuclear cell Sensitivity and Specificity Monocytes 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Limit of Detection Neoplasms Genetics pharmacodynamics Humans Lymphocytes Thymidine phosphorylase Capecitabine Chromatography High Pressure Liquid chemistry.chemical_classification Thymidine Phosphorylase peripheral blood mononuclear cells (PBMCs) General Medicine Molecular biology Thymine 030104 developmental biology Enzyme chemistry 030220 oncology & carcinogenesis Toxicity Leukocytes Mononuclear Molecular Medicine Thymidine Ex vivo
Zdroj:	Nucleosides and Nucleotides, 37(8), 436. Taylor and Francis Ltd.
ISSN:	1532-2335 0732-8311
Popis:	The enzyme thymidine phosphorylase (TP) is important for activation of capecitabine and 5-fluorouracil. Assessment of TP phenotype might be suitable for identification of patients at risk of fluoropyrimidine-induced toxicity. In this paper, we describe the development and validation an assay for TP activity in peripheral blood mononuclear cells (PBMCs). The assay was based on ex vivo conversion of the TP substrate thymidine to thymine. The amount of thymine formed was determined by high-performance liquid chromatography - ultraviolet detection (HPLC-UV) with 5-bromouracil as internal standard. Lymphocytes and monocytes were purified from isolated PBMCs to examine cell-specific TP activity. TP activity in PBMCs demonstrated Michaelis-Menten kinetics. The lower limit of quantification was 2.3 µg PBMC protein and assay linearity was demonstrated up to 22.7 µg PBMC protein. Within-day and between-day precisions were ≤9.2% and ≤6.0%, respectively. Adequate stability TP activity was demonstrated after long-term storage of PBMC dry pellets and lysates at -80 °C. In monocytes, TP activity was approximately 3 times higher than in lymphocytes. Clinical applicability was demonstrated in samples that were collected from five cancer patients. A simple, precise and sensitive HPLC-UV assay for quantification of TP activity in PBMCs was developed that can be applied for clinical research.
Databáze:	OpenAIRE
Externí odkaz:	https://explore.openaire.eu/search/publication?articleId=doi_dedup___::e49c22f933c9c74d1a85bd32fdcf5a6b https://pubmed.ncbi.nlm.nih.gov/30285552 Zobrazit plný text záznamu Plný text ve formátu PDF Plný text ve formátu HTML
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