title: A new sensitive and quantitative chemiluminescent assay to monitor intracellular xanthine oxidase activity for rapid screening of inhibitors in living endothelial cells.
abstract: Xanthine oxidase (XO) is an important enzyme, expressed at high levels in the vasculature in endothelial cells, that catalyzes the hydroxylation of hypoxanthine to xanthine and xanthine to uric acid. Excessive production of uric acid results in hyperuricemia linked to gout and cardiovascular diseases. Testing inhibition of XO is important for detection of potentially effective drugs or natural products that could be used to treat diseases caused by increased XO activity. In the present study, for the first time, we developed an in vitro chemiluminescent bioassay to determine XO activity in living endothelial cells and the IC50 value of oxypurinol, the active metabolite of the inhibitor drug allopurinol. Intracellular XO activity was measured in less than 20 min with a luminol/catalyst-based chemiluminescence assay able to measure XO with a limit of 0.4 μU/mL. Oxypurinol addition to 5 × 103cells (ranging from 5.0 to 0.0 μM) caused a linear decrease in XO activity, with an IC50 of 1.0 ± 0.5 μM. The detection system developed was low-cost, rapid, reproducible, and easily miniaturizable so suitable to be used on small quantities of cells.
authors.affiliation: Department of Chemistry "Giacomo Ciamician", Alma Mater Studiorum - University of Bologna, Bologna, Italy. cristiana.caliceti@unibo.it.|Istituto Nazionale Biostrutture e Biosistemi (INBB), Rome, Italy. cristiana.caliceti@unibo.it.|Centro Interdipartimentale di Ricerca Industriale Energia e Ambiente (CIRI EA), Alma Mater Studiorum - University of Bologna, Bologna, Italy. cristiana.caliceti@unibo.it.
authors.forename: C
authors.initials: C
authors.lastname: Caliceti
authors.affiliation: Department of Chemistry "Giacomo Ciamician", Alma Mater Studiorum - University of Bologna, Bologna, Italy.|Centro Interdipartimentale di Ricerca Industriale Energia e Ambiente (CIRI EA), Alma Mater Studiorum - University of Bologna, Bologna, Italy.
authors.forename: D
authors.initials: D
authors.lastname: Calabria
authors.affiliation: Department of Chemistry "Giacomo Ciamician", Alma Mater Studiorum - University of Bologna, Bologna, Italy.|Istituto Nazionale Biostrutture e Biosistemi (INBB), Rome, Italy.|Centro Interdipartimentale di Ricerca Industriale Energia e Ambiente (CIRI EA), Alma Mater Studiorum - University of Bologna, Bologna, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Roda
mesh_terms: D000493:Allopurinol / Q000737:chemistry / Q000494:pharmacology; D001681:Biological Assay; D003593:Cytoplasm / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D057230:Limit of Detection; D008163:Luminescent Measurements / Q000379:methods*; D008165:Luminol / Q000737:chemistry; D010084:Oxidation-Reduction; D010117:Oxypurinol / Q000494:pharmacology; D014527:Uric Acid / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014969:Xanthine Oxidase / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D014527:Uric Acid; D008165:Luminol; D000493:Allopurinol; D014969:Xanthine Oxidase; D010117:Oxypurinol
keywords: Chemiluminescence; Inhibitors; Quantitative intracellular assay; Vascular endothelial cells; Xanthine oxidase