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A new sensitive and quantitative chemiluminescent assay to monitor intracellular xanthine oxidase activity for rapid screening of inhibitors in living endothelial cells

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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 × 103 cells (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.

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Acknowledgments

We are grateful to Prof. M. Guardigli and M. Mirasoli for the critical review of this manuscript.

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Authors and Affiliations

  1. Department of Chemistry “Giacomo Ciamician”, Alma Mater Studiorum - University of Bologna, Bologna, Italy

    C. Caliceti, D. Calabria & A. Roda

  2. Istituto Nazionale Biostrutture e Biosistemi (INBB), Rome, Italy

    C. Caliceti & A. Roda

  3. Centro Interdipartimentale di Ricerca Industriale Energia e Ambiente (CIRI EA), Alma Mater Studiorum - University of Bologna, Bologna, Italy

    C. Caliceti, D. Calabria & A. Roda

Authors
  1. C. Caliceti
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  2. D. Calabria
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  3. A. Roda
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Corresponding author

Correspondence to C. Caliceti.

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Fundings source

This work was supported by the Istituto Nazionale Biostrutture e Biosistemi (INBB), Roma, Italy. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Published in the topical collection Highlights of Analytical Chemical Luminescence with guest editors Aldo Roda, Hua Cui, and Chao Lu.

C. Caliceti and D. Calabria contributed equally to this work.

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Caliceti, C., Calabria, D. & Roda, A. A new sensitive and quantitative chemiluminescent assay to monitor intracellular xanthine oxidase activity for rapid screening of inhibitors in living endothelial cells. Anal Bioanal Chem 408, 8755–8760 (2016). https://doi.org/10.1007/s00216-016-9763-x

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  • DOI: https://doi.org/10.1007/s00216-016-9763-x

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