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Licensed Unlicensed Requires Authentication Published by De Gruyter November 29, 2008

Isotope tracing enhancement of chemiluminescence assays for nitric oxide research

Julia Cornelius, Tuan Tran, Nicole Turner, Abigail Piazza, Lauren Mills, Ryan Slack, Sean Hauser, J. Steven Alexander, Matthew B. Grisham, Martin Feelisch and Juan Rodriguez
From the journal Biological Chemistry

Abstract

Chemiluminescence assays are used widely for the detection of nitric oxide (NO)-derived species in biological fluids and tissues. Here, we demonstrate that these assays can be interfaced with mass-sensitive detectors for parallel determination of isotopic abundance. Results obtained with tri-iodide and ascorbic acid-based reductive assays indicate that mass spectrometric detection enables NO isotope-tracing experiments to be carried out to a limit of detectability of a few picomoles, a sensitivity similar to that of standard gas phase chemiluminescence methods. The advantage afforded by mass spectrometric detection is demonstrated using the murine macrophage cell line J774, which is shown here to reduce 15NO3- to 15NO2- under anoxic conditions. The particular combination of an analytical and cellular system described here may hold promise for future characterization of the enzymatic pathways contributing to mammalian nitrate reductase activity, without background interference from 14NO2- derived from other sources.


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These authors contributed equally to this work.

Corresponding author

Received: 2008-9-7
Accepted: 2008-10-22
Published Online: 2008-11-29
Published in Print: 2009-2-1

©2009 by Walter de Gruyter Berlin New York