Removal of Ammonia Interference in the Redox Chemiluminescence Assay of Nitric Oxide

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Abstract
Nitric oxide (NO) is now recognized as an important mediator that regulates a number of physiological functions. The most sensitive chemical method for its quantitation is via a redox-chemiluminescence detector (RCD), which can detect sub-picomole quantities of NO. In a microsomal metabolism study involving nitroglycerin, we observed that NH3 could produce a chemiluminescence signal that interfered with the assay of NO, although the RCD sensitivity toward NH3 was, on a molar basis, approximately 2700× less than that toward NO. Incorporation of a Porapak® T column either completely removed NH3 RCD signal (< 40 nmole), or caused a chromatographic separation between NH3 and NO (> 40 nmole of NH3). The presence of the polymer packing did not affect the sensitivity of detector response toward NO. The applicability of this separation method was validated in a biochemical study in which microsomes from bovine coronary artery smooth muscle cells were incubated under conditions that would produce NO as well as NH3 the latter probably through protein degradation. The separation method described appears to be useful in safeguarding artifactual contamination from NH3 in the redox chemiluminescence assay of NO.