Inaccuracies of Nitric Oxide Measurement Methods in Biological Media

Abstract

Despite growing reports on the biological action of nitric oxide (NO) as a function of NO payload, the validity of such work is often questionable due to the manner in which NO is measured and/or the solution composition in which NO is quantified. To highlight the importance of measurement technique for a given sample type, NO produced from a small molecule NO donor (N-diazeniumdiolated l-proline, PROLI/NO) and a NO-releasing xerogel film were quantified in a number of physiological buffers and fluids, cell culture media, and bacterial broth using the Griess assay, a chemiluminescence analyzer, and an amperometric NO sensor. Despite widespread use, the Griess assay proved to be inaccurate for measuring NO in many of the media tested. In contrast, the chemiluminescence analyzer provided superb kinetic information in most buffers, but was impractical for NO analysis in proteinaceous media. The electrochemical NO sensor enabled greater flexibility across the various media with potential for spatial resolution, albeit at lower than expected NO totals versus either the Griess assay or chemiluminescence. The results of this study highlight the importance of measurement strategy for accurate NO analysis and reporting NO-based biological activity.

Antibacterial Action of Nitric Oxide-Releasing Chitosan Oligosaccharides against Pseudomonas aeruginosa under Aerobic and Anaerobic Conditions

“…NO-selective electrochemical sensors were fabricated in house as previously reported (43). Briefly, polished polycrystalline Pt disk electrodes (2 mm) sealed in Kel-F (CH Instruments, Austin, TX) were coated with an NO-selective membrane prepared by mixing MTMOS (60 l), ethanol (300 l), 17 FTMS (15 l), water (80 l), and 0.5 M hydrochloric acid (5 l) for 1 h at 25°C.…”

“…While NO release from macromolecular scaffolds is generally measured in deoxygenated medium, measuring the amount of bioavailable (i.e., nonscavenged) NO is critical for elucidating the biocidal dose-response relationship of NO under the intended conditions, as NO is rapidly scavenged by oxygen and proteins in biological media (45). Unfortunately, foaming associated with nutrient-rich medium makes chemiluminescence detection difficult and irreproducible (43). Thus, we turned to amperometric NO detection to carry out NO measurements in broth (LB).…”

“…As might be expected, both the total NO payload and the release duration of COS-NO measured via amperometry were reduced compared to chemiluminescence detection. These decreases are common for electrochemical sensors that are based on the diffusion of NO to the working electrode and the inherent loss of NO to the ambient atmosphere (43).…”

An Improved Assay for Measuring Low Levels of Nitric Oxide in Cultured Pulmonary Myofibroblasts

“…Moreover, in this study we have also refined the technique by using PBS as the assay solvent thus reducing background noise from interfering species. Matrix effects are well known in cell-based assays and are of paramount concern when measuring very low levels of analyte from cells in culture; a recent study that compared NO quantification from an NO-releasing film using spectrophotometric, chemiluminescent, or amperometric detection methods found that NO measured in cell culture media or serum was always significantly lower than that measured in PBS [30].…”

New Iridoids from <i>Scrophularia ningpoensis</i>

“…The anti-inflammatory activity of the isolated compounds 1-11 were evaluated through inhibition of NO production in lipopolysaccharide-induced RAW264.7 macrophage cells using the Griess method 25) and experiments were carried out as our previous descriptions. 26) Minocycline was used as a positive control.…”