Accurate nitric oxide measurements from donors in cell media: identification of scavenging agents

Nitric oxide (NO) is an essential messenger in human physiology, mediating cellular processes ranging from proliferation to apoptosis. The effects of NO are concentration dependent, and control over the instantaneous amount of NO available to cells is essential for determining the therapeutic NO dosages for various applications. As such, the development of NO therapeutic materials relies on accurate quantitative NO measurements that provide both total NO release from the NO donor as well as instantaneous NO concentrations. On the basis of the complexity of the cell media environment, inaccurate NO reporting often occurs for in vitro studies. These inaccuracies result from using inert media such as phosphate buffer saline (PBS), failing to account for the reactivity of media components. In this work, we describe a method for directly quantifying the instantaneous and total amounts of NO from commonly used NO donors in commercially available cell media routinely used for endothelial and neural cell lines. A riboflavin-tryptophan complex found in the media was identified as the major scavenger of NO in the cell media and likely reacts with NO via a radical-radical reaction. This finding significantly impacts the amount of available NO. The scavenging effects are concentration dependent on the riboflavin-tryptophan complex and the NO release rate from the NO donor. The results of this study provide insights on the exogenous amounts of NO that are present in cell media and may provide an explanation for differences in NO dosages between buffer experiments and in vitro and in vivo studies.