A new method for monitoring nitric oxide production using Teflon membrane microdialysis

Abstract

The measurement of nitric oxide (NO) by electron spin resonance (ESR) is complicated by potentially toxic spin-trapping agents, which may affect the NO-producing cells per se and/or cause artifacts and systemic side effects. These problems can be addressed by preventing direct interaction between the agent and the biological system. In the present study, we utilized Teflon as a barrier between the spin trap and the living cell, since the material is permeable to gas only. Our aim was to investigate if NO could diffuse across the membrane in sufficient amounts to be trapped and quantified by ESR. We used standard microdialysis equipment and specially designed dialysis probes, or tubing, with Teflon membranes. Sodium nitroprusside was used as a NO donor and Fe– N-dithiocarboxysarcosine (Fe(DTCS) 2) as a spin trap. NO readily diffuses through Teflon and could be quantified in concentrations considerably below 50 nM in a reproducible and accurate manner. In cell cultures of activated murine macrophages, NO synthesis from iNOS could be monitored and we noted a huge increase in NO concentration by superoxide dismutase. We conclude that spin trapping of NO by Fe(DTCS) 2 across Teflon membranes is an attractive approach for quantifying and monitoring nitric oxide production without interfering with cell viability.