Is the Real In Vivo Nitric Oxide Concentration Pico or Nano Molar? Influence of Electrode Size on Unstirred Layers and NO Consumption

OBJECTIVE: There is a debate if the [NO] required to influence vascular smooth muscle is below 50 nM or much higher. Electrodes with 30 μm and larger diameter report [NO] below 50 nM, whereas those with diameters of <10-12 μm report hundreds of nM. This study examined how size of electrodes influenced [NO] measurement due to NO consumption and unstirred layer issues. METHODS: Electrodes were 2 mm disk, 30 μm × 2 mm carbon fiber, and single 7 μm diameter carbon fiber within open tip microelectrode, and exposed 7 μm carbon fiber of ~15 μm to 2 mm length. RESULTS: All electrodes demonstrated linear calibrations with sufficient stirring. As stirring slowed, 30 μm and 2 mm electrodes reported much lower [NO] due to unstirred layers and high NO consumption. The three 7 μm microelectrodes had minor stirring issues. With limited stirring with NO present, 7 μm open tip microelectrodes advanced toward 30 μm and 2 mm electrodes experienced dramatically decreased current within 10-50 μm of the larger electrodes due to high NO consumption. None of the 7 μm microelectrodes interacted. CONCLUSIONS: The data indicate large electrodes underestimate [NO] due to excessive NO consumption under conditions where unstirred layers are unavoidable and true microelectrodes are required for valid measurements.