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Nitric oxide (NO) is involved in many biological functions, including blood pressure regulation, the immune response, and neurotransmission. However, excess production of NO can lead to the generation of reactive nitrogen species and nitrosative stress and has been linked to several neurodegenerative diseases and cardiovascular disorders. Because NO is short-lived and generally difficult to detect, its primary stable degradation product, nitrite, is frequently monitored in its place. In this paper, an improved method using microchip electrophoresis with electrochemical detection (ME-EC) was developed for the separation and detection of nitrite in cell lysates. A separation of nitrite from several electroactive cell constituents and interferences was optimized, and the effect of sample and buffer conductivity on peak efficiency was explored. It was found that the addition of 10 mM NaCl to the run buffer caused stacking of the nitrite peak and improved limits of detection. A platinum black working electrode was also evaluated for the detection of nitrite and other electroactive cellular species after electrophoretic separation. The use of a modified platinum working electrode resulted in 2.5-, 1.7-, and 7.2-fold signal enhancement for nitrite, ascorbic acid, and hydrogen peroxide, respectively, and increased the sensitivity of the method for nitrite twofold. The optimized ME-EC method was used to compare nitrite production by native and lipopolysaccharide-stimulated RAW 264.7 macrophage cells., Competing Interests: Conflicts of interest There are no conflicts to declare.

Carnosine, a dipeptide found in a variety of tissues, is believed to possess antioxidant properties. It serves as a scavenger of reactive nitrogen and oxygen species (RNOS), which are important stress mediators of pro-inflammatory conditions and can lead to macrophage activation. In this study, intracellular concentrations of carnosine in murine RAW 264.7 macrophage cells were determined using microchip electrophoresis with laser-induced fluorescence detection following derivatization with naphthalene-2,3-dicarboxaldehyde and cyanide. The method was linear from 25 nM to 5 μM with a limit of detection in cell lysate samples of 65 nM. Using the method of standard additions, the basal intracellular content of carnosine in macrophage cells was determined to be 0.079 ± 0.02 nmol/10 6 cells. The uptake of carnosine by these cells was then investigated under both physiological and pro-inflammatory conditions. There was a 2.8-fold increase in carnosine uptake for macrophages exposed to lipopolysaccharide and interferon-γ prior to incubation, compared to the controls. This suggests that macrophages may use carnosine uptake as a defense mechanism under pro-inflammatory conditions. Future studies will investigate the role of the carnosine transporter in carnosine uptake and its possible correlation with cell morphological changes observed after stimulation., Competing Interests: The authors have declared no conflict of interest.