Optimization of a microchip electrophoresis method with electrochemical detection for the determination of nitrite in macrophage cells as an indicator of nitric oxide production.

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.

Microchip electrophoresis with laser-induced fluorescence detection for the determination of the ratio of nitric oxide to superoxide production in macrophages during inflammation.

It is well known that excessive production of reactive oxygen and nitrogen species is linked to the development of oxidative stress-driven disorders. In particular, nitric oxide (NO) and superoxide (O2•-) play critical roles in many physiological and pathological processes. This article reports the use of 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate and MitoSOX Red in conjunction with microchip electrophoresis and laser-induced fluorescence detection for the simultaneous detection of NO and O2•- in RAW 264.7 macrophage cell lysates following different stimulation procedures. Cell stimulations were performed in the presence and absence of cytosolic (diethyldithiocarbamate) and mitochondrial (2-methoxyestradiol) superoxide dismutase (SOD) inhibitors. The NO/O2•- ratios in macrophage cell lysates under physiological and proinflammatory conditions were determined. The NO/O2•- ratios were 0.60 ± 0.07 for unstimulated cells pretreated with SOD inhibitors, 1.08 ± 0.06 for unstimulated cells in the absence of SOD inhibitors, and 3.14 ± 0.13 for stimulated cells. The effect of carnosine (antioxidant) or Ca2+ (intracellular messenger) on the NO/O2•- ratio was also investigated. Graphical Abstract Simultaneous detection of nitric oxide and superoxide in macrophage cell lysates.

Carnosine modulates nitric oxide in stimulated murine RAW 264.7 macrophages.

Excess nitric oxide (NO) production occurs in several pathological states, including neurodegeneration, ischemia, and inflammation, and is generally accompanied by increased oxidative/nitrosative stress. Carnosine [ß-alanine-histidine (ß-Ala-His)] has been reported to decrease oxidative/nitrosative stress-associated cell damage by reducing the amount of NO produced. In this study, we evaluated the effect of carnosine on NO production by murine RAW 264.7 macrophages stimulated with lipopolysaccharides + interferon-γ. Intracellular NO and intracellular and extracellular nitrite were measured by microchip electrophoresis with laser-induced fluorescence and by the Griess assay, respectively. Results showed that carnosine causes an apparent suppression of total NO production by stimulated macrophages accompanied by an unexpected simultaneous drastic increase in its intracellular low toxicity endproduct, nitrite, with no inhibition of inducible nitric oxide synthase (iNOS). ESI-MS and NMR spectroscopy in a cell-free system showed the formation of multiple adducts (at different ratios) of carnosine-NO and carnosine-nitrite, involving both constituent amino acids (ß-Ala and His) of carnosine, thus providing a possible mechanism for the changes in free NO and nitrite in the presence of carnosine. In stimulated macrophages, the addition of carnosine was also characterized by changes in the expression of macrophage activation markers and a decrease in the release of IL-6, suggesting that carnosine might alter M1/M2 macrophage ratio. These results provide evidence for previously unknown properties of carnosine that modulate the NO/nitrite ratio of stimulated macrophages. This modulation is also accompanied by changes in the release of pro-inflammatory molecules, and does not involve the inhibition of iNOS activity.

Indirect detection of superoxide in RAW 264.7 macrophage cells using microchip electrophoresis coupled to laser-induced fluorescence.

Superoxide, a naturally produced reactive oxygen species (ROS) in the human body, is involved in many pathological and physiological signaling processes. However, if superoxide formation is left unregulated, overproduction can lead to oxidative damage to important biomolecules, such as DNA, lipids, and proteins. Superoxide can also lead to the formation of peroxynitrite, an extremely hazardous substance, through its reaction with endogenously produced nitric oxide. Despite its importance, quantitative information regarding superoxide production is difficult to obtain due to its high reactivity and low concentrations in vivo. MitoHE, a fluorescent probe that specifically reacts with superoxide, was used in conjunction with microchip electrophoresis (ME) and laser-induced fluorescence (LIF) detection to investigate changes in superoxide production by RAW 264.7 macrophage cells following stimulation with phorbol 12-myristate 13-acetate (PMA). Stimulation was performed in the presence and absence of the superoxide dismutase (SOD) inhibitors, diethyldithiocarbamate (DDC) and 2-metoxyestradiol (2-ME). The addition of these inhibitors resulted in an increase in the amount of superoxide specific product (2-OH-MitoE(+)) from 0.08 ± 0.01 fmol (0.17 ± 0.03 mM) in native cells to 1.26 ± 0.06 fmol (2.5 ± 0.1 mM) after PMA treatment. This corresponds to an approximately 15-fold increase in intracellular concentration per cell. Furthermore, the addition of 3-morpholino-sydnonimine (SIN-1) to the cells during incubation resulted in the production of 0.061 ± 0.006 fmol (0.12 ± 0.01 mM) of 2-OH-MitoE(+) per cell on average. These results demonstrate that indirect superoxide detection coupled with the use of SOD inhibitors and a separation method is a viable method to discriminate the 2-OH-MitoE(+) signal from possible interferences.

Microchip electrophoresis with amperometric detection method for profiling cellular nitrosative stress markers.

The overproduction of nitric oxide (NO) in cells results in nitrosative stress due to the generation of highly reactive species such as peroxynitrite and N2O3. These species disrupt the cellular redox processes through the oxidation, nitration, and nitrosylation of important biomolecules. Microchip electrophoresis (ME) is a fast separation method that can be used to profile cellular nitrosative stress through the separation of NO and nitrite from other redox-active intracellular components such as cellular antioxidants. This paper describes a ME method with electrochemical detection (ME-EC) for the separation of intracellular nitrosative stress markers in macrophage cells. The separation of nitrite, azide (interference), iodide (internal standard), tyrosine, glutathione, and hydrogen peroxide (neutral marker) was achieved in under 40 s using a run buffer consisting of 7.5 to 10 mM NaCl, 10 mM boric acid, and 2 mM TTAC at pH 10.3 to 10.7. Initially, NO production was monitored by the detection of nitrite (NO2(-)) in cell lysates. There was a 2.5- to 4-fold increase in NO2(-) production in lipopolysaccharide (LPS)-stimulated cells. The concentration of NO2(-) inside a single unstimulated macrophage cell was estimated to be 1.41 mM using the method of standard additions. ME-EC was then used for the direct detection of NO and glutathione in stimulated and native macrophage cell lysates. NO was identified in these studies based on its migration time and rapid degradation kinetics. The intracellular levels of glutathione in native and stimulated macrophages were also compared, and no significant difference was observed between the two conditions.