Abstract
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.
Highlights
Nitric oxide (NO) is an important molecule involved in cellular signaling and platelet regulation
For the analysis of cell lysate samples by microchip electrophoresis with electrochemical detection (ME-EC), the initial experiments employed a background electrolyte consisting of 10 mM borate with 2 mM tetradecyltrimethylammonium chloride (TTAC) at pH 10
When cell lysate samples were analyzed, it was found that nitrite comigrated with an interferent peak that was present on the filters used for sample preparation
Summary
Nitric oxide (NO) is an important molecule involved in cellular signaling and platelet regulation. It is generated in vivo from arginine via the enzyme nitric oxide synthase (NOS). 1 A specific form of NOS, inducible nitric oxide synthase (iNOS), is activated to produce nitric oxide as part of the immune response.. The resulting NO is capable of reacting with cellular components and can disrupt biological function.. Peroxynitrite can cause nitration of proteins, peroxidation of lipids, and cleavage of the phosphate backbone of DNA.. Peroxynitrite can cause nitration of proteins, peroxidation of lipids, and cleavage of the phosphate backbone of DNA.2–4 These biomolecular modifications have been implicated in the pathology of several neurodegenerative and cardiovascular diseases.. It is generated in vivo from arginine via the enzyme nitric oxide synthase (NOS). 1 A specific form of NOS, inducible nitric oxide synthase (iNOS), is activated to produce nitric oxide as part of the immune response. The resulting NO is capable of reacting with cellular components and can disrupt biological function. Under conditions of inflammation, NO can react with superoxide to form peroxynitrite. Peroxynitrite can cause nitration of proteins, peroxidation of lipids, and cleavage of the phosphate backbone of DNA. These biomolecular modifications have been implicated in the pathology of several neurodegenerative and cardiovascular diseases. It is important to have robust analytical methods that are capable of monitoring reactive nitrogen and oxygen species (RNOS) in biological systems
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