Related Experiment Video
Updated: May 1, 2026
08:41
Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications
Published on: July 14, 2017
8.5K
Microchip electrophoresis with amperometric detection method for profiling cellular nitrosative stress markers.
Dulan B Gunasekara1, Joseph M Siegel, Giuseppe Caruso
1Ralph N. Adams Institute for Bioanalytical Chemistry, University of Kansas, 2030 Becker Drive, Lawrence, KS 66047, USA. slunte@ku.edu.
The Analyst
|April 15, 2014
Summary
This study developed a microchip electrophoresis method to detect nitric oxide (NO) and nitrite in macrophage cells, revealing increased nitrite levels after lipopolysaccharide stimulation. The method efficiently profiles cellular nitrosative stress markers.
Area of Science:
- Analytical Chemistry
- Cell Biology
- Biochemistry
Background:
- Overproduction of nitric oxide (NO) leads to nitrosative stress, disrupting cellular redox balance via reactive nitrogen species.
- Profiling intracellular redox-active molecules is crucial for understanding cellular stress responses.
- Microchip electrophoresis (ME) offers a rapid separation technique for analyzing complex biological samples.
Purpose of the Study:
- To develop and validate a microchip electrophoresis with electrochemical detection (ME-EC) method for analyzing nitrosative stress markers in macrophage cells.
- To quantify nitric oxide (NO) and nitrite (NO2-) production in response to cellular stimulation.
- To assess intracellular glutathione levels in native and stimulated macrophages.
Main Methods:
- A microchip electrophoresis (ME) system with electrochemical detection (EC) was employed for rapid separation.
- The method separated key analytes including nitrite, tyrosine, glutathione, and hydrogen peroxide in under 40 seconds.
- Standard additions were used to quantify intracellular nitrite concentration in single macrophage cells.
Main Results:
- Lipopolysaccharide (LPS) stimulation resulted in a 2.5- to 4-fold increase in nitrite (NO2-) production in macrophage cell lysates.
- The intracellular concentration of nitrite in unstimulated macrophages was estimated at 1.41 mM.
- Direct detection of NO and glutathione was achieved; no significant difference in glutathione levels was observed between native and stimulated cells.
Conclusions:
- The developed ME-EC method is effective for rapid profiling of intracellular nitrosative stress markers in macrophage cells.
- The study demonstrates a significant increase in nitrite production upon LPS stimulation, indicating elevated NO metabolism.
- ME-EC provides a valuable tool for investigating cellular redox dynamics and the impact of nitrosative stress.
