Simultaneous determination of malondialdehyde and 3-nitrotyrosine in cultured human hepatoma cells by liquid chromatography-mass spectrometry
Language English Country England, Great Britain Media print-electronic
Document type Journal Article
Grant support
Long-term organisation development plan 1011
Ministry of Defence of the Czech Republic
SV/FVZ201410
Ministry of Education, Youth and Sports of the Czech Republic
PubMed
30051494
DOI
10.1002/bmc.4349
Knihovny.cz E-resources
- Keywords
- 3-nitrotyrosine, HepG2 cells, liquid chromatography-mass spectrometry, malondialdehyde, oxidative stress,
- MeSH
- Hep G2 Cells MeSH
- Chromatography, Liquid methods MeSH
- Humans MeSH
- Limit of Detection MeSH
- Malondialdehyde analysis MeSH
- Liver Neoplasms metabolism MeSH
- Oxidative Stress MeSH
- Reproducibility of Results MeSH
- Tandem Mass Spectrometry methods MeSH
- Tyrosine analogs & derivatives analysis MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- 3-nitrotyrosine MeSH Browser
- Malondialdehyde MeSH
- Tyrosine MeSH
Although reactive oxygen/nitrogen species (ROS/RNS) have a fundamental role in physiological processes, enhanced ROS/RNS production induced by exogenous sources, including drugs and other xenobiotics, may result in serious damage to biomolecules. Oxidative/nitrosative stress is being intensively investigated and might be responsible for a variety of health side effects. The present liquid chromatography-tandem mass spectrometry (LC-MS/MS) method provides reliable and accurate simultaneous measurement of malondialdehyde (MDA) and 3-nitrotyrosine (3-NT) in cultured human hepatoma (HepG2) cells. Sample preparation process involving ultrasonic homogenization, alkaline hydrolysis of protein-bound MDA and 3-NT, deproteination, derivatization of MDA by 2,4-dinitrophenylhydrazine and solid-phase extraction was optimized, ensuring the isolation and purification of desired analytes. Additionally, nonprotein thiols and nonprotein disulfides were measured using HPLC-UV. The established lower limit of quantification (0.025 nmol/mL for MDA; 0.0125 nmol/mL for 3-NT) allowed their LC-MS/MS determination in HepG2 cells exposed to model oxidizing agent, tert-butyl hydroperoxide (t-BOOH). The results show significant changes in MDA and 3-NT concentrations and alterations in thiol redox-state in dependence on the t-BOOH concentration and duration of its incubation in HepG2 cells. Concurrent evaluation of oxidative/nitrosative stress biomarkers in the in vitro model may significantly facilitate assessment of toxicity of newly developed drugs in preclinical trials and thus improve their safety profile.
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