title: In vitro/in vivo screening of oxidative homeostasis and damage to DNA, protein, and lipids using UPLC/MS-MS.
abstract: Multiple analytical methods are required to comprehensively assess oxidative homeostasis and specific damage to macromolecules. Our aim was to develop a straightforward strategy for the fast assessment of global oxidative status and specific damage to DNA, proteins, and lipids. To this end, an analytical method, based on ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS/MS), was developed and validated for the quantification of 16 oxidative stress (OS) biomarkers. Some of these markers were unstable; thus, an easy sample treatment procedure, including fractionation and derivatization, was set up. The method was validated according to Food and Drug Administration (FDA) guidelines, and it provided good results in terms of intra- and inter-day precision (≤17.2 and 16 %, respectively), accuracy (relative error measurement between -16.6 and 19.8 %), and linearity (R (2) > 0.994). The approach was applied to determine the oxidative insult provoked to cultured rat hepatocytes by cumene hydroperoxide and to analyze the liver and serum samples from patients diagnosed with nonalcoholic steatohepatitis. In both studies, significant differences were found if compared to the corresponding control groups; interestingly, ophthalmic acid was shown as an OS biomarker in both models for the first time. A key advantage of the novel approach in comparison with former multi-method approaches is that now a single method is applied to assess the 16 OS biomarkers. Its comprehensive capacity to profile oxidative homeostasis and damage in both in vitro and clinical samples has been illustrated, which indicates that the proposed approach is a good choice to evaluate whether OS is involved in physiological signals, diseases, or toxic events and to what extent.
authors: Unidad de Hepatología Experimental, Instituto de Investigación Sanitaria - Fundación Hospital La Fe, CIBERehd, Centro de Investigaciones Biomédicas en Red de Enfermedades Hepáticas y Digestivas, FIS, 46026, Valencia, Spain.
authors: Aitor
authors: A
authors: Carretero
authors: Zacarías
authors: Z
authors: León
authors: Juan Carlos
authors: JC
authors: García-Cañaveras
authors: Angela
authors: A
authors: Zaragoza
authors: María José
authors: MJ
authors: Gómez-Lechón
authors: María Teresa
authors: MT
authors: Donato
authors: Agustín
authors: A
authors: Lahoz
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004249:DNA Damage; D005982:Glutathione Transferase / Q000032:analysis; D022781:Hepatocytes / Q000378:metabolism; D006706:Homeostasis; D006801:Humans; D008055:Lipids / Q000737:chemistry*; D008099:Liver / Q000378:metabolism*; D008297:Male; D008315:Malondialdehyde / Q000032:analysis; D065626:Non-alcoholic Fatty Liver Disease / Q000175:diagnosis / Q000378:metabolism; D018384:Oxidative Stress; D010100:Oxygen / Q000737:chemistry*; D010477:Perfusion; D011506:Proteins / Q000737:chemistry*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry
chemical_list: D015415:Biomarkers; D008055:Lipids; D011506:Proteins; D008315:Malondialdehyde; D005982:Glutathione Transferase; D010100:Oxygen