Determination of biomarkers of protein oxidation in tissue and plasma

Oxidative and nitrosative stress cause changes in proteins which can alter their structure and/or function. However, these changes especially in specific amino acid residues have proven to be reliable biomarkers of oxidative stress and inflammation. The aim of this study was to develop and validate a rapid method for the quantification of five selected biomarkers orto-tyrosine (o-tyr), meta-tyrosine (m-tyr), 3NO2-tyrosine (3NO2-tyr), 3I-tyrosine (3I-tyr) and 3Chloro-tyrosine (3Cl-tyr) in liver, brain and plasma, thus providing a snapshot of the oxidative stress status of the organism. The extraction and cleanup method entails protein precipitation, followed by digestion with pronase. Biomarker quantification is carried out using an Ultra Performance Liquid Chromatography-tandem Mass Spectrometry employing positive electrospray ionization and a reversed phase chromatographic separation. Liver, brain and plasma samples from hypoxic (FiO2: 8%) vs. normoxic newborn piglets (n=5) were studied employing the developed analytical method. m-tyrosine/phenylalanine, orto-tyrosine/phenylalanine, 3Cl-tyrosine/p-tyrosine and 3I-tyrosine/p-tyrosine ratios in liver of hypoxic animals were significantly increased as compared to normoxic. Although no significant differences were found for brain and plasma samples, a clear tendency to increased ratios was observed under hypoxic conditions. This analytical tool has proofed its suitability for the analysis of tissue and plasma samples from newborn piglets. The analysis of biomarkers of protein oxidation in bigger cohorts will be the topic of future studies with the aim of gaining a deeper insight into the mechanisms of oxidation-derived protein modification caused during hypoxia.