Prolonged and intense physical exercise enhances the production of reactive oxygen species (ROS) due to the imbalance between pro-oxidant factors and antioxidant defenses. This condition, known as oxidative stress, not only causes fatigue but also mitochondrial damage. The measurement of oxidative damage biomarkers, such as isoprostanes, seems promising to assess muscle fatigue and prevent overtraining. Isoprostanes are produced by the non-enzymatic peroxidation of arachidonic acid catalyzed by ROS, as well as by the esterification of cell membrane lipids. Thanks to the rapid diffusion equilibrium across membranes of salivary glands, monitoring of these exercise-related oxidative stress indicators in oral fluid could become a valid alternative to traditional blood and urine assays. The less obtrusive sampling allows multiple collections from a same subject even during a single session of physical activity. In this work, an innovative analytical procedure, based on micro-extraction by packed sorbent (MEPS) coupled to liquid chromatography-tandem mass spectrometry (UHPLC-ESI(-)-MS/MS), was developed for the determination of F2-isoprostanes (i.e. 15-F2t-IsoP, 5-F2t-IsoP and 5-epi-5-F2t-IsoP) in oral fluid. Oral fluid sample underwent protein precipitation with acetonitrile before clean-up and pre-concentration of the analytes by the semi-automated, fast and reliable MEPS procedure. Extracted samples were analyzed in less than 10 minutes by UHPLC-ESI(-)-MS/MS. The analytical method was validated and showed satisfactory analytical performances for all the analytes. The procedure was suitable for the determination of F2-isoprostanes in oral fluid samples collected from ten healthy volunteers performing incremental exercise test on a cycle ergometer at constant pedaling cadence (70 rpm). The initial cycling power (60 W) increased of about 20 W every three minutes until exhaustion or impossibility to maintain the pace.
Analysis of isoprostanes in oral fluid using micro extraction by packed sorbent coupled to liquid chromatography-tandem mass spectrometry to monitor oxidative stress in healthy adults performing physical activity
Silvia Ghimenti
Primo
;Denise Biagini;Tommaso Lomonaco;Shaula Antoni;Fabio Di Francesco;Roger Fuoco
2019-01-01
Abstract
Prolonged and intense physical exercise enhances the production of reactive oxygen species (ROS) due to the imbalance between pro-oxidant factors and antioxidant defenses. This condition, known as oxidative stress, not only causes fatigue but also mitochondrial damage. The measurement of oxidative damage biomarkers, such as isoprostanes, seems promising to assess muscle fatigue and prevent overtraining. Isoprostanes are produced by the non-enzymatic peroxidation of arachidonic acid catalyzed by ROS, as well as by the esterification of cell membrane lipids. Thanks to the rapid diffusion equilibrium across membranes of salivary glands, monitoring of these exercise-related oxidative stress indicators in oral fluid could become a valid alternative to traditional blood and urine assays. The less obtrusive sampling allows multiple collections from a same subject even during a single session of physical activity. In this work, an innovative analytical procedure, based on micro-extraction by packed sorbent (MEPS) coupled to liquid chromatography-tandem mass spectrometry (UHPLC-ESI(-)-MS/MS), was developed for the determination of F2-isoprostanes (i.e. 15-F2t-IsoP, 5-F2t-IsoP and 5-epi-5-F2t-IsoP) in oral fluid. Oral fluid sample underwent protein precipitation with acetonitrile before clean-up and pre-concentration of the analytes by the semi-automated, fast and reliable MEPS procedure. Extracted samples were analyzed in less than 10 minutes by UHPLC-ESI(-)-MS/MS. The analytical method was validated and showed satisfactory analytical performances for all the analytes. The procedure was suitable for the determination of F2-isoprostanes in oral fluid samples collected from ten healthy volunteers performing incremental exercise test on a cycle ergometer at constant pedaling cadence (70 rpm). The initial cycling power (60 W) increased of about 20 W every three minutes until exhaustion or impossibility to maintain the pace.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
