Assessment of a standardized ROS production profile in humans by electron paramagnetic resonance.
Despite the growing interest in the role of reactive oxygen species (ROS) in health and disease, reliable quantitative noninvasive methods for the assessment of oxidative stress in humans are still lacking. EPR technique, coupled to a specific spin probe (CMH: 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine) is here presented as the method of choice to gain a direct measurement of ROS in biological fluids and tissues. The study aimed at demonstrating that, differently from currently available "a posteriori" assays of ROS-induced damage by means of biomolecules (e.g., proteins and lipids) spin-trapping EPR provides direct evidence of the "instantaneous" presence of radical species in the sample and, as signal areas are proportional to the number of excited electron spins, lead to absolute concentration levels. Using a recently developed bench top continuous wave system (e-scan EPR scanner, Bruker) dealing with very low ROS concentration levels in small (50 μL) samples, we successfully monitored rapid ROS production changes in peripheral blood of athletes after controlled exercise and sedentary subjects after antioxidant supplementation. The correlation between EPR results and data obtained by various enzymatic assays (e.g., protein carbonyls and thiobarbituric acid reactive substances) was determined too. Synthetically, our method allows reliable, quick, noninvasive quantitative determination of ROS in human peripheral blood.
Dipartimento di Fisiopatologia Medico-Chirurgica e dei Trapianti, Università di Milano, Via Fratelli Cervi 93, 20090 Segrate, Italy.
Simona
S
Mrakic-Sposta
Maristella
M
Gussoni
Michela
M
Montorsi
Simone
S
Porcelli
Alessandra
A
Vezzoli
D000975:Antioxidants / Q000008:administration & dosage / Q000494:pharmacology; D015415:Biomarkers / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods* / Q000592:standards*; D015444:Exercise; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D018384:Oxidative Stress / Q000187:drug effects; D050050:Protein Carbonylation / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012015:Reference Standards; D015203:Reproducibility of Results; D017392:Thiobarbituric Acid Reactive Substances / Q000378:metabolism; D008063:Thioctic Acid / Q000008:administration & dosage / Q000494:pharmacology; D013997:Time Factors; D055815:Young Adult
D000975:Antioxidants; D015415:Biomarkers; D017382:Reactive Oxygen Species; D017392:Thiobarbituric Acid Reactive Substances; D008063:Thioctic Acid