An HPLC method forin vivo quantitation of oxygen free radicals using spin and chemical traps in biological systems

Summary

We describe here anin vivo method for direct and simultaneous determination and quantitation of the oxygen free radicals (OFR) superoxide (O₂ ⁻) and hydroxy (OH) radicals in biological tissue and blood of 2 week-old swine. Our method utilizes OFR trapping techniques, a spin trap 5,5-dimethyl-1-pyrroline-n-oxide (DMPO), 50 mg/kg, for O₂ ⁻ and a chemical trap, Na salicylate, (SA, 100 mg/kg) for OH, was infused into the right atrium or pulmonary artery of two-week old swine (n=12). The OFR contents of coronary sinus (CS) blood and left ventricular (LV) tissue (quick frozen at 77°K) were measured by an HPLC method developed by us (Waters 590 solvent delivery system, using Waters electrochemical 460 EC detector, and 740 data module) at +0.6V. The DMPO-O₂ ⁻ (measured as DMPO-OH) adduct assay was performed with a mobile phase consisting of 0.03 M citric acid, 0.05 M NaOH and 8.5% acetonitrile (Ph 5.1) at a flow rate of 1 ml/min through a Waters Resolve 5 μ C₁₈ column. The salicylate-OH products (2,5 and 2,3 dihydroxy benzoic acids, DHBA) were assayed using mobile phase of 0.03 M Na citrate, 0.03 M Na acetate, with N₂ bubbled (pH 3.6) at a flow rate of 0.8 ml/min through a 5μ Resolve C₁₈ column. The detected peak for DMPO-O₂ ⁻ adduct (9.5 min) was standardized with a hypoxanthine (HX) and xanthine oxidase (XO) mixture and the salicylate-OH products (11.5 min) were standardized with HX, XO and FeCl₃. Forin vitro experiments, the blood/tissue samples were immediately (<30 sec) incubated directly with 100 mM DMPO and/or 200 mM salicylate for 1 min, vortexed and injected for HPLC analysis. Superoxide dismutase (1 μM) and DMSO (10 mM) scavenged O₂ ⁻ and OH adduct peaks by 77 and 80% respectively. The coefficient of variation for DMPO-O₂ ⁻ adduct was ±12.6% and for salicylate-OH adduct was ±10.9% (n=12). The normal LV tissue levels determined for O₂ ⁻ and OH were 0.41 and 0.32 nm/g wet weight, respectively. (In blood, the OFR contents were very small: 0.09 and 0.06 nm/ml, respectively.) This method is very specific and sensitive, 50 pm for O₂ ⁻ and 0.2 pm for OH radicals.