ISSN:
1612-1112
Keywords:
Column liquid chromatography
;
Oxygen free radicalsin vivo
;
Spin traps
;
Chemical traps
;
Biological fluid and tissue
Source:
Springer Online Journal Archives 1860-2000
Topics:
Chemistry and Pharmacology
Notes:
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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF02270443
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