TY - JOUR
T1 - A comparative study of EPR spin trapping and cytochrome c reduction techniques for the measurement of superoxide anions
AU - Sanders, Scherer P.
AU - Harrison, Stephen J.
AU - Kuppusamy, Periannan
AU - Sylvester, J. T.
AU - Zweier, Jay L.
N1 - Funding Information:
Acknowledgements -- We express our gratitude to Brenda L. Jordan for preparation of the manuscript. This work was supported by the National Institutes of Health Grant HL41970, American Lung Association of Maryland Award, and American Heart Association Award 91012800.
PY - 1994/6
Y1 - 1994/6
N2 - Superoxide anions (O2.-) generated by the reaction of xanthine with xanthine oxidase were measured by the reduction of cytochrome c and by electron paramagnetic resonance (EPR) spectroscopy using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Studies were performed to determine the relative sensitivities of these two techniques for the measurement of O2.-. Mixtures of xanthine, xanthine oxidase, DMPO generated two adducts, a transient DMPO-OOH and a smaller but longer-lived DMPO-OH. Both adducts were inhibited by superoxide dismutase (SOD), demonstrating they originated from O2.-, and were also significantly decreased when the experiments were performed using unchelated buffers, suggesting that metal ion impurities in unchelated buffers alter the formation or degradation of DMPO-adducts. O2.-, generated by concentrations of xanthine as low as 0.05 μM, were detectable using EPR spin trapping. In contrast, mixtures of xanthine, xanthine oxidase, and cytochrome c measured spectrophotometrically at 550 nm demonstrated that concentrations of xanthine above 1 μM were required to produce measurable levels of reduced cytochrome c. These studies demonstrate that spin trapping using DMPO was at least 20-fold more sensitive than the reduction of cytochrome c for the measurement of superoxide anions. However, at levels of superoxide generation where cytochrome c provides a linear measurement of production, EPR spin trapping may underestimate radical production, probably due to degradation of DMPO radical adducts.
AB - Superoxide anions (O2.-) generated by the reaction of xanthine with xanthine oxidase were measured by the reduction of cytochrome c and by electron paramagnetic resonance (EPR) spectroscopy using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Studies were performed to determine the relative sensitivities of these two techniques for the measurement of O2.-. Mixtures of xanthine, xanthine oxidase, DMPO generated two adducts, a transient DMPO-OOH and a smaller but longer-lived DMPO-OH. Both adducts were inhibited by superoxide dismutase (SOD), demonstrating they originated from O2.-, and were also significantly decreased when the experiments were performed using unchelated buffers, suggesting that metal ion impurities in unchelated buffers alter the formation or degradation of DMPO-adducts. O2.-, generated by concentrations of xanthine as low as 0.05 μM, were detectable using EPR spin trapping. In contrast, mixtures of xanthine, xanthine oxidase, and cytochrome c measured spectrophotometrically at 550 nm demonstrated that concentrations of xanthine above 1 μM were required to produce measurable levels of reduced cytochrome c. These studies demonstrate that spin trapping using DMPO was at least 20-fold more sensitive than the reduction of cytochrome c for the measurement of superoxide anions. However, at levels of superoxide generation where cytochrome c provides a linear measurement of production, EPR spin trapping may underestimate radical production, probably due to degradation of DMPO radical adducts.
KW - Cytochrome c
KW - EPR spectroscopy
KW - Free radicals
KW - Spin trapping
KW - Superoxide anion
UR - http://www.scopus.com/inward/record.url?scp=0028232679&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0028232679&partnerID=8YFLogxK
U2 - 10.1016/0891-5849(94)90190-2
DO - 10.1016/0891-5849(94)90190-2
M3 - Article
C2 - 8070678
AN - SCOPUS:0028232679
SN - 0891-5849
VL - 16
SP - 753
EP - 761
JO - Free Radical Biology and Medicine
JF - Free Radical Biology and Medicine
IS - 6
ER -