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Review
. 2007 Apr;49(4):717-27.
doi: 10.1161/01.HYP.0000258594.87211.6b. Epub 2007 Feb 12.

Measurement of reactive oxygen species in cardiovascular studies

Sergey Dikalov  1 , Kathy K Griendling, David G Harrison
Affiliations
Review

Measurement of reactive oxygen species in cardiovascular studies

Sergey Dikalov et al. Hypertension. 2007 Apr.
No abstract available

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Figures

Figure 1
Figure 1
Detection of O2•− by reduction of ferricytochrome c. A, Various enzymes (shown as an example is xanthine oxidase) can generate electrons, which can directly reduce cytochrome c and also reduce oxygen leading to O2•− formation. Superoxide, in turn, can donate its electron to ferricytochrome c, leading to formation of ferrocytochrome c. SOD is used to differentiate between a direct reduction of ferricytochrome c vs reduction mediated by O2•−. B, Reduction of ferricytochrome c can be detected at 550 nm absorbance, using 540 and 560 nm as isosbestic points.
Figure 2
Figure 2
The typical ESR spectrometer (A) consisting of a microwave generator, a resonator, and magnets. ESR detects the absorption of microwave energy on the transition of unpaired electrons in an applied magnetic field (B). Because the electron has a magnetic moment, it acts like a compass when it placed in a magnetic field (B). It will have a state of lowest energy when the moment of the electron is aligned with the magnetic field (↑) and a state of highest energy when it aligned against the magnetic field (↓). The energy of the unpaired electron, therefore, is a function of the magnetic field: E=±0.5g μBB, where “g” is the g-factor constant, “μB” is the Bohr magneton constant, and “B” is the magnetic field.
Figure 3
Figure 3
Detection of superoxide by ESR. A, Spin trapping of O2•− by spin trap EMPO produces superoxide radical adduct EMPO/OOH, which is decomposed by glutathione peroxidase (GPx) into OH-adduct and reduced by flavin enzymes and ascorbate into ESR silent hydroxylamine. B, The reaction of O2•− with spin probe CMH produces stable nitroxide radical, which can be conveniently detected in frozen samples.
Figure 4
Figure 4
Detection of O2•− by DHE and HPLC. A, The reaction of O2•− with DHE produces 2-hydroxyethidium (2-HE+). B, HPLC tracing of untreated control mouse aortic tissue incubated with 50 μmol/L of DHE for 30 minutes. C, HPLC tracing of angiotensin II–infused mouse (0.7 mg/kg per day for 14 days) aortic tissue incubated with 50 μmol/L of DHE for 30 minutes. Angiotensin II significantly increases production of the O2•−-specific product 2-HE+.
Figure 5
Figure 5
Detection of hydrogen peroxide with DCFH-DA and N-acetyl-3,7-dihydroxyphenoxazine (Amplex Red). A, DCFH-DA is cleaved by intracellular esterases to DCFH and oxidized by ROS to the highly fluorescent molecule DCF. B, Nonfluorescent Amplex Red is oxidized by the horseradish peroxidase and H2O2 to highly fluorescent resorufin.
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References

    1. Harrison DG, Dikalov S. Oxidative events in cell and vascular biology. In: Re R, DiPette D, Schiffrin E, Sowers J, editors. Molecular Mechanisms in Hypertension. 1st ed. Taylor & Francis Medical Books; Abingdon, United Kingdom: 2006. pp. 297–320.
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