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Vascular Medicine Institute

University of Pittsburgh

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EPR (Electron Paramagnetic Resonance) Spectrometry

EPR is used to identify and quantify various reactive biomolecules possessing unpaired electrons (free radicals). As the half-life of most free radical species ranges from milliseconds to minutes, not all species are detectable directly. Therefore, we utilize spin traps and probes, molecules that react with free radicals and are subsequently transformed to more stable and thus EPR-detectable free radicals with reactant-specific signature spectra. EPR1
EPR2

For example, we routinely utilize EPR and EPR spin trapping to detect the following radical species in cells, tissues and plasma:

O2•-   (superoxide anion)
OH  (hydroxyl radical)
NO  (nitric oxide)
Asc•-  (ascorbyl radical)
L/LO/LOO  (lipid peroxidation-derived, alkyl, alkoxyl and peroxyl radicals)

 

 

 

Hydroethidine Oxidation (DHE)

Hydroethidine (HE), commonly referred to as DHE, can be oxidized by cells and tissues to several fluorescent products including ethidium. However, only one of these oxidation products is produced specifically by the reaction of HE with superoxide (O2•-), 2-hydroxy ethidium (2-OH-E+). This method is highly sensitive and specific for O2•-. Using HPLC-based electrochemical detection methods, we can quantify low nM levels of 2-OH-E+ extracted from cells and tissues. In addition, this assay can be used to follow O2•- formation in mitochondria using a triphenylphosphonium derivative of HE, Mito-HE (MitoSOX).
DHE

 

 

 

Immunospin Trapping

A combination of EPR spin trapping and immunohistochemistry, immunospin trapping is a recently developed technique for assessing overall free radical formation in tissues. Using the EPR spin trap 5,5-Dimethyl-1-Pyrroline N-Oxide (DMPO) and a commercially available polyclonal antibody raised to DMPO-octanoic acid, we can detect protein free radical formation in vitro and in vivo. Protein-derived free radicals form covalent bonds with DMPO effectively sequestering the spin trap and thus making it readily available for antibody detection (see below).
IST

 

 

 

High-Throughput Assay Systems

PLATE-BASED ASSAYS
Detection and quantification in whole cells in culture as well as in cell-free systems using spectrophotometric, chemiluminescence and fluorescence techniques in a 96- or 384-well format.
  • superoxide anion
  • hydrogen peroxide
  HTS2
HTS1  

 

ASSAYS FOR SUPEROXIDE
Cytochrome C
  • Classic method for detection of O2.-
  • Detection of extracellular O2.-
  • Not very sensitive ¨ Good for cell-free assays
cyto-c

 

ASSAYS FOR H2O2
Amplex Red
  • Extracellular detection of  H2O2
  • Fluorescence assay [540nm (excitation) and 580 nm (emission)]
amplex red
Homovanillic Acid (HVA)
  • End-point
  • Specific for H2O2
  • Fluorescence assay [315nm (excitation) and 425 nm (emission)]
hva

 

CELL-BASED HTS ASSAYS FOR INITIAL DRUG SCREENINGS

L-012

  • Chemiluminescence assay
  • Detection of sueroxide and peroxynitrite
  • Very sensitive
  • Ideal for high throughput assays
l-012
Luminol
  • Chemiluminescence assay
  • Detection of superoxide and H2O2
  • Undergoes redox cycling
  • Suited for high throughput assays
luminol

*Results further confirmed with Cytochrome C, Amplex Red, and EPR

 

 

 

Oxygen Consumption

Oxygen Consumption Oxygen is requisite for the formation of ROS thus; assessment of O2 consumption can be used as an indirect method of rates of ROS formation. Using polarographic “Clark-type” electrodes (WPI and YSI) in multiple closed-system variable-temperature platforms, we can monitor real-time rates of O2 consumption from cells and tissues. ROS formation can be indirectly determined by stimulated oxygen consumption rates that are greater than rates from normal metabolism.