First 11 pages of thesis. - OPUS - Universität Würzburg

2.2 Methods
2.2.1 Detection of free radicals in the vasculature
The measurement of vascular free radical production is difficult for
several reasons. Since radicals are very short lived, they usually do not occur at
high concentrations in the biological environment. Low intracellular steady-state
concentrations of superoxide result from the balance between endogenous
partial reduction of oxygen to superoxide and the scavenging of superoxide by
highly efficient cytoplasmic and mitochondrial SOD, resulting in intracellular
superoxide concentrations which rarely exceed 1 nmol/L. Extra cellular release
of small proportions of intracellularly formed superoxide may occur via anion
channels. In addition, superoxide levels formed from plasma membrane bound
oxidases are maintained at low local concentrations due to extra cellular fluid
components, including low molecular weight oxidant scavengers and the
heparin binding extracellular (EC)-SOD. Similarly, the intracellular
concentrations of nitric oxide depend on the balance between the rate of
formation from L-arginine and the reaction of nitric oxide with superoxide,
resulting in peroxynitrite formation. Thus, the relatively short half-life (seconds)
of these radicals and the efficient systems which evolved to scavenge radicals
require that any detection technique must be sensitive enough to effectively
compete with these intracellular and extra cellular antioxidant components for
reaction with the substance in question.
Some of the currently available methods include chemiluminescence
techniques, fluorescent based assays, enzymatic assays and electron spin
resonance. Reduction of ferricytochrome c has been used to measure rates of
formation of superoxide by numerous enzymes, tissue extracts, and whole cells.
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