First 11 pages of thesis. - OPUS - Universität Würzburg

spectra 190 . Addition of exogenous iron-dithiocarbamates and nitronyl nitroxides
has also been used to detect nitric oxide formation 191 . Endogenous ascorbate is
oxidized to ascorbate radical which can be directly detected at room
temperature 192 . An emerging approach to the use of ESR in vascular biology
has been the use of cyclic hydroxylamines. These molecules are not spin traps,
in that they do not “trap” radicals, but they are oxidized to form very stable
radicals with half-lives of several hours, permitting ESR detection. The spin
probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine.HCl (CMH)
is oxidized by superoxide and peroxynitrite to form CM . radical. This molecule
has successfully been used with intact cells and in vivo to detect reactive
oxygen species released into the circulation. Thus, the cyclic hydroxylamines
and similar compounds are very useful for the detection of superoxide and other
reactive oxygen species in vascular tissues. Overall, ESR has proven to be
useful as a free radical detection strategy. In this respect superoxide detection
by ESR was proven to be 20 times more sensitive than the cytochrome C
reduction assay.
2.2.2 Measurement of vascular nitric oxide production by
electron paramagnetic spin trapping
Mice were injected with 100 U of heparin, anaesthetized with Avertin (10
mg/kg, i.p.) after 5 minutes and dissected on a styrofoam board. The aorta was
removed rapidly and was cleaned from the perivascular fatty tissues while being
maintained at 4°C in chilled KHB on a cold plate. The aorta was cut into 2mm
rings and the rings were placed in each well of a 12 well plate containing 1.5 ml
KHB. For the preparation of the Fe-(DETC)2 spin trap, 4.5 mg of FeSO4 (1.6
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