First 11 pages of thesis. - OPUS - Universität Würzburg

ings were prepared as mentioned above. The aortas were cut into 2 mm rings
and five rings were placed in 100 µl KHB containing 25 µM deferoxamine and 5
µM DETC in each well of a 98 well plate. Deferoxamine and DETC stock
solutions were prepared by dissolving the compounds in KHB. The reagents
were freshly prepared everyday. Samples were incubated for 1hour at 37°C
with the spin probe, CMH (250 µM). Superoxide production was assessed by
pre incubating aortic rings with PEG-SOD (100 U/ml) parallel to CMH for 1 hour.
The experimental procedure was carried out according to a previously
published protocol 193 . The instrumental settings for superoxide measurements
are as follows: Centre field: 3388 G. Sweep width: 132 G. Microwave frequency:
9.497 GHz. Microwave power: 1.25 mW. Modulation Amplitude: 1.63 G.
Modulation frequency: 86 kHz. Time constant: 40.96 ms. Conversion Time:
10.24 ms. Number of scans: 50. The intensity of the ESR signal was normalized
to the protein content of the sample.
We chose to use the cyclic hydroxylamine CMH because of its higher
stability when compared to the widely used nitrone spin traps DMPO and
DEPMO 194 . Furthermore, CMH is more sensitive and reliable than the nitrone
spin traps. In particular CMH has a higher scavenging efficacy for superoxide,
compared to CP-H, which is another cyclic hydroxylamine.The higher stability of
CM . even in the presence of reducing agents like ascorbate or glutathione 195
and the ability of CMH to penetrate cells are additionally advantageous.
- 50 -