A fluorescent assay of brain lipid peroxidation was used for
screening new antioxidants for the prevention of neurodegeneration
caused by free radicals. Incubation of rat brain homogenates
led to a temperature-dependent increase in production of fluorescent
adducts of peroxidized poly-unsaturated fatty acids; it was
inhibited completely by lowering the incubation temperature
to 4°C. This tissue disruption-induced brain lipid peroxidation
at 37°C was blocked by deferoxamine (IC
50 = 0.3 µM)
and EDTA; it was augmented by adding submicromolar iron and
hemoglobin. Ferrous ion's pro-oxidative activities were five
times more potent than ferric ion. Micromolar manganese completely
inhibited lipid peroxidation, confirming earlier unexpected
in vivo reports. Trolox and vitamin C suppressed brain lipid
peroxidation with IC
50 values of 20 and 500 µM, respectively.
U-78517F was approximately 20 times more potent than Trolox.
17ß-Estradiol, hydralazine,
S-nitrosoglutathione and
3-hydroxybenzylhydrazine were as potent as Trolox. Melatonin,
glutathione,
-lipoic acid and
l-deprenyl were about 20 times
less potent than Trolox. Surprisingly,
N-tert-butyl-
-phenylnitrone
was a weak antioxidant. Furthermore, this procedure can also
detect pro-oxidative side effects of vitamin C, oxidized glutathione,
penicillamine and Angeli's salt. The present results obtained
from this selective fluorescent assay are consistent with earlier
reports that iron complexes promote while manganese inhibits
brain lipid peroxidation caused by cell disruption.
S-Nitrosoglutathione,
melatonin, 17ß-estradiol, and manganese have been
successfully tested in cell/animal models for their potential
neuroprotective effects. In conclusion, monitoring fluorescent
adducts of peroxidizing polyunsaturated fatty acids in brain
homogenates is a simple, quantitative method for studying iron-dependent
brain lipid peroxidation and for screening of potential neuroprotective
antioxidants in both
in vitro and
in vivo preparations.
