ABSTRACT-Amylold is a 39-to 43-amino-add neurotoxic peptide that aggregtes to form the core of Ahemer disease-sociated senile (amyloid) plaques. No EPR Spectrscopy and Spin Trapping. 8APs were solubilized to 1.0 mg/ml by addition of buffer [150 mM phosphatebuffered saline at pH 7.4 (PBS) or Hepes at pH 7.4] to lyophilized powder. BSA was solubilized similarly, to concentrations of 1-62 mg/ml. Buffer used for spin trapping contained 50 mM N-tert-butyl-a-phenylnitrone (PBN) (Sigma or Aldrich). In studies designed to inhibit putative metalcatalyzed reactions, the chelator deferoxamine mesylate or EDTA (Sigma) was dissolved to 10 mM in PBS/PBN prior to peptide addition. In a further attempt to nullify buffer-borne metals, PBS/PBN was stirred overnight with Chelex 100, a nonspecific metal-chelating agent.Peptide solutions were aliquoted into a 300-A4 aqueous quartz EPR flat cell that was subsequently sealed at both ends and immersed in a 3rC water bath for 0-135 hr and removed periodically for EPR analysis. EPR spectra were acquired on a Bruker (Billerica, MA) 300 EPR spectrometer equipped with computerized data acquisition software. Instrumental parameters were microwave power = 20 mW, modulation amplitude = 0.% G, gain = 1 x i1W, and conversion time =

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Four biomarkers of neuronal protein oxidation [W/S ratio of MAL‐6 spin‐labeled synaptosomes, phenylhydrazine‐reactive protein carbonyl content, glutamine synthetase (GS) activity, creatine kinase (CK) activity] in three brain regions [cerebellum, inferior parietal lobule (IPL), and hippocampus (HIP)] of Alzheimer's disease (AD)‐demented and age‐matched control subjects were assessed. These endpoints indicate that AD brain protein may be more oxidized than that of control subjects. The W/S ratios of AD hippocampal and inferior parietal synaptosomes are 30 and 46% lower, respectively, than corresponding values of tissue isolated from control brain; however, the difference between the W/S ratios of AD and control cerebellar synaptosomes is not significant. Protein carbonyl content is increased 42 and 37% in the Alzheimer's HIP and IPL regions, respectively, relative to AD cerebellum, whereas carbonyl content in control HIP and IPL is similar to that of control cerebellum. GS activity decreases an average of 27% in the AD brain; CK activity declines by 80%. The brain regional variation of these oxidation‐sensitive biomarkers corresponds to established histopathological features of AD (senile plaque and neurofibrillary tangle densities) and is paralleled by an increase in immunoreactive microglia. These data indicate that senile plaque‐dense regions of the AD brain may represent environments of elevated oxidative stress.