HPLC with electrochemical array detection (HPLC-ECD) was used to quantify 3,3Ј-dityrosine (diTyr) and 3-nitrotyrosine (3-NO 2 -Tyr) in four regions of the human brain that are differentially affected in Alzheimer's disease (AD). DiTyr and 3-NO 2 -Tyr levels were elevated consistently in the hippocampus and neocortical regions of the AD brain and in ventricular cerebrospinal fluid (VF), reaching quantities five-to eightfold greater than mean concentrations in brain and VF of cognitively normal subjects. Uric acid, a proposed peroxynitrite scavenger, was decreased globally in the AD brain and VF. The results suggest that AD pathogenesis may involve the activation of oxidant-producing inflammatory enzyme systems, including nitric oxide synthase.Key words: HPLC; nitrotyrosine; dityrosine; Alzheimer's disease; protein oxidation; inflammation The Alzheimer's disease (AD) brain exhibits region-specific patterns of amyloid plaque deposition, neurofibrillary tangle (NFT) accumulation, and neuron death. The limbic system and association areas of the neocortex show the most pronounced histopathological alterations in AD, whereas cortical somatosensory and cerebellar neurons are relatively spared (Pearson et al., 1985;Henderson and Finch, 1989;Braak and Braak, 1994). Recent models of AD attempt to link disease progression with an inflammatory component combined with increased oxidative stress (Rogers et al., 1996). C lassical hallmarks of inflammation such as edema and neutrophil infiltration are not acknowledged characteristics of the AD brain, although numerous correlates of inflammation are present. Acute-phase reactants such as C-reactive protein, major histocompatibility complex glycoproteins, complement, monocyte chemoattractants, interleukin-1, and interleukin-6 are elevated in AD brain in spatial association with neuritic plaques (Griffin et al., 1989McGeer et al., 1989;Bauer et al., 1991;Strauss et al., 1992;C arpenter et al., 1993; Wood et al., 1993;Iwamoto et al., 1994;Mrak et al., 1995;Pereira et al., 1996;Rogers et al., 1996;Sheng et al., 1996). Reactive microglia, f unctionally similar to monocytes, are increased in the AD brain and concentrate near senile plaques (McGeer et al., 1987;Haga et al., 1989;Itagaki et al., 1989; Carpenter et al., 1993;MacKenzie et al., 1995).Enhanced oxidative stress in the AD brain is manifested by increases in protein carbonyl content and lipid and DNA oxidation products and by inactivation of sensitive enzymes (Oliver et al., 1987;C. Smith et al., 1991C. Smith et al., , 1992Mecocci et al., 1993;Balazs and Leon, 1994;Chen et al., 1994;Hensley et al., 1995; Lovell et al., 1995;M. Smith et al., 1996;Butterfield et al., 1997;Lyras et al., 1997;Sayre et al., 1997). Correlation between oxidative and inflammatory biomarkers has not been achieved in the AD brain, although the activation of an inflammatory response might, in large part, explain AD brain oxidation. For instance, activated microglia release superoxide (O 2 ⅐ Ϫ ) and hydrogen peroxide (H 2 O 2 ) (Colton et al., 1994), whe...

Mitochondrial complexes I, II, and III were studied in isolated brain mitochondrial preparations with the goal of determining their relative abilities to reduce O2 to hydrogen peroxide (H2O2) or to reduce the alternative electron acceptors nitroblue tetrazolium (NBT) and diphenyliodonium (DPI). Complex I and II stimulation caused H2O2 formation and reduced NBT and DPI as indicated by dichlorodihydrofluorescein oxidation, nitroformazan precipitation, and DPI‐mediated enzyme inactivation. The O2 consumption rate was more rapid under complex II (succinate) stimulation than under complex I (NADH) stimulation. In contrast, H2O2 generation and NBT and DPI reduction kinetics were favored by NADH addition but were virtually unobservable during succinate‐linked respiration. NADH oxidation was strongly suppressed by rotenone, but NADH‐coupled H2O2 flux was accelerated by rotenone. α‐Phenyl‐N‐tert‐butyl nitrone (PBN), a compound documented to inhibit oxidative stress in models of stroke, sepsis, and parkinsonism, partially inhibited complex I‐stimulated H2O2 flux and NBT reduction and also protected complex I from DPI‐mediated inactivation while trapping the phenyl radical product of DPI reduction. The results suggest that complex I may be the principal source of brain mitochondrial H2O2 synthesis, possessing an “electron leak” site upstream from the rotenone binding site (i.e., on the NADH side of the enzyme). The inhibition of H2O2 production by PBN suggests a novel explanation for the broad‐spectrum antioxidant and antiinflammatory activity of this nitrone spin trap.

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