Chronic high dose L-DOPA alone or in combination with the COMT inhibitor entacapone does not increase oxidative damage or impair the function of the nigro-striatal pathway in normal cynomologus monkeys.

Parkinson's disease (PD) is characterised by a loss of pigmented dopaminergic neurones in the zona compacta of substantia nigra. The mechanisms underlying nigral cell death remain unknown but may involve oxidative damage. There has been concern that L-DOPA treatment may accelerate nigral pathology in PD through chemical and enzymatic oxidation to reactive oxygen species. In the present study, we examined tissues from normal macaque monkeys treated for 13 weeks with high doses of L-DOPA (in combination with the peripheral decarboxylase inhibitor, carbidopa) and/or the COMT inhibitor, entacapone. Plasma was analysed for changes in protein carbonyls as a marker of oxidative damage to protein. Cortical tissue was examined for changes in levels of protein carbonyls, lipid peroxidation and oxidative damage to DNA. The integrity of the nigro-striatal pathway was assessed by nigral tyrosine hydroxylase mRNA levels and specific [(3)H]mazindol binding to dopaminergic terminals in caudate-putamen. No alterations in plasma protein carbonyls were observed in any treatment group. An increase was found in the levels of protein carbonyls, lipid peroxidation and 5-OH uracil, but not other products of oxidative DNA damage, in cerebral cortex of monkeys treated with L-DOPA plus carbidopa or with L-DOPA plus carbidopa and entacapone but this was only statistically significant in the latter group. There was no change in nigral tyrosine hydroxylase mRNA levels or specific striatal [(3)H]mazindol binding in brain tissue from monkeys treated with either L-DOPA plus carbidopa or L-DOPA plus carbidopa and entacapone. The results show that in the normal monkeys L-DOPA does not provoke marked oxidative damage even at high doses, and that there is little or no potentiation of its effects by entacapone. Neither L-DOPA plus carbidopa nor L-DOPA plus carbidopa and entacapone led to obvious damage to the nigro-striatal pathway.

Oxidative damage to proteins, lipids, and DNA in cortical brain regions from patients with dementia with Lewy bodies.

Dementia with Lewy bodies (DLB) forms the second most common pathological subgroup of dementia after Alzheimer's disease. The present study compares the levels of oxidative damage to proteins, lipids, and DNA bases in cortical brain areas from patients with DLB with levels in matched control tissues. Overall, there was a trend for protein carbonyl levels to be increased in all areas, but a significant difference was found only in the parietal and temporal lobes. No differences were observed in the levels of lipid peroxidation. Measurement of products of damage to DNA bases showed increased levels of thymine glycol, 8-hydroxyguanine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 5-hydroxycytosine, 5-hydroxyuracil, 5-hydroxymethyluracil, and xanthine. Xanthine levels were increased in the DLB group in the parietal, occipital, and temporal lobes, indicating that peroxynitrite or other deaminating species may be involved. The finding of increased protein carbonyls and increased DNA base products in cortical regions from DLB patients indicates that oxidative stress may play a role in DLB.

Artefacts in HPLC detection of 3-nitrotyrosine in human brain tissue.

An HPLC method was used for quantification of 3-nitrotyrosine (3-NT) in human postmortem brain tissue. A peak with similar retention time to 3-NT was detected in brain tissue from patients with Parkinson's disease, Huntington's chorea, multiple system atrophy, and Alzheimer's disease but not in control tissue. The peak was lost on reduction with dithionite, a criterion often used to identify 3-NT. Tissue from the same neurodegenerative diseases was analysed by HPLC using a photodiode array detector in series with an amperometric electrochemical detector, but the peak was found not to be 3-NT. The absorbance spectrum, fragmentation pattern on mass spectroscopy, and electrochemical profile of this peak do not match authentic 3-NT. A search of the mass spectroscopy databases failed to reveal its identity. The presence of this closely eluting, dithionite-reducible peak could confound analysis of human tissues for 3-NT. In vitro experiments showed that high concentrations of peroxynitrite were needed to achieve detectable levels of 3-NT in human brain tissue.

An assessment of oxidative damage to proteins, lipids, and DNA in brain from patients with Alzheimer's disease.

Oxidative stress may contribute to neuronal loss in Alzheimer's disease (AD). The present study compares the levels of oxidative damage to proteins, lipids, and DNA bases from seven different brain areas of AD and matched control tissues by using a range of techniques. No differences in levels of lipid peroxidation were found in any of the brain regions by using two different assay systems. Overall, there was a trend for protein carbonyl levels to be increased in AD in frontal, occipital, parietal, and temporal lobe, middle temporal gyrus, and hippocampus, but a significant difference was found only in the parietal lobe. Gas chromatography-mass spectrometry was used to measure products of damage to all four DNA bases. Increased levels of some (8-hydroxyadenine, 8-hydroxyguanine, thymine glycol, Fapy-guanine, 5-hydroxyuracil, and Fapy-adenine), but not all, oxidized DNA bases were observed in parietal, temporal, occipital, and frontal lobe, superior temporal gyrus, and hippocampus. The baseline level of oxidative DNA damage in the temporal lobe was higher than in other brain regions in both control and AD brain. The finding of increased oxidative damage to protein and DNA strengthens the possibility that oxidative damage may play a role in the pathogenesis of AD in at least some key brain regions.

Oxidative damage and motor neurone disease difficulties in the measurement of protein carbonyls in human brain tissue.

It has been suggested in the literature that elevated oxidative protein damage, measured as protein carbonyls, is present in the nervous system of patients with sporadic motor neurone disease (MND). However, the actual reported levels of brain protein carbonyls vary over a wide range. We show here that this is probably due to the use of the different protocols for the carbonyl assay; results differ depending on when the dinitrophenylhydrazine reagent is added and at what stage in the procedure protein is assayed for the calculation of carbonyls on a unit protein basis. Using a range of different procedures, we were unable to confirm reports of elevated protein carbonyls in motor cortex from brains of patients with MND. We also measured thiobarbituric acid-reactive material in the brain samples using an HPLC-based TBA test in the presence of butylated hydroxytoluene. In general, there was no significant elevation of TBARS in MND motor cortex. However, four patients showed values higher than any of the control patients (both 'normal' control and 'disease control'). There was no correlation of TBARS with protein carbonyl values. We suggest that oxidative damage in motor cortex in sporadic MND, if it occurs, may be confined to a small group of patients and may affect different molecular targets in each patient.