Increased oxidative damage to DNA in an animal model of amyotrophic lateral sclerosis

Substantial evidence suggest that oxidative damage may play a role in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS). We examined levels of 8-Hydroxy-2'-deoxyguanosine (8OH2'dG) in the nuclear DNA from the spinal cord, frontal cortex, striatum and cerebellum from G93A mice at 60, 90, and 120 days of age. We also used in vivo microdialysis to measure free levels of 8OH2'dG and 8-Hydroxyguanine (8OHG) at the same time points in the frontal cortex of G93A mice. Increased 8OH2'dG DNA levels were observed in the spinal cord (at 60, 90 and 120 days), in the cortex (at 90, and 120 days), and in the striatum (at 120 days), as compared to age-matched littermate controls. No significant changes were found in the cerebellum at any of the time points studied. Free levels of 8OH2'dG in the cortex of G93A mice were increased, as compared to control mice, at 90 and 120 days. Free levels of 8OHG were found to be significantly higher at 120 days of age in control mice than in G93A mice. These results provide evidence that in this model of ALS oixidative DNA-damage is increased and base excision-repair may be deficient.     

A carbon column-based liquid chromatography electrochemical approach to routine 8-hydroxy-2'-deoxyguanosine measurements in urine and other biologic matrices: a one-year evaluation of methods.

8-Hydroxy-2'-deoxyguanosine (8OH2'dG) is a principal stable marker of hydroxyl radical damage to DNA. It has been related to a wide variety of disorders and environmental insults, and has been proposed as a useful systematic marker of oxidative stress. Analytic procedures for 8OH2'dG in DNA digests are well established; however, routine measurement of free 8OH2'dG in other body fluids such as urine or plasma has been problematic. This has hindered its evaluation as a general clinical, therapeutic monitoring, or environmental assessment tool. Therefore, we developed a liquid chromatography electrochemical column-switching system based on the use of the unique purine selectivity of porous carbon columns that allows routine accurate measurement of 8OH2'dG in a variety of biologic matrices. This paper describes the rationale of the system design and the protocols developed for 8OH2'dG in urine, plasma, cerebrospinal fluid, tissue, DNA, saliva, sweat, kidney dialysis fluid, foods, feces, culture matrix, and microdialysates. Concentrations in both human and animal body fluids and tissues are reported. The system performance is discussed in the context of a 1-year evaluation of the methods applied to approximately 3600 samples, using internal quality control and external blind testing to determine long-term accuracy. The methods are reliable and accurate, and therefore should prove useful in assessing the role and utility of oxidative DNA damage in aging and human illness.     

Urinary 8-hydroxy-2'-deoxyguanosine,a metabolite of oxidized DNA,is not elevated in HIV patients on combination antiretroviral therapy

Mitochondrial toxicity of nucleoside analogues has been proposed to be the etiology of a range of side-effects from antiretroviral therapy of HIV infection. In this study, urinary 8-hydroxy-2'-deoxyguanosine (8OH2'dG), a metabolite of oxidized DNA, was measured to determine if antiretroviral therapy leads to oxidative damage to DNA. A cross-sectional study was carried out measuring urinary 8OH2'dG in three groups of HIV-infected patients: (1) antiretroviral medication na?ve, (2) patients on antiretroviral medications without lipodystrophy and (3) patients on antiretroviral medications with lipodystrophy. Twenty-five patients were enrolled in each group. The mean spot urinary 8OH2'dG measurements per mg creatinine for the three groups were: antiretroviral na?ve 4.27 +/- 0.61 (ng 8OH2'dG/mg creatinine +/- SEM), on antiretroviral medications without lipodystrophy 2.88 +/- 0.26, and on antiretroviral medications with lipodystrophy 3.27 +/- 0.30. The differences between the means of the three groups is not statistically significant (p = 0.055), and these results are not significantly different from reported values for healthy controls [A carbon column-based liquid chromatography electrochemical approach to routine 8-hydroxy-2-deoxyguanosine measurements in urine and other biologic matrices: a one-year evaluation of methods. Free Radical Biology and Medicine 27 (1999) 647-666].     

Chronic consumption of a flavanol- and procyanindin-rich diet is associated with reduced levels of 8-hydroxy-2'-deoxyguanosine in rat testes

Cocoa can contain a high concentration of flavanols and procyanidins which have been reported to have strong antioxidative activity. In the present study, male Sprague-Dawley rats were fed diets containing 0, 0.5, 1, or 2% cocoa rich in flavanols for two weeks. Blood, liver, heart and testes were collected and analyzed for markers of oxidative damage. Plasma epicatechin concentrations, 8-hydroxy-2'-deoxyguanosine (8OH2'dG), and oxidized and reduced glutathione were quantitated by HPLC with electrochemical detection. Plasma F(2)-isoprostanes were measured using an enzyme immunoassay. Plasma epicatechin concentrations increased in a dose-dependant fashion according to the amount of cocoa in the diet (128 nM-790 nM). Cocoa supplementation was associated with lower than normal concentrations of 8OH2'dG in the testes (0.590 + 0.40 vs. 0.328 + 0.29; p < 0.05). Liver and heart 8OH2'dG levels were unaffected by dietary treatment. In erythrocytes, the glutathione pool was significantly less oxidized in the cocoa fed group compared to controls (p < 0.05). In liver and testes, no differences in superoxide dismutase activities were detected. Concentrations of plasma F(2)-isoprostanes and thiobarbituric acid reactive substances were similar in all groups. These results support the concept that a diet rich in flavanols and procyanidins can improve oxidant defense and reduce tissue markers for oxidative stress, although these effects can be tissue specific.     

Evidence of Increased Oxidative Damage in Both Sporadic and Familial Amyotrophic Lateral Sclerosis

Abstract: Some cases of autosomal dominant familial amyotrophic lateral sclerosis (FALS) are associated with mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1), suggesting that oxidative damage may play a role in ALS pathogenesis. To further investigate the biochemical features of FALS and sporadic ALS (SALS), we examined markers of oxidative damage to protein, lipids, and DNA in motor cortex (Brodmann area 4), parietal cortex (Brodmann area 40), and cerebellum from control subjects, FALS patients with and without known SOD mutations, SALS patients, and disease controls (Pick's disease, progressive supranuclear palsy, diffuse Lewy body disease). Protein carbonyl and nuclear DNA 8-hydroxy-2'-deoxyguanosine (OH⁸dG) levels were increased in SALS motor cortex but not in FALS patients. Malondialdehyde levels showed no significant changes. Immunohistochemical studies showed increased neuronal staining for hemeoxygenase-1, malondialdehyde-modified protein, and OH⁸dG in both SALS and FALS spinal cord. These studies therefore provide further evidence that oxidative damage may play a role in the pathogenesis of neuronal degeneration in both SALS and FALS.     

Increased oxidative damage to DNA in a transgenic mouse model of Huntington's disease

Mitochondrial dysfunction and oxidative damage may play a role in the pathogenesis of Huntington's disease (HD). We examined concentrations of 8-hydroxy-2-deoxyguanosine (OH(8)dG), a well-established marker of oxidative damage to DNA, in a transgenic mouse model of HD (R6/2). Increased concentrations of OH(8)dG were found in the urine, plasma and striatal microdialysates of the HD mice. Increased concentrations were also observed in isolated brain DNA at 12 and 14 weeks of age. Immunocytochemistry showed increased OH(8)dG staining in late stages of the illness. These results suggest that oxidative damage may play a role in the pathogenesis of neuronal degeneration in the R6/2 transgenic mouse model of HD.     

Cystatin C: a candidate biomarker for amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurologic disease characterized by progressive motor neuron degeneration. Clinical disease management is hindered by both a lengthy diagnostic process and the absence of effective treatments. Reliable panels of diagnostic, surrogate, and prognostic biomarkers are needed to accelerate disease diagnosis and expedite drug development. The cysteine protease inhibitor cystatin C has recently gained interest as a candidate diagnostic biomarker for ALS, but further studies are required to fully characterize its biomarker utility. We used quantitative enzyme-linked immunosorbent assay (ELISA) to assess initial and longitudinal cerebrospinal fluid (CSF) and plasma cystatin C levels in 104 ALS patients and controls. Cystatin C levels in ALS patients were significantly elevated in plasma and reduced in CSF compared to healthy controls, but did not differ significantly from neurologic disease controls. In addition, the direction of longitudinal change in CSF cystatin C levels correlated to the rate of ALS disease progression, and initial CSF cystatin C levels were predictive of patient survival, suggesting that cystatin C may function as a surrogate marker of disease progression and survival. These data verify prior results for reduced cystatin C levels in the CSF of ALS patients, identify increased cystatin C levels in the plasma of ALS patients, and reveal correlations between CSF cystatin C levels to both ALS disease progression and patient survival.     

Combination of neurofilament heavy chain and complement C3 as CSF biomarkers for ALS

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and ultimately fatal neurodegenerative disease with an average survival of 3 years from symptom onset. Rapid and conclusive early diagnosis is essential if interventions with disease-modifying therapies are to be successful. Cytoskeletal modification and inflammation are known to occur during the pathogenesis of ALS. We measured levels of cytoskeletal proteins and inflammatory markers in the CSF of ALS, disease controls and healthy subjects. We determined threshold values for each protein that provided the optimal sensitivity and specificity for ALS within a training set, as determined by receiver operating characteristic analysis. Interestingly, the optimal assay was a ratio of the levels for phosphorylated neurofilament heavy chain and complement C3 (pNFH/C3). We next applied this assay to a separate test set of CSF samples to verify our results. Overall, the predictive pNFH/C3 ratio identified ALS with 87.3% sensitivity and 94.6% specificity in a total of 71 ALS subjects, 52 disease control subjects and 40 healthy subjects. In addition, the level of CSF pNFH correlated with survival of ALS patients. We also detected increased pNFH in the plasma of ALS patients and observed a correlation between CSF and plasma pNFH levels within the same subjects. These findings support large-scale prospective biomarker studies to determine the clinical utility of diagnostic and prognostic signatures in ALS.     

Increased levels of F2-isoprostanes following aneurysmal subarachnoid hemorrhage in humans

Subarachnoid hemorrhage (SAH) resulting from aneurysmal rupture is the major cause of nontraumatic SAH. We hypothesized that oxidative stress could be increased following aneurysmal SAH due to hemoglobin release and ischemia-reperfusion injury and that may further contribute to poor outcome. We collected plasma and cerebrospinal fluid (CSF) samples from 11 non-SAH controls and 15 aneurysmal SAH patients for up to 10 days after surgery and investigated status of oxidative stress in patients. Results showed that mean or peak levels of F(2)-isoprostanes (F(2)-IsoPs), a specific marker of lipid peroxidation, and total nitrate/nitrite, metabolites of nitric oxide and peroxynitrite, in CSF and plasma were significantly higher in SAH patients than in controls. First-day levels were also higher in CSF, but not in plasma, in SAH patients. Moreover, mean and peak levels of CSF F(2)-IsoPs were positively correlated with poor outcome or severity of clinical conditions in patients. Furthermore, levels of retinol, delta-tocopherol, beta+gamma-tocopherol, lutein, beta-carotene, and coenzyme Q(10) in plasma were significantly lower in SAH patients than in controls. Our results indicate that oxidative damage may play important roles in the severity and complications of aneurysmal SAH and suggest that means to suppress lipid peroxidation may be beneficial in improving the outcome of aneurysmal SAH.     

Levels of 8-oxo-dGsn and 8-oxo-Gsn in random urine are consistent with 24 h urine in healthy subjects and patients with renal disease

Oxidatively generated damage to nucleic acids may play an important role in the pathophysiological processes of a variety of diseases. 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGsn) and 8-oxo-7,8-dihydroguanosine (8-oxo-Gsn) are oxidatively generated products of DNA and RNA, respectively. Our previous studies have suggested that the amounts of 8-oxo-dGsn and 8-oxo-Gsn in urine were considerably higher than other body fluid or tissue. The aim of this study was to investigate whether 8-oxo-dGsn and 8-oxo-Gsn levels in random urine samples are consistent with those in 24?h urine samples in healthy subjects and patients with renal disease. A total of 16 healthy subjects and 104 renal disease patients were enrolled in this study, and their random and 24?h urine samples were collected. The levels of urinary 8-oxo-dGsn and 8-oxo-Gsn were quantified by LC-MS/MS and corrected by creatinine. Regardless of healthy subjects or renal disease patients, the levels of oxidised nucleosides in random urine samples were consistent with 24?h urine samples. Regardless of the age bracket, there is no significant difference between random samples and 24?h urine samples. In conclusion, 8-oxo-dGsn and 8-oxo-Gsn levels in random urine samples could replace those in 24?h urine samples, and were considered as the representative of the level of systemic oxidative stress for the whole day.     

Analysis of smoking and LPO in ALS

Smoking has been suggested as one of the risk factor for amyotrophic lateral sclerosis (ALS) development. In order to investigate whether adverse effects of cigarette smoke in ALS have any association with increase in oxidative stress, disease severity, lipid hydroperoxides (LPO) and superoxide dismutase-1 (SOD1) levels were measured in biofluids of smoker and never smoker ALS patients and clinically correlated. Serum and CSF from sporadic ALS patients (n = 50) diagnosed with El Escorial criteria were collected in the study. Serum (n = 50) and CSF (n = 42) were also collected from normal healthy controls. The LPO levels were estimated using commercially available kits. Enzyme-linked immunosorbent assays (ELISAs) were used to quantitate SOD1. Their levels were further analyzed among smoker and never smoker subjects. Significantly elevated LPO in sera and CSF of ALS patients were observed (p < 0.05). There was considerably increased LPO in sera and CSF of smoker ALS subjects matched with disease severity as compared to never smoker ALS (p < 0.05). ALS group did not show any alteration in SOD1 when compared to controls (p > 0.05). In addition, no change has been observed in SOD1 levels in ALS subjects who smoke (p > 0.05). Increased LPO and unaltered SOD1 in ALS patients may suggest the neuro-pathological association of LPO with ALS disease independent of SOD1. With current findings, it may be proposed that LPO levels might constitute as probable biomarker for smoker ALS patients, however, it cannot be concluded without larger gender matched studies. Additional investigations are needed to determine whether LPO upregulation is primary or secondary to motor neuron degeneration in ALS.     

Transforming growth factor-Beta 1 (tgf-Beta 1) in patients with amyotrophic lateral sclerosis

Previous investigations have shown that the transforming growth factor-beta 1 (TGF-beta 1) may protect neurons against excitotoxic and oxidative damage and may inhibit apoptosis. The aim of this study was to investigate the role of TGF-beta 1 in patients with amyotrophic lateral sclerosis (ALS). The study involved 24 ALS patients and 15 control group people. The ALS patients were divided into groups according to their clinical status, and duration of ALS. The TGF-beta 1 in the serum and cerebrospinal fluid (CSF) was measured by the enzyme-linked immunosorbent assay (ELISA). Results showed that TGF-beta 1 concentrations in the serum, and CSF in the whole group of ALS patients did not differ from those of the controls, but the serum TGF-beta 1 concentration was significantly higher in ALS patients with a terminal clinical status than in controls. The TGF-beta 1 concentration was significantly higher in the CSF of the patients, with a long duration of ALS, than in the patients with a short duration of ALS, and there was a significant positive correlation between the CSF TGF-beta 1 and the duration of ALS. TGF-beta 1 may play a role in neurodegeneration of ALS, and may be an indicator of the duration of the disease.     

The onset of lipid peroxidation in rheumatoid arthritis: consequences and monitoring

Several epidemiological studies propose the association of rheumatoid arthritis (RA) with oxidative stress. The aim of this study was to estimate the possible onset of systemic lipid peroxidation in RA patients and its relevance for pathophysiology and monitoring of RA. Seventy-three patients with RA and 73 healthy subjects were included in the study. Lipid peroxidation was estimated by the measurement of 4-hydroxynonenal (4-HNE), 4-hydroxyhexenal, malondialdehyde, acrolein, crotonaldehyde, 4-oxononenal, and isoprostanes (8-isoPGF_2α) levels. Cytosolic phospholipase A₂ (cPLA₂), platelet-activating factor acetylhydrolase (PAF-AH) and glutathione peroxidase (GSH-Px) activities and vitamin E levels were also determined. In parallel, the plasma levels of phospholipid arachidonic acid (AA), linoleic acid (LA), and 4-HNE-protein adducts were monitored. Plasma of RA patients had increased vitamin E levels, but decreased GSH-Px activity and phospholipid AA and LA levels when compared to levels of the healthy subjects. The levels of aldehydes were significantly increased in the plasma of the RA patients and even more in urine. Significant increases in HNE-modified protein adducts was observed for the first time in plasma of RA patients, while the activities of PAF-AH and cPLA₂ were decreased. The 8-isoPGF_2α levels were 9-fold higher in plasma and 3-fold higher in urine of RA patients and were related to the severity of disease. The levels of lipid peroxidation products in plasma and in urine suggest the relationship between lipid peroxidation and the development of RA. Additionally, urine 8-isoPGF_2α, plasma 4-HNE and 4-HNE-protein adducts appear to be convenient biomarkers to monitor progression of this autoimmune disease.     

DNA oxidative damage and strand breaks in young healthy individuals: a gender difference and the role of life style factors

The aim of this study was to analyze background levels of DNA damage in young (19-31 years) non-smoking individuals and to correlate damage to gender and life style. DNA single strand breaks (SSB) and alkali labile sites (ALS) were measured in 99 subjects living in Stockholm, Sweden. Further, oxidative DNA damage was analyzed using the DNA repair glycosylase FPG as well as HPLC-ECD for specific analysis of 8-oxo-7,8-dihydro-2'deoxyguanosine (8-oxodG). We found that males had higher (P < 0.001) levels of SSB + ALS than females, but no difference was seen for oxidative lesions. There was no correlation between FPG sites and 8-oxodG. For females, there was a positive correlation between FPG levels and body mass index and a negative correlation between SSB + ALS and fruit intake. We conclude that the background level of oxidative DNA damage, analyzed with improved methods, is low and that gender, fruit intake and BMI can affect DNA damage.     

Clinical perspective on oxidative stress in sporadic amyotrophic lateral sclerosis

Sporadic amyotrophic lateral sclerosis (ALS) is one of the most devastating neurological diseases; most patients die within 3 to 4 years after symptom onset. Oxidative stress is a disturbance in the pro-oxidative/antioxidative balance favoring the pro-oxidative state. Autopsy and laboratory studies in ALS indicate that oxidative stress plays a major role in motor neuron degeneration and astrocyte dysfunction. Oxidative stress biomarkers in cerebrospinal fluid, plasma, and urine are elevated, suggesting that abnormal oxidative stress is generated outside of the central nervous system. Our review indicates that agricultural chemicals, heavy metals, military service, professional sports, excessive physical exertion, chronic head trauma, and certain foods might be modestly associated with ALS risk, with a stronger association between risk and smoking. At the cellular level, these factors are all involved in generating oxidative stress. Experimental studies indicate that a combination of insults that induce modest oxidative stress can exert additive deleterious effects on motor neurons, suggesting that multiple exposures in real-world environments are important. As the disease progresses, nutritional deficiency, cachexia, psychological stress, and impending respiratory failure may further increase oxidative stress. Moreover, accumulating evidence suggests that ALS is possibly a systemic disease. Laboratory, pathologic, and epidemiologic evidence clearly supports the hypothesis that oxidative stress is central in the pathogenic process, particularly in genetically susceptive individuals. If we are to improve ALS treatment, well-designed biochemical and genetic epidemiological studies, combined with a multidisciplinary research approach, are needed and will provide knowledge crucial to our understanding of ALS etiology, pathophysiology, and prognosis.     

Oxidative stress in ALS: Key role in motor neuron injury and therapeutic target

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder characterized by death of motor neurons leading to muscle wasting, paralysis, and death, usually within 2–3 years of symptom onset. The causes of ALS are not completely understood, and the neurodegenerative processes involved in disease progression are diverse and complex. There is substantial evidence implicating oxidative stress as a central mechanism by which motor neuron death occurs, including elevated markers of oxidative damage in ALS patient spinal cord and cerebrospinal fluid and mutations in the antioxidant enzyme superoxide dismutase 1 (SOD1) causing approximately 20% of familial ALS cases. However, the precise mechanism(s) by which mutant SOD1 leads to motor neuron degeneration has not been defined with certainty, and the ultimate trigger for increased oxidative stress in non-SOD1 cases remains unclear. Although some antioxidants have shown potential beneficial effects in animal models, human clinical trials of antioxidant therapies have so far been disappointing. Here, the evidence implicating oxidative stress in ALS pathogenesis is reviewed, along with how oxidative damage triggers or exacerbates other neurodegenerative processes, and we review the trials of a variety of antioxidants as potential therapies for ALS.     

Decreased plasma and cerebrospinal fluid ascorbate levels in patients with septic encephalopathy

Septic encephalopathies rapidly affect brain function without the involvement of a specific area causing a broad range of reversible neurologic symptoms. Capillary leakage including dysfunction of the blood-brain barrier has been proposed as a potential pathogenic mechanism in this entity. We tested the hypothesis that oxidative stress measured in plasma and cerebrospinal fluid (CSF) of patients suffering from septic encephalopathy could be linked to the neurologic symptoms of the disease. The neurologic symptoms of eleven patients with septic encephalopathy were described semiquantitatively through a score system. The ascorbate levels were significantly lower in both plasma and CSF from patients with septic encephalopathy than controls, and in CSF but not plasma this decrease correlated with the severity of neurologic symptoms. No significant changes were found for alpha-tocopherol. Our findings suggest that the short-term oxidative stress may be an important factor in the development of septic encephalopathy, possibly through dysregulation of the blood-brain barrier.     

Free Copper,Ferroxidase and SOD1 Activities,Lipid Peroxidation and NO<Subscript><Emphasis Type="Italic">x</Emphasis></Subscript> Content in the CSF. A Different Marker Profile in Four Neurodegenerative Diseases

The understanding of oxidative damage in different neurodegenerative diseases could enhance therapeutic strategies. Our objective was to quantify lipoperoxidation and other oxidative products as well as the activity of antioxidant enzymes and cofactors in cerebrospinal fluid (CSF) samples. We recorded data from all new patients with a diagnosis of either one of the four most frequent neurodegenerative diseases: Parkinson’s disease (PD), Alzheimer’s disease (AD), Huntington’s disease (HD) and lateral amyotrophic sclerosis (ALS). The sum of nitrites and nitrates as end products of nitric oxide (NO) were increased in the four degenerative diseases and fluorescent lipoperoxidation products in three (excepting ALS). A decreased Cu/Zn-dependent superoxide dismutase (SOD) activity characterized the four diseases. A significantly decreased ferroxidase activity was found in PD, HD and AD, agreeing with findings of iron deposition in these entities, while free copper was found to be increased in CSF and appeared to be a good biomarker of PD.     

Proteomic profiling of cerebrospinal fluid identifies biomarkers for amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is characterized by degeneration of motor neurons. We tested the hypothesis that proteomic analysis will identify protein biomarkers that provide insight into disease pathogenesis and are diagnostically useful. To identify ALS specific biomarkers, we compared the proteomic profile of cerebrospinal fluid (CSF) from ALS and control subjects using surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF-MS). We identified 30 mass ion peaks with statistically significant (p < 0.01) differences between control and ALS subjects. Initial analysis with a rule-learning algorithm yielded biomarker panels with diagnostic predictive value as subsequently assessed using an independent set of coded test subjects. Three biomarkers were identified that are either decreased (transthyretin, cystatin C) or increased (carboxy-terminal fragment of neuroendocrine protein 7B2) in ALS CSF. We validated the SELDI-TOF-MS results for transthyretin and cystatin C by immunoblot and immunohistochemistry using commercially available antibodies. These findings identify a panel of CSF protein biomarkers for ALS.     

Cerebrospinal fluid levels of free 3-nitrotyrosine are not elevated in the majority of patients with amyotrophic lateral sclerosis or Alzheimer's disease

The mechanisms behind the degeneration of neurons in diseases such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS) are not fully understood. However, oxidation of certain amino acid residues in proteins may contribute to cell injury and some of these oxidized amino acids may also be suitable as biomarkers for oxidative injury. Therefore, it is suggested that the reaction between peroxynitrite (ONOO(-)) and tyrosine in vivo can be monitored by monitoring the formation of 3-nitrotyrosine (3-NT). In this work, a newly developed gas chromatographic-mass spectrometric method was applied to human cerebrospinal fluid (CSF). The free 3-NT levels were determined in the CSF from 19 controls, 17 patients with AD and 14 patients with ALS. The levels of free 3-NT in the CSF were considerably lower than those previously reported. The majority of the patients with AD or ALS had free 3-NT levels in the same range as seen in the control individuals and only a few patients showed increased levels of free 3-NT.