8-hydroxy-2--deoxyguanosine and Amyotrophic-Lateral-Sclerosis

Accurate patient stratification into prognostic categories and targeting Amyotrophic Lateral Sclerosis (ALS)-associated pathways may pave the way for promising trials. We evaluated blood-based prognostic indicators using an array of pathological markers. Plasma samples were collected as part of a large, phase III clinical trial (Mitotarget/TRO19622) at months 1, 6, 12 and 18. The ALSFRS-r score was used as a proxy of disease progression to assess the predictive value of candidate biological indicators. First, established clinical predictors were evaluated in all 512 patients. Subsequently, pathologic markers, such as proxies of neuronal integrity (Neurofilament light chain and phosphorylated heavy chain), DNA oxidation (8-oxo-2'-desoxyguanosine), lipid peroxidation (4-hydroxy-2-nonenal, isoprostane), inflammation (interleukin-6) and iron status (ferritin, hepcidin, transferrin) were assessed in a subset of 109 patients that represented the whole cohort. Markers of neuronal integrity, DNA and lipid oxidation, as well as iron status at baseline are accurate predictors of disability at 18-month follow-up. The composite scores of these markers in association with established clinical predictors enable the accurate forecasting of functional decline. The identified four biomarkers are all closely associated with 'ferroptosis', a recently discovered form of programmed cell death with promising therapeutic targets. The predictive potential of these pathophysiology-based indicators may offer superior patient stratification for future trials, individualised patient care and resource allocation.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Aldehydes; Amyotrophic Lateral Sclerosis; Biomarkers; Disease Progression; Female; Ferritins; Ferroptosis; Follow-Up Studies; Humans; Iron; Isoprostanes; Lipid Peroxidation; Male; Middle Aged; Neurofilament Proteins; Neurons; Predictive Value of Tests; Prognosis

Objective Lifestyle changes may play an important role in the incidence reduction and delay of onset age of amyotrophic lateral sclerosis (ALS) in the Koza/Kozagawa/Kushimoto (K) area. The aim of this study was to evaluate recent lifestyle changes in the K area and to investigate the relationships between lifestyle and oxidative stress among the residents. Methods We conducted a medical checkup for elderly residents in the K area and the control area and evaluated the urinary 8-OHdG levels, cognitive function test scores and metal contents in serum and scalp hair, coupled with a lifestyle questionnaire survey between 2010 and 2015. Results Recent lifestyle changes among the K residents, including a decrease in the Japanese pickle consumption, increase in fresh vegetable consumption and decrease in farm work, were evaluated in this study. Low consumption of Japanese pickles, high consumption of fresh vegetables, rare farm work and low levels of 8-OHdG/creatinine were all associated with high scores in the cognitive function tests. Frequent farm work and consumption of Japanese pickles was associated with high contents of transition metals, such as Mn, Al and V, in the scalp hair. Conclusion These lifestyle changes among residents in the K area may be associated with their oxidative stress.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Age of Onset; Aged; Aged, 80 and over; Amyotrophic Lateral Sclerosis; Deoxyguanosine; Diet; Exercise; Female; Hair; Humans; Incidence; Japan; Life Style; Male; Oxidative Stress; Scalp; Vegetables

Pathophysiological mechanisms that contribute to neurodegeneration in Amyotrophic Lateral Sclerosis (ALS) include oxidative stress and inflammation. We conducted a preliminary study to explore these mechanisms, to discuss their link in ALS, and to determine the feasibility of incorporating this combined analysis into current biomarkers research.. We enrolled 10 ALS patients and 10 controls. We measured the activities of glutathione peroxidase, glutathione reductase, superoxyde dismutase (SOD), and the levels of serum total antioxidant status (TAS), malondialdehyde (MDA), 8-hydroxy-2'-deoxyguanosine (8-OHdG), and glutathione status (e.g. glutathione disulfide, GSSG/reduced glutathione, GSH). We analysed the concentrations of homocysteine, several cytokines, vitamins and metals by standard methods used in routine practice.. There was a significant decrease in TAS levels (p=0.027) and increase in 8-OHdG (p=0.014) and MDA (p=0.011) levels in ALS patients. We also observed a significantly higher GSSG/GSH ratio (p=0.022), and IL-6 (p=0.0079) and IL-8 (p=0.009) concentrations in ALS patients. Correlations were found between biological and clinical markers (homosysteine vs. clinical status at diagnosis, p=0.02) and between some biological markers such as IL-6 vs. GSSG/GSH (p=0.045) or SOD activity (p=0.017).. We confirmed the systemic alteration of both the redox and the inflammation status in ALS patients, and we observed a link with some clinical parameters. These promising results encourage us to pursue this study with collection of combined oxidative stress and inflammatory markers.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aged; Aged, 80 and over; Amyotrophic Lateral Sclerosis; Biomarkers; Cytokines; Deoxyguanosine; Female; Glutathione Peroxidase; Glutathione Reductase; Homocysteine; Humans; Inflammation; Male; Malondialdehyde; Middle Aged; Oxidative Stress; Pilot Projects; Severity of Illness Index; Statistics as Topic

The implication of lipid peroxidation in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) derive from high abundance of peroxidation-prone polyunsaturated fatty acids in central nervous system and its relatively low antioxidant content. In the present work, we evaluated the effect of dietary changes aimed to modify fatty acid tissular composition in survival, disease onset, protein, and DNA oxidative modifications in the hSODG93A transgenic mice, a model of this motor neuron disease. Both survival and clinical evolution is dependent on dietary fatty acid unsaturation and gender, with high unsaturated diet, leading to loss of the disease-sparing effect of feminine gender. This was associated with significant increases in protein carbonyl and glycoxidative modifications as well as non-nuclear 8-oxo-dG, a marker of mitochondrial DNA oxidation. Comparison of these data with γH2AX immunostaining, a marker of DNA damage response, suggests that the highly unsaturated diet-blunted mitochondrial-nuclear free radical dependent crosstalk, since increased 8-oxo-dG was not correlated with increased DNA damage response. Paradoxically, the highly unsaturated diet led to lower peroxidizability but higher anti-inflammatory indexes. To sum up, our results demonstrate that high polyunsaturated fatty acid content in diets may accelerate the disease in this model. Further, these results reinforce the need for adequately defining gender as a relevant factor in ALS models, as well as to use structurally characterized markers for oxidative damage assessment in neurodegeneration.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Amyotrophic Lateral Sclerosis; Animals; Biomarkers; Deoxyguanosine; Dietary Fats; Disease Models, Animal; DNA Damage; DNA Repair; DNA, Mitochondrial; Fats, Unsaturated; Fatty Acids, Unsaturated; Female; Free Radicals; Glycosylation; Histones; Inflammation; Lipid Peroxidation; Male; Mice; Mice, Transgenic; Nerve Degeneration; Oxidative Stress; Point Mutation; Protein Carbonylation; Protein Processing, Post-Translational; Recombinant Fusion Proteins; Sex Characteristics; Superoxide Dismutase; Superoxide Dismutase-1

Increasing evidence indicates that oxidative stress is an important mechanism underlying motor neuron (MN) degeneration in amyotrophic lateral sclerosis (ALS). Mitochondrial DNA (mtDNA) is highly susceptible to oxidative damage and has little potential for repair, although mitochondrial transcription factor A (TFAM) plays essential roles in maintaining mitochondrial DNA by reducing oxidative stress, promoting mtDNA transcription, and regulating mtDNA copy number. To analyze a possible therapeutic effect of TFAM on ALS pathology, double transgenic mice overexpressing G93A mutant SOD1 (G93ASOD1) and human TFAM (hTFAM) were newly generated in the present study. Rotarod scores were better in G93ASOD1/hTFAM double-Tg mice than G93ASOD1 single-Tg mice at an early symptomatic stage, 15 and 16 weeks of age, with a 10% extension of the onset age in double-Tg mice. The number of surviving MNs was 30% greater in double-Tg mice with end-stage disease, at 19 weeks, with remarkable reductions in the amount of the oxidative stress marker 8-OHdG and the apoptotic marker cleaved caspase 3 and with preserved COX1 expression. Double-immunofluorescence study showed that hTFAM was expressed specifically in MNs and microglia in the spinal cords of double-Tg mice. The present study suggests that overexpression of TFAM has a potential to reduce oxidative stress in MN and delay onset of the disease in ALS model mice. © 2012 Wiley Priodicals, Inc.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Amyotrophic Lateral Sclerosis; Animals; Caspase 3; Cell Count; Choline O-Acetyltransferase; Deoxyguanosine; Disease Models, Animal; DNA-Binding Proteins; Electron Transport Complex IV; Gene Expression Regulation; Humans; Kaplan-Meier Estimate; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mitochondrial Proteins; Motor Neurons; Nerve Tissue Proteins; Spinal Cord; Superoxide Dismutase; Transcription Factors

While free radicals and inflammation constitute major routes of neuronal injury occurring in amyotrophic lateral sclerosis (ALS), neither antioxidants nor non-steroidal anti-inflammatory drugs have shown significant efficacy in human clinical trials. We examined the possibility that concurrent blockade of free radicals and prostaglandin E(2) (PGE(2))-mediated inflammation might constitute a safe and effective therapeutic approach to ALS. We have developed 2-hydroxy-5-[2-(4-trifluoromethylphenyl)-ethylaminobenzoic acid] (AAD-2004) as a derivative of aspirin. AAD-2004 completely removed free radicals at 50 nM as a potent spin-trapping molecule and inhibited microsomal PGE(2) synthase-1 (mPGES-1) activity in response to both lipopolysaccharide-treated BV2 cell with IC(50) of 230 nM and recombinant human mPGES-1 protein with IC(50) of 249 nM in vitro. In superoxide dismutase 1(G93A) transgenic mouse model of ALS, AAD-2004 blocked free radical production, PGE(2) formation, and microglial activation in the spinal cords. As a consequence, AAD-2004 reduced autophagosome formation, axonopathy, and motor neuron degeneration, improving motor function and increasing life span. In these assays, AAD-2004 was superior to riluzole or ibuprofen. Gastric bleeding was not induced by AAD-2004 even at a dose 400-fold higher than that required to obtain maximal therapeutic efficacy in superoxide dismutase 1(G93A). Targeting both mPGES-1-mediated PGE(2) and free radicals may be a promising approach to reduce neurodegeneration in ALS and possibly other neurodegenerative diseases.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Amyotrophic Lateral Sclerosis; Analysis of Variance; Animals; Anti-Inflammatory Agents, Non-Steroidal; Aspirin; Calcium-Binding Proteins; Cerebral Cortex; Deoxyguanosine; Dinoprostone; Disease Models, Animal; Encephalitis; Free Radical Scavengers; Free Radicals; Gene Expression Regulation; Humans; Ibuprofen; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microfilament Proteins; Microglia; Motor Neurons; Oxidative Stress; Riluzole; Spinal Cord; Sulfasalazine; Superoxide Dismutase; Tyrosine

Hippocampal neurogenesis in the subgranular zone (SGZ) of dentate gyrus (DG) occurs throughout life and is regulated by pathological and physiological processes. The role of oxidative stress in hippocampal neurogenesis and its response to exercise or neurodegenerative diseases remains controversial. The present study was designed to investigate the impact of oxidative stress, treadmill exercise and sex on hippocampal neurogenesis in a murine model of heightened oxidative stress (G93A mice). G93A and wild type (WT) mice were randomized to a treadmill running (EX) or a sedentary (SED) group for 1 or 4 wk. Immunohistochemistry was used to detect bromodeoxyuridine (BrdU) labeled proliferating cells, surviving cells, and their phenotype, as well as for determination of oxidative stress (3-NT; 8-OHdG). BDNF and IGF1 mRNA expression was assessed by in situ hybridization. Results showed that: (1) G93A-SED mice had greater hippocampal neurogenesis, BDNF mRNA, and 3-NT, as compared to WT-SED mice. (2) Treadmill running promoted hippocampal neurogenesis and BDNF mRNA content and lowered DNA oxidative damage (8-OHdG) in WT mice. (3) Male G93A mice showed significantly higher cell proliferation but a lower level of survival vs. female G93A mice. We conclude that G93A mice show higher hippocampal neurogenesis, in association with higher BDNF expression, yet running did not further enhance these phenomena in G93A mice, probably due to a 'ceiling effect' of an already heightened basal levels of hippocampal neurogenesis and BDNF expression.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Amyotrophic Lateral Sclerosis; Animals; Bromodeoxyuridine; Cell Proliferation; Cell Survival; Dentate Gyrus; Deoxyguanosine; Disease Models, Animal; Exercise Test; Female; Gene Expression Regulation; Hippocampus; Intercellular Signaling Peptides and Proteins; Male; Mice; Neurogenesis; Neurons; Oxidative Stress; Phenotype; Physical Conditioning, Animal; RNA, Messenger; Sex Characteristics; Tyrosine

Mutations in the gene encoding cytosolic Cu,Zn-superoxide dismutase (SOD1) have been linked to familial amyotrophic lateral sclerosis (FALS). However the molecular mechanisms of motor neuron death are multi-factorial and remain unclear. Here we examined DNA damage, p53 activity and apoptosis in SH-SY5Y human neuroblastoma cells transfected to achieve low-level expression of either wild-type or mutant Gly(93)-->Ala (G93A) SOD1, typical of FALS. DNA damage was investigated by evaluating the levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and DNA strand breaks. Significantly higher levels of DNA damage, increased p53 activity, and a greater percentage of apoptotic cells were observed in SH-SY5Y cells transfected with G93A SOD1 when compared to cells overexpressing wild-type SOD1 and untransfected cells. Western blot, FACS, and confocal microscopy analysis demonstrated that G93A SOD1 is present in the nucleus in association with DNA. Nuclear G93A SOD1 has identical superoxide dismutase activity but displays increased peroxidase activity when compared to wild-type SOD1. These results indicate that the G93A mutant SOD1 association with DNA might induce DNA damage and trigger the apoptotic response by activating p53. This toxic activity of mutant SOD1 in the nucleus may play an important role in the complex mechanisms associated with motor neuron death observed in ALS pathogenesis.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Amyotrophic Lateral Sclerosis; Apoptosis; Cell Nucleus; Chromatin; Deoxyguanosine; DNA Damage; Humans; Immunoenzyme Techniques; Lipid Peroxidation; Neuroblastoma; Superoxide Dismutase; Superoxide Dismutase-1; Tumor Cells, Cultured; Tumor Suppressor Protein p53

There is strong evidence from studies in humans and animal models to suggest the involvement of energy metabolism defects in neurodegenerative diseases. Uridine, a pyrimidine nucleoside, has been suggested to be neuroprotective in neurological disorders by improving bioenergetic effects, increasing ATP levels and enhancing glycolytic energy production. We assessed whether uridine treatment extended survival and improved the behavioral and neuropathological phenotype observed in G93A-ALS mice. In vitro and in vivo pharmacokinetic analyses in mutant SOD models provided optimal dose and assurance that uridine entered the brain. A dose-ranging efficacy trial in G93A mice was performed using survival, body weight, open-field analysis, and neuropathology as outcome measures. Urinary levels of 8-hydroxy-2'-deoxyguanosine, identifying DNA oxidative damage, were measured and used as a pharmacodynamic biomarker. Uridine administration significantly extended survival in a dose-dependent manner in G93A mice, while improving the behavioral and neuropathological phenotype. Uridine increased survival by 17.4%, ameliorated body weight loss, enhanced motor performance, reduced gross lumbar and ventral horn atrophy, attenuated lumbar ventral horn neuronal cell death, and decreased reactive astrogliosis. Consistent with a therapeutic effect, uridine significantly reduced urinary 8-hydroxy-2'-deoxyguanosine in G93A mice. These data suggest that uridine may be a therapeutic candidate in ALS patients.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Amyotrophic Lateral Sclerosis; Animals; Anterior Horn Cells; Behavior, Animal; Body Weight; Deoxyguanosine; Disease Models, Animal; Dose-Response Relationship, Drug; Energy Metabolism; Humans; Kaplan-Meier Estimate; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neuroprotective Agents; Random Allocation; Rats; Rats, Sprague-Dawley; Spinal Cord; Superoxide Dismutase; Survival Rate; Uridine

We aimed to investigate oxidative stress biomarkers in a cross-sectional pilot study of 50 participants with sporadic ALS (SALS) compared to 46 control subjects. We measured urinary 8-oxodeoxyguanosine (8-oxodG), urinary 15-F(2t)-isoprostane (IsoP), and plasma protein carbonyl by ELISA methods. We also determined if ELISA measurement of 8-oxodG could be validated against measures from high-pressure liquid chromatography coupled with electrochemical detection, the current standard method. We found that 8-oxodG and IsoP levels adjusted for creatinine were significantly elevated in SALS participants. These differences persisted after age and gender were controlled in regression analyses. These markers are highly and positively correlated with each other. 8-oxodG measured by the two techniques from the same urine sample were positively correlated (p<.0001). Protein carbonyl was not different between SALS participants and controls. In conclusion, using ELISA, we confirmed that certain oxidative stress biomarkers were elevated in SALS participants. ELISA may be reliable and thus useful in epidemiology studies requiring large numbers of samples to determine the significance of increased oxidative stress markers in SALS. Further studies are required.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aged; Amyotrophic Lateral Sclerosis; Biomarkers; Blood Proteins; Cross-Sectional Studies; Deoxyguanosine; Dinoprost; Enzyme-Linked Immunosorbent Assay; Female; Humans; Male; Middle Aged; Oxidative Stress; Pilot Projects; Reactive Oxygen Species

To investigate the possibility that mitochondrial oxidative damage or oxidative DNA damage or both contribute to the neurodegenerative process of sporadic amyotrophic lateral sclerosis (sALS), this study used high-performance liquid chromatography with an electrochemical detector to measure the concentrations of the reduced and oxidized forms of coenzyme Q10 (CoQ10) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in the cerebrospinal fluid (CSF) of 17 patients with sALS and 17 age-matched controls with no neurological diseases. The percentage of oxidized CoQ10 in the CSF of sALS patients was greater than that in the CSF of controls (p<0.002) and was negatively correlated with the duration of illness (rho=-0.61, p<0.01). The concentration of 8-OHdG in the CSF of sALS patients was greater than that in the CSF of controls (p<0.005) and was positively correlated with the duration of illness (rho=0.53, p<0.005). The percentage of oxidized CoQ10 was correlated with the concentrations of 8-OHdG in the CSF of sALS patients (rho=-0.53, p<0.05). These results suggest that both mitochondrial oxidative damage and oxidative DNA damage play important roles in the pathogenesis of sporadic amyotrophic lateral sclerosis.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aged; Amyotrophic Lateral Sclerosis; Apoptosis; Case-Control Studies; Chromatography; Deoxyguanosine; DNA Damage; Humans; Middle Aged; Mitochondria; Neurodegenerative Diseases; Oxidation-Reduction; Oxidative Stress; Oxygen; Time Factors

Hydroxyl radical, ascorbate free radical, superoxide dismutase (SOD) activities, Cu,Zn-SOD protein, Mn-SOD protein, 8-hydroxy-2' -deoxyguanosine (8-OHdG) and metals were compared in red blood cells (RBC), plasma and/or cerebrospinal fluid (CSF) between patients with sporadic amyotrophic lateral sclerosis (SALS), familial ALS (FALS) showing the Leu126Ser mutation in the Cu, Zn-SOD gene and controls. In patients with FALS or SALS, concentrations of hydroxyl radical in blood and ascorbate free radical and 8-OHdG in CSF were higher than control group values, while SOD activities in RBC and CSF were lower. In contrast, Cu, Zn-SOD protein concentrations in RBC were low only in FALS patients. Concentrations of Cu in CSF of SALS patients were higher than in controls. Thus, the pathogenesis of increased oxidative stress differs between SALS patients and FALS patients with a mutant Leu126Ser SOD1 gene.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aged; Amyotrophic Lateral Sclerosis; Case-Control Studies; Deoxyguanosine; Erythrocytes; Female; Hematologic Tests; Humans; Leucine; Male; Metals; Middle Aged; Mutation; Oxidative Stress; Reference Values; Serine; Superoxide Dismutase

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.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Amyotrophic Lateral Sclerosis; Animals; Brain Chemistry; Chromatography, High Pressure Liquid; Deoxyguanosine; Disease Models, Animal; DNA Damage; Guanine; Humans; Male; Mice; Mice, Transgenic; Microdialysis; Oxidative Stress; Spinal Cord

To assess a role for oxidative stress in the pathogenesis of amyotrophic lateral sclerosis (ALS), we analyzed the immunohistochemical localization of 8-hydroxy2'-deoxyguanosine (OHdG) as a nucleic acid oxidation product, acrolein-protein adduct and 4-hydroxy-2-nonenal (HNE)-protein adduct as lipid peroxidation products, Nepsiloncarboxymethyl-lysine (CML) as a lipid peroxidation or protein glycoxidation product, pentosidine as a protein glycoxidation product, and imidazolone and pyrraline as nonoxidative protein glycation products in the spinal cord of three familial ALS patients with superoxide dismutase(SOD 1) A4V mutation, six sporadic ALS patients, and six age-matched control individuals. The spinal cord sections of the control cases did not show any distinct immunoreactivities for these examined products. In the familial ALS cases, intense immunoreactivities for pyrraline and CML were confined to the characteristic Lewy body-like hyaline inclusions, and imidazolone immunoreactivity was located in the cytoplasm of the residual motor neurons. No significant immunoreactivities for other examined products were detected in the familial ALS spinal cords. In the sporadic ALS cases, intense immunoreactivities for pentosidine, CML and HNE-protein adduct were seen in the cytoplasm of the degenerated motor neurons, and OHdG immunoreactivity was located in the cell nuclei of the residual neurons and glial cells. The present results indicate that oxidative reactions are involved in the disease processes of sporadic ALS, while there is no evidence for increased oxidative damage except for CML deposition in the familial ALS spinal cords. Furthermore, it is likely that the accumulation of pyrraline and imidazolone supports a nonoxidative mechanism in SOD1-related motor neuron degeneration.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Acrolein; Adult; Aged; Aldehydes; Amyotrophic Lateral Sclerosis; Arginine; Deoxyguanosine; Glycosylation; Humans; Imidazoles; Lipid Peroxidation; Lysine; Male; Middle Aged; Motor Neurons; Oxidative Stress; Spinal Cord; Superoxide Dismutase

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.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Amyotrophic Lateral Sclerosis; Animals; Biomarkers; Cerebral Palsy; Cerebrospinal Fluid; Chromatography, Liquid; Deoxyguanosine; DNA; DNA Damage; Electrochemistry; Humans; Oxidative Stress; Parkinson Disease; Reference Standards; Reproducibility of Results; Sensitivity and Specificity

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.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Aged; Aged, 80 and over; Amyotrophic Lateral Sclerosis; Autopsy; Biomarkers; Brain; Brain Diseases; Cerebellum; Deoxyguanosine; DNA Damage; Energy Metabolism; Female; Humans; Male; Malondialdehyde; Middle Aged; Motor Cortex; Oxidative Stress; Parietal Lobe; Reference Values; Superoxide Dismutase

Topics: 8-Hydroxy-2'-Deoxyguanosine; Amyotrophic Lateral Sclerosis; Deoxyguanosine; DNA; DNA Damage; Free Radicals; Humans; Neurons; Reference Values; Spinal Cord