Oxidative Stress-Induced Damage to RNA and DNA and Mortality in Individuals with Psychiatric Illness.

Importance: All-cause mortality and the risk for age-related medical disease is increased in individuals with psychiatric illness, but the underlying biological mechanisms are not known. Oxidative stress on nucleic acids (DNA and RNA; NA-OXS) is a molecular driver of aging and a potential pathophysiological mechanism in a range of age-related disorders. Objective: To study the levels of markers of NA-OXS in a large cohort of community-dwelling individuals with and without psychiatric illness and to evaluate their association with prospective all-cause mortality. Design, Setting, and Participants: This cohort study used a combined cohort of participants from 2 population-based health studies: the Danish General Suburban Population Study (January 2010 to October 2013) and nondiabetic control participants from the Vejle Diabetes Biobank study (March 2007 to May 2010). Individual history of psychiatric illness was characterized using register data on psychiatric diagnoses and use of psychotropic drugs before baseline examination. Urinary markers of systemic RNA (8-oxo-7,8-dihydroguanosine [8-oxoGuo]) and DNA (8-oxo-7,8-dihydro-2'-deoxyguanosine [8-oxodG]) damage from oxidation were measured by ultraperformance liquid chromatography-tandem mass spectrometry. Cox proportional hazard regression models were applied for survival analyses, using register-based all-cause mortality updated to May 2023. The follow-up time was up to 16.0 years. Exposures: History of psychiatric illness. Main Outcomes and Measures: Mortality risk according to psychiatric illness status and 8-oxoGuo or 8-oxodG excretion level. Results: A total of 7728 individuals were included (3983 [51.5%] female; mean [SD] age, 58.6 [11.9] years), 3095 of whom (40.0%) had a history of psychiatric illness. Mean (SD) baseline 8-oxoGuo was statistically significantly higher in individuals with psychiatric illness than in those without (2.4 [1.2] nmol/mmol vs 2.2 [0.9] nmol/mmol; P < .001), whereas 8-oxodG was not. All-cause mortality was higher in the psychiatric illness group vs the no psychiatric illness group (hazard ratio [HR], 1.44; 95% CI, 1.27-1.64; P < .001) and increased sequentially with each increasing tertile of 8-oxoGuo excretion in both groups to an almost doubled risk in the psychiatric illness/high 8-oxoGuo group compared to the no psychiatric illness/low 8-oxoGuo reference group (HR, 1.99; 95% CI, 1.58-2.52; P < .001). These results persisted after adjustment for a range of potential confounders and in a sensitivity analysis stratified for sex. Conclusions and Relevance: This study establishes systemic oxidative stress-induced damage to RNA as a potential mechanism in the accelerated aging observed in psychiatric disorders and urinary 8-oxoGuo as a potentially useful marker of mortality risk in individuals with psychiatric illness.

Urinary 8-oxoGuo as a potential novel evaluation index for patients with nephrotic syndrome.

Urinary 8-oxo-7,8-dihydroguanosine (8-oxoGuo) and 8-oxo-7,8-dihydro-2'- deoxyguanosine (8-oxodGuo) are considered biomarkers of oxidative stress, and patients with nephrotic syndrome have been reported to have increased oxidative stress levels. In this study, we aimed to assess the value of 8-oxoGuo and 8-oxodGuo as novel biomarkers to evaluate the severity of nephrotic syndrome. In total, 107 patients with nephrotic syndrome and 116 healthy controls were recruited for this study. The concentrations of urinary 8-oxoGuo and 8-oxodGuo were measured using isotope-labeled liquid chromatography with tandem mass spectrometry. Urinary creatinine was used to regulate 8-oxoGuo and 8-oxodGuo concentrations. Urinary 8-oxoGuo and 8-oxoGuo/Cr levels in patients with nephrotic syndrome were significantly higher than those in healthy control participants. 8-oxoGuo/Cr showed a positive correlation with the 24 h urinary total protein (UTP) and UTP levels and negative correlations with serum total protein and albumin levels. After treatment, urinary 8-oxoGuo and 8-oxoGuo/Cr levels were significantly lower in the group with a low 24 h-UTP value (<3.5 g/d) than in the high value group. 8-oxoGuo can be used as a feasible and reliable biomarker for the assessment of nephrotic syndrome.HighlightsUrinary 8-oxoGuo level was significantly increased in patients with nephrotic syndrome.Urinary 8-oxoGuo level increased with an increase in plasma protein and a decrease in urine protein.Urinary 8-oxoGuo level decreased with nephrotic syndrome remission when urinary microalbumin showed no significant change.Urinary 8-oxoGuo level can be used as novel biomarkers of nephrotic syndrome.

An in silico kinetic model of 8-oxo-7,8-dihydro-2-deoxyguanosine and 8-oxo-7,8-dihydroguanosine metabolism from intracellular formation to urinary excretion.

Oxidatively generated DNA damage is of paramount importance in a wide range of physiological and pathophysiological processes. Urinary 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) is often used as an outcome marker in studies on the role of oxidatively generated DNA damage, but its exact relation to intracellular damage levels and variations in DNA repair have been unclear. Using a new approach of quantitative kinetic modeling inspired by pharmacokinetics, we find evidence that in steady state - i.e. when systemic consequences of given change in damage or cellular removal rates have stabilized - the urinary excretion of 8-oxodG is closely correlated to rates of damage and intracellular 8-oxodG levels, but independent of the rate of cellular removal. Steady state was calculated to occur within approximately 12 h. A similar pattern was observed in a model of the corresponding RNA marker 8-oxo-7,8-dihydroguanosine (8-oxoGuo), but with steady-state occurring slower (up to 5 d). These data have significant implications for the planning of studies and interpretation of data involving urinary 8-oxodG/8-oxoGuo excretion as outcome.HighlightsThe kinetics of 8-oxodG/8-oxoGuo formation, removal and excretion were simulated in silico.The model was based on existing data on 8-oxodG/8-oxoGuo levels and removal/excretion rates.Intracellular 8-oxodG/8-oxoGuo was closely correlated with urinary excretion in steady state.Changes in removal rates did not influence urinary excretion of 8-oxodG/8-oxoGuo.

Urinary 8-oxo-7,8-dihydroguanosine levels are elevated in HCV-infected patients.

HCV patients are usually under substantial oxidative stress because of viral infection. A total of 177 patients with HCV infection and 198 age- and sex-matched healthy controls were enrolled in this study. We evaluated the urinary levels of 8-oxo-7, 8-dihydro-2'deoxyguanosine (8-oxodGuo) and 8-oxo-7, 8-dihydroguanosine (8-oxoGuo) in patients with HCV infection and explored the factors affecting the urinary 8-oxodGuo or 8-oxoGuo levels. Biomarkers of liver function, cancer, and inflammation were determined. Nonparametric correlations were used to evaluate the correlation between 8-oxoGuo or 8-oxodGuo and various laboratory biochemical indicators. Results showed that the levels of urinary 8-oxoGuo both in male and female patients with HCV infection were significantly higher than those in healthy controls (both p < 0.0001), while the urinary 8-oxodGuo levels only in male patients with HCV infection were significantly higher than those in healthy controls (p < 0.01). Urinary 8-oxoGuo was significantly associated with the white blood cell count, C-reactive protein level, and 8-oxodGuo level (p = 0.016, p = 0.003, and p = 0.000, respectively). Urinary 8-oxodGuo was significantly associated with the white blood cell count and 8-oxoGuo level (p = 0.018 and p = 0.000, respectively). A regression equation of urinary 8-oxoGuo or 8-oxodGuo was also established using the biomarkers in plasma. The results suggested that patients with a high C-reactive protein level are likely to have high urinary 8-oxoGuo levels as well, which may be useful for assessing the level of inflammation and oxidative stress in HCV patients.Supplemental data for this article is available online at https://doi.org/10.1080/15257770.2021.1961272 .

The effect of liraglutide and sitagliptin on oxidative stress in persons with type 2 diabetes.

Glucagon-like peptide 1 receptor agonists have shown cardioprotective effects which have been suggested to be mediated through inhibition of oxidative stress. We investigated the effect of treatment with a glucagon-like peptide 1 receptor agonist (liraglutide) on oxidative stress measured as urinary nucleic acid oxidation in persons with type 2 diabetes. Post-hoc analysis of two independent, randomised, placebo-controlled and double-blinded clinical trials. In a cross-over study where persons with type 2 diabetes and microalbuminuria (LIRALBU, n = 32) received liraglutide (1.8 mg/day) or placebo for 12 weeks in random order, separated by 4 weeks of wash-out. In a parallel-grouped study where obese persons with type 2 diabetes (SAFEGUARD, n = 56) received liraglutide (1.8 mg/day), sitagliptin (100 mg/day) or placebo for 12 weeks. Endpoints were changes in the urinary markers of DNA oxidation (8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG)) and RNA oxidation [8-oxo-7,8-dihydroguanosine (8-oxoGuo)]. In LIRALBU, we observed no significant differences between treatment periods in urinary excretion of 8-oxodG [0.028 (standard error (SE): 0.17] nmol/mmol creatinine, p = 0.87) or of 8-oxoGuo [0.12 (0.12) nmol/mmol creatinine, p = 0.31]. In SAFEGUARD, excretion of 8-oxodG was not changed in the liraglutide group [2.8 (- 8.51; 15.49) %, p = 0.62] but a significant decline was demonstrated in the placebo group [12.6 (- 21.3; 3.1) %, p = 0.02], resulting in a relative increase in the liraglutide group compared to placebo (0.16 nmol/mmol creatinine, SE 0.07, p = 0.02). Treatment with sitagliptin compared to placebo demonstrated no significant difference (0.07 (0.07) nmol/mmol creatinine, p = 0.34). Nor were any significant differences for urinary excretion of 8-oxoGuo liraglutide vs placebo [0.09 (SE: 0.07) nmol/mmol creatinine, p = 0.19] or sitagliptin vs placebo [0.07 (SE: 0.07) nmol/mmol creatinine, p = 0.35] observed. This post-hoc analysis could not demonstrate a beneficial effect of 12 weeks of treatment with liraglutide or sitagliptin on oxidatively generated modifications of nucleic acid in persons with type 2 diabetes.

Treatment of Hyperthyroidism Reduces Systemic Oxidative Stress, as Measured by Markers of RNA and DNA Damage.

BACKGROUND: Whole-body oxidative stress can be estimated by the urine excretion of oxidized guanosine species, 8-oxo-7,8-dihydroguanosine (8-oxoGuo) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), derived from RNA and DNA, respectively. These oxidative stress markers are not well explored in thyroid disorders. OBJECTIVE: We aimed to determine whether treatment of hyperthyroid patients affects the levels of these oxidative stress markers. METHODS: Urinary excretion of 8-oxoGuo and 8-oxodG was measured in 51 hyperthyroid patients (toxic nodular goiter [TNG], n = 30; Graves disease [GD], n = 21) before or shortly after initiation of therapy and when stable euthyroidism had been achieved for at least 12 months. RESULTS: Adjusting for age, the baseline urinary excretion of oxidative stress markers correlated positively with plasma thyroxine (8-oxoGuo, P = 0.002; 8-oxodG, P = 0.021) and was significantly higher in GD than in TNG patients (P = 0.001 for both oxidative stress markers). Restoration of euthyroidism significantly affected the excretion of the oxidative stress markers. In TNG, 8-oxoGuo decreased from geometric mean 2.11 nmol/mmol creatinine (95% CI, 1.85-2.39) to 1.91 nmol/mmol (95% CI, 1.67-2.19; P = 0.001), while 8-oxodG decreased from 1.65 nmol/mmol (95% CI, 1.41-1.93) to 1.48 nmol/mmol (95% CI, 1.27-1.74; P = 0.026). In GD, 8-oxoGuo decreased from 2.25 nmol/mmol (95% CI, 1.95-2.59) to 1.79 nmol/mmol (95% CI, 1.63-1.97; P = 0.0003), while 8-oxodG decreased from 2.02 nmol/mmol (95% CI, 1.73-2.38) to 1.54 nmol/mmol (95% CI, 1.31-1.81; P = 0.001). In the euthyroid state, there were no differences between groups. CONCLUSION: Restoration of euthyroidism in patients with hyperthyroidism significantly decreased the systemic oxidative stress load by 10% to 25%. Our findings may help to explain the higher morbidity and mortality linked to hyperthyroid diseases, as shown in observational studies.

Short-term biological variation and Reference change values of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine and 8-oxo-7,8-dihydroguanosine.

AIM: The DNA and RNA oxidative damage products urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-odGsn) and 8-oxo-7,8-dihydroguanosine (8-oGsn) have potential use in clinical practice. However, biological variation (BV) and reference change values (RCVs) have not been established. The aim of this study was to establish the short-term between-subject BV(CVG ), within-subject BV(CVI ), and RCVs for urinary 8-odGsn and 8-oGsn. METHODS: First-morning midstream urine specimens were collected from 20 apparently healthy subjects(ten males and ten females) on five consecutive days. 8-odGsn and 8-oGsn were measured using LC-MS/MS, while urine creatinine (U-Cr) was also measured to correct their results. A two-level nested ANOVA was used to estimate the CVI and CVG. RESULTS: The values of CVG for 8-odGsn, 8-odGsn/U-Cr, 8-oGsn, and 8-oGsn/U-Cr were 31.2%, 39.6%, 35.3%, and 28.8%, respectively, while CVI for them were 40.5%, 9.0%, 33.5%, and 12.1%, respectively. The RCVs for 8-odGsn, 8-odGsn/U-Cr, 8-oGsn, and 8-oGsn/U-Cr were 112.5%, 26.7%, 93.7%, and 36.5%, respectively. CONCLUSION: BV and RCVs were firstly established for 8-oxo-dGsn and 8-oGsn, and can be used in clinical practice.

Effects of a highly controlled carbohydrate-reduced high-protein diet on markers of oxidatively generated nucleic acid modifications and inflammation in weight stable participants with type 2 diabetes; a randomized controlled trial.

Carbohydrate-restricted diets are increasingly recognized as options for dietary management of type 2 diabetes mellitus (T2DM). We investigated the effects of a carbohydrate-reduced high-protein (CRHP) and a conventional diabetes (CD) diet on oxidative stress and inflammation in weight stable individuals with T2DM. We hypothesized that the CRHP diet would improve markers of oxidatively generated RNA and DNA modifications as well as inflammatory parameters. Thirty participants with T2DM were randomized to 6 weeks of CRHP or CD dietary treatment (30/50 energy percentage (E%) carbohydrate, 30/17E% protein, 40/33E% fat), followed by a cross-over to the opposite diet for a subsequent 6-week period. All meals were provided during the study and body weight was controlled. Diurnal urine samples were collected after 4 weeks on each diet and oxidatively generated RNA and DNA modifications were measured as 8-oxo-7,8-dihydroguanosine (8-oxoGuo) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), respectively. Fasting concentrations of soluble urokinase plasminogen activator receptor, high-sensitivity C-reactive protein, tumor necrosis factor alpha and interleukin-6 were measured before and after 6 weeks of interventions. Compared with the CD diet, the CRHP diet increased 24-hour urinary excretion of 8-oxoGuo by 9.3% (38.6 ± 12.6 vs. 35.3 ± 11.0 nmol/24 h, p = .03), whereas 8-oxodG did not differ between diets (24.0 ± 9.5 vs. 24.8 ± 11.1 nmol/24 h, p = .17). Changes in plasma inflammatory parameters did not differ between CRHP and CD diets, all p ≥ .2. The clinical implications of increased RNA oxidation following a CRHP diet as well as long-term effects of carbohydrate-restriction on markers of oxidatively generated nucleic acid modifications should be a field of future study.

Targeted and untargeted metabolomics applied to occupational exposure to hyperbaric atmosphere.

Occupational exposure to hyperbaric atmosphere occurs in workers who carry out their activity in environments where breathing air pressure is at least 10% higher than pressure at sea level, and operations can be divided in Dry or Wet activities. The increased air pressure implies the formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), consumption of antioxidants and reduction of antioxidant enzyme activity, causing lipid peroxidation, DNA and RNA damage. The present study was aimed to establish the relation between hyperbaric exposure and metabolic changes due to ROS unbalance, by means of the determination of urinary biomarkers of oxidatively generated damage to DNA and RNA during a controlled diving session. The investigated biomarkers were 8-oxo-7,8-dihydroguanine (8-oxoGua), 8-oxo-7,8-dihydroguanosine (8-oxoGuo), and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo). The experimental session involved six experienced divers subjected to 3 atmospheres absolute for 30 minutes in two different experiments, in both dry and wet conditions. Urine samples were collected at t = 0 (before exposure) and 30 (end of exposure),90, 240, 480 and 720 minutes. The concentration of 8-oxoGua, 8-oxoGuo, and 8-oxodGuo was determined by isotopic dilution high performance liquid chromatography (HPLC-MS/MS). In all subjects there is an increase of the urinary excretion of 8oxo-Guo and 8oxo-dGuo, in both conditions, after 1.5 - 4 hours from the start of the experiment, and that the values tend to return to the baseline after 12 hours. Besides that, also the nucleic magnetic resonance (NMR)-based untargeted metabolomics was employed for the same objective on the same samples, confirming a different metabolic response in the subjects exposed to dry or wet conditions. In particular, the observed hypoxanthine urinary level increases during the underwater hyperbaric exposure, in agreement with the trend observed for 8-oxoGuo and 8-oxodGuo levels. Present results confirmed the relationship between exposure and oxidative stress and depicted a clear temporal trend of the investigated biomarkers. Due to the possible negative consequences of oxidative stress on workers, present research shows a new line in term of risk prevention.

A potential urinary biomarker to determine frailty status among older adults.

BACKGROUND: Association of oxidative stress biomarkers with aging and several age-related diseases is well documented. However, the possible role of these factors on frailty status in older adults has not been extensively studied. OBJECTIVE: To evaluate whether urinary 8-oxo-7,8-dihydroguanosine (8-oxo-Gsn), a biomarker of RNA oxidative damage, was independently associated with frailty. METHODS: In this cross-sectional analysis, frailty phenotype was assessed among 230 participants living in a senior community. Participants received a comprehensive geriatric assessment. Serum high-sensitivity C-reactive protein (hsCRP), white blood cell count (WBC), urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine, and 8-oxo-Gsn were measured. RESULTS: Participants' mean age was 83.9 ± 4.4 years. In total, 33 % were frail, 45 % were pre-frail, and 22 % were non-frail. Urinary 8-oxo-Gsn, serum hsCRP, and WBC were significantly higher in the frail group than in the non-frail and pre-frail groups (p-values < 0.05). Adjusting for age, sex, and Charlson comorbidity index, statistically significant positive associations with frailty were observed for urinary 8-oxo-Gsn (odds ratio [OR]: 1.70, 95 % confidence interval [CI]: 0.264-0.732) and hsCRP (OR: 1.337, 95 % CI: 0.089-0.412). Urinary 8-oxo-Gsn of 3.175 µmol/mol had the optimal predictive value for frailty, with an area under the receiver operating characteristic curve (AUC) of 0.72 (95 % CI: 0.649-0.788). The prediction probability combining urinary 8-oxo-Gsn and a simple question evaluating exhaustion had the optimal predictive value for frailty, with an AUC of 0.90 (p < 0.001, 95 % CI: 0.85-0.95). CONCLUSION: Urinary 8-oxo-Gsn level was independently associated with frailty. This urinary biomarker may be a promising indicator of frailty.

Development of a gas chromatography-mass spectrometry method for breast cancer diagnosis based on nucleoside metabolomes 1-methyl adenosine, 1-methylguanosine and 8-hydroxy-2'-deoxyguanosine.

Metabolomes are small molecule metabolites (<1000 Da) produced by cellular processes. Metabolomes are close counterparts to the genome, transcriptome and proteome. The aim of this study was to develop a method to detect and quantify candidate nucleoside metabolomes 1-methyl adenosine (1-MA), 1-methylguanosine (1-MG) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in the urine of patients with breast cancer using gas chromatography-mass spectrometry (GC-MS). The method was applied to urine specimens from patients with breast cancer (n = 56) and benign breast tumors (n = 22), as well as from healthy females (n = 20). The relative standard deviations of precision and repeatability analysis were <10%, and recoveries ranged from 88.5 to 105.6%. Limits of detection were 0.014, 0.012, and 0.018 mg/L for 1-MA, 1-MG and 8-OHdG, respectively. The lower limits of quantitation were 0.056, 0.048 and 0.072 mg/L, respectively. There were significant differences in concentrations of candidate metabolomes between patients with cancer and the healthy individuals, especially for those in the early stages of the disease (p < 0.001). No significant differences were observed between the benign and healthy groups. In conclusion, a reliable GC-MS method for the detection and quantification of 1-MA, 1-MG, and 8-OHdG metabolomes in urine has been developed.

8-Hydroxyguanosine as a possible RNA oxidative modification marker in urine from colorectal cancer patients: Evaluation by ultra performance liquid chromatography-tandem mass spectrometry.

Oxidative RNA damage has been found to be associated with a variety of diseases, and 8-hydroxyguanosine (8-OHG) is a typical marker of oxidative modification of RNA. This guanosine modification is an emerging biomarker for disease detection and determination of 8-OHG in human urine is favored because it is noninvasive to patients. However, due to its poor ionization efficiency in mass spectrometry and trace amount in urine, accurate quantification of this modified nucleoside is still challenging. Herein, a rapid, accurate, sensitive and robust method using solid-phase extraction (SPE) combined with isotope dilution ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was developed for detection of this oxidative RNA modification in human urine. The limit of detection can reach 1.5 fmol and the method exhibits good precision on intra-day (1.8-3.3%) and inter-day (0.6-1.2%) analyses. Satisfactory recovery (87.5-107.2%) at three spiked levels was achieved by using HLB cartridge for urine pretreatment. Using this method, we quantified 8-OHG in urine from 65 colorectal cancer (CRC) patients and 76 healthy volunteers. The measured level of urinary 8-OHG for CRC patients and healthy controls is 1.91 ± 0.63 nmol/mmol creatinine and 1.33 ± 0.35 nmol/mmol creatinine, respectively. We found the content of 8-OHG in urine was raised in CRC patients patients, implying this oxidative RNA modification marker could act as a potential noninvasive indicator for early screening of CRC. In addition, this study will make contributions to the investigations of the influences of oxidative stress on the formation and development of CRC.

Levels of Urinary Biomarkers of Oxidatively Generated Damage to DNA and RNA in Different Groups of Workers Compared to General Population.

(1) Background: The products of guanine oxidation in DNA and RNA excreted in urine are 8-oxo-7,8-dihydroguanine (8-oxoGua), 8-oxo-7,8-dihydroguanosine (8-oxoGuo), and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo). Despite intra and inter-individual variability, it is possible to identify situations that significantly increase the levels of these compounds when comparing urinary concentrations of some workers to those of the general population. (2) Methods: urines from gasoline pump attendants (58 from Saudi Arabia and 102 from Italy), 24 workers of a fiberglass reinforced plastics plant, 17 painters and 6 divers were analyzed by HPLC/MS-MS. To test the individual variability, two subjects provided daily samples for one month, and 132 urine samples from the general population were analyzed. (3) Results: We summarized the results for each biomarker, and found the following were statistically higher than in the general population: 8-oxoGua in fiberglass and Italian gasoline workers; 8-oxodGuo in fiberglass and both Saudi Arabian and Italian gasoline workers; 8-oxoGuo in fiberglass workers, both Saudi Arabian and Italian gasoline workers, and painters after the working shift. (4) Conclusions: these results confirm that both 8-oxodGuo and 8-oxoGuo are valuable biomarkers for occupational exposures to dangerous chemicals and seem to suggest that 8-oxoGuo, related to RNA oxidation, is a suitable biomarker to evaluate short term, reversible effects of occupational exposures even within the health-based limit values.

Classification and Temporal Variability in Urinary 8-oxodG and 8-oxoGuo: Analysis by UHPLC-MS/MS.

Oxidative stress damage has been found to be associated with exposure of children to environmental pollutants, but there are few data on the variability of urinary oxidative stress biomarkers and the accuracy of biomarker concentration classification. We performed a longitudinal study in Chinese school-aged children to investigate the variability of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo) concentrations and the ability of a single first morning urine sample to assess accuracy and sensitivity of biomarkers concentration classification. After adjusting for both creatinine and specific gravity, we characterized the distribution and reproducibility of repeated measurement of 8-oxodG and 8-oxoGuo by using intraclass correlation coefficients (ICCs) derived from linear mixed model and performed surrogate category analyses to determine whether a single spot sample could accurately classify 8-oxodG and 8-oxoGuo levels. Results indicated that the geometric mean (GM) concentrations of 8-oxodG and 8-oxoGuo were 3.865 ng/mL and 5.725 ng/mL, respectively. High variability of 8-oxodG and 8-oxoGuo was observed in the single spot first morning urine sample (ICC = 0.25 and 0.18, respectively). Three repeated urinary specimens achieved sensitivity of 0.87 for 8-oxodG and 0.83 for 8-oxoGuo in low tertile and sensitivity of 0.78 in high tertile. But classification in medium tertile was less accurate for both 8-oxodG and 8-oxoGuo. In conclusion, high variability of urinary 8-oxodG and 8-oxoGuo levels results in repeated samplings needed for accurate classification.

Prevention by L-carnitine of DNA damage induced by 3-hydroxy-3-methylglutaric and 3-methylglutaric acids and experimental evidence of lipid and DNA damage in patients with 3-hydroxy-3-methylglutaric aciduria.

3-hydroxy-3-methylglutaric aciduria (HMGA) is an inherited disorder of the leucine catabolic pathway in which occurs a deficiency of the 3-hydroxy-3-methylglutaryl-CoA lyase enzyme. Therefore, the organic acids 3-hydroxy-3-methylglutaric (HMG) and 3-methylglutaric (MGA), mainly, accumulate in tissues of affected patients. Lately, much attention has been focused on free radicals as mediators of tissue damage in human diseases, causing lipid peroxidation, protein oxidation and DNA damage. The treatment of this disease is based in a restricted protein ingest and supplementation with l-carnitine (LC), an antioxidant and detoxifying agent. In the present work, we investigated the in vitro oxidative damage to DNA induced by the accumulation of organic acids and oxidative stress parameters in vivo of patients with 3-HMG, as well as the effect of the recommended therapy. The in vitro DNA damage was analyzed by the alkaline comet assay in leukocytes incubated with HMG and MGA (1 mM, 2.5 mM and 5 mM) and co-incubated with LC (90 µM and 150 µM). The in vivo urinary 15-F2t-isoprostane levels and urinary oxidized guanine species were measured by ELISA kits in patient's urine before and after the treatment with LC. HMG and MGA induced a DNA damage index (DI) significantly higher than that of the control group. The DI was significantly reduced in the presence of LC. It was also verified a significant increase of oxidized guanine species and urinary isoprostane levels, biomarker of oxidative DNA damage and lipid peroxidation respectively, in patients before treatment. After the treatment and supplementation with LC, patients presented significantly lower levels of those biomarkers. Analyzing the data together, we can conclude that HMGA patients present oxidative lipid and DNA damage, which is induced by HMG and MGA, and the antioxidant therapy with LC can prevent that kind of injuries.

Identification and quantification of isoguanosine in humans and mice.

Isoguanine (2-hydroxyadenine), considered to be a non-natural nucleobase has, however, been shown to occur in the croton bean, butterfly wings and a mollusk. For the first time, to the best of our knowledge, we report the identification of isoguanosine (2-hydroxyadenosine), the ribonucleoside, in humans and mouse. Isoguanosine is identified and quantified in RNA from mouse liver samples and in human urine and cerebrospinal fluid. Isoguanine could not be detected as the 2'-deoxyribonucleoside in mouse liver DNA. It could be speculated that the source of isoguanosine was formation from adenosine during oxidative stress in the body. However, the urinary concentrations of isoguanosine and the levels in the liver found here by using isotope dilution liquid chromatography-tandem mass spectrometry are identical to or exceed those of 8-oxo-7,8-dihydro-2'-deoxyguanosine and 8-oxo-7,8-dihydro-guanosine. Guanine is the nucleobase that is oxidized the easiest, so it appears spectacular that the levels of isoguanosine are higher than the levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine and 8-oxo-7,8-dihydro-guanosine. It also appears intriguing that it was only possible to detect the ribonucleoside isoguanosine and not the 2'-deoxyribonucleoside. These observations could indicate that the isoguanosine found is not formed by oxidative stress and could have biological functions.

2',3'-cGMP exists in vivo and comprises a 2',3'-cGMP-guanosine pathway.

The discovery in 2009 that 2',3'-cAMP exists in biological systems was rapidly followed by identification of 2',3'-cGMP in cell and tissue extracts. To determine whether 2',3'-cGMP exists in mammals under physiological conditions, we used ultraperformance LC-MS/MS to measure 2',3'-cAMP and 2',3'-cGMP in timed urine collections (via direct bladder cannulation) from 25 anesthetized mice. Urinary excretion rates (means ± SE) of 2',3'-cAMP (15.5 ± 1.8 ng/30 min) and 2',3'-cGMP (17.9 ± 1.9 ng/30 min) were similar. Mice also excreted 2'-AMP (3.6 ± 1.1 ng/20 min) and 3'-AMP (9.5 ± 1.2 ng/min), hydrolysis products of 2',3'-cAMP, and 2'-GMP (4.7 ± 1.7 ng/30 min) and 3'-GMP (12.5 ± 1.8 ng/30 min), hydrolysis products of 2',3'-cGMP. To validate that the chromatographic signals were from these endogenous noncanonical nucleotides, we repeated these experiments in mice (n = 18) lacking 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), an enzyme known to convert 2',3'-cyclic nucleotides to their corresponding 2'-nucleotides. In CNPase-knockout mice, urinary excretions of 2',3'-cAMP, 3'-AMP, 2',3'-cGMP, and 3'-GMP were increased, while urinary excretions of 2'-AMP and 2'-GMP were decreased. Infusions of exogenous 2',3'-cAMP increased urinary excretion of 2',3'-cAMP, 2'-AMP, 3'-AMP, and adenosine, whereas infusions of exogenous 2',3'-cGMP increased excretion of 2',3'-cGMP, 2'-GMP, 3'-GMP, and guanosine. Together, these data suggest the endogenous existence of not only a 2',3'-cAMP-adenosine pathway (2',3'-cAMP → 2'-AMP/3'-AMP → adenosine), which was previously identified, but also a 2',3'-cGMP-guanosine pathway (2',3'-cGMP → 2'-GMP/3'-GMP → guanosine), observed here for the first time. Because it is well known that adenosine and guanosine protect tissues from injury, our data support the concept that both pathways may work together to protect tissues from injury.

Reduced levels of modified nucleosides in the urine of autistic children. Preliminary studies.

The aim of this study was to investigate and compare the levels of concentration of modified nucleosides in the urine of autistic and healthy children. The compounds have never been analyzed before. The levels of nucleosides in the urine of both groups were determined by validated high performance liquid chromatography coupled to mass spectrometry (LC-MS/MS) method using multiple reaction monitoring (MRM) mode. Chromatographic separation was achieved with HILIC column and tubercidin was used as the internal standard for the quantification of urinary nucleosides. The within run accuracy and precision ranged from 89 to 106% and from 0.8% to 4.9%, respectively. Lower levels of O-methylguanosine, 7-methylguanosine, 1-methyladenosine, 1-methylguanine, 7-methylguanine and 3-methyladenine in the urine of 22 children with autism, aged 3 to 16 were observed. The differences were not observed in 20 healthy volunteers, in a similar age group. These findings show that modified nucleosides there are metabolic disturbances and nutritional deficiencies in autistic children.

The effect of structured personal care on RNA oxidation: A 19-year follow-up of the randomized trial Diabetes Care in General Practice (DCGP).

AIMS: The urinary marker of RNA oxidation, 8­oxo­7,8­dihydroguanosine (8-oxoGuo), but not the corresponding marker of DNA oxidation, 8­oxo­7,8­dihydro­2'­deoxyguanosine (8-oxodG), is a prognostic biomarker in patients with type 2 diabetes (T2D). The aim of the present study was to investigate the effect of structured personal care (individualized multifactorial treatment) versus standard care on RNA oxidation level in patients with T2D and to assess if the effect of structured personal care on all-cause and diabetes-related mortality was modified by RNA oxidation level. METHODS: Urine samples were analyzed for 8-oxoGuo/8-oxodG from 1381 newly diagnosed T2D patients from the cluster randomized trial Diabetes Care in General Practice cohort, and 970 patients were reexamined after six years of intervention. RESULTS: The yearly variation in RNA oxidation levels were not significantly different between the structured personal care group and standard care group. The effect of treatment on all-cause and diabetes-related mortality was not modified by the level of RNA oxidation. No changes in DNA oxidation were seen. CONCLUSIONS: Structured personal care does not influence RNA oxidation level nor is it better for patients with high RNA oxidation level. Thus, structured personal care may not impact the disease-related aspects identified by RNA oxidation level in T2D patients.