Quantitative determination of urinary 8-hydroxy-2'-deoxyguanosine level in healthy Japanese volunteers

8-hydroxy-2'-deoxyguanosine (8-OHdG), as a measure of oxidative stress, was measured in healthy Japanese volunteers using an ELISA (New 8-OHdG Check, JICA). Analysis of daytime spot urine of 83 healthy male subjects and smoking habit, exercise and age revealed significant correlation only between the urinary level of 8-OHdG and age. As the inter-individual variation of 8-OHdG of the daytime spot urine was relatively high, we next determined inter-and intra-individual variation of 5 healthy volunteers. The levels of 8-OHdG/creatinine in morning spot urine significantly correlated with 8-OHdG levels in 24-h pool urine. Thus, a morning spot urine sample can be used for the measurement of 8-OHdG instead of inconvenient 24-h sampling.     

Longitudinal study of urinary 8-hydroxy-2'-deoxyguanosine excretion in healthy adults

Numerous studies have investigated the urinary excretion of 8-hydroxy-2'-deoxyguanosine (8-OHdG) as a biomarker for the assessment of oxidative DNA damage in humans. In this study, we performed six consecutive series of measurement of urinary levels of 8-OHdG in 68 healthy probands, in order to provide information on the intra- and inter-individual variability of 8-OHdG and to estimate the influence of smoking, age, sex, body weight and body mass index (BMI) on the excretion of 8-OHdG. The intraindividual coefficient of variation (CV) of urinary 8-OHdG/24h ranged from 0.18 to 1.06 (mean CV=0.48). Women excreted significantly lower amounts of 8-OHdG/24h than men, but the difference lost its significance when the body weight or urinary creatinine were used as covariates. By multiple linear regression analysis significant correlations between the mean individual levels of 8-OHdG/24h excretion and urinary creatinine (r_p = 0.61), and cotinine (r_p = 0.27) have been observed, whereas no statistically significant effect of age, body weight and BMI was found. The 8-OHdG/creatinine ratio was found to be significantly increased in 23 smokers (1.95 ± 0.40 μmol/mol) opposed to 45 non-smoking probands (1.62 ± 0.50 μmol/mol), which is in good agreement with previously published data. No effect of passive smoking on the excretion of 8-OHdG was found. From our data we conclude that the intraindividual variability of urinary 8-OHdG excretion has been underestimated so far, indicating that values of 8-OHdG measured by single spot monitoring are not representative for individual base levels.     

Oxidative stress in very low birth weight infants as measured by urinary 8-OHdG

Very low birth weight (VLBW) infants can be subjected to oxidative stress in the course of intensive care. We measured 8-hydroxydeoxyguanosine (8-OHdG), a biomarker of oxidative stress, and estimated the degree of oxidative stress in such infants. We also examined if the administered oxygen was related to oxidative stress. Urine samples of 50 Japanese VLBW infants [birth weights: 956.3+/-277.6g, and gestational ages: 28.0+/-2.6 weeks (mean +/- SD)] were collected on various postnatal days and 8-OHdG levels were determined using an ELISA kit. Sixteen term infants served as normal controls. As body weights at sampling increased, the average levels of urinary 8-OHdG decreased. 8-Hydroxydeoxyguanosine levels were: infants under 1000g, 29.5+/-16.4 micromol/mol creatinine (n = 24); 1000-1500g, 23.8+/-14.9 (n = 12); over 1500g, 16.1+/-8.5 (n = 14); and control, 10.9+/-7.2 (n = 16). Significant differences were found between <1000g group and > or = 1500g group (p = 0.0030), <1000g group and control (p < 0.0001), and 1000-1500g group and control (p = 0.0108). Also as postconceptional age at sampling increased, the average levels of 8-OHdG decreased. 8-Hydroxydeoxyguanosine levels were: infants before 252 days (36 weeks) of postconception: 27.4+/-15.5 micromol/mol creatinine (n = 34); after 252 days, 18.2+/-12.5 (n = 16). Differences between <252 days group and control (p < 0.0001), and <252 days group and > or = 252 days groups (p = 0.0253) were statistically significant. Among the three groups based on ambient oxygen concentration (21%, 22-29%, and > or = 30%) there was no significant difference (p = 0.417). The more premature the infants were, the more intense was the oxidative stress, hence, it is the prematurity rather than the administered oxygen which causes oxidative stress in VLBW infants. Drury et al. ["Urinary 8-hydroxydeoxyguanosine in infants and children" Free Radic. Res. 28 (1998) 423-4281 measured urinary 8-OHdG of 28 infants (24-40 weeks gestation) and found no gestation or birthweight related differences. This discrepancy seemed to be because of difference in birth weights and sampling period of the subjects.     

Quantification of urinary o,o'-dityrosine, a biomarker for oxidative damage to proteins, by high performance liquid chromatography with triple quadrupole tandem mass spectrometry. A comparison with ion-trap tandem mass spectrometry

We recently described an isotope dilution reversed-phase liquid chromatography-atmospheric pressure chemical ionization-ion-trap-tandem mass spectrometry (HPLC-APCI-MS/MS) method for the quantitative determination of oxidized amino acids in human urine, including o,o'-dityrosine, a specific marker of protein oxidation. In the present study, we investigated the possibility to use a triple quadrupole instrument for the analysis of this biomarker in urine. The two instruments were compared in terms of sensitivity, specificity and reproducibility. Results showed that the triple quadrupole instrument reaches 2.5-fold higher sensitivity (LOD=0.01 microM) compared to the previously used ion-trap instrument. Precision of the present assay is as follows: in-day variation is 4.6% and inter-day variation is 17%. The currently developed method was applied to a group of smoker urine samples. The mean urinary o,o'-dityrosine concentration was 0.08+/-0.01 microM. Expressed per urinary creatinine concentration, this corresponds to 10.1+/-0.4 micromol/mol creatinine. This is comparable to the previously reported values of 5.8+/-0.3 micromol/mol creatinine in non-smokers night-time urines, and 12.3+/-5 micromol/mol creatinine in day-time urines measured by the ion-trap instrument.     

Monitoring urinary excretion of 5-hydroxymethyluracil for assessment of oxidative DNA damage and repair

Urinary excretion of oxidized nucleobases and nucleosides has been used as a biomarker of oxidative DNA damage and repair. Most studies have focused on the measurements of 8-oxo-7,8-dihydro-2'-deoxyguanosine; however, the urinary levels of other DNA modifications may represent useful indicators of oxidative stress. We developed a method for the determination of 5-hydroxymethyluraciI (5-HMUra), consisting of the separation of the modified base in urine by HPLC and quantification by GC/MS in the selective ion monitoring mode. This experimental approach was subsequently validated in human samples, with the effect of storage and the inter- and intra-individual variations in 5-HMUra excretion being evaluated. Results showed that 5-HMUra is stable in samples frozen at-80 °C for at least 4 months. Inter-individual variations in 5-HMUra excretion were observed when the results were expressed either as nmoles excreted per kg per day (1.2-2.4) or corrected by creatinine values (7.2-12.2 nmoles 5-HMUra per mmoles creatinine). Intra-individual variability was low, varying slightly at different time collections for several individuals. Differences in the excretion of 5-HMUra in urine collected at three different 8-h intervals during the day were not significant and, in particular, the levels of 5-HMUra calculated from the overnight or the 24-h samples were highly correlated. These results indicate that monitoring urinary levels of 5-HMUra could be a suitable indicator of oxidative damage in human studies.     

Monitoring urinary excretion of 5-hydroxymethyluracil for assessment of oxidative DNA damage and repair

Abstract

Urinary excretion of oxidized nucleobases and nucleosides has been used as a biomarker of oxidative DNA damage and repair. Most studies have focused on the measurements of 8-oxo-7,8-dihydro-2′-deoxyguanosine; however, the urinary levels of other DNA modifications may represent useful indicators of oxidative stress. We developed a method for the determination of 5-hydroxymethyluraciI (5-HMUra), consisting of the separation of the modified base in urine by HPLC and quantification by GC/MS in the selective ion monitoring mode. This experimental approach was subsequently validated in human samples, with the effect of storage and the inter- and intra-individual variations in 5-HMUra excretion being evaluated. Results showed that 5-HMUra is stable in samples frozen at-80 °C for at least 4 months. Inter-individual variations in 5-HMUra excretion were observed when the results were expressed either as nmoles excreted per kg per day (1.2–2.4) or corrected by creatinine values (7.2–12.2 nmoles 5-HMUra per mmoles creatinine). Intra-individual variability was low, varying slightly at different time collections for several individuals. Differences in the excretion of 5-HMUra in urine collected at three different 8-h intervals during the day were not significant and, in particular, the levels of 5-HMUra calculated from the overnight or the 24-h samples were highly correlated. These results indicate that monitoring urinary levels of 5-HMUra could be a suitable indicator of oxidative damage in human studies.     

Urinary 8-Hydroxydeoxyguanosine in Infants and Children

Several diseases of prematurity are thought to be related to oxidative injury and many of the available markers are unsatisfactory. An assay was developed using HPLC with electrochemical detection for the quantitation of urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) as a proposed indicator for oxygen-derived free radical injury to DNA in preterm infants.

A median value of 3.79 pmol/mol creatinine was obtained for normal children (2–15 years old, n = 14). Urinary 8-OHdG excretion in neonates ranged from 0–99μmol/mol creatinine. There were no gestation or birthweight related differences in urinary 8-OHdG, and no correlation with urinary malondialdehyde. Mean 8-OHdG excretion increased with postnatal age (r= 0.80, p < 0.0001, n = 15), mirroring the growth velocity curve. These changes could also be due to changes in the activity of the enzyme responsible for 8-OHdG excision.

Urinary 8-OHdG levels are unlikely to accurately reflect oxygen derived free radical activity given the strength of the relationship with growth.     

Comparison of an oxidative stress biomarker "urinary8-hydroxy-2'-deoxyguanosine," between smokers and non-smokers

A great deal of effort has been made on the effect of oxidative stress for smokers. What seems to be lacking, however, is its evidence. Analyzing 1076 participants (age 35.9 +/- 12.9, urinary8-OHdG Mean +/- S.D., 11.4 +/- 6.7, n = 1076), our study found the significant increase in a biomarker of DNA damage urinary 8-OHdG/creatinine among smokers (7.75 +/- 2.8 ng/ml x CRE (n = 154) and 7.36 +/- 2.5 ng/ml x CRE (n = 627) (p < 0.05), Relative Risk = 2.9 (1.4-6.2) sex and age +/- 2 matching 105 male smokers and non-smokers. There was no significance on the comparison between female smokers and non-smokers. Smokers have significantly decreased serum alpha-tocopherol (1012 +/- 455, 1152 +/- 857, p < 0.03). The amount of serum ascorbate did not change. Smokers lowered serum HDL-cholesterol compared to non-smokers (59.3 +/- 11.8, 63.9 +/- 13.3, p < 0.05). The result of oxidative stress profile (OSP) also indicated that the increase of oxidative stress to smokers (p < 0.05). The calculated value of oxygen radical absorbance capacity (ORAC) of the meal for subjects was 1600 ORAC units.     

Simultaneous determination of tyrosine, phenylalanine and deoxyguanosine oxidation products by liquid chromatography-tandem mass spectrometry as non-invasive biomarkers for oxidative damage

We developed an isotope dilution HPLC-atmospheric pressure chemical ionization-tandem mass spectrometry (HPLC-APCI-MS/MS) method for the simultaneous determination of p-tyrosine, phenylalanine, o,o'-dityrosine, m-tyrosine, o-tyrosine, 3-chlorotyrosine and 3-nitrotyrosine and 8-hydroxy-2'-deoxyguanosine (8-OHdG) that requires no extensive sample pre-treatment. p-[(2)H(4)]Tyrosine and o,o'-[(2)H(6)]dityrosine were used as internal standards. Calibration curves of the method were linear (r(2)=0.990-0.999) over a concentration range of 0.03-10 microM for o-tyrosine; 0.04-10 microM for 3-nitrotyrosine and 3-chlorotyrosine; 0.05-10 microM for o,o'-dityrosine; and for m-tyrosine; 1.0-100 microM for p-tyrosine and for phenylalanine; and 0.01-10 microM for 8-OHdG. The detection limits were from 0.025 to 0.05 microM for the tyrosine derivatives; 0.01 microM for 8-OHdG; and 0.5 microM for p-tyrosine and for phenylalanine, respectively. Within-day coefficients of variation (CV) for spiked human urine samples ranged from 2.7 to 7.0%, except for 8-OHdG (13.7%). Between-day variations ranged from 7.9 to 13.0%, except for o-tyrosine (CV = 18.2%), and for 8-OHdG (CV = 24.7%).The background levels of p-tyrosine, phenylalanine, o,o'-dityrosine, and o-tyrosine in morning urine of eight healthy volunteers were 3890+/-590, 3420+/-730, 5.8+/-0.3, and 9.2+/-1.5 micromol/mol creatinine, respectively. Using the present HPLC-APCI-MS/MS method, the urinary background levels of m-tyrosine, 3-chlorotyrosine, 3-nitrotyrosine and 8-OHdG were below the limit of detection.     

Analysis of 8-hydroxy-2'-deoxyguanosine in urine using high-performance liquid chromatography-electrospray tandem mass spectrometry

8-hydroxy-2'-deoxyguanosine (8-OHdG) is a widely used biomarker of oxidative stress in research related to DNA, protein damage as well as lipid peroxidation. HPLC-MS/MS with electrospray ionization (ESI) and the use of isotopically labelled 8-OHdG as an internal standard allows a simple quantification of 8-OHdG in urine samples. HPLC separation utilized the peak cutting technique and a 1.5 mmx120 mm analytical anion exchange column. Novel method entails only minimal sample handling including the addition of a buffer and an internal standard followed by centrifugation before the samples are ready for analysis. The levels of 8-OHdG in human urine samples (n=246) varied from 0.16 to 16.48 microg/L and the corresponding creatinine-normalized values were ranged from 0.49 to 14.27 microg of 8-OHdG/g creatinine. The correlation between the developed HPLC-MS/MS method and the existing HPLC-EC method was good with an R2 value of 0.8707.     

Comparison of 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels using mass spectrometer and urine albumin creatinine ratio as a predictor of development of diabetic nephropathy

Abstract Background. Measurement of urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) has recently become more popular as a means of assessing oxidative stress in the human body. The aim of this study is to compare the levels of urine 8-OHdG in patients with type 2 diabetes with and without nephropathy and to evaluate its role as a biochemical marker for distinguishing these patients from healthy and patients without complications. Methods. For this purpose, 52 patients with type 2 diabetes mellitus (32 with nephropathy (DMN), 20 without nephropathy (DM)) and 20 healthy control subjects (C) were included in this study. The urine concentrations of 8-OHdG were measured by modified LC-MS/MS method and compared with the first morning voiding urine albumin/creatinine ratio (UACR) and HbA1c values of the same patients. Results. The concentrations of urine 8-OHdG in DMN and DM patients were higher than those of the control subjects (3.47?±?0.94, 2.92?±?1.73, 2.1?±?0.93 nmol/mol creatinine, respectively). But there was no statistical difference between DMN and DM (p =?0.115). There is significant correlation between urinary 8-OHdG and UACR (r =?0.501, p 相似文献   

Evaluation of a multi-parameter biomarker set for oxidative damage in man: increased urinary excretion of lipid, protein and DNA oxidation products after one hour of exercise

The objective of the present study was to evaluate a comprehensive set of urinary biomarkers for oxidative damage to lipids, proteins and DNA, in man. Eighteen moderately trained males (mean age 24.6+/-0.7) exercised 60min at 70% of maximal O2 uptake on a cycle ergometer. Urine fractions for 12 h were collected 1 day before, and for 3 consecutive days after exercise. As biomarkers of lipid peroxidation, 8 aldehydes (i.e. propanal, butanal, pentanal, hexanal, heptanal, octanal, nonanal and malondialdehyde-MDA)and acetone were analyzed in urines by gas chromatography with electron capture detection (GC-ECD). As a biomarker of protein oxidation, o,o'-dityrosine was analyzed in urine samples by a recently developed isotope dilution HPLC-atmospheric pressure chemical ionization (APCI)-tandem-mass spectrometry (HPLC-APCI-MS/MS) methodology. As a biomarker of oxidative DNA damage, urinary excretion of 8-hydroxy-2'-deoxyguanosine (8-OHdG) was measured by an ELISA method. On the day of exercise, significant increases were observed in urinary excretions of acetone (p < 0.025, n = 18) and butanal (p < 0.01, n = 18) in the 12h daytime fractions compared to the daytime fraction before exercise. The urinary acetone excretion was also significantly (p < 0.05) increased on the 1st day after exercise. Octanal and nonanal were increased in the daytime urine fraction on the 2nd day after exercise. However, these increases were of borderline significance (p = 0.09 and p = 0.07, respectively). Significantly elevated urinary o,o'-dityrosine amounts were observed in the daytime fraction on the day of exercise (p < 0.025) and on the 1st day after exercise (p = 0.07) compared to the before exercise daytime fraction. Excretion of urinary 8-OHdG was statistically significantly increased in the daytime fractions on the day of exercise (p = 0.07) and on the 1st day after exercise (p < 0.025) compared to before exercise daytime fraction. Increases in urinary excretions of acetone, propanal, pentanal, MDA and 8-OHdG significantly correlated with training status (hours of exercise/week) of the volunteers, while o,o'-dityrosine did not. To our knowledge, the present study is the first to evaluate a multi-parameter non-invasive biomarker set for damage to three main cellular targets of ROS. It shows that 1 h of exercise may already induce oxidative damage in moderately trained individuals and that the chosen urinary biomarkers are sensitive enough to monitor such damage.     

Analysis of 8-hydroxydeoxyguanosine among workers exposed to diesel particulate exhaust: comparison with urinary metabolites and PAH air monitoring

Oxidative DNA damage and repair, as measured by 8-hydroxy-2'-deoxyguanosine (8-OHdG) in urine and DNA samples were studied in association with work-related diesel exhaust exposure among garage and waste collection workers. Seasonal variations of the urinary 8-OHdG levels in pre- and two post-workshift urine samples of 29 exposed workers and 36 control persons were evaluated. The mean±SE levels of post-workshift 8-OHdG (μmol/mol crea) were 1.52±0.44 in winter and 1.61±0.33 in summer for the exposed workers, and 1.56±0.61 in winter and 1.43±0.49 in summer for the controls, respectively. No significant difference in the urinary 8-OHdG levels between exposed workers and control subjects in winter (p=0.923) and summer (p=0.350) was observed. A linear mixed model, adjusted for years of employment, age, ex/non-smoking and BMI, indicated no significant dose exposure-relationships between the urinary 8-OHdG and 15 PAH air concentrations nor between the 8-OHdG and 7 PAH monohydroxy-metabolites analyzed in the same workers. 8-OHdG was also analyzed in the mononuclear cell DNA of 19 exposed and 18 control subjects. The mean value of 8-OHdG/non-modified 2'-deoxyguanosine (8-OHdG/105 dG±SE) were 4.89±0.17 for the exposed and 4.11±0.16 for the control persons, which showed no correlation with the urinary 8-OHdG levels (r=0.01, n=28, P=0.96). The PAH exposure at workplaces was mainly composed of volatile compounds, particularly naphthalene, suggesting low exposure through the respiratory tract and a low effect of PAH in ROS induction.     

Association of hepatitis virus infection,alcohol consumption and plasma vitamin A levels with urinary 8-hydroxydeoxyguanosine in chemical workers

Urinary 8-hydroxydeoxyguanosine (8-OHdG) DNA adduct has been used as a biomarker in epidemiological studies. However, the determinants for urinary 8-OHdG have not been clearly identified. We tested urinary 8-OHdG levels in 205 male workers who had been exposed to vinyl chloride monomer (VCM). Epidemiological information was obtained by an interviewer-administered questionnaire. Hepatitis B surface antigen (HBsAg) and anti-hepatitis C antibody (anti-HCV) were also determined by immunoassay. Plasma antioxidants including Vitamins A and E, alpha- and beta-carotenes were assayed by high performance liquid chromatography. Median of urinary 8-OHdG level was 9.8 ng/mg creatinine (range, 1.4-60.1). Multiple linear regression analysis showed that alcohol drinkers had higher urinary 8-OHdG than those who did not, but there was no dose-response between the amount of alcohol consumption and urinary 8-OHdG. Workers with positive HBsAg, anti-HCV and elevated plasma Vitamin A level were independently associated with higher levels of urinary 8-OHdG, whereas age, smoking, body mass index, plasma alpha- and beta-carotenes, Vitamin E levels, or VCM exposure did not show such an association. The results suggest that active inflammation of hepatitis B and C, alcohol consumption and higher Vitamin A level can induce oxidative stress. Thus, we conclude that potential determinants need to be considered in epidemiological studies when urinary 8-OHdG is used as a biomarker.     

Comparison of 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels using mass spectrometer and urine albumin creatinine ratio as a predictor of development of diabetic nephropathy

Abstract

Background. Measurement of urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) has recently become more popular as a means of assessing oxidative stress in the human body. The aim of this study is to compare the levels of urine 8-OHdG in patients with type 2 diabetes with and without nephropathy and to evaluate its role as a biochemical marker for distinguishing these patients from healthy and patients without complications. Methods. For this purpose, 52 patients with type 2 diabetes mellitus (32 with nephropathy (DMN), 20 without nephropathy (DM)) and 20 healthy control subjects (C) were included in this study. The urine concentrations of 8-OHdG were measured by modified LC-MS/MS method and compared with the first morning voiding urine albumin/creatinine ratio (UACR) and HbA1c values of the same patients. Results. The concentrations of urine 8-OHdG in DMN and DM patients were higher than those of the control subjects (3.47?±?0.94, 2.92?±?1.73, 2.1?±?0.93 nmol/mol creatinine, respectively). But there was no statistical difference between DMN and DM (p =?0.115). There is significant correlation between urinary 8-OHdG and UACR (r =?0.501, p <?0.001). According to ROC analysis, the AUC value of HbA1c was higher than the value of the AUC of 8-OHdG (0.882 and 0.771, respectively). Conclusions. This study shows that the urine 8-OHdG levels increase in diabetic patients. However, urinary 8-OHdG is not a useful clinical marker, compared with UACR, to predict the development of diabetic nephropathy in diabetic patients.     

Oxidative stress early in pregnancy and pregnancy outcome

The objectives of this study were to determine whether oxidative stress early in pregnancy influenced pregnancy outcome. A combination of assays were used for exogenous and endogenous anti-oxidants together with two well accepted biomarkers for oxidative stress, the urinary excretion of 8-iso-PGF(2alpha) (a biomarker marker for lipid oxidation, n=508) and 8-oxo-7,8 dihydro-2 deoxyguanosine (8-OHdG, a biomarker for DNA oxidation, n=487). The two biomarkers tracked different pregnancy outcomes. Isoprostanes were associated with an increased risk of pre-eclampsia and a decreased proportion of female births. In contrast, 8-OHdG tracked lower infant birthweight and shortened gestation duration. Birth defects were associated with low levels of 8-OHdG.     

Accurate quantification of dimethylamine (DMA) in human urine by gas chromatography-mass spectrometry as pentafluorobenzamide derivative: evaluation of the relationship between DMA and its precursor asymmetric dimethylarginine (ADMA) in health and disease

Dimethylamine [DMA, (CH(3))(2)NH)] is abundantly present in human urine. Main sources of urinary DMA have been reported to include trimethylamine N-oxide, a common food component, and asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthesis. ADMA is excreted in the urine in part unmetabolized and in part after hydrolysis to DMA by dimethylarginine dimethylaminohydrolase (DDAH). Here we describe a GC-MS method for the accurate and rapid quantification of DMA in human urine. The method involves use of (CD(3))(2)NH as internal standard, simultaneous derivatization with pentafluorobenzoyl chloride and extraction in toluene, and selected-ion monitoring of m/z 239 for DMA and m/z 245 for (CD(3))(2)NH in the electron ionization mode. GC-MS analysis of urine samples from 10 healthy volunteers revealed a DMA concentration of 264+/-173 microM equivalent to 10.1+/-1.64 micromol/mmol creatinine. GC-tandem MS analysis of the same urine samples revealed an ADMA concentration of 27.3+/-15.3 microM corresponding to 1.35+/-1.2 micromol/mmol creatinine. In these volunteers, a positive correlation (R=0.83919, P=0.0024) was found between urinary DMA and ADMA, with the DMA/ADMA molar ratio being 10.8+/-6.2. Elevated excretion rates of DMA (52.9+/-18.5 micromol/mmol creatinine) and ADMA (3.85+/-1.65 micromol/mmol creatinine) were found by the method in 49 patients suffering from coronary artery disease, with the DMA/ADMA molar ratio also being elevated (16.8+/-12.8). In 12 patients suffering from end-stage liver disease, excretion rates of DMA (47.8+/-19.7 micromol/mmol creatinine) and ADMA (5.6+/-1.5 micromol/mmol creatinine) were found to be elevated, with the DMA/ADMA molar ratio (9.17+/-4.2) being insignificantly lower (P=0.46). Between urinary DMA and ADMA there was a positive correlation (R=0.6655, P<0.0001) in coronary artery disease, but no correlation (R=0.27339) was found in end-stage liver disease.     

Neopterin in renal cell carcinoma: inhalational administration of interleukin-2 is not accompanied by a rise of urinary neopterin.

Inhalational administration of interleukin-2 (IL-2) is effective in controlling renal cell carcinoma (RCC) lung metastases with minimal toxicity. Neopterin is an indicator of systemic immune activation in metastatic cancer and is increased after systemic IL-2 administration. Urinary neopterin was investigated in 13 patients with metastatic RCC and 18 controls. In seven patients, urinary neopterin was followed before and after treatment with inhalational IL-2. Neopterin was measured by high-performance liquid chromatography and creatinine was determined by Jaffé reaction. Urinary neopterin was significantly increased in patients with metastatic RCC compared to controls (257 +/- 263 micromol/mol creatinine vs. 110 +/- 41 micromol/mol creatinine; Mann-Whitney U-test, p < 0.05). Median survival was significantly longer in patients with urinary neopterin <173 micromol/mol creatinine compared to patients with neopterin > or = 173 micromol/mol creatinine (698 vs. 245 days; log-rank test, p < 0.05). No significant increase was observed after inhalational IL-2 therapy (147 +/- 101 vs. 153 +/- 54 micromol/mol creatinine). We conclude that urinary neopterin is increased in patients with metastatic RCC, and higher neopterin concentrations are indicative of poor prognosis. The absence of an increase in urinary neopterin after inhalational IL-2 therapy is in accord with the lack of significant systemic toxicity.     

Muscle soreness-induced reduction in force generation is accompanied by increased nitric oxide content and DNA damage in human skeletal muscle

We examined the effect of exercise-induced muscle soreness on maximal force generation, tissue nitric oxide (NO) and 8-hydroxydeoxyguanosine (8-OHdG) content in human skeletal muscle. Female volunteers were assigned to control (C) and muscle soreness (MS) groups (n = 6 in each). MS group performed 200 eccentric muscle actions of the rectus femoris to induce muscle soreness. Maximal force generation was measured 24 h before and after exercise in both groups. Needle biopsy samples were assayed for NO content with electron spin resonance spectroscopy after ex vivo spin trapping, and 8-OHdG content were measured with an enzyme-linked immuno assay. Maximal force decreased by 11+/-5.4% (p < .05) 24 h after exercise in MS group. Muscle soreness increased NO and 8-OHdG contents from their control values of 0.39+/-0.08 arbitrary units and 0.035+/-0.004 pmol/micromol DNA to 0.96+/-0.05 (p < .05) arbitrary units and 0.044+/-0.005 (p < .05) pmol/micromol DNA, respectively. This is the first demonstration that muscle soreness-induced decrease in maximal force generation is a result of an increase in muscular NO content and associated with enhanced formation of 8-OHdG in human skeletal muscle.     

Cigarette smokers have decreased lymphocyte and platelet alpha-tocopherol levels and increased excretion of the gamma-tocopherol metabolite gamma-carboxyethyl-hydroxychroman (gamma-CEHC)

Cigarette smoking is associated with increased oxidative stress and increased risk of degenerative disease. As the major lipophilic antioxidant, requirements for vitamin E may be higher in smokers due to increased utilisation. In this observational study we have compared vitamin E status in smokers and non-smokers using a holistic approach by measuring plasma, erythrocyte, lymphocyte and platelet alpha- and gamma-tocopherol, as well as the specific urinary vitamin E metabolites alpha- and gamma-carboxyethyl-hydroxychroman (CEHC). Fifteen smokers (average age 27 years, smoking time 7.5 years) and non-smokers of comparable age, gender and body mass index (BMI) were recruited. Subjects completed a 7-day food diary and on the final day they provided a 24 h urine collection and a 20 ml blood sample for measurement of urinary vitamin E metabolites and total vitamin E in blood components, respectively. No significant differences were found between plasma and erythrocyte alpha- and gamma-tocopherol in smokers and non-smokers. However, smokers had significantly lower alpha-tocopherol (mean+/-SD, 1.34+/-0.31 micromol/g protein compared with 1.94+/-0.54, P = 0.001) and gamma-tocopherol (0.19+/-0.04 micromol/g protein compared with 0.26+/-0.08, P = 0.026) levels in their lymphocytes, as well as significantly lower alpha-tocopherol levels in platelets (1.09+/-0.49 micromol/g protein compared with 1.60+/-0.55, P = 0.014; gamma-tocopherol levels were similar). Interestingly smokers also had significantly higher excretion of the urinary gamma-tocopherol metabolite, gamma-CEHC (0.49+/-0.25mg/g creatinine compared with 0.32+/-0.16, P = 0.036) compared to non-smokers, while their alpha-CEHC (metabolite of alpha-tocopherol) levels were similar. There was no significant difference between plasma ascorbate, urate and F2-isoprostane levels. Therefore in this population of cigarette smokers (mean age 27 years, mean smoking duration 7.5 years), alterations to vitamin E status can be observed even without the more characteristic changes to ascorbate and F2-isoprostanes. We suggest that the measurement of lymphocyte and platelet vitamin E may represent a valuable biomarker of vitamin E status in relation to oxidative stress conditions.