Quantitative metabolic profiling of urinary eicosanoids for clinical phenotyping.

The eicosanoids are a family of lipid mediators of pain and inflammation involved in multiple pathologies, including asthma, hypertension, cancer, atherosclerosis, and neurodegenerative diseases. These signaling mediators act locally, but are rapidly metabolized and transported to the systemic circulation as a mixture of primary and secondary metabolites. Accordingly, urine has become a useful readily accessible biofluid for monitoring the endogenous synthesis of these molecules. Herein, we present the validation of a rapid, repeatable, and precise method for the extraction and quantification of 32 eicosanoid urinary metabolites by LC-MS/MS. For 12 out of 17 deconjugated glucuronide eicosanoids, there was no improvement in recovered signal. These metabolites cover the major synthetic pathways, including prostaglandins, leukotrienes, and isoprostanes. The method linearity was >0.99 for all metabolites analyzed, the limit of detection ranged from 0.05-5 ng/ml, and the average extraction recoveries were >90%. All analytes were stable for at least three freeze/thaw cycles. The method was formatted for large-scale analysis of clinical cohorts, and the long-term repeatability was demonstrated over 15 months of acquisition, evidencing high precision (CV <15%, except for tetranorPGEM and 2,3-dinor-11ß-PGF2α, which were <30%). The presented method is suitable for focused mechanistic studies as well as large-scale clinical and epidemiological studies that require repeatable methods capable of producing data that can be concatenated across multiple cohorts.

Wood dust and urinary 15-F2t isoprostane in Italian industry workers.

Wood dust is one of the most common occupational exposures, with about 3.6 million of workers in the wood industry in Europe. Wood particles can deposit in the nose and the respiratory tract and cause adverse health effects. Occupational exposure to wood dust has been associated with malignant tumors of the nasal cavity and paranasal sinuses. The induction of oxidative stress and the generation of reactive oxygen species through activation of inflammatory cells could have a role in the carcinogenicity of respirable wood dust. Therefore, we conducted a cross-sectional study to evaluate the prevalence of urinary 15-F2t isoprostane (15-F2t-IsoP), a biomarker of oxidative stress and peroxidation of lipids, in 123 wood workers compared to 57 unexposed controls living in Tuscany region, Italy. 15-F2t-IsoP generation was measured by ELISA. The main result of the present study showed that a statistically significant excess of this biomarker occurred in the workers exposed to 1.48 mg/m3 of airborne wood dust with respect to the unexposed controls. The overall mean ratio (MR) between the workers exposed to wood dust and the controls was 1.36, 95% Confidence Interval (C.I.) 1.18-1.57, after correction for age and smoking habits. A significant increment of 15-F2t-IsoP (43%) was observed in the smokers as compared to the non-smokers. The urinary excretion of 15-F2t-IsoP was significantly associated with co-exposure to organic solvents, i.e., MR of 1.41, 95% C.I. 1.17-1.70, after adjustment for age and smoking habits. A 41% excess was observed in long-term wood workers, 95% C.I. 1.14-1.75. Multivariate regression analysis showed that the level of 15-F2t-IsoP was linearly correlated to the length of exposure, regression coefficient (ß) = 0.244 ±â€¯0.002 (SE). The overall increment by exposure group persisted after stratification for smoking habits. For instance, in smokers, a 53% excess was detected in the wood workers as compared to the controls, 95% C.I. 1.23-1.91. Our data support the hypothesis that oxidative stress and lipid peroxidation can have a role in the toxicity of wood dust F2-IsoP measure can be a tool for the evaluation of the effectiveness of targeted interventions aimed to reduce exposures to environmental carcinogens.

Relationship Between Alternative Resuscitation Strategies, Host Response and Injury Biomarkers, and Outcome in Septic Shock: Analysis of the Protocol-Based Care for Early Septic Shock Study.

OBJECTIVES: The Protocol-based Care for Early Septic Shock trial found no differences across alternative resuscitation strategies in all-cause mortality. A separate aim was to determine whether differences in resuscitation strategies affected trajectories of biomarkers of key pathways associated with downstream clinical outcomes of sepsis and whether there were differences in survival across treatment arms for patients with different baseline biomarker profiles. DESIGN: Secondary analysis of a large randomized clinical trial. SETTING: Thirty-one U.S. hospitals. PATIENTS: Six hundred twenty-eight patients with septic shock. INTERVENTIONS: Two resuscitation protocols versus usual care. MEASUREMENTS AND MAIN RESULTS: We measured a panel of biomarkers representing four pathophysiologic domains: "inflammation" (tumor necrosis factor, interleukin-6, and -10); "coagulation" (D-dimers, thrombin-antithrombin complex); "oxidative stress" (urine isoprostane); and "tissue hypoxia" (lactate) at 0, 6, 24, and 72 hours after treatment. We analyzed whether alternative resuscitation strategies affected biomarker trajectories over 72 hours and whether effects on 90-day hospital mortality varied by baseline (time 0) biomarker profiles-both using regression models with interaction terms for treatment arms. For all baseline biomarkers, higher concentrations were associated with increased risk of death by 90 days. However, there was no significant effect of treatment assignment on subsequent biomarker trajectories. We did find evidence for heterogeneity of treatment effect of protocol-based care on mortality for patients with different baseline [interleukin-6] and [interleukin-6] × [interleukin-10] profiles, whereas patients with the lowest quartiles fared better with protocol-based care (odds ratios, 0.32 [0.13-075]; p = 0.01 and 0.32 [0.14-0.73]; p = 0.01, respectively). CONCLUSIONS: In patients with septic shock, alterations in inflammation, coagulation, oxidative stress, and tissue hypoxia are common and associated with adverse outcomes but are not influenced by protocol-based resuscitation compared with usual care. However, contrary to expectation, protocol-based resuscitation appeared to be superior in patients with lower concentrations of inflammatory biomarkers. The mechanisms responsible for this effect are unclear.

Oxidative Stress and Breast Cancer Risk in Premenopausal Women.

BACKGROUND: Detrimental effects of oxidative stress are widely recognized, but induction of apoptosis and senescence may also have benefits for cancer prevention. Recent studies suggest oxidative stress may be associated with lower breast cancer risk before menopause. METHODS: We conducted a nested case-control study (N = 457 cases, 910 controls) within the NIEHS Sister Study cohort of 50,884 women. Premenopausal women ages 35-54 were eligible for selection. We matched controls 2:1 to cases on age and enrollment year and were breast cancer-free at the time of the corresponding case's diagnosis. Oxidative stress was measured by urinary F2-isoprostane and metabolite (15-F2t-isoprostane-M) concentrations. Odds ratios (OR) and 95% confidence intervals (CIs) were calculated with multivariable conditional logistic regression. RESULTS: After multivariable adjustment for body mass index (BMI) and other potential confounders, the OR for breast cancer comparing the >90th (≥2.94 ng/mgCr) to <25th percentile (1.01 ng/mgCr) was 1.1 (CI: 0.65, 1.7) for F2-isoprostane and 0.70 (CI: 0.43, 1.1) for the metabolite. Higher metabolite concentrations were associated with lower breast cancer risk among women who were also premenopausal (353 cases, OR: 0.59, CI: 0.34, 1.0) or <46 years (82 cases, OR: 0.15, CI: 0.06, 0.42) at diagnosis. ORs for the metabolite and breast cancer were inverse among women with BMI 18.5-24.9 kg/m (OR: 0.47, CI: 0.18, 1.2, 208 cases) and >30 kg/m (OR: 0.71, CI: 0.30, 1.7, 107 cases), but not among women with BMI 25-29.9 kg/m (OR: 0.98, CI: 0.39, 2.5, 138 cases). CONCLUSIONS: Together with other studies, our results support a possible inverse association between oxidative stress and premenopausal breast cancer risk.

Urinary phthalate and phthalate alternative metabolites and isoprostane among couples undergoing fertility treatment.

BACKGROUND: Epidemiological data suggest associations between phthalate exposures to a variety of adverse reproductive outcomes including reduced sperm quality and reproductive success. While mechanisms of these associations are not fully elucidated, oxidative stress has been implicated as a potential mediator. We examined associations of urinary metabolites of phthalates and phthalate alternative plasticizers with oxidative stress among couples seeking fertility treatment. METHODS: Seventeen urinary plasticizer metabolites and 15-F2t isoprostane, a biomarker of oxidative stress, were quantified in spot samples from 50 couples seeking fertility treatment who enrolled in the Sperm Environmental Epigenetics and Development Study during 2014-2015. RESULTS: In multivariable analyses, percent change in isoprostane was positively associated with interquartile range increases for the oxidative metabolites of di-2-ethylhexyl phthalate, [mono-2-ethyl-5-hydroxyhexyl phthalate (MEHHP; 20.0%, p=0.02), mono-2-ethyl-5-oxohexyl phthalate (MEOHP; 24.1%, p=0.01), and mono-2-ethyl-5-carboxypentyl phthalate (MECPP; 24.1%, p=0.004)], mono-isobutyl phthalate (MiBP; 17.8%, p=0.02), mono-hydroxyisobutyl phthalate (MHiBP; 27.5%, p=0.003), and cyclohexane-1,2-dicarboxylic acid mono-hydroxy-isononyl ester (MHINCH; 32.3%, p=0.002). Stratification of participants by sex revealed that isoprostane was positively associated with MHiBP (41.4%, p=0.01) and monocarboxy-isononyl phthalate (MCNP; 26.0%, p=0.02) among females and MEOHP (35.8%, p=0.03), MiBP (29.2%, p=0.01), MHiBP (34.7%, p=0.007) and MHINCH (49.0%, p=0.002) among males. CONCLUSIONS: Our results suggest that exposure to phthalates and phthalate replacements are associated with higher levels of oxidative stress in a sex-specific manner. Additional studies are needed to replicate our findings and to examine the potential health implications of the use of phthalates and alternative phthalates in consumer end products.

Fermented goat milk improves antioxidant status and protects from oxidative damage to biomolecules during anemia recovery.

BACKGROUND: Iron deficiency anemia (IDA) is one of the most common nutritional problems in the world, and it is accepted that reactive oxygen species (ROS) production is altered during IDA. The aim of this study was to assess the influence of fermented goat and cow milks on enzymatic antioxidant activities and gene expression, and their role in protecting from oxidative damage during anemia recovery. RESULTS: After feeding the fermented milks-based diets (cow or goat), a significant elevation of some antioxidant endogenous enzymes was found, together with an increase in total antioxidant status (TAS), and a decrease in 8-hydroxy-2'-deoxyguanosine (8-OHdG) was recorded in animals consuming fermented goat milk-based diet. In contrast, DNA strand breaks, hydroperoxides, 15-F2t-isoprostanes and protein carbonyl groups were lower in some tissues in animals fed fermented goat milk-based diet, revealing an improvement in both systemic and cellular antioxidant activity of plasma and tissues due to fermented goat milk consumption. CONCLUSION: Fermented goat milk consumption induces a protective increase in TAS together with lower oxidative damage biomarkers, revealing that the milk protects main cell bioconstituents (lipids, protein, DNA, prostaglandins) from evoked oxidative damage during anemia recovery. © 2016 Society of Chemical Industry.

Oxidative stress and inflammation in mucopolysaccharidosis type IVA patients treated with enzyme replacement therapy.

Mucopolysaccharidosis type IVA (MPS IVA) is an inborn error of glycosaminoglycan (GAG) catabolism due to the deficient activity of N-acetylgalactosamine-6-sulfate sulfatase that leads to accumulation of the keratan sulfate and chondroitin 6-sulfate in body fluids and in lysosomes. The pathophysiology of this lysosomal storage disorder is not completely understood. The aim of this study was to investigate oxidative stress parameters, pro-inflammatory cytokine and GAG levels in MPS IVA patients. We analyzed urine and blood samples from patients under ERT (n=17) and healthy age-matched controls (n=10-15). Patients presented a reduction of antioxidant defense levels, assessed by a decrease in glutathione content and by an increase in superoxide dismutase activity in erythrocytes. Concerning lipid and protein damage, it was verified increased urine isoprostanes and di-tyrosine levels and decreased plasma sulfhydryl groups in MPS IVA patients compared to controls. MPS IVA patients showed higher DNA damage than control group and this damage had an oxidative origin in both pyrimidine and purine bases. Interleukin 6 was increased in patients and presented an inverse correlation with GSH levels, showing a possible link between inflammation and oxidative stress in MPS IVA disease. The data presented suggest that pro-inflammatory and pro-oxidant states occur in MPS IVA patients even under ERT. Taking these results into account, supplementation of antioxidants in combination with ERT can be a tentative therapeutic approach with the purpose of improving the patient's quality of life. To the best of our knowledge, this is the first study relating MPS IVA patients with oxidative stress.

Increased intake of fruits and vegetables in overweight subjects: effects on body weight, body composition, metabolic risk factors and dietary intake.

A diet rich in fruits and vegetables has been associated with several health benefits. However, the effects on body weight (BW) and metabolic markers are not fully known. The present study investigated the effects of increased intake of fruits and vegetables in overweight and obese men and women on dietary habits, anthropometry and metabolic control. In a 16-week controlled intervention, thirty-four men and thirty-four women aged 35-65 years (BMI>27 kg/m2) were randomised to an intervention (IN) or a reference (RG) group. All participants received general dietary advice, and subjects in the IN group received fruits and vegetables for free, of which ≥500 g had to be eaten daily. BW, waist circumference (WC), sagittal abdominal diameter (SAD), plasma insulin, blood glucose, glycated Hb (HbA1c), serum lipids, blood pressure, plasminogen activator inhibitor-1 activity, urinary isoprostane (iso-8-PGF 2α) and serum carotenoids were measured. Diet was assessed using 3-d weighed food records. In all, thirty subjects in the IN group and thirty-two in the RG group completed the intervention. Intake of fruits and vegetables doubled in the IN group, whereas intake of fruits increased in the RG group. Serum α- and ß-carotene concentrations and intakes of folate and vitamin C increased significantly in the IN group. Energy intake, BW, WC and SAD decreased significantly in both groups. Supine systolic blood pressure decreased significantly in the IN group, with no between-group differences. No significant changes were observed for other metabolic markers. Provision of fruits and vegetables led to substantially increased intakes, with subsequent favourable changes in anthropometry and insulin levels, which tended to be more pronounced in the IN group. The observed improvements may, in combination with improved nutritional markers, have health benefits in the long term.

Oxidative stress in relation to diet and physical activity among premenopausal women.

Higher levels of oxidative stress, as measured by F2-isoprostanes, have been associated with chronic diseases such as CVD and some cancers. Improvements in diet and physical activity may help reduce oxidative stress; however, previous studies regarding associations between lifestyle factors and F2-isoprostane concentrations have been inconsistent. The aim of this cross-sectional study was to investigate whether physical activity and intakes of fruits/vegetables, antioxidant nutrients, dietary fat subgroups and alcohol are associated with concentrations of F2-isoprostane and the major F2-isoprostane metabolite. Urinary F2-isoprostane and its metabolite were measured in urine samples collected at enrolment from 912 premenopausal women (aged 35-54 years) participating in the Sister Study. Physical activity, alcohol consumption and dietary intakes were self-reported via questionnaires. With adjustment for potential confounders, the geometric means of F2-isoprostane and its metabolite were calculated according to quartiles of dietary intakes, alcohol consumption and physical activity, and linear regression models were used to evaluate trends. Significant inverse associations were found between F2-isoprostane and/or its metabolite and physical activity, vegetables, fruits, vitamin C, α-carotene, vitamin E, ß-carotene, vitamin A, Se, lutein+zeaxanthin and long-chain n-3 fatty acids. Although trans fats were positively associated with both F2-isoprostane and its metabolite, other dietary fat subgroups including SFA, n-6 fatty acids, n-3 fatty acids, MUFA, PUFA, short-chain n-3 fatty acids, long-chain n-3 fatty acids and total fat were not associated with either F2-isoprostane or its metabolite. Our findings suggest that lower intake of antioxidant nutrients and higher intake of trans fats may be associated with greater oxidative stress among premenopausal women.

Retinoid and carotenoid status in serum and liver among patients at high-risk for liver cancer.

BACKGROUND: Approximately 2.7 million Americans are chronically infected with hepatitis C virus (HCV). HCV patients with cirrhosis form the largest group of persons at high risk for hepatocellular carcinoma (HCC). Increased oxidative stress is regarded as a major mechanism of HCV-related liver disease progression. Deficiencies in retinoid and carotenoid antioxidants may represent a major modifiable risk factor for disease progression. This study aims to identify key predictors of serum antioxidant levels in patients with HCV, to examine the relationship between retinoid/carotenoid concentrations in serum and hepatic tissue, to quantify the association between systemic measures of oxidative stress and antioxidant status, and to examine the relationship between retinoids and stellate cell activation. METHODS: Patients undergoing liver biopsy (n = 69) provided fasting blood, fresh tissue, urine and completed a diet history questionnaire. Serum and questionnaire data from healthy volunteers (n = 11), normal liver tissue from public repositories and patients without liver disease (n = 11) were also collected. Urinary isoprostanes, serum and tissue retinoid concentrations were obtained by UHPLC-MS-MS. Immunohistochemistry for αSMA was performed on FFPE sections and subsequently quantified via digital image analysis. Associations between urinary isoprostanes, αSMA levels, and retinoids were assessed using Spearman correlation coefficients and non-parametric tests were utilized to test differences among disease severity groups. RESULTS: There was a significant inverse association between serum retinol, lycopene, and RBP4 concentrations with fibrosis stage. Serum ß-carotene and lycopene were strongly associated with their respective tissue concentrations. There was a weak downward trend of tissue retinyl palmitate with increasing fibrosis stage. Tissue retinyl palmitate was inversely and significantly correlated with hepatic αSMA expression, a marker for hepatic stellate cell activation (r = -0.31, P < 0.02). Urinary isoprostanes levels were inversely correlated with serum retinol, ß-carotene, and RBP4. CONCLUSIONS: A decrease in serum retinol, ß-carotene, and RBP4 is associated with early stage HCV. Retinoid and carotenoid levels decline as disease progresses, and our data suggest that this decline occurs early in the disease process, even before fibrosis is apparent. Measures of oxidative stress are associated with fibrosis stage and concurrent antioxidant depletion. Vitamin A loss is accompanied by stellate cell activation in hepatic tissue.

Effects of an antioxidant beverage on biomarkers of oxidative stress in Alzheimer's patients.

PURPOSE: The purpose of the study was to test whether daily consumption of a beverage with high antioxidant power, combining extracts of green tea and apple over a period of 8 months, would affect blood and urinary concentrations of biomarkers of oxidative stress in Alzheimer's patients. METHODS: The study included 100 subjects, 48 of them were Alzheimer's patients, aged 76.5 ± 3.5 years, and 52 were control subjects, aged 79 ± 4 years, without dementia. Three blood and urine samples were taken from each participant, the first (T i) before starting the antioxidant or placebo beverage intake, the second (T m) 4 months after the antioxidant or placebo beverage intake and the third (T f) 8 months after the antioxidant or placebo beverage intake, and concentrations of biomarkers of oxidative stress were measured on serum, lysed erythrocytes or urine by UV-Vis spectrophotometry or by competitive in vitro enzyme-linked immunosorbent assay, according to the parameter analyzed. RESULTS: The administration of the antioxidant beverage to the Alzheimer's patients prevented the decrease in total antioxidant status in the moderate phase of the disease (T i = 1.40 ± 0.10 mmol/L vs T f = 1.20 ± 0.08 mmol/L), increased values of glutathione peroxidase and superoxide dismutase in initial (165 and 24 % respectively) and moderate phase (75 and 85 % respectively), and prevented the increase in protein carbonyls in moderate phase (T i = 0.17 ± 0.07 nmol/mg protein vs T f = 0.21 ± 0.06 nmol/mg protein), with a significant decrease in protein carbonyls since the fourth month of the intake in initial phase (T m = 0.21 ± 0.06 nmol/mg protein vs T f = 0.11 ± 0.05 nmol/mg protein). CONCLUSION: Our results suggest that antioxidant beverage could be used as a natural complementary therapy for alleviate or decrease the oxidative stress effects in the stages of Alzheimer's disease.

Beneficial effect of refined red palm oil on lipid peroxidation and monocyte tissue factor in HCV-related liver disease: a randomized controlled study.

BACKGROUND: A large amount of endotoxin can be detected in the peripheral venous blood of patients with liver cirrhosis, contributing to the pathogenesis of hepatotoxicity because of its role in oxidative stress. The present study aimed to test the effect of the supplementation with red palm oil (RPO), which is a natural oil obtained from oil palm fruit (Elaeis guineensis) rich in natural fat-soluble tocopherols, tocotrienols and carotenoids, on lipid peroxidation and endotoxemia with plasma endotoxin-inactivating capacity, proinflammatory cytokines profile, and monocyte tissue factor in patients with chronic liver disease. METHODS: The study group consisted of sixty patients (34 males and 26 females; mean age 62 years, range 54-75) with Child A/B, genotype 1 HCV-related cirrhosis without a history of ethanol consumption, randomly enrolled into an 8-week oral daily treatment with either vitamin E or RPO. All patients had undergone an upper gastrointestinal endoscopy 8 months before, and 13 out of them showed esophageal varices. RESULTS: Both treatments significantly decreased erythrocyte malondialdehyde and urinary isoprostane output, only RPO significantly affected macrophage-colony stimulating factor and monocyte tissue factor. Liver ultrasound imaging did not show any change. CONCLUSIONS: RPO beneficially modulates oxidative stress and, not least, downregulates macrophage/monocyte inflammatory parameters. RPO can be safely advised as a valuable nutritional implementation tool in the management of chronic liver diseases.

Association of abnormal erythrocyte morphology with oxidative stress and inflammation in metabolic syndrome.

In carrying out their role of free radical scavenging, erythrocytes become damaged due to oxidation of membrane lipids and proteins. Such damage may change the morphology of the erythrocytes. The present study aims to demonstrate change in erythrocyte morphology in MetS and associate the changes with increased oxidative stress and inflammation that were shown in our recent study. One hundred participants were recruited from a rural town of Australia. Whole blood viscosity, erythrocyte aggregation, erythrocyte deformability, lipid profile and blood sugar level, oxidative stress markers (erythrocyte reduced glutathione, superoxide dismutase, urinary isoprostanes) and inflammatory markers (high sensitivity C-reactive protein) were measured. Erythrocyte morphological study was performed by scanning electron microscopy. Recruited participants were classified into MetS and non-MetS following the National Cholesterol Education Program Adult Treatment Panel III definition. Data were analyzed by IBM SPSS 20 software. The mean percentages of biconcave cells were decreased whereas acanthocytes, stomatocytes and echinocytes were increased in MetS group compared to healthy controls. Morphologically abnormal erythrocytes were significantly correlated with oxidative stress and chronic inflammation markers. Free radicals generated in increased concentration in MetS seem to damage erythrocyte changing its morphology which possibly could affect other hemorheological parameters.

Urban air and tobacco smoke as conditions that increase the risk of oxidative stress and respiratory response in youth.

BACKGROUND: Air pollution and tobacco smoke can induce negative effects on the human health and often leads to the formation of oxidative stress. OBJECTIVE: The purpose of this study was to clarify the role of the urbanization degree and of passive exposure to tobacco smoke in the formation of oxidative stress. Thus, a group of non-smoking adolescents was recruited among those who live and attend school in areas with three different population densities. To each subject a spot of urine was collected to quantify 15-F2t isoprostane as a marker of oxidative stress and cotinine as a marker of passive exposure to tobacco smoke. Furthermore, respiratory functionality was also measured. RESULTS: Multiple linear regression analysis results showed a direct correlation (p<0.0001) of 15-F2t isoprostane with both the urbanization and passive smoke. Lung function parameters proved significantly lower for the subjects living in the most populous city of Torino. CONCLUSION: This remarks the negative effect that urbanization has on the respiratory conditions. Lastly, lung functionality presented a low inverse correlation with 15-F2t isoprostane, suggesting an independent mechanism than that of the urban factor.

Antioxidant supplements reduced oxidative stress and stabilized liver function tests but did not reduce inflammation in a randomized controlled trial in obese children and adolescents.

Oxidative stress and low-grade systemic inflammation may contribute to the pathogenesis of obesity-induced comorbidities, including nonalcoholic fatty liver disease. Increasing intake of dietary antioxidants might be beneficial, but there are few data in obese children. To examine the effect of antioxidant supplementation on biomarkers of oxidative stress, inflammation, and liver function, we randomly assigned overweight or obese children and adolescents (n = 44; mean ± SD age: 12.7 ± 1.5 y) participating in a lifestyle modification program to a 4-mo intervention with daily antioxidants (vitamin E, 400 IU; vitamin C, 500 mg; selenium, 50 µg) or placebo. We measured anthropometrics, antioxidant status, oxidative stress (F(2)-isoprostanes, F(2)-isoprostane metabolites), inflammation, liver enzymes, fasting insulin and glucose, and lipid profile at baseline and endpoint. There was a significant treatment effect of antioxidant supplementation on antioxidant status [α-tocopherol, ß = 23.2 (95% CI: 18.0, 28.4); ascorbic acid, ß = 70.6 (95% CI: 51.7, 89.4); selenium, ß = 0.07 (95% CI: 0.01, 0.12)] and oxidative stress [8-iso-prostaglandin F2α, ß = -0.11 (95% CI: -0.19, -0.02)] but not on any of the inflammatory markers measured. There was a significant treatment effect on alanine aminotransferase [ß = -0.13 (95% CI: -0.23, -0.03)], a trend toward a significant effect on aspartate aminotransferase [ß = -0.04 (95% CI: -0.09, 0.01)], and no significant effect on γ-glutamyltransferase [ß = -0.03 (95% CI: -0.11, 0.06)]. In summary, antioxidant supplementation for 4 mo improved antioxidant-oxidant balance and modestly improved liver function tests; however, it did not reduce markers of systemic inflammation despite significant baseline correlations between oxidative stress and inflammation. The study was registered at clinicaltrials.gov as NCT01316081.

Reduced atherosclerotic burden in subjects with genetically determined low oxidative stress.

OBJECTIVE: NADPH oxidase, one of the most important enzymes producing reactive oxidant species, is suggested to play a role in experimental atherosclerosis, but its role in human atherosclerosis is still unclear. We hypothesized that a reduced activity of NADPH oxidase might be linked to a reduced atherosclerotic burden. METHODS AND RESULTS: Thirty-one women carriers of hereditary deficiency of NOX2, the catalytic subunit of NADPH oxidase, were matched for sex and age with 31 controls and 31 obese women. Flow-mediated dilation and intima-media thickness, 2 surrogate markers of atherosclerosis, serum activity of NOX2, urinary isoprostanes, serum levels of nitrite/nitrate, and platelet production of isoprostanes and nitrite/nitrate were determined. Compared with controls (5.7±3.0% and 0.60±0.11 mm), carriers of NOX2 deficiency had higher flow-mediated dilation (9.2±5.0%; P<0.001) and lower intima-media thickness (0.50±0.11 mm; P=0.002), whereas obese women had lower flow-mediated dilation (3.2±2.1%; P=0.007) and higher intima-media thickness (0.71±0.15 mm; P<0.001). Compared with controls, carriers of NOX2 deficiency had lower urinary isoprostanes (132.6±87.3 versus 82.3±46.0 pg/mg creatinine; P=0.007) and serum NOX2 activity (24.9±19.3 versus 12.8±11.9 pg/mL; P=0.004) and higher serum nitrite/nitrate (23.8±7.6 versus 30.5±6.3 µmol/L; P<0.001), whereas obese women had higher urinary isoprostanes (132.6±87.3 versus 182.2±84.6 pg/mg creatinine; P=0.008) and serum NOX2 activity (24.9±19.3 versus 36.1±18.6 pg/mL; P=0.008) and lower serum nitrite/nitrate (23.8±7.6 versus 12.6±4.2 µmol/L; P<0.001). Flow-mediated dilation correlated with intima-media thickness (r=-0.433; P<0.001), serum NOX2 activity (r=-325; P<0.001), and urinary isoprostanes (r=-0.314; P=0.002). Ex vivo study showed that, compared with controls, platelets from carriers of NOX2 deficiency had lower isoprostanes (P<0.001) and higher nitrite/nitrate (P<0.001), whereas platelets from obese women had higher isoprostanes (P<0.001) and lower nitrite/nitrate (P=0.013). CONCLUSIONS: The study shows reduced atherosclerotic burden in carriers of NOX2 deficiency, suggesting that oxidative stress generated by this enzymatic pathway is implicated in human atherosclerosis.

Analysis of lipid peroxidation biomarkers in extremely low gestational age neonate urines by UPLC-MS/MS.

Extremely low gestational age neonates (ELGAN) frequently require the use of oxygen supply in the delivery room leading to systemic inflammation and oxidative stress that are responsible for increased morbidity and mortality. The objective of this study was to establish reference ranges of a set of representative isoprostanes and prostaglandins, which are stable biomarkers of lipid peroxidation often correlated with oxidative stress-related disorders. First, a quantitative ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated. The proposed analytical method was tailored for its application in the field of neonatology, enabling multi-analyte detection in non-invasive, small-volume urine samples. Then, the lipid peroxidation product concentrations in a total of 536 urine samples collected within the framework of two clinical trials including extremely low gestational age neonates (ELGAN) were analyzed. The access to a substantially large number of samples from this very vulnerable population provided the chance to establish reference ranges of the studied biomarkers. Up to the present, and for this population, this is the biggest reference data set reported in literature. Results obtained should assist researchers and pediatricians in interpreting test results in future studies involving isoprostanes and prostaglandins, and could help assessing morbidities and evaluate effectiveness of treatment strategies (e.g., different resuscitation conditions) in the neonatal field.

Acetaminophen attenuates lipid peroxidation in children undergoing cardiopulmonary bypass.

OBJECTIVE: Hemolysis, occurring during cardiopulmonary bypass, is associated with lipid peroxidation and postoperative acute kidney injury. Acetaminophen inhibits lipid peroxidation catalyzed by hemeproteins and in an animal model attenuated rhabdomyolysis-induced acute kidney injury. This pilot study tests the hypothesis that acetaminophen attenuates lipid peroxidation in children undergoing cardiopulmonary bypass. DESIGN: Single-center prospective randomized double-blinded study. SETTING: University-affiliated pediatric hospital. PATIENTS: Thirty children undergoing elective surgical correction of a congenital heart defect. INTERVENTIONS: Patients were randomized to acetaminophen (OFIRMEV [acetaminophen] injection; Cadence Pharmaceuticals, San Diego, CA) or placebo every 6 hours for four doses starting before the onset of cardiopulmonary bypass. MEASUREMENT AND MAIN RESULTS: Markers of hemolysis, lipid peroxidation (isofurans and F2-isoprostanes), and acute kidney injury were measured throughout the perioperative period. Cardiopulmonary bypass was associated with a significant increase in free hemoglobin (from a prebypass level of 9.8 ± 6.2 mg/dL to a peak of 201.5 ± 42.6 mg/dL postbypass). Plasma and urine isofuran and F2-isoprostane concentrations increased significantly during surgery. The magnitude of increase in plasma isofurans was greater than the magnitude in increase in plasma F2-isoprostanes. Acetaminophen attenuated the increase in plasma isofurans compared with placebo (p = 0.02 for effect of study drug). There was no significant effect of acetaminophen on plasma F2-isoprostanes or urinary makers of lipid peroxidation. Acetaminophen did not affect postoperative creatinine, urinary neutrophil gelatinase-associated lipocalin, or prevalence of acute kidney injury. CONCLUSION: Cardiopulmonary bypass in children is associated with hemolysis and lipid peroxidation. Acetaminophen attenuated the increase in plasma isofuran concentrations. Future studies are needed to establish whether other therapies that attenuate or prevent the effects of free hemoglobin result in more effective inhibition of lipid peroxidation in patients undergoing cardiopulmonary bypass.

Effects of stimulation of autophagy on the urinary excretion of biomarkers of the oxidative status.

Autophagy, a health-promoting lysosomal degradation pathway that controls the quality of the cytoplasm by eliminating protein aggregates and damaged organelles including 8-OHdG-rich mitochondria, is under investigation as a target for prevention and/or treatment of several human diseases and decelerating aging. Stimulation of autophagy was shown to rescue older liver cells from accumulation of 8-OHdG-rich mitochondria and to increase urinary 8-OHdG levels. Urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) is a recently recommended biomarker for monitoring oxidative status over time. In order to rule out the possibility that the in vivo stimulation of autophagy may cause an increase in the oxidative status, in this study we compared the effects of the stimulation of autophagy by two different procedures (the administration of antilipolytic drug and everolimus, an mTOR inhibitor in clinical use) on the urinary levels of 8-OHdG and 15-isoprostane F2t, another well-known biomarker of the oxidative status. Results show that both procedures increased the urinary 8-OHdG levels without any change in urinary 15-isoprostane F2t; this increase in urinary 8-OHdG levels after the antilipolytic drug was fully suppressed by the simultaneous injection of glucose to make rats transiently incompetent for the endocrine stimulation of autophagy. Conclusions are that the in vivo stimulation of autophagy does not affect the oxidative status and that the increasing effect on urinary 8-OHdG may be secondary to an increased degradation of previously accumulated 8-OHdG-rich (mt)DNA. The authors are aware that findings may open the way to a safe, easy, highly desirable non-invasive test for successful in vivo activation of autophagy after pharmacological stimulation.

Polycystic ovary syndrome has no independent effect on vascular, inflammatory or thrombotic markers when matched for obesity.

INTRODUCTION: Previous studies investigating cardiovascular (CV) risk in obese women with polycystic ovary syndrome (PCOS) have been potentially confounded by not adequately accounting for body weight. OBJECTIVE: To assess if PCOS increases CV risk independently in young obese women by examining carotid intima-media wall thickness (cIMT) and platelet function. DESIGN: A case-control study comparing women with PCOS (n = 21) to age (32·8 ± 7·2 vs 33·5 ± 6·7 years), and weight (100·9 ± 16·7 vs 99·3 ± 14·7 kg)-matched controls (n = 19). Platelet function was examined by flow cytometry, clot structure and fibrinolysis by turbidimetric assays and endothelial function by ELISA and post ischaemic reactive hyperaemia. RESULTS: The PCOS group had higher testosterone 1·2 ± 0·3 vs 0·9 ± 0·3 nmol/l (P = 0·01), HOMA-IR 2·5 ± 1·7 vs 1·7 ± 1·0 (P = 0·08), impaired glucose regulation 33·3% vs 5·3% (P = 0·02), and urinary isoprostane 16·0 ± 4·4 vs 11·8 ± 7·1 ng/ml (P = 0·04) compared to controls. Mean cIMT 0·5 ± 0·05 vs 0·48 ± 0·06 mm (P = 0·36), and basal platelet surface expression (percentage of positive cells) of P-selectin 0·52 ± 0·3 vs 0·43 ± 0·23 (P = 0·40) and fibrinogen binding 0·97 ± 0·4 vs 0·83 ± 0·3 (P = 0·48) did not significantly differ between the PCOS and control groups respectively. Furthermore, platelets sensitivity to stimulation with adenosine-5'-diphosphate or inhibition with prostacyclin, clot structure and fibrinolytic efficiency ex vivo, endothelial reactive hyperaemic index (RHI), inflammation (hsCRP) and adhesion markers (sE-selectin, sP-selectin, sVCAM-1 and sICAM-1) were not significantly different between the two groups. CONCLUSIONS: PCOS appeared not to independently increase atherothrombotic risk when matched for obesity. It is likely that any excess CV risk in young obese women with PCOS can either be attributed to obesity or is not yet apparent at this early stage of the condition.