Oxidative Stress Metabolites Research Articles

Skin- and Plasmaautofluorescence in hemodialysis with glucose-free or glucose-containing dialysate

BackgroundHaemodialysis (HD) patients suffer from an increased risk of cardiovascular disease (CVD). Skin autofluorescence (SAF) is a strong marker for CVD. SAF indirectly measures tissue advanced glycation end products (AGE) being cumulative metabolites of oxidative stress and cytokine-driven inflammatory reactions. The dialysates often contain glucose.MethodsAutofluorescence of skin and plasma (PAF) were measured in patients on HD during standard treatment (ST) with a glucose-containing dialysate (n = 24). After that the patients were switched to a glucose-free dialysate (GFD) for a 2-week period. New measurements were performed on PAF and SAF after 1 week (M1) and 2 weeks (M2) using GFD. Nonparametric paired statistical analyses were performed between each two periods.ResultsSAF after HD increased non-significantly by 1.2% while when a GFD was used during HD at M1, a decrease of SAF by 5.2% (p = 0.002) was found. One week later (M2) the reduction of 1.6% after the HD was not significant (p = 0.33). PAF was significantly reduced during all HD sessions. Free and protein-bound PAF decreased similarly whether glucose containing or GFD was used. The HD resulted in a reduction of the total PAF of approximately 15%, the free compound of 20% and the protein bound of 10%. The protein bound part of PAF corresponded to approximately 56% of the total reduction. The protein bound concentrations after each HD showed the lowest value after 2 weeks using glucose-free dialysate (p < 0.05). The change in SAF could not be related to a change in PAF.ConclusionsWhen changing to a GFD, SAF was reduced by HD indicating that such measure may hamper the accumulation and progression of deposits of AGEs to protein in tissue, and thereby also the development of CVD. Glucose-free dialysate needs further attention. Protein binding seems firm but not irreversible.Trial registrationISRCTN registry: ISRCTN13837553. Registered 16/11/2016 (retrospectively registered).

LC-MS-Based Metabolic Fingerprinting of Aqueous Humor.

Aqueous humor (AH) is a transparent fluid which fills the anterior and posterior chambers of the eye. It supplies nutrients and removes metabolic waste from avascular tissues in the eye. Proper homeostasis of AH is required to maintain adequate intraocular pressure as well as optical and refractive properties of the eye. Application of metabolomics to study human AH may improve knowledge about the molecular mechanisms of eye diseases. Until now, global analysis of metabolites in AH has been mainly performed using NMR. Among the analytical platforms used in metabolomics, LC-MS allows for the highest metabolome coverage. The aim of this study was to develop a method for extraction and analysis of AH metabolites by LC-QTOF-MS. Different protocols for AH preparation were tested. The best results were obtained when one volume of AH was mixed with one volume of methanol : ethanol (1 : 1). In the final method, 2 µL of extracted sample was analyzed by LC-QTOF-MS. The method allowed for reproducible measurement of over 1000 metabolic features. Almost 250 metabolites were identified in AH and assigned to 47 metabolic pathways. This method is suitable to study the potential role of amino acids, lipids, oxidative stress, or microbial metabolites in development of ocular diseases.

Mechanisms of Improvement of Erythropoiesis with Low Dose Deferasirox in Low Risk Myelodysplastic Syndromes

Introduction: Myelodysplastic syndromes (MDS) are a group of heterogeneous clonal stem cell disorder characterized by dysplasia leading to ineffective hematopoiesis and evolution to acute myeloid leukemia. Anemia is the most frequent cytopenia in MDS and the majority of patients requires red blood cell (RBC) transfusion resulting in the development of iron overload (IO). Deferasirox (DFX) became a standard treatment of IO in MDS and seems to have positive effects on hematopoiesis in some MDS patients leading to reduction of RBC transfusion or even transfusion independence (Gatterman, 2012, Maurillo, 2015). In this study, our aims were to decipher the intrinsic mechanisms explaining the potential positive effects of DFX on erythroid differentiation in low risk MDS.Methods: We report our data about erythroid differentiation, cell cycle, apoptosis and cell signaling pathways concerning CD34+ hematopoietic stem progenitor cells (HSPCs) from low risk MDS samples in a 2-step erythroid differentiation liquid culture with low dose DFX.Results: We observed a higher proliferation rate at the end of the cell culture procedure with DFX 3µM versus control condition (p=0,038). We did not find these effects with DFX > 5µM. This higher proliferation rate is due to the combination of less apoptotic cells and more cycling cells. At day 10 of the culture (D10) (n=7), there were less apoptotic cells for DFX 3μM condition (DFX 3): 13,8% versus 17,5% for control condition (p=0,0325). This lower apoptosis rate was more relevant at D14 (n=9): 19,1% versus 24,7% for control condition (p=0,007). There were more cycling cells with DFX 3 at D10 (n=6): fewer cells were blocked at the beginning of the cell cycle in subG0-G0-G1 phases (68,9% versus 77,6%) (p=0,0001) and more cells (30,5% versus 21,9%) were engaged in cell cycle (S-G2-M) (p=0,0001) for DFX 3. Regarding clonogenic assays (n=10), there was an increased number of CFU-E colonies for DFX 3 versus control condition with on average 72,7 (15-261) and 46,5 (3-174,5) colonies respectively (p=0,04). We did not see any effect of DFX on erythroid differentiation. The percentages of proerythroblasts, basophilic and orthochromatic erythroblasts were similar for DFX 3 and control at D5, D10 and D14 of the cell culture regardless the techniques (flow cytometry or morphological analyses by cytospin).These functional effects were partially related to iron chelation of DFX. As expected, we saw a lower iron intracellular concentration (-12,7%) by mass spectrometry with DFX 3 (p=0,019). Nevertheless this chelation effect was not sufficient to activate iron regulation metabolism (IRP-IRE) in REMSA assay, indeed we saw no difference in IRP activity between DFX 3 and control condition. Moreover, low dose of DFX had a protector effect against mitochondrial reactive oxygen species at D14 (n=9; p=0,03). We aim to complete the Redox profile by analysis of some oxidative stress metabolites like malonaldehyde, carbonyl groups, γH2AX and oxidized form of glutathione.Then, we have investigated which signaling pathways were responsible for these functional effects by flow cytometry (FC) and immunofluorescence (IF) microscopy. By FC, PI3K/AKT, mTOR and MAPK/ERK were not over-activated in DFX 3. However,we found an increased nuclear translocation of NFκB in DFX3 (p=0,0004) by IF. Using RT-qPCR microarrays, we have analyzed the expression of 84 gene targets of NFκB. Five genes were upregulated in DFX 3 with a fold change >2 compared to control: BCL2A1, BIRC3, TNFRSF1B, EGFR, IL1B. This gene profile leads in silico to an anti-apoptotic signal (PCVIz®).To demonstrate a link between the ROS protection effects of DFX and the NFκB activation pathway, we have realized a cellular model of inhibition of thioredoxin (TRX), a ROS regulated protein, by three siRNA anti-TRX1. TRX1 in its reduced form can activate NFκB. By inhibiting TRX1, we hypothesize that NFκB pathway cannot be activated and that could impede cell proliferation. Definitive results will be presented at ASH.Conclusions: Our study described the pro-proliferative effects of low dose of DFX on erythroid progenitors in low risk MDS patients in vitro via a mechanism of mitochondrial ROS protection and NFκB activation. Those data highlight the potential interest of an early introduction of iron chelation therapy with low dose DFX (around 5mg/kg/d corresponding to a plasmatic level of 3µM) in the disease course of transfused low risk MDS patients. DisclosuresMeunier:Novartis: Research Funding. Park:Novartis: Research Funding.

Pre-storage conditioning ameliorates the negative impact of 1-methylcyclopropene on physiological injury and modifies the response of antioxidants and γ-aminobutyrate in ‘Honeycrisp’ apples exposed to controlled-atmosphere conditions

Controlled atmosphere (CA) storage limits ethylene-related ripening of apple fruit, but can promote the development of CA-related injury. CA-related injury can be exacerbated by the ethylene antagonist, 1-methylcyclopropene (1-MCP). The main objective of this study was to investigate the impact of pre-storage conditioning, 1-MCP and elevated CO2 on the development of CA-related injury and oxidative stress metabolites in ‘Honeycrisp’ (Malus×domestica Borkh.) apple fruit. Upon harvest, fruit were treated with or without 1μLL−1 1-MCP for 24h, and then transferred to CA (2.5kPa O2 and 2.5kPa or 0.03kPa CO2) at 3°C (no conditioning) or conditioned at 10°C for 5d prior to CA. Apples were CA-stored for up to 35 weeks. CA-related injury occurred with storage under both CA regimes, regardless of 1-MCP and conditioning, whereas 1-MCP exacerbated the negative impact of elevated CO2 on this disorder, approximating an 80% incidence, which was coincident with a 400% increase in γ-aminobutyrate (GABA). By comparison, pre-storage conditioning reduced the negative impact of 1-MCP on the incidence of CA-related injury at 2.5kPa CO2 by nearly 80%, and this was associated with a dramatically lower GABA concentration. Overall, rapid declines in total ascorbate and ascorbate/dehydroascorbate ratios were evident with storage, regardless of treatment. Transient declines in total glutathione and glutathione/glutathione disulphide ratios were more rapid and sustained for longer periods of time in conditioned than non-conditioned fruit. There were dramatic shifts in the levels of phosphorylated and non-phosphorylated pyridine dinucleotides with storage duration, regardless of treatment, resulting in elevated ratios of NADPH/NADP+ and NADH/NAD+ after 35 weeks, especially for conditioned fruit subjected to elevated CO2. Canonical powered partial least squares analysis revealed that metabolite profiles changed with storage period and treatment, and were most divergent during elevated CO2 storage. Moreover, CA-related injury was strongly associated with changes in GABA, and moderately linked to total glutathione and glutathione redox status.

Hearing, reactive metabolite formation, and oxidative stress in cochleae after a single acute overdose of acetaminophen: an in vivo study

Context: Although the liver is the primary target organ in acetaminophen (APAP) toxicity, other organs are affected. Previous data suggested that chronic APAP abuse can be ototoxic and the mechanism involves APAP-induced oxidative stress and reactive metabolite (N-acetyl-p-benzoquinone imine, NAPQI)-induced endoplasmic reticulum stress. However, the effect of a single acute overdose on hearing has not been tested.Objectives: To determine if a single acute APAP overdose causes hearing damage, and to explore possible mechanisms of APAP ototoxicity.Materials and methods: Male C57BL/6 J mice were treated with a single human-relevant overdose of APAP (300 mg APAP per kg bodyweight). Blood, liver and cochleae were harvested at 0, 2, 6 and 24 h post-APAP. In some mice, auditory brainstem responses (ABRs) to a range of frequencies were measured at 24 h. The furosemide plus kanamycin (FS/K) model of drug ototoxicity was used as a positive control for hearing loss. NAPQI formation after APAP was assessed by measuring glutathione depletion and covalent protein binding, and oxidative stress was assessed by measuring glutathione disulfide.Results: There was no evidence of reactive metabolite formation or hearing loss after a single overdose of APAP at a clinically relevant dose. However, there was a transient increase in oxidative stress.Discussion: Although a single acute overdose was not ototoxic, there was evidence of oxidative stress which may support a role for oxidative stress in hearing loss due to chronic APAP abuse.Conclusion: A single human-relevant acute overdose of APAP causes transient oxidative stress in cochleae but not hearing loss.

Biomarkers of Flutamide-Bioactivation and Oxidative Stress In Vitro and In Vivo.

The nonsteroidal androgen-receptor antagonist flutamide is associated with hepatic injury. Oxidative stress and reactive metabolite formation are considered contributing factors to liver toxicity. Here we have used flutamide as a model drug to study the generation of reactive drug metabolites that undergo redox cycling to induce oxidative stress (OS) in vitro and in vivo. Lipid peroxidation (LPO) markers, as well as genes regulated by the redox-sensitive Nrf2 pathway, have been identified as surrogates for the characterization of OS. These markers and metabolism biomarkers for drug bioactivation have been investigated to characterize drug-induced hepatic damage. Rat hepatocytes and in vivo studies showed that several LPO markers, namely the isoprostanes 15R-PD2, dihydro keto PE2, and iPF(2α)-VI, as well as hydroxynonenal mercapturic acid metabolites, had increased significantly by 24 hours after flutamide treatment from 4.9 to 15.3-fold in hepatocytes and from 2.6 to 31.0-fold in rat plasma. Induction of mRNA expression levels for Nrf2-regulated genes was evident as well, with heme oxygenase 1, glutathione-S-transferase π1 and NAD(P)H dehydrogenase showing a 3.6-, 4.1-, and 1.9-fold increase in hepatocytes and 5.6-, 7.5-, and 94.1-fold in rat liver. All effects were observed at drug concentrations that did not show overt liver toxicity. Addition of an in situ hydrogen peroxide-generating system to in vitro experiments demonstrated the formation of a reactive di-imine intermediate as the responsible metabolic pathway for the generation of OS. The dataset suggests that hepatic oxidative stress conditions can be mediated via metabolic activation and can be monitored with suitable biomarkers preceding the terminal damage.

Hepatic non-parenchymal cells: Master regulators of alcoholic liver disease?

Chronic alcohol consumption is one of the most common causes of the progression of alcoholic liver disease (ALD). In the past, alcohol-mediated hepatocyte injury was assumed to be a significantly major cause of ALD. However, a huge number of recent and brilliant studies have demonstrated that hepatic non-parenchymal cells including Kupffer cells, hepatic stellate cells, liver sinusoidal endothelial cells and diverse types of lymphocytes play crucial roles in the pathogenesis of ALD by producing inflammatory mediators such as cytokines, oxidative stress, microRNA, and lipid-originated metabolites (retinoic acid and endocannabinoids) or by directly interacting with parenchymal cells (hepatocytes). Therefore, understanding the comprehensive roles of hepatic non-parenchymal cells during the development of ALD will provide new integrative directions for the treatment of ALD. This review will address the roles of non-parenchymal cells in alcoholic steatosis, inflammation, and liver fibrosis and might help us to discover possible therapeutic targets and treatments involving modulating the non-parenchymal cells in ALD.

Protection against Methotrexate Induced Hepato-Renal Toxicity in Rats by Zinc and its Combination with Vitamin C and Vitamin E

Methotrexate, a well-known cytotoxic chemotherapy, anti-folate, immunosuppressant and effectively treats all major disorders like cancer, psoriasis, refractory rheumatoid arthritis, etc. It is widely and mostly used drug having multitherapy properties and well tolerated, however, its cytotoxic nature leads to many serious side effects, including hepato-renal toxicity, so it is necessary to reduce its side effect and get its benefit. Among all hypothesis, oxidative stress and toxic metabolite are the noticeable cause of toxicity. The present study investigated the protective effects of Zinc alone and its combination with vitamin C and vitamin E against the methotrexate induced hepato-renal toxicity in male albino rats. Zinc is a well-known potent anti-oxidant that scavenges the free radical in tissues. Vitamin C and vitamin E are water soluble and insoluble antioxidant, respectively. In conclusion, the rats treated with zinc alone and its combination with vitamin C and vitamin E had a significant decrease in the level of creatinine, urea, total bilirubin, SGOT, SGPT in blood serum when compared with methotrexate treated rats. Similarly the activity of catalase and reduced glutathione were enhanced and lipid peroxidase was decreased. Histopathology report confirmed the hepato-renal protectivity of Zinc and its combination with vitamin C and vitamin E by restoring the normal architecture of liver and kidney.

Elevation of Derivatives of Reactive Oxygen Metabolites Elevated in Young "Disaster Responders" in Hypertension due to Great East Japan Earthquake.

There have been very few studies on serum biomarkers associated with hypertension in disaster situations. We assessed biomarkers associated with disaster-related hypertension (DRH) due to the Great East Japan Earthquake of March 2011.We collected blood samples from members of the Japan Self Defense Forces (JSDF) (n = 77) after completing disaster relief operations. We divided them into two groups based on systolic blood pressure. We defined DRH as either systolic blood pressure greater than 140 mmHg or diastolic blood pressure greater than 90 mmHg at the time of completing missions.In subjects with DRH, the mean blood pressure was 143.5 ± 5.0/99.5 ± 2.4 mmHg. Height and body weight measurements were slightly greater in the DRH group but the differences were not significant, and age was significantly higher in the DRH group. There were no differences in serum biochemical tests including metabolic markers, sulfur-containing amino acids, and cytokines. Among nitric oxide-related amino acids, asymmetric dimethylarginine (ADMA) was lower in the DRH group than in the normotension group (0.40 ± 0.02 versus 0.31 ± 0.02 μmol/L P = 0.04). The serum oxidative stress metabolite levels (d-ROMs; indicators of active oxygen metabolite products) were significantly higher in the DRH group (273.6 ± 6.08 versus 313.5 ± 13.7 U.CARR P = 0.016). Using multivariable regression analysis, d-ROMs levels were particularly predictive for DRH.Oxidative stress is associated with DRH in responders to the disaster of the Great East Japan Earthquake.

Effects of the herb mixture, DTS20, on oxidative stress and plasma alcoholic metabolites after alcohol consumption in healthy young men

BackgroundThis study was designed to investigate the effect of a herbal mixture extract (DTS20) on the attenuation of oxidative stress and hangover after alcohol consumption in healthy volunteers. MethodsDTS20 consists of Viscum album L. (40%), Lycium chinense L. (30%), Inonotus obliquus (20%), and Acanthopanax senticosus H. (10%). We recruited healthy, nonsmoking, adult men volunteers aged between 21 years and 30 years to participate in a crossover trial. Twenty participants received either one package of placebo with 200mL water or DTS20 with 200mL water. Thirty minutes later, the volunteers ingested one bottle of Soju, which is a commercially available liquor (19% alcohol in 360mL). ResultsVolunteers received the opposite treatment after a 1-week washout period. DTS20 is mainly composed of sugars (564.5mg/g) and polyphenol (28.2mg/g). Alcohol levels in the DTS20 group were significantly lower than the control group at 2hours after drinking Soju (p<0.05). Acetaldehyde levels in the DTS20 group tended to be lower than the control group at 2hours after drinking Soju, but was not significantly different. The antioxidant activity level was also significantly different between the control and DTS20 group 2hours after drinking Soju (p<0.05). No differences in plasma alanine transaminase or aspartate transaminase levels were observed between plasma levels before drinking and 2hours after drinking Soju in the control group. ConclusionIt was concluded that DTS20 reduced oxidative stress and hangover by mitigating plasma alcohol concentrations and elevating antioxidative activity in healthy male adults.

Influence of green tea catechins on oxidative stress metabolites at rest and during exercise in healthy humans

ObjectivesThe aim of this study was to investigate the effects of green tea catechins (GTC) on oxidative stress metabolites in healthy individuals while at rest and during exercise. The effects investigated included response to fat metabolism, blood lactate concentrations, and rating of perceived exertion. MethodsIn a paralleled, crossover, randomized controlled study, 16 trained male gymnastic students were randomly divided into two groups. The rest group (n = 8; GTC-NEX) received a single dose of 780 mg GTC with water but no exercise; the exercise group (n = 8; GTC-EX) received a similar dose of GTC but were instructed to exercise. This was followed by a crossover study with similar exercise regime as a placebo group (PL-EX) that received water only. Blood samples were collected at baseline and after 60 and 120 min of GTC intake. Oxidative stress blood biomarkers using the diacron reactive oxygen metabolite (d-ROMs) and biological antioxidant potential (BAP) tests; urinary 8-hydroxydeoxyguanosine (8-OHdG); 8-OHdG/creatinine; and blood lactate concentrations were analyzed. During the cycle ergometer exercise, volume of maximal oxygen uptake, volume of oxygen consumption, volume of carbon dioxide, and respiratory exchange ratio were measured from a sample of respiratory breath gas collected during low, moderate, and high intensity exercising, and the amount of fat burning and sugar consumption were calculated. Analysis of variance was used to determine statistical significance (P < 0.05) between and among the groups. ResultsLevels of postexercise oxidative stress metabolites BAP and d-ROMs were found significant (P < 0.0001) in the PL-EX and GTC-EX groups, and returned to pre-exercise levels after the recovery period. Levels of d-ROMs showed no significant difference from baseline upon GTC intake followed by resting and a resting recovery period in the GTC-NEX group. BAP levels were significant upon GTC intake followed by resting (P = 0.04), and after a resting recovery period (P = 0.0006) in the GTC-NEX group. Urinary 8-OHdG levels were significant (P < 0.005) for all groups after the recovery period. A significant difference was noticed between the ratios of resting BAP to d-ROMs and exercise-induced BAP to d-ROMs (P = 0.022) after 60 min of GTC intake, as well as resting 8-OHdG and exercise-induced 8-OHdG levels (P = 0.004) after the recovery period. Oxidative potentials were higher when exercise was performed at low to moderate intensity, accompanied by lower blood lactate concentration and higher amounts of fat oxidation. ConclusionsThe results of the present study indicate that single-dose consumption of GTC influences oxidative stress biomarkers when compared between the GTC-NEX and GTC-EX groups, which could be beneficial for oxidative metabolism at rest and during exercise, possibly through the catechol-O-methyltransferase mechanism that is most often cited in previous studies.

Individual Shrink Wrapping of Zucchini Fruit Improves Postharvest Chilling Tolerance Associated with a Reduction in Ethylene Production and Oxidative Stress Metabolites.

We have studied the effect of individual shrink wrapping (ISW) on the postharvest performance of refrigerated fruit from two zucchini cultivars that differ in their sensitivity to cold storage: Sinatra (more sensitive) and Natura (more tolerant). The fruit was individually shrink wrapped before storing at 4°C for 0, 7 and 14 days. Quality parameters, ethylene and CO2 productions, ethylene gene expression, and oxidative stress metabolites were assessed in shrink wrapped and non-wrapped fruit after conditioning the fruit for 6 hours at 20°C. ISW decreased significantly the postharvest deterioration of chilled zucchini in both cultivars. Weight loss was reduced to less than 1%, pitting symptoms were completely absent in ISW fruit at 7 days, and were less than 25% those of control fruits at 14 days of cold storage, and firmness loss was significantly reduced in the cultivar Sinatra. These enhancements in quality of ISW fruit were associated with a significant reduction in cold-induced ethylene production, in the respiration rate, and in the level of oxidative stress metabolites such as hydrogen peroxide and malonyldialdehyde (MDA). A detailed expression analysis of ethylene biosynthesis, perception and signaling genes demonstrated a downregulation of CpACS1 and CpACO1 genes in response to ISW, two genes that are upregulated by cold storage. However, the expression patterns of six other ethylene biosynthesis genes (CpACS2 to CpACS7) and five ethylene signal transduction pathway genes (CpCTR1, CpETR1, CpERS1, CpEIN3.1 and CpEN3.2), suggest that they do not play a major role in response to cold storage and ISW packaging. In conclusion, ISW zucchini packaging resulted in improved tolerance to chilling concomitantly with a reduction in oxidative stress, respiration rate and ethylene production, as well as in the expression of ethylene biosynthesis genes, but not of those involved in ethylene perception and sensitivity.

Dietary Glucosinolates Sulforaphane, Phenethyl Isothiocyanate, Indole-3-Carbinol/3,3'-Diindolylmethane: Anti-Oxidative Stress/Inflammation, Nrf2, Epigenetics/Epigenomics and In Vivo Cancer Chemopreventive Efficacy.

Glucosinolates are a group of sulfur-containing glycosides found in many plant species, including cruciferous vegetables such as broccoli, cabbage, brussels sprouts, and cauliflower. Accumulating evidence increasingly supports the beneficial effects of dietary glucosinolates on overall health, including as potential anti-cancer agents, because of their role in the prevention of the initiation of carcinogenesis via the induction of cellular defense detoxifying/antioxidant enzymes and their epigenetic mechanisms, including modification of the CpG methylation of cancer-related genes, histone modification regulation and changes in the expression of miRNAs. In this context, the defense mechanism mediated by Nrf2-antioxidative stress and anti-inflammatory signaling pathways can contribute to cellular protection against oxidative stress and reactive metabolites of carcinogens. In this review, we summarize the cancer chemopreventive role of naturally occurring glucosinolate derivatives as inhibitors of carcinogenesis, with particular emphasis on specific molecular targets and epigenetic alterations in in vitro and in vivo human cancer animal models.

Electrochemical Detection of Nitric Oxide and Peroxynitrite Anion in Microchannels at Highly Sensitive Platinum-Black Coated Electrodes. Application to ROS and RNS Mixtures prior to Biological Investigations

The electrochemical detection of nitric oxide (NO) and peroxynitrite anion (ONOO−) was investigated at Pt-black electrodes in microchannels. Owing to the high reactivity of these species under conditions close to physiological media, kinetic parameters were determined before evaluating the detection performances from synthetic solutions. Highly sensitive and stable Pt-black electrodes allowed detection limits down to 30nM (NO) and 40nM (ONOO−) to be reached with very high sensitivities. As NO and ONOO− are two relevant biological molecules involved in oxidative stress their simultaneous detections with other major molecules like hydrogen peroxide and nitrite were also performed and validated experimentally at pH 8.4. These results demonstrated that relative ROS/RNS contents in synthetic mixtures can be easily assessed at selected detection potentials. Beyond the interest of using small volumes in microfluidic channels, optimization of detection requires precise conditions easy to implement. These were delineated to lead in microdevices to high-performances detection of oxidative stress metabolites either from analytes or from the production of a few living cells like macrophages.

Covalent Thiol Adducts Arising from Reactive Intermediates of Cocaine Biotransformation

Exposure to cocaine results in the depletion of hepatocellular glutathione and macromolecular protein binding in humans. Such cocaine-induced responses have generally been attributed to oxidative stress and reactive metabolites resulting from oxidative activation of the cocaine tropane nitrogen. However, little conclusive data exists on the mechanistic pathways leading to protein modification or the structure and specificity of cocaine-derived adduction products. We now report a previously uncharacterized route of cocaine bioactivation leading to the covalent adduction of biological thiols, including cysteine and glutathione. Incubation of cocaine with biological nucleophiles in an in vitro biotransformation system containing human liver microsomes identified a monooxygenase-mediated event leading to the oxidation of, and subsequent sulfhydryl addition to, the cocaine aryl moiety. Adduct structures were confirmed using ultra-high performance liquid chromatography coupled to high resolution, high mass accuracy mass spectrometry. Examination of assays containing transgenic bactosomes expressing single human cytochrome P450 isoforms determined the role of P450s 1A2, 2C19, and 2D6 in the oxidation process resulting in adduct formation. P450-catalyzed aryl epoxide formation and subsequent attack by free nucleophilic moieties is consistent with the resulting adduct structures, mechanisms of formation, and the empirical observation of multiple structural and stereo isomers. Analogous adduction mechanisms were maintained across all sulfhydryl-containing nucleophile models examined; N-acetylcysteine, glutathione, and a synthetic cysteine-containing hexapeptide. Predictive in silico calculations of molecular reactivity and electrophilicity/nucleophilicity were compared to the results of in vitro assay incubations in order to better understand the adduction process using the principles of hard and soft acid and base (HSAB) theory. This study elucidated a novel metabolic pathway that may be of particular significance to the clinical and forensic toxicology of cocaine and provides analytical tools and methods that can be applied to the determination of these conjugates in humans, opening a new area of research on cocaine biotransformation and toxicology.

Metabolomics Reveals Metabolic Alterations by Intrauterine Growth Restriction in the Fetal Rabbit Brain

BackgroundIntrauterine Growth Restriction (IUGR) due to placental insufficiency occurs in 5–10% of pregnancies and is a major risk factor for abnormal neurodevelopment. The perinatal diagnosis of IUGR related abnormal neurodevelopment represents a major challenge in fetal medicine. The development of clinical biomarkers is considered a promising approach, but requires the identification of biochemical/molecular alterations by IUGR in the fetal brain. This targeted metabolomics study in a rabbit IUGR model aimed to obtain mechanistic insight into the effects of IUGR on the fetal brain and identify metabolite candidates for biomarker development.Methodology/Principal FindingsAt gestation day 25, IUGR was induced in two New Zealand rabbits by 40–50% uteroplacental vessel ligation in one horn and the contralateral horn was used as control. At day 30, fetuses were delivered by Cesarian section, weighed and brains collected for metabolomics analysis. Results showed that IUGR fetuses had a significantly lower birth and brain weight compared to controls. Metabolomics analysis using liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) and database matching identified 78 metabolites. Comparison of metabolite intensities using a t-test demonstrated that 18 metabolites were significantly different between control and IUGR brain tissue, including neurotransmitters/peptides, amino acids, fatty acids, energy metabolism intermediates and oxidative stress metabolites. Principle component and hierarchical cluster analysis showed cluster formations that clearly separated control from IUGR brain tissue samples, revealing the potential to develop predictive biomarkers. Moreover birth weight and metabolite intensity correlations indicated that the extent of alterations was dependent on the severity of IUGR.ConclusionsIUGR leads to metabolic alterations in the fetal rabbit brain, involving neuronal viability, energy metabolism, amino acid levels, fatty acid profiles and oxidative stress mechanisms. Overall findings identified aspargine, ornithine, N-acetylaspartylglutamic acid, N-acetylaspartate and palmitoleic acid as potential metabolite candidates to develop clinical biomarkers for the perinatal diagnosis of IUGR related abnormal neurodevelopment.

Metabolomics in pneumonia and sepsis: an analysis of the GenIMS cohort study.

To determine the global metabolomic profile as measured in circulating plasma from surviving and non-surviving patients with community-acquired pneumonia (CAP) and sepsis. Random, outcome-stratified case-control sample from a prospective study of 1,895 patients hospitalized with CAP and sepsis. Cases (n=15) were adults who died before 90days, and controls (n=15) were adults who survived, matched on demographics, infection type, and procalcitonin. We determined the global metabolomic profile in the first emergency department blood sample using non-targeted mass-spectrometry. We derived metabolite-based prognostic models for 90-day mortality. We determined if metabolites stimulated cytokine production by differentiated Thp1 monocytes in vitro, and validated metabolite profiles in mouse liver and kidney homogenates at 8h in cecal ligation and puncture (CLP) sepsis. We identified 423 small molecules, of which the relative levels of 70 (17%) were different between survivors and non-survivors (p≤0.05). Broad differences were present in pathways of oxidative stress, bile acid metabolism, and stress response. Metabolite-based prognostic models for 90-day survival performed modestly (AUC=0.67, 95% CI 0.48, 0.81). Five nucleic acid metabolites were greater in non-survivors (p≤0.05). Of these, pseudouridine increased monocyte expression of TNFα and IL1β versus control (p<0.05). Pseudouridine was also increased in liver and kidney homogenates from CLP mice versus sham (p<0.05 for both). Although replication is required, we show the global metabolomic profile in plasma broadly differs between survivors and non-survivors of CAP and sepsis. Metabolite-based prognostic models had modest performance, though metabolites of oxidative stress may act as putative damage-associated molecular patterns.

Dose–response to 3 months of quercetin-containing supplements on metabolite and quercetin conjugate profile in adults

Quercetin, a flavonol in fruits and vegetables, has been demonstrated to have antioxidant, anti-inflammatory and immunomodulating influences. The purpose of the present study was to determine if quercetin, vitamin C and niacin supplements (Q-500 = 500 mg/d of quercetin, 125 mg/d of vitamin C and 5 mg/d of niacin; Q-1000 = 1000 mg/d of quercetin, 250 mg/d of vitamin C and 10 mg/d of niacin) would alter small-molecule metabolite profiles and serum quercetin conjugate levels in adults. Healthy adults (fifty-eight women and forty-two men; aged 40-83 years) were assigned using a randomised double-blinded placebo-controlled trial to one of three supplement groups (Q-1000, Q-500 or placebo). Overnight fasted blood samples were collected at 0, 1 and 3 months. Quercetin conjugate concentrations were measured using ultra-performance liquid chromatography (UPLC)-MS/MS, and metabolite profiles were measured using two MS platforms (UPLC-quadrupole time-of-flight MS (TOFMS) and GC-TOFMS). Statistical procedures included partial least square discriminant analysis (PLS-DA) and linear mixed model analysis with repeated measures. After accounting for age, sex and BMI, quercetin supplementation was associated with significant shifts in 163 metabolites/quercetin conjugates (false discovery rate, P<0·05). The top five metabolite shifts were an increase in serum guaiacol, 2-oxo-4-methylthiobutanoic acid, allocystathionine and two bile acids. Inflammatory and oxidative stress metabolites were not affected. PLS-DA revealed a clear separation only between the 1000 mg/d and placebo groups (Q(2)Y = 0·763). The quercetin conjugate, isorhamnetin-3-glucuronide, had the highest concentration at 3 months followed by quercetin-3-glucuronide, quercetin-3-sulphate and quercetin diglucuronide. In human subjects, long-term quercetin supplementation exerts disparate and wide-ranging metabolic effects and changes in quercetin conjugate concentrations. Metabolic shifts were apparent at the 1000 mg/d dose; further research is required to understand the health implications of these shifts.

Oxidative stress and nitric oxide in polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is a common endocrine disorder affecting 5–10% of premenopausal women. In addition to its well-established reproductive effects, PCOS is a metabolic disorder characterised by insulin resistance, hyperandrogenism and dyslipidaemia but the effects of these disturbances on oxidative stress and nitric oxide metabolism are not known. We thus sought to compare measures of oxidative stress and nitric oxide metabolites in patients with PCOS and healthy volunteers (HV). Preliminary data from banked frozen plasma samples obtained from a phenotypically detailed cohort of PCOS patients (age 29.4 ± 6.6 years, BMI 33.2 ± 7.4 kg/m 2 ) and age/BMI-matched HV showed similar nitrite (PCOS [ n = 25] 153 ± 16.57 nmol/l, HV [ n = 17] 187.8 ± 26.59nmol/l, p = NS) but lower nitrate levels in subjects with PCOS (PCOS [ n = 33] 7.3 ± 1.92 μmol/l, HV [ n = 27] 24.54 ± 2.59μmol/l, p < 0.001). The PCOS and control group displayed similar lipid profiles (TC: 4.79 ± 0.1 and 4.5 ± 0.06, HDL: 1.35 ± 0.1 and 1.38 ± 0.05 and LDL: 2.93 ± 0.15 and 2.76 ± 0.15, respectively). Plasma nitrite correlated inversely with LDL concentrations in subjects with PCOS ( r = −0.531, p < 0.05) but not in HVs. There were no significant relationships between plasma nitrate and lipid levels. Inspired by these results, data will be presented at this meeting from a fresh group of subjects in which these parameters are compared in the context of an increased free radical formation (detected as direct spin-trapping of lipid-derived radicals in patient blood) and ORAC status that result from dysfunctional lipid metabolism within PCOS. Abbreviatons: PCOS, polycystic ovary syndrome; HV, healthy volunteers; NS, non-significant; TC, total cholesterol; HDL, high-density lipoprotein; LDL, low-density lipoprotein; ORAC, oxygen radical absorbance capacity.

Exposure of Arabidopsis thaliana to excess Zn reveals a Zn-specific oxidative stress signature

Zinc (Zn) is an essential micronutrient for plants, but accumulation of excess Zn causes oxidative stress, even though the element is not redox-active. An oxidative stress signature, consisting of multiple oxidative stress related parameters, is indicative of disturbance of redox homeostasis and signaling, but has not been determined after exposure to excess Zn. To reveal general and Zn-specific effects, changes in oxidative stress related gene expression, enzyme activities and metabolites were determined after 24h exposure of Arabidopsis thaliana plants to a concentration range of 0, 100, 250 or 500μM excess ZnSO4, and the oxidative stress signature compared to the ones reported previously for cadmium or copper exposure in the same experimental system. Zn accumulated in both roots and leaves, which led to a disturbed redox homeostasis as evident from increased H2O2 levels and altered glutathione redox state. In roots, magnesium uptake was affected and glutathione levels decreased due to reduced glutathione reductase activity and increased phytochelatin production. In leaves, mRNA levels of all chloroplast-localized superoxide dismutases (SOD) were downregulated, indicating that the converse regulation by Cu availability of FeSOD (FSD) and Cu/ZnSOD genes (CSDs) was disturbed. Furthermore, an opposite stress-dependent transcriptional regulation of the different loci of a miRNA species was observed: excess Zn repressed primary transcripts of the MIR398a gene, but increased MIR398b and MIR398c transcription. Also, accumulation of mRNA was inhibited for CSD2, but stimulated for CSD1. MiR398 has been reported to downregulate CSD1 and CSD2 mRNA. Here, the opposite effect on CSD1 and CSD2 mRNA accumulation under Zn stress in the leaves could be explained if CSD1 in the leaves would be regulated exclusively by miR398a, and not by miR398b/c under these conditions. The results support the existence of Zn-specific signal transduction pathways influencing anti-oxidative responses and are useful as a starting point for genetic screens identifying upstream metal-specific sensing and signaling mechanisms.