Perioperative redox changes in patients undergoing hepato-pancreatico-biliary cancer surgery.

BACKGROUND: Tissue injury induces inflammation and the surgical stress response, which are thought to be central to the orchestration of recovery or deterioration after surgery. Enhanced formation of reactive oxygen and nitrogen species accompanies the inflammatory response and triggers separate but integrated reduction/oxidation (redox) pathways that lead to oxidative and/or nitrosative stress (ONS). Quantitative information on ONS in the perioperative period is scarce. This single-centre exploratory study investigated the effects of major surgery on ONS and systemic redox status and their potential associations with postoperative morbidity. METHODS: Blood was collected from 56 patients at baseline, end of surgery (EoS) and the first postoperative day (day-1). Postoperative morbidity was recorded using the Clavien-Dindo classification and further categorised into minor, moderate and severe. Plasma/serum measures included markers of lipid oxidation (thiobarbituric acid-reactive substances; TBARS, 4-hydroxynonenal; 4-HNE, 8-iso-prostaglandin F2⍺; 8-isoprostanes). Total reducing capacity was measured using total free thiols (TFTs) and ferric-reducing ability of plasma (FRAP). Nitric oxide (NO) formation/metabolism was measured using cyclic guanosine monophosphate (cGMP), nitrite, nitrate and total nitroso-species (RxNO). Interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF-⍺) were measured to evaluate inflammation. RESULTS: Both oxidative stress (TBARS) and nitrosative stress (total nitroso-species) increased from baseline to EoS (+14%, P = 0.003 and +138%, P < 0.001, respectively), along with an increase in overall reducing capacity (+9%, P = 0.03) at EoS and protein-adjusted total free thiols (+12%, P = 0.001) at day-1 after surgery. Nitrite, nitrate and cGMP concentrations declined concomitantly from baseline to day-1. Baseline nitrate was 60% higher in the minor morbidity group compared to severe (P = 0.003). The increase in intraoperative TBARS was greater in severe compared to minor morbidity (P = 0.01). The decline in intraoperative nitrate was more marked in the minor morbidity group compared to severe (P < 0.001), whereas the cGMP decline was greatest in the severe morbidity group (P = 0.006). CONCLUSION: In patients undergoing major HPB surgery, intraoperative oxidative and nitrosative stress increased, with a concomitant increase in reductive capacity. Baseline nitrate was inversely associated with postoperative morbidity, and the hallmarks of poor postoperative outcome include changes in both oxidative stress and NO metabolism.

Nitric oxide and long-term outcomes after kidney transplantation: Results of the TransplantLines cohort study.

Impaired endogenous nitric oxide (NO) production may contribute to graft failure and premature mortality in kidney transplant recipients (KTR). We investigated potential associations of 24-h urinary NOx (NO3- + NO2-) excretion (uNOx) with long-term outcomes. uNOx was determined by HPLC and GC-MS in 698 KTR and in 132 kidney donors before and after donation. Additionally, we measured urinary nitroso species (RXNO) by gas-phase chemiluminescence. Median uNOx was lower in KTR compared to kidney donors (688 [393-1076] vs. 1301 [868-1863] before donation and 1312 [982-1853] µmol/24 h after donation, P < 0.001). During median follow-up of 5.4 [4.8-6.1] years, 150 KTR died (61 due to cardiovascular disease) and 83 experienced graft failure. uNOx was inversely associated with all-cause mortality (HR per doubling of uNOx: 0.84 [95% CI 0.75-0.93], P < 0.001) and cardiovascular mortality (HR 0.78 [95% CI 0.67-0.92], P = 0.002). The association of uNOx with graft failure was lost when adjusted for renal function (HR per doubling of uNOx: 0.89 [95% CI 0.76-1.05], P = 0.17). There were no significant associations of urinary RXNO with outcomes. Our study suggests that KTR have lower NO production than healthy subjects and that lower uNOx is associated with a higher risk of all-cause and cardiovascular mortality.

Dietary Nitrate Supplementation Does Not Alter Exercise Efficiency at High Altitude - Further Results From the Xtreme Alps Study.

Introduction: Nitrate supplementation in the form of beetroot juice (BRJ) ingestion has been shown to improve exercise tolerance during acute hypoxia, but its effect on exercise physiology remains unstudied during sustained terrestrial high altitude exposure. We hypothesized that performing exercise at high altitude would lower circulating nitrate and nitrite levels and that BRJ ingestion would reverse this phenomenon while concomitantly improving key determinants of aerobic exercise performance. Methods: Twenty seven healthy volunteers (21 male) underwent a series of exercise tests at sea level (SL, London, 75 m) and again after 5-8 days at high altitude (HA, Capanna Regina Margherita or "Margherita Hut," 4,559 m). Using a double-blind protocol, participants were randomized to consume a beetroot/fruit juice beverage (three doses per day) with high levels of nitrate (∼0.18 mmol/kg/day) or a nitrate-depleted placebo (∼11.5 µmoles/kg/day) control drink, from 3 days prior to the exercise trials until completion. Submaximal constant work rate cycle tests were performed to determine exercise efficiency and a maximal incremental ramp exercise test was undertaken to measure aerobic capacity, using breath-by-breath pulmonary gas exchange measurements throughout. Concentrations of nitrate, nitrite and nitrosation products were quantified in plasma samples collected at 5 timepoints during the constant work rate tests. Linear mixed modeling was used to analyze data. Results: At both SL and HA, plasma nitrate concentrations were elevated in the nitrate supplementation group compared to placebo (P < 0.001) but did not change throughout increasing exercise work rate. Delta exercise efficiency was not altered by altitude exposure (P = 0.072) or nitrate supplementation (P = 0.836). V̇O2peak decreased by 24% at high altitude (P < 0.001) and was lower in the nitrate-supplemented group at both sea level and high altitude compared to placebo (P = 0.041). Dietary nitrate supplementation did not alter other peak exercise variables or oxygen consumption at anaerobic threshold. Circulating nitrite and S-nitrosothiol levels unexpectedly rose in a few individuals right after cessation of exercise at high altitude. Conclusion: Whilst regularly consumed during an 8 days expedition to terrestrial high altitude, nitrate supplementation did not alter exercise efficiency and other exercise physiological variables, except decreasing V̇O2peak. These results and those of others question the practical utility of BRJ consumption during prolonged altitude exposure.

Dietary nitrate supplementation to enhance exercise capacity in hypoxic COPD: EDEN-OX, a double-blind, placebo-controlled, randomised cross-over study.

RATIONALE: Dietary nitrate supplementation improves skeletal muscle oxygen utilisation and vascular endothelial function. We hypothesised that these effects might be sufficient to improve exercise performance in patients with COPD and hypoxia severe enough to require supplemental oxygen. METHODS: We conducted a single-centre, double-blind, placebo-controlled, cross-over study, enrolling adults with COPD who were established users of long-term oxygen therapy. Participants performed an endurance shuttle walk test, using their prescribed oxygen, 3 hours after consuming either 140 mL of nitrate-rich beetroot juice (BRJ) (12.9 mmol nitrate) or placebo (nitrate-depleted BRJ). Treatment order was allocated (1:1) by computer-generated block randomisation. MEASUREMENTS: The primary outcome was endurance shuttle walk test time. The secondary outcomes included area under the curve to isotime for fingertip oxygen saturation and heart rate parameters during the test, blood pressure, and endothelial function assessed using flow-mediated dilatation. Plasma nitrate and nitrite levels as well as FENO were also measured. MAIN RESULTS: 20 participants were recruited and all completed the study. Nitrate-rich BRJ supplementation prolonged exercise endurance time in all participants as compared with placebo: median (IQR) 194.6 (147.5-411.7) s vs 159.1 (121.9-298.5) s, estimated treatment effect 62 (33-106) s (p<0.0001). Supplementation also improved endothelial function: NR-BRJ group +4.1% (-1.1% to 14.8%) vs placebo BRJ group -5.0% (-10.6% to -0.6%) (p=0.0003). CONCLUSION: Acute dietary nitrate supplementation increases exercise endurance in patients with COPD who require supplemental oxygen. Trial registration number ISRCTN14888729.

Exercise Training Induces a Shift in Extracellular Redox Status with Alterations in the Pulmonary and Systemic Redox Landscape in Asthma.

Redox dysregulation and oxidative stress have been implicated in asthma pathogenesis. Exercise interventions improve symptoms and reduce inflammation in asthma patients, but the underlying mechanisms remain unclear. We hypothesized that a personalised exercise intervention would improve asthma control by reducing lung inflammation through modulation of local and systemic reactive species interactions, thereby increasing antioxidant capacity. We combined deep redox metabolomic profiling with clinical assessment in an exploratory cohort of six female patients with symptomatic asthma and studied their responses to a metabolically targeted exercise intervention over 12 weeks. Plasma antioxidant capacity and circulating nitrite levels increased following the intervention (p = 0.028) and lowered the ratio of reduced to oxidised glutathione (p = 0.029); this was accompanied by improvements in physical fitness (p = 0.046), symptoms scores (p = 0.020), quality of life (p = 0.046), lung function (p = 0.028), airway hyperreactivity (p = 0.043), and eosinophilic inflammation (p = 0.007). Increased physical fitness correlated with improved plasma antioxidant capacity (p = 0.019), peak oxygen uptake and nitrite changes (p = 0.005), the latter also associated with reductions in peripheral blood eosinophil counts (p = 0.038). Thus, increases in "redox resilience" may underpin the clinical benefits of exercise in asthma. An improved understanding of exercise-induced alterations in redox regulation offers opportunities for greater treatment personalisation and identification of new treatment targets.

Plasma Nitrate Levels Are Related to Metabolic Syndrome and Are Not Altered by Treatment with DPP-4 Inhibitor Linagliptin: A Randomised, Placebo-Controlled Trial in Patients with Early Type 2 Diabetes Mellitus.

The depletion of nitrate and nitrite, stable nitric oxide (NO) end-products, promotes adipose tissue dysfunction and insulin resistance (IR). Dipeptidyl peptidase-4 (DPP-4) inhibitors have the potentially beneficial side effect of increasing NO availability. In this study, nitrate and nitrite levels and the effects of DPP-4 inhibitor linagliptin were investigated in relation to metabolic syndrome (MetS) markers. Treatment-naive patients with early type 2 diabetes mellitus (T2DM) (n = 40, median age 63 IQR (55-67) years, 63% male, mean HbA1c 45 ± 4.4 mmol/mol) were randomized (1:1) to linagliptin (5 mg/day) or placebo. MetS-related markers (body mass index (BMI), triglycerides, HOMA-IR, gamma-glutamyltransferase (GGT), C-reactive protein (CRP), and adiponectin), plasma levels of nitrate, nitrite, total free thiols (TFT) and vegetable intake were estimated at baseline and after 4 and 26 weeks of treatment. Plasma nitrate, but not nitrite, correlated positively with vegetable intake (r = 0.38, p = 0.018) and was inversely associated with HOMA-IR (r = -0.44, p = 0.006), BMI (r = -0.35, p = 0.028), GGT (r = -0.37, p = 0.019) and CRP (r = -0.34, p = 0.034). The relationship between nitrate and HOMA-IR remained significant after adjusting for BMI, CRP, vegetable intake and GGT. With stable vegetable intake, nitrate and nitrite, TFT, adipokines and CRP did not change after 26 weeks of linagliptin treatment. While plasma nitrate is inversely associated with MetS, linagliptin treatment does not significantly influence nitrate and nitrite concentrations, oxidative stress, adipose tissue function and systemic inflammation.

Red Blood Cell and Endothelial eNOS Independently Regulate Circulating Nitric Oxide Metabolites and Blood Pressure.

BACKGROUND: Current paradigms suggest that nitric oxide (NO) produced by endothelial cells (ECs) through endothelial nitric oxide synthase (eNOS) in the vessel wall is the primary regulator of blood flow and blood pressure. However, red blood cells (RBCs) also carry a catalytically active eNOS, but its role is controversial and remains undefined. This study aimed to elucidate the functional significance of RBC eNOS compared with EC eNOS for vascular hemodynamics and nitric oxide metabolism. METHODS: We generated tissue-specific loss- and gain-of-function models for eNOS by using cell-specific Cre-induced gene inactivation or reactivation. We created 2 founder lines carrying a floxed eNOS (eNOSflox/flox) for Cre-inducible knockout (KO), and gene construct with an inactivated floxed/inverted exon (eNOSinv/inv) for a Cre-inducible knock-in (KI), which respectively allow targeted deletion or reactivation of eNOS in erythroid cells (RBC eNOS KO or RBC eNOS KI mice) or in ECs (EC eNOS KO or EC eNOS KI mice). Vascular function, hemodynamics, and nitric oxide metabolism were compared ex vivo and in vivo. RESULTS: The EC eNOS KOs exhibited significantly impaired aortic dilatory responses to acetylcholine, loss of flow-mediated dilation, and increased systolic and diastolic blood pressure. RBC eNOS KO mice showed no alterations in acetylcholine-mediated dilation or flow-mediated dilation but were hypertensive. Treatment with the nitric oxide synthase inhibitor Nγ-nitro-l-arginine methyl ester further increased blood pressure in RBC eNOS KOs, demonstrating that eNOS in both ECs and RBCs contributes to blood pressure regulation. Although both EC eNOS KOs and RBC eNOS KOs had lower plasma nitrite and nitrate concentrations, the levels of bound NO in RBCs were lower in RBC eNOS KOs than in EC eNOS KOs. Reactivation of eNOS in ECs or RBCs rescues the hypertensive phenotype of the eNOSinv/inv mice, whereas the levels of bound NO were restored only in RBC eNOS KI mice. CONCLUSIONS: These data reveal that eNOS in ECs and RBCs contribute independently to blood pressure homeostasis.

Divergent trajectories of cellular bioenergetics, intermediary metabolism and systemic redox status in survivors and non-survivors of critical illness.

BACKGROUND: Numerous pathologies result in multiple-organ failure, which is thought to be a direct consequence of compromised cellular bioenergetic status. Neither the nature of this phenotype nor its relevance to survival are well understood, limiting the efficacy of modern life-support. METHODS: To explore the hypothesis that survival from critical illness relates to changes in cellular bioenergetics, we combined assessment of mitochondrial respiration with metabolomic, lipidomic and redox profiling in skeletal muscle and blood, at multiple timepoints, in 21 critically ill patients and 12 reference patients. RESULTS: We demonstrate an end-organ cellular phenotype in critical illness, characterized by preserved total energetic capacity, greater coupling efficiency and selectively lower capacity for complex I and fatty acid oxidation (FAO)-supported respiration in skeletal muscle, compared to health. In survivors, complex I capacity at 48 h was 27% lower than in non-survivors (p = 0.01), but tended to increase by day 7, with no such recovery observed in non-survivors. By day 7, survivors' FAO enzyme activity was double that of non-survivors (p = 0.048), in whom plasma triacylglycerol accumulated. Increases in both cellular oxidative stress and reductive drive were evident in early critical illness compared to health. Initially, non-survivors demonstrated greater plasma total antioxidant capacity but ultimately higher lipid peroxidation compared to survivors. These alterations were mirrored by greater levels of circulating total free thiol and nitrosated species, consistent with greater reductive stress and vascular inflammation, in non-survivors compared to survivors. In contrast, no clear differences in systemic inflammatory markers were observed between the two groups. CONCLUSION: Critical illness is associated with rapid, specific and coordinated alterations in the cellular respiratory machinery, intermediary metabolism and redox response, with different trajectories in survivors and non-survivors. Unravelling the cellular and molecular foundation of human resilience may enable the development of more effective life-support strategies.

Circulating biomarkers of nitric oxide bioactivity and impaired muscle vasoreactivity to exercise in adults with uncomplicated type 1 diabetes.

AIMS/HYPOTHESIS: Early compromised endothelial function challenges the ability of individuals with type 1 diabetes to perform normal physical exercise. The exact mechanisms underlying this vascular limitation remain unknown, but may involve either formation or metabolism of nitric oxide (NO), a major vasodilator, whose activity is known to be compromised by oxidative stress. METHODS: Muscle microvascular reactivity (near-infrared spectroscopy) to an incremental exhaustive bout of exercise was assessed in 22 adults with uncomplicated type 1 diabetes (HbA1c 64.5 ± 15.7 mmol/mol; 8.0 ± 1.4%) and in 21 healthy individuals (18-40 years of age). NO-related substrates/metabolites were also measured in the blood along with other vasoactive compounds and oxidative stress markers; measurements were taken at rest, at peak exercise and after 15 min of recovery. Demographic characteristics, body composition, smoking status and diet were comparable in both groups. RESULTS: Maximal oxygen uptake was impaired in individuals with type 1 diabetes compared with in healthy participants (35.6 ± 7.7 vs 39.6 ± 6.8 ml min-1 kg-1, p < 0.01) despite comparable levels of habitual physical activity (moderate to vigorous physical activity by accelerometery, 234.9 ± 160.0 vs 280.1 ± 114.9 min/week). Compared with non-diabetic participants, individuals with type 1 diabetes also displayed a blunted exercise-induced vasoreactivity (muscle blood volume at peak exercise as reflected by ∆ total haemoglobin, 2.03 ± 5.82 vs 5.33 ± 5.54 µmol/l; interaction 'exercise' × 'group', p < 0.05); this was accompanied by lower K+ concentration (p < 0.05), reduced plasma L-arginine (p < 0.05)-in particular when HbA1c was high (mean estimation: -4.0, p < 0.05)-and lower plasma urate levels (p < 0.01). Nonetheless, exhaustive exercise did not worsen lipid peroxidation or other oxidative stress biomarkers, and erythrocytic enzymatic antioxidant resources were mobilised to a comparable extent in both groups. Nitrite and total nitrosation products, which are potential alternative NO sources, were similarly unaltered. Graphical abstract CONCLUSIONS/INTERPRETATION: Participants with uncomplicated type 1 diabetes displayed reduced availability of L-arginine, the essential substrate for enzymatic nitric oxide synthesis, as well as lower levels of the major plasma antioxidant, urate. Lower urate levels may reflect a defect in the activity of xanthine oxidase, an enzyme capable of producing NO from nitrite under hypoxic conditions. Thus, both canonical and non-canonical NO production may be reduced. However, neither of these changes exacerbated exercise-induced oxidative stress. TRIAL REGISTRATION: clinicaltrials.gov NCT02051504.

Effects of dietary nitrate supplementation on microvascular physiology at 4559 m altitude - A randomised controlled trial (Xtreme Alps).

Native highlanders (e.g. Sherpa) demonstrate remarkable hypoxic tolerance, possibly secondary to higher levels of circulating nitric oxide (NO) and increased microcirculatory blood flow. As part of the Xtreme Alps study (a randomised placebo-controlled trial of dietary nitrate supplementation under field conditions of hypobaric hypoxia), we investigated whether dietary supplementation with nitrate could improve NO availability and microvascular blood flow in lowlanders. Plasma measurements of nitrate, nitrite and nitroso species were performed together with measurements of sublingual (sidestream dark-field camera) and forearm blood flow (venous occlusion plethysmography) in 28 healthy adult volunteers resident at 4559 m for 1 week; half receiving a beetroot-based high-nitrate supplement and half receiving an identically-tasting low nitrate 'placebo'. Dietary supplementation increased plasma nitrate concentrations 4-fold compared to the placebo group, both at sea level (SL; 19.2 vs 76.9 µM) and at day 5 (D5) of high altitude (22.9 vs 84.3 µM, p < 0.001). Dietary nitrate supplementation also significantly increased both plasma nitrite (0.78 vs. 0.86 µM SL, 0.31 vs. 0.41 µM D5, p = 0.03) and total nitroso product (11.3 vs. 19.7 nM SL, 9.7 vs. 12.3 nM D5, p < 0.001) levels both at sea level and at 4559 m. However, plasma nitrite concentrations were more than 50% lower at 4559 m compared to sea level in both treatment groups. Despite these significant changes, dietary nitrate supplementation had no effect on any measured read-outs of sublingual or forearm blood flow, even when environmental hypoxia was experimentally reversed using supplemental oxygen. In conclusion, dietary nitrate supplementation does not improve microcirculatory function at 4559 m.

Long-lasting blood pressure lowering effects of nitrite are NO-independent and mediated by hydrogen peroxide, persulfides, and oxidation of protein kinase G1α redox signalling.

AIMS: Under hypoxic conditions, nitrite (NO2-) can be reduced to nitric oxide (NO) eliciting vasorelaxation. However, nitrite also exerts vasorelaxant effects of potential therapeutic relevance under normal physiological conditions via undetermined mechanisms. We, therefore, sought to investigate the mechanism(s) by which nitrite regulates the vascular system in normoxia and, specifically, whether the biological effects are a result of NO generation (as in hypoxia) or mediated via alternative mechanisms involving classical downstream targets of NO [e.g. effects on protein kinase G1α (PKG1α)]. METHODS AND RESULTS: Ex vivo myography revealed that, unlike in thoracic aorta (conduit vessels), the vasorelaxant effects of nitrite in mesenteric resistance vessels from wild-type (WT) mice were NO-independent. Oxidants such as H2O2 promote disulfide formation of PKG1α, resulting in NO- cyclic guanosine monophosphate (cGMP) independent kinase activation. To explore whether the microvascular effects of nitrite were associated with PKG1α oxidation, we used a Cys42Ser PKG1α knock-in (C42S PKG1α KI; 'redox-dead') mouse that cannot transduce oxidant signals. Resistance vessels from these C42S PKG1α KI mice were markedly less responsive to nitrite-induced vasodilation. Intraperitoneal (i.p.) bolus application of nitrite in conscious WT mice induced a rapid yet transient increase in plasma nitrite and cGMP concentrations followed by prolonged hypotensive effects, as assessed using in vivo telemetry. In the C42S PKG1α KI mice, the blood pressure lowering effects of nitrite were lower compared to WT. Increased H2O2 concentrations were detected in WT resistance vessel tissue challenged with nitrite. Consistent with this, increased cysteine and glutathione persulfide levels were detected in these vessels by mass spectrometry, matching the temporal profile of nitrite's effects on H2O2 and blood pressure. CONCLUSION: Under physiological conditions, nitrite induces a delayed and long-lasting blood pressure lowering effect, which is NO-independent and occurs via a new redox mechanism involving H2O2, persulfides, and PKG1α oxidation/activation. Targeting this novel pathway may provide new prospects for anti-hypertensive therapy.

Inorganic nitrate and nitrite supplementation fails to improve skeletal muscle mitochondrial efficiency in mice and humans.

BACKGROUND: Inorganic nitrate, abundant in leafy green vegetables and beetroot, is thought to have protective health benefits. Adherence to a Mediterranean diet reduces the incidence and severity of coronary artery disease, whereas supplementation with nitrate can improve submaximal exercise performance. Once ingested, oral commensal bacteria may reduce nitrate to nitrite, which may subsequently be reduced to nitric oxide during conditions of hypoxia and in the presence of "nitrite reductases" such as heme- and molybdenum-containing enzymes. OBJECTIVE: We aimed to explore the putative effects of inorganic nitrate and nitrite on mitochondrial function in skeletal muscle. METHODS: Mice were subjected to a nitrate/nitrite-depleted diet for 2 wk, then supplemented with sodium nitrate, sodium nitrite, or sodium chloride (1 g/L) in drinking water ad libitum for 7 d before killing. Skeletal muscle mitochondrial function and expression of uncoupling protein (UCP) 3, ADP/ATP carrier protein (AAC) 1 and AAC2, and pyruvate dehydrogenase (PDH) were assessed by respirometry and Western blotting. Studies were also undertaken in human skeletal muscle biopsies from a cohort of coronary artery bypass graft patients treated with either sodium nitrite (30-min infusion of 10 µmol/min) or vehicle [0.9% (wt:vol) saline] 24 h before surgery. RESULTS: Neither sodium nitrate nor sodium nitrite supplementation altered mitochondrial coupling efficiency in murine skeletal muscle, and expression of UCP3, AAC1, or AAC2, and PDH phosphorylation status did not differ between the nitrite and saline groups. Similar results were observed in human samples. CONCLUSIONS: Sodium nitrite failed to improve mitochondrial metabolic efficiency, rendering this mechanism implausible for the purported exercise benefits of dietary nitrate supplementation. This trial was registered at clinicaltrials.gov as NCT04001283.

Characterising nitric oxide-mediated metabolic benefits of low-dose ultraviolet radiation in the mouse: a focus on brown adipose tissue.

AIMS/HYPOTHESIS: Exposure to sunlight has the potential to suppress metabolic dysfunction and obesity. We previously demonstrated that regular exposure to low-doses of ultraviolet radiation (UVR) reduced weight gain and signs of diabetes in male mice fed a high-fat diet, in part via release of nitric oxide from skin. Here, we explore further mechanistic pathways through which low-dose UVR exerts these beneficial effects. METHODS: We fed mice with a luciferase-tagged Ucp1 gene (which encodes uncoupling protein-1 [UCP-1]), referred to here as the Ucp1 luciferase transgenic mouse ('Thermomouse') a high-fat diet and examined the effects of repeated exposure to low-dose UVR on weight gain and development of metabolic dysfunction as well as UCP-1-dependent thermogenesis in interscapular brown adipose tissue (iBAT). RESULTS: Repeated exposure to low-dose UVR suppressed the development of glucose intolerance and hepatic lipid accumulation via dermal release of nitric oxide while also reducing circulating IL-6 (compared with mice fed a high-fat diet only). Dietary nitrate supplementation did not mimic the effects of low-dose UVR. A single low dose of UVR increased UCP-1 expression (by more than twofold) in iBAT of mice fed a low-fat diet, 24 h after exposure. However, in mice fed a high-fat diet, there was no effect of UVR on UCP-1 expression in iBAT (compared with mock-treated mice) when measured at regular intervals over 12 weeks. More extensive circadian studies did not identify any substantial shifts in UCP-1 expression in mice exposed to low-dose UVR, although skin temperature at the interscapular site was reduced in UVR-exposed mice. The appearance of cells with a white adipocyte phenotype ('whitening') in iBAT induced by consuming the high-fat diet was suppressed by exposure to low-dose UVR in a nitric oxide-dependent fashion. Significant shifts in the expression of important core gene regulators of BAT function (Dio2, increased more than twofold), fatty acid transport (increased Fatp2 [also known as Slc27a2]), lipolysis (decreased Atgl [also known as Pnpla2]), lipogenesis (decreased Fasn) and inflammation (decreased Tnf), and proportions of macrophages (increased twofold) were observed in iBAT of mice exposed to low-dose UVR. These effects were independent of nitric oxide released from skin. CONCLUSIONS/INTERPRETATION: Our results suggest that non-burning (low-dose) UVR suppresses the BAT 'whitening', steatotic and pro-diabetic effects of consuming a high-fat diet through skin release of nitric oxide, with some metabolic and immune pathways in iBAT regulated by UVR independently of nitric oxide.

Blueberries improve biomarkers of cardiometabolic function in participants with metabolic syndrome-results from a 6-month, double-blind, randomized controlled trial.

BACKGROUND: Anthocyanin-rich blueberry intake is associated with reduced type 2 diabetes and cardiovascular disease (CVD) risk in prospective studies, although long-term randomized controlled trials (RCTs) have not been conducted in at-risk populations. OBJECTIVE: In the longest-duration RCT to date, we examined the effect of 6-mo blueberry intake on insulin resistance and cardiometabolic function in metabolic syndrome. METHODS: A double-blind, parallel RCT (n = 115; age 63 ± 7 y; 68% male; body mass index 31.2 ± 3.0 kg/m2) was conducted, which fed 2 dietarily achievable blueberry intakes [equivalent to 1/2 and 1 cup/d (75/150 g)] compared with matched placebo. Insulin resistance was assessed via the homeostasis model assessment of insulin resistance (primary endpoint) and confirmed by [6-6-2H2]-glucose-labeled, 2-step hyperinsulinemic clamp (n = 20). Clinically relevant cardiometabolic endpoints [including flow-mediated dilatation, augmentation index, lipoprotein status (by nuclear magnetic resonance spectroscopy), and nitric oxide (NO)-related metabolite assay] and anthocyanin metabolism were assessed. RESULTS: A daily intake of 1 cup of blueberries improved endothelial function (flow-mediated dilatation: +1.45%; 95% CI: 0.83%, 2.1%; P = 0.003), systemic arterial stiffness (augmentation index: -2.24%; 95% CI: -3.97%, -0.61%; P = 0.04) and attenuated cyclic guanosine monophosphate concentrations. In statin nonusers (n = 71), elevated high-density lipoprotein cholesterol (+0.08 mmol/L; P = 0.03), high-density lipoprotein particle density (+0.48n, ×10-6; P = 0.002) and apolipoprotein A-I (+0.05 g/L; P = 0.01) concentrations were observed following the 1-cup/d intervention. Treatment compliance was 94.1% (wrapper returns) and total concentrations of anthocyanin-derived phenolic acid metabolites significantly increased, dose-dependently, in serum and 24-h urine (P < 0.01 and P < 0.001, respectively). Insulin resistance, pulse wave velocity, blood pressure, NO, and overall plasma thiol status were unaffected. Likewise, a half cup per day had no effect on any biomarkers. CONCLUSIONS: Despite insulin resistance remaining unchanged we show, to our knowledge, the first sustained improvements in vascular function, lipid status, and underlying NO bioactivity following 1 cup blueberries/d. With effect sizes predictive of 12-15% reductions in CVD risk, blueberries should be included in dietary strategies to reduce individual and population CVD risk. This study was registered at clinicaltrials.gov as NCT02035592.

Reactive Species Interactome Alterations in Oocyte Donation Pregnancies in the Absence and Presence of Pre-Eclampsia.

In pregnancy, maternal physiology is subject to considerable adaptations, including alterations in cardiovascular and metabolic function as well as development of immunological tolerance towards the fetus. In an oocyte donation pregnancy, the fetus is fully allogeneic towards the mother, since it carries both oocyte donor antigens and paternal antigens. Therefore, oocyte donation pregnancies result in an immunologically challenging pregnancy, which is reflected by a higher-than-normal risk to develop pre-eclampsia. Based on the allogeneic conditions in oocyte donation pregnancies, we hypothesized that this situation may translate into alterations in concentration of stable readouts of constituents of the reactive species interactome (RSI) compared to normal pregnancies, especially serum free thiols, nitric oxide (NO) and hydrogen sulfide (H2S) related metabolites. Indeed, total free thiol levels and nitrite (NO2-) concentrations were significantly lower whereas protein-bound NO and sulfate (SO42-) concentrations were significantly higher in both oocyte donation and naturally conceived pregnancies complicated by pre-eclampsia. The increased concentrations of nitrite observed in uncomplicated oocyte donation pregnancies suggest that endothelial NO production is compensatorily enhanced to lower vascular tone. More research is warranted on the role of the RSI and bioenergetic status in uncomplicated oocyte donation pregnancies and oocyte donation pregnancies complicated by pre-eclampsia.

Rapid free thiol rebound is a physiological response following cold-induced vasoconstriction in healthy humans, primary Raynaud and systemic sclerosis.

Raynaud's phenomenon (RP) is often the first sign of systemic sclerosis (SSc). Molecular mechanisms involved are incompletely understood, but reactive oxygen, nitrogen, and sulfur species are thought to play an important role in the pathogenesis of SSc. Free thiol groups play a protective role against oxidative stress and may represent an attractive therapeutic target. We aimed to investigate the effects of hypothermia-induced vasoconstriction on the responsiveness of redox-related markers. Thirty participants (n = 10/group [SSc, primary Raynaud's phenomenon (PRP), healthy controls (HC)]) were included in this study. Fingertip photoelectric plethysmography was performed during a standardized cooling and recovery experiment. Venous blood was collected at four predetermined time points. Free thiols, NO-derived species (nitros(yl)ated species, nitrite, nitrate), sulfate and endothelin-1 were measured. Lower baseline concentrations of free thiols were observed in PRP and SSc patients (HC: 5.87 [5.41-5.99] µmol/g; PRP: 5.17 [4.74-5.61]; SSc 5.28 [4.75-5.80], P = 0.04). Redox-related markers remained unchanged during cooling. However, an unexpected increase in systemic free thiol concentrations was observed in all groups during the recovery phase. The response of this marker differed between groups, with a higher increase found in SSc patients (HC Δ = 1.30 [1.48-1.17]; PRP Δ = 1.04 [1.06-1.03]; SSc Δ = 1.72 [1.13-1.49], P = 0.04). NO-derived species, sulfate and endothelin-1 levels remained unchanged throughout the recovery phase. This exploratory study sheds light on the rapid responsiveness of systemic free thiol concentrations following reperfusion, which may reflect overall redox balance. The robust response to reperfusion in SSc patients suggests that reductive systems involved in this response are functionally intact in these patients.

Pushing arterial-venous plasma biomarkers to new heights: A model for personalised redox metabolomics?

The chemical and functional interactions between Reactive Oxygen (ROS), Nitrogen (RNS) and Sulfur (RSS) species allow organisms to detect and respond to metabolic and environmental stressors, such as exercise and altitude exposure. Whether redox markers and constituents of this 'Reactive Species Interactome' (RSI) differ in concentration between arterial and venous blood is unknown. We hypothesised that such measurements may provide useful insight into metabolic/redox regulation at the whole-body level and would be consistent between individuals exposed to identical challenges. An exploratory study was performed during the Xtreme Alps expedition in 2010 in which four healthy individuals (2 male, 2 female) underwent paired arterial and central venous blood sampling before, during and after performance of a constant-work-rate cardiopulmonary exercise test, at sea level and again at 4559 m. Unexpectedly, plasma total free thiol and free cysteine concentrations remained substantially elevated at altitude throughout exercise with minimal arteriovenous gradients. Free sulfide concentrations changed only modestly upon combined altitude/exercise stress, whereas bound sulfide levels were lower at altitude than sea-level. No consistent signal indicative of the expected increased oxidative stress and nitrate→nitrite→NO reduction was observed with 4-hydroxynonenal, isoprostanes, nitrate, nitrite, nitroso species and cylic guanosine monophosphate. However, the observed arteriovenous concentration differences revealed a dynamic pattern of response that was unique to each participant. This novel redox metabolomic approach of obtaining quantifiable 'metabolic signatures' to a defined physiological challenge could potentially offer new avenues for personalised medicine.

Lower limb ischemic preconditioning combined with dietary nitrate supplementation does not influence time-trial performance in well-trained cyclists.

OBJECTIVES: Dietary nitrate (NO3-) supplementation and ischaemic preconditioning (IPC) can independently improve exercise performance. The purpose of this study was to explore whether NO3- supplementation, ingested prior to an IPC protocol, could synergistically enhance parameters of exercise. DESIGN: Double-blind randomized crossover trial. METHODS: Ten competitive male cyclists (age 34±6years, body mass 78.9±4.9kg, V⋅O2peak 55±4 mLkgmin-1) completed an incremental exercise test followed by three cycling trials comprising a square-wave submaximal component and a 16.1km time-trial. Oxygen uptake (V⋅O2) and muscle oxygenation kinetics were measured throughout. The baseline (BASE) trial was conducted without any dietary intervention or IPC. In the remaining two trials, participants received 3×5min bouts of lower limb bilateral IPC prior to exercise. Participants ingested NO3--rich gel (NIT+IPC) 90min prior to testing in one trial and a low NO3- placebo in the other (PLA+IPC). Plasma NO3- and nitrite (NO2-) were measured immediately before and after application of IPC. RESULTS: Plasma [NO3-] and [NO2-] were higher before and after IPC in NIT+IPC compared to BASE (P<0.001) but did not differ between BASE and PLA+IPC. There were no differences in V⋅O2 kinetics or muscle oxygenation parameters between trials (all P>0.4). Performance in the time-trial was similar between trials (BASE 1343±72s, PLA+IPC 1350±75s, NIT+IPC 1346±83s, P=0.98). CONCLUSIONS: Pre-exercise IPC did not improve sub-maximal exercise or performance measures, either alone or in combination with dietary NO3- supplementation.

Pharmacological preconditioning with inhaled nitric oxide (NO): Organ-specific differences in the lifetime of blood and tissue NO metabolites.

BACKGROUND: Endogenous nitric oxide (NO) may contribute to ischemic and anesthetic preconditioning while exogenous NO protects against ischemia-reperfusion (I/R) injury in the heart and other organs. Why those beneficial effects observed in animal models do not always translate into clinical effectiveness remains unclear. To mitigate reperfusion damage a source of NO is required. NO inhalation is known to increase tissue NO metabolites, but little information exists about the lifetime of these species. We therefore sought to investigate the fate of major NO metabolite classes following NO inhalation in mice in vivo. METHODS: C57BL/6J mice were exposed to 80 ppm NO for 1 h. NO metabolites were measured in blood (plasma and erythrocytes) and tissues (heart, liver, lung, kidney and brain) immediately after NO exposure and up to 48 h thereafter. Concentrations of S-nitrosothiols, N-nitrosamines and NO-heme products as well as nitrite and nitrate were quantified by gas-phase chemiluminescence and ion chromatography. In separate experiments, mice breathed 80 ppm NO for 1 h prior to cardiac I/R injury (induced by coronary arterial ligation for 1 h, followed by recovery). After sacrifice, the size of the myocardial infarction (MI) and the area at risk (AAR) were measured. RESULTS: After NO inhalation, elevated nitroso/nitrosyl levels returned to baseline over the next 24 h, with distinct multi-phasic decay profiles in each compartment. S/N-nitroso compounds and NO-hemoglobin in blood decreased exponentially, but remained above baseline for up to 30min, whereas nitrate was elevated for up to 3hrs after discontinuing NO breathing. Hepatic S/N-nitroso species concentrations remained steady for 30min before dropping exponentially. Nitrate only rose in blood, liver and kidney; nitrite tended to be lower in all organs immediately after NO inhalation but fluctuated considerably in concentration thereafter. NO inhalation before myocardial ischemia decreased the ratio of MI/AAR by 30% vs controls (p = 0.002); only cardiac S-nitrosothiols and NO-hemes were elevated at time of reperfusion onset. CONCLUSIONS: Metabolites in blood do not reflect NO metabolite status of any organ. Although NO is rapidly inactivated by hemoglobin-mediated oxidation in the circulation, long-lived tissue metabolites may account for the myocardial preconditioning effects of inhaled NO. NO inhalation may afford similar protection in other organs.

Nrf2 Deficiency Unmasks the Significance of Nitric Oxide Synthase Activity for Cardioprotection.

The transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a key master switch that controls the expression of antioxidant and cytoprotective enzymes, including enzymes catalyzing glutathione de novo synthesis. In this study, we aimed to analyze whether Nrf2 deficiency influences antioxidative capacity, redox state, NO metabolites, and outcome of myocardial ischemia reperfusion (I/R) injury. In Nrf2 knockout (Nrf2 KO) mice, we found elevated eNOS expression and preserved NO metabolite concentrations in the aorta and heart as compared to wild types (WT). Unexpectedly, Nrf2 KO mice have a smaller infarct size following myocardial ischemia/reperfusion injury than WT mice and show fully preserved left ventricular systolic function. Inhibition of NO synthesis at onset of ischemia and during early reperfusion increased myocardial damage and systolic dysfunction in Nrf2 KO mice, but not in WT mice. Consistent with this, infarct size and diastolic function were unaffected in eNOS knockout (eNOS KO) mice after ischemia/reperfusion. Taken together, these data suggest that eNOS upregulation under conditions of decreased antioxidant capacity might play an important role in cardioprotection against I/R. Due to the redundancy in cytoprotective mechanisms, this fundamental antioxidant property of eNOS is not evident upon acute NOS inhibition in WT mice or in eNOS KO mice until Nrf2-related signaling is abrogated.