Reduction in blood pressure following acute dietary nitrate ingestion is correlated with increased red blood cell S-nitrosothiol concentrations.

Dietary nitrate (NO3-) supplementation can enhance nitric oxide (NO) bioavailability and lower blood pressure (BP) in humans. The nitrite concentration ([NO2-]) in the plasma is the most commonly used biomarker of increased NO availability. However, it is unknown to what extent changes in other NO congeners, such as S-nitrosothiols (RSNOs), and in other blood components, such as red blood cells (RBC), also contribute to the BP lowering effects of dietary NO3-. We investigated the correlations between changes in NO biomarkers in different blood compartments and changes in BP variables following acute NO3- ingestion. Resting BP was measured and blood samples were collected at baseline, and at 1, 2, 3, 4 and 24 h following acute beetroot juice (∼12.8 mmol NO3-, ∼11 mg NO3-/kg) ingestion in 20 healthy volunteers. Spearman rank correlation coefficients were determined between the peak individual increases in NO biomarkers (NO3-, NO2-, RSNOs) in plasma, RBC and whole blood, and corresponding decreases in resting BP variables. No significant correlation was observed between increased plasma [NO2-] and reduced BP, but increased RBC [NO2-] was correlated with decreased systolic BP (rs = -0.50, P = 0.03). Notably, increased RBC [RSNOs] was significantly correlated with decreases in systolic (rs = -0.68, P = 0.001), diastolic (rs = -0.59, P = 0.008) and mean arterial pressure (rs = -0.64, P = 0.003). Fisher's z transformation indicated no difference in the strength of the correlations between increases in RBC [NO2-] or [RSNOs] and decreased systolic blood pressure. In conclusion, increased RBC [RSNOs] may be an important mediator of the reduction in resting BP observed following dietary NO3- supplementation.

Cellular Pre-Adaptation to the High O2 Concentration Used in Standard Cell Culture Confers Resistance to Subsequent H2O2-Induced Cell Death.

The addition of hydrogen peroxide (H2O2) to cultured cells is widely used as a method to modulate redox-regulated cellular pathways, including the induction of programmed cell death in cell culture experiments and the testing of pro- and antioxidant compounds. Here, we assessed the effect on the cellular response to H2O2 of pre-adapting squamous cell carcinoma cells (A431) to the standard cell culture oxygenation of 18.6% O2, compared to cells pre-adapted to a physiological skin O2 concentration (3.0% O2). We showed that cells pre-adapted to 18.6% O2 resisted H2O2-induced cell death compared to cells pre-adapted to 3.0% O2 for 96 h prior to treatment with H2O2. Moreover, the enzymatic activities of catalase and glutathione reductase, as well as the protein expression levels of catalase, were higher in cells pre-adapted to 18.6% O2 compared to cells pre-adapted to 3.0% O2. H2O2-resistant cells, pre-adapted to 18.6% O2, exhibited increased nuclear Nrf-2 levels. It is concluded that A431 cells pre-adapted to standard cell culture oxygenation conditions resist H2O2-induced cell death. This effect may be related to their heightened activation of Nrf-2.

STING-Triggered CNS Inflammation in Human Neurodegenerative Diseases.

BACKGROUND: Some neurodegenerative diseases have an element of neuroinflammation that is triggered by viral nucleic acids, resulting in the generation of type I interferons. In the cGAS-STING pathway, microbial and host-derived DNA bind and activate the DNA sensor cGAS, and the resulting cyclic dinucleotide, 2'3-cGAMP, binds to a critical adaptor protein, stimulator of interferon genes (STING), which leads to activation of downstream pathway components. However, there is limited work demonstrating the activation of the cGAS-STING pathway in human neurodegenerative diseases. METHODS: Post-mortem CNS tissue from donors with multiple sclerosis (n = 4), Alzheimer's disease (n = 6), Parkinson's disease (n = 3), amyotrophic lateral sclerosis (n = 3) and non-neurodegenerative controls (n = 11) were screened by immunohistochemistry for STING and relevant protein aggregates (e.g., amyloid-ß, α-synuclein, TDP-43). Human brain endothelial cells were cultured and stimulated with the STING agonist palmitic acid (1-400 µM) and assessed for mitochondrial stress (release of mitochondrial DNA into cytosol, increased oxygen consumption), downstream regulator factors, TBK-1/pIRF3 and inflammatory biomarker interferon-ß release and changes in ICAM-1 integrin expression. RESULTS: In neurodegenerative brain diseases, elevated STING protein was observed mainly in brain endothelial cells and neurons, compared to non-neurodegenerative control tissues where STING protein staining was weaker. Interestingly, a higher STING presence was associated with toxic protein aggregates (e.g., in neurons). Similarly high STING protein levels were observed within acute demyelinating lesions in multiple sclerosis subjects. To understand non-microbial/metabolic stress activation of the cGAS-STING pathway, brain endothelial cells were treated with palmitic acid. This evoked mitochondrial respiratory stress up to a ~2.5-fold increase in cellular oxygen consumption. Palmitic acid induced a statistically significant increase in cytosolic DNA leakage from endothelial cell mitochondria (Mander's coefficient; p < 0.05) and a significant increase in TBK-1, phosphorylated transcription factor IFN regulatory factor 3, cGAS and cell surface ICAM. In addition, a dose response in the secretion of interferon-ß was observed, but it failed to reach statistical significance. CONCLUSIONS: The histological evidence shows that the common cGAS-STING pathway appears to be activated in endothelial and neural cells in all four neurodegenerative diseases examined. Together with the in vitro data, this suggests that the STING pathway might be activated via perturbation of mitochondrial stress and DNA leakage, resulting in downstream neuroinflammation; hence, this pathway may be a target for future STING therapeutics.

S-nitrosothiols, and other products of nitrate metabolism, are increased in multiple human blood compartments following ingestion of beetroot juice.

Ingested inorganic nitrate (NO3⁻) has multiple effects in the human body including vasodilation, inhibition of platelet aggregation, and improved skeletal muscle function. The functional effects of oral NO3⁻ involve the in vivo reduction of NO3⁻ to nitrite (NO2⁻) and thence to nitric oxide (NO). However, the potential involvement of S-nitrosothiol (RSNO) formation is unclear. We hypothesised that the RSNO concentration ([RSNO]) in red blood cells (RBCs) and plasma is increased by NO3⁻-rich beetroot juice ingestion. In healthy human volunteers, we tested the effect of dietary supplementation with NO3⁻-rich beetroot juice (BR) or NO3⁻-depleted beetroot juice (placebo; PL) on [RSNO], [NO3⁻] and [NO2⁻] in RBCs, whole blood and plasma, as measured by ozone-based chemiluminescence. The median basal [RSNO] in plasma samples (n = 22) was 10 (5-13) nM (interquartile range in brackets). In comparison, the median values for basal [RSNO] in the corresponding RBC preparations (n = 19) and whole blood samples (n = 19) were higher (p < 0.001) than in plasma, being 40 (30-60) nM and 35 (25-80) nM, respectively. The median RBC [RSNO] in a separate cohort of healthy subjects (n = 5) was increased to 110 (93-125) nM after ingesting BR (12.8 mmol NO3⁻) compared to a corresponding baseline value of 25 (21-31) nM (Mann-Whitney test, p < 0.01). The median plasma [RSNO] in another cohort of healthy subjects (n = 14) was increased almost ten-fold to 104 (58-151) nM after BR supplementation (7 × 6.4 mmol of NO3⁻ over two days, p < 0.01) compared to PL. In conclusion, RBC and plasma [RSNO] are increased by BR ingestion. In addition to NO2⁻, RSNO may be involved in dietary NO3⁻ metabolism/actions.

Oxidative stress in autoimmune rheumatic diseases.

The management of patients with autoimmune rheumatic diseases such as rheumatoid arthritis (RA) remains a significant challenge. Often the rheumatologist is restricted to treating and relieving the symptoms and consequences and not the underlying cause of the disease. Oxidative stress occurs in many autoimmune diseases, along with the excess production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). The sources of such reactive species include NADPH oxidases (NOXs), the mitochondrial electron transport chain, nitric oxide synthases, nitrite reductases, and the hydrogen sulfide producing enzymes cystathionine-ß synthase and cystathionine-γ lyase. Superoxide undergoes a dismutation reaction to generate hydrogen peroxide which, in the presence of transition metal ions (e.g. ferrous ions), forms the hydroxyl radical. The enzyme myeloperoxidase, present in inflammatory cells, produces hypochlorous acid, and in healthy individuals ROS and RNS production by phagocytic cells is important in microbial killing. Both low molecular weight antioxidant molecules and antioxidant enzymes, such as superoxide dismutase, catalase, glutathione peroxidase, and peroxiredoxin remove ROS. However, when ROS production exceeds the antioxidant protection, oxidative stress occurs. Oxidative post-translational modifications of proteins then occur. Sometimes protein modifications may give rise to neoepitopes that are recognized by the immune system as 'non-self' and result in the formation of autoantibodies. The detection of autoantibodies against specific antigens, might improve both early diagnosis and monitoring of disease activity. Promising diagnostic autoantibodies include anti-carbamylated proteins and anti-oxidized type II collagen antibodies. Some of the most promising future strategies for redox-based therapeutic compounds are the activation of endogenous cellular antioxidant systems (e.g. Nrf2-dependent pathways), inhibition of disease-relevant sources of ROS/RNS (e.g. isoform-specific NOX inhibitors), or perhaps specifically scavenging disease-related ROS/RNS via site-specific antioxidants.

Relationship Between Urinary Nitrate Excretion and Blood Pressure in the InChianti Cohort.

BACKGROUND: Inorganic nitrate from the oxidation of endogenously synthesized nitric oxide (NO) or consumed in the diet can be reduced to NO via a complex enterosalivary circulation pathway. The relationship between total nitrate exposure by measured urinary nitrate excretion and blood pressure in a large population sample has not been assessed previously. METHODS: For this cross-sectional study, 24-hour urinary nitrate excretion was measured by spectrophotometry in the 919 participants from the InChianti cohort at baseline and blood pressure measured with a mercury sphygmomanometer. RESULTS: After adjusting for age and sex only, diastolic blood pressure was 1.9 mm Hg lower in subjects with ≥2 mmol urinary nitrate excretion compared with those excreting <1 mmol nitrate in 24 hours: systolic blood pressure was 3.4 mm Hg (95% confidence interval (CI): -3.5 to -0.4) lower in subjects for the same comparison. Effect sizes in fully adjusted models (for age, sex, potassium intake, use of antihypertensive medications, diabetes, HS-CRP, or current smoking status) were marginally larger: systolic blood pressure in the ≥2 mmol urinary nitrate excretion group was 3.9 (CI: -7.1 to -0.7) mm Hg lower than in the comparison <1 mmol excretion group. CONCLUSIONS: Modest differences in total nitrate exposure are associated with lower blood pressure. These differences are at least equivalent to those seen from substantial (100 mmol) reductions in sodium intake.

On the mechanism by which dietary nitrate improves human skeletal muscle function.

Inorganic nitrate is present at high levels in beetroot and celery, and in green leafy vegetables such as spinach and lettuce. Though long believed inert, nitrate can be reduced to nitrite in the human mouth and, further, under hypoxia and/or low pH, to nitric oxide. Dietary nitrate has thus been associated favorably with nitric-oxide-regulated processes including blood flow and energy metabolism. Indeed, the therapeutic potential of dietary nitrate in cardiovascular disease and metabolic syndrome-both aging-related medical disorders-has attracted considerable recent research interest. We and others have shown that dietary nitrate supplementation lowers the oxygen cost of human exercise, as less respiratory activity appears to be required for a set rate of skeletal muscle work. This striking observation predicts that nitrate benefits the energy metabolism of human muscle, increasing the efficiency of either mitochondrial ATP synthesis and/or of cellular ATP-consuming processes. In this mini-review, we evaluate experimental support for the dietary nitrate effects on muscle bioenergetics and we critically discuss the likelihood of nitric oxide as the molecular mediator of such effects.

Nitrite/nitrate detection in serum based on dual-plate generator-collector currents in a microtrench.

A dual-electrode sensor is developed for rapid detection of nitrite/nitrate at micromolar levels in phosphate buffer media and in dilute horse serum without additional sample pre-treatment. A generator-collector configuration is employed so that on one electrode nitrate is reduced to nitrite and on the second electrode nitrite is oxidised back to nitrate. The resulting redox cycle gives rise to a specific and enhanced current signal which is exploited for sensitive and reliable measurement of nitrite/nitrate in the presence of oxygen. The electrode design is based on a dual-plate microtrench (approximately 15 µm inter-electrode gap) fabricated from gold-coated glass and with a nano-silver catalyst for the reduction of nitrate. Fine tuning of the phosphate buffer pH is crucial for maximising collector current signals whilst minimising unwanted gold surface oxidation. A limit of detection of 24 µM nitrate and a linear concentration range of 200-1400 µM is reported for the microtrench sensor in phosphate buffer and dilute horse serum. Relative standard deviations for repeat measurements were in the range 1.8-6.9% (n=3) indicating good repeatability in both aqueous and biological media. Preliminary method validation against the standard chemiluminescence method used in medical laboratories is reported for nitrate analysis in serum.