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.

Independent and interactive associations of dietary nitrate and salt intake with blood pressure and cognitive function: a cross-sectional analysis in the InCHIANTI study.

Blood pressure (BP) control is a key target for interventions to reduce cognitive decline. This cross-sectional study explored associations between objective (24-hour urine excretion) and subjective (food frequency questionnaire [FFQ]) measures of dietary sodium and nitrate intakes with cognitive function and resting BP in the InCHIANTI cohort. Baseline data from 989 participants aged >50 years were included. In fully adjusted models, participants with concurrent high nitrate and low sodium (Odds Ratio (OR)=0.49, 95%CI 0.32-0.76, p = 0.001) and high nitrate and high sodium (OR = 0.49, 95%CI 0.32-0.77, p = 0.002) 24-hour urinary concentrations had lower odds of high BP than participants with low nitrate and high sodium concentrations. We found no significant associations between sodium and nitrate intakes (24-hour urinary concentrations and FFQ) and poor cognitive performance. Urinary nitrate excretion was associated with lower BP and results appeared to be independent of sodium intake. Further analyses in longitudinal studies are required to substantiate these findings.

The Fabp5/calnexin complex is a prerequisite for sensitization of mice to experimental autoimmune encephalomyelitis.

We previously showed that calnexin (Canx)-deficient mice are desensitized to experimental autoimmune encephalomyelitis (EAE) induction, a model that is frequently used to study inflammatory demyelinating diseases, due to increased resistance of the blood-brain barrier to immune cell transmigration. We also discovered that Fabp5, an abundant cytoplasmic lipid-binding protein found in brain endothelial cells, makes protein-protein contact with the cytoplasmic C-tail domain of Canx. Remarkably, both Canx-deficient and Fabp5-deficient mice commonly manifest resistance to EAE induction. Here, we evaluated the importance of Fabp5/Canx interactions on EAE pathogenesis and on the patency of a model blood-brain barrier to T-cell transcellular migration. The results demonstrate that formation of a complex comprised of Fabp5 and the C-tail domain of Canx dictates the permeability of the model blood-brain barrier to immune cells and is also a prerequisite for EAE pathogenesis.

Urinary nitrate concentration as a marker for kidney transplant rejection.

BACKGROUND: Early identification and treatment of kidney transplant rejection episodes is vital to limit loss of function and prolong the life of the transplanted kidney and recipient. Current practice depends on detecting a creatinine rise. A biomarker to diagnose transplant rejection at an earlier time point than current practice, or to inform earlier decision making to biopsy, could be transformative. It has previously been shown that urinary nitrate concentration is elevated in renal transplant rejection. Nitrate is a nitric oxide (NO) oxidation product. Transplant rejection upregulates NO synthesis via inducible nitric oxide synthase leading to elevations in urinary nitrate concentration. We have recently validated a urinary nitrate concentration assay which could provide results in a clinically relevant timeframe. Our aim was to determine whether urinary nitrate concentration is a useful tool to predict renal transplant rejection in the context of contemporary clinical practice. METHODS: We conducted a prospective observational study, recruiting renal transplant participants over an 18-month period. We made no alterations to the patients' clinical care including medications, immunosuppression, diet and frequency of visits. We collected urine samples from every clinical attendance. We assessed the urinary nitrate to creatinine ratio (uNCR) between patient groups: routine attendances, biopsy proven rejection, biopsy proven no rejection and other call backs. uNCR was examined over time for those with biopsy proven transplant rejection. These four groups were compared using an ANOVA test. RESULTS: A total of 2656 samples were collected. uNCR during biopsy proven rejection, n = 15 (median 49 µmol/mmol, IQR 23-61) was not significantly different from that of routine samples, n = 164 (median 55 µmol/mmol, IQR 37-82) (p = 0.55), or biopsy proven no rejection, n = 12 (median 39 µmol/mmol, IQR 21-89) (P = 0.77). Overall uNCR was highly variable with no diagnostic threshold for kidney transplant rejection. Furthermore, within-patient uNCR was highly variable over time, and thus it was not possible to produce individualised patient thresholds to identify rejection. The total taking Tacrolimus was 204 patients, with no statistical difference between the uNCR of all those on Tacrolimus, against those not, p = 0.18. CONCLUSION: The urinary nitrate to creatinine ratio is not a useful biomarker for renal transplant rejection.

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.

Engagement of people with multiple sclerosis to enhance research into the physiological effect of hyperbaric oxygen therapy.

BACKGROUND: Thousands of people with multiple sclerosis (MS) have used self-administered oxygen therapy in the UK. Clinical trials have been performed, with scant evidence that people with MS have been consulted to explore how they benefit from or how to optimize this treatment. The conventional MS disease disability scores used in trials seldom reflect the effects individuals report when using oxygen therapy to treat their symptoms. METHODS: Three people with MS and the manager of an MS Centre formed a public involvement group and collaborated with clinicians and scientists to inform a lab-based study to investigate the physiological effects of oxygen therapy on microvascular brain endothelial cells. RESULTS: People with MS often use oxygen therapy at a later stage when their symptoms worsen and only after using other treatments. The frequency of oxygen therapy sessions and hyperbaric pressure is individualized and varies for people with MS. Despite direct comparisons of efficacy proving difficult, most individuals are exposed to 100% O2 at 1.5 atmosphere absolute (ATA; 1140 mmHg absolute) for 60 min. In a laboratory-based study human brain endothelial cells were exposed in vitro to 152 mmHg O2 for 60 min with and without pressure, as this equates to 20% O2 achievable via hyperbarics, which was then replicated at atmospheric pressure. A significant reduction in endothelial cells ICAM-1 (CD54) implicated in inflammatory cell margination across the blood brain barrier was observed under oxygen treatment. CONCLUSIONS: By collaborating with people living with MS, we were able to design laboratory-based experimental protocols that replicate their treatment regimens to advance our understanding of the physiological effects of hyperbaric oxygen treatment on brain cells and their role in neuroinflammation.

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.