Pharmacokinetics and Pharmacodynamics of Inorganic Nitrate in Heart Failure With Preserved Ejection Fraction.

RATIONALE: Nitrate-rich beetroot juice has been shown to improve exercise capacity in heart failure with preserved ejection fraction, but studies using pharmacological preparations of inorganic nitrate are lacking. OBJECTIVES: To determine (1) the dose-response effect of potassium nitrate (KNO3) on exercise capacity; (2) the population-specific pharmacokinetic and safety profile of KNO3 in heart failure with preserved ejection fraction. METHODS AND RESULTS: We randomized 12 subjects with heart failure with preserved ejection fraction to oral KNO3 (n=9) or potassium chloride (n=3). Subjects received 6 mmol twice daily during week 1, followed by 6 mmol thrice daily during week 2. Supine cycle ergometry was performed at baseline (visit 1) and after each week (visits 2 and 3). Quality of life was assessed with the Kansas City Cardiomyopathy Questionnaire. The primary efficacy outcome, peak O2-uptake, did not significantly improve (P=0.13). Exploratory outcomes included exercise duration and quality of life. Exercise duration increased significantly with KNO3 (visit 1: 9.87, 95% confidence interval [CI] 9.31-10.43 minutes; visit 2: 10.73, 95% CI 10.13-11.33 minute; visit 3: 11.61, 95% CI 11.05-12.17 minutes; P=0.002). Improvements in the Kansas City Cardiomyopathy Questionnaire total symptom (visit 1: 58.0, 95% CI 52.5-63.5; visit 2: 66.8, 95% CI 61.3-72.3; visit 3: 70.8, 95% CI 65.3-76.3; P=0.016) and functional status scores (visit 1: 62.2, 95% CI 58.5-66.0; visit 2: 68.6, 95% CI 64.9-72.3; visit 3: 71.1, 95% CI 67.3-74.8; P=0.01) were seen after KNO3. Pronounced elevations in trough levels of nitric oxide metabolites occurred with KNO3 (visit 2: 199.5, 95% CI 98.7-300.2 µmol/L; visit 3: 471.8, 95% CI 377.8-565.8 µmol/L) versus baseline (visit 1: 38.0, 95% CI 0.00-132.0 µmol/L; P<0.001). KNO3 did not lead to clinically significant hypotension or methemoglobinemia. After 6 mmol of KNO3, systolic blood pressure was reduced by a maximum of 17.9 (95% CI -28.3 to -7.6) mm Hg 3.75 hours later. Peak nitric oxide metabolites concentrations were 259.3 (95% CI 176.2-342.4) µmol/L 3.5 hours after ingestion, and the median half-life was 73.0 (interquartile range 33.4-232.0) minutes. CONCLUSIONS: KNO3 is potentially well tolerated and improves exercise duration and quality of life in heart failure with preserved ejection fraction. This study reinforces the efficacy of KNO3 and suggests that larger randomized trials are warranted. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT02256345.

Heart Failure, Left Ventricular Remodeling, and Circulating Nitric Oxide Metabolites.

BACKGROUND: Stable plasma nitric oxide (NO) metabolites (NOM), composed predominantly of nitrate and nitrite, are attractive biomarkers of NO bioavailability. NOM levels integrate the influence of NO-synthase-derived NO production/metabolism, dietary intake of inorganic nitrate/nitrite, and clearance of NOM. Furthermore, nitrate and nitrite, the most abundant NOM, can be reduced to NO via the nitrate-nitrite-NO pathway. METHODS AND RESULTS: We compared serum NOM among subjects without heart failure (n=126), subjects with heart failure and preserved ejection fraction (HFpEF; n=43), and subjects with heart failure and reduced ejection fraction (HFrEF; n=32). LV mass and extracellular volume fraction were measured with cardiac MRI. Plasma NOM levels were measured after reduction to NO via reaction with vanadium (III)/hydrochloric acid. Subjects with HFpEF demonstrated significantly lower unadjusted levels of NOM (8.0 µmol/L; 95% CI 6.2-10.4 µmol/L; ANOVA P=0.013) than subjects without HF (12.0 µmol/L; 95% CI 10.4-13.9 µmol/L) or those with HFrEF (13.5 µmol/L; 95% CI 9.7-18.9 µmol/L). There were no significant differences in NOM between subjects with HFrEF and subjects without HF. In a multivariable model that adjusted for age, sex, race, diabetes mellitus, body mass index, current smoking, systolic blood pressure, and glomerular filtration rate, HFpEF remained a predictor of lower NOM (ß=-0.43; P=0.013). NOM did not correlate with LV mass, or LV diffuse fibrosis. CONCLUSIONS: HFpEF, but not HFrEF, is associated with reduced plasma NOM, suggesting greater endothelial dysfunction, enhanced clearance, or deficient dietary ingestion of inorganic nitrate. Our findings may underlie the salutary effects of inorganic nitrate supplementation demonstrated in recent clinical trials in HFpEF.

Neutralizing Th2 inflammation in neonatal islets prevents ß-cell failure in adult IUGR rats.

Intrauterine growth restriction (IUGR) leads to development of type 2 diabetes (T2D) in adulthood. The mechanisms underlying this phenomenon have not been fully elucidated. Inflammation is associated with T2D; however, it is unknown whether inflammation is causal or secondary to the altered metabolic state. Here we show that the mechanism by which IUGR leads to the development of T2D in adulthood is via transient recruitment of T-helper 2 (Th) lymphocytes and macrophages in fetal islets resulting in localized inflammation. Although this immune response is short-lived, it results in a permanent reduction in islet vascularity and impaired insulin secretion. Neutralizing interleukin-4 antibody therapy given only in the newborn period ameliorates inflammation and restores vascularity and ß-cell function into adulthood, demonstrating a novel role for Th2 immune responses in the induction and progression of T2D. In the neonatal stage, inflammation and vascular changes are reversible and may define an important developmental window for therapeutic intervention to prevent adult-onset diabetes.

Interaction with phospholipids modulates alpha-synuclein nitration and lipid-protein adduct formation.

Intracellular aggregates of alpha-syn (alpha-synuclein) represent pathoanatomical hallmarks of neurodegenerative disorders (synucleinopathies). The molecular mechanisms underlying alpha-syn aggregation into filamentous inclusions may involve oxidation and nitration of the protein. Whereas the effects of oxidants and nitrating species on soluble alpha-syn have been studied in detail, the effect of these reactive species on alpha-syn associated with lipids is still unknown. In the present paper, we report that alpha-syn bound to small unilamellar liposomes composed of phosphatidylcholine/phosphatidic acid is resistant to oxidation and nitration when compared with soluble alpha-syn. Additionally, increasing concentrations of unsaturated fatty acids diminished the oxidation and nitration of alpha-syn upon exposure to fluxes of peroxynitrite (8-20 microM x min(-1)). To investigate the effect of oxidized lipids on alpha-syn, the protein was incubated with the bifunctional electrophile 4-HNE [4-hydroxy-2(E)-nonenal]. MS analysis showed the formation of three major products corresponding to the native protein and alpha-syn plus one or two 4-HNE molecules. Trypsin digestion of the modified protein followed by peptide 'finger-printing' revealed that 4-HNE modified the peptide E46GVVHGVATVAEK58. Further analysis of the peptides with liquid chromatography-tandem MS identified the modified residue as His50. The data indicate that the association of alpha-syn with biological membranes protects the protein from oxidation and nitration and thus diminishes the formation of protein molecules capable of forming aggregates. However, products of lipid peroxidation can also modify alpha-syn, generating novel protein adducts that could serve as biomarkers for documenting oxidative processes in human as well as animal and cellular models of alpha-syn aggregation and pathology.