Effects of sodium nitrite supplementation on vascular function and 2 related small metabolite signatures in middle - aged and older adults 3 4 Allison

34 Insufficient nitric oxide (NO) bioavailability plays an important role in endothelial dysfunction and 35 arterial stiffening with aging. Supplementation with sodium nitrite, a precursor of NO, 36 ameliorates age-related vascular endothelial dysfunction and arterial stiffness in mice, but 37 effects on humans, including the metabolic pathways altered, are unknown. The purpose of this 38 study was to determine the safety, feasibility and efficacy of oral sodium nitrite supplementation 39 for improving vascular function in middle-aged and older adults, and to identify related 40 circulating metabolites. Ten weeks of sodium nitrite (80 or 160 mg/day, capsules, TheraVasc, 41 Inc., randomized, placebo-control, double-blind) increased plasma nitrite acutely (5to 15-fold, 42 p<0.001 vs. placebo) and chronically (p<0.10), and was well-tolerated without symptomatic 43 hypotension or clinically-relevant elevations in blood methemoglobin. Endothelial function, 44 measured by brachial artery flow-mediated dilation, increased 45-60% vs. baseline (p<0.10) 45 without changes in body mass or blood lipids. Measures of carotid artery elasticity (ultrasound 46 and applanation tonometry) improved (decreased β-stiffness index, increased cross-sectional 47 compliance, p<0.05) without changes in brachial or carotid artery blood pressure. Aortic pulse 48 wave velocity was unchanged. Nitrite-induced changes in vascular measures were significantly 49 related to 11 plasma metabolites identified by untargeted analysis. Baseline abundance of 50 multiple metabolites, including glycerophospholids and fatty acyls, predicted vascular changes 51 with nitrite. This study provides evidence that sodium nitrite supplementation is well-tolerated, 52 increases plasma nitrite concentrations, improves endothelial function and lessens carotid artery 53 stiffening in middle-aged and older adults, perhaps by altering multiple metabolic pathways, 54 thereby warranting a larger clinical trial. 55 56 57 58 59 by 10.0.33.1 on July 2, 2017 http://jaysiology.org/ D ow nladed fom Introduction 60 61 Cardiovascular diseases (CVD) remain the leading cause of mortality in modern 62 societies and advancing age is the primary risk factor for CVD (26). With the rapidly changing 63 demographics of aging and record numbers of older adults, the burden of disease is projected 64 to increase to unprecedented levels in the near future (14). As such, it is of upmost importance 65 to establish evidence-based interventions to prevent or delay the development and progression 66 of CVD. 67 As much as 80% of all CVD are associated with dysfunction and disorders of arteries, 68 namely stiffening of the large elastic arteries and vascular endothelial dysfunction (24, 26). 69 Several mechanisms are responsible for these changes, with reductions in the bioavailability of 70 the vascular-protective molecule, nitric oxide (NO) being among the most important (7, 33, 34). 71 The nitrite anion (inorganic nitrite), once considered an inert byproduct of NO metabolism, is 72 now recognized as a cytoprotective molecule and the major storage form of NO in tissues. 73 Through a one-step reduction to NO, nitrite augments circulating and tissue NO bioavailability 74 and also acts as an independent signaling molecule, giving it substantial therapeutic potential 75 for the prevention and treatment of arterial aging and CVD (34). Short-term supplementation 76 with sodium nitrite reverses aortic stiffness and endothelial dysfunction in old mice (10, 35), and 77 results of recent studies examining dietary nitrate in adults with hypertension (18) or 78 cardiovascular risk factors (31) or sodium nitrite in patients with chronic clinical diseases (25) 79 suggest possible benefits of these approaches in humans. However, presently there is no 80 evidence supporting the use of chronic nitrite supplementation for improving impaired baseline 81 arterial function in otherwise healthy middle-aged and older adults in the context of maintaining 82 optimal vascular health and related CVD risk profiles. 83 Accordingly, we performed a study to determine the feasibility, safety, dosing and 84 efficacy of nitrite supplementation on arterial function in healthy middle-aged and older adults 85 prior to conducting a larger clinical trial. We hypothesized that 10 weeks of oral sodium nitrite 86 by 10.0.33.1 on July 2, 2017 http://jaysiology.org/ D ow nladed fom supplementation (80 or 160 mg/d) would reduce age-related vascular endothelial dysfunction 87 and arterial stiffness in this group. The study was designed to test key aspects necessary for 88 conducting a future larger-scale trial, including the ability to recruit, retain and successfully 89 collect and analyze required data in this population; tolerability and adherence with the 90 intervention; and efficacy and optimal dosing of nitrite supplementation for improving vascular 91 function. This study was considered successful if >85% of randomized subjects had analyzable 92 plasma nitrite and vascular measures at baseline and Week 10 of the intervention, and no 93 serious adverse events occurred. Finally, to gain novel molecular insight into the possible 94 pathways by which nitrite may be acting, we also performed an unbiased analysis of the plasma 95 metabolome to identify circulating analytes and metabolites that were related to improvements 96 in vascular function with treatment. 97 Materials and Methods 98 This double-blind, placebo-controlled, randomized parallel-design study was conducted 99 at the University of Colorado Boulder Clinical Translational Research Center (CTRC) and blood 100 was assayed at the Colorado Clinical Translational Sciences Institute CTRC Core Lab, Mayo 101 Medical Laboratories and Boulder Community Hospital. 102 Subjects 103 Men and post-menopausal women ages 50 to 79 years of age were recruited from 104 Boulder and the surrounding communities. All subjects were free of cardio-metabolic diseases, 105 including peripheral arterial disease (ankle-brachial index >0.90), as assessed by graded 106 exercise test, blood analyses, medical history and general physical examination by a physician. 107 Subjects demonstrated impaired endothelial function, defined as a brachial artery flow-mediated 108 dilation value <7%. Subjects were excluded if they had glucose-6-phosphate dehydrogenase 109 (G6PD) deficiency (kinetic spectrophotometry), systolic blood pressure <100 mmHg, blood 110 methemoglobin (MetHb) >2% (ABL825 co-oximeter, Radiometer America, Inc., Brea, CA), 111 hypersensitivity to nitrates or nitrites, a medical history of anemia or clotting disorders, active 112 by 10.0.33.1 on July 2, 2017 http://jaysiology.org/ D ow nladed fom malignancy or infection, severe obesity (body mass index >40 kg/m) or were taking 113 medications that interacted with nitrite or altered vascular function (i.e., PDE-5 inhibitors, 114 systemic β-adrenergic blockers, tricyclic antidepressants, meperidine, nitrates, anticoagulants, 115 calcium channel blockers, hormone replacement therapy, imitrex, sumatriptan, ACE inhibitors, 116 ANG-II receptor blockers or diurectics). No subjects took nitrateor nitrite-related supplements. 117 To increase the generalizability of the study, some subjects were enrolled even if they took 118 certain medications or dietary supplements as long as they maintained their intake throughout 119 the study and refrained from taking medications or supplements 12 hours prior to vascular 120 measurements. The following prescription drugs were taken by some subjects: statins (Placebo 121 n=2, Nitrite 80 mg/d n=1), selective serotonin reuptake inhibitors (Nitrite 80 mg/d n=1, Nitrite 122 160 mg/d n=1), proton pump inhibitors (Placebo n=1, Nitrite 80 mg/d n=1, Nitrite 160 mg/d n=3), 123 bupropion (Placebo n=1), finasteride (Nitrite 160 mg/d n=1), inhaled fluticasone/salmeterol 124 (Nitrite 80 mg/d n=1), inhaled levosalbutamol (Nitrite 80 mg/d n=1), levothyroxine (Placebo n=1, 125 Nitrite 80 mg/d n=1), lorazepam (Nitrite 80 mg/d n=1), nasal mometasone furoate monohydrate 126 (Placebo n=2), tamsulosin (Placebo n=1), ophthalmic timolol (Placebo n=1) and zolpidem 127 (Nitrite 80 mg/d n=1). Sixteen subjects took no medications and 13 took no dietary 128 supplements. 129 All procedures were approved by the Institutional Review Board at the University of 130 Colorado Boulder. The nature, benefits and risks of the study were explained to all participants, 131 and their written informed consent was obtained before participation. This study was approved 132 by the Food and Drug Administration (IND111401) and registered on ClinicalTrials.gov 133 (NCT02022670). 134 Randomization and Intervention 135 Subjects were randomized using a blocked randomization scheme stratified for sex 136 (male vs. female) and age (50-65 years vs. 66-79 years) to avoid a potential imbalance amongst 137 groups for these two key baseline characteristics. Placebo or sodium nitrite capsules (40 mg or 138 by 10.0.33.1 on July 2, 2017 http://jaysiology.org/ D ow nladed fom 80 mg, TheraVasc, Inc., Cleveland, OH) were taken orally twice a day (morning and evening) 139 for 10 weeks. Subjects were educated by a CTRC registered dietician about foods containing 140 moderate and high levels of nitrate or nitrite (e.g., spinach, kale, beets), and subjects were 141 instructed to maintain a constant dietary intake of these foods and to avoid beets and beetroot 142 juice throughout the study. Sample size was estimated using an effect size of 0.74 from 143 published data in humans assessing the effects of oral nitrates on blood pressure (21, 36, 44), 144 brachial artery flow-mediated dilation after forearm ischemia (44) and work from our laboratory 145 (29). 146 Procedures 147 Subject Characteristics and Clinical Blood Assays. 148 Body mass index and waist and hip circumferences were measured by anthropometry 149 as previously described (42). Arterial blood pressure and heart rate were measured over the 150 brachial artery during rest in a supine position using a semi-automated device (Dynamap XL, 151 Johnson & Johnson, Raritan, NJ). 152 Fasting plasma glucose was measured by enzymatic methods (Roche Diagnostic 153 Systems, Somerville, NJ) and fasting plasma insulin by radioimmunoassay (Diagnostic Systems 154 Laboratory, Webster, TX). Insulin resistance was estimated with the homeostasis model of 155 insulin resistance (HOMA-IR) by the formula [fasting plasma glucose (mg/dl) x fasting plasma 156 insulin (μU/ml)]/405 (22). The HOMA-IR has been validated against other measures of insulin 157 sensitivity (e.g., intravenous glucose tolerance test) as a reliable estimate of insulin sensitivity 158 (3). Fasting serum blood lipid concentrations were determined using standard assays. High159 sensitivity C-reactive protein (serum, immunoturbidimetric method, Beckman Coulter), oxidized 160 low-density lipoprotein (EDTA-treated plasma, ALPCO Diagnostics, ELISA), total antioxidant 161 status (serum, Randox Laboratories, inhibition of the increase in absorbance by oxidized 2162 acrylamideo-2-methylpropane sulfonic acid over three minutes), glutathione perioxidase 163 (NaHep-treated whole blood, Randox Laboratories, oxidative method), norepinephrine (EGTA 164 by 10.0.33.1 on July 2, 2017 http://jaysiology.org/ D ow nladed fom and glutathione-treated serum, BioRad Laboratories, HPLC), renin activity (EDTA-treated 165 plasma, DiaSorin, RIA), aldosterone (serum, Diagnostic Products Corporation, RIA), endothelin166 1 (EDTA and aprotinin-treated plasma, Penisula Labs, antibody method), cortisol (serum, 167 Beckman Coulter, one-step competitive assay), free fatty acids (serum, WaKo Chemicals USA, 168 enzymatic method), leptin (serum, Beckman Coulter, RIA) and adiponectin (serum, Milipore 169 RIA) were analyzed at the Colorado Translational Sciences Institute CTRC Core Lab. 170 Angiotensin II (EDTAand bestastin-treated plasma, Evaluating Data, RIA) and epinephrine 171 (EGTA and glutathione-treated serum, BioRad Laboratories, HPLC) were also measured, but 172 are not reported because the majority of samples were below detection limits. 173 Feasibility, Safety and Tolerability. 174 Subject adherence to the intervention was measured by pill count. Retention of subjects 175 and adverse events (Table 4) were documented by the CTRC and reported to the Institutional 176 Review Board and Food and Drug Administration. Tolerability and side effects of the 177 intervention were determined by symptom questionnaire, blood pressure and blood MetHb. A 178 cut-off value of 12% blood MetHb or a drop of >20 mmHg systolic blood pressure were 179 instituted as stopping criteria. Subjects ingested their first capsule in the laboratory under 180 supervision. Because of the potential hypotensive and methemoglobinemia-inducing properties 181 seen with higher doses of nitrite or nitrates (30), seated and postural blood pressure measures, 182 blood sampling for MetHb levels and a symptom questionnaire were administered before, 15 183 min, 30 min, 60 min and 120 min after initial capsule ingestion. Six hours later, pulse co184 oximetry (Rad-57 Rainbow SET, Masimo Corporation, Irvine, CA) was performed to measure 185 MetHb levels instead of a blood draw. Subjects were monitored for adverse events again on 186 Day 2, Week 1, Week 2, Week 4 and Week 10. These measures were performed by staff who 187 were not involved in vascular data acquisition or analyses to ensure blinding of investigators. 188 Plasma Nitrite. 189 by 10.0.33.1 on July 2, 2017 http://jaysiology.org/ D ow nladed fom To determine the acute effects of our capsules on circulating nitrite concentrations, 190 plasma was collected before and 30 minutes after capsule ingestion in a subset of subjects 191 (Placebo n=7, Nitrite 80 mg/d n=6, Nitrite 160 mg/d n=9). The timing of this blood sample was 192 based upon pharmacokinetic data from the manufacturer for capturing peak plasma 193 concentrations (12). Plasma was also collected in all subjects at Week 10 of the intervention, 194 >12 hours after capsule ingestion to determine the non-acute effects of supplementation on 195 circulating nitrite concentrations. 196 Prior to coming into contact with blood samples or preservative solution, pipette tips and 197 microcentrifuge tubes were washed with fresh Millipore-filtered double-distilled water (ddH2O) in 198 triplicate to remove any nitrate or nitrite residue on labware. Whole blood was collected with 199 syringes and tubes from the same lot (rinsing with dH2O was not possible). In a darkened room, 200 whole blood was slowly added by syringe to prefilled tubes with freshly-made (day of) N201 Ethylmaleimide/EDTA (10 mM/2.5 mM final concentrations, respectively), inverted several times 202 for adequate mixing, centrifuged, plasma aliquoted and flash frozen in liquid nitrogen, and 203 stored at -60 degrees C until shipped on dry ice to a laboratory for analysis. Nitrite 204 concentrations were measured with a dedicated high-throughput HPLC system (ENO-20, 205 EiCom Corporation, San Diego, CA) using reverse phase chromatography and diazo-coupling 206 for spectrophotometric detection. The system operates over a wide range of concentrations 207 with high sensitivity (5, 6). 208 Vascular Outcomes. 209 All vascular measures were performed >12 hours after ingesting the last dose of sodium 210 nitrite, and 4 to 24 hours after ingesting food, alcohol or caffeine or performing exercise (13, 41). 211 The same investigator [AED], who was blinded to the subject group assignment, performed all 212 data acquisition and analyses. 213 Brachial artery flow-mediated dilation was assessed using high-resolution 214 ultrasonography (Xario XG, Toshiba America Medical Systems, Inc., Tustin, CA) before and 215 by 10.0.33.1 on July 2, 2017 http://jaysiology.org/ D ow nladed fom after reactive hyperemia, as described previously (9, 11). Flow-mediated dilation was measured 216 on multiple occasions at baseline (average of three visits) and at 10 weeks (average of two 217 visits) to account for day-to-day variation in vascular function (13). Flow-mediated dilation was 218 expressed as millimeter change (mmΔ) and percentage change (%Δ) from baseline diameter 219 (13). Shear rate was calculated as 8 x mean velocity/occlusion diameter using pulsed Doppler 220 signals (angle of insonation <70 degrees) and a sample volume over the entire width of the 221 artery during the first 10 velocity envelopes after cuff release. Arterial diameters and blood 222 velocities were captured and analyzed by Vascular Research Tools 5 software version 5.10.9 223 (Medical Imaging Applications, LLC, Coralville, IA) equipped with Top Performance Analysis 224 Integrated System with imager and frame grabber (DICOM, Rosslyn, VA) and vascular ECG225 gating module (University of Iowa, Iowa City, IA). The coefficient of variation for baseline and 226 peak brachial diameter in our laboratory is 0.3% and 0.6%, respectively. 227 Local stiffness/elasticity at the carotid artery was measured as previously described (37). 228 Briefly, the subject’s right common carotid artery maximal (i.e., end-systolic) and minimal (i.e., 229 end-diastolic) diameters were recorded by B-mode ultrasound (Xario XG, Toshiba America 230 Medical Systems, Inc., Tustin, CA) for >10 heart cycles (mean±SE, 31±2). Carotid blood 231 pressure was subsequently measured via applanation tonometry of the carotid artery with a 232 custom transducer (Non-Invasive Hemodynamics Workstation, Cardiovascular Engineering, 233 Inc., Norwood, MA) (40). Carotid artery cross-sectional compliance was calculated as 234 (3.141592*((2*carotid diastolic diameter)*(carotid systolic-diastolic diameter)+(carotid systolic235 diastolic diameter))/4*(carotid pulse pressure)) (41). β-stiffness index was calculated as 236 (Ln(carotid systolic blood pressure/carotid diastolic blood pressure))/(carotid systolic diameter237 diastolic diameter)/carotid diastolic diameter) (15). β-stiffness index is proposed to be a blood 238 pressure-independent index of arterial stiffness (15). 239 Central (carotid to femoral artery) and peripheral (carotid to radial artery) pulse wave 240 velocity and augmentation index at the carotid and radial arteries were determined by sequential 241 by 10.0.33.1 on July 2, 2017 http://jaysiology.org/ D ow nladed fom applanation tonometry with ECG gating of the R wave (Non-Invasive Hemodynamics 242 Workstation, Cardiovascular Engineering, Inc., Norwood, MA) (40) at each artery (an average of 243 10 heart cycles) by a blinded investigator [AED]. Distances between the carotid artery and 244 suprasternal notch, radial artery and suprasternal notch and femoral artery and suprasternal 245 notch were measured on the body surface with a tape measure or caliper. Pulse wave velocity 246 and augmentation index were calculated via software imbedded in the Non-Invasive 247 Hemodynamics Workstation using the equation distance/transit time and Δpressure/pulse 248 pressure x 100, respectively (40). 249 Metabolomics. 250 EDTA-treated plasma was collected before and at Week 10 of the intervention, frozen 251 and stored at -80°C until analysis. Compounds were extracted from samples using an 252 established custom technique that yields molecules from four major classes (lipid, protein, 253 carbohydrate and nucleic acids) (23, 32, 46). Aqueous and lipid fractions were analyzed using 254 Liquid Chromatography-Mass Spectrometry (LC-MS, 6210 ESI-TOF, Agilent Technologies, Inc., 255 Santa Clara, CA) and output was analyzed using commercial and custom software (1, 46). The 256 unbiased analysis involved assessment of metabolites from major metabolic pathways. Quality 257 control included duplicate samples and analysis of a pooled plasma sample containing labeled 258 or exogenous negative or positive spike-in controls, resulting in false discovery rates <3%. 259 Spectral data was extracted and aligned based on mass and retention time using Profinder 260 software (Agilent). Metabolites were annotated with IDBrowser in Mass Profiler Professional 261 (MPP, Agilent) using a combination of available databases: METLIN Metabolite Database, 262 Human Metabolome Database (HMDB), LIPID Metabolites And Pathways Strategy (LIPID 263 MAPS) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Metabolites identified as 264 being significantly altered by treatment were confirmed using MS/MS (6520 Q-TOF, Agilent) and 265 matched to the NIST 14 MS/MS library. Chemical formula generation was attempted for all 266 molecules that remained unidentified in both LC-MS and LC-MS/MS analyses. 267 by 10.0.33.1 on July 2, 2017 http://jaysiology.org/ D ow nladed fom Statistical Analyses 268 Data are expressed as means±SE. Statistical analyses were performed with IBM SPSS 269 (version 22) and G*Power 3.1 software. Unless otherwise indicated, missing values or outliers 270 (Grubb’s test) were replaced with the group mean. Differences amongst groups in baseline 271 subject characteristics were determined using One-Way ANOVA (p<0.05). Because this study 272 was designed to determine dosage and sample size for a larger clinical trial, significance was 273 set at p<0.10 for group differences (38). Sodium nitrite treatments were compared with placebo 274 using a mixed-model ANOVA (within factor, time; between factor, treatment group), with 275 contrast set to changes observed in placebo controls. In the case of a significant time x 276 treatment interaction, paired samples t-tests were performed (p<0.05). 277 After metabolomics data were filtered using MPP software (Agilent), small molecules not 278 present in at least 50% of any one group at baseline or Week 10 were excluded to reduce the 279 probability of false discovery, resulting in 3,063 detected small molecules for subsequent 280 analyses. Molecular concentrations were transformed to a log2 scale to assess their total 281 magnitude of change. To detect metabolites significantly altered by treatment, small molecules 282 were required to withstand a two-fold change filter and remain significant after a paired t-test 283 (p<0.05), while not being significantly altered (p<0.05) in the placebo group. Significantly 284 altered molecules were then modeled independently in linear regression models within each 285 treatment group to assess their individual association with vascular functional outcomes. 286 To assess whether baseline metabolomic signatures predict responsiveness to nitrite, 287 baseline log2 transformed concentrations of all 3,063 molecules were tested with independent 288 linear regression models within each dosage group. Only metabolites that were significantly 289 associated with responsiveness to nitrite and did not show an interaction with dosage level (80 290 or 160 mg/d) were included. Metabolites were subsequently grouped by class using the HMDB 291 database to determine which metabolic pathways were most involved in our analysis. A subject 292 was designated “responsive to the intervention” if they exhibited a 1% improvement from 293 by 10.0.33.1 on July 2, 2017 http://jaysiology.org/ D ow nladed fom baseline for flow-mediated dilation, an 8% improvement in carotid artery cross-sectional 294 compliance and a 15% improvement in β-stiffness index measures, changes shown to be 295 predictive of mortality or CVD events (16, 43). 296 Results 297 The average age of subjects was 62±1 years (range 50-77). Subjects were healthy and 298 there were no significant differences amongst groups for clinical characteristics at baseline 299 (Tables 1 and 2). Anthropometrics, brachial artery blood pressure, fasting blood lipids, glucose 300 and insulin remained unchanged by the intervention. Circulating analytes, including C-reactive 301 protein and endothelin-1, did not significantly change (Table 3). 302 Feasibility, Safety and Tolerability. 303 Of the 87 subjects assessed for eligibility, 34 subjects were randomized (39% enrollment 304 rate; Figure 1). Subjects were non-Hispanic Caucasian (84%), non-Hispanic Black/African (6%) 305 or Hispanic (10%). With only two subjects dropping out, there was a 94% completion rate for 306 the study. Subject adherence to the intervention was favorable, with 71% not missing taking 307 any capsules. Of the nine subjects who missed taking a capsule, four subjects missed one, 308 three subjects missed two and two subjects missed taking three capsules during the 10-week 309 intervention. Acquisition of data was successful for brachial artery flow-mediated dilation (97% 310 analyzable), brachial artery shear rate (94% analyzable), arterial stiffness measures (94-97% 311 analyzable) and blood analytes (71-100% analyzable). 312 No severe adverse events occurred (Table 4). Both doses of nitrite were well-tolerated 313 without symptomatic hypotension or clinically-relevant elevations in blood methemoglobin 314 (maximal value=1.5%). Treatment-emergent adverse events, all of which were expected, were 315 mild. Headache, nausea, fatigue, dizziness/lightheadedness, asymptomatic orthostatic 316 intolerance and dry mouth were reported. One subject, who had a history of migraines, 317 reported headaches as her reason for dropping out of the Nitrite 160 mg/d group. Another 318 by 10.0.33.1 on July 2, 2017 http://jaysiology.org/ D ow nladed fom subject dropped out of the Placebo group complaining of dizziness. No symptoms caused 319 subjects to drop out of the Nitrite 80 mg/d group. 320 Plasma Nitrite. 321 Oral sodium nitrite acutely increased plasma nitrite 5to 15-fold at 30 minutes after 322 capsule ingestion for both doses (Figure 2A). At Week 10 of the intervention, plasma nitrite 323 remained modestly elevated in the bloodstream more than 12 hours after last capsule ingestion 324 (Figure 2B). 325 Vascular Outcomes. 326 Brachial artery flow-mediation dilation increased after 10 weeks of nitrite 327 supplementation for both doses (Figure 3A, 3C). Other brachial artery parameters (Table 5), 328 including blood pressure, remained unchanged except Nitrite 80 mg/d increased the absolute 329 change in arterial diameter (Figure 3B, 3D). Aortic stiffness, as measured by carotid to femoral 330 pulse wave velocity, and wave reflection, as measured by augmentation index, were unchanged 331 (Table 6). Local stiffness measures at the carotid artery had significant time x treatment 332 interactions and, β-stiffness index was reduced from baseline in the Nitrite 80 mg/d group, 333 suggesting a possible de-stiffening effect of nitrite on the carotid artery (Figure 4A-D, Table 6). 334 Metabolomics. 335 To determine whether alterations in the metabolome are associated with changes in 336 measures of vascular function, metabolites shown to change with treatment were tested for an 337 association with functional outcomes. For subjects receiving Nitrite 80 mg/d, changes in flow338 mediated dilation were related to increases in two molecules, LysoPE(18:3(9Z,12Z,15Z)/0:0) 339 and DG(28:2), and decreases in N,N-Dimethyl-L-valine, 5-Aminoimidazole-4-carboxamide-1-1340 βD-ribofuranosyl 5'-monophosphate and two unknown metabolites (Table 7). For subjects 341 receiving the higher dose of nitrite, increases in L-Glutamine and PC(18:1/18:1) and decreases 342 in LysoPC(20:5) were significantly related to improvements in flow-mediated dilation. Increases 343 in an unknown compound was significantly related to improvements in carotid artery cross344 by 10.0.33.1 on July 2, 2017 http://jaysiology.org/ D ow nladed fom sectional compliance, whereas changes in β-stiffness index were significantly associated with 345 increases in PE(30:0) and one unknown compound. 346 To determine the ability of the plasma metabolome to predict an individual’s response to 347 nitrite supplementation, baseline metabolomic profiles were compared with changes in vascular 348 function after sodium nitrite supplementation (summary shown in Table 8; individual metabolites 349 shown in Long Data Set Tables 1-3). In total, 11 known metabolites populating three metabolic 350 pathways, comprised of benzene, fatty acyl and glycerophospholipid metabolism, were 351 significantly associated with responsiveness to intervention for measures of flow-mediated 352 dilation. β-stiffness index outcomes were significantly associated with baseline concentrations 353 of 73 metabolites from 11 metabolic pathways, including azoline, carboxylic acid, fatty acid and 354 fatty acyl, glycerolipid, glycerophospholipid, isoprenoid, N-arylamide, nucleoside, sphingolipid 355 and steroid metabolism. Lastly, an individual’s responsiveness to nitrite treatment with regards 356 to carotid artery cross-sectional compliance was significantly associated with baseline 357 concentrations of 80 metabolites residing in 11 metabolic pathways, involving amine, carboxylic 358 acid, fatty acid, fatty acyl, glycerolipid, glycerophospholipid, isoprenoid, prenol lipid, sphingolipid, 359 steroid and sterol lipid metabolites. 360 Discussion 361 The primary objectives of this study were to determine our ability to recruit, retain and 362 successfully collect and analyze data in this population, and to determine the tolerability, 363 efficacy and optimal dosage of chronic sodium nitrite supplementation for improving age-related 364 impairments in endothelial function and large elastic artery stiffness. We showed that brachial 365 artery flow-mediated dilation, a measure of endothelial function, increased with both doses of 366 nitrite and that the elastic properties of the carotid artery, estimated by local measures of 367 ultrasonography and tonometry, also improved. Moreover, changes in select metabolites were 368 associated with changes in vascular function and concentrations of several metabolites at 369 baseline predicted responsiveness to supplementation, providing initial insight into the pathways 370 by 10.0.33.1 on July 2, 2017 http://jaysiology.org/ D ow nladed fom that may be involved in the nitrite-related vascular improvements. Finally, we determined that 371 both doses of nitrite were safe and well-tolerated and that the lower dose of nitrite was sufficient 372 to elicit these important vascular changes, solidifying the dosing protocol for a future clinical 373 trial. 374 Interest in sodium nitrite as a treatment for CVD was rekindled in recent years after 375 chronic oral supplementation was shown to protect the heart from ischemia-reperfusion injury in 376 preclinical models (4, 8, 17) and infusion produced transient vasodilation of conduit and 377 resistance arteries in humans (34). Of special interest was the ability of nitrite to dilate arteries 378 without producing tolerance (27), a phenomenon that results in a drug becoming ineffective over 379 time, and nitrite’s ability to be stored in tissues until reduced under physiological conditions by a 380 one-step reaction to NO in a manner independent of the enzyme endothelial nitric oxide 381 synthase (eNOS) (5). As with CVD, advancing age is accompanied by a pro-oxidant milieu, in 382 which NO bioavailability is reduced due to decreased synthesis by eNOS and/or sequestration 383 of produced NO by the free radical superoxide (34). Therefore, oral nitrite supplementation is 384 an attractive intervention to prevent or reduce age-related endothelial dysfunction. In the 385 present study, we found that both doses of nitrite improved flow-mediated dilation, a measure of 386 endothelial-dependent dilation that has been shown to predict future cardiovascular events and 387 mortality (47, 48). To our knowledge, this is the first study to show beneficial effects of non388 acute (sustained) supplementation with sodium nitrite on conduit artery health in middle-aged 389 and older, but otherwise healthy, adults with impaired baseline endothelial function. Other 390 recent studies have shown that four weeks of oral nitrate, an inorganic anion that is converted 391 by bacteria in the gut to nitrite, improves flow-mediated dilation in patients with hypertension 392 (18) or older adults with elevated risk factors for CVD (31). In contrast, one study found that 10 393 weeks of sodium nitrite did not alter endothelial function in patients with diabetes and peripheral 394 arterial disease (25). 395 by 10.0.33.1 on July 2, 2017 http://jaysiology.org/ D ow nladed fom Stiffening of the large elastic arteries has been linked to CVD and is predictive of 396 mortality and disease (24, 43). Distending pressure, NO bioavailability and wall structure are 397 some of the major contributors to arterial stiffness (40). In the present study, there was a 398 significant time x treatment interaction for local arterial stiffness and compliance at the carotid 399 artery, but aortic stiffness was unchanged by chronic nitrite supplementation. No prior studies 400 have determined the effects of oral nitrite or nitrate on large elastic artery stiffness in healthy 401 adults, or on carotid artery elasticity in any population. Two previous studies in humans have, 402 however, reported that four weeks of supplementation with oral nitrate (dietary or capsules) 403 decreases aortic stiffness in patients with elevated CVD risk factors (18, 31). Although these 404 studies were not designed to determine the mechanisms behind these changes in stiffness, the 405 authors speculated that decreases in blood pressure may be involved since both studies 406 included patients with hypertension who had significant decreases in brachial artery blood 407 pressure in response to dietary nitrate. In our study, blood pressure, measured both centrally 408 (carotid artery) and peripherally (brachial artery) did not change. Instead, we detected a 409 significant time x treatment effect for carotid diameter in the nitrite-supplemented groups. This, 410 along with the increases in endothelial function in the brachial artery, and increased 411 concentrations of nitrite in the blood, suggests that NO bioavailability likely was increased by our 412 intervention. NO lowers vascular smooth muscle tone, especially in more muscular arteries like 413 the carotid artery (vs. the less muscular aorta) (40), possibly explaining the discrepant effects of 414 nitrite supplementation on arterial stiffness in these two vascular beds. 415 Both doses of our sodium nitrite capsules increased plasma nitrite concentrations 416 robustly at 30 minutes and modestly at 12 hours after capsule ingestion, in agreement with their 417 known pharmacokinetic profile (12). As for the underlying mechanisms by which physiological 418 changes occurred, blood lipids, glucose, blood pressure and body mass were not altered, 419 suggesting that changes in traditional risk factors were not responsible for the improvements in 420 vascular function with our intervention. To gain further mechanistic insight, we determined the 421 by 10.0.33.1 on July 2, 2017 http://jaysiology.org/ D ow nladed fom changes in circulating metabolites with treatment. Recent studies have shown that 422 “metabolomic signatures” can act as biomarkers and predictors of clinical disease and response 423 to medications (2, 20, 28), making them an exciting new tool in clinical research and medicine. 424 Our metabolomics analyses revealed combinations of specific molecular classes that were 425 significantly associated with our vascular measures with nitrite supplementation. Of all the 426 pathways, glycerophospholipids such as phosphatidylcholine (PC), lyso-PC, 427 phosphatidylethanolamine (PE) and lyso-PE were the most frequently altered class. Decreases 428 in lyso-PC(20:5) and PC(18:1/118:1) were associated with nitrite-induced increases in brachial 429 artery flow-mediated dilation. Lyso-PC(20:5) has been previously shown to be elevated in older 430 rodents (39) and humans (19) and is implicated in atherosclerotic processes (2, 45). 431 Furthermore, glycerophospholipids were the most abundant class of metabolites from blood 432 samples at baseline that predicted an individual’s response to nitrite for all three vascular 433 outcome measures, suggesting that this pathway may be central to changes in physiological 434 function with nitrite supplementation. Another class of metabolites, fatty acyls, was also 435 predictive for carotid artery stiffness measures. These metabolites are involved in lipid oxidation 436 and thus contribute to pathways involved in CVD (2). To our knowledge, this is the first study to 437 determine the metabolic pathways associated with nitrite-induced changes in vascular function. 438 The information derived from this novel technique will help to determine the metabolomic 439 pathways of focus for future studies and may help predict individuals who will respond favorably 440 to treatment with sodium nitrite. 441 Limitations of the current study include the diverse nature of the plasma metabolome, 442 which makes it difficult to positively identify every metabolite detected, although metabolites are 443 continually being confirmed and can potentially be validated at a later date in a larger trial. We 444 also acknowledge that our study involved mostly Caucasians and healthy, middle-aged and 445 older adults. Whether sodium nitrite is well-tolerated and improves vascular function in other 446 populations, including adults with cardiovascular or kidney disease, remains to be determined. 447 by 10.0.33.1 on July 2, 2017 http://jaysiology.org/ D ow nladed fom In conclusion, we found that sodium nitrite supplementation was safe and well-tolerated 448 in healthy middle-aged and older adults with few side effects, particularly in the lower dose 449 (Nitrite 80 mg/d) group. Vascular function was improved by both doses of nitrite, especially in 450 the Nitrite 80 mg/d group and several classes of metabolites explained the variance of vascular 451 function with our intervention. Therefore, a large clinical trial using the lower dose of nitrite is 452 warranted to determine the effects of sodium nitrite on age-related endothelial dysfunction and 453 arterial stiffening. Overall, this study adds to the increasing evidence that sodium nitrite 454 supplementation is a promising nutraceutical for the treatment of age-related processes and 455 multiple diseases. 456 Acknowledgements 457 The authors thank Rachelle E. Kaplon, Talia Strahler, Anaheed Little, Eric Chung, Molly 458 McNamara, Molly Nowlan, Sierra D. Hill, Rick Reisdorph and the staff of the University of 459 Colorado Boulder CTRC for their technical assistance. 460 Grants 461 This work was supported by National Institutes of Health awards R37 AG013038 (DRS), 462 T32 AG000279 (JNJ, LCJ, AED), R21 HL107105 (DRS, AED) and CTRC Grant UL1 463 RR025780.464Disclosures465Tony Giordano is President and CEO of TheraVasc, Inc. and Nathan Bryan is co-466 founder and CSO of Neogenis Labs, Inc. Nathan Bryan also receives royalties from patents467from the University of Texas Health Science Center in Houston.468References4691. 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Changes in plasma nitrite a) 30 minutes (time x treatment p<0.001; subset of616subjects: Placebo n=7, Nitrite 80 mg/d n=6, Nitrite 160 mg/d n=9) and b) 12 to 18 hours617(time x treatment p=0.070) after capsule ingestion at baseline (Base) and Week 10 (Wk10).618*p<0.05 vs. group base.619620Figure 3. Brachial artery a) flow-mediated dilation (time x treatment p=0.063), b) absolute621change in diameter (time x treatment p=0.099) and individual responses for c) flow-622mediated dilation and d) absolute change in diameter at baseline (Base) and Week 10623(Wk10). *p<0.05 vs. group base.624625Figure 4. Carotid artery a) β-stiffness index (time x treatment p=0.005), b) cross-sectional626compliance (time x treatment p=0.035) and individual responses for c) β-stiffness index627and d) cross-sectional compliance at baseline (Base) and Week 10 (Wk10). *p<0.05 vs.628group base.629630631by10.0.33.1onJuly2,2017http://jaysiology.org/Downladedfom Table 1. Subject Characteristics.632PlaceboNitrite 80 mg/dNitrite 160 mg/d Baseline Week 10 Baseline Week 10 Baseline Week 10Age, years62±3--60±2--63±2--Males/Females, n6/4--5/5--6/5--Body mass, kg75±3 75±371±3 71±370±3 71±3Body mass index, kg/m 26±1 26±124±1 25±124±1 24±1Waist circumference, cm 83±3 83±382±3 83±379±3 79±3Waist:hip ratio, U0.82±0.03 0.82±0.03 0.84±0.03 0.85±0.03 0.80±0.03 0.80±0.03SBP, mmHg121±5 119±3116±4 119±4122±4 124±3DBP, mmHg73±2 72±172±2 74±274±3 73±2Heart rate, bpm57±3 57±253±2 56±259±2 59±3Data are means±SE; SBP, systolic blood pressure; DBP, diastolic blood pressure.633634635636Table 2. Clinical Blood Characteristics.637PlaceboNitrite 80 mg/dNitrite 160 mg/d Baseline Week 10 Baseline Week 10 Baseline Week 10Total cholesterol, mg/dL 164±7 158±7 175±8 174±9180±6 183±8HDL-cholesterol, mg/dL 59±6 58±658±4 60±457±4 59±4LDL-cholesterol, mg/dL 90±5 87±4 100±6 101±7109±6 108±8Triglycerides, mg/dL79±8 67±570±6 70±1375±6 80±9Glucose, mg/dL94±1 91±290±2 90±193±1 91±2Insulin, μU/mL8±1 10±29±18±18±17±1HOMA-IR, U1.9±0.2 2.2±0.4 2.0±0.2 1.8±0.3 1.8±0.2 1.6±0.2HOMA-IR, homeostasis model assessment-insulin resistance.638639 by10.220.33.1onJuly12,2017http://jap.physiology.org/Downloadedfrom Table 3. Circulating Factors.640PlaceboNitrite 80 mg/dNitrite 160 mg/d Baseline Week 10 Baseline Week 10 Baseline Week 10C-reactive protein, mg/L0.59±0.15 0.59±0.09 0.79±0.22 0.61±0.13 0.50±0.07 0.56±0.08Oxidized LDL, U/L43±342±349±448±451±350±2Total antioxidant status, mmol/L 1.37±0.03 1.37±0.04 1.41±0.03 1.41±0.03 1.36±0.04 1.40±0.04Glutathione peroxidase, U/L 8181±1054 8180±1123 7109±380 7158±447 7392±762 7631±682Norepinephrine, pg/mL347±38 342±46367±57 296±54329±42 255±23Renin activity, ng/mL/hr0.28±0.07 0.26±0.07 0.33±0.05 0.34±0.10 0.26±0.06 0.24±0.03Aldosterone, ng/dL4.7±0.7 4.9±0.94.1±0.7 4.0±0.06 5.3±1.0 3.7±0.5Endothelin-1, pg/mL6.0±0.3 5.8±0.46.4±0.3 5.7±0.35.6±0.2 5.7±0.3Cortisol, μg/mL9.9±0.8 9.8±1.08.3±0.9 8.4±1.29.1±1.0 9.1±1.0Free fatty acids, μmol/L454±61 411±60497±27 446±34497±30 472±51Leptin, ng/mL8.6±2.2 8.0±2.28.0±2.6 7.8±2.46.8±2.0 8.7±3.0Adiponectin, μg/mL10.8±1.3 10.7±1.4 10.8±1.4 11.5±1.5 10.5±1.8 10.7±1.5641Table 4. Safety and Tolerability.642643644645646647648649650651652653654655656Placebo Nitrite80 mg/dNitrite160 mg/dAverage Peak Blood Methemoglobin, % 0.68±0.04 0.74±0.06 1.10±0.06Peak Blood Methemoglobin, %1.01.11.5Treatment-Emergent Adverse Events, nHeadache103Nausea100Fatigue022Dizziness/lightheadedness112Asymptomatic orthostatic intolerance111Dry mouth100Subjects with ≥1 Adverse Events, n244Drop-outs, n101 by10.220.33.1onJuly12,2017http://jap.physiology.org/Downloadedfrom Table 5. Brachial Artery Parameters.657PlaceboNitrite 80 mg/dNitrite 160 mg/d Baseline Week 10 Baseline Week 10Baseline Week 10Baseline Arterial Diameter, mm 3.70±0.15 3.66±0.16 3.59±0.17 3.45±0.123.67±0.16 3.60±0.15Peak Arterial Diameter, mm3.79±0.16 3.81±0.14 3.68±0.12 3.74±0.163.76±0.14 3.78±0.16Time to Peak Diameter, s52±559±643±545±347±643±4Shear Rate, s1743±177 1901±149 1872±79 2104±1451832±148 2010±184Nitrite 80 mg/d (n=8).658659660Table 6. Arterial Stiffness Parameters.661PlaceboNitrite 80 mg/dNitrite 160 mg/d Baseline Week 10 Baseline Week 10Baseline Week 10Carotid systolic pressure, mmHg122±6125±4121±7 124±8125±4127±7Carotid pulse pressure, mmHg56±559±457±760±860±563±6Carotid artery diameter at end-diastole, mm† 6.55±0.16 6.59±0.13 6.66±0.32 6.44±0.33* 6.58±0.12 6.54±0.12Carotid change in diameter, mm†0.34±0.03 0.34±0.02 0.37±0.04 0.46±0.06* 0.40±0.03 0.39±0.0.03Carotid augmentation index, %25±319±520±3 18±519±217±2Radial augmentation index, %-10±6 -10±5-11±5 -11±4-13±5-17±3Carotid to femoral PWV, cm/s‡782±35 774±36 780±55 845±75946±62942±77Carotid to radial PWV, cm/s1062±35 1040±30 967±27 1002±391014±25 1014±34*p<0.05 vs. group base; †p<0.05 for time x treatment; ‡Placebo (n=9).662663664665666667668 by10.220.33.1onJuly12,2017http://jap.physiology.org/Downloadedfrom Table 7. Changes in Metabolites Explaining Variance in Vascular Function.669Classβ StandardError P-valueFlow-mediated dilationNitrite 80 mg/dayDG(28:2)glycerolipids-0.646 0.045 0.044N,N-Dimethyl-L-valineamino acids-0.744 0.103 0.014LysoPE(18:3(9Z,12Z,15Z)/0:0)glycerophospholipids 0.649 0.050 0.042C19 H50 N11 O13 P Sunknown-0.810 0.238 0.0055-Aminoimidazole-4-carboxamide-1-βD-ribofuranosyl 5'-monophosphateimidazole ribonucleosidesand ribonucleotides -0.734 0.361 0.016C35 H62 N O15 Punknown-0.866 0.297 0.001Nitrite 160 mg/dayL-Glutaminecarboxylic acid andderivatives-0.771 0.071 0.009LysoPC(20:5)glycerophospholipids -0.672 0.084 0.033PC(18:1/18:1)glycerophospholipids -0.680 0.398 0.031β-stiffness indexNitrite 160 mg/dayC34 H53 N6 O2 Punknown0.785 0.167 0.007PE(30:0)glycerophospholipids 0.634 0.592 0.049Cross-sectional complianceNitrite 160 mg/dayC34 H53 N6 O2 Punknown-0.721 0.001 0.019 by10.220.33.1onJuly12,2017http://jap.physiology.org/Downloadedfrom Table 8. Metabolites from Blood Collected at Baseline that Predict an670Individual’s Vascular Response to Nitrite.671Molecular ClassNumber of MetabolitesPredicting Within EachPathwayFlow-mediated dilationBenzene and substituted derivatives1Glycerophospholipids10Unknown15β-stiffness indexAzolines1Carboxylic acids and derivatives3Fatty acids and conjugates6Fatty acyls20Glycerolipids5Glycerophospholipids27Isoprenoids1N-arylamides1Nucleoside and nucleotide analogues1Sphingolipids4Steroids and steroid derivatives4Unknown85Cross-sectional complianceAmines1Carboxylic acids and derivatives5Fatty acids and conjugates5Fatty acyls16Glycerolipids6Glycerophospholipids33Isoprenoids1Prenol lipids1Sphingolipids4Steroids and steroid derivatives7Sterol lipids1Unknown81672by10.0.33.1onJuly2,2017http://jaysiology.org/Downladedfom