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Abstract
Dietary NO3− (nitrate) and NO2− (nitrite) support ˙NO (nitric oxide) generation and downstream vascular signaling responses. These nitrogen oxides also generate secondary nitrosating and nitrating species that react with low molecular weight thiols, heme centers, proteins, and unsaturated fatty acids. To explore the kinetics of NO3−and NO2−metabolism and the impact of dietary lipid on nitrogen oxide metabolism and cardiovascular responses, the stable isotopes Na15NO3 and Na15NO2 were orally administered in the presence or absence of conjugated linoleic acid (cLA). The reduction of 15NO2− to 15NO was indicated by electron paramagnetic resonance spectroscopy detection of hyperfine splitting patterns reflecting 15NO-deoxyhemoglobin complexes. This formation of 15NO also translated to decreased systolic and mean arterial blood pressures and inhibition of platelet function. Upon concurrent administration of cLA, there was a significant increase in plasma cLA nitration products 9- and 12-15NO2-cLA. Coadministration of cLA with 15NO2− also impacted the pharmacokinetics and physiological effects of 15NO2−, with cLA administration suppressing plasma NO3−and NO2−levels, decreasing 15NO-deoxyhemoglobin formation, NO2−inhibition of platelet activation, and the vasodilatory actions of NO2−, while enhancing the formation of 9- and 12-15NO2-cLA. These results indicate that the biochemical reactions and physiological responses to oral 15NO3−and 15NO2−are significantly impacted by dietary constituents, such as unsaturated lipids. This can explain the variable responses to NO3−and NO2−supplementation in clinical trials and reveals dietary strategies for promoting the generation of pleiotropic nitrogen oxide-derived lipid signaling mediators.
Clinical Trial Registration—URL: http://www.clinicaltrials.gov. Unique identifier: NCT01681836.
Introduction
Inorganic NO2− (nitrite) undergoes reactions that yield vasodilatory products that contribute to blood pressure (BP) regulation and hypoxic signaling.1,2 The entero-salivary microbiome-mediated reduction of NO3−(nitrate) to NO2−and ˙NO (nitric oxide) in mammals also promotes vascular relaxation and other pleiotropic pharmacological responses.3–6 These reactions of NO3−/NO2−/˙NO both complement L-arginine/˙NO synthase/˙NO/cGMP signaling and expand the spectrum of nitrogen oxide intermediates that instigate signaling responses beyond the activation of guanylate cyclase. This new understanding contrasts with the previous perspective that tissue NO3−and NO2− represented potentially toxic and relatively stable ˙NO oxidation products.
There are an abundance of metabolic and inflammatory reactions that NO3−and NO2−undergo that can differentially impact downstream signaling responses1,2,4 (Figure 1A). Although NO3−is formed upon the oxidation of ˙NO by oxyhemoglobin, much greater endogenous NO3−levels are achieved by the ingestion of dietary NO3−sources, including leafy green vegetables, roots (eg, beets), and fruits.7 Once consumed, NO3−is readily reduced to NO2−by commensal oral bacterial nitrate reductase activities.8 Although the majority of NO3−is reduced in the oral cavity by commensal bacteria, xanthine oxidoreductase can reduce NO3− to NO2−.9 Up to 25% of plasma NO3− can be stored and concentrated by the salivary glands, thus, providing a facile route for entero-salivary recycling.10–13 Also acquired from both endogenous ˙NO oxidation reactions and dietary sources, the more intrinsically-reactive NO2− can undergo multiple reactions, such as protonation to HNO2(nitrous acid) in the low pH environments of the gastrointestinal tract, inflammatory foci, and the mitochondrial intermembrane compartment. Nitrite is also absorbed into the circulation and tissues where it can react with (1) deoxyhemoglobin and other heme proteins and molybdopterin-containing enzymes to form ˙NO14–17 or (2) heme peroxidases to yield the nitrating species nitrogen dioxide (˙NO2).18 During intermediary metabolism and inflammatory responses, partially reduced oxygen species, such as superoxide (O2˙−), H2O2(hydrogen peroxide), and lipid peroxyl radicals will also undergo a variety of reactions with ˙NO and NO2− to accelerate the rates of production of nitrosating (addition of ˙NO) and nitrating (addition of ˙NO2) species.19 ˙NO, NO2−, and HNO2 will also undergo a variety of reactions that lead to the generation of symmetric and asymmetric dinitrogen trioxide (ONONO and OONNO).20,21 These unstable intermediates rapidly decay to yield secondary nitrosating and nitrating species, as well as ˙NO. These and other redox reactions are responsible for propagating downstream signaling and pathogenic responses that are a consequence of oxidative, nitrosative, and nitrative post-translational protein modification (PTM) reactions.
Trial schemes and NO2− signaling pathways. A, Multiple metabolic and inflammatory reactions yield nitrogen dioxide. •NO2(nitration) and NO+(nitrosation) reactions produce an array of bioactive nitrogen oxide products, including NO2-FA (fatty acid nitroalkene derivatives) and S-nitrosothiol derivatives that have incompletely-characterized biological activities. B, To understand the metabolism of single doses of heavy nitrogen labeled sodium NO3− and NO2− and to characterize their signaling pathways and physiological effects, crossover drug design studies were conducted. Ten healthy volunteers were randomized into 1 of the 2 subject cohorts to receive a single dose of each study drug, oral 15N-labeled sodium NO3− and NO2−, in random order, separated by a washout period. Five of the 10 healthy volunteers who completed trial 1 returned and were randomized to receive a single dose of each drug, oral 15N-labeled sodium NO3− and NO2− plus conjugated linoleic acid, in random order, separated by a washout period in trial 2. C, To track NO2− metabolism in vivo, MetHb (methemoglobin), 15NO-Hb (15NO˙ ligand to deoxyhemoglobin), RS-NO (S-nitrosothiols), and 15NO2-FA (fatty acid nitroalkene derivatives) formation were examined. EPR indicates electron paramagnetic resonance spectroscopy.
Human studies and animal models reveal that ≤15 day courses of inorganic and dietary NO3− and NO2− administration induce robust responses that include decreased BP,1,2,11,22 hypoxic vasodilation,1,23,24 modulation of mitochondrial function under hypoxic or exercise stress,25,26 prevention of endothelial dysfunction,27 and inhibition of platelet aggregation.28,29 It remains uncertain as to whether these effects are mediated by ˙NO, secondary nitrating and nitrosating products or a combination thereof that will induce functionally-significant PTMs, such as thiol S-nitrosation or thiol alkylation.30,31
One class of nitrogen oxide metabolites are electrophilic NO2-FA (fatty acid nitroalkene derivatives),32 formed by radical addition reactions of ˙NO2 with alkenyl carbons of unsaturated fatty acids. Once formed, these lipid electrophiles rapidly react with the nucleophilic amino acid cysteine, and to a lesser extent histidine, residues via reversible Michael addition.33 Endogenously present at low nM concentrations in healthy human plasma and urine, the rates and extents of production of NO2-FA can be increased by an array of metabolic and inflammatory-related nitration reactions (Figure 1C).34 Upon the PTM of functionally-significant hyperreactive cysteine residues in critical enzymes and transcriptional regulatory proteins, NO2-FA influence gene expression and inflammatory responses.35 For example, specific proinflammatory and BP regulation enzymes are directly inhibited by NO2-FA, including xanthine oxidoreductase, cyclooxygenase-2, and soluble epoxide hydrolase.36–38 Notably, NO2-FA mediate pleiotropic signaling actions: this includes the PTM of p65 that inhibits nuclear factor-κB to inhibit proinflammatory cytokine expression, the activation of heat shock factor-1 dependent heat shock protein expression, the partial agonism of peroxisome proliferator-activating receptor-γ, and the activation of nuclear factor (erythroid derived) 2-regulated anti-inflammatory gene expression.39–43
The most prevalent endogenous NO2-FA, nitro-conjugated linoleic acid (NO2-cLA), is detected as both the free acid, complex lipid-esterified, and as protein adducts.34,44 cLA (predominantly 18:2, cis-9, trans-11) is a dietary polyunsaturated fatty acid prevalent in dairy products, meats, and plants. Endogenous generation of cLA in humans occurs by the isomerization of bis-allylic linoleic acid to a conjugated diene and by the desaturation of oleic acid to cLA, with both reactions catalyzed by the gut microbiome.45 Importantly, the external flanking carbons of the conjugated diene of cLA are 3 to 4 orders of magnitude more reactive than bis-allylic linoleic acid.44 This promotes facile addition reactions of radical species, such as ˙NO2 to yield NO2-cLA. It will always be a daunting challenge to dissect which products of nitrogen oxide metabolism, such as ˙NO, metal-NO complexes, RS-NO (S-nitrosothiols), or NO2-FA, are proximally responsible for modulating the physiological actions of nitrogen oxides in humans, such as the regulation of inflammatory responses, BP regulation, and platelet function.
We hypothesized that ingested NO3−is reduced to the redox-active metabolite NO2− in vivo, which is then metabolized to both ˙NO and NO2-cLA. Because ˙NO and NO2-cLA are both signaling molecules known to modulate BP and platelet function,3,38,46–50 we evaluated nitrogen oxide levels and clinical responses on consumption of oral 15NO3− and 15NO2− by healthy adults, with and without cLA supplementation. The use of US Food and Drug Administration (FDA) Investigational New Drug–approved (IND #115926) oral formulations of 15NO3− and 15NO2− allowed for specific metabolite tracking in vivo, and demonstrated that 15NO-Hb (15NO˙ ligand to deoxyhemoglobin) and 15NO2-cLA formation primarily occurred after 15NO2− consumption and that the physiological responses and nitrogen oxide product profiles were strongly influenced by the concomitant presence of cLA.
Materials and Methods
Study Design: 2 Pharmacokinetic Studies of 15NO3− and 15NO2− Metabolism Were Conducted, Without (Trial 1) and With cLA (Trial 2) Supplementation
In trial 1, 10 subjects were enrolled. To understand the precise mechanism of how the metabolites signal and exert their effects, these subjects were invited to return to participate in a modified pharmacokinetic study, trial 2, to serve as a direct paired control. Five subjects completed trial 2. Subjects were randomized in both trials as shown in Figure 1B. All study subjects were 18 to 60 years of age and had a normal BP defined as systolic BP (SBP) ≤130 and diastolic BP (DBP) ≤85 mm Hg. The study was approved by the University of Pittsburgh Institutional Review Board and the US FDA for use of these investigational new drugs. Before performing any of the research study procedures or interventions, subjects provided written informed consent, and procedures were followed in accordance with institutional guidelines.
The selected drug doses were 1 g Na15NO3− (11.8 mmol) and 20 mg Na15NO2− (0.29 mmol) with and without 3 g cLA. With each drug dose, plasma samples were collected for pharmacokinetic analysis of NO3− and NO2−, and MetHb (methemoglobin) was assessed using noninvasive co-oximetry (Masimo Corp, Irvine, CA), at times 0 (baseline or trough before study drugs), 0.5, 1, 2, 3, 6, and 24 hours postdrug administration. Before the start of each pharmacokinetic study visit, subjects fasted for 10 to 12 hours. BP and mean arterial pressure were measured for 30 to 45 minutes to ensure subjects were at a steady state before the time-0 mean arterial pressure (MAP) and administration of the study drugs. BP, MAP, respiratory rate, and heart rate monitoring were measured every 15 minutes during the first 2 hours, then at 3, 6, and 24 hours postdrug administration.
To track NO2− metabolism in vivo (Figure 1C), pharmacokinetic evaluations were used to examine total (15N and 14N) plasma NO3−, NO2−, and RS-NO (S-nitrosothiols) by gas-phase reductive ozone-based chemiluminescence detection. 15NO was differentiated from endogenous 14NO in blood using electron paramagnetic resonance spectroscopy via the formation of the 15NO-Hb. This iron-nitrosyl paramagnetic species has a distinctive hyperfine absorbance measured by electron paramagnetic resonance, which produces a doublet for NO-Hb labeled with 15N. To elucidate the overall metabolic fate of 15NO3− and 15NO2− without and with cLA, plasma and urinary 15NO2-cLA were differentiated from endogenous 14NO2-cLA using liquid chromatography-tandem mass spectrometry (LC-MS/MS) as previously described.44 A detailed description of the urinary measures is published separately.34 Platelet activity was monitored at time 0, 6, and 24 hours after 15N drug with and without cLA. Comprehensive methods are described in the online-only Data Supplement.
Statistical Analysis
Detailed statistical analyses for each data set are included in the individual figure legends. Where differences existed between trial drug treatments, post hoc multiple comparisons were performed using Bonferroni correction. Measurements shown represent mean±SEM. A P value <0.05 was considered significant.
Results
15NO3−/15NO2− Pharmacokinetics With and Without cLA
Ten healthy adult volunteers (baseline characteristics in Table S1 in the online-only Data Supplement) who fulfilled the inclusion/exclusion criteria were randomized into 1 of the 2 subject cohorts of 5 subjects to receive a single dose of each study drug, oral Na15NO3− (1 g), and Na15NO2− (20 mg) in random order (Figure 1B). Pharmacokinetic visits were separated by a 3- to 7-day washout period to ensure washout of nitrogen oxides and metabolites. Total plasma NO3− concentrations were significantly increased after 15NO3− dosing (peak level of 769±38 μM; P<0.001; Figure 2A). On dosing with 15NO2−, total plasma NO3− concentrations rose for 3 hours compared with baseline (P<0.001; Figure 2B). When the same subjects received oral 15NO3−, total plasma NO2− concentrations increased for 6 hours compared with baseline (P<0.01; Figure 2C). In contrast, when subjects received oral 15NO2−, total plasma NO2− concentrations rapidly peaked at 0.5 hours (5.5±0.7 μM; P<0.001; Figure 2D) and returned to basal concentrations after 3 hours.
15NO3− and 15NO2− pharmacokinetics without and with conjugated linoleic acid (cLA). A, After oral 15NO3−, plasma NO3− concentrations increase at all time points compared with baseline. B, After oral 15NO2−, plasma NO3− concentrations rise through 3 h compared with baseline. C, After oral 15NO3−, plasma NO2− concentrations increase through 6 h compared with baseline. D, After oral 15NO2−, plasma NO2− concentrations increase at 0.5 h and return to baseline after 6 h. E, When plasma NO3− concentrations were examined after oral 15NO3− treatment alone compared with 15NO3− with cLA, lower plasma NO3− concentrations were achieved with 15NO3− plus cLA. F, A trend toward lower plasma NO2− concentrations was seen through 1 h with 15NO2− plus cLA compared with 15NO2− alone. Repeated measures ANOVA with time as the within-subject effect was used to evaluate response to drug treatment for the end point measures in A through D. 2×2 repeated measures ANOVA with time as the within-subject effect and trial drug(s) (without vs with cLA) as the between-subject effect was used to compare the end point measures between trial 1 and trial 2 in E through F.
Five of the 10 healthy volunteers who completed trial 1 returned for trial 2 and were randomized to receive a single dose of each study drug, Na15NO3− (1 g) and Na15NO2− (20 mg), plus 3 g cLA, in random order, separated by a 3- to 7-day washout period so that metabolic responses could be compared with those from trial 1 (Figure 1B). Total plasma NO3− concentrations were lower after 15NO3−+cLA at 1 hour (473.2±72.4 μM; Figure 2E, closed circles) compared with 15NO3− supplementation (743.1±72.2 μM; P<0.01; Figure 2E, open circles). Peak plasma NO3− concentration occurred at 2 hours for 15NO3−+cLA administration and 1 hour for 15NO3− alone. No significant differences in plasma NO2− concentrations were observed for 15NO3− treatment alone versus 15NO3−+cLA (Figure S1). Whereas total plasma NO2− concentrations increased over time after 15NO2−+cLA (P=0.001), a trend toward lower total plasma NO2− concentrations was seen at 0.5 and 1 hour (Figure 2F, closed circles) when compared with 15NO2− alone (P=0.059; Figure 2F, open circles). Peak plasma NO2− concentration was 3.0±0.5 μM for 15NO2−+cLA and 4.6±1.0 μM for 15NO2− at 0.5 hours.
Formation of MetHb and 15NO-Hb
Further metabolism of 15NO2− was also reflected by MetHb, 15NO-Hb, plasma RS-NO, and 15NO2-cLA generation (Figure 1C). After 15NO3− administration, no significant increase in MetHb was observed (Figure 3A). After 15NO2− dosing, a small but significant increase in MetHb occurred from baseline to 1 hour (baseline, 1.2±0.1%; peak at 1 hour, 1.6±0.1%; P<0.001; Figure 3B) and returned to baseline by 3 hours. cLA supplementation did not impact MetHb levels (not shown). The metabolism of 15NO2− to 15NO was measured by the formation of 15NO-Hb in red blood cells sedimented from venipuncture blood samples. A representative electron paramagnetic resonance spectra (gray) shows the hyperfine splitting characteristic of 15NO-Hb from one subject (raw data in black; Figure 3C). There was no evidence for the formation of 14NO-Hb (Figure S2B). 15NO-Hb formation was only detected between 0.5 and 1 hour after 15NO2− supplementation (Figure 3D). 15NO-Hb derivatives were not detectable after 15NO2−+cLA and 15NO3−+cLA administrations (not shown).
NO2− signaling and MetHb (methemoglobin) and 15NO-Hb (15NO˙ ligand to deoxyhemoglobin) formation. A, After oral 15NO3−, no significant increase in MetHb occurs. B, After oral 15NO2−, a significant rise in MetHb occurs through 1 h. C, A representative EPR spectra (gray) shows the characteristic hyperfine splitting of 15NO-Hb from 1 subject after oral 15NO2− (raw tracings shown in black). The fit is composed of 42% pentacoordinate 15N alphanitrosyl Hb, 21% hexacoordinate alphanitrosyl Hb, and 38% betanitrosyl Hb. Inclusion of pentacoordinate 14N alphanitrosyl Hb does not substantially improve the fit (Figure S3B). D, 15NO-Hb formation is detected with oral 15NO2− treatment only at 0.5 to 1 h, with no 15NO-Hb detected with oral 15NO3− treatment. Repeated measures ANOVA with time as the within-subject effect was used to evaluate response to drug treatment for the end point measures in A and B. N.D. indicates not detected; and NS, not significant
Formation of RS-NO
Representative plasma RS-NO traces are shown at baseline and for peak concentrations of RS-NO at 0.5 hours after 15NO2− dosing, followed by an additional time point after 15NO2− treatment (Figure 4A). 15NO2− consumption significantly increased plasma RS-NO concentrations (baseline, 3.2±1.0 nmol/L; peak concentration of 6.1±1.0 nmol/L at 0.5 hours; P<0.05; Figure 4B) and returned to basal concentrations after 3 hours. In contrast, after 15NO3− dosing, there was no significant impact on RS-NO concentration (Figure S3A, open circles). There was also no impact of cLA supplementation on plasma RS-NO concentrations after dosing in combination with either 15NO3− or 15NO2− compared with 15NO3− or 15NO2− alone (Figure S3A and S3B).
NO2− signaling and RS-NO (S-nitrosothiols). A, Representative plasma RS-NO traces at baseline and the time when peak RS-NO concentrations were detected with oral 15NO2− followed by a time point after 15NO2− treatment. B, After oral 15NO2−, plasma RS-NO concentrations increase significantly, but after 3 h, approach baseline. Repeated measures ANOVA with time as the within-subject effect was used to evaluate response to drug treatment for the end point measure in B.
Formation of NO2-cLA
HPLC-MS/MS analysis of the structural regioisomers, β-oxidation metabolites, Michael addition products and pharmacokinetics of 15NO3− and 15NO2−-induced fatty acid nitration in trials 1 and 2 has been reported.34 Endogenous plasma 14NO2-cLA concentrations averaged 1.58±0.2 nmol/L after 15NO3− and 15NO2− and rose significantly to 2.84±0.24 nmol/L with cLA supplementation. Mean plasma 15NO2-cLA concentrations were maximal at 24 hours after 15NO3−+cLA (3.06±1.79 nmol/L), and 15NO2−+cLA supplementation resulted in maximal 15NO2-cLA concentrations that were sustained between 1 and 6 hours (6.61±4.84 to 4.86±2.39 nmol/L). These 15NO2−-related levels paralleled the levels of free plasma cLA. These data support that free cLA concentrations are limiting for fatty acid nitration. A representative LC-ESI-MS/MS (liquid chromatography–electrospray ionization–tandem mass spectrometry) chromatogram of 1 volunteer’s plasma lipid extract (Figure 5) shows the 15NO2-cLA regioisomers generated on dosing with 15NO 2−+cLA.34
NO2− signaling and NO2-cLA (nitro-conjugated linoleic acid) formation. A representative LC-ESI-MS/MS chromatogram of plasma lipid extract shows 15NO2-cLA present in plasma of volunteers treated with oral 15NO2− plus cLA. Adapted from Delmastro-Greenwood et al34 with permission of the publisher. Copyright © 2015, Elsevier Inc. Authorization for this adaptation has been obtained both from the owner of the copyright in the original work and from the owner of copyright in the translation or adaptation.
Physiological Responses to 15NO3− and 15NO2− Supplementation With and Without cLA Supplementation
In trial 1, oral 15NO2− dosing induced vasodilation, decreasing systolic (Δ−12.1±3.3 mm Hg; P<0.001), diastolic (Δ−8.1±2.0 mm Hg; P=0.002), and MAP (Δ−10.4±1.2 mm Hg; P=0.001; Figure 6A and B, open circles) with no differences in heart rate (data not shown). In this 15NO2−-dosed cohort, the reduction in SBP and MAP correlated with the change in plasma NO3− concentration (Figure S4A and S4C). Also, the greater the reduction in DBP at 1 hour, the greater the rise in total NO3− concentration at 0.5 hours (Figure S4B). Notably, the significant decreases in SBP and MAP that persisted for ≤2 hours after subjects were treated with 15NO2− were abolished on coadministration of cLA in trial 2 (Figure 6A and 6B, closed circles).
Physiological effects and NO2− signaling without and with conjugated linoleic acid (cLA). In trial 1 only, oral 15NO2− dosing induced vasodilation, decreasing systolic (Δ−12.1±3.3 mm Hg; P<0.001; A, open circles), diastolic (Δ−8.1±2.0 mm Hg; P=0.002), and MAP (Δ−10.4±1.2 mm Hg; P=0.001; B, open circles). Significant decreases in systolic blood pressure (SBP) (A) and mean arterial pressure (MAP) (B) but not diastolic blood pressure that persisted through 1.5 h after subjects were treated with oral 15NO2− alone (open circles) were abolished by cLA supplementation (closed circles). C, Inhibition of the percentage of platelet activation in whole blood was observed 6 hours after 15NO2− dosing (P=0.02; open bars), however, no differences in the percentage of platelet activation were detected when comparing oral 15NO2− treatment alone (open bars) vs oral 15NO2− with cLA (dark bars) for 24 h. 2×2 repeated measures ANOVA with time as the within-subject effect and trial drug(s) (without vs with cLA) as the between-subject effect was used to compare the end point measures between trial 1 and trial 2 in A through C.
Inhibition of platelet activation in whole blood was observed 6 hours after 15NO2− dosing (baseline, 33.2±7.1%; 6 hours postdosing, 7.8±1.6%; P=0.02; Figure 6C, open bars)—an effect that was eliminated when subjects were dosed with 15NO2−+cLA, (Figure 6C, dark bars). After 15NO3− dosing in all 10 subjects who completed trial 1, there was significant inhibition of platelet activation 6 hours after 15NO3− administration (baseline, 17.0±4.1%; 6 hours postdosing, 9.0±1.7%; P=0.029; Figure S5A). With coadministration of 15NO3−+cLA in trial 2, there was no significant inhibition of platelet activation in whole blood at 6 hours (baseline, 27.5±12.3%; 6 hours postdosing, 20.0±7.5%; Figure S5B).
In subjects given oral 15NO3−, plasma 15NO2− concentrations were significantly increased from 1 to 2 hours post-dosing (Figure S1, open circles), with a maximum of 1.40 µmol/L reached. In trial 1, oral 15NO3− dosing induced a small reduction in DBP at 3 hours compared with baseline (ΔDBP=−6.3±1.2 mm Hg; P=0.003; Figure S6B, open circles) with no significant change in SBP and MAP (Figure S6A and S6C, open circles) or heart rate (data not shown). No significant correlations were noted between changes in SBP, DBP, or MAP and absolute NO3− or NO2− concentrations after 15NO3− administration (not shown). After 15NO3−+cLA dosing, there were no significant changes in SBP, DBP, and MAP (Figure S6A through S6C, closed circles) or heart rate (data not shown).
Discussion
There are a panoply of physiological reactions that nitrogen oxides can undergo, all uniquely influenced by factors such as changes in pH, oxygen tension, CO2/H2CO3 levels, rates of production of reactive inflammatory mediators, and redox-reactive metalloprotein levels. The relative significance of these mitigating factors will change as basal metabolism transitions to metabolic stress and inflammatory responses. This will critically impact the specific reactions that occur between ˙NO/NO2−, molecular oxygen, partially reduced oxygen species, and metal centers. In the context of ˙NO signaling, the physiological outcomes of these reactions are manifested by the diversion of ˙NO away from canonical guanylate cyclase activation and cGMP-dependent signaling responses to the generation of a spectrum of highly reactive and transient secondary oxidizing, nitrosating, and nitrating products (Figure 1A).51 At high concentrations, these reactive products may be pathogenic, but under physiological conditions, these chemically-reactive intermediates (1) represent a metastable ˙NO reserve that still signals via guanylate cyclase activation and cGMP-dependent mechanisms and (2) react with and modify the structure and function of cell targets (eg, unsaturated fatty acids and proteins). These latter reactions instigate an array of PTMs and non–cGMP-dependent signaling responses.31 In particular, the nucleophilic amino acid cysteine confers proteins with a sensitivity for reduction-oxidation (redox)-induced PTMs that include cysteine oxidation, glutathionylation, S-nitrosation, and alkylation upon Michael addition by electrophilic species.52 These PTMs intimately link metabolic and inflammatory status with changes in cell and organ function because many enzymes, receptors, and transcriptional regulatory proteins that regulate metabolism and inflammation are endowed with functionally-significant hyperreactive cysteine moieties. Herein, we report that oral coadministration of 3 g of the polyunsaturated fatty acid cLA with 15NO2− significantly redirects the vasodilatory and platelet inhibitory actions of NO2− to alternative pathways.
Nitrite and its reduction product ˙NO are typically viewed to induce vasodilation via cGMP-dependent mechanisms.1,2 In the human brachial artery, forearm blood flow increases after NO2− infusion, in concert with a simultaneous rise in ˙NO formation.1 Abundant clinical studies have demonstrated significant reductions in BP in healthy adults after both single ingestion,11,53 repetitive 15-day22,54,55 courses of NO3−-rich foods56 or consumption of a traditional Japanese diet,57 another rich source of dietary NO3−. Various dosages of pure NO3− and NO2− preparations also significantly reduced BP in healthy adults.53,58,59 These human studies have largely been limited to testing single or short-term doses of NO3− or NO2−, with no evaluation of downstream metabolites. Animal models also recapitulate canonical vasodilatory and BP lowering effects of ˙NO stemming from NO2− and NO3−.60,61 There are no statistically significant differences between RS-NO concentrations with 15NO2− alone versus 15NO2−+cLA, with peak concentrations of 15NO-Hb being ≈150× greater than peak concentrations of RS-NO. Thus, these human NO2− pharmacokinetic data collectively demonstrate elevated plasma 15NO2− concentrations (Figure 2D) and 15NO2− reduction to 15NO (Figure 3D) in vivo as the most likely mediators of the simultaneous systolic and mean arterial BP reductions upon 15NO2− administration (Figure 6A and 6B, open circles). Oral administration of 15NO2− in concert with cLA blunted hemodynamic responses (Figure 6A and 6B, closed circles), with no detectable formation of 15NO-Hb adducts. The lack of BP response with cLA addition does not indicate that the 15NO2− drug dose is not vasodilating, but that cLA addition is reducing the nitrite concentrations enough to reduce the BP lowering extreme. A reduction in BP requires a systemic increase in blood flow (or a decrease in systemic vascular resistance—SVR) that cannot be compensated by an increase in stroke volume and heart rate (MAP−central venous pressure [CVP]=cardiac output [CO]×SVR). We have previously measured vasodilation in the human forearm at nitrite concentrations as low as 150 to 200 nmol/L (0.1–0.2 μM).1
Oral administration of single doses of 15NO3− and 15NO2− gave detectable increases in plasma nitrogen oxide concentrations, with each nitrogen oxide displaying unique pharmacokinetic and metabolite profiles. Moreover, coadministration of cLA significantly affected these parameters. Oral 15NO3− reached its highest plasma NO3− concentration 1 hour after dosing and yielded a peak concentration of plasma NO2− 2 to 6 hours after dosing (Figure 2A and 2C). This concentration of plasma NO2− with oral 15NO3− was not sufficient to increase MetHb, to yield detectable red blood cell 15NO-Hb levels (Figure 3A and 3D), or to induce (Figure S6A and S6C) or sustain (Figure S6B) reductions in BP parameters. Dosing of 15NO3− in concert with cLA suppressed peak plasma NO3− levels by ≈30% (Figure 2E, closed circles). For oral 15NO2−, the highest plasma concentration of NO2− was measured 0.5 hours after dosing (Figure 2D), giving a modest but significant increase in plasma NO3− metabolite concentrations 2 to 3 hours later (Figure 2B). Notably, the coadministration of cLA with 15NO2− also suppressed peak plasma NO2− concentrations by ≈30% (Figure 2F, closed circles).
Nitrite reacts with oxyHb to form NO3− and MetHb and with deoxyhemoglobin to ultimately form NO-Hb16. On NO2− protonation to HNO2 and reaction with ˙NO, both symmetric (ONONO) and asymmetric (OONNO) dinitrogen trioxide intermediates are generated, serving as proximal mediators of biomolecule nitrosation and nitration.20,62 After 15NO2− administration, evidence of all of these reactions was noted, with a significant 25% increase in MetHb levels, the formation of ≈1 µmol/L paramagnetic 15NO-Hb species, and a 3 nmol/L increase in mean plasma protein RS-NO levels 0.5 hours post-dosing (Figures 3B, 3D, 4A, and 4B). With cLA supplementation, mean plasma NO2-cLA concentration increased by ≈5 nM34. Only a few studies have reported changes in plasma ˙NO concentrations at baseline and after dietary NO3− or NO2− supplementation. For example, after a single 5.6 mmol dose of NO3−-rich beetroot juice,53 plasma cGMP—an indicator of ˙NO generation—increased within 3 hours. After a single 24 mmol dose of KNO3, plasma cGMP increased within 3 hours and remained elevated through 24 hours.53
After entero-salivary reduction of NO3− to NO2−, NO2− is readily protonated (pKa=3.4) to HNO2 in the acidic gastric compartment. This promotes a redirection of NO2− chemistry where HNO2 in turn gives rise to secondary N2O3 species that mediate nitrosation, nitration, and oxidation of susceptible targets. These reactions can be blunted by administering proton-pump inhibitors, such as esomeprazole, thus, inhibiting gastric acid secretion and NO2− protonation to HNO2—a pH response that typifies the pharmacological action of proton pump inhibitors.63 Esomeprazole also inhibited the BP-lowering effects of nitrite. The more basic conditions in the stomach after esomeprazole administration limited the protonation of NO2− to HNO2—a species that undergoes both dismutation and ˙NO reactions to yield the nitrosating and nitrating products symmetric and asymmetric dinitrogen trioxide (ONONO and ONNOO).20 The pathways leading to NO2-cLA formation first require entero-salivary reduction of NO3− to NO2−. In the present study, where 15NO2− and 15NO3− were orally consumed in the presence of cLA, stomach pH would either remain the same or be lowered by the acidic cLA (pKa=4.0). The suppression of detectable plasma NO3− and NO2− by ≈30% with cLA coadministration indicated that the greater availability and reactivity of cLA favored its nitration, thus, supporting the generation of NO2-cLA derivatives rather than the accumulation of NO3− and NO2− in plasma and presumably other tissue compartments. Our present report supports the concept that the addition of cLA and its facile nitration by NO2−-derived species is a consequence of these acid-catalyzed reactions. Our data indicate that this concomitantly leads to an attenuation of ˙NO-forming reactions that would otherwise result in the vasodilation and inhibition of platelet function that was observed when 15NO2− alone was administered. There may also have been an impact of cLA on gut nitrogen oxide absorption or microbial NO3− reduction. The small increase in plasma RS-NO derivatives observed in the 15NO2−-treated cohort in trial 1 at 0.5 hours postadministration (Figure 4B) was not significantly different compared with trial 2 with addition of cLA (Figure S3B). Dosing of volunteers with 15NO3− with cLA had no impact on plasma RS-NO levels (Figure S3A).
In healthy, hypertensive, and high body mass index subjects, cLA supplementation (3–6.8 g/d; 5–26 weeks) compared with control or placebo displays no significant impact on BP in humans.64–67 Herein, we observed that the decreased BP induced by 15NO2− was abrogated by concomitant cLA administration (Figure 6A and 6B). In addition to promoting a shift in nitrosative and nitrative chemistries, cLA might also be modulating NO2− responses by impacting endogenous ˙NO production.68–70 The present data do not diminish the significance of heme proteins, such as deoxyHb, in mediating reactions of NO2− that lead to changes in vascular function. Rather, our results further affirm the impact that other biomolecules, such as conjugated diene-containing fatty acids that readily undergo radical addition reactions, can have on nitrogen oxide reaction pathways and downstream signaling responses. This is manifested by the fact that Fe-NO Hb levels in 15NO2−-treated subjects are decreased to undetectable levels with 15NO2− + cLA administration (electron paramagnetic resonance analysis has a limit of quantification of ≈500 nmol/L for Fe-NO complexes). These findings indicate that cLA supplementation and other dietary constituents have the ability to modulate the biochemical reactions of NO2−, the trafficking of downstream nitrogen oxide metabolites and the physiological concentrations of this pluripotent signaling mediator. Moreover, a more chronic exposure to elevated cLA and fat in the diet has the potential to also influence the entero-salivary microbiome and its impact on nitrogen oxide metabolism.71
Along with vasodilatory actions, ˙NO formed by the reduction of NO2− inhibits platelet function, including platelet adhesion to the endothelium47 and platelet aggregation.72 In contrast, partially reduced oxygen species, such as O2˙− and H2O2, propagate platelet activation.73,74 The mechanism by which ˙NO affects platelet activity is by activating cGMP-dependent signaling. Dietary NO3− sources (beetroot juice or KNO3−) reduce platelet activation or aggregation,11,29,75,76 and NO2− in the presence of deoxygenated red blood cells inhibit platelet activation or aggregation.16,28,29,77–80 Herein, we confirm that 15NO2− inhibits human platelets in vivo, and note that the inhibition of platelet activation occurred in whole blood for 6 hours after 15NO2− dosing, with the inhibition of platelet activation still evident well beyond the physiological plasma NO2−peak and the time of 15NO-Hb detection. Previous in vitro studies revealed that 2 synthetic NO2-FA, nitro-linoleic acid, and nitro-arachidonic acid (at high concentrations in buffered saline) inhibited platelet aggregation and activation, respectively.81,82 In the more clinically-relevant study herein, 15NO2− administration in vivo decreased platelet activation in whole blood at 6 hours. The whole-blood basal platelet activation levels of subjects were moderately increased, which are attributed to an artifact of the venipuncture sampling. There may be a threshold plasma NO2− concentration where sufficient ˙NO formation subsequently reduces platelet activation with 15NO2− dosing, whereas oral 15NO2− and 15NO3− in combination with cLA diverted nitrogen oxide metabolites to support the formation of 15NO2-cLA, thus, consistently attenuating NO2− and NO3− inhibition of platelet activation.
Study Limitations and Strengths
The data presented here cannot exclude dietary cLA contribution during trial 1 and dietary NO3− and NO2− contributions also influencing baseline plasma NO3− and NO2− concentrations in both trials. Additionally, no formal power calculations were performed as these were exploratory phase I studies. Although this study may be underpowered to detect an effect of 15NO3− on BP, given that there were significant reductions in all BP parameters with 15NO2−, the abolishment of the 15NO2− BP effect with the addition of cLA can be assumed to be because of the impact of cLA and not insufficient power. Traditional human NO3− intakes of 40 to 100 mg/day,83,84 NO2− intakes of 0 to 20 mg/day85 and cLA intakes of 150 to 200 mg/day86 are low, but daily intakes of ≤2.6 g of cLA are reported in the literature. Therefore, it may be difficult to achieve all 3 of these intakes simultaneously through diet on a daily basis, so as to be comparable with those provided in capsule formulations herein. However, our goal with cLA supplementation was to define whether the biochemical reactions and physiological responses to oral 15NO3− and 15NO2− were modulated by cLA. Our investigations are strengthened by using paired pharmacokinetic studies in the same subjects so direct comparisons could be made using 15N sodium NO3− and NO2− with and without cLA supplementation to track ˙NO metabolism and the source of ˙NO and NO2-cLA species formation in vivo with each drug.
Perspectives
The oral administration of cLA altered the metabolic fate of oral 15NO3− and 15NO2− and modulated the ˙NO-signaling and vasodilatory properties of these nitrogen oxides. The cGMP-dependent signaling actions of NO3− and NO2− can be further transformed by digestive reactions, the entero-salivary microbiome and metalloprotein-catalyzed reactions to yield ˙NO and additional nitrogen oxides that elicit redox signaling responses via nitration, nitrosation, and oxidation reactions. One would expect that the spectrum of nitrogen oxide products formed will depend on diet, metabolic and inflammatory status, acidic microenvironments, and NO3−-reducing salivary bacteria. The concurrent administration of cLA in the diet decreased peak plasma NO3− and NO2− concentrations, enhanced the formation of 15NO2-cLA, and decreased detectable ˙NO concentrations in the vascular compartment, thus, effectively diverting nitrosative reactions to nitration events. This diversion of nitrosative reactions to nitrative chemistries resulted in the increased generation of 15NO2-FA, with concomitant attenuation of the inhibition of platelet activation and abrogation of the vasodilatory properties of NO2−. This affirms that the metabolic and the physiological responses to oral 15NO3− and 15NO2− can be significantly modulated by the composition of the diet, particularly with meat and dairy products that are rich in cLA.
Acknowledgments
We thank Ram Agarwal, Deborah Sperling, and Judy Starling for their work in the development of the oral sodium nitrate and nitrite capsules; Nydia Chien for FDA Investigational New Drug submission oversight; Sean Barrett for study drug dispensing; and Alex Despines, Sergey Ivanovich, and Yinna Wang for their technical assistance in the clinical trial. We also thank Andreas Perlegas for help with electron paramagnetic resonance analyses.
Sources of Funding
The research was supported by 5K12-HD052892, UL1-TR000005, and UL1-RR024153 (University of Pittsburgh Clinical and Translational Science Institute), the McKamish Family Foundation and the Institute for Transfusion Medicine and the Hemophilia Center of Western Pennsylvania (K.S. Hughan), R01-HL064937 (B.A. Freeman), R37-HL058115 (B.A. Freeman), P01-HL103455 (M.T. Gladwin and B.A. Freeman), and R37-HL058091 (D.B. Kim-Shapiro).
Disclosures
M.T. Gladwin and D.B. Kim-Shapiro are coinventors on a US government patent for the use of sodium nitrite for the treatment of cardiovascular diseases and have received royalties from the US government. B.A. Freeman and S.G. Wendell acknowledge interest in Complexa, Inc. The other authors report no conflicts.
Footnotes
Current address for B. Goodpaster: Translational Research Institute for Metabolism and Diabetes, Florida Hospital, Sanford-Burnham Medical Research Institute, Orlando, FL.
The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA.117.09016/-/DC1.
- Received February 2, 2017.
- Revision received March 2, 2017.
- Accepted June 14, 2017.
- © 2017 American Heart Association, Inc.
References
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- 3.↵
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- 5.↵
- 6.↵
- 7.↵
- 8.↵
- 9.↵
- 10.↵
- 11.↵
- 12.↵
- 13.↵
- 14.↵
- 15.↵
- 16.↵
- 17.↵
- 18.↵
- 19.↵
- 20.↵
- 21.↵
- 22.↵
- 23.↵
- 24.↵
- 25.↵
- 26.↵
- 27.↵
- 28.↵
- 29.↵
- 30.↵
- 31.↵
- 32.↵
- 33.↵
- 34.↵
- 35.↵
- 36.↵
- 37.↵
- 38.↵
- 39.↵
- 40.↵
- 41.↵
- 42.↵
- 43.↵
- 44.↵
- 45.↵
- 46.↵
- 47.↵
- 48.↵
- 49.↵
- 50.↵
- 51.↵
- 52.↵
- 53.↵
- 54.↵
- 55.↵
- 56.↵
- 57.↵
- 58.↵
- 59.↵
- 60.↵
- 61.↵
- 62.↵
- 63.↵
- 64.↵
- 65.↵
- 66.↵
- 67.↵
- 68.↵
- 69.↵
- 70.↵
- 71.↵
- 72.↵
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- 74.↵
- 75.↵
- 76.↵
- 77.↵
- 78.↵
- 79.↵
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- 84.↵
- 85.↵
- 86.↵
Novelty and Significance
What Is New?
We developed oral capsule formulations of NO3− and NO2− with the stable [15N] isotope allowing for specific metabolite tracking in vivo and to investigate the human vascular responses.
What Is Relevant?
Oral 15NO2− consumption resulted in ˙NO formation, vasodilation, and acute inhibition of platelet activation.
cLA with oral 15NO3−/NO2− diverted metabolic products to 15NO2-cLA.
Addition of dietary cLA with oral 15NO2− decreased ˙NO formation and vasodilation and attenuated the acute inhibition of platelet activation.
Summary
Concurrent cLA in the diet altered the metabolic fate of oral 15NO3− and 15NO2− with decreased plasma NO3−/NO2− levels, enhanced formation of 15NO2-FA species and decreased ˙NO formation. This diversion of the downstream reactions of NO3− and NO2− to NO2-cLA attenuated the inhibition of platelet activation and abrogated the vasodilatory properties of NO2−.
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