Nitrite and nitrate-dependent generation of anti-inflammatory fatty acid nitroalkenes

Abstract

A gap in our understanding of the beneficial systemic responses to dietary constituents nitrate (NO3(-)), nitrite (NO2(-)) and conjugated linoleic acid (cLA) is the identification of the downstream metabolites that mediate their actions. To examine these reactions in a clinical context, investigational drug preparations of (15)N-labeled NO3(-) and NO2(-) were orally administered to healthy humans with and without cLA. Mass spectrometry analysis of plasma and urine indicated that the nitrating species nitrogen dioxide was formed and reacted with the olefinic carbons of unsaturated fatty acids to yield the electrophilic fatty acid, nitro-cLA (NO2-cLA). These species mediate the post-translational modification (PTM) of proteins via reversible Michael addition with nucleophilic amino acids. The PTM of critical target proteins by electrophilic lipids has been described as a sensing mechanism that regulates adaptive cellular responses, but little is known about the endogenous generation of fatty acid nitroalkenes and their metabolites. We report that healthy humans consuming (15)N-labeled NO3(-) or NO2(-), with and without cLA supplementation, produce (15)NO2-cLA and corresponding metabolites that are detected in plasma and urine. These data support that the dietary constituents NO3(-), NO2(-) and cLA promote the further generation of secondary electrophilic lipid products that are absorbed into the circulation at concentrations sufficient to exert systemic effects before being catabolized or excreted.

Keywords: Conjugated linoleic acid; Diet; Nitrate; Nitrite; Nitro-fatty acid; Nitrogen metabolism; Redox signaling.

Figure 1

Trial I and II study design. Trial I: volunteers were randomized to receive either ¹⁵NO₂⁻ (20 mg) or ¹⁵NO₃⁻ (1 gm) and blood samples were collected at t = 0, 0.5, 1, 2, 3, 6, and 24 h. After a 7 day washout period, volunteers returned to receive the other oxide of nitrogen. In Trial II, the same ¹⁵NO₂⁻ or ¹⁵NO₃⁻ administration and blood collection protocol was implemented, along with cLA (3 gm) supplementation and urine collection at t = 0, 6, and 24 h.

Figure 2

Dietary supplementation of nitrite, nitrate and conjugated linoleic acid supports NO₂-cLA formation detected in plasma. Oral ¹⁵NO₂⁻ (20 mg) or ¹⁵NO₃⁻ (1 gm) was ingested ± cLA (3 gm). (A) LC-MS/MS chromatograms of plasma lipid extract show endogenous ¹⁴NO₂-cLA (MRM, 326/46) present at each time point and ¹⁵NO₂-cLA (MRM, 327/47) formed following ¹⁵NOx supplementation for ¹⁵NO₂⁻ at 2 h and ¹⁵NO₃⁻ at 24 h. (B) ¹⁴NO₂-cLA remains at basal levels over 24 h after ¹⁵NO₂⁻ (1.0 ± 0.3 nM) or ¹⁵NO₃⁻ supplementation (2.1 ± 0.7 nM). (C) Only ¹⁵NO₃⁻ administration led to detectable ¹⁵NO₂-cLA at 24 h (range = 0 to 25.3 nM, 0.14 nM median). (D) ¹⁴NO₂-cLA remained at basal levels after cLA supplementation (+¹⁵NO₂⁻, 2.6 ± 0.8 nM) and (+¹⁵NO₃⁻, 3.1 ± 0.9 nM). (E) ¹⁵NO₂⁻ + cLA resulted in early and significant increases from 1–6 h in ¹⁵NO₂-cLA (1 h range = 0 to 25.7 nM, 2.5 nM median and 6 h range = 0 to 11.5 nM, 3.3 nM median). Following ¹⁵NO₃⁻ + cLA, ¹⁵NO₂-cLA was detectable at 2 h and highest at 24 h (range = 0 to 9.9 nM, 1.6 nM median). n=5 volunteers.

Figure 3

Plasma levels of free cLA and NO₂⁻. (A) Free (10E,12Z)-cLA and (9Z,11E)-cLA levels measured from plasma over time in Trial II. The mean represents a combination of values from ¹⁵NO₃⁻ and ¹⁵NO₂⁻ supplementation. (9Z,11E)-cLA is found endogenously whereas (10E, 12Z)-cLA is solely derived from the supplement. Both isomers of free cLA peaked at 3 h with (9Z,11E)-cLA levels ranging from 530 to 1350 nM with a mean of 858 ± 138 nM. (10E,12Z)-cLA ranging from 270 to 710 nM with a mean of 430 ± 77.8 nM. (B) Representative chromatograms of the PTAD-derivatized (9Z,11E)-cLA, (10E,12Z)-cLA, and (11Z,13E)-cLA internal standard in plasma. (C) The ¹⁴N and ¹⁵N contribution to total plasma NO₂⁻ concentration after ¹⁵NO₂⁻ or ¹⁵NO₃⁻ supplementation in Trial I based on the mean NO₂⁻ value (Fig S1A). (D) The ¹⁴N and ¹⁵N contribution to total plasma NO₂⁻ concentration after ¹⁵NO₂⁻ + cLA or ¹⁵NO₃⁻ + cLA supplementation in Trial II based on the mean NO₂⁻ value (Fig S1B). LC-MS/MS was used to differentiate between the contributions of ¹⁴NOx and ¹⁵NOx species to overall NO₂⁻ levels and the mean concentration for each time point was used for both (B) and (C). The mean NO₂⁻ concentration ± SEM can be found in Supplementary Fig. S1 for n=5 volunteers.

Figure 4

Urinary NO₂-cLA and NO₂⁻ concentrations. Urine was collected at 0, 6, and 24 h following oral ¹⁵NO₂⁻+ cLA or ¹⁵NO₃⁻ + cLA administration. (A) ¹⁴NO₂-cLA levels in urine are highest at baseline and decrease over time. (B) Urinary ¹⁵NO₂-cLA was greatest 6 h after ¹⁵NO₂⁻ + cLA consumption (range = 0 to 55.0 pmol/mg Cre, 10.7 pmol/mg Cre median) and 24 h after ¹⁵NO₃⁻ + cLA consumption (range = 3.8 to 44.3 pmol/mg Cre, 12.5 pmol/mg Cre median). (C) The ¹⁴N and ¹⁵N contribution to total urinary NO₂⁻ concentration after ¹⁵NO₂⁻ + cLA or ¹⁵NO₃⁻ + cLA supplementation in Trial II based on the mean NO₂⁻ value (Supplementary Fig. S1C). n=5 volunteers.

Figure 5

Formation and signaling of NO₂-cLA. Nitrate (NO₃⁻) and nitrite (NO₂⁻) are dietary sources of nitrogen dioxide (•NO₂). Nitrate is reduced to nitrite by entero-salivary bacteria. Nitrite, combined with the low pH of the stomach favors •NO₂ formation via nitrous acid (HNO₂) generation. Various oxides of nitrogen can form from the decomposition of HNO₂ in the gut, including ^•NO₂. In inflammation, ^•NO₂ can arise from the protonation of NO₂⁻ to nitrous acid (HNO₂) or NO₂⁻ oxidation by heme peroxidases. Another significant mechanism of ^•NO₂ formation involves peroxynitrite (ONOO⁻), peroxynitrous acid (ONOOH), which are formed through a reaction of ^•NO and superoxide (O₂^•−). These species can readily diffuse through the membrane to mediate unsaturated fatty acid nitration and oxidation via homolysis of ONOOH to ^•NO₂ and ^•OH. Peroxynitrite also reacts with CO₂ to form nitrosoperoxocarbonate (ONOOCO₂) and like HNO₂, this compound can undergo homolytic scission to form ^•NO₂. Nitrogen dioxide reacts with the π electrons of alkenes via an addition reaction and a reaction with a second ^•NO₂ results in the reformation of the double bond. Conjugated diene containing PUFAs, such as cLA, are especially susceptible to nitration, as opposed to methylene-interrupted species. The endogenous production and exogenous administration of electrophilic fatty acids targets multiple redox-sensing transcriptional regulators. It has been previously demonstrated that nitroalkenes (a) putatively bind HSP70, releasing HSF-1 and thus driving HSF-1-dependent gene transcription; (b) covalently adduct Keap1, causing dissociation from and translocation of Nrf2 to induce ARE gene transcription, and (c) modify the p65 subunit of NF-κB, sustaining inhibition by IκB and blocking p50/p65-dependent gene transcription. In the nucleus, (d) nitroalkenes covalently bind and act as partial PPARγ agonists, stimulating gene transcription.