Conjugated Linoleic Acid Modulates Clinical Responses to Oral Nitrite and Nitrate

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.

Keywords: blood pressure; heme; lipids; nitric oxide; pharmacokinetics; platelet activation; sodium nitrite.

Figures

Figure 1. Trial schemes and NO2− signaling pathways

(A) Multiple metabolic and inflammatory reactions yield nitrogen dioxide. Nitration (·NO2) and nitrosation (NO+) reactions produce an array of bioactive nitrogen oxide products, including NO2-FA 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 physiologic effects, cross-over drug design studies were conducted. Ten healthy volunteers were randomized into one of two 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 (cLA), in random order, separated by a washout period in Trial 2. (C) To track NO2− metabolism in vivo, methemoglobin (MetHb), 15NO bound to the heme of hemoglobin (15NO-Hb), RS-NO and 15NO2-fatty acid (FA) formation were examined.

Figure 2. 15NO3− and 15NO2− PK without and with cLA

(A) Following oral 15NO3−, plasma NO3− concentrations increase at all time points compared to baseline. (B) Following oral 15NO2−, plasma NO3− concentrations rise through 3 hr compared to baseline. (C) Following oral 15NO3−, plasma NO2− concentrations increase through 6 hr compared to baseline. (D) Following oral 15NO2−, plasma NO2− concentrations increase at 0.5 hr and return to baseline after 6 hr. (E) When plasma NO3− concentrations were examined following oral 15NO3− treatment alone compared to 15NO3− with cLA, lower plasma NO3− concentrations were achieved with 15NO3− plus cLA. (F) A trend towards lower plasma NO2− concentrations was seen through 1 hr with 15NO2− plus cLA compared to 15NO2− alone. Repeated measures ANOVA with time as the within-subject effect was used to evaluate response to drug treatment for the endpoint measures in A–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 endpoint measures between Trial 1 and Trial 2 in E–F.

Figure 3. NO2− signaling and methemoglobin and 15NO-Hb formation

(A) Following oral 15NO3−, no significant increase in methemoglobin (MetHb) occurs. (B) Following oral 15NO2−, a significant rise in MetHb occurs through 1 hr. (C) A representative EPR spectra (red) shows the characteristic hyperfine splitting of 15NO-Hb from one subject following oral 15NO2− (raw tracings shown in blue). 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 (Fig. S3B). (D) 15NO-Hb formation is detected with oral 15NO2− treatment only at 0.5 to 1 hours, with no 15NO-Hb detected with oral 15NO3− treatment. NS = not significant, N.D. = not detected. Repeated measures ANOVA with time as the within-subject effect was used to evaluate response to drug treatment for the endpoint measures in A–B.

Figure 4. NO2− signaling and RS-NO

(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) Following oral 15NO2−, plasma RS-NO concentrations increase significantly, but after 3 hr, approach baseline. Repeated measures ANOVA with time as the within-subject effect was used to evaluate response to drug treatment for the endpoint measure in B.

Figure 5. NO2− signaling and NO2-cLA 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 with permission from Delmastro-Greenwood et al. Nitrite and Nitrate-Dependent Generation of Fatty Acid Nitroalkenes. Free Radic Biol Med. 2015;89:333–341.).

Figure 6. Physiologic effects and NO2− signaling without and with cLA

Significant decreases in SBP (A) and MAP (B) but not DBP that persisted through 1.5 hr after subjects were treated with oral 15NO2− alone (open circles) were abolished by cLA supplementation (closed circles). (C) No differences in the % of platelet activation were detected when comparing oral 15NO2− treatment alone (open bars) versus oral 15NO2− with cLA (dark bars) over 24 hr. 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 endpoint measures between Trial 1 and Trial 2 in A–C.