Nitro-Fatty Acid Logistics: Formation, Biodistribution, Signaling, and Pharmacology

Abstract

In addition to supporting cellular energetic demands and providing building blocks for lipid synthesis, fatty acids (FAs) are precursors of potent signaling molecules. In particular, the presence of conjugated double bonds on the fatty-acyl chain provides a preferential target for nitration generating nitro-FAs (NO₂-FAs). The formation of NO₂-FAs is a nonenzymatic process that requires reactive nitrogen species and occurs locally at the site of inflammation or during gastric acidification. NO₂-FAs are electrophilic and display pleiotropic signaling actions through reversible protein alkylation. This review focuses on the endogenously formed NO₂-FAs' mechanism of absorption, systemic distribution, signaling, and preclinical models. Understanding the dynamics of these processes will facilitate targeted dietary interventions and further the current pharmacological development aimed at low-grade inflammatory diseases.

Keywords: endogenous formation; inflammation; nitro-fatty acid; signaling; systemic distribution.

Figure 1.. Overview of NO₂-FA Formation, Distribution, Metabolism, Signaling, and Protection.

NO₂-FAs are mainly formed in the gastric compartment and, upon absorption and distribution, exert protective effects by activating antioxidant, anti-inflammatory, and antifibrotic signaling pathways. NO₂-FAs are finally metabolized and eliminated, at least partially, through kidney filtration. Abbreviations: HSP, ; NF-κB, nuclear factor-κB; NO₂-FA, nitro-fatty acid; Keap1, ; Nrf2, nuclear factor (erythroid-derived 2)-like 2; TG, triglyceride.

Figure 2.. Mechanism of Biologically Relevant Nitration Reactions.

(A) Tyrosine nitration: a one-electron oxidation of tyrosine leads to the formation of a tyrosyl radical, which is stabilized by resonance. A radical–radical reaction between the tyrosyl radical and ^•NO₂ results in the formation of nitrotyrosine. Nitrotyrosine is not reactive and exerts its biological activity by inducing protein conformational changes induced by charge and spatial modifications. (B) Formation of 8-NO₂-cGMP: 8-nitro-cGMP is formed upon the activity of guanylate cyclase on NO₂-GTP. GTP levels in cells largely exceed the amount of cGMP and as a consequence are the preferred biological substrate for purine nitration. Nitration of GTP is initiated by one-electron oxidation to form a radical that can be stabilized by resonance. As for the tyrosyl radical, a radical-radical reaction with ^•NO₂ forms NO₂-GTP. 8-NO₂-cGMP reacts slowly and irreversibly with thiols with the concomitant release of nitrite. (C) Nitration of monounsaturated and bis-allylic FAs: this nitration mechanism is inefficient as the initial addition of ^•NO₂ results in an unstable radical. The radical intermediate reverses back to the alkene with the elimination of ^•NO₂, usually resulting in cis–trans isomerization of the double bond. This reaction renders NO₂-FAs only in the presence of high concentrations of ^•NO₂. (D) Nitration of conjugated dienes: ^•NO₂ adds to the double bond and the resulting radical is also stabilized by resonance. This radical is then oxidized to form an NO₂-FA with conjugated double bonds. The resulting molecule contains two electrophilic carbon, and thiols can add reversibly via Michael addition reaction to the β and δ carbon. Abbreviations: NO₂-FA, nitro-fatty acid.

Figure 3.. Formation and Biodistribution of NO₂-FA.

NO₂-CLA formation is promoted under the acidic conditions found in the stomach through the reaction of dietary nitrite-derived radicals and CLA (usually found esterified in triglycerides). For therapeutic purposes, NO₂-FAs can also be administered orally as a drug as is the case for CXA-10 (Complexa Inc. – currently undergoing Phase II clinical trials). Through the activity of lipases, triglycerides are hydrolyzed and NO₂-FAs are released and absorbed together with other FAs by enterocytes, packed into chylomicrons as triglycerides and moved into the circulation via the lymphatic system. (A) Once the chylomicrons reach the capillaries, the NO₂-FAs are cleaved off the triglycerides by the activity of lipoprotein lipases in a process that requires the presence of docking protein GPIHBP1. The released NO₂-FAs can bind to fatty acid transporters (e.g., CD36) or diffuse into the endothelial cells and reach the parenchymal cells, such as cardiomyocytes. Heart, kidney, muscle, liver, and adipose tissue are among the main targets. Once the NO₂-FAs reach the target cells, they participate in signaling pathways via post-translational modifications of proteins, are metabolized, and then enzymatically inactivated. (B) Hydrophilic metabolites of NO₂-Fas, including dicarboxylic acid derivatives, β-oxidation products, mercapturic acids, and cysteine adducts, are filtered in the kidney and eliminated in the urine. (C) NO₂-FAs that reach the adipose tissue are re-esterified into triglycerides for storage. Degradation products formed during tissue metabolism and NO₂-FAs released from adipocytes through ATGL activity reach the circulation where they bind to albumin and are transported and delivered to the liver for excretion or filtered into urine by the kidneys. (D) Alternatively, NO₂-FAs that reach the liver can be reincorporated into triglycerides, assembled in the endoplasmic reticulum and Golgi compartments into VLDL particles, and mature VLDL particles containing Apo B, E, andCIII released to circulation and distributed systemically to target tissues. This initiates a new cycle of delivery, signaling, inactivation, metabolism, and elimination. Finally, the liver clears remaining LDL particles and remnant chylomicrons through selective uptake and breakdown. Abbreviations: Apo, apolipoprotein; ATGL, adipose triglyceride lipase; GPIHBP1, glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1; IDL, intermediate density lipoprotein; LDL, low density lipoprotein; LPL, lipoprotein lipase; NO₂-CLA, nitro-conjugated linoleic acid; NO₂-FA, nitro-fatty acid; OAT, ; TG, triglyceride; VLDL, very low density lipoprotein.