Nitro‐fatty Acids Occur in Human Plasma in the Picomolar Range: a Targeted Nitro‐lipidomics GC–MS/MS Study

Abstract

First studies on the occurrence of nitrated fatty acids in plasma of healthy subjects revealed basal concentrations of 600 nM for free/nonesterified nitro-oleic acid (NO(2)-OA) as measured by liquid chromatography tandem mass spectrometry (LC-MS/MS). We recently showed by a gas chromatography tandem mass spectrometry (GC-MS/MS) method the physiological occurrence of two isomers, i.e., 9-NO(2)-OA and 10-NO(2)-OA, at mean basal plasma concentrations of 880 and 940 pM, respectively. In consideration of this large discrepancy we modified our originally reported method by replacing solid-phase extraction (SPE) by solvent extraction with ethyl acetate and by omitting the high-performance liquid chromatography (HPLC) step for a more direct detection and with the potential for lipidomics studies. Intra-assay imprecision and accuracy of the modified method in human plasma were 1-34% and 91-221%, respectively, for added NO(2)-OA concentrations in the range 0-3,000 pM. This method provided basal plasma concentrations of 306 +/- 44 pM for 9-NO(2)-OA and 316 +/- 33 pM for 10-NO(2)-OA in 15 healthy subjects. Nitro-arachidonic acid and nitro-linolenic acid were not detectable in the plasma samples. In summary, our studies show 9-NO(2)-OA and 10-NO(2)-OA as endogenous nitrated fatty acids in human plasma in the pM range; HPLC is recommendable as a sample clean-up step for reliable quantification of nitro-oleic acids by GC-MS/MS.

Characterization and quantification of endogenous fatty acid nitroalkene metabolites in human urine

“…Initial concentrations of free and esterifi ed NO 2 -FA were initially reported to be in the high nM range ( 14 ). Subsequent studies support that 9-NO 2 -OA and 10-NO 2 -OA are present in the free form in plasma at concentrations of approximately 1 nM ( 33 ). In line with this, 9-NO 2 -CLA and 12-NO 2 -CLA are the most abundant LA-derived nitrated species that have been detected at present, with plasma concentrations also approaching 1 nM in healthy human donors ( 1 ).…”

“…Fatty acid nitroalkene derivatives are produced via reactions associated with infl ammation, metabolic acidosis, and gastric acidifi cation. Free and esterifi ed nitrofatty acid derivatives have been detected in human and animal plasma, low density lipoproteins, inflammatory mediator-activated macrophages and rodent heart, and isolated cardiac mitochondria after ischemia and reperfusion ( 14,17,(31)(32)(33).…”

Covalent Peroxisome Proliferator-activated Receptor γ Adduction by Nitro-fatty Acids

“…The covalent nature of NO 2 -FA binding should result in a leftshift of the EC 50 value over time, as more and more of the receptor becomes adducted. GST-purified PPAR␥ LBD (0.5 nM) was incubated with fluorescent ligand and the ligand of interest, and then terbium emission and FRET signals were measured at various times during reactions (3,10,20, and 60 min). This revealed a decreased EC 50 value over time for OA-NO 2 , indicating that the dissociation rate of OA-NO 2 from PPAR␥ is much lower than for Rosi or the nonelectro- philic homolog of OA-NO 2 , nitro-stearic acid (Table 2).…”

“…Nitroalkene derivatives of oleic acid (OA-NO 2 ) and linoleic acid (LNO 2 ) have been detected in healthy human blood and murine cardiac tissue. The levels of free/unesterified OA-NO 2 are ϳ1-3 nM in human plasma (9,10), with OA-NO 2 produced at increased rates and present at higher concentrations during inflammatory and metabolic stress (11)(12)(13). The signaling actions of NO 2 -FA are primarily ascribed to the electrophilic olefinic carbon situated ␤ to the electron-withdrawing NO 2 substituent, facilitating kinetically rapid and reversible Michael addition with nucleophilic amino acids (i.e.…”

Modulation of Nitro-fatty Acid Signaling

“…Consequently, nitroalkenes reversibly react with both low and high molecular weight nucleophiles by Michael addition to exert redox-regulated signaling and modulate Cys-and Zn-Cys-dependent protein function (7,8). The levels of free nitroalkenes detected in healthy volunteers are 0.72 nM and 9.9 pmol/mg creatinine NO 2 -CLA for human plasma and urine, respectively (4,9,10). During acute metabolic and inflammatory stress, free nitroalkene tissue levels can increase in murine hearts from undetectable to 9.5 and 17.3 nM for NO 2 -OA and NO 2 -CLA respectively, following an episode of focal cardiac ischemia-reperfusion (11).…”