Red cell membrane and plasma linoleic acid nitration products: synthesis, clinical identification, and quantitation

Abstract

Nitric oxide (*NO) and its reactive metabolites mediate the oxidation, nitration, and nitrosation of DNA bases, amino acids, and lipids. Here, we report the structural characterization and quantitation of two allylic nitro derivatives of linoleic acid (LNO(2)), present as both free and esterified species in human red cell membranes and plasma lipids. The LNO(2) isomers 10-nitro-9-cis, 12-cis-octadecadienoic acid and 12-nitro-9-cis, 12-cis-octadecadienoic acid were synthesized and compared with red cell and plasma LNO(2) species based on chromatographic elution and mass spectral properties. Collision-induced dissociation fragmentation patterns from synthetic LNO(2) isomers were identical to those of the two most prevalent LNO(2) positional isomers found in red cells and plasma. By using [(13)C]LNO(2) as an internal standard, red cell free and esterified LNO(2) content was 50 +/- 17 and 249 +/- 104 nM, respectively. The free and esterified LNO(2) content of plasma was 79 +/- 35 and 550 +/- 275 nM, respectively. Nitrated fatty acids, thus, represent the single largest pool of bioactive oxides of nitrogen in the vasculature, with a net LNO(2) concentration of 477 +/- 128 nM, excluding buffy coat cells. These observations affirm that basal oxidative and nitrating conditions occur in healthy humans to an extent that is sufficient to induce abundant membrane and lipoprotein-fatty acid nitration. Given that LNO(2) is capable of mediating cGMP and non-cGMP-dependent signaling reactions, fatty acid nitration products are species representing the convergence of ()NO and oxygenated lipid cell-signaling pathways.

Fig. 1.

Characterization of LNO₂ by GC MS. (A) LNO₂ methyl esters were resolved by using a 100-m fused silica column and detected by total ion-count monitoring after EI ionization. Peak 1 (38.75 min) corresponds to linoleic acid nitrated at the 12-carbon (C12), and Peak 2 (39.00 min) corresponds nitration on the 10-carbon (C10). (B) LNO₂ and HPLC-separated positional isomers of LNO₂ were derivatized to their PFB esters, resolved on a 30-m CP-Sil 8CB MS, and detected by using NICI GC-MS. Peaks 1 and 2 correspond to C12 and C10 isomers, respectively. These species account for 90–95% of the total peak areas. (C) EI spectra were obtained from the peaks shown in A. Unique fragments (namely, m/z = 196 for C10, and m/z = 250 and m/z = 282 for C12) were detected that enabled structural identification of the isomers.

Fig. 2.

Characterization of LNO₂ by ESI triple-quadrupole MS. (A) Synthetic LNO₂ positional isomers were separated by HPLC and detected by MS/MS in the MRM mode by monitoring the 324/277 collision-induced dissociation transition (graph 1). No other peaks were detected in the chromatogram. The same method was used to resolve LNO₂ isomers present in the total lipid extract from 1 ml of packed red blood cells (graph 2) and plasma (graph 3). (B) EPI analysis of Peak 1, revealing fragments unique to the C12 positional isomer of LNO₂, m/z = 196 and m/z =157, plus fragments common to all LNO₂ isomers (m/z = 233, 244, 277, 293, and 306) (graph 1). EPI analyses of Peak 1 for red cell (graph 2) and plasma (graph 3) lipid extracts gave similar fragmentation patterns. All identifying fragments from graph 1 are present in graphs 2 and 3. (C). In addition to the common fragment ions of LNO₂, peak 2 displayed unique fragments m/z = 228 and m/z = 168 (graph 1). These fragments, particularly m/z = 228, indicate nitration of the C10 of LNO₂. EPI spectra from Peak 2 of resolved red cell (graph 2) and plasma (graph 3) lipid extracts gave fragmentation patterns similar to the synthetic standard.

Fig. 3.

Quantitative analysis of red blood cell and plasma LNO₂. (A) We added [¹³C]LNO₂ to lipid extractions as an internal standard to quantitate the free and esterified LNO₂ content of red cells and plasma obtained from healthy human volunteers. The mean age of the five female and five male subjects was 34 years. The endogenous LNO₂ isomers coeluted with the added ¹³C-labeled LNO₂ internal standard, with the internal standard differentiated from endogenous LNO₂ by monitoring its unique m/z = 342/295 MRM transition. (B) The internal standard curve for LNO₂ reveals linear detector responses over five orders of magnitude. The limit of quantitation (LOQ) for LNO₂, as defined by 10 times the SD of the noise, is ≈0.3 fmol (≈100 fg) injected on column.