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

Abstract

The oxidation and nitration of unsaturated fatty acids transforms cell membrane and lipoprotein constituents into mediators that regulate signal transduction. The formation of 9-NO2-octadeca-9,11-dienoic acid and 12-NO2-octadeca-9,11-dienoic acid stems from peroxynitrite- and myeloperoxidase-derived nitrogen dioxide reactions as well as secondary to nitrite disproportionation under the acidic conditions of digestion. Broad anti-inflammatory and tissue-protective responses are mediated by nitro-fatty acids. It is now shown that electrophilic fatty acid nitroalkenes are present in the urine of healthy human volunteers (9.9 ± 4.0 pmol/mg creatinine); along with electrophilic 16- and 14-carbon nitroalkenyl β-oxidation metabolites. High resolution mass determinations and coelution with isotopically-labeled metabolites support renal excretion of cysteine-nitroalkene conjugates. These products of Michael addition are in equilibrium with the free nitroalkene pool in urine and are displaced by thiol reaction with mercury chloride. This reaction increases the level of free nitroalkene fraction >10-fold and displays a K(D) of 7.5 × 10(-6) M. In aggregate, the data indicates that formation of Michael adducts by electrophilic fatty acids is favored under biological conditions and that reversal of these addition reactions is critical for detecting both parent nitroalkenes and their metabolites. The measurement of this class of mediators can constitute a sensitive noninvasive index of metabolic and inflammatory status.

Keywords: Michael addition; electrophile; nitration; nitro-fatty acid.

Fig. 1.

CLA nitration products in human urine. The extracted urine sample (black line, MRM 324.2/46) was mixed at three different ratios with IS (gray line, 100 nM ¹⁵NO₂-CLA, MRM 325.2/47). Five percent of ¹⁵NO₂-CLA in 95% of urine (A), 50% of ¹⁵NO₂-CLA in 50% of urine (B), and 95% of ¹⁵NO₂-CLA in 5% of urine (C). Urine-derived NO₂-CLA displayed retention times between NO₂-[¹³C₁₈]LA (D) and NO₂-[¹³C₁₈]OA (E).

Fig. 2.

Nitrated fatty acids in urine are electrophilic. Human urine extract was analyzed by HPLC-ESI-MS/MS in the MRM mode to detect NO₂-CLA and its β-oxidation metabolites. A: Representative chromatogram showing the detection of NO₂-CLA and its β-oxidation products in human urine. NO₂-FAs were followed as precursors of ions with m/z 46. B: Treatment with excess BME leads to the complete consumption of NO₂-FAs as evidenced by the disappearance of the 46 amu precursor peaks from their original retention times. Intrinsic gas phase instability of addition products results in in-source fragmentation (β elimination reaction), consistent with the neutral loss of BME during the ionization process. C: Detection of the corresponding BME adducts after neutral loss of 78 amu. D: Chemical structure of 9-NO₂-CLA and 12-NO₂-CLA (upper structures) and the four possible isomeric structures (10-BME-9-NO₂-CLA, 12-BME-9-NO₂-CLA, 11-BME-12-NO₂-CLA, and 9-BME-12-NO₂-CLA) that are formed upon reaction with BME.

Fig. 3.

Confirmation of NO₂-CLA β-oxidation products in human urine. NO₂-FA acids obtained from urine (black lines) were compared with the NO₂-CLA metabolites obtained from effluent of ¹⁵NO₂-CLA Langendorff-perfused isolated rat hearts (dashed lines). A: Comparative chromatographic profile of NO₂-CLA (MRM 324.2/46). High resolution MS/MS data on peaks 1 and 2 indicate 12-NO₂-CLA and 9-NO₂-CLA respectively. B: Comparative chromatographic profile of NO₂-CLA (MRM 296.2/46). Peaks 1 and 2 indicate dinor-10-NO₂-CLA and dinor-7-NO₂-CLA respectively. C: Comparative chromatographic profile of tetranor-NO₂-CLA (MRM 268.1/46). Peaks 1 and 2 indicate tetranor-8-NO₂-CLA and tetranor-5-NO₂-CLA respectively.

Fig. 4.

Correlation between NO₂-CLA levels and its metabolites in healthy human urine. A strong correlation (R > 0.94) was observed between the levels of NO₂-CLA and its β-oxidation products (dinor-NO₂-CLAand tetranor NO₂-CLA) in urine samples obtained from healthy volunteers. a.u., arbitrary units; AUC, area under the curve.

Fig. 5.

Detection of cysteine conjugates of nitro-fatty acids in urine. A: Chromatographic profiles of urinary Cys-NO₂-CLA and its β-oxidation metabolites (upper panels) and the products of the reaction of synthetic ¹⁵NO₂-CLA heart metabolites with cysteine (lower panels). B: High resolution MS³ spectral data of urine-derived Cys-NO₂-CLA (upper panel) and ¹⁵NO₂-CLA heart metabolites reacted with cysteine (lower panel). Inserts show corresponding MS/MS data following the neutral loss of HNO₂. All measurements were performed in the positive ion mode. C: Chemical structures of the four possible positional isomers of Cys-NO₂-CLA (12-Cys-9-NO₂-CLA, 10-Cys-9-NO₂-CLA, 9-Cys-12-NO₂-CLA, and 11-Cys-12-NO₂-CLA).

Fig. 6.

Reversibility of Cys-NO₂-CLA Michael adducts in urine. Cys-¹⁵NO₂-CLA (100 nM) was added to either methanol or urine and incubated for 120 min at 37°C. Whereas no free ¹⁵NO₂-CLA could be detected in methanol (A), the presence of free ¹⁵NO₂-CLA together with decreased levels of Cys-¹⁵NO₂-CLA indicated that a new equilibrium was established in the urine sample (B). Cys-¹⁵NO₂-CLA was measured in the positive ion mode (MRM 448.3/400.2) and free ¹⁵NO₂-CLA in the negative ion mode (MRM 325.2/47).

Fig. 7.

Equilibrium displacement of addition products in urine. A dynamic equilibrium governs the relative concentration of the addition products and the free NO₂-FAs in urine. Incubation of urine samples in the presence of HgCl₂ (10 mM) for 30 min at 37°C, resulted in a marked shift toward the formation of free nitroalkenes (A) and a complete loss of the cysteine addition products (B). A.U.C., area under the curve.

Fig. 8.

Equilibrium constant determination for NO₂-OA reaction with cysteine. A: Representative spectral changes observed upon cumulative additions of cysteine to NO₂-OA. Insert: Reference spectra utilized for spectral deconvolution analysis. B: Dose-dependent changes in free and conjugated NO₂-OA concentration upon cysteine addition (expressed as the thiolate form). C: One-site binding plot showing progressive saturation of Cys-NO₂-OA formation in the presence of increasing doses of cysteinate. Dissociation constants were calculated by nonlinear regression assuming a one-site specific binding model (R² 0.964).