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

• Published in: Lipids 2009-09, Vol.44 (9), p.855-865
• Main Authors: Tsikas, Dimitrios, Zoerner, Alexander A, Mitschke, Anja, Gutzki, Frank-Mathias
• Format: Article
• Language: English
• Subjects: Biomedical and Life Sciences; blood chemistry; blood plasma; chemical analysis; Fatty acids; Fatty Acids - blood; Gas chromatography; Gas Chromatography-Mass Spectrometry - methods; GC–MS/MS; HPLC; human health; human physiology; Humans; Interferences; LC–MS/MS; Life Sciences; Lipidology; Lipidomics; Liquid chromatography; Mass spectrometry; Medical Biochemistry; Medicinal Chemistry; Microbial Genetics and Genomics; Neurochemistry; Nitration; nitrification; Nitro Compounds - blood; Nutrition; oleic acid; Original Article; Quantification; Sensitivity and Specificity; Solvent extraction; spectral analysis; Stable isotopes; Tandem Mass Spectrometry - methods; Validation
• ISSN: 0024-4201; 1558-9307
• DOI: 10.1007/s11745-009-3332-4

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 (NO₂-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-NO₂-OA and 10-NO₂-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 NO₂-OA concentrations in the range 0-3,000 pM. This method provided basal plasma concentrations of 306 ± 44 pM for 9-NO₂-OA and 316 ± 33 pM for 10-NO₂-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-NO₂-OA and 10-NO₂-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.