Tsikas Dimitrios, Zoerner Alexander A, Mitschke Anja, Gutzki Frank-Mathias
Institute of Clinical Pharmacology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625, Hannover, Germany.
Lipids. 2009 Sep;44(9):855-65. doi: 10.1007/s11745-009-3332-4. Epub 2009 Aug 22.
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.
对健康受试者血浆中硝化脂肪酸的首次研究表明，通过液相色谱串联质谱法（LC-MS/MS）测定，游离/未酯化的硝基油酸（NO(2)-OA）的基础浓度为600 nM。我们最近通过气相色谱串联质谱法（GC-MS/MS）发现了两种异构体即9-NO(2)-OA和10-NO(2)-OA的生理存在，其平均基础血浆浓度分别为880和940 pM。考虑到这一巨大差异，我们对最初报道的方法进行了改进，用乙酸乙酯溶剂萃取取代固相萃取（SPE），并省略高效液相色谱（HPLC）步骤以实现更直接的检测，并具有脂质组学研究的潜力。在人血浆中，对于添加浓度在0 - 3000 pM范围内的NO(2)-OA，改进方法的批内不精密度和准确度分别为1 - 34%和91 - 221%。该方法在15名健康受试者中测得9-NO(2)-OA的基础血浆浓度为306±44 pM，10-NO(2)-OA为316±33 pM。血浆样本中未检测到硝基花生四烯酸和硝基亚麻酸。总之，我们的研究表明9-NO(2)-OA和10-NO(2)-OA是人体血浆中pM范围内的内源性硝化脂肪酸；对于通过GC-MS/MS可靠定量硝基油酸，推荐使用HPLC作为样品净化步骤。
