Simultaneous quantitative profiling of 20 isoprostanoids from omega-3 and omega-6 polyunsaturated fatty acids by LC-MS/MS in various biological samples

Abstract

Isoprostanoids are a group of non-enzymatic oxygenated metabolites of polyunsaturated fatty acids. It belongs to oxylipins group, which are important lipid mediators in biological processes, such as tissue repair, blood clotting, blood vessel permeability, inflammation and immunity regulation. Recently, isoprostanoids from eicosapentaenoic, docosahexaenoic, adrenic and α-linolenic namely F3-isoprostanes, F4-neuroprostanes, F2-dihomo-isoprostanes and F1-phytoprostanes, respectively have attracted attention because of their putative contribution to health. Since isoprostanoids are derived from different substrate of PUFAs and can have similar or opposing biological consequences, a total isoprostanoids profile is essential to understand the overall effect in the testing model. However, the concentration of most isoprostanoids range from picogram to nanogram, therefore a sensitive method to quantify 20 isoprostanoids simultaneously was formulated and measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The lipid portion from various biological samples was extracted prior to LC-MS/MS evaluation. For all the isoprostanoids LOD and LOQ, and the method was validated on plasma samples for matrix effect, yield of extraction and reproducibility were determined. The methodology was further tested for the isoprostanoids profiles in brain and liver of LDLR(-/-) mice with and without docosahexaenoic acid (DHA) supplementation. Our analysis showed similar levels of total F2-isoprostanes and F4-neuroprostanes in the liver and brain of non-supplemented LDLR(-/-) mice. The distribution of different F2-isoprostane isomers varied between tissues but not for F4-neuroprostanes which were predominated by the 4(RS)-4-F4t-neuroprostane isomer. DHA supplementation to LDLR(-/-) mice concomitantly increased total F4-neuroprostanes levels compared to F2-isoprostanes but this effect was more pronounced in the liver than brain.

Keywords: Dihomo-isoprostanes; Isoprostanes; Mass spectrometry; Neuroprostanes; Oxidative stress; Phytoprostanes; Quantification; ROS.

Figure 1

Chemical structure of isoprostanes derived from non-enzymatic oxidation of n-6 PUFA, adrenic acid (AdA) and arachidonic acid (AA), and n-3 PUFA, α-linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) measured in this study.

Figure 2

Total ion current chromatogram of IsoP: with PA derivatization (A), with HP derivatization (B), and without derivatization (C) of the same sample mix.

Figure 3

Chromatogram of selected reaction monitoring (SRM) of metabolites from each PUFA A: adrenic acid, B: arachidonic acid, C: α-linolenic acid, D: eicosapentaenoic acid and E: docosahexaenoic acid.

Figure 4

Optimum chromatographic separation of diastereoisomers for 8-F_3t-IsoP (a) and 8-epi-8-F_3t-IsoP (b). The diastereoisomers of 5-F_2t-IsoP (c) and 5-epi-5-F_2t-IsoP (d) were unable to resolve as well in the chromatographic analysis.

Figure 5

Chromatogram of selected reaction monitoring (SRM) of metabolites detected in human plasma : 15-F₂t-IsoP, 15-epi-15-F₂t-IsoP (A) ; 5-F₂t-IsoP, 5-epi-5-F₂t-IsoP (B) ; 10-F₄t-NeuroP, 10-epi-10-F₄t-NeuroP (C) ; 4(RS)-F₄t-NeuroP (D) ; internal standard d₄-15-F_2t-IsoP (E).

Figure 6

Isoprostanoids levels in the liver and brain of LDLR^−/− mice given either oleic acid rich sunflower oil (Ctrl, n=3) or a mixture of oleic acid rich sunflower oil and DHA rich tuna oil providing 2% of energy as DHA (DHA, n=3). The sum of F₂-IsoPs and sum of F₄-NeuroPs represents the ‘total’ sum of the isomers measured for the respective group.

Figure 7

Correlations between the levels of AA or DHA and the corresponding isoprostanoids (i.e. sum of F₂-IsoPs and sum of F₄-NeuroPs) in the liver (A and B).