Abstract
Polyunsaturated fatty acids (PUFAs) and eicosanoids are important mediators of inflammation. The functional role of eicosanoids in metabolic-syndrome-related diseases has been extensively studied. However, their role in neuroinflammation and the development of neurodegenerative diseases is still unclear. The aim of this study was the development of a sample pretreatment protocol for the simultaneous analysis of PUFAs and eicosanoids in mouse liver and brain. Liver and brain samples of male wild-type C57BL/6J mice (11–122 mg) were used to investigate conditions for tissue rinsing, homogenization, extraction, and storage. A targeted liquid chromatography–negative electrospray ionization tandem mass spectrometry method was applied to quantify 7 PUFAs and 94 eicosanoids. The final pretreatment protocol consisted of a 5-min homogenization step by sonication in 650 μL n-hexane/2-propanol (60:40 v/v) containing 2,6-di-tert-butyl-4-methylphenol at 50 μg/mL. Homogenates representing 1 mg tissue were extracted in a single step with n-hexane/2-propanol (60:40 v/v) containing 0.1% formic acid. Autoxidation was prevented by addition of 2,6-di-tert-butyl-4-methylphenol at 50 μg/mL and keeping the samples at 4 °C during sample preparation. Extracts were dried under nitrogen and reconstituted in liquid chromatography eluent before analysis. Recovery was determined to range from 45% to 149% for both liver and brain tissue. Within-run and between-run variability ranged between 7% and 18% for PUFAs and between 1% and 24% for eicosanoids. In liver, 7 PUFAs and 15 eicosanoids were quantified; in brain, 6 PUFAs and 21 eicosanoids had significant differences within the brain substructures. In conclusion, a robust and reproducible sample preparation protocol for the multiplexed analysis of PUFAs and eicosanoids by liquid chromatography–tandem mass spectrometry in liver and discrete brain substructures was developed.
Highlights
Eicosanoids are potent lipid mediators of inflammation, synthesized from polyunsaturated fatty acids (PUFAs) via cyclooxygenase (COX), lipoxygenases, and cytochrome P450
Well-established lipid extraction protocols based on liquid–liquid extraction according to Bligh and Dyer or Folch [15,16,17,18] are limited because of the distribution of PUFAs and eicosanoids in both the water-rich upper layer and the chloroform-rich lower layer
Methyl tert-butyl ether (MTBE) of pro analysis quality was obtained from Carl Roth (Karlsruhe, Germany). 2-Butanol of extra-pure quality was purchased from Thermo Fisher Scientific (Waltham, MA, USA). 2,6-Di-tert-butyl-4-methylphenol (BHT) of gas chromatography quality and EDTA of ACS reagent grade were purchased from Sigma-Aldrich (St Louis, MO, USA)
Summary
Eicosanoids are potent lipid mediators of inflammation, synthesized from polyunsaturated fatty acids (PUFAs) via cyclooxygenase (COX), lipoxygenases, and cytochrome P450. Special requirements for tissue analysis include the investigation of conditions for a reproducible homogenization of different tissue structures. Protocols for the analysis of selected PUFAs or eicosanoids in brain or liver tissue for subsequent liquid chromatography (LC)–tandem mass spectrometry (MS/MS) analysis have been published [20,21,22,23,24,25,26,27,28,29,30,31,32]. Little is known about the preanalytical requirements of eicosanoid analysis in tissue [33, 34] and defined sample pretreatment protocols; a systematic investigation of all influencing and disturbing factors has not yet been done
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