Determination of the metabolites, eicosanoids derived from polyunsaturated fatty acids in Drosophila tissue with high performance liquid chromatography tandem mass spectrometry
Eicosanoids, which mediate various physiological and pathophysiological processes, are mainly formed from C20 polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA,20:4n-6) and eicosapentaenoic acid (EPA,20:5n - 3) through cyclooxygenases (COX), lipoxygenases (LOXs) and cytochrome P450 (C...
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| Main Authors: | , |
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| Format: | Article |
| Language: | English |
| Published: |
Zhejiang University Press
2016-01-01
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| Series: | 浙江大学学报. 农业与生命科学版 |
| Subjects: | |
| Online Access: | https://www.academax.com/doi/10.3785/j.issn.1008-9209.2015.03.232 |
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| Summary: | Eicosanoids, which mediate various physiological and pathophysiological processes, are mainly formed from C20 polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA,20:4n-6) and eicosapentaenoic acid (EPA,20:5n - 3) through cyclooxygenases (COX), lipoxygenases (LOXs) and cytochrome P450 (CYPs) pathways, including prostaglandins (PGs), thromboxanes (TXs) and the lipoxin/leukotriene family of eicosanoids such as hydroperoxyeicosatetraenoic acids (HPETEs), hydroxyeicosatetraenoic acids (HETEs), and epoxyeicosatrienoic acids (EETs). Vast knowledge of eicosanoids stems from works in mammals. Lipid signaling that complicates our understanding of fatty acid signaling is highly complex and fine-tuned in mammal species. Fortunately, the small genetic model Drosophila melanogaster is considered to be an ideal model to investigate the flexible nature of eicosanoids signaling pathways. However, it seems that Drosophila possess a special lipid metabolic system which is different from mammals. Thus, before studying the physiological mechanism of eicosanoids by using Drosophila, it is necessary to clarify its metabolic characteristics to C20 PUFAs based on the detection of eicosanoids in Drosophila. Therefore, this study is aimed to develop a high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for the determination of eicosanoids in Drosophila.Fifteen metabolites of AA and EPA produced by human in COX, LOX and CYP pathway were chosen, and each pathway contained 1 or 2 metabolites as delegates to ensure the representative of the method. These eicosanoids included PGF<sub>2α</sub>, PGE<sub>2</sub>, PGF<sub>3α</sub>, PGE<sub>3</sub>, 15 (S)-HETE, LTB<sub>4</sub>, 15 (S)-HEPE,11 (12)-EET,20-HETE,17 (18)-EpETE,17,18-DiHETE,15 (S)-HpEPE,15 (S)-HpETE, PGH<sub>2</sub> and 5 (S)-HpETE, and PGE2-d<sub>4</sub>, 15 (S)-HETE-d<sub>8</sub> and 20-HETE-d<sub>6</sub> were used as internal standards. Samples were prepared by solid phase extraction and separated on an Endeavorsil<sup>TM</sup> C<sub>18</sub> column (100 mm × 2.1 mm,1.8 μm). The analytes were detected by using multiple reaction monitoring (MRM) in a negative electrospray ion mode. The HPLC-MS/MS method to analyze 15 selected eicosanoids in Drosophila qualitatively and quantitatively was established by optimizing the sample pretreatment and the detection conditions.It was found that the recoveries were significantly influenced by the pH of sample adjusted by 1 mol/L sodium acetate buffer containing 5% methanol, and the optimized pH was at 6. The response values of analytes separated on a mobile phase of ultrapure water with 0.1% formic acid-acetonitrile were higher than these of ultrapure water with 0.1% formic acid-methanol, ultrapure water-acetonitrile or ultrapure water-methanol. To reduce the matrix interference, the blank Drosophila matrix was used to prepare the standard working solution. Obtained results showed that the calibration curves were of good linearity for the 15 metabolites in the range of 2.5-100 ng/mL with the correlation coefficient (r) of 0.991. The limits of detection and quantitation were about 0.1-2.6 ng/g and 0.3-8.7 ng/g, respectively. Spiked recovery experiments showed that both recoveries (89.3%-111.5%) and relative standard deviations (1.0%-15.0%) met the requirements of analytical methods.In conclusion, our study has established a simple, specific and sensitive method that suitable for the determination of eicosanoids in Drosophila which serves an approach to clarify the metabolic characteristics of Drosophila to C20 PUFAs. |
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| ISSN: | 1008-9209 2097-5155 |