Division of Computational and Systems Medicine, Department of Surgery and Cancer, Imperial College London , Sir Alexander Fleming Building, Exhibition Road, London SW7 2AZ, United Kingdom.
Department of Medicine, Imperial College London , St. Mary's Campus, London, United Kingdom.
Anal Chem. 2015 Dec 1;87(23):11721-31. doi: 10.1021/acs.analchem.5b02794. Epub 2015 Nov 18.
Lipid mediators, highly bioactive compounds synthesized from polyunsaturated fatty acids (PUFAs), have a fundamental role in the initiation and signaling of the inflammatory response. Although extensively studied in isolation, only a limited number of analytical methods offer a comprehensive coverage of the oxylipin synthetic cascade applicable to a wide range of human biofluids. We report the development of an ultrahigh-performance liquid chromatography-electrospray ionization triple quadrupole mass spectrometry (UHPLC-MS) assay to quantify oxylipins and their PUFA precursors in 100 μL of human serum, plasma, urine, and cell culture supernatant. A single 15 min UHPLC run enables the quantification of 43 oxylipins and 5 PUFAs, covering pro and anti-inflammatory lipid mediators synthesized across the cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP450) pathways. The method was validated in multiple biofluid matrixes (serum, plasma, urine, and cell supernatant) and suppliers, ensuring its suitability for large scale metabonomic studies. The approach is accurate, precise, and reproducible (RSD < 15%) over multiple days and concentrations. Very high sensitivity is achieved with limits of quantification inferior to picograms for the majority of analytes (0.05-125 pg) and linear range spanning up to 5 orders of magnitude. This enabled the quantification of the great majority of these analytes at their low endogenous level in human biofluids. We successfully applied the procedure to individuals undergoing a fasting intervention; oxylipin profiles highlighted significantly altered PUFA and inflammatory profiles in accordance with previously published studies as well as offered new insight on the modulation of the biosynthetic cascade responsible for the regulation of oxylipins.
脂质介质是一类由多不饱和脂肪酸（PUFAs）合成的高生物活性化合物，在炎症反应的启动和信号传递中具有重要作用。尽管它们在单独研究时已经得到了广泛的研究，但只有少数分析方法能够全面涵盖适用于广泛人体生物流体的氧化脂合成级联。我们报告了一种超高效液相色谱-电喷雾电离三重四极杆质谱（UHPLC-MS）测定法的开发，该方法可用于定量分析 100 μL 人血清、血浆、尿液和细胞培养上清液中的氧化脂及其 PUFAs 前体。单次 15 分钟 UHPLC 运行即可定量 43 种氧化脂和 5 种 PUFAs，涵盖了 COX、LOX 和 CYP450 途径中合成的促炎和抗炎脂质介质。该方法在多种生物流体基质（血清、血浆、尿液和细胞上清液）和供应商中进行了验证，确保了其在大规模代谢组学研究中的适用性。该方法具有准确性、精密度和重现性（RSD < 15%），可在多天和多个浓度范围内使用。大多数分析物（0.05-125 pg）的检测限非常低，灵敏度非常高，线性范围可跨越 5 个数量级。这使得能够在人体生物流体中以其低内源性水平定量检测出这些分析物中的绝大多数。我们成功地将该程序应用于接受禁食干预的个体；氧化脂谱突出显示了与先前发表的研究一致的 PUFAs 和炎症特征的显著改变，并且为负责调节氧化脂的生物合成级联的调节提供了新的见解。
