Winterhoff Moritz, Chen Fangfang, Sahini Nishika, Ebensen Thomas, Kuhn Maike, Kaever Volkhard, Bähre Heike, Pessler Frank
TWINCORE Centre for Experimental and Clinical Infection Research, 30625 Hannover, Germany.
Helmholtz Centre for Infection Research (HZI), 38124 Braunschweig, Germany.
Metabolites. 2021 Apr 26;11(5):270. doi: 10.3390/metabo11050270.
Itaconate is derived from the tricarboxylic acid (TCA) cycle intermediate -aconitate and links innate immunity and metabolism. Its synthesis is altered in inflammation-related disorders and it therefore has potential as clinical biomarker. Mesaconate and citraconate are naturally occurring isomers of itaconate that have been linked to metabolic disorders, but their functional relationships with itaconate are unknown. We aimed to establish a sensitive high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay for the quantification of itaconate, mesaconate, citraconate, the pro-drug 4-octyl-itaconate, and selected TCA intermediates. The assay was validated for itaconate, mesaconate, and citraconate for intra- and interday precision and accuracy, extended stability, recovery, freeze/thaw cycles, and carry-over. The lower limit of quantification was 0.098 µM for itaconate and mesaconate and 0.049 µM for citraconate in 50 µL samples. In spike-in experiments, itaconate remained stable in human plasma and whole blood for 24 and 8 h, respectively, whereas spiked-in citraconate and mesaconate concentrations changed during incubation. The type of anticoagulant in blood collection tubes affected measured levels of selected TCA intermediates. Human plasma may contain citraconate (0.4-0.6 µM, depending on the donor), but not itaconate or mesaconate, and lipopolysaccharide stimulation of whole blood induced only itaconate. Concentrations of the three isomers differed greatly among mouse organs: Itaconate and citraconate were most abundant in lymph nodes, mesaconate in kidneys, and only citraconate occurred in brain. This assay should prove useful to quantify itaconate isomers in biomarker and pharmacokinetic studies, while providing internal controls for their effects on metabolism by allowing quantification of TCA intermediates.
衣康酸衍生自三羧酸（TCA）循环中间体顺乌头酸，它将先天免疫与代谢联系起来。其合成在炎症相关疾病中会发生改变，因此具有作为临床生物标志物的潜力。中康酸和柠康酸是衣康酸的天然异构体，它们与代谢紊乱有关，但其与衣康酸的功能关系尚不清楚。我们旨在建立一种灵敏的高效液相色谱 - 串联质谱（HPLC-MS/MS）测定法，用于定量衣康酸、中康酸、柠康酸、前药4-辛基衣康酸以及选定的TCA中间体。该测定法针对衣康酸、中康酸和柠康酸进行了日内和日间精密度与准确度、延长稳定性、回收率、冻融循环以及残留的验证。在50 μL样品中，衣康酸和中康酸的定量下限为0.098 μM，柠康酸为0.049 μM。在加标实验中，衣康酸在人血浆和全血中分别在24小时和8小时内保持稳定，而加标的柠康酸和中康酸浓度在孵育过程中发生变化。采血管中抗凝剂的类型会影响选定TCA中间体的测量水平。人血浆可能含有柠康酸（0.4 - 0.6 μM，取决于供体），但不含衣康酸或中康酸，全血的脂多糖刺激仅诱导产生衣康酸。这三种异构体在小鼠器官中的浓度差异很大：衣康酸和柠康酸在淋巴结中含量最高，中康酸在肾脏中含量最高，而脑中仅含有柠康酸。该测定法在生物标志物和药代动力学研究中定量衣康酸异构体时应会很有用，同时通过允许定量TCA中间体为其对代谢的影响提供内部对照。
