Establishment, Validation, and Initial Application of a Sensitive LC-MS/MS Assay for Quantification of the Naturally Occurring Isomers Itaconate, Mesaconate, and Citraconate

Abstract

Itaconate is derived from the tricarboxylic acid (TCA) cycle intermediate cis-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.

Citraconate inhibits ACOD1 (IRG1) catalysis, reduces interferon responses and oxidative stress, and modulates inflammation and cell metabolism

“…We identified citraconate as the first endogenous ACOD1 inhibitor, but the physiological consequences at the organismal level remain unknown. In healthy mice, we previously found the highest citraconate levels in lymph nodes 5 . Although the cell type(s) harbouring citraconate remain unknown, it is tempting to speculate that it may modulate immune processes in lymph nodes by inhibiting ACOD1.…”

“…L6511) and 400 U ml −1 human IFN-γ (PeproTech, catalogue no. 300-02) for 0, 6, 12, 18, 24, 30 and 48 h and then extracted with 1 ml of ice-cold extraction buffer as described previously 5 .…”

“… 3 , 4 ) and has been detected in human biofluids, cells and tissues in a variety of inflammatory and infectious diseases as well as in rodent models of human diseases (summarized in ref. 5 ). Information about the origin and catabolism of mesaconate and citraconate in eukaryotes is sparse.…”

“…There is growing evidence that increased levels of mesaconate or citraconate can be associated with human metabolic diseases (summarized in ref. 5 ). However, it remains to be clarified whether these increased concentrations are pathophysiologically relevant or whether they merely represent epiphenomena of derangements in metabolism.…”

“…In humans, there is no information about the tissue distribution of mesaconate or citraconate. However, in a screen of organs from healthy mice, we have recently shown that mesaconate occurs in lymph nodes, and citraconate in lymph nodes and spleen, raising the possibility of some function in immunity 5 .

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