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.

Keywords: CAD; Irg1; Krebs cycle; biomarker; cis-aconitate decarboxylase; citraconate; itaconate; mass spectrometry; mesaconate; metabolism.

Figure 1

Detection of itaconate and isomers by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). (A)—Chemical structures of tricarboxylic acid (TCA) cycle intermediates, itaconic acid isomers and derivatives. Compounds that can be quantified by the HPLC-MS/MS assay are highlighted in black. (B)—HPLC-MS/MS ion chromatogram of cis-aconitate, itaconic acid and isomers, and 4-octyl itaconate at the lower limit of quantification (LLOQ) (245 fmol on column for cis-aconitate and citraconate; 490 fmol on column for itaconate and mesaconate and 100 fmol on column for 4-octyl-itaconate). (C)—HPLC-MS/MS ion chromatograms of the employed internal standards. (D)—HPLC-MS/MS ion chromatograms of all analytes included in the method are highlighted in black. 4-octyl-itaconate eluted after applying the methanol gradient (dashed line in grey); trans-aconitate (grey) could be identified but was not quantified in this assay.

Figure 2

Itaconate levels in LPS/IFNγ−stimulated whole blood. (A)—Itaconate and mesaconate were detectable in whole blood (WB) and plasma after 4 h of stimulation, but could not be quantified reliably, since concentrations were <LLOQ. Citraconate levels were significantly higher, but not affected by LPS/IFNγ−stimulation. Plasma was obtained from Li-heparin anticoagulated blood collection tubes. Dashed line marks LLOQ. Samples from donor A; n = 3 aliquots from the same blood draw; mean ± SEM; n/d: not detected. Concentrations of TCA intermediates from this experiment are shown in Figure S1. (B)—Itaconate levels were quantifiable in blood leukocytes present in buffy coats from donor A upon LPS/IFNγ-stimulation; n = 3; mean ± SEM; n/d: not detected Citrate and lactate are shown to reveal the shift towards aerobic glycolysis. (C)—Comparison of TCA metabolite levels in EDTA and Li-heparin blood stimulated with LPS/IFNγ for 4 h. Itaconate was quantifiable in both preparations of stimulated blood, but mesaconate and citraconate levels were not detectable. TCA metabolite levels varied greatly when comparing EDTA and Li-heparin blood while citraconate and mesaconate levels were <LLOQ. Samples from donor B, n = 2 replicates from the same blood draw; mean; n/d: not detected.

Figure 3

Itaconate, mesaconate, and citraconate concentrations in extracted mouse organs of 44 to 46 weeks old C57BL/6J wild-type (WT) mice. Itaconate was most abundant in organs related to the immune system while mesaconate was enriched in organs responsible for metabolism and secretion/excretion. Citraconate was also highest in lymph nodes and was the only isomer that could be quantified in brain; n = 4 male and n = 6 female mice; mean ± SEM; values < LLOQ were considered missing values.