13c3 lactic acid

¹³C₃-lactic acid, ¹³C₂-oxalic acid, ²H₃-sacrosine, ²H₈-valine, ¹³C₃-dihydroxyacetone, ²H₁₀-isoleucine, ¹³C₄-fumaric acid, ¹³C₄-malic acid, ²H₃-aspartic acid, ¹³C₅-glutamic acid, ¹³C₆-4-hydroxybenzoic acid, ²H₃-lauric acid, ¹³C₅-ribose, ¹³C₂-taurine, ²H₄-citric acid, ²H₇-ornithine, ¹³C₆-tyrosine, ¹³C₆-dopa, ²H₆-kynurenine, ²H₈-cystamine, and ¹³C₁₁-tryptophan were purchased from Cambridge Isotope Laboratories, Inc. (MA, USA). ²H₃-2-hydorxybutyric acid and 2-isopropylmalic acid were purchased from CDN isotopes (Quebec, CA) and Sigma Aldrich (Tokyo, Japan), respectively. The compounds were dissolved in methanol, as shown in Supplementary Table 3, and then the obtained solution was used as an extraction solution. A standard alkane series mixture (C7 to C33) and octafluoronaphthalene (OFN) were purchased from Restek Co. (PA, USA) and Shimadzu Co. (Kyoto, Japan), respectively. Methoxyamine hydrochloride and N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA), which were used for the derivatization, were obtained from Sigma-Aldrich and GL Science (Tokyo, Japan), respectively.

Detection of TCA metabolites was supported by Lipidall Technologies Company Limited, China. Itaconic acid and TCA cycle metabolites were extracted from mouse spinal cord tissue using acetonitrile: water (1:1) and derivatized using 3-nitrophenylhdyrazones. The content of itaconic acid and TCA was analyzed using Jasper HPLC coupled to a Sciex 4500 MD system. Briefly, itaconic acid and TCA were separated on a Phenomenex Kinetex C18 column (100 x 2.1 mm, 2.6 µm) using 0.1% formic acid in water as mobile phase A and 0.1% formic acid in acetonitrile as mobile phase B. d4-succinic acid, d4-citric acid, d3-malic acid, 13C-3-lactic acid, d3-pyruvic acid, d4-fumaric acid used as quantitative internal standards were purchased from Cambridge Isotope Laboratories.