D3 malic acid

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.

Fresh lung tissue was washed with ice-cold PBS and immediately frozen in liquid nitrogen. Three samples per group were collected and sent for a TCA organic acid (OA) metabolomics’ analysis (LipidALL Technologies, Changzhou, China). The organic acid involved in the TCA cycle were extracted from murine lung tissue using acetonitrile: water (1:1) and derivatized using 3-nitrophenylhdyrazone, then were analyzed on a Jasper HPLC coupled to a Sciex 4500 MD system. In brief, individual organic acids were separated on a Phenomenex Kinetex C18 column (100 × 2.1 mm, 2.6 μm) using 0.1% formic acid (Sigma) in water as the mobile phase A and 0.1% formic acid in acetonitrile (Sigma) as the mobile phase B. d⁴-succinic acid, d⁴-citric acid, d³-malic acid, ¹³C-3-lactate acid, d³-pyruvate acid and d⁴-fumarate acid purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA) were used as internal standards for the quantitation.