Determination of organic acid content
MCW (Methanol-Chloroform-Water)-method was used to extract tissue metabolites, according to Ma et al. (2017). Weigh 50~100 mg (Fresh weight, Fw) tissue powder (kept in liquid nitrogen) to a precooled plastic 2 mL centrifuge tube. Add 0.7 mL 7:3 Methanol-Chloroform (-20℃) to the sample plus 20 µL PIPES (300 µmol/L) as the internal standard (IS). Then, add two metal beads to homogenize the samples on the grinding mill at a frequency of 40 repetitions/s for 5 min. The mixture is incubated at -20℃ for 2 h with a shaking every other 30 min. Subsequently, add 0.56 mL ice-cold ddH2O to the mixture, vigorously vortex the tube and centrifuge for 10 min at 8000 rpm at 4℃. Transfer the upper, aqueous methanol-H2O phase to a new precooled 2 mL centrifuge tube and add 0.56 mL ice-cold ddH2O to the residue and repeat the extraction step; then combine the second upper phase with the first one. The viscous, high-molecular mass components are filtered by 0.45 µM cellulose acetate centrifuge filters. Dry the extracts with N2 at room temperature (~24℃) and re-dissolve the dry extract with 200 µL ddH2O, as the concentrated samples. Finally, dilute the concentrated samples 20 times with ddH2O (10 µL concentrated sample +190 µL ddH2O) and the samples for quantifying is ready. LC-MS/MS (TSQ Quantum™ Access MAX, Thermo Scientific™, America) was used to inspect organic acids content (Ma et al. 2014, 2017).