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).