Lactic acid

Organic acids were determined according to the procedure described by Chen and Zhang (2007) (link) with modifications. A 1 g freeze-dried sample was homogenized in 10 mL of a phosphoric acid solution (0.1 mL/100 mL) for 2 min and then centrifuged at 10,000g for 10 min at 4 °C. The above procedure was repeated once, and the supernatants were combined and diluted to a volume of 25 mL with the above phosphoric acid solution. The extract solution was filtered through a 0.22 μm membrane prior to HPLC analysis. The HPLC instrument and column used were the same as those mentioned in Section 2.3.2. The HPLC conditions were as follows: the mobile phase comprised 0.1 % H₃PO₄-methanol (v/v = 97:3), the detection wavelength was 210 nm, the flow rate was 1 mL/min, and the column temperature was 30 °C. Each organic acid was analyzed by comparison with the retention times and peak areas of standards, namely, succinic acid (≥98 %), malic acid (≥98 %), and lactic acid (≥98 %) (Beijing Solarbio Science & Technology Co., Ltd).

The potato amylose was purchased from Sigma Chemical Co. (St. Louis, MO, USA). The maize amylopectin was purchased from Macklin Biochemical Technology Co. (Shanghai, China). Standards of oxalic acid, malic acid and tartaric acid were purchased from Yuanye Biotechnology Co., Ltd. (Shanghai, China), and lactic acid and citric acid were purchased from Solarbio Technology Co. (Beijing, China). Other reagents were analytical grade.

The levels of glucose, lactic acid and ATP in the PASMCs were assessed using glucose (cat. no. BC2505; Beijing Solarbio Science & Technology Co., Ltd.), lactic acid (cat. no. BC2235; Beijing Solarbio Science & Technology Co., Ltd.) and ATP (cat. no. BC0305; Beijing Solarbio Science & Technology Co., Ltd.) assay kits, respectively, according to the manufacturer's protocols. Briefly, lysis buffer extracted from the PASMCs was added to a 96-well plate, and the absorbance values were measured to assess the concentrations of glucose, lactic acid and ATP. The rates of glucose consumption and lactic acid production and the amount of cellular ATP were normalized against the protein concentrations.