5 13c glutamine

Estradiol benzoate was purchased from Aladdin. Oxytocin injection and soybean oil was purchased from Maclean. Guizhi Fuling Capsules are provided by Kangyuan Pharmaceutical. 5-13C-glutamine (Cambridge Isotope Laboratories, Andover, MA, United States); PGF2α kit, PGE2 kit and β-EP kit were purchased from Nanjing Jiancheng Bioengineering Research Institute.

Stable isotope internal standard 5-¹³C-glutamine was purchased from Cambridge Isotope Laboratories (Andover, MA, USA). Myristic-1,2-¹³C2 acid, methoxamine hydrochloride (purity 98%), and pyridine (≥99.8% GC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). N-methyl-trimethylsilyl-trifluoroacetamide (MSTFA) and 1% trimethylchlorosilane (TMCS) were provided by Pierce Chemical (Rockford, IL, USA). Methanol, acetonitrile, and n-heptane were of HPLC grade and obtained from Merck (Darmstadt, Germany). Purified water was produced by a Milli-Q system (Millipore, Bedford, MA, USA). Ammonium acetate (purity 98.0%) and ammonia solution (25%, w/w) were purchased from Aladdin (Shanghai, China) and Nanjing Chemical Reagent (Nanjing, China), respectively.

RPMI 1640 medium was purchased from Gibco. Fetal Bovine Serum (FBS), penicillin, and streptomycin were purchased from HyClone. GM-CSF was purchased from PeproTech. Ammonium acetate, LPS, oligomycin, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), rotenone, and antimycin A were purchased from Sigma. BBL™ Thioglycollate Medium was purchased from BD Biosciences, US. [5-13C]glutamine was obtained from Cambridge Isotope Laboratories. HPLC grade ammonium hydroxide, acetonitrile, and methanol were purchased from Fisher Scientific. Deionized water was produced by a Milli-Q system.

Livers isolated from each group were homogenized and mixed with 80% (v/v) methanol solution containing 15 μg/mL of 5‐¹³C‐glutamine (Cambridge Isotope Laboratories) as the internal standard. After centrifugation at 16 000 g for 10 minutes, the supernatant was collected and evaporated to dryness. The residue was dissolved, centrifuged and prepared for liquid chromatography‐quadrupole/time‐of‐flight mass spectrometry (LC‐Q/TOF‐MS)‐based metabolomics. Subsequent metabonomic analysis was performed as previously described.¹⁵ MultiQuant 2.0 analytical software (AB SCIEX, USA) was used for data analysis, and the quantification of samples was carried out with the peak area under protein concentration correction. The overall difference between groups was investigated with partial least squares discriminant analysis using SIMCA‐P software (Sweden). Furthermore, 3D sparse partial least squares discriminant analysis and heatmaps were generated at MetaboAnalyst (http://www.metaboanalyst.ca).