Smart metabolites database

A liver metabolomic analysis was performed using GC/MS as described previously (15) with some modifications. In brief, freeze-fractured liver samples (approximately 20 mg) were suspended in 250 μl of methanol-chloroform-water (2•5:1:1) and 5 μl of 1 mg/ml 2-isopropylmalic acid as an internal standard and homogenised using a polytron homogeniser (Microtec; China). Samples were subsequently mixed in a shaker at 1200 rpm at 37°C for 30 min and centrifuged at 16 000 g at 4°C for 5 min. Next, 160 μl of the supernatant was mixed with 200 μl of distilled water and vortexed, followed by centrifugation at 16 000 g at 4°C for 5 min; subsequently, 250 μl of the supernatant was dried under a vacuum using a centrifugal evaporator (RD-400; Yamato Scientific). Dried samples were pre-treated, derivatised and analysed by GC/MS (GCMS QP2010-Ultra; Shimadzu) within 24 h of derivatisation as described by Qiao et al. (15) . The Shimadzu Smart Metabolites Database (Shimadzu) was used to identify metabolites. The DNA content in the liver was analysed using the method by Labarca & Paigen (16) . The relative metabolite content was calculated as follows: the peak area of each metabolite was divided by that of the internal standard, 2-isopropylmalic acid, on the same chromatogram. It was further divided by the DNA content. The level for each metabolite in the control liver was set at 100.