3 trimethylsilyl propionic 2 2 3 3 d4 acid sodium salt tsp

All chemicals used for the buffer solution were purchased from Sigma-Aldrich except for the ²H₂O which was purchased from Cortecnet and the 3-(trimethylsilyl)propionic-2,2,3,3-d₄ acid sodium salt (TSP) from Cambridge Isotope Laboratories Inc. 96-well plates and NMR tubes were purchased from Bruker Biospin Ltd. (Germany).
Aliquots of 0.5 ml urine per sample were thawed overnight at 4°C. Cellular components and other insoluble material were then spun down by centrifugation for 10 min at 3184 g and 4°C and the supernatants were transferred into 96-well plates. 270 μL of urine from each sample were mixed with 30 μL buffer solution in ²H₂O (pH = 7.4) containing 1.5 M K₂HPO₄, 2 mM NaN₃ and 4 mM of TSP-2,2,3,3-d₄ as an internal standard and chemical shift reference (0.4 mM final concentration in each sample). Finally, 165 μL of each urine-buffer mixture were transferred to 3 mm SampleJet NMR tubes and placed in refrigerated racks (6°C) of a SampleJet system until the NMR measurements. Both mixing of urine with buffer and transfer of the mixture to NMR tubes were performed by two 215 Gilson liquid handler robots and controlled by the SampleTrack software (Bruker Biospin Ltd.).

Lead nitrate (Pb(NO₃)₂), sodium lauryl sulfate, creatinine anhydrous, propionic acid, lithium d-, and l-lactate, acetylacetone, 37% formaldehyde, cimetidine, indoleacetic acid, methylumbelliferone, and methylumbelliferyl N-acetyl-β-d-glucosaminide were purchased from Sigma-Aldrich Fine Chemicals Inc. (MO, USA). Formic acid, HPLC grade acetonitrile (ACN), ethanol and methanol (MeOH) were purchased from Merck (Darmstadt, Germany). Hippuric acid, 2,2′-dipyridyl disulfate (DPDS), triphenylphosphine (TPP) and 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). 5-Aminolevulinic acid hydrochloride and guanidinoacetic acid were purchased from Alfa Aesar (MA, USA). Sulbactam was purchased from Cayman Chemical (Michigan, USA). Acetic acid was purchased from Hayashi Pure Chemical Ind.,Ltd. (Osaka, Japan). Trifluoroacetic acid (TFA) was purchased from Riedel-de Haën (Seelze, Germany). 3-(Trimethylsilyl)propionic-2,2,3,3-d₄ acid sodium salt (TSP) was purchased from Cambridge Isotope Laboratories (MA, USA). 1,1-Dimethylbiguanide hydrochloride (metformin) was purchased from Santa Cruz Biotechnology, Inc. (Texas, USA). Protein Assay Dye Reagent was purchased from Bio-Rad Laboratories (California, USA).