Deuterated water

All samples were stored at −80 °C until metabolomic analysis with NMR spectroscopy was performed. All tissues were homogenized before the analyses. In brief, the frozen tissue pieces were weighed and placed into pre-cooled (dry ice) homogenization tubes. Ice-cold extraction solvent (10 volumes of ethanolic solution (EtOH:H₂O, 77:23, v/v) was added to each tube in dry ice and the tissues were then homogenized by homogenizator (Ika Homogenizer T10, Sigma-Aldrich, Milan, Italy) three times over 30 s with 30 s pause intervals to ensure constant temperatures during homogenization. Then, the samples were ultra-sonicated at 20 kHz using a ultrasonic disintegrator MK2 (exponential probe, 8 µm peak-to-peak; MSE Scientific Instruments, Crawley, UK) and centrifuged at 14,000× g for 30 min. This method has been proven to have the same efficiency as the more common perchloric extract method, with the added benefit of a simpler experimental procedure [16 (link)]. The supernatant was then freeze-dried two times in a Savant RTV 4104 freeze dryer (Mildford, ME), and the residue resuspended in 0.7 mL of deuterated water (Sigma-Aldrich, Milan, Italy) containing 0.1 mM of 3 (trimethylsilyl)-propionic-2,2,3,3-d4 acid sodium salt (TSP) as an internal standard.

The following chemical reagents were used in this work:-

Benzoic acid (C₆H₅COOH) (“chemically pure” grade), used without additional purification. The purity of reagents was declared by the manufacturer >99% by weight.

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β-cyclodextrin (C₄₂H₇₀O₃₅) from Sigma-Aldrich Saint (Louis, MO, USA) with a CD content of ≥99% was used without additional purification;

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Dimethylsulfoxide (C₂H₆OS) was purified by distillation according to the method [75 ] before use. The DMSO content was 99.4 wt.%. The residual water content in the organic solvents used was taken into account when preparing the solutions.

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Deuterated water and dimethylsulphoxide (atomic fraction of deuterium more than 99.9%) were purchased from Sigma-Aldrich USA. Ethyl alcohol (C₂H₅OH) (96% by vol.) was purified by distillation at the atmospheric pressure.

The solutions were prepared by the weight method according to exact weights. The analytical scales of the brand AUW220D (SHIMADZU) were used. For the preparation of solutions, fresh bidistillate water was used.

Mice were fed ad libitum and ip. injected with deuterated water (Sigma) (20 μl per gram body weight). Mice were killed 6 h later and liver tissue and whole blood collected. Palmitate content was analyzed using gas chromatography-mass spectrometry. We determined the percentage contribution of newly made fatty acid using the equation: percentage of newly made fatty acid = (total ²H-labeled fatty acid/(²H-labeled body water × n)) × 100, where n is the number of exchangeable hydrogens, assumed to equal 22 for palmitate. We determined the absolute amount of newly synthesized fatty acids by multiplying the percentage of newly made fatty acids by the concentration of the total fatty acids^{53 (link)}.