Sodium 3 trimethylsilyl 2 2 3 3 d4 propionate tsp

Sodium 3-trimethylsilyl [2,2,3,3-d4]-propionate (TSP) and deuterium oxide (D₂O, 99.9% D) were purchased from Cambridge Isotope Laboratories, Inc. (Cambridge, MA, USA). Sodium azide (NaN₃), K₂HPO₄·3H₂O and KH₂PO₄, all at analytical grade, were obtained from Sigma Aldrich (Milano, Italy).

Control and treated NHAIDH1mut, U87IDH1mut, and NHAIDH1wt cells were extracted using the dual-phase extraction method as previously described 52 (link). Briefly, ~3.0 × 10⁷ cells were trypsinized, centrifuged and vortexed in 10 mL of ice-cold methanol followed by 10 mL each of ice-cold chloroform and ice-cold water. Following phase separation, the aqueous phase was lyophilized and resuspended in 400 μL of deuterium oxide (Cambridge Isotope Laboratories, USA). 5 mM sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 (TSP) (Cambridge Isotope Laboratories, USA) was placed in a coaxial insert and used as an external chemical shift and quantification reference. Aqueous phase spectra (90° flip angle (FA), 3 s repetition time (TR), 384 scans) were acquired using a 500 MHz Bruker Avance spectrometer (Bruker Biospin, Germany). Prior to Fourier transformation, a line broadening of 0.3 Hz was applied to FIDs. All detectable metabolites were then quantified by peak integration following deconvolution of overlapping peaks using Mnova (MestreLab Research, Spain). Peak integrals were corrected for saturation and Nuclear Overhauser effect (NOE) correction factors and normalized to cell number as previously published 39 (link).

Approximately 1x10⁷ cells were extracted using the dual-phase extraction method as previously described [21 (link)]. Briefly, cells were trypsinized, centrifuged and vortexed in 10 ml of ice-cold methanol followed by 10 ml each of ice-cold chloroform and ice-cold water. Following phase separation, the aqueous phase was lyophilized and resuspended in 400 μL of deuterium oxide (Cambridge Isotope Laboratories, Andover, MA, USA)-based potassium phosphate buffer at pH 7. 5 mM sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 (TSP) (Cambridge Isotope Laboratories, Andover, MA, USA) placed in a coaxial insert was used as an external chemical shift and quantification reference.
Spectra were recorded using a 600 MHz Bruker Avance spectrometer (Bruker Biospin, Rheinstetten, Germany). Spectra were acquired using a 90° pulse, 3 second relaxation delay, 8 dummy scans, 16K data points, and 256 acquisitions (acquisition time ≈13 min). Water suppression was achieved using the 1D water presaturation ZGPR sequence. Prior to Fourier transformation, the FIDs were multiplied by an exponential weight function corresponding to a line broadening of 0.3 Hz.