NMR SAMPLE PREPARATION

2.6.1. Removal of multivalent ion contaminants and pH adjustment

Interfering polyvalent metallic ions were removed by treating the samples with a chelating ion exchange resin (Chelex-100, Bio Rad Laboratories, Richmond, USA)

[62,74], Chelex operates better in a slightly alkaline environment, and it thoroughly removes traces of metals, without altering the concentration of non-metallic ions.

The pH of the supernatant (obtained as described in Sections 2.4.2 and 2.5.3) was immediately adjusted to slightly alkaline (pH >7.5) with 3 M KOH. The supernatant was further mixed with 5 mg Chelex per 0.2 ml sample, and left at room temperature for 1 hour, occasionally stirring gently. Its pH was further adjusted to 8.5-8.9 by treatment with 3 M KOH, then 1 M KOH for fine pH adjustment. A centrifugation step (3000g for 5 min.) followed, in order to remove the precipitated KCIO4, and the traces of Chelex resin from the sample. Samples were kept frozen (-20PC) until they were lyophilized.

2.6.2. Lvophilization and preparation of the NMR sample

Frozen samples were freeze-dried (Edwards High Vacuum International, BOC, Crawley, UK) in order to remove all the water content. The lyophilized samples were kept at -20°C, and each sample for NMR analysis was prepared the same day NMR spectroscopy was performed. The lyophilized powder was redissolved in 0.5- 0.7 ml deuterium oxide (D2O; Goss Scientific Instruments Ltd., Ingatestone, UK). As an internal concentration and chemical shift standard, 3-trimethylsilyl- tetradeuterosodium propionate (TSP; Goss Scientific Instruments Ltd.) was added to each NMR sample. Various volumes of the standard (1 mM TSP in D2O) were added to individual preparations, according to the amount of biological material present in each sample: 25 /^l-50 ^1 were enough for cell extracts, while for tissue preparations 50 /d-100 jLcl of the standard TSP solution were appropriate. Finally, the pH of the sample was adjusted to 8.9 with diluted solutions of DCl or NaOD (Aldrich, Gillingham, UK). A centrifugation step was usually necessary to remove the residual KCIO4 precipitate (3000g for 5 min.).

2.7. *H-NMR SPECTROSCOPY

Specific metabolite profiles from all the cell and tissue extracts examined were obtained by 'H-NMR spectroscopy. All spectra were acquired on a Varian Unity- plus NMR system (Varian Associates Inc., NMR Instruments, Palo Alto, CA)

operating at a proton frequency of 500 MHz, at 26°-30°C. Samples were placed into borosilicate NMR tubes (5 mm or 2 mm diameter, Wilmad Glass Company Inc., Buena, NJ, USA).

2.7.1. Single-pulse spectra

Single-pulse ^H-NMR spectra (approaching full relaxation) were acquired with 45® pulses applied every 5 s. The sample spinning rate was 20 Hz, spectral width 5000 Hz and data size 32K points. The residual water signal (HOD) was suppressed by applying a second radiofrequency pulse at the frequency of the water peak in the delay between acquisitions. For a satisfactory signal/noise ratio 512 or 1024 scans were accumulated and Fourier transformed with a line broadening of 0.2-0.3 Hz.

Metabolite identification in the one-dimensional spectra was carried out by:

1). chemical shift and coupling pattern of metabolites as described in the literature [61,62,74];

2). comparison with spectra of metabolites in known concentrations obtained at the same pH and spectroscopic conditions;

3). two-dimensional spectroscopic methods.

4). spiking of compounds to obtain assignments of unknown peaks

The following metabolites were identified from their strongest and best resolved resonances: /?-hydroxybutyrate - (j8-HB) - 7CH3 1.19 ppm (doublet); Threonine (Thr) - 7CH3 1.30 ppm (doublet); Lactate (Lac) -CH3 1.34 ppm (doublet); Alanine (Ala) - CH3 1.47 ppm (doublet); Acetate (Ace) -CH31.92 ppm (singlet); N-acetylaspartate (NAA) -NCOCH3 2.02 ppm (singlet); N-acetylaspartylglutamate (NAAG) -NCOCH3

2.05 ppm (singlet); 7-aminobutyric acid (GABA) - aCU2 2.30 ppm (triplet); Glutamate (Glu) - 7CH2 2.34 ppm (triplet); Succinate (Sue) -CHg 2.41 ppm (singlet); Glutamine (Gin) - 7CH2 2.44 ppm (triplet); Aspartate (Asp) -CH2 2.56 and 2.75 ppm (double doublet); Creatine (Cr) -NCH3 3.04 ppm (singlet); Taurine (Tau) -SCH2

3.08 ppm (triplet) and -NCH2 3.42 ppm (triplet); Choline -N(CH3)3 3.21 ppm (singlet); Phosphorylcholine (PC) -N(CH3)3 3.22 ppm (singlet); Glycerophosphorylcholine (GPC) -N(CH3)3 3.23 ppm (singlet); Glycine (Gly) -CH2

Metabolite quantification Amounts of metabolites present in samples were calculated from their intensities in ’H-NMR spectra, by reference to the internal standard TSP after baseline correction. The calculations for metabolite quantification were based on the fact that the intensity of a given signal in the proton spectrum is proportional to the amount of compound and number of protons contributing to that signal.

2.7.2 Two-dimensional (2D) spectroscopy

Two-dimensional spectroscopy was performed in order to aid the assignment of certain resonances in single-pulse spectra. All 2D experiments were performed non­ spinning to avoid long-term instability in the acquisition.

Homonuclear 2D-J-couvled spectroscopy (2D-J spectroscopy) This technique was used to separate chemical shifts from spin-spin coupling constants, and to determine the true multiplicity of couplings. Homonuclear J-resolved 2D spectra were acquired in spin-echo experiments with a sequence of 9QP pulses applied on the observed nucleus (^H), followed by a 180° pulse. For each of the 64 tl increments, 64 transients were collected with a repetition time of 1.5 s, spectral width 5000 Hz and data size 2K points. In the indirectly detected dimension (FI) the digitization was IK number of points, which was then zero-filled to 2K, and the spectral width was 50 Hz in order to include the greatest likely 3-bond proton couplings. Post-processing of the J-resolved spectra required a rotation by 45° to align the J-coupled multiplet components in the second dimension, followed by symmetrization of the data.

Homonuclear correlated spectroscopy (2D-COSY) This technique was used for identification of coupled resonances in the ID spectrum. Absolute value, and where time allowed, phase-sensitive COSY spectra were acquired either using a standard phase cycling regime, or alternatively with the application of pulsed field gradients (PFG; 20 G/cm, duration 2 ms, rise and fall time 100 ixs) to select coherence pathways [75]. Typically 192 transients were collected for each of 256 tl increments (repetition time = 1.2 s; spectral width = 5000 H^. Data were collected into 2K data points in F2, and IK in FI, which was then zero-filled to 2K. Sine-bell (for absolute- value COSY) and gaussian (for phase-sensitive COSY) weighting functions were applied before the Fourier transform in each dimension. The COSY plots were

symmetrised after initial inspection of the data for cross peaks of unequal intensity above and below the diagonal.