1H Nuclear Magnetic Resonance (NMR) Spectroscopy

Sample preparation was performed as previously described^51–53. Briefly, plasma samples were defrosted on ice and centrifuged at 17,000 xg for 5 min at 4 °C. An equal volume of plasma was aliquoted (100 μL) into a fresh tube and diluted to 600 μL in a 75 mM phosphate buffer (5:1 disodium phosphate Na₂HPO₄, monosodium phosphate NaH₂PO₄ in 100% D₂O, pH 7.4). The volume of plasma used was limited by the volume of plasma collected, and the volume chosen was the maximum volume available for 90% of samples.

Lyophilized brain tissue samples were resuspended in 600 μL of phosphate buffer (0.2 M Na₂HPO₄, 0.043 NaH₂PO₄, in 100% D₂O with 0.05 wt % 3-trimethylsilylpropanoic acid (TSP)).

¹H NMR spectra were acquired using a 700-MHz Bruker AVII spectrometer operating at 16.4 T equipped with a ¹H (13C/15 N) TCI cryoprobe⁵². Sample temperature was stable at 310 K. ¹H NMR spectra were acquired using a one-dimensional (1D) Nuclear Overhauser Effect Spectroscopy (NOESY) pre-saturation scheme for attenuation of the water resonance with a 2 s pre-saturation⁵². An additional sequence, the spin-echo Carr-Purcell-Meiboom-Gill (CPMG) sequence, was used for plasma samples to suppress broad signals arising from large molecular weight plasma components with a τ interval of 400 μs, 80 loops, 32 data collections, an acquisition time of 1.5 s, a relaxation delay of 2 s, and a fixed receiver gain⁵¹. CPMG spectra provide a measurement of small molecular weight metabolites and mobile side chains of lipoproteins in the plasma sample and were used for all further analysis of plasma samples.