In vivo 1H NMR spectroscopy

The detection of NAD⁺ by ¹H MRS requires the use of short echo-time methods to minimize T₂-related losses and the avoidance of water perturbation to eliminate saturation transfer effects on NAD⁺. The sequence previously employed on rat brain was composed of 3D LASER localization with an echo-time of 14 ms (10). A direct translation to the human brain at 7 T is not feasible as limitations in maximum and average RF amplitudes would lengthen the LASER echo-time to a minimum of 50 ms. As a result the pulse sequence was transformed into a 1D LASER method (TR/TE = 1,500/18 ms) selecting a 20 mm slice parallel to the surface coil (Fig. 1A) through the occipital cortex. Slice selection was achieved with a pair of 2 ms adiabatic full passage (AFP, hyperbolic secant modulation (12), bandwidth = 5.0 kHz) refocusing pulses. Spatial localization in the X and Z directions are provided by the human head anatomy. The actual brain volume over all volunteers amounted to 96 ± 12 mL as evaluated from multi-slice MR images. The brain volume was not corrected for the coil reception profile. The contribution from muscle was < 5% on all volunteers. Significant lipid signal from skull and scalp tissue was present, but was inconsequential for NAD⁺ detection as lipids do not have NMR resonances beyond 5.5 ppm. Signal excitation was achieved with a minimum-phase 8.0 ms Shinnar-Le Roux pulse (13) of 0.775 kHz bandwidth centered on 8.8 ppm. As water is minimally perturbed, no additional forms of water suppression were required. ¹H MR spectra were acquired as 2,048 complex points over a 12 kHz spectral width with NA = 640 for a total acquisition time of 16 min.

¹H MR detection of NAD⁺ on human brain at 7 T. (A) MR image displaying the human brain, an 80 mm diameter surface coil transceiver (bottom white line) and a 20 mm thick slice for NAD⁺ detection. The bottom of the slice is typically set immediately above the sagittal sinus, as shown. (B) ¹H MR spectrum acquired from human brain (TR/TE = 1,500/18 ms, NA = 640, approximate volume = 100 mL) displaying the three NAD⁺ H2, H4 and H6 resonances in addition to a NAA amide resonance and various signals from adenosine, ATP, macromolecules (MM) and other, undefined compounds. Besides a minor water resonance at 4.7 ppm (~0.5% of full intensity) the rest of the spectrum is devoid of signal. (C) Representative ¹H MR spectra from four subjects displaying visually good reproducibility. To allow better visualization of the NAD⁺ resonances, the vertical scale has increased 5 times relative to (B) and the signal ‘hump’ between 7.8 and 8.6 ppm has been truncated.

Absolute quantification of NAD⁺ requires an internal concentration reference. In addition to the NAA amide singlet at ~7.85 ppm, total creatine was also pursued as a concentration reference. Since the lipids signals from skull and scalp overwhelm the spectrum at TE = 18 ms, creatine spectra were acquired at 50, 75, 100, 150 and 200 ms. Following spectral fitting the creatine intensity at TE = 0 ms was obtained via extrapolation.