Intracellular redox state revealed by in vivo ³¹P MRS measurement of NAD⁺ and NADH contents in brains

Abstract

Purpose Nicotinamide adenine dinucleotide (NAD), in oxidized (NAD+) or reduced (NADH) form, plays key roles in cellular metabolism. Intracellular NAD+/NADH ratio represents the cellular redox state; however, it is difficult to measure in vivo. We report here a novel in vivo 31P MRS method for noninvasive measurement of intracellular NAD concentrations and NAD+/NADH ratio in the brain. Methods It uses a theoretical model to describe the NAD spectral patterns at a given field for quantification. Standard NAD solutions and independent cat brain measurements at 9.4 T and 16.4 T were used to evaluate this method. We also measured T1 values of brain NAD. Results Model simulation and studies of solutions and brains indicate that the proposed method can quantify submillimolar NAD concentrations with reasonable accuracy if adequate 31P MRS signal-to-noise ratio and linewidth were obtained. The NAD concentrations and NAD+/NADH ratio of cat brains measured at 16.4 T and 9.4 T were consistent despite the significantly different T1 values and NAD spectra patterns at two fields. Conclusion This newly established 31P MRS method makes it possible for the first time to noninvasively study the intracellular redox state and its roles in brain functions and diseases, and it can potentially be applied to other organs.

In vivo NAD assay reveals the intracellular NAD contents and redox state in healthy human brain and their age dependences

“…Based on the MR physics, known molecular structures, and chemical shift information of the NAD + and NADH, the exact pattern of the NAD spectrum, including the chemical shifts and peak ratios of different NAD + resonances at a given field strength, can be precisely predicted (17,24). With this information, one can fit all of the NAD + and NADH resonances together; hence, the statistical power for achieving reliable quantification for the low-concentration metabolites will be much higher than fitting them independently.…”

“…By using the α-ATP resonance signal as an internal standard and assuming [ATP] = 2.8 mM in the normal brain tissue (17,21,22), we were able to noninvasively obtain quantitative data for the human brain on 17 healthy subjects spanning the ages of 21-68 y old. The results yielded the following intracellular concentrations: Chemical Shift (ppm) 6 2 -2 -6 -14 -10 -18…”

“…Thus, both NAD + and NADH can be quantified simultaneously by matching the in vivo NAD spectra with the model-simulated spectra. It has been shown that this approach works well in animal brains at ultrahigh fields of 9.4 and 16.4 T (17). In this study, we further exploit the feasibility and potential of this novel approach for noninvasive assessment of intracellular NAD + and NADH contents and NAD + /NADH redox state in healthy human brains using a 7-T human MR scanner and ultimately, studying their roles in human aging.…”

“…Recently, we have developed a novel magnetic resonance (MR) -based quantification approach that uses a high-field MR scanner to obtain the endogenous 31 P MR signals of the NAD molecules in intact animal brains (17). Distinct from earlier 31 P MR spectroscopy (MRS) studies that reported total NAD contents (18)(19)(20), our approach is able to resolve the MR signal of NADH from that of NAD + by taking advantage of their specific spectroscopic characteristics at a given magnetic field strength that can be precisely predicted based on a theoretical model (17).…”

“…Distinct from earlier 31 P MR spectroscopy (MRS) studies that reported total NAD contents (18)(19)(20), our approach is able to resolve the MR signal of NADH from that of NAD + by taking advantage of their specific spectroscopic characteristics at a given magnetic field strength that can be precisely predicted based on a theoretical model (17). Thus, both NAD + and NADH can be quantified simultaneously by matching the in vivo NAD spectra with the model-simulated spectra.…”