NAD is an essential metabolite that exists in NAD + or NADH form in all living cells. Despite its critical roles in regulating mitochondrial energy production through the NAD + /NADH redox state and modulating cellular signaling processes through the activity of the NAD + -dependent enzymes, the method for quantifying intracellular NAD contents and redox state is limited to a few in vitro or ex vivo assays, which are not suitable for studying a living brain or organ. Here, we present a magnetic resonance (MR) -based in vivo NAD assay that uses the high-field MR scanner and is capable of noninvasively assessing NAD + and NADH contents and the NAD + /NADH redox state in intact human brain. The results of this study provide the first insight, to our knowledge, into the cellular NAD concentrations and redox state in the brains of healthy volunteers. Furthermore, an age-dependent increase of intracellular NADH and age-dependent reductions in NAD + , total NAD contents, and NAD + /NADH redox potential of the healthy human brain were revealed in this study. The overall findings not only provide direct evidence of declined mitochondrial functions and altered NAD homeostasis that accompany the normal aging process but also, elucidate the merits and potentials of this new NAD assay for noninvasively studying the intracellular NAD metabolism and redox state in normal and diseased human brain or other organs in situ.redox state | NAD | in vivo 31 P MR spectroscopy | human brain | aging N AD, a multifunctional metabolite found in all living cells, has been the interest of many scientific investigations since its discovery in the early 20th century (1). NAD is known to convert between its oxidized NAD + and reduced NADH forms during the breakdown of nutrients; hence, the intracellular NAD + /NADH redox state reflects the metabolic balance of the cell in generating ATP energy through oxidative phosphorylation in mitochondria and/or glycolysis in cytosol (2). More recently, after several protein families associated with cell survival were found to use NAD + as their main substrate with activities also regulated by the availability of the NAD + , the full extent of the NAD's function as a metabolic regulator began to unfold (3-5). A growing number of studies have indicated that NAD + can modulate metabolic signaling pathways and mediate important cellular processes, including calcium homeostasis, gene expression, aging, degeneration, and cell death; therefore, the cellular NAD could serve as a therapeutic target for treating various metabolic or age-related diseases and promoting longevity (6-12).Despite the critical relevance of the intracellular NAD metabolism to human health and diseases, assessment of NAD contents and NAD + /NADH redox state is extremely challenging. Only a few invasive techniques based on biochemical assays or autofluorescence methods have been used to analyze tissue samples or cell extracts (13,14). However, during the preparation of such ex vivo sample, the NAD + and NADH contents are likely altered, beca...

Quantitative assessment of cerebral glucose consumption rate (CMR) and tricarboxylic acid cycle flux (V) is crucial for understanding neuroenergetics under physiopathological conditions. In this study, we report a novel in vivo Deuterium (H) MRS (DMRS) approach for simultaneously measuring and quantifying CMR and V in rat brains at 16.4 Tesla. Following a brief infusion of deuterated glucose, dynamic changes of isotope-labeled glucose, glutamate/glutamine (Glx) and water contents in the brain can be robustly monitored from their well-resolved H resonances. Dynamic DMRS glucose and Glx data were employed to determine CMR and V concurrently. To test the sensitivity of this method in response to altered glucose metabolism, two brain conditions with different anesthetics were investigated. Increased CMR (0.46 vs. 0.28 µmol/g/min) and V (0.96 vs. 0.6 µmol/g/min) were found in rats under morphine as compared to deeper anesthesia using 2% isoflurane. This study demonstrates the feasibility and new utility of the in vivo DMRS approach to assess cerebral glucose metabolic rates at high/ultrahigh field. It provides an alternative MRS tool for in vivo study of metabolic coupling relationship between aerobic and anaerobic glucose metabolisms in brain under physiopathological states.

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.