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

Abstract

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.

Keywords: NAD; aging; human brain; in vivo 31P MR spectroscopy; redox state.

Fig. 1.

In vivo ³¹P MR spectra of two representative subjects at ages (A) 36 and (B) 52 y old. They were obtained within 16 min from human occipital lobe, where the ³¹P coil (5 cm in diameter) sensitivity profile was verified by in vivo ³¹P chemical shift imaging in a previous study (19). Insets display the expanded spectra in the chemical shift range from −9.0 to −11.5 ppm with the original in vivo ³¹P signals (gray) and the total signals (red) of α-ATP and NAD determined by the model fitting. The individual fitting components of α-ATP (blue), NAD⁺ (black), and NADH (green) and the residual signal of the fitting are also shown. The quantification results of NAD⁺/NADH ratio (RX) of (A) 4.8 and (B) 3.4 indicate a lower redox state for the older subject. PDE, phosphodiester; PME, phosphomonoester.

Fig. 2.

Simulated and in vivo ³¹P MR spectra of the human brain at 7 T. (A) Model-simulated spectra of NAD⁺ quartet, NADH singlet, total NAD, and combined α-ATP and NAD signals with an HLW of 18 Hz and an NAD⁺/NADH RX of 3.35. (B) Experimentally measured in vivo ³¹P MR spectra of human occipital lobe processed with 10-Hz line broadening that have the same HLW and RX values as the simulated spectra in A. The individual and combined in vivo spectra of NAD⁺, NADH, and total NAD were obtained by subtracting corresponding fitting components from the original brain spectra.

Fig. 3.

Age dependences of (A) intracellular NAD⁺, NADH, and total NAD concentrations and (B) NAD⁺/NADH RP observed in healthy human brains. The open symbols represent individual subject data, and the filled symbols display the average data from three age groups of younger (21–26 y old; n = 7), middle (33–36 y old; n = 4), and older (59–68 y old; n = 6) subjects.

Fig. 4.

A simplified scheme summaries the major functions of intracellular NAD: NAD as a coenzyme in regulating the glucose–oxygen metabolic balance that controls the cerebral ATP energy production through the NAD⁺/NADH redox state (Left), and NAD⁺ as a cosubstrate in modulating the activities of the NAD⁺-consuming enzymes that mediate the cellular signaling processes through the availability of cellular NAD⁺ (Right). The abnormality in either one or both of these functions could lead to mitochondrial dysfunction, neurodegeneration, or age-related metabolic disorders.