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Edited by Marcus E. Raichle, Washington University in St. Louis, St. Louis, MO, and approved January 27, 2015 (received for review September 16, 2014)
NAD is an essential metabolite that exists in NAD+ or NADH form In addition, NAD+ does not fluoresce and thus, cannot be detected
in all living cells. Despite its critical roles in regulating mitochon- by fluorometry (16). Clearly, a nondestructive detection and quan-
drial energy production through the NAD+/NADH redox state and tification method is highly desired to investigate the NAD contents
modulating cellular signaling processes through the activity of the and redox state in the human body or intact animal models.
NAD+-dependent enzymes, the method for quantifying intracellu- Recently, we have developed a novel magnetic resonance (MR)
lar NAD contents and redox state is limited to a few in vitro or ex -based quantification approach that uses a high-field MR scanner
vivo assays, which are not suitable for studying a living brain or to obtain the endogenous 31P MR signals of the NAD molecules in
organ. Here, we present a magnetic resonance (MR) -based in vivo intact animal brains (17). Distinct from earlier 31P MR spectros-
NAD assay that uses the high-field MR scanner and is capable copy (MRS) studies that reported total NAD contents (1820), our
of noninvasively assessing NAD+ and NADH contents and the approach is able to resolve the MR signal of NADH from that of
NAD+/NADH redox state in intact human brain. The results of this NAD+ by taking advantage of their specific spectroscopic charac-
study provide the first insight, to our knowledge, into the cellular teristics at a given magnetic field strength that can be precisely
NAD concentrations and redox state in the brains of healthy vol- predicted based on a theoretical model (17). Thus, both NAD+ and
unteers. Furthermore, an age-dependent increase of intracellular NADH can be quantified simultaneously by matching the in vivo
NADH and age-dependent reductions in NAD+, total NAD contents, NAD spectra with the model-simulated spectra. It has been shown
and NAD+/NADH redox potential of the healthy human brain were that this approach works well in animal brains at ultrahigh fields of
revealed in this study. The overall findings not only provide direct 9.4 and 16.4 T (17). In this study, we further exploit the feasibility
evidence of declined mitochondrial functions and altered NAD ho- and potential of this novel approach for noninvasive assessment of
meostasis that accompany the normal aging process but also, eluci- intracellular NAD+ and NADH contents and NAD+/NADH redox
date the merits and potentials of this new NAD assay for non- state in healthy human brains using a 7-T human MR scanner and
invasively studying the intracellular NAD metabolism and redox ultimately, studying their roles in human aging.
state in normal and diseased human brain or other organs in situ.
Results
redox state | NAD | in vivo 31
P MR spectroscopy | human brain | aging In Vivo 31
P MRS of Human Brain at 7 T. Fig. 1 displays the in vivo
31
P MR spectra acquired from the occipital lobes in two repre-
6 2 -2 -6 -10
Chemical Shift (ppm)
-14 -18
vivo 31P MRS in the human brain.
By using the -ATP resonance signal as an internal standard and
B PCr
Residual
assuming [ATP] = 2.8 mM in the normal brain tissue (17, 21, 22),
Fitting
we were able to noninvasively obtain quantitative data for the
human brain on 17 healthy subjects spanning the ages of 2168 y
-ATP
old. The results yielded the following intracellular concentrations:
-ATP NADH
[NAD+] = 0.30 0.02 mM or 0.27 0.02 mol/g (after a unit
Original
PME & NAD+ -ATP conversion using a brain tissue density of 1.1 g wet brain tissue per
Pi PDE
1 mL), [NADH] = 0.06 0.01 mM or 0.06 0.01 mol/g, and total
-9.5 -10.5 (ppm)
NAD ([NAD]total = [NAD+] + [NADH]) = 0.37 0.02 mM or
6
6
4
2 -2
6
-6
8 10
-10
12 14
-14
16 18
-18
20
11) ensured reliable spectral fitting and NAD quantification. NADH NADH
Although it is difficult to determine the actual SNRs of NAD & NAD+ 40
& NAD+
and NADH because of their complex spectral patterns and signal
overlapping, we were able to measure the SNR of 17 2 (n = 11)
20
NADH
NAD+ and NADH signals, respectively. & NAD+ & NAD+
10
NADH
model-fitted (Fig. 1, red traces) spectra within the chemical shift
4
10
NAD+ NAD+
the in vivo NAD spectra to the NAD model simulation at 5
(a quartet with different chemical shifts and peak ratios that are -9 -10 -11 -9 -10 -11
field-dependent) and NADH (a singlet with a field-independent Chemical Shift (ppm) Chemical Shift (ppm)
chemical shift) as well as their combined resonance signals at 7 T
with different resonance linewidths (LWs; e.g., LW = 8, 16, and Fig. 2. Simulated and in vivo 31P MR spectra of the human brain at 7 T.
24 Hz) and a typical NAD+/NADH ratio of four were exempli- (A) Model-simulated spectra of NAD+ quartet, NADH singlet, total NAD,
and combined -ATP and NAD signals with an HLW of 18 Hz and an NAD+/NADH
fied in Fig. S1. Fig. S1 illustrates how the NAD spectra evolve
RX of 3.35. (B) Experimentally measured in vivo 31P MR spectra of human
with increasing resonance LWs, which mimics the sample con- occipital lobe processed with 10-Hz line broadening that have the same
ditions from an ex vivo solution toward in vivo tissue. Therefore, HLW and RX values as the simulated spectra in A. The individual and
in an intact brain, the five sharp peaks that characterize the spec- combined in vivo spectra of NAD + , NADH, and total NAD were obtained
trum of NAD+ and NADH mixture merge into an apparent doublet by subtracting corresponding fitting components from the original brain
shown in Fig. 2, which also includes the -ATP resonance in the spectra.
cerebral metabolic rate of oxygen (CMRO2). These processes plexes IV) activities observed in aged mice brains (38), which is in
are regulated by the intracellular NAD+/NADH redox state, line with another human brain study showing an 30% reduction
which determines the metabolic balance between the cellular in both neuronal oxidative glucose metabolism and neurotrans-
carbohydrate and oxygen metabolisms in supporting the brain mission cycling rates in elderly people (39). A similar reduction
ATP energy under normal or diseased condition (2, 29, 30). (40%) of mitochondrial oxidative and phosphorylation activities
We hypothesize that the intracellular NAD+ /NADH RX could was also observed in the human skeletal muscle during healthy
provide a single valuable measure directly linking to the met- aging, suggesting that the decline in mitochondrial functionality
abolic oxygen-to-glucose index defined by the CMRO2/CMRglc should be a global phenomenon in the aging process (40).
ratio, which is an essential biomarker of the neuroenergetics for In addition to governing energy homeostasis through regulat-
various brain states (31). ing the NAD+/NADH redox state, the biosynthesis and catabo-
In this study, we found a gradual but significant decline in the lism of NAD+ can influence the activity of various enzymes that
human brain NAD+/NADH RX (or RP) associated with a de- mediates metabolic processes in the cell. As shown in Fig. 4, Right,
creasing NAD+ level and an increasing NADH level in the process NAD+ could also function as a cosubstrate for several classes
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