MRS-based NAD+ quantification: Responsibility of a novel
MRS-based method quantifying NAD+ in vivo

With increasing prevalence of cardiometabolic disease, the challenge of the
coming years is to intervene at an earlier stage in the pathogenesis of such
disease and discover new therapeutic targets. To identify and monitor the
mechanisms responsible for blunted cardiometabolic health in humans, and
progression to metabolic disease such as diabetes, non-invasive imaging methods
are needed to investigate metabolism dynamically. Standard Magnetic Resonance
Spectroscopy (MRS) methodology is nowadays commonly used to examine readily
detectable biochemical compounds (like ectopic lipids). However, by dedicated
design of novel*MRS sequences, more unique metabolites key in the development
of metabolic*disorders, can be visualized and quantified*non-invasively.
Animal studies indicate that Nicotinamide Adenine Dinucleotide (NAD+), by being
an activator of many intracellular enzymes and a co-regulator of mitochondrial
function and biogenesis, may well play a role in regulating metabolic health.
Human data on the relevance of NAD+ is still very scarce because investigation
in humans requires rapidly processed tissue samples obtained via invasive
procedures (muscle biopsies). Consequently, kinetic information is particularly
difficult to obtain. 31P-MRS at high magnetic field (7T) was used to quantify
NAD+ and NADH in the brain (Zhu, Lu et al. 2015). But at clinical field
strength, the spectral resolution is lower, resulting in overlapping peaks of
NAD+, NADH and ATP signals.
Therefore, to ensure robust quantification in muscle, design of tailored
sequences for NAD+ detection are warranted in order to achieve the simultaneous
suppression of ATP signals that are overlapping with NAD and enable
quantification at clinical field strength.

The objective of this study is to validate a novel MRS-based method to quantify
NAD+ by selectively suppressing the coupled α-ATP spin system, which is
overlapping with the NAD+/NADH signals in vivo. For NAD+/NADH quantification to
be valuable in metabolic research, it should be possible to pick up changes in
response to physiological stimuli. It is well known that NAD+ is increased upon
fasting and ischemia. Therefore, in the current protocol, the measurement of
NAD+ will be validated after such interventions. Furthermore, the in vivo
measurements will be compared to NAD+ and NADH quantification in muscle
biopsies.

Proof of principle study with two short interventions making use of pre/post
design.

ischemia (8 minutes) and prolonged fasting (36h)

This study carries no benefits for the subjects and carries minor risks for the
subjects. The major burdens consist of a moderate time commitment,
staying fasted for a prolonged time and multiple muscle biopsies and blood
sampling.