'VEMA-trial: MRS based NAD+ quantification. Responsibility of a novel MRS-based method quantifying NAD+ in vivo'

Rationale: 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 (3T), 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.

Objective: The objective of this study is to investigate whether differences over time are able to determine with 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, exercise and ischemia. Therefore, in the current protocol, the measurement of NAD+ will be applied after such interventions. Furthermore, the in vivo measurements will be compared to NAD+ and NADH quantification in muscle biopsies to explorative investigate whether similar results are found as in vivo.

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

Study population: Two groups of respectively 8 and 12 healthy young lean subjects will be included.
Intervention (if applicable): One group with 8 subjects will undergo an ischemia protocol during a MRS measurement. The other group will consist of 12 subjects who will remain fasted for 36 hours and MRS will be performed before, during and after the fasting period. Before/after intervention, also muscle biopsies will be taken.

Main study parameters/endpoints: NAD+ concentration as measured by 31P-MRS before/after intervention. Secondary endpoint is NAD+ as determined in muscle biopsies.

Nature and extent of the burden and risks associated with participation, benefit and group relatedness: 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.

Primary Objective:
The primary objective of this study is to determine whether the physiological increase of NAD+ induced by either ischemia or prolonged fasting can be measured with 31P-MRS in healthy lean subjects.

Exploratory Objective(s):
An exploratory objective of this study is to investigate whether the NAD+ levels measured in vivo with 31P-MRS are comparable with the ex vivo blood and muscle NAD+ levels.

Ischemia-part: 1 day, 4 hours
Fasting-part: 1.5 day, 36 hours

One of the two subject groups will undergo a ischemia protocol combined with MRS measurements (“ischemia” group).
The other group will be included to investigate whether differences over time are able to determine with the MRS-method by monitoring the fasting-induced increase in NAD+ concentration (“fasting” group). MRS measurements will be performed at 4 time points, at the start of the fasting period and after 12-, 24- and 36 hours of fasting. After every MRS measurement, blood will be drawn. After the first and after the last MRS measurement, a muscle biopsy will be taken.

Ischemia group:
In order to increase NAD+ concentrations, a MRI compatible blood pressure cuff will be secured around the thigh of the subject for rapid inflation (ischemia) and subsequently deflation during a MRS measurement to quantify NAD+ levels. Inflation of the cuff will be performed for 8 minutes (cuff pressure inflated to 50 mmHg above resting systolic pressure determined prior to the start of the measurement). The two days before the test days, volunteers will refrain from strenuous exercise and food intake will be standardized by providing a standardized breakfast and lunch.

Fasting group:
Furthermore, twelve young lean subjects will be included to investigate whether differences over time are able to determine with the MRS-method by monitoring the fasting-induced increase in NAD+ concentrations. For very controlled conditions and to monitor substrate oxidation by indirect calorimetry, subjects will enter so-called respiration chambers in the evening of day 1. Until the morning of day 3, subjects will stay in the chamber and only consume water. MRS measurements will be performed every 12 hours (evening before entering the respiration chamber, morning of day 2, late afternoon of day 2 and morning of day 3 (before breakfast)). In the evening of day 1 and the morning of day 3, a muscle biopsy will be taken to exploratively investigate whether similar results are found as in vivo, see figure 3 for scheme of tests. The day before the test days, volunteers will refrain from strenuous exercise. Food intake will be standardized by asking subjects to consume a light lunch and stay fasted thereafter until they come to the university at 17.30. After the evening meal (provided at the university) subjects will stay fasted overnight and the following day and night.

Note: due to the outbreak of the COVID-19 pandemic the fasting group was not performed in this study.