Background and aims
Nicotinamide adenosine dinucleotide (NAD) plays an important role in energy metabolism. NAD+ forms a redox couple with NADH and a high concentration of NAD⁺ as well as a high NAD⁺/NADH ratio are strongly associated with metabolic health. In contrast, decreased NAD⁺ bioavailability is reported in both ageing and obese humans as well as in diabetic mice. Currently, assessment of NAD metabolites in muscle relies on (invasive) biopsies. Determination of NAD metabolites using standard ³¹P-MRS is not possible in muscle, since the resonance of α-ATP is overlapping and thereby masking the NAD resonances. Here, we developed a non-invasive phosphorus magnetic resonance spectroscopy (³¹P-MRS) technique to determine NAD metabolites in vivo. The newly developed MRS sequence was validated in vivo by measuring the NAD⁺/H levels in resting state and during ischemia, as it is well known that ischemia decrease NAD⁺/NADH levels.

Materials and methods
We applied a homonuclear BIRD (HB) filter to suppress the α-ATP resonance, allowing quantification of NAD⁺/NADH. For validation, we included 8 young healthy lean participants and NAD⁺/NADH were measured in the lower leg in rest and during eight minutes of ischemia using this new ³¹P-MRS technique. Ischemia was induced by inflating an upper leg cuff to a pressure of 50 mmHg above systolic blood pressure.

Results
The HB filter succeeded in suppressing the α-ATP resonance by 85% and allowed quantification of NAD⁺/NADH levels. Validation measurements with acute ischemia revealed an acute reduction in NAD⁺ signal intensity (from 93 ± 4 to 78 ± 4 [AU], p<0.05) and tended to reduce the ratio NAD⁺/NADH (from 3.41 ± 0.71 to 2.21 ± 0.29, p=0.08), in line with previous research in muscle biopsies.

Conclusion
We developed a state-of-the-art ³¹P-MRS technique enabling non-invasive quantification of NAD metabolites. Applying this technique during ischemia, we could detect the expected changes in NAD+ and NADH, showing that physiological changes in NAD metabolites can be detected by dedicated ³¹P-MRS. This provides new non-invasive means for future metabolic research on the relevance of NAD metabolism in type 2 diabetes.