Mitochondrial NAD(P)H In vivo: Identifying Natural Indicators of Oxidative Phosphorylation in the 31P Magnetic Resonance Spectrum

Abstract

Natural indicators provide intrinsic probes of metabolism, biogenesis and oxidative protection. Nicotinamide adenine dinucleotide metabolites (NAD(P)) are one class of indicators that have roles as co-factors in oxidative phosphorylation, glycolysis, and anti-oxidant protection, as well as signaling in the mitochondrial biogenesis pathway. These many roles are made possible by the distinct redox states (NAD(P)+ and NAD(P)H), which are compartmentalized between cytosol and mitochondria. Here we provide evidence for detection of NAD(P)+ and NAD(P)H in separate mitochondrial and cytosol pools in vivo in human tissue by phosphorus magnetic resonance spectroscopy (31P MRS). These NAD(P) pools are identified by chemical standards (NAD+, NADP+, and NADH) and by physiological tests. A unique resonance reflecting mitochondrial NAD(P)H is revealed by the changes elicited by elevation of mitochondrial oxidation. The decline of NAD(P)H with oxidation is matched by a stoichiometric rise in the NAD(P)+ peak. This unique resonance also provides a measure of the improvement in mitochondrial oxidation that parallels the greater phosphorylation found after exercise training in these elderly subjects. The implication is that the dynamics of the mitochondrial NAD(P)H peak provides an intrinsic probe of the reversal of mitochondrial dysfunction in elderly muscle. Thus, non-invasive detection of NAD(P)+ and NAD(P)H in cytosol vs. mitochondria yields natural indicators of redox compartmentalization and sensitive intrinsic probes of the improvement of mitochondrial function with an intervention in human tissues in vivo. These natural indicators hold the promise of providing mechanistic insight into metabolism and mitochondrial function in vivo in a range of tissues in health, disease and with treatment.

SS‐31 and NMN: Two paths to improve metabolism and function in aged hearts

“…While limited by signal to noise and high variance, the novel MRS method that we describe here presents an exciting new means to analyze metabolic changes in response to a stimulus in live mice. We believe that the mitochondrial NAD(P)H peak we have described consists primarily of NADH based on its dynamic behavior here and in our prior study in which it was first described (Conley et al, 2016), although we are unable to definitively say to what degree NADPH contributes to the peak. Taking all of the above results into consideration, we propose the following model to contextualize the differences in metabolic shifts seen between the mouse groups with the increase in cardiac workload.…”

“…While limited by signal to noise and high variance, the novel MRS method that we describe here presents an exciting new means to analyze metabolic changes in response to a stimulus in live mice. We believe that the mitochondrial NAD(P)H peak we have described consists primarily of NADH based on its dynamic behavior here and in our prior study in which it was first described (Conley et al, 2016 (Chavez et al, 2019).…”

“…This measurement provides an assessment of how effectively the heart can respond to increased work by determining the degree to which In order to analyze how less abundant metabolites in the heart respond to increased cardiac workload in these mice, peaks corresponding to chemical standards of α-ATP, UDP-glucose, NAD + , TA B L E 1 Significant age-related metabolomics changes in mouse hearts NADH, NADP + , and NADPH were fit to the experimental spectra for quantification using a custom model in OriginPro software (OriginLab, Northampton, MA). An additional peak corresponding to mitochondrial NAD(P)H was also fit to the data, based upon our prior study demonstrating the presence of this peak in vivo, which has an altered chemical shift relative to cytosolic NAD(P)H due to the unique environment of the mitochondrial matrix (Conley, Ali, Flores, Jubrias, & Shankland, 2016). A representative fit of experimental data using this model is shown in Figure 4c.…”

Mitochondrial oxidative capacity and NAD+ biosynthesis are reduced in human sarcopenia across ethnicities

“…Collectively, the MEMOSA study results provide a genome-wide molecular resource of mechanisms and biomarkers of pathological muscle aging across ethnicities. Our work establishes loss of mitochondrial oxidative capacity as a major mechanism of sarcopenia which can be assessed non-invasively using 31P imaging as a biomarker 60 , and provides a strong rationale for intervention trials targeting muscle mitochondrial bioenergetics to manage sarcopenia in older people.…”

Signaling and Regulation Through the NAD+and NADP+Networks

“…Although 31 P NMR resonances of NADP + and NADPH are riding at the foot of the -ATP peak, their resonances and their compartmental ratio can be distinguished in vivo in human tight muscle by careful extraction of the nicotinamide nucleotide phosphate peak region and measuring their magnitude changes upon changes in mitochondrial population density and fitness of the test subject upon exercise training (24).…”