Intracellular redox state revealed by in vivo 31P MRS measurement of NAD+ and NADH contents in brains

Nicotinamide adenine dinucleotide (NAD), as an important coenzyme mediating cytosolic and mitochondrial biochemical processes, plays a key role in energy metabolism in all living cells (1,2). The conversion between its reduced form (NADH) and oxidized form (NAD+) occurs in all redox reactions catalyzed by NADH-dependent dehydrogenase, including the major metabolic pathways of glycolysis, tricarboxylic acid cycle, and electron transport chain. Besides regulating bioenergetics and maintaining mitochondrial function, accumulating evidence has suggested that NAD+ and NADH are also involved in other biological activities (3,4) such as calcium homeostasis (5,6), antioxidation and oxidative stress, gene expression, immunological functions, aging (7), and cell death (2,8). Several redox pairs, i.e., NAD+ and NADH, the oxidized and reduced forms of nicotinamide adenine dinucleotide phosphate (NADP) and those of glutathione, are involved in establishing the intracellular redox state. However, the significantly higher intracellular level of NAD under physiological conditions (4,9) makes NAD the dominant component in regulating intracellular redox state. Therefore, the concentration ratio of NAD+ and NADH (defined as RX = [NAD+]/[NADH]) is widely accepted as a major representation of the intracellular redox state; and its changes have been linked to alterations in energy metabolism under various physiopathological conditions including aging (10), diabetes, stroke, cancer, and epilepsy (11–13). To date, there have been only two approaches used to assess the intracellular NAD+/NADH redox state: one is based on biochemical analysis and the other relies on the autofluorescence signal of the intracellular NADH. The biochemical analysis uses high-performance liquid chromatography (14), capillary electrophoresis (15), or enzymatic cycling assays (13), to measure the NAD+ and NADH contents and, thus, the NAD+/NADH ratio. This approach detects the total amount of cellular NAD+ or NADH content, including free and protein-bound forms, without distinguishing their subcellular compartmentation. Since the biochemical approaches require tissue biopsy and extraction, they are not suitable for longitudinal studies in intact organs. On the other hand, by measuring weak endogenous fluorescence signals, the autofluorescence approach can monitor the intracellular level of NADH, mainly from the mitochondria in living cells (16–19). However, this approach suffers from low detection sensitivity, limited penetration and difficulty in detecting the NADH signal from deep tissue or organs, NADH unrelated artifacts, and potential cell injury resulting from ultraviolet irradiation. Moreover, the autofluorescence approach is unable to detect the NAD+ form and it is difficult to provide absolute quantification of intracellular NADH concentration (16,17). These drawbacks have restricted its utility in quantitatively determining the intracellular NAD contents and the redox ratio in vivo. Although studies using novel genetically encoded fluorescent sensors have reported improved sensitivity of NADH detection (20,21), a noninvasive approach suitable for in vivo applications aiming to quantify intracellular NAD+ and NADH contents is still absent. In vivo 31P MRS has been used to study cellular metabolism in intact cells and organs since the late 1970s (22), and it could be, in principle, a good candidate for studying cellular NAD metabolism and redox state since both NAD+ and NADH molecules have two phosphate spins. Unfortunately, although NAD signals have been identified in 31P spectra in vivo decades ago, it was generally believed that low concentration NAD metabolites were below the level of in vivo NMR detection. Another challenge is the difficulty in resolving individual NAD+ and NADH resonances from their overlapped signals in an in vivo 31P spectrum, particularly, at relatively low fields. In this study, by taking the advantages of high/ultra-high fields, we developed a novel 31P MRS-based quantification method capable of directly measuring and quantifying the intracellular NAD+ and NADH concentrations and their ratio in vivo. This approach is based on a theoretical NMR model for a description of the NAD phosphorus resonance signals and spectral patterns at a given field strength. By least-square fitting the model to the partially overlapped resonances of NAD+, NADH, and α-ATP in a 31P spectrum, the NAD+ and NADH concentrations and the RX value can be determined. To test and evaluate this novel NAD quantification method, the following studies were designed and conducted: (i) model simulations mimicking the 31P MR spectra obtained under various measurement conditions; (ii) rigorous tests using a series of NAD standard solutions with known NAD+ and NADH concentrations and varied RX, pH, or free [Mg2+]; (iii) independent measurements of anesthetized healthy cat brains at two different magnetic fields (9.4 T vs. 16.4 T). The longitudinal (T1) and apparent transverse ( T2∗) relaxation times of NAD+ and NADH in cat brains were also measured at 9.4 T and 16.4 T. Our results indicate, for the first time, that it is possible to reliably measure the intracellular NAD+ and NADH concentrations in an intact brain. The significance of this advancement includes providing noninvasive means for intracellular redox state evaluation in normal or diseased brains and the potential for translation to human application.