Nicotinamide adenine dinucleotide (NAD⁺) is known to play an important role in the cellular metabolism as a coenzyme for electron transfer enzymes. In addition, reduction-oxidation (redox) reactions in particular have the involvement of NAD⁺ (oxidized) and NADH (reduced). The reactions carried out by NAD play a pivotal role in glycolysis and mitochondrial metabolism and the small changes in its level may lead to impairment in cellular function and survival^1,2. Thus, in vivo monitoring of NAD⁺ levels can be used to study the oxidative stress levels related to various physiological conditions. Currently, in vivo detection of NAD species (NAD⁺ + NADH) is possible only via ³¹P NMR spectroscopy which is limited by inability to separate oxidized and reduced forms³. Very recently, ¹H MR spectroscopy has been shown to detect NAD+ in the brain although it is limited by the spatial resolution and SNR⁴. Chemical Exchange Saturation Transfer (CEST) technique has been utilized to image metabolites and macromolecules in vivo based on the property of exchangeable protons with water. CEST has been recently applied to image various molecules in vivo such as glutamate⁵, creatine⁶, myo-inositol⁷ and glucosaminoglycan⁸. In the current study, we characterized the CEST effect from NAD+ at different saturation pulse power and saturation duration in vitro. The initial results are discussed.High resolution NMR spectroscopy of NAD⁺ and NADH solutions (200 mM, pH 2.8) was performed on 9.4T vertical bore scanner (Bruker, Germany) at 5 and 37 ºC with a single pulse-acquire spectroscopy with TR=4 s, number of averages=32. For CEST imaging, NAD⁺ solution (20 mM) was prepared in phosphate buffer saline (PBS) and adjusted to a pH 7. The CEST parameters were optimized at 9.4T MRI scanner (Agilent Technologies, USA). During the course of experiment temperature was maintained at 37ºC. The sequence parameters were: slice thickness=10 mm, GRE flip angle=5, GRE readout TR=5.6 ms, TE=2.7 ms, FOV=20×20 mm², matrix size=128×128. CEST images from 0 to 5 ppm were collected in step size of 0.2 ppm at different saturation pulse power (B₁) (4.7, 7.05 and 9.39 µT) and saturation durations (1, 2, 3, 4 and 5 s). B₀ correction was done by acquiring WASSR images at 0.24 μT from -1 to +1 ppm in steps of 0.1 ppm, using the same parameters as CEST. Z-spectra were plotted using the normalized image intensity as a function of the resonance offset of the saturation pulse. CEST maps were computed using the equation CEST=100×[(S_-ve – S_+ve)/S₀] where S_-ve and S_+ve are the B₀ corrected MR signals acquired while saturating at -3.9 ppm and+3.9 ppm from water resonance, while S₀ is the image obtained without application of any saturation pulse. The CEST contrast map was further corrected for any B₁ inhomogeneity.The high resolution ₁H NMR spectra of NAD_⁺ showed the clear resonance from amine group (–NH₂) at 3 ppm and amide group (–CONH₂) at 3.9 ppm from water at low temperature (Fig. 1A) which was found to be broadened at 37 ºC due to fast exchange related peak broadening (Fig. 1B). The spectrum from NADH at low temperature shows that there is no exchangeable peak in the compound at 3.9 ppm (Fig 1C). The z-spectrum and asymmetry plot from 10 mM NAD₊ solution at 37 °C with B₁ parameters 2.35 µT and 3 seconds shows CEST effect from –OH (~0.5-1.5 ppm), –NH₂ (3 ppm) and amide (3.9 ppm) groups (Fig 2A,B), while the ¹H MR spectrum at the same temperature was unable to observe the amide peak due to exchange related broadening (Fig 1A, inset). Figure 3A depicts the CEST contrast from amide group of NAD⁺ at different B₁ (4.7, 7.05 and 9.39 µT) at 1 sec saturation duration. Increase in both B₁ and saturation duration resulted in increased CEST contrast (Fig. 3B). The next step is to monitor the change in the brain and muscular NAD⁺ concentration in normal and pathological conditions using the optimized saturation parameters.