Alanine is a non-essential amino acid which is manufactured in the body mostly from pyruvate via the reductive amination known to play an important role in the metabolism and alanine cycle. Altered metabolic phenotype is a hallmark of tumor development and glycolysis has been reported to be of high importance¹. However, new data using hyperpolarized ¹³C pyruvate suggests the myc-driven pre-tumor condition in which the production of alanine from pyruvate predominates the glycolysis (Fig1)². This begs for an imaging technique to monitor metabolic changes in the body with a focus on alanine as a biomarker for the detection of myc-driven cancers. Chemical Exchange Saturation Transfer (CEST) technique offers a principle to image metabolites and macromolecules in vivo based on the property of exchangeable protons with water³. CEST phenomenon has been recently exploited to image various molecules in vivo such as creatine⁴, glutamate⁵, glucosaminoglycan⁶ and so on. In the current study, we have characterized the CEST effect from alanine at different saturation pulse power and saturation duration in vitro. In addition, concentration dependence of alanine CEST is studied. The initial results are discussed.For CEST imaging, alanine solution (30 mM) was prepared in phosphate buffer saline (PBS) and adjusted to a pH 7. For the concentration dependence of CEST, alanine solutions of varying concentrations, 2, 4, 6, 8, and 10 mM, were prepared in phosphate buffer saline and adjusted to a pH 7. CEST imaging was performed on 9.4T MRI scanner (Agilent, USA). The CEST parameters were optimized at 9.4T MRI scanner (Agilent, USA). During the course of experiment temperature was maintained at 37 ºC. 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) (4.69, 7.05, 9.38, 11.73 and 14.08 µT) and saturation durations (1, 2, 3 and 4 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 ppm and+3 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 z-spectrum of alanine solution shown in Fig 2A depicts the CEST contrast from alanine at B₁ parameters 7.05 µT and 1 sec saturation duration. CEST assymetry plot from alanine showing the CEST contrast at 3 ppm (Fig 2B). The CEST maps shown in figure 3A show the CEST contrast from 10 mM alanine solution with different B₁ and 1 second saturation duration. Both increase in B₁ and saturation duration resulted in increased CEST contrast from alanine (Fig 3 B). A linear relationship between alanine concentration and CEST contrast was observed at B1 7.05 µT and saturation duration 2 sec. Further standardization for the estimation of contribution of other metabolites to the alanine CEST contrast are underway. The next step is to monitor the change in the alanine concentration after intravenous injection of glucose/pyruvate in a pre-clinical model of myc-driven tumor using the optimized saturation parameters for in vivo CEST MRI.