Assignment of the molecular origins of CEST signals at 2 ppm in rat brain

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Chemical exchange saturation transfer (CEST) has the potential for molecular MR imaging of specific resonances with higher sensitivity than conventional proton magnetic resonance spectroscopy (1H MRS) 1. In CEST imaging, an irradiation radiofrequency (RF) pulse is applied at the frequency offsets of exchangeable protons of solute molecules, and the subsequent chemical exchange between those saturated protons and bulk water reduces the magnetization of the measured water signal, which provides a way to indirectly detect the solute molecules. Because water is significantly more abundant than the solutes, the detection sensitivity to exchanging protons is magnified—especially when long irradiation pulses are used.
Creatine is an essential metabolite for energy production, and a sensitive method for imaging creatine distribution in biological tissues would be of great interest, such as for studying brain or muscle metabolism. Traditionally, creatine has been measured via 1H MRS, although it suffers from several limitations such as low spatial resolution and sensitivity for in vivo studies. Recently, CEST at 2 ppm (CEST@2ppm) from water in brain has been suggested as an indicator of creatine distribution 4. Studies on samples containing the main brain tissue metabolites at their physiological concentrations and pH showed that creatine dominates CEST@2ppm compared with other compounds 6. This suggests that CEST@2ppm could provide a method to image creatine distributions specifically with high spatial‐resolution and high sensitivity. However, biological tissues also contain a large variety of macromolecules that also have exchangeable protons. Some protons, such as those in proteins with arginine side chains, have similar chemical shifts (2 ppm) and chemical exchange rates (500 to 1000 s−17) as those of creatine, and thus may not be easily distinguished from creatine using CEST. Potentially, such proteins may also contribute to CEST@2ppm and thereby decrease the specificity of CEST@2ppm for detecting creatine. Unfortunately, a comprehensive investigation of the relative contributions from creatine and proteins has not been reported. One challenge is that it is difficult to mimic the arginine residues of proteins at physiological concentration using simple model phantoms, as there are many types of proteins that contain different proportions of arginine residues in biological tissues.
Here, we used dialysis to selectively remove creatine and other small molecules from samples of brain homogenates in an attempt to measure the CEST@2ppm of the residual solutes. Dialysis is a classic laboratory technique that relies on selective diffusion of molecules across a semipermeable membrane to separate molecules based on membrane pore size. A sample and a buffer solution are placed on opposite sides of a dialysis membrane, and sample molecules that are smaller than the pores pass through the membrane, whereas large molecules are retained. Because of the large size difference between creatine and most proteins, this enables the simple separation of creatine and other small molecules from brain‐tissue samples, and in turn provides us with an opportunity to quantitatively evaluate the relative contributions of creatine and proteins to CEST@2ppm.

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