Role of the cholesterol hydroxyl group in the chemical exchange saturation transfer signal at −1.6 ppm

Chemical exchange saturation transfer (CEST) can provide metabolite‐weighted images in the clinical setting; therefore, understanding the origin of each CEST signal is essential to revealing the changes in diseases at the molecular level, which would provide further insight for diagnoses and treatments. The CEST signal at −1.6 ppm is attributed to the choline methyl group of phosphatidylcholines. The methyl groups have no exchangeable protons, so the corresponding CEST signals must result from the relayed nuclear Overhauser effect (rNOE); however, the detailed mechanism remains unclear. Cholesterol is a major component of biological membranes, and its content is closely related to the dynamics and phases of these lipids. However, cholesterol has a hydroxyl group, which could participate in proton exchange to complete the rNOE process. In this study, we used liposomes containing cholesterol and its analogs (5α‐cholestane and progesterone), which presumably have similar capabilities of influencing lipid bilayers, and found that the steroid hydroxyl group is the key to inducing the rNOE at −1.6 ppm. Our results suggest that the origin of the rNOE at −1.6 ppm likely requires an intermolecular NOE between the proton of the choline methyl group and that of the cholesterol hydroxyl group, and a chemical exchange between the cholesterol hydroxyl group and bulk water. However, the phenomenon in which the rNOE at −1.6 ppm appears when the cholesterol concentration is high seems to contradict the in vivo results, suggesting a more complicated mechanism associated with the rNOE at −1.6 ppm in biological membranes. The origin of the relayed nuclear Overhauser effect (rNOE) at −1.6 ppm in the z‐spectra of chemical exchange saturation transfer (CEST) experiments is still unclear. The z‐spectra of DPPC liposomes containing cholesterol and its analog 5α‐cholestane show that the hydroxyl group of the cholesterol is the key to inducing this rNOE. We hypothesize that observing this signal requires the NOE between choline methyl on the phosphatidylcholines and cholesterol hydroxyl and the chemical exchange between cholesterol hydroxyl and water.