  Citation: Albatany, M.; Meakin, S.; Bartha, R. Brain pH Measurement Using AACID CEST MRI Incorporating the 2 ppm Amine Resonance. Tomography 2022, 8, 730–739. https://doi.org/10.3390/ tomography8020060 Academic Editor: Emilio Quaia Received: 19 December 2021 Accepted: 1 March 2022 Published: 9 March 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Article Brain pH Measurement Using AACID CEST MRI Incorporating the 2 ppm Amine Resonance Mohammed Albatany 1 , Susan Meakin 2 and Robert Bartha 1,3, * 1 Centre of Functional and Metabolic Mapping, Robarts Research Institute, The University of Western Ontario, London, ON N6A 3K7, Canada; malbatan@uwo.ca 2 Department of Biochemistry, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, ON N6A 3K7, Canada; smeakin@uwo.ca 3 Department of Medical Biophysics, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, ON N6A 3K7, Canada * Correspondence: rbartha@robarts.ca; Tel.: +1-(519)931-5777 (ext. 24039) Abstract: Many pathological conditions lead to altered intracellular pH (pH i ) disrupting normal cellular functions. The chemical exchange saturation transfer (CEST) method, known as Amine and Amide Concentration Independent Detection (AACID), can produce image contrast that is predominantly dependent on tissue intracellular pH i . The AACID value is linearly related to the ratio of the 3.5 ppm amide CEST effect and the 2.75 ppm amine CEST effect in the physiological range. However, the amine CEST effect at 2 ppm is often more clearly defined in vivo, and may provide greater sensitivity to pH changes. The purpose of the current study was to compare AACID measurement precision utilizing the 2.0 and 2.75 ppm amine CEST effects. We hypothesized that the 2.0 ppm amine CEST resonance would produce measurements with greater sensitivity to pH changes. In the current study, we compare the range of the AACID values obtained in 24 mice with brain tumors and in normal tissue using the 2 ppm and 2.75 ppm amine resonances. All CEST data were acquired on a 9.4T MRI scanner. The AACID measurement range increased by 39% when using the 2 ppm amine resonance compared to the 2.75 ppm resonance, with decreased measurement variability across the brain. These data indicate that in vivo pH measurements made using AACID CEST can be enhanced by incorporating the 2 ppm amine resonance. This approach should be considered for pH measurements made over short intervals when no changes are expected in the concentration of metabolites that contribute to the 2 ppm amine resonance. Keywords: Brain pH; AACID; CEST MRI; 2 ppm amine resonance; cancer; glioblastoma multiforme 1. Introduction Intracellular pH plays an important role in many physiological processes, including apoptosis, cell proliferation, and protein interactions, and it is altered in several disease states. In cancer, altered intracellular and extracellular pH gradients can lead to drug resistance [1,2]. Chemical exchange saturation transfer (CEST) can produce image contrast that is dependent on tissue pH [3–6], and can be used to non-invasively study cellular pH under various conditions. More specifically, amide proton transfer (APT) efficiency varies with pH, providing sensitivity to this physiological parameter. Although the measurement of the APT CEST effect depends on several factors including the amide proton concentra- tion, water concentration, and the relaxivity (R 1 ) of bulk water [6], this contrast has been successfully used to identify ischemic tissue following acute stroke [7–12] and to study cancer [13–17]. In previous work [18] using 9.4T MRI, we have demonstrated that the ratio of the 3.5 ppm amide CEST effect to the 2.75 ppm amine CEST effect varies linearly with pH in the physiological range, and is largely independent of protein concentration and tem- perature [18]. This ratiometric CEST method called Amine and Amide Concentration Tomography 2022, 8, 730–739. https://doi.org/10.3390/tomography8020060 https://www.mdpi.com/journal/tomography