pH-sensitive amide proton transfer (APT) MRI provides a surrogate metabolic biomarker that complements the widely-used perfusion and diffusion imaging. However, the endogenous APT MRI is often calculated using the asymmetry analysis (MTRasym), which is susceptible to an inhomogeneous shift due to concomitant semisolid magnetization transfer (MT) and nuclear overhauser (NOE) effects. Although the intact brain tissue has little pH variation, white and gray matter appears distinct in the MTRasym image. Herein we showed that the heterogeneous MTRasym shift not related to pH highly correlates with MT ratio (MTR) and longitudinal relaxation rate (R-1w), which can be reasonably corrected using the multiple regression analysis. Because there are relatively small MT and R-1w changes during acute stroke, we postulate that magnetization transfer and relaxation-normalized APT (MRAPT) analysis increases MRI specificity to acidosis over the routine MTRasym image, hence facilitates ischemic lesion segmentation. We found significant differences in perfusion, pH and diffusion lesion volumes (P < 0.001, ANOVA). Furthermore, MRAPT MRI depicted graded ischemic acidosis, with the most severe acidosis in the diffusion lesion (-1.05 +/- 0.29%/s), moderate acidification within the pH/diffusion mismatch (i.e., metabolic penumbra, -0.67 +/- 0.27%/s) and little pH change in the perfusion/pH mismatch (i.e., benign oligemia, -0.04 +/- 0.14%/s), providing refined stratification of ischemic tissue injury. (C) 2016 Elsevier Inc. All rights reserved.