Iopromide was diluted with PBS to concentrations of 270, 178, 135, 90, 45, and 22.5 mM, and were adjusted to 6.47 pH units with concentrated sodium hydroxide. All CEST experiments were performed using a 600 MHz Varian Inova NMR spectrometer with an inverse cryoprobe. Samples were analyzed at 37.3°C. The temperature was calibrated by measuring the separation of resonances of neat methanol and ethylene glycol samples between 25°C and 40°C (22 (link)). The probe was manually tuned to each sample, and the 90° pulse time was measured to initially estimate the saturation power. The exact saturation power was obtained from the Bloch fitting. CEST spectra were acquired with a continuous-wave saturation pulse and saturation frequencies set at 10 to −10 ppm in 0.2 ppm increments, and each scan was averaged four times. For the first set of studies, saturation times ranging between 0.5 and 6 sec were tested with a relaxation delay from 5.5 to 0 seconds to maintain a total time of 6 seconds for the pre-acquisition period. These studies were conducted with a saturation power of 5.2 and 13.14 μT and a sample with a concentration of 178 mM. For the second set of studies, saturation powers ranging between 0.325 and 7.618 μT were tested with a saturation time of 6 sec and with no additional relaxation delay, using a sample with a concentration of 178 mM. For the third set of studies, samples with a series of concentrations were tested with saturation time of 6 seconds and saturation power of approximately 3.2 μT.
The Bloch-McConnell equations modified for chemical exchange were fit to experimental CEST spectrum (Eq. [12] ) (8 (link),23 (link)). The integrating factor method was used to solve the Bloch equations. The lsqcurvefit routine with the trust-region-reflective algorithm was used to initially fit the CEST spectra, which resulted in rough parameter estimates. The nlinfit routine with the Levenburg-Marquardt algorithm was then used to fit the CEST spectra to obtain the final parameter estimates. The Jacobian from the nlinfit routine was used to determine confidence intervals for fitted parameters.
Other CEST studies with MRI scanners have accounted for B0 and B1 inhomogeneities (23 (link),24 (link)). To account for B0 and B1 inhomogeneities in our studies, each point of an experimental CEST spectrum was assumed to represent a Gaussian-shaped point spread function, which was modeled during the fitting process. However, the fitting results with and without the point spread function were identical, indicating that accounting for B0 and B1 inhomogeneities was unnecessary with data generated from our NMR spectrometer. Subsequent analyses were conducted without incorporating the point spread function. The fitting process estimated values for the T1w, T2w, T1A, and T2A relaxation times of the amide protons of the agent. As previously reported, these fitted estimates of the CEST agent’s relaxation times are inconsequential to the fitting process (8 (link)). Furthermore, to validate the fitting method, fitted parameters were estimated using all but one experimental CEST spectrum, then the remaining experimental spectrum was compared to a simulated spectrum using the fitted parameters. In all cases, the remaining experimental spectrum and simulated spectrum showed an outstanding match.
Lorentzian line shape fitting was used to measure the CEST effect (MS) for each amide from each experimental spectrum as previously described, using spectra normalized from 0% to 100% water signal (which set M0 equal to 100%) (19 (link)). The fittings were performed with Matlab v2011a using the trust-region-reflective algorithm to reach a convergence criterion of 10−16. The QUEST, QUESP, QUESPT, LB-QUESP, EH-QUESP, HW-QUESP, LB-Conc, EH-Conc, and HW-Conc methods were used to estimate kex rates from these CEST measurements determined from the Lorentzian line shape fittings.
The Bloch-McConnell equations modified for chemical exchange were fit to experimental CEST spectrum (
Other CEST studies with MRI scanners have accounted for B0 and B1 inhomogeneities (23 (link),24 (link)). To account for B0 and B1 inhomogeneities in our studies, each point of an experimental CEST spectrum was assumed to represent a Gaussian-shaped point spread function, which was modeled during the fitting process. However, the fitting results with and without the point spread function were identical, indicating that accounting for B0 and B1 inhomogeneities was unnecessary with data generated from our NMR spectrometer. Subsequent analyses were conducted without incorporating the point spread function. The fitting process estimated values for the T1w, T2w, T1A, and T2A relaxation times of the amide protons of the agent. As previously reported, these fitted estimates of the CEST agent’s relaxation times are inconsequential to the fitting process (8 (link)). Furthermore, to validate the fitting method, fitted parameters were estimated using all but one experimental CEST spectrum, then the remaining experimental spectrum was compared to a simulated spectrum using the fitted parameters. In all cases, the remaining experimental spectrum and simulated spectrum showed an outstanding match.
Lorentzian line shape fitting was used to measure the CEST effect (MS) for each amide from each experimental spectrum as previously described, using spectra normalized from 0% to 100% water signal (which set M0 equal to 100%) (19 (link)). The fittings were performed with Matlab v2011a using the trust-region-reflective algorithm to reach a convergence criterion of 10−16. The QUEST, QUESP, QUESPT, LB-QUESP, EH-QUESP, HW-QUESP, LB-Conc, EH-Conc, and HW-Conc methods were used to estimate kex rates from these CEST measurements determined from the Lorentzian line shape fittings.
