Steffen Goerke

Inverse Z-spectrum analysis for spillover-, MT-, and T1-corrected steady-state pulsed CEST-MRI – application to pH-weighted MRI of acute stroke

Endogenous chemical exchange saturation transfer (CEST) effects are always diluted by competing effects, such as direct water proton saturation (spillover) and semi‐solid macromolecular magnetization transfer (MT). This leads to unwanted T2 and MT signal contributions that lessen the CEST signal specificity to the underlying biochemical exchange processes. A spillover correction is of special interest for clinical static field strengths and protons resonating near the water peak. This is the case for all endogenous CEST agents, such as amide proton transfer, –OH‐CEST of glycosaminoglycans, glucose or myo‐inositol, and amine exchange of creatine or glutamate. All CEST effects also appear to be scaled by the T1 relaxation time of water, as they are mediated by the water pool. This forms the motivation for simple metrics that correct the CEST signal.

Characterization of creatine guanidinium proton exchange by water-exchange (WEX) spectroscopy for absolute-pH CEST imaging in vitro

Chemical exchange saturation transfer (CEST) enables indirect detection of small metabolites in tissue by MR imaging. To optimize and interpret creatine‐CEST imaging we characterized the dependence of the exchange‐rate constant ksw of creatine guanidinium protons in aqueous creatine solutions as a function of pH and temperature T in vitro. Model solutions in the low pH range (pH = 5–6.4) were measured by means of water‐exchange (WEX)‐filtered 1H NMR spectroscopy on a 3 T whole‐body MR tomograph. An extension of the Arrhenius equation with effective base‐catalyzed Arrhenius parameters yielded a general expression for ksw(pH, T). The defining parameters were identified as the effective base‐catalyzed rate constant kb,eff(298.15 K) = (3.009 ± 0.16) × 109 Hz l/mol and the effective activation energy EA,b,eff = (32.27 ± 7.43) kJ/mol at a buffer concentration of cbuffer = (1/15) M. As expected, a strong dependence of ksw on temperature was observed. The extrapolation of the exchange‐rate constant to in vivo conditions (pH = 7.1, T = 37 °C) led to the value of the exchange‐rate constant ksw = 1499 Hz. With the explicit function ksw(pH, T) available, absolute‐pH CEST imaging could be realized and experimentally verified in vitro. By means of our calibration method it is possible to adjust the guanidinium proton exchange‐rate constant ksw to any desired value by preparing creatine model solutions with a specific pH and temperature. Copyright

MR imaging of protein folding in vitro employing Nuclear-Overhauser-mediated saturation transfer

MR Z‐spectroscopy allows enhanced imaging contrast on the basis of saturation transfer between the proton pools of cellular compounds and water, occurring via chemical exchange (chemical exchange saturation transfer, CEST) or dipole–dipole coupling (nuclear Overhauser effect, NOE). In previous studies, signals observed in the aliphatic proton region of Z‐spectra have been assigned to NOEs between protons in water molecules and protons at the surface of proteins. We investigated a possible relationship between the signal strength of NOE peaks in Z‐spectra obtained at B0 = 7 T and protein structure. Here, we report a correlation of NOE‐mediated saturation transfer with the structural state of bovine serum albumin (BSA), which was monitored by fluorescence spectroscopy. Encouraged by CEST signal changes observed in tumor tissue, our observation also points to a possible contrast mechanism for MRI sensitive to the structural integrity of proteins in cells. Therefore, protein folding should be considered as an additional property affecting saturation transfer between water and proteins, in combination with the microenvironment and physiological quantities, such as metabolite concentration, temperature and pH. Copyright

Downfield-NOE-suppressed amide-CEST-MRI at 7 Tesla provides a unique contrast in human glioblastoma

The chemical exchange saturation transfer (CEST) effect observed in brain tissue in vivo at the frequency offset 3.5 ppm downfield of water was assigned to amide protons of the protein backbone. Obeying a base‐catalyzed exchange process such an amide‐CEST effect would correlate with intracellular pH and protein concentration, correlations that are highly interesting for cancer diagnosis. However, recent experiments suggested that, besides the known aliphatic relayed‐nuclear Overhauser effect (rNOE) upfield of water, an additional downfield rNOE is apparent in vivo resonating as well around +3.5 ppm. In this study, we present further evidence for the underlying downfield‐rNOE signal, and we propose a first method that suppresses the downfield‐rNOE contribution to the amide‐CEST contrast. Thus, an isolated amide‐CEST effect depending mainly on amide proton concentration and pH is generated.

Signature of protein unfolding in chemical exchange saturation transfer imaging.

Chemical exchange saturation transfer (CEST) allows the detection of metabolites of low concentration in tissue with nearly the sensitivity of MRI with water protons. With this spectroscopic imaging approach, several tissue‐specific CEST effects have been observed in vivo. Some of these originate from exchanging sites of proteins, such as backbone amide protons, or from aliphatic protons within the hydrophobic protein core. In this work, we employed CEST experiments to detect global protein unfolding. Spectral evaluation revealed exchange‐ and NOE‐mediated CEST effects that varied in a highly characteristic manner with protein unfolding tracked by fluorescence spectroscopy. We suggest the use of this comprehensive spectral signature for the detection of protein unfolding by CEST, as it relies on several spectral hallmarks. As proof of principle, we demonstrate that the presented signature is readily detectable using a whole‐body MR tomograph (B0 = 7 T), not only in denatured aqueous protein solutions, but also in heat‐shocked yeast cells. A CEST imaging contrast with the potential to detect global protein unfolding would be of particular interest regarding protein unfolding as a marker for stress, ageing, and disease. Copyright

Quantitative pulsed CEST-MRI using Ω-plots.

Chemical exchange saturation transfer (CEST) allows the indirect detection of dilute metabolites in living tissue via MRI of the tissue water signal. Selective radio frequency (RF) with amplitude B1 is used to saturate the magnetization of protons of exchanging groups, which transfer the saturation to the abundant water pool. In a clinical setup, the saturation scheme is limited to a series of short pulses to follow regulation of the specific absorption rate (SAR). Pulsed saturation is difficult to describe theoretically, thus rendering quantitative CEST a challenging task. In this study, we propose a new analytical treatment of pulsed CEST by extending a former interleaved saturation–relaxation approach. Analytical integration of the continuous wave (cw) eigenvalue as a function of the RF pulse shape leads to a formula for pulsed CEST that has the same structure as that for cw CEST, but incorporates two form factors that are determined by the pulse shape. This enables analytical Z‐spectrum calculations and permits deeper insight into pulsed CEST. Furthermore, it extends Dixons Ω‐plot method to the case of pulsed saturation, yielding separately, and independently, the exchange rate and the relative proton concentration. Consequently, knowledge of the form factors allows a direct comparison of the effect of the strength and B1 dispersion of pulsed CEST experiments with the ideal case of cw saturation. The extended pulsed CEST quantification approach was verified using creatine phantoms measured on a 7 T whole‐body MR tomograph, and its range of validity was assessed by simulations. Copyright

Aggregation-induced changes in the chemical exchange saturation transfer (CEST) signals of proteins

Chemical exchange saturation transfer (CEST) is an MRI technique that allows mapping of biomolecules (small metabolites, proteins) with nearly the sensitivity of conventional water proton MRI. In living organisms, several tissue‐specific CEST effects have been observed and successfully applied to diagnostic imaging. In these studies, particularly the signals of proteins showed a distinct correlation with pathological changes. However, as CEST effects depend on various properties that determine and affect the chemical exchange processes, the origins of the observed signal changes remain to be understood. In this study, protein aggregation was identified as an additional process that is encoded in the CEST signals of proteins. Investigation of distinct proteins that are involved in pathological disorders, namely amyloid beta and huntingtin, revealed a significant decrease of all protein CEST signals upon controlled aggregation. This finding is of particular interest with regard to diagnostic imaging of patients with neurodegenerative diseases that involve amyloidogenesis, such as Alzheimers or Huntingtons disease. To investigate whether the observed CEST signal decrease also occurs in heterogeneous mixtures of aggregated cellular proteins, and thus prospectively in tissue, heat‐shocked yeast cell lysates were employed. Additionally, investigation of different cell compartments verified the assignment of the protein CEST signals to the soluble part of the proteome. The results of in vitro experiments demonstrate that aggregation affects the CEST signals of proteins. This observation can enable hypotheses for CEST imaging as a non‐invasive diagnostic tool for monitoring pathological alterations of the proteome in vivo.

Proton transfer pathways, energy landscape, and kinetics in creatine-water systems.

We study the exchange processes of the metabolite creatine, which is present in both tumorous and normal tissues and has NH2 and NH groups that can transfer protons to water. Creatine produces chemical exchange saturation transfer (CEST) contrast in magnetic resonance imaging (MRI). The proton transfer pathway from zwitterionic creatine to water is examined using a kinetic transition network constructed from the discrete path sampling approach and an approximate quantum-chemical energy function, employing the self-consistent-charge density-functional tight-binding (SCC-DFTB) method. The resulting potential energy surface is visualized by constructing disconnectivity graphs. The energy landscape consists of two distinct regions corresponding to the zwitterionic creatine structures and deprotonated creatine. The activation energy that characterizes the proton transfer from the creatine NH2 group to water was determined from an Arrhenius fit of rate constants as a function of temperature, obtained from harmonic transition state theory. The result is in reasonable agreement with values obtained in water exchange spectroscopy (WEX) experiments.

Assessing the predictability of IDH mutation and MGMT methylation status in glioma patients using relaxation-compensated multi-pool CEST MRI at 7.0 Tesla

Background Early identification of prognostic superior characteristics in glioma patients such as isocitrate dehydrogenase (IDH) mutation and O6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation status is of great clinical importance. The study purpose was to investigate the non-invasive predictability of IDH mutation status, MGMT promoter methylation, and differentiation of low-grade versus high-grade glioma (LGG vs HGG) in newly diagnosed patients employing relaxation-compensated multipool chemical exchange saturation transfer (CEST) MRI at 7.0 Tesla. Methods Thirty-one patients with newly diagnosed glioma were included in this prospective study. CEST MRI was performed at a 7T whole-body scanner. Nuclear Overhauser effect (NOE) and isolated amide proton transfer (APT; downfield NOE-suppressed APT = dns-APT) CEST signals (mean value and 90th signal percentile) were quantitatively investigated in the whole tumor area with regard to predictability of IDH mutation, MGMT promoter methylation status, and differentiation of LGG versus HGG. Statistics were performed using receiver operating characteristic (ROC) and area under the curve (AUC) analysis. Results were compared with advanced MRI methods (apparent diffusion coefficient and relative cerebral blood volume ROC/AUC analysis) obtained at 3T. Results dns-APT CEST yielded highest AUCs in IDH mutation status prediction (dns-APTmean = 91.84%, P < 0.01; dns-APT90 = 97.96%, P < 0.001). Furthermore, dns-APT metrics enabled significant differentiation of LGG versus HGG (AUC: dns-APTmean = 0.78, P < 0.05; dns-APT90 = 0.83, P < 0.05). There was no significant difference regarding MGMT promoter methylation status at any contrast (P > 0.05). Conclusions Relaxation-compensated multipool CEST MRI, particularly dns-APT imaging, enabled prediction of IDH mutation status and differentiation of LGG versus HGG and should therefore be considered as a non-invasive MR biomarker in the diagnostic workup.

Chemical exchange saturation transfer MRI serves as predictor of early progression in glioblastoma patients

Purpose To prospectively investigate chemical exchange saturation transfer (CEST) MRI in glioblastoma patients as predictor of early tumor progression after first-line treatment. Experimental Design Twenty previously untreated glioblastoma patients underwent CEST MRI employing a 7T whole-body scanner. Nuclear Overhauser effect (NOE) as well as amide proton transfer (APT) CEST signals were isolated using Lorentzian difference (LD) analysis and relaxation compensated by the apparent exchange-dependent relaxation rate (AREX) evaluation. Additionally, NOE-weighted asymmetric magnetic transfer ratio (MTRasym) and downfield-NOE-suppressed APT (dns-APT) were calculated. Patient response to consecutive treatment was determined according to the RANO criteria. Mean signal intensities of each contrast in the whole tumor area were compared between early-progressive and stable disease. Results Pre-treatment tumor signal intensity differed significantly regarding responsiveness to first-line therapy in NOE-LD (p = 0.0001), NOE-weighted MTRasym (p = 0.0186) and dns-APT (p = 0.0328) contrasts. Hence, significant prediction of early progression was possible employing NOE-LD (AUC = 0.98, p = 0.0005), NOE-weighted MTRasym (AUC = 0.83, p = 0.0166) and dns-APT (AUC = 0.80, p = 0.0318). The NOE-LD provided the highest sensitivity (91%) and specificity (100%). Conclusions CEST derived contrasts, particularly NOE-weighted imaging and dns-APT, yielded significant predictors of early progression after fist-line therapy in glioblastoma. Therefore, CEST MRI might be considered as non-invasive tool for customization of treatment in the future.