Synopsis

In this study, we present a method for absolute pH mapping using the guanidyl CEST signal. The method is an extension of a previous approach that allowed compensating for various concomitant effects other than pH, but additionally required measuring the amide signal. By optimizing the pulse shape of the pulsed presaturation the pH sensitivity of the guanidyl signal could now be shifted to the physiologically relevant range around pH 7.1. The shift of sensitivity was verified experimentally in a multi-pH creatine phantom at 9.4T. Thus, CEST-based pH mapping with exceptional specificity is now also possible using only the guanidyl signal.

Introduction

Intracellular pH (pH_i) is a valuable biomarker for assessment of cancer, as pH_i is known to be increased in tumors.¹ Recently we demonstrated, that non-invasive mapping of pH_i in tumors is feasible by a combination of the amide (Δω=3.5ppm) and guanidyl (Δω=2.0ppm) CEST signal at 9.4T.² In this method, concomitant effects, such as concentration changes and spillover dilution, were compensated using a ratiometric approach^3–5 in combination with the relaxation-compensated inverse metric MTR_Rex.^6–8

However, with this approach, stable pH mapping in the physiological range of pH 7 to 7.5 was only feasible using a combination of the amide and guanidyl signal. Therefore, the aim of this study was to further develop the method, so that only the guanidyl signal is needed for reliable pH mapping. This was achieved by using a pulsed presaturation introducing a new parameter – the pulse shape – which enables shifting the pH sensitivity of the guanidyl signal to the physiological pH range. As a model system for the guanidyl signal, creatine model solutions were used. Using only the guanidyl signal makes the entire method more robust and, moreover, the pulsed presaturation enables the translation of the method to whole-body MR scanners and, thus, prospective application in humans.

Methods

The previous method for absolute pH mapping² is based on the assumption of a base-catalyzed exchange rate$~k_{ex}(pH)=k_{c}\cdot{10^{pH}}$, with the exchange rate characterizing constant k_c. Thus, pH mapping is possible via$~pH=log_{10}\big(\frac{k_{ex}}{k_{c}}\big)~$by measuring k_ex and calibration of k_c. For a pulsed presaturation with Gaussian-shaped pulses, MTR_Rex is:⁹
$$MTR_{Rex}^{pulsed}=\frac{1}{Z_{lab}}-\frac{1}{Z_{ref}}=DC\cdot{c_{1}}\cdot{\frac{f_{s}k_{ex}}{R_{1w}}}\cdot{\frac{(\gamma{B_{1}^{2}})}{(\gamma{B_{1}^{2}})+k_{ex}(k_{ex}+R_{2s})\cdot{c_{2}^{2}}}}\quad\quad\quad\quad[Eq~1]$$ with the transversal relaxation rate R_2s of the CEST pool, the duty cycle DC, and the form factors c₁ and c₂ representing the shape of the pulses. For continuous-wave (cw) presaturation, DC=c₁=c₂=1. In this study, c₂ was calculated¹⁰ via$~c_{2}=\frac{(\int_{0}^{t_{p}}B_{1}(t)dt)^{2}}{t_{p}\int_{0}^{t_{p}}B_{1}^{2}(t)dt}~$and MTR_Rex of the creatine guanidyl signal via Z_lab=Z(2ppm) and Z_ref=Z(-2ppm).

Application of a ratiometric approach at two different B₁$~CEST_{ratio}^{pulsed}=\frac{MTR_{Rex}^{pulsed}(B_{1,high})}{MTR_{Rex}^{pulsed}(B_{1,low})}~$and using R_2s=100Hz enables measurement of k_ex.² Importantly, from this theory the parameter c₂ (i.e. the pulse shape) enables shifting the pH sensitivity of the CEST_ratio to higher pH. To translate the measured k_ex to pH, k_c needs to be calibrated. The calibration of k_c is determined experimentally by finding the maximum of MTR_Rex(pH) (Eq1), which is a symmetric function around position$~pH_{max}=log_{10}\big(\frac{\gamma{B_{1}}}{k_{c}c_{2}}\big)$, enabling a stable fitting procedure.¹¹

Creatine model solutions at different pH (6.0 to 8.2) were investigated. All measurements were performed on a 9.4T small-animal MR scanner (Bruker BioSpec) and stabilized at 37.0±0.1°C. For CEST-MRI a custom-built 2D FISP image readout (0.5×0.5x2mm³) with cw or pulsed (c₂ between 0.36 and 0.70) presaturation with mean B₁=0.6,1.0,1.4µT of duration 12s (DC=1) was applied. Data were corrected for B₀ and B₁ inhomogeneities¹² using a WASABI scan¹³.

Results and Discussion

No relevant broadening of the guanidyl peak was observed for Z-spectra as a function of c₂ (Figure 1B). Therefore, c₂ introduces a new possibility of shifting the pH sensitivity to higher pH without interfering peak broadening. In contrast, shifting the pH sensitivity by increasing B_1,high is impracticable for in vivo applications due to excessive peak broadening and the impeded reliable extraction of the guanidyl signal from other superimposing signals in the in vivo Z-spectrum.

The pH dependency of MTR_Rex(pH) showed the expected symmetric shape in coherence with theory (Eq1, Figure 2). For pulsed presaturations, a shift towards higher pH was observed for decreasing c₂ (i.e. sharper pulses, Figure 1C). Experimental determination of pH_max enabled calculating a mean k_c(c₂) of 89±3µHz (Figure 3A) corresponding to k_ex=1120Hz at pH 7.1, in good agreement with literature.^14,15 A slight trend was observed for k_c(c₂), which, though, was smaller than the B₁ dispersion of k_c for each c₂ (errors in Figure 3A). Accordingly, for each c₂ the corresponding k_c was used for calculation of pH. In addition, the CEST_ratio(c₂) showed a shift towards higher pH (Figure 3B), thus verifying the ability to shift the pH sensitivity (i.e. the position of the steepest slope of CEST_ratio(pH)) by tuning the pulse shape. Importantly, the range of highest pH sensitivity could be shifted up to the physiologically relevant range of pH 7 to 7.5 using c₂=0.42.

With the optimal c₂ in hand, pH maps were calculated for cw and c₂=0.42 (Figure 4A,B). An excellent correlation of titrated pH (pH_titrated) and pH measured by CEST-MRI (pH_CEST) was observed for cw and pulsed presaturation in the pH range of 6.5–7.5 (cw: r=0.9922,p<0.001 and c₂=0.42: r=0.9953,p<0.001) (Figure 4C,D). In contrast to cw, the significantly smaller fluctuations in pH values (std in Figure 4C,D) for the optimized pulses demonstrate the improved pH sensitivity above pH 7 (Figure 4E).

Conclusion

In this study, the pulse shape of the presaturation was identified as a new parameter to shift the pH sensitivity of the guanidyl signal to the physiological pH range. Applicability of the presented method was demonstrated in creatine model solutions at 9.4T, showing reliable calculation of guanidyl-based pH maps in the range of pH 6.5 to 7.5. Consequently, by eliminating the need for the amide signal, this makes the previous pH mapping method more robust. Moreover, the pulsed presaturation enables the translation of the pH mapping method to whole-body MR scanners and, thus, prospective application in humans.

Acknowledgements

We gratefully thank the German Research Foundation (DFG; GO 2172/1-1) for financial support.

Conflict of Interest:
The German Cancer Research Center (DKFZ) maintains a research collaboration agreement with Bruker BioSpin MRI GmbH. Bruker did not have any influence on the direction or contents of this manuscript.
The authors declare that they have no competing interests.

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