Chemical Exchange Saturation Transfer for Lactate-Weighted Imaging at 3 T MRI: Comprehensive In Silico, In Vitro, In Situ, and In Vivo Evaluations

Round 1

Reviewer 1 Report

In this manuscript, Radke et al. present an elaborate study to optimize lactate-weighted CEST imaging at clinically relevant field strength (3 T) and subsequently apply the optimized protocol to assess lactate-related changes in the CEST signal of the calf muscle after exercise. The optimized protocol could be adapted to investigate changes in the CEST effect of lactate in other diseases with potential application in tumor imaging. The manuscript is well-written and the analysis was performed with great care. However, the authors need to investigate and discuss potential changes in the pH as a possible confounder to observations and interpretations. I recommend major revisions to the manuscript before acceptance (see detailed comments below).

General comment

The manuscript is submitted to the Special Issue: Quantitative Imaging in Oncology and I do understand that this may be beyond the scope of this study and depends on the availability of appropriate patients, but an example case of a tumor patient applying the optimized sequence could emphasize the utility for Oncology. This could additionally validate with MRS scans in the same patient.

Major comments

1. Line 168: How did the authors control for pH changes and was the pH value measured in the phantom?
2. Line 213: Again, how did the authors warrant stable pH values. I would invite the authors to include pH measurements for the different lactate Ampuwa solutions.
3. 292: The authors need to include a phantom scan with a pH variation. This should be done at in vivo realistic concentrations and T = 37 covering realistic pH shifts. This is needed to justify their interpretation of the in vivo data.
4. Line 427.: The authors need to discuss the possible effects of pH changes due to increased concentrations of lactate which introduce an acidity shift and higher observable MTRasym values in the lactate and creatine regions (see also DeBrosse 2016 Figure 2). This discussion needs to be supplemented by appropriate in vitro experiments. As it is quite possible that the overserved changes are a combination of pH shift and increased lactate concentrations.

Minor comments

1. Line 2: I would suggest using the term lactate-weighted imaging, as there are possible confounding factors such pH sensitivity of the CEST signal so referring to the image as just lactate may be overstating the abilities of the method.
2. Line 21: CEST imaging of lactate is optimized not the CEST effect itself.
3. Line 32: The authors state in the abstract that concentration changes were detected in vitro, in situ, and in vivo. This conclusion may be warranted for the in vitro and in situ case where the environment is fully pH controlled. For in vivo, the changes could also be related to pH alteration due to high acidity with increasing lactate concentrations which should be clarified in the abstract.
4. Line 129: Replace sequence with acquisition or protocol as WASSR is just a variant of a CEST experiment. Also, WASSR is measuring the absolute water frequency and its shift due to B0 inhomogeneities and not B0 itself. Please rephrase throughout the manuscript.
5. Line 271.: Please include z-spectra and MTRasym curves for all three scenarios (in vitro, in situ, and in vivo) to give insight into the expected data quality/appearance.
6. Line 469 Changes were observed in the lactate-weighted signal not lactate itself. Please clarify.

Author Response

Responses to the Reviewers‘ Comments

The authors thank the reviewers for their careful revision of the manuscript and valuable comments, which are addressed and considered in the revised version of the manuscript. Please note that all changes made to the manuscript have been highlighted using the “Track changes”-mode in Microsoft Word and are detailed in this document, too. Please note that line numbers refer to the main text body of the revised document.

Reply to Reviewer 1

R1 General Comment: “The manuscript is submitted to the Special Issue: Quantitative Imaging in Oncology and I do understand that this may be beyond the scope of this study and depends on the availability of appropriate patients, but an example case of a tumor patient applying the optimized sequence could emphasize the utility for Oncology. This could additionally validate with MRS scans in the same patient.“

Authors’ Response: We agree with the reviewer that applying our optimized sequence to an example patient would enhance the manuscript. Due to coronavirus determinations at our institution, patients such as cancer patients who belong to risk groups for severe disease are excluded from research measurements. Therefore, the ethical principles we use exclude the survey of patients. Thus, we have limited the manuscript to the applications of in vitro, in situ, and in vivo models.

R1 Major Comment #1: “Line 168: How did the authors control for pH changes and was the pH value measured in the phantom?”

Authors’ Response: We agree with the reviewer that, as explained in the introduction, the pH also influences the measured CEST effect in addition to the fractional concentration. In the previous in vitro experiments, pH was not monitored.

Authors’ Action: Taking into account this valuable comment, new in vitro experiments were performed and reported in the manuscript. A pH of 7.3 was adjusted with PBS and the lactate concentration was systematically varied. The passage in the manuscript reads as follows:

“Preparation of phantoms: For the concentration-dependent phantom study, calcium L-(+)-lactate hydrate (Carl ROTH, Karlsruhe, Germany) was dissolved in phosphate-buffered saline (ROTI®Cell PBS, Carl ROTH) at pH 7.3 (pH value of a muscle at rest [41]), and phantoms were prepared with different concentrations of lactate, i.e., 0, 5, 10, 20, and 40 mM lactate. (lines 169 ff)

R1 Major Comment #2: Line 213: Again, how did the authors warrant stable pH values. I would invite the authors to include pH measurements for the different lactate Ampuwa solutions.

Authors’ Response and Action: Another reasonable concern raised by the Reviewer. As explained in the previous comment, the phantom measurements for the different lactate concentrations were again performed under a PBS buffered pH of 7.3.

R1 Major Comment #3: Line 292: The authors need to include a phantom scan with a pH variation. This should be done at in vivo realistic concentrations and T = 37 covering realistic pH shifts. This is needed to justify their interpretation of the in vivo data.

Authors’ Response: We agree with the reviewer that an investigation of the pH value by means of in vitro experiments is essential for more accurate classification of the in vivo experiments.

Authors’ Action:  In accordance with the reviewer's valuable suggestion to investigate the influence of altered pH on the CEST effect in vitro, we systematically varied pH using PBS. The sections in the manuscript read as follows:

1. Materials and Methods

“For the pH-dependent phantom study, 40 mM lactate samples were prepared in PBS solutions with pH-values of 7.6 7.3, 7.0, and 6.7.” (lines 169 ff)

2. Results

“We observed an increase in MTRasym value from 0.56% to 0.61% at a temperature of 37°C and a decrease in pH from 7.3 to 7.0. However, when the pH was lowered further (pH = 6.7), the CEST effect decreased substantial, as it did when the pH was increased to 7.6. We monitored the temperature before (T = 37.1 °C) and after the measurement (T = 36.7°C).“ (lines 307 ff)

3. Discussion

“Furthermore, the in-vitro experiments showed that pH changes in a physiological range of 7.0 - 7.3 can be neglected in contrast to the change in lactate value. The results we observed are consistent with those of DeBrosse et al. In both studies, the LATEST effect is maximized at a pH value of 7.0 [16].“ (lines 424ff)

“However, it should be kept in mind that in addition to changes in fractional lactate concentration, changes in pH also influence the observed LATEST effect. Street et al. demonstrated a change in pH from 7.38 (resting) to 7.04 (post-exercise) [41]. In the in-vitro experiments, we observed a slight increase in the LATEST effect from 0.56% to 0.61% between pH values of 7.3 and 7.0 at a temperature of 37 °C. In comparison, the LATEST effect increased from 0.03% (0 mM lactate) to 0.56% (40 mM lactate). Thus, MTRasym levels measured immediately after exercise could be slightly elevated due to the pH change, but this does not explain the substantial changes. The change in lactate concentration should therefore be considered a substantial factor.” (lines 447ff)

R1 Major Comment #4: Line 427: The authors need to discuss the possible effects of pH changes due to increased concentrations of lactate which introduce an acidity shift and higher observable MTRasym values in the lactate and creatine regions (see also DeBrosse 2016 Figure 2). This discussion needs to be supplemented by appropriate in vitro experiments. As it is quite possible that the overserved changes are a combination of pH shift and increased lactate concentrations.

Authors’ Response: We thank the reviewer for this critical comment, which allows us to examine the MTRasym values measured in vivo with respect to the influence of pH and lactate changes.

Authors‘ Action: As elaborated in our previous comments, the in vitro experiments were extended to measurements of the lactate effect under systematic variation of pH concentrations. We amended the discussion section based on the acquired results. The paragraph in the Discussion now reads:

“Furthermore, the in-vitro experiments showed that pH changes in a physiological range of 7.0 - 7.3 can be neglected compared to the change of lactate levels. Our results are consistent with those of DeBrosse et al.. In both studies, the LATEST effect is maximized at a pH value of 7.0 [16].“ (lines 424ff)

“However, it should be kept in mind that in addition to changes in fractional lactate concentration, changes in pH also influence the observed LATEST effect. Street et al. demonstrated a change in pH from 7.38 (resting) to 7.04 (post-exercise) [41]. In the in-vitro experiments, we observed a slight increase in the LATEST effect from 0.56% to 0.61% between pH values of 7.3 and 7.0 at a temperature of 37 °C. In comparison, the LATEST effect increased from 0.03% (0 mM lactate) to 0.56% (40 mM lactate). Thus, MTRasym levels measured immediately after exercise could be slightly elevated due to the pH change, but this does not explain the substantial changes. The change in lactate concentration should therefore be considered a substantial factor.” (lines 447ff)

R1 Minor comment #1: Line 2: I would suggest using the term lactate-weighted imaging, as there are possible confounding factors such pH sensitivity of the CEST signal so referring to the image as just lactate may be overstating the abilities of the method.

Authors’ Response and Action: Again, the reviewer has a good point, and the title has been revised to read:

“Chemical exchange saturation transfer for lactate-weighted imaging at 3 T MRI: Comprehensive in-silico, in-vitro, in-situ, and in-vivo evaluations“ (line 2)

R1 Minor comment #2: Line 21: CEST imaging of lactate is optimized not the CEST effect itself.

Authors’ Response and Action: Again, we agree with the reviewer that the wording we used is incorrect. We thank the reviewer and have revised the relevant sentence. The sentence now reads as follows:

“Based on in-silico, in-vitro, in-situ, and in-vivo evaluations, this study aims to establish and optimize the chemical exchange saturation transfer (CEST) imaging of lactate (Lactate-CEST - LATEST).“ (line 20ff)

R1 Minor comment #3: Line 32: The authors state in the abstract that concentration changes were detected in vitro, in situ, and in vivo. This conclusion may be warranted for the in vitro and in situ case where the environment is fully pH controlled. For in vivo, the changes could also be related to pH alteration due to high acidity with increasing lactate concentrations which should be clarified in the abstract.

Authors’ Response: We agree with the reviewer that the in-vivo experiments do not allow only the lactate concentration to change without changing other confounding effects such as pH concentration.

Authors‘ Action: We, therefore, revised the passage in the abstract and explicitly referred to the fact that the variation of the lactate concentration was achieved by muscular strain. The fact that changes in temperature and pH always accompany athletic exertion was then explicitly taken up again in the discussion. The two amended passages read as follows:

Abstract: “The optimized sequences were used to image variable lactate concentrations in vitro (using phan-tom measurements), in situ (using nine human cadaveric lower leg specimens), and in vivo (using four healthy volunteers after exertional exercise) that were then statistically analyzed using the non-parametric Friedman test and Kendall Tau-b rank correlation.“ (lines 23ff)

Disucssion: “However, it should be kept in mind that in addition to changes in fractional lactate concentration, changes in pH also influence the observed LATEST effect. Street et al. demonstrated a change in pH from 7.38 (resting) to 7.04 (post-exercise) [41]. In the in-vitro experiments, we observed a slight increase in the LATEST effect from 0.56% to 0.61% between pH values of 7.3 and 7.0 at a temperature of 37 °C. In comparison, the LATEST effect increased from 0.03% (0 mM lactate) to 0.56% (40 mM lactate). Thus, MTRasym levels measured immediately after exercise could be slightly elevated due to the pH change, but this does not explain the substantial changes. The change in lactate concentration should therefore be considered a substantial factor.“ (lines 447 ff)

R1 Minor comment #4: Line 129: Replace sequence with acquisition or protocol as WASSR is just a variant of a CEST experiment. Also, WASSR is measuring the absolute water frequency and its shift due to B0 inhomogeneities and not B0 itself. Please rephrase throughout the manuscript.

Authors’ Response and Action: Indeed, the two formulations are irritating. Following the reviewer's suggestion, we have replaced "sequence" with "acquisition" in the appropriate places. In addition, we have clarified in the manuscript that WASSR is not used to determine B0 itself, but only B0 inhomogeneities. The sentences now read as follows:

“The MRI protocol included standard morphologic sequences, i.e., coronal T2-weighted (T2w) turbo spin-echo (TSE) and sagittal T1-weighted (T1w) TSE sequences. Further MR mapping acquisitions were acquired to map relaxation times:“ (lines 117ff)

“For B₀ inhomogeneity correction, a Water Saturation Shift Referencing (WASSR) acquisition was performed with t_p = 25 ms, t_d = 25 ms, n_p = 1, B₁ = 0.2 µT, 62 dynamics, and Δω=1 ppm; otherwise, the same sequence parameters as for the CEST sequence were used. Further image parameters are indicated in Table 1.” (lines 129ff)

R1 Minor comment #5: Line 271.: Please include z-spectra and MTRasym curves for all three scenarios (in vitro, in situ, and in vivo) to give insight into the expected data quality/appearance.

Authors‘ Respons: We thank the reviewer for the helpful comment and fully agree that the presentation of Z-spectra and MTRasym curves supports the assessment of data quality.

Authors‘ Action: In accordance with the reviewer's suggestion, we have added a figure that additionally shows the changes Z-spectra and MTRasym curves for all 3 scenarios. The figure attached in the supplementary material is as follows:

Supplementary Figure 2: Visualization of representative Z-spectra and the resulting MTRasym curves under systematic variation of lactate concentration in vitro (A, B), in situ (C, D), and in vivo (E, F).

R1 Minor comment #6: Line 469 Changes were observed in the lactate-weighted signal not lactate itself. Please clarify.

Authors‘ Respons and Action: We thank the reviewer for his critical assessment of the wording and agree that it can be misunderstood. We have revised the wording in the relevant sentence and it now reads as follows:

”Finally, we were

Responses to the Reviewers‘ Comments

The authors thank the reviewers for their careful revision of the manuscript and valuable comments, which are addressed and considered in the revised version of the manuscript. Please note that all changes made to the manuscript have been highlighted using the “Track changes”-mode in Microsoft Word and are detailed in this document, too. Please note that line numbers refer to the main text body of the revised document.

Reply to Reviewer 1

R1 General Comment: “The manuscript is submitted to the Special Issue: Quantitative Imaging in Oncology and I do understand that this may be beyond the scope of this study and depends on the availability of appropriate patients, but an example case of a tumor patient applying the optimized sequence could emphasize the utility for Oncology. This could additionally validate with MRS scans in the same patient.“

Authors’ Response: We agree with the reviewer that applying our optimized sequence to an example patient would enhance the manuscript. Due to coronavirus determinations at our institution, patients such as cancer patients who belong to risk groups for severe disease are excluded from research measurements. Therefore, the ethical principles we use exclude the survey of patients. Thus, we have limited the manuscript to the applications of in vitro, in situ, and in vivo models.

R1 Major Comment #1: “Line 168: How did the authors control for pH changes and was the pH value measured in the phantom?”

Authors’ Response: We agree with the reviewer that, as explained in the introduction, the pH also influences the measured CEST effect in addition to the fractional concentration. In the previous in vitro experiments, pH was not monitored.

Authors’ Action: Taking into account this valuable comment, new in vitro experiments were performed and reported in the manuscript. A pH of 7.3 was adjusted with PBS and the lactate concentration was systematically varied. The passage in the manuscript reads as follows:

“Preparation of phantoms: For the concentration-dependent phantom study, calcium L-(+)-lactate hydrate (Carl ROTH, Karlsruhe, Germany) was dissolved in phosphate-buffered saline (ROTI®Cell PBS, Carl ROTH) at pH 7.3 (pH value of a muscle at rest [41]), and phantoms were prepared with different concentrations of lactate, i.e., 0, 5, 10, 20, and 40 mM lactate. (lines 169 ff)

R1 Major Comment #2: Line 213: Again, how did the authors warrant stable pH values. I would invite the authors to include pH measurements for the different lactate Ampuwa solutions.

Authors’ Response and Action: Another reasonable concern raised by the Reviewer. As explained in the previous comment, the phantom measurements for the different lactate concentrations were again performed under a PBS buffered pH of 7.3.

R1 Major Comment #3: Line 292: The authors need to include a phantom scan with a pH variation. This should be done at in vivo realistic concentrations and T = 37 covering realistic pH shifts. This is needed to justify their interpretation of the in vivo data.

Authors’ Response: We agree with the reviewer that an investigation of the pH value by means of in vitro experiments is essential for more accurate classification of the in vivo experiments.

Authors’ Action:  In accordance with the reviewer's valuable suggestion to investigate the influence of altered pH on the CEST effect in vitro, we systematically varied pH using PBS. The sections in the manuscript read as follows:

1. Materials and Methods

“For the pH-dependent phantom study, 40 mM lactate samples were prepared in PBS solutions with pH-values of 7.6 7.3, 7.0, and 6.7.” (lines 169 ff)

2. Results

“We observed an increase in MTRasym value from 0.56% to 0.61% at a temperature of 37°C and a decrease in pH from 7.3 to 7.0. However, when the pH was lowered further (pH = 6.7), the CEST effect decreased substantial, as it did when the pH was increased to 7.6. We monitored the temperature before (T = 37.1 °C) and after the measurement (T = 36.7°C).“ (lines 307 ff)

3. Discussion

“Furthermore, the in-vitro experiments showed that pH changes in a physiological range of 7.0 - 7.3 can be neglected in contrast to the change in lactate value. The results we observed are consistent with those of DeBrosse et al. In both studies, the LATEST effect is maximized at a pH value of 7.0 [16].“ (lines 424ff)

“However, it should be kept in mind that in addition to changes in fractional lactate concentration, changes in pH also influence the observed LATEST effect. Street et al. demonstrated a change in pH from 7.38 (resting) to 7.04 (post-exercise) [41]. In the in-vitro experiments, we observed a slight increase in the LATEST effect from 0.56% to 0.61% between pH values of 7.3 and 7.0 at a temperature of 37 °C. In comparison, the LATEST effect increased from 0.03% (0 mM lactate) to 0.56% (40 mM lactate). Thus, MTRasym levels measured immediately after exercise could be slightly elevated due to the pH change, but this does not explain the substantial changes. The change in lactate concentration should therefore be considered a substantial factor.” (lines 447ff)

R1 Major Comment #4: Line 427: The authors need to discuss the possible effects of pH changes due to increased concentrations of lactate which introduce an acidity shift and higher observable MTRasym values in the lactate and creatine regions (see also DeBrosse 2016 Figure 2). This discussion needs to be supplemented by appropriate in vitro experiments. As it is quite possible that the overserved changes are a combination of pH shift and increased lactate concentrations.

Authors’ Response: We thank the reviewer for this critical comment, which allows us to examine the MTRasym values measured in vivo with respect to the influence of pH and lactate changes.

Authors‘ Action: As elaborated in our previous comments, the in vitro experiments were extended to measurements of the lactate effect under systematic variation of pH concentrations. We amended the discussion section based on the acquired results. The paragraph in the Discussion now reads:

“Furthermore, the in-vitro experiments showed that pH changes in a physiological range of 7.0 - 7.3 can be neglected compared to the change of lactate levels. Our results are consistent with those of DeBrosse et al.. In both studies, the LATEST effect is maximized at a pH value of 7.0 [16].“ (lines 424ff)

“However, it should be kept in mind that in addition to changes in fractional lactate concentration, changes in pH also influence the observed LATEST effect. Street et al. demonstrated a change in pH from 7.38 (resting) to 7.04 (post-exercise) [41]. In the in-vitro experiments, we observed a slight increase in the LATEST effect from 0.56% to 0.61% between pH values of 7.3 and 7.0 at a temperature of 37 °C. In comparison, the LATEST effect increased from 0.03% (0 mM lactate) to 0.56% (40 mM lactate). Thus, MTRasym levels measured immediately after exercise could be slightly elevated due to the pH change, but this does not explain the substantial changes. The change in lactate concentration should therefore be considered a substantial factor.” (lines 447ff)

R1 Minor comment #1: Line 2: I would suggest using the term lactate-weighted imaging, as there are possible confounding factors such pH sensitivity of the CEST signal so referring to the image as just lactate may be overstating the abilities of the method.

Authors’ Response and Action: Again, the reviewer has a good point, and the title has been revised to read:

“Chemical exchange saturation transfer for lactate-weighted imaging at 3 T MRI: Comprehensive in-silico, in-vitro, in-situ, and in-vivo evaluations“ (line 2)

R1 Minor comment #2: Line 21: CEST imaging of lactate is optimized not the CEST effect itself.

Authors’ Response and Action: Again, we agree with the reviewer that the wording we used is incorrect. We thank the reviewer and have revised the relevant sentence. The sentence now reads as follows:

“Based on in-silico, in-vitro, in-situ, and in-vivo evaluations, this study aims to establish and optimize the chemical exchange saturation transfer (CEST) imaging of lactate (Lactate-CEST - LATEST).“ (line 20ff)

R1 Minor comment #3: Line 32: The authors state in the abstract that concentration changes were detected in vitro, in situ, and in vivo. This conclusion may be warranted for the in vitro and in situ case where the environment is fully pH controlled. For in vivo, the changes could also be related to pH alteration due to high acidity with increasing lactate concentrations which should be clarified in the abstract.

Authors’ Response: We agree with the reviewer that the in-vivo experiments do not allow only the lactate concentration to change without changing other confounding effects such as pH concentration.

Authors‘ Action: We, therefore, revised the passage in the abstract and explicitly referred to the fact that the variation of the lactate concentration was achieved by muscular strain. The fact that changes in temperature and pH always accompany athletic exertion was then explicitly taken up again in the discussion. The two amended passages read as follows:

Abstract: “The optimized sequences were used to image variable lactate concentrations in vitro (using phan-tom measurements), in situ (using nine human cadaveric lower leg specimens), and in vivo (using four healthy volunteers after exertional exercise) that were then statistically analyzed using the non-parametric Friedman test and Kendall Tau-b rank correlation.“ (lines 23ff)

Disucssion: “However, it should be kept in mind that in addition to changes in fractional lactate concentration, changes in pH also influence the observed LATEST effect. Street et al. demonstrated a change in pH from 7.38 (resting) to 7.04 (post-exercise) [41]. In the in-vitro experiments, we observed a slight increase in the LATEST effect from 0.56% to 0.61% between pH values of 7.3 and 7.0 at a temperature of 37 °C. In comparison, the LATEST effect increased from 0.03% (0 mM lactate) to 0.56% (40 mM lactate). Thus, MTRasym levels measured immediately after exercise could be slightly elevated due to the pH change, but this does not explain the substantial changes. The change in lactate concentration should therefore be considered a substantial factor.“ (lines 447 ff)

R1 Minor comment #4: Line 129: Replace sequence with acquisition or protocol as WASSR is just a variant of a CEST experiment. Also, WASSR is measuring the absolute water frequency and its shift due to B0 inhomogeneities and not B0 itself. Please rephrase throughout the manuscript.

Authors’ Response and Action: Indeed, the two formulations are irritating. Following the reviewer's suggestion, we have replaced "sequence" with "acquisition" in the appropriate places. In addition, we have clarified in the manuscript that WASSR is not used to determine B0 itself, but only B0 inhomogeneities. The sentences now read as follows:

“The MRI protocol included standard morphologic sequences, i.e., coronal T2-weighted (T2w) turbo spin-echo (TSE) and sagittal T1-weighted (T1w) TSE sequences. Further MR mapping acquisitions were acquired to map relaxation times:“ (lines 117ff)

“For B₀ inhomogeneity correction, a Water Saturation Shift Referencing (WASSR) acquisition was performed with t_p = 25 ms, t_d = 25 ms, n_p = 1, B₁ = 0.2 µT, 62 dynamics, and Δω=1 ppm; otherwise, the same sequence parameters as for the CEST sequence were used. Further image parameters are indicated in Table 1.” (lines 129ff)

R1 Minor comment #5: Line 271.: Please include z-spectra and MTRasym curves for all three scenarios (in vitro, in situ, and in vivo) to give insight into the expected data quality/appearance.

Authors‘ Respons: We thank the reviewer for the helpful comment and fully agree that the presentation of Z-spectra and MTRasym curves supports the assessment of data quality.

Authors‘ Action: In accordance with the reviewer's suggestion, we have added a figure that additionally shows the changes Z-spectra and MTRasym curves for all 3 scenarios. The figure attached in the supplementary material is as follows:

Supplementary Figure 2: Visualization of representative Z-spectra and the resulting MTRasym curves under systematic variation of lactate concentration in vitro (A, B), in situ (C, D), and in vivo (E, F).

R1 Minor comment #6: Line 469 Changes were observed in the lactate-weighted signal not lactate itself. Please clarify.

Authors‘ Respons and Action: We thank the reviewer for his critical assessment of the wording and agree that it can be misunderstood. We have revised the wording in the relevant sentence and it now reads as follows:

”Finally, we were able to detect exertional exercise-induced lactate-weighted signal in vivo using our optimized CEST sequence, which may allow quantification of altered lactate levels using non-invasive MRI across a broad spectrum of diseases in the future.” (lines 500ff)

able to detect exertional exercise-induced lactate-weighted signal in vivo using our optimized CEST sequence, which may allow quantification of altered lactate levels using non-invasive MRI across a broad spectrum of diseases in the future.” (lines 500ff)

Author Response File: Author Response.docx

Reviewer 2 Report

Title: Chemical exchange saturation transfer imaging of lactate at 3T MRI: Comprehensive in-silico, in-vitro, in-situ, and in-vivo evaluations 
Authors: Karl Ludger Radke, et al. 
Journal: tomography-1673615 

The feasibility of LATEST imaging at a clinical field strength of 3 T was demonstrated with in vitro and in vivo imaging of human volunteers.

This manuscript consists of a non-structured abstract with keywords, 5 sections (introduction, materials & methods with 8 subsections, results with 4 subsections, discussion, and conclusions) on 14 pages of single-spaced text with 5 embedded figures and 3 tables. There are 51 references and 1 equation. No URL is cited. A supplement is provided with 2 figures and their captions. 

LATEST is a promising approach to measure lactate in humans, so this study to establish the feasibility for human studies is timely and important. A LATEST pulse sequence was implemented and optimized on a common variety of clinical 3T MRI scanner (Siemens Magnetom Prisma). 

The "Data Availability Statement" on page 14 is an acknowledgment but provides no information on data sharing. Specifically, the availability of data sets and the LATEST 3T pulse sequence used in this study are not described. No URL is cited. This section should be replaced with pertinent information.

On page 3, "our prototype LATEST CEST sequence" is mentioned. Is this available for sharing? 

MR image analysis was performed with a Python script. Is this available for sharing? MATLAB scripts were used for tissue property analysis. (section 2.7) Are these scripts available for sharing? What R modules were used for statistical analysis? Are these available for sharing? 

Ampuwa is mentioned in the last paragraph of page 6. Should this be identified as Ampuwa® (Fresenius Kabi Deutschland GmbH, www.fresenius-kabi.de)?

The Bloch-McConnell simulation, https://github.com/cest-sources/BM_sim_fit/, was used in this study but the recommended reference was not cited: 

Zaiss, M., Angelovski, G., Demetriou, E., McMahon, M. T., Golay, X. and Scheffler, K. (2017), QUESP and QUEST revisited – fast and accurate quantitative CEST experiments. Magn. Reson. Med. doi:10.1002/mrm.26813

None of the examinations were repeated, so test-retest reproducibility of the measurements was not evaluated. Seven limitations were listed in the last paragraph of the Discussion. This list is appropriate for the manuscript.

The literature review is appropriate and up-to-date. 

Overall, this manuscript introduces a comprehensive feasibility study of LATEST imaging with in silico, in vitro, in situ, and in vivo measurements with and without exercise. The in vitro study incorporates both phantom and cadaver studies. The results were compared with prior reports to provide insight into the intrinsic performance of LATEST. In situ and in vivo measurements were effectively compared. Longitudinal measurements in 4 human subjects after exercise demonstrate moderate variability and help establish LATEST as a feasible metric for in vivo studies. Data and methods sharing should be specified in detail to ensure that this study can be independently reproduced and applied. 

Author Response

Responses to the Reviewers‘ Comments

The authors thank the reviewers for their careful revision of the manuscript and valuable comments, which are addressed and considered in the revised version of the manuscript. Please note that all changes made to the manuscript have been highlighted using the “Track changes”-mode in Microsoft Word and are detailed in this document, too. Please note that line numbers refer to the main text body of the revised document.

Reply to Reviewer 2

R2 General Comment: “Overall, this manuscript introduces a comprehensive feasibility study of LATEST imaging with in silico, in vitro, in situ, and in vivo measurements with and without exercise. The in vitro study incorporates both phantom and cadaver studies. The results were compared with prior reports to provide insight into the intrinsic performance of LATEST. In situ and in vivo measurements were effectively compared. Longitudinal measurements in 4 human subjects after exercise demonstrate moderate variability and help establish LATEST as a feasible metric for in vivo studies. Data and methods sharing should be specified in detail to ensure that this study can be independently reproduced and applied. 

Authors’ Response: We want to thank the reviewer for taking the time to review our manuscript and for the general appreciation and insightful and constructive comments. Please read our responses to each comment below, where we address each comment separately.

R2 Comment #1: The "Data Availability Statement" on page 14 is an acknowledgment but provides no information on data sharing. Specifically, the availability of data sets and the LATEST 3T pulse sequence used in this study are not described. No URL is cited. This section should be replaced with pertinent information.”

Authors’ Response and Action: In the light of the Reviewer’s justified comment, we have revised the passage regarding data availability. The passage now reads as follows:

"Data and evaluation scripts can be provided by the authors upon reasonable request." (line 528f)

R2 comment #2: On page 3, "our prototype LATEST CEST sequence" is mentioned. Is this available for sharing?

Authors’ Response: We appreciate the helpful comment regarding the availability of the sequence we used. For the CEST experiments we performed, we used a WIP sequence provided by Siemens Healthineers; this sequence can be requested from Siemens and made available for research purposes.

Authors‘ Action: In line with the reviewer's suggestions, we have added the manufacturer designation to the first mention of the CEST sequence we used, and the amended section now reads:

“For T1 mapping, an inversion recovery TSE sequence was used with seven inversion times (TIs: 25-3000 ms), while for T2 mapping, a spin-echo (SE) sequence was acquired with 16 different echo times (TEs: 9.7-164.9 ms) as well as a LATEST CEST sequence (WIP 816 A, Siemens Healthineers).” (line 119 ff)

R2 comment #3: MR image analysis was performed with a Python script. Is this available for sharing? MATLAB scripts were used for tissue property analysis. (section 2.7) Are these scripts available for sharing? What R modules were used for statistical analysis? Are these available for sharing?

Authors‘ Response and Action: We thank the reviewer for the helpful suggestions. The Python and MATLAB scripts we used are not currently freely available on GitHub or any other platform. As the the reviewer, we also see the free availability of data and analysis tools as essential for free research; we have therefore revised our "Data Availability Statement". Furthermore, we have added the R packages used. The sections in the manuscript read as follows:

“Statistical analyses were performed by K.L.R. in R (v4.0.3, R Foundation for Statistical Computing) [44] using the packages “pgirmess” [45], “pysch” [46], and “ggpubr” [47]” (line 257f)

“Data and evaluation scripts can be provided by the authors upon reasonable request.“ (line 528f)

R2 comment #4: Ampuwa is mentioned in the last paragraph of page 6. Should this be identified as Ampuwa® (Fresenius Kabi Deutschland GmbH, www.fresenius-kabi.de)?

Authors‘ Response and Action: We thank the reviewer for the critical review. As correctly identified by the reviewer, Ampuwa (Fresenius Kabi Deutschland GmbH, www.fresenius-kabi.de) was used for in situ and in vitro measurements. Taking into account the suggestions of reviewer 1, the in vitro experiments were revised without Ampuwa and now read:

“Preparation of phantoms: For the concentration-dependent phantom study, calcium L-(+)-lactate hydrate (Carl ROTH, Karlsruhe, Germany) was dissolved in phosphate-buffered saline (ROTI®Cell PBS, Carl ROTH) at pH 7.3 (pH value of a muscle at rest [41]), and phantoms were prepared with different concentrations of lactate, i.e., 0, 5, 10, 20, and 40 mM lactate. For the pH-dependent phantom study, 40 mM lactate samples were prepared in PBS solutions with pH-values of 7.6 7.3, 7.0, and 6.7.” (lines 169 ff)

R2 comment #5: The Bloch-McConnell simulation, https://github.com/cest-sources/BM_sim_fit/, was used in this study but the recommended reference was not cited: Zaiss, M., Angelovski, G., Demetriou, E., McMahon, M. T., Golay, X. and Scheffler, K. (2017), QUESP and QUEST revisited – fast and accurate quantitative CEST experiments. Magn. Reson. Med. doi:10.1002/mrm.26813

Authors‘ Response and Action: This is another helpful comment for which we would like to thank the reviewer. Following the reviewer's suggestion, we have included the corresponding reference.

R2 comment #6: None of the examinations were repeated, so test-retest reproducibility of the measurements was not evaluated. Seven limitations were listed in the last paragraph of the Discussion. This list is appropriate for the manuscript.

Authors‘ Response and Action: We agree with the reviewer that the experiments we performed were done without test-retest reproducibility experiments. We have therefore included this in the section on limitations of the discussion which reads:

“Seventh, we did not perform reproducibility measurements and test-retest experiments due to our standardization of the study regions.“ (line 491 ff)

R2 comment #7: The literature review is appropriate and up-to-date. 

Authors‘ Response: We thank the reviewer for critically reviewing the literature we used. In line with comment "R2 comment 5", we have extended the bibliography.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors carefully addressed all my comments and supplied additional measurements and figures which substantiate the findings presented in the manuscript. These included additional phantom measurements to investigate the variability of the CEST effect upon pH changes. These experiments were sufficient to justify the interpretation of the in vivo results. Additionally, z-Spectra and MTRasym curves were supplied to give insight into the data quality. Reasonable pushback was given about the inclusion of tumor patients to further present the applicability of the method as this was not possible due to the local IRB and patients available at the institution.

I have no further comments and the manuscript should be published in the current version.