Indexes of Fat Oxidation from Ramp vs. Graded Incremental Protocols in Postmenopausal Women

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

I would like to thank the authors for their article. Below are my comments.

1. The English language needs to be improved.
2. What is the originality and the novelty of the current study? The authors should clearly state the abovementioned points in the last paragraph of the introduction.
3. How was the sample size defined? Since the number of 17 participants is relatively small.
4. The materials and methods section states that: ''Total physical activity was calculated and expressed as metabolic equivalents (met⋅wk-1) based on the International Physical Activity Questionnaire - short form.'' The IPAQ should be described in the materials, and the authors should provide a table of relevant results.
5. The materials and methods section needs to be better organized.
6. I suggest the authors consider adding a last paragraph in the discussion section, providing the strengths and limitations of their study.

Comments on the Quality of English Language

Accept after major revisions

Author Response

We thank the reviewer for the time and effort devoted to revising our work. We are glad that the reviewer finds merit in the manuscript, and we have done our best to address all the constructive criticism that was provided.

Comment 1: The English language needs to be improved.
Response: Thanks for allowing us to improve the manuscript. All the authors have revised the sentences/words throughout it. Moreover, the affiliated University of Verona offered a Grammarly check as a service. We hope that the reviewer will accept our careful revisions, and we are confident that the quality of the English does not now limit the reader's understanding of the research.

Comment 2: What is the originality and the novelty of the current study? The authors should clearly state the above mentioned points in the last paragraph of the introduction.

Response: The reviewer’s comment made us realize we had not adequately emphasized the novelty of this study that resign on:

• Ramp incremental test can accurately detect MFO and FATmax in postmenopausal women
• The time- and cost-efficiency of ramp incremental testing can facilitate the use of these indexes of metabolic flexibility in research and clinical setting

The point mentioned above was better organized and rephrased for the clarity of the reader. lines 103-107:
“Therefore, given the importance of characterizing the potential role of fat oxidation as an index of metabolic flexibility in the female population, especially after the menopause transition, we aimed to extend and clarify the results of previous studies by testing a different population of postmenopausal women. To this aim, we compared the performance of a time-efficient ramp to the traditional graded, incremental testing for determining MFO and FAT_max.”

We hope this statement better describes the novelty of this study.

Comment 3: How was the sample size defined? Since the number of 17 participants is relatively small.

Response: Thanks for allowing us to clarify that. We add this information in the statistics section from Line 241: Power analysis was conducted a priori, based on the expected SD of fat oxidation reported during constant load exercise in previous articles as the primary variable (Meyer et al 2010). To identify significant differences, with an α error of 0.20 and a statistical power (1 − β) of 0.80, an n value of 17 subjects was necessary.

 After the study ended, we also performed a full compromise power analysis in hindsight on Maximal fat oxidation (G*Power results appended below for your convenience), which tends to exclude a type 2 error.

-- Thursday, November 30, 2023 -- 12:53:20

4) t tests - Means: Difference between two dependent means (matched pairs)

5) Analysis: Compromise: Compute implied α & power

6) Input: Tail(s) = Two

7) Effect size dz = 0.525

8) β/α ratio = 1

9) Total sample size = 17

10) Output: Noncentrality parameter δ = 2.1646305

11) Critical t = 1.3301621

12) Df = 16

13) α err prob = 0.2021153

14) β err prob = 0.2021153

15) Power (1-β err prob) = 0.7978847

 

Comment 4:  The materials and methods section states that: ''Total physical activity was calculated and expressed as metabolic equivalents (met⋅wk-1) based on the International Physical Activity Questionnaire - short form.'' The IPAQ should be described in the materials, and the authors should provide a table of relevant results.

Response: 

We agree with the reviewer, IPAQ description has been now better described and moved to the protocol section. Lines 142-148:

 “In addition, the participant's physical activity levels were assessed using the short form of the International Physical Activity Questionnaire  (Craig et al., 2003). The questionnaire was administered in a self-report format, with participants providing information about the frequency, duration, and intensity of physical activities over the past seven days, as well as sedentary behavior. Total physical activity was calculated and expressed as metabolic equivalents (met⋅wk-1), allowing for the categorization of participants’ physical activity as low, moderate, or high as described elsewhere (Craig et al., 2003). “

 Furthermore, a table with the IPAQ results, together with an Overview of anagraphic, anthropometrics, and fitness variables, has been added.

Comment 5: The materials and methods section needs to be better organized.
Response: The reviewers are correct. Materials and Methods have now been better organized. 

Comment 6: I suggest the authors consider adding a last paragraph in the discussion section, providing the strengths and limitations of their study.
Response: We thank the reviewer for the suggestion. A limitations section has been added at the end of the manuscript from lines 440-449 (and reported below for the reviewer's convenience).

5. Limitations:

While it is plausible that overnight fasting may increase test-retest reliability through increased homogeneity of substrate availability as well as facilitate the comparison with previous work, we opted instead for a standardized meal to resemble the ecological conditions of a standard cardiorespiratory testing session while also containing an important source of variations. Indeed, the main aim of this study was to evaluate the accuracy of the ramp vs. graded incremental approach in determining fat metabolism performed in the same preanalytical and ecological conditions. Notably, the observed within-subject variability between subsequent tests on FO, FATmax, and MFO was similar to that described in the existing literature (10-25%) and compatible with our choice of using a standard meal rather than a “fasting night” (Croci et al., 2014; Meyer et al., 2009).

--> Regarding adding the strengths of our study, we thought that it would be a repetition of what is written along the discussion (line 352-355; line 427-429 and line 442-443). Thus, we are reluctant to repeat this information in the discussion, which is already somewhat long. We hope the reviewer will accept our view. We reported below for reviewer’s convenience:

Line 352-355: Furthermore, the present study provided the first data on the direct comparison between ramp incremental and graded protocol in postmenopausal women

Line 427-429: In the present study, the correction strategy adopted to adjust the power output derived from the ramp test could explain the observed similar power output at FAT_max among protocols compared with Michalik's work.

Line 442: we opted instead for a standardized meal to resemble the ecological conditions of a standard cardiorespiratory testing session,

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

Teso and colleagues sought to determine indexes of fat oxidation from ramp vs. graded incremental protocols in postmenopausal women. The authors collected a substantial amount of data. They argue that the ramp protocol offers a valid alternative to the graded protocol for identifying the maximal rate of fat oxidation and the relative exercise intensity at which it occurs. The study design and analytical methods are appropriate, and I agree that the data of this study are useful for prescribing exercise therapy. However, there are some concerns regarding interpreting the results. Additionally, there are a few areas in the paper that could benefit from further attention to enhance its clarity and precision.

 

Major comments

1.         As illustrated in Figure 2E, there seems to be a discrepancy between the fat oxidation data obtained from the ramp and graded protocols, although the difference may not be statistically significant. Accordingly, there is a difference of ±10% between the limit of agreement of FATmax for the ramp and graded protocols (Figure 3). The authors write that Bland-Altman analysis showed no significant bias between measures but relatively low precision. Even if there is no statistically significant difference, it is important to carefully consider the impact of different loading methods on the resulting outcomes. I kindly request that you consider this point in greater depth to avoid readers' misinterpretation.

 

2.         A more detailed discussion of the results from the perspective of postmenopausal women should be needed.

 

3.         The limitations of the study should be described in the Discussion section.

 

4.         The practical implications, particularly the second and third points, are somewhat exaggerated. It is challenging to reach such a conclusion based on the results of this study alone.

 

5.         There are some inappropriate citations in the Introduction and Discussion sections.

It would seem that there are a few instances where the reference numbers do not correspond with the information presented (ex. p2 line 91, p10 lines 359-360 and 370).

Although the authors wrote "Four previous studies" on page 9 line 317, only two references are cited. In addition, ref. 31 is a commentary article.

Add the reference number in the sentence (p10 lines 336-337).

 

6.         The LLOA and ULOA values for VO2max in Table 2 may be incorrect.

 

 

Minor comments

1.         Words used in the title are not included in the keywords.

2.         It would be helpful for readers if you could provide details about the equipment and statistical analysis software utilized in this study.

3.         Explain Figure 1 in the text.

4.         Add units to both the vertical and horizontal axes in Figure 3.

5.         There are no data on the respiratory exchange ratio mentioned in the notes in Table 1.

Author Response

The reviewer summarised very clearly the aim and main findings of our study, and we are glad that she/he finds merit in it. We have done our best to address all the constructive criticism that was provided

Comment 1:  As illustrated in Figure 2E, there seems to be a discrepancy between the fat oxidation data obtained from the ramp and graded protocols, although the difference may not be statistically significant. Accordingly, there is a difference of ±10% between the limit of agreement of FATmax for the ramp and graded protocols (Figure 3). The authors write that Bland-Altman analysis showed no significant bias between measures but relatively low precision. Even if there is no statistically significant difference, it is important to carefully consider the impact of different loading methods on the resulting outcomes. I kindly request that you consider this point in greater depth to avoid readers' misinterpretation.

Response:  The reviewer raises a valid point regarding the discrepancy in fat oxidation data between the ramp and graded protocols despite the lack of statistical significance. The relatively large limits of agreement for both MFO (0.08 to 0.19 g/min) and FATmax (-7.8% to +10.6% VO2max) highlight a substantial degree of variability between the two methods (or on the intrinsic measures of FOx). We agree with the reviewers and tried our best to avoid readers’ misinterpretation of the conclusions. Two paragraphs have been added to the discussion. Here are the detailed:

• MFO and FO discrepancies:

The difference in MFO between the ramp and graded protocols can be explained by the difference in the speed of adjustment (i.e. the kinetics) of the V̇O₂ and V̇CO₂ upon metabolic transitions. In the moderate and heavy exercise domain, both signals typically display an exponential rise, reaching a steady-state within 3-5 min. However, due to the 20 times higher diffusion coefficient of CO₂ compared to O₂, V̇CO₂ typically displays a faster kinetic. Therefore, the resulting R displays a transitory peak in the first 2-3 min of exercise eventually adjusting to a lower steady-state value within 5 min. Moreover, the speed of adjustment of V̇O₂ and V̇CO₂ is affected by the fitness level of the subjects, with slower kinetics characterizing less fit individuals. Since the estimate of fat oxidation through indirect calorimetry depends on the product of g⋅l^-1 (derived from R) and l⋅min^-1 (derived from V̇O₂), the duration of the exercise stage will affect the estimate, theoretically the more so in less fit subjects (characterized by a slower speed of adjustment of V̇O₂).

A full explanation has now been added to the discussion!

• FATmax low precision LLOA ULOA:

Should be noted that FATmax (and FO) have inherent sources of variability. Diurnal variation, Physical activity, Dietary intake, hydration status, psychological factors. Even if we tried to control some of these aspects (standardized meal, hydration, and time of the day), it is possible that some factors independent of the protocols might affect the FO curve. Moreover, while it is plausible that overnight fasting may increase test-retest reliability through increased homogeneity of substrate availability as well as facilitate the comparison with previous work, we opted instead for a standardized meal to resemble the ecological conditions of a standard cardiorespiratory testing session, while also containing an important source of variations. Indeed, the main aim of this study was to evaluate the accuracy of the ramp vs. graded incremental approach in determining fat metabolism performed in the same preanalytical and ecological conditions. However, please note that the observed within-subject variability between subsequent tests on FO, FAT_max, and MFO was similar to that described in the existing literature (10-25%) and compatible with our choice of using a standard meal rather than a “fasting night” (Croci et al., 2014; Meyer et al., 2009).

We add a synthesized explanation of this in the limitation section.

Comment 2: A more detailed discussion of the results from the perspective of postmenopausal women should be needed.
Response: A paragraph has been added to lines 340 -352 (and reported here below for the reviewer's convenience):
The growing research interest in women's metabolic profile and flexibility after menopause is driven by the increasing prevalence of metabolic disorders in this population [9], [41]. Changes in female sex hormone concentrations after menopause have been associated with increased metabolic and cardiovascular risk factors as well as neurodegenerative diseases and osteoporosis [41], [42], [43], [44]. These risks include insulin resistance, visceral adiposity, and dyslipidemia, which is associated with metabolic inflexibility and disorders such as type 2 diabetes [41], [45], [46]. MFO is a key marker of metabolic flexibility in individuals with metabolic disorders as it represents the body's capacity to utilize fat as a predominant fuel source during physical activity [15]. For these reasons, recent articles are investigating how exercise interventions can enhance fat oxidation to mitigate the adverse metabolic effects associated with menopause [42], [47], [48].

 

Comment 3: The limitations of the study should be described in the Discussion section.
Response: We thank the reviewer for the suggestion. A limitations section has been added at the end of the manuscript from lines 439-449 (and reported below for the reviewer's convenience).

5. Limitations

While it is plausible that overnight fasting may increase test-retest reliability through increased homogeneity of substrate availability as well as facilitate the comparison with previous work, we opted instead for a standardized meal to resemble the ecological conditions of a standard cardiorespiratory testing session while also containing an important source of variations. Indeed, the main aim of this study was to evaluate the accuracy of the ramp vs. graded incremental approach in determining fat metabolism performed in the same preanalytical and ecological conditions. Notably, the observed within-subject variability between subsequent tests on FO, FATmax, and MFO was similar to that described in the existing literature (10-25%) and compatible with our choice of using a standard meal rather than a “fasting night” (Croci et al., 2014; Meyer et al., 2009).

Comment 4: The practical implications, particularly the second and third points, are somewhat exaggerated. It is challenging to reach such a conclusion based on the results of this study alone.
Response: We thank the reviewer. The conclusion has now been revised!

Comment 5:
a) There are some inappropriate citations in the Introduction and Discussion sections.

We agree with the reviewer. Some mistakes may come from multiple versions of the manuscripts.
We double-checked the order of the references and detected inappropriate citations.
Citations are replaced with more appropriate ones in lines 46, 50-51, 55, 58, 72, 80, 94, 408, 413

b) It would seem that there are a few instances where the reference numbers do not correspond with the information presented (ex. p2 line 91, p10 lines 359-360 and 370).

Thanks for pointing out the mistakes! References have now been corrected.

c) Although the authors wrote "Four previous studies" on page 9 line 317, only two references are cited. In addition, ref. 31 is a commentary article.

Thanks for spotting the mistakes! We added the two other missing studies on page 9 line 336.
Ref 31 is a commentary article that presents normative data (MFO and FATmax) from 160 individuals (101 women). For this reason, we consider that relevant for making comparisons with our postmenopausal participants.

d) Add the reference number in the sentence (p10 lines 336-337).
Thanks! Added!

Comment 6: The LLOA and ULOA values for VO2max in Table 2 may be incorrect.
Response: The LLOA and ULOA were in mL instead of L. Thanks for spotting the mistake! Corrected. For the same reason, VO2 in Table 1 has also been revised.

MINOR COMMENTS:

Comment 1:  Words used in the title are not included in the keywords.
Response: We are not sure what question is being asked. We used different keywords to enhance the article's discoverability (broaden the scope of indexing). By using a diverse set of keywords, we can ensure that our work is accessible to an audience using variable search terms, thereby increasing the article’s visibility and impact.

Comment 2: It would be helpful for readers if you could provide details about the equipment and statistical analysis software utilized in this study.
Response: We agree with the reviewer. Statistical software (G*power for power analysis and Sigmaplot 14.0) was added to the statistical analysis. Moreover, all the used equipment has been added to the methods.

Comment 3: Explain Figure 1 in the text.
Response: Added in line 206

Comment 4: Add units to both the vertical and horizontal axes in Figure 3.
Response: Units added in Figure 3

Comment 5: There are no data on the respiratory exchange ratio mentioned in the notes in Table 1 Response: Thanks for spotting the mistake! Notes have now been corrected!

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors significantly improved their manuscript. No more comments to add.

Author Response

Comments 1: The authors significantly improved their manuscript. No more comments to add.
Response:  We are glad the reviewer finds that we have responded satisfactorily to most of the criticism.

We once more thank the reviewer for the constructive criticism and we think that the manuscript is now improved.

 

Reviewer 2 Report

Comments and Suggestions for Authors

I am grateful for your comprehensive responses to my comments. The manuscript has undergone a substantial revision and is now of a much higher standard.

Comments

1.         I suggest that "However" in line 386 should be deleted and "smaller" in line 387 should be changed to small.

2.         The reference number in line 406 may not be [24] but [23].

3.         Please correct “post-menopausal” to “postmenopausal” in lines 405, 409, and 411.

4.         It is best to include limitations in the last paragraph of the discussion section.

Author Response

We are glad that the reviewer finds that we have responded satisfactorily to almost all the comments. Once more, we thank the reviewer for the constructive criticism that was aimed at improving our manuscript.

Comment 1: I suggest that "However" in line 386 should be deleted and "smaller" in line 387 should be changed to small
Response: We agree with the reviewer. However has now been removed and smaller corrected.

Comment 2: The reference number in line 406 may not be [24] but [23].
We thank the reviewer for spotting the mistakes. Changes have been made.

Comment 3: Please correct “post-menopausal” to “postmenopausal” in lines 405, 409, and 411
Corrected! We thank the reviewer again!

Comment 4: It is best to include limitations in the last paragraph of the discussion section.
Limitations have now been included in the discussion section (Lines: 431-441)