Determination of the Protein and Amino Acid Content of Fruit, Vegetables and Starchy Roots for Use in Inherited Metabolic Disorders

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Comments

 

Overall, a well written paper, giving greater insight into the amino acid/ protein content of fruits and vegetables in the use of rare IMDs in which diet is limited. I only have a few comments

 

 

Would be useful to see the correlation graphs even if in the supplementary section- this clearly allows the reader to visualise the correlations of AA v protein content- might need to choose a few graphs

 

Would the authors be able to expand on the other AA conditions: what new recommendations would they make for HCU, MSUD  HT1 and UCD using this new analysis, what revisions would be suggested to current practice.  

 

How will they in cooperate these new findings into clinical practice.

 

In PKU where most of the analysis has been done specifically on Phe content of fruits and vegetables,  this has expanded the diet widely how will this work expand the diet for other conditions. It would be helpful to have this debate. I appreciate it might mean that some other sections be removed in the discussion to ensure the discussion is not too long

 

 

Other comments

1.        Starchy roots would be good to have a definition of these please

2.        Line 55 maintenance -would be good to qualify this maintenance of what?

3.        Line 58 qualify … Internationally protein is counted, as the amino acid content of many foods are unknown hence a proxy measurement is used – this study allows a more accurate intake of Phe, Met, Leu

4.        Could argue line 75/6 that although the simplified approach  gives flexibility could also underestimate foods that could be allowed in the diet. For example avocado is allowed freely in PKU although would need to be counted if using protein, bananas allowed freely in PKU but counted if using protein, mushrooms etc. These anomalies could be useful to tease out

5.        Could your aim also include allowing more fruits, vegetables freely if the AA content was low ?

6.        Examples would be good if possible cruciferous vegetables or alternative Brassicas, similarly for starchy roots (carrots, parsnips). Might be good to have this explanation further up in the paragraph mentioned in line 160

7.        Why were multiple samples of certain foods used and not for others?

8.        Line 218 might need to qualify this line for individual foods, large variations in AA content per g of protein were evident and give examples. Would this be expected?

9.        In the supplementary table it might be good to put what the current protein equivalent is for each AA e.g. 50mg of phe = 1 g protein, 20mg Met etc

10.  References some need attention e.g. Alex Pinto should be Pinto A etc

 

Author Response

Dear reviewer,

Many thanks for taking the time to review our article and provide your feedback. 

Here is our responses. We hope this meets your expectations.

Comment 1: Would be useful to see the correlation graphs even if in the supplementary section.

Reply 1: We have included graphs for protein versus individual amino acids as Table S8

Comment 2: Would the authors be able to expand on the other AA conditions: what new recommendations would they make for HCU, MSUD  HT1 and UCD using this new analysis, what revisions would be suggested to current practice.  Line 'Internationally, thresholds for protein and amino acid content below which foods can be allowed without restriction need be considered for these other IMDs' now included. 

Comment 3: How will they in cooperate these new findings into clinical practice. Reply 2: As above, these thresholds will be incorporated into the development of educational resources for individual disorders. 

 

In PKU where most of the analysis has been done specifically on Phe content of fruits and vegetables,  this has expanded the diet widely how will this work expand the diet for other conditions. It would be helpful to have this debate. I appreciate it might mean that some other sections be removed in the discussion to ensure the discussion is not too long. Line 'Internationally, thresholds for protein and amino acid content below which foods can be allowed without restriction need be considered for these other IMDs' now included. 

Other comments

1. Starchy roots would be good to have a definition of these please. Definition included on line ___ 'defined as plants which store edible starch material in subterranean stems, roots, rhizomes, corms, and tubers' 
2. Line 55 maintenance -would be good to qualify this maintenance of what? Changed to maintenance of lean body mass
3. Line 58 qualify … Internationally protein is counted, as the amino acid content of many foods are unknown hence a proxy measurement is used – this study allows a more accurate intake of Phe, Met, Leu. Edited line ___ to read, 'while the simplified diet approach is a practical method to monitor protein as a proxy of AA intake'
4. Could argue line 75/6 that although the simplified approach  gives flexibility could also underestimate foods that could be allowed in the diet. For example avocado is allowed freely in PKU although would need to be counted if using protein, bananas allowed freely in PKU but counted if using protein, mushrooms etc. These anomalies could be useful to tease out -
   Added to the discussion-lines '

   It is important to note that using protein as a proxy of amino acid intake may underestimate foods that can be allowed in the diet of those with AA related IMDs. For example, foods should as beetroot, butternut squash and mangetout would need to be counted in PKU if using protein values observed but can be allowed freely based on the accurate Phe data obtained in this study.'

    
5. Could your aim also include allowing more fruits, vegetables freely if the AA content was low ? Added as an aim: A further aim of this study is to expand on the number of fruits, vegetables and starchy roots that are allowed freely in AA related IMDs and UCDs, provided that their AA and protein content are sufficiently low using new data from this study.
6. Examples would be good if possible cruciferous vegetables or alternative Brassicas, similarly for starchy roots (carrots, parsnips). Might be good to have this explanation further up in the paragraph mentioned in line 160 -  Table 1 clearly defines what groups the vegetables were in. Feels the table labels this more clearly than including in the text. 
7. Why were multiple samples of certain foods used and not for others? We did multiples samples of food we had a big question over i.e. if they needed to be included or excluded in the counting of exchanges i.e. the orange sweet potato and the avocado. The cost of the analysis was the reason we did not do it for others. 
8. Line 218 might need to qualify this line for individual foods, large variations in AA content per g of protein were evident and give examples. Would this be expected? Add line 'for example, in ‘Cruciferous vegetables, Phe content per g protein, varied from 17 mg for ‘mooli (daikon), white, raw’ to 49 mg for ‘Cabbage, york, boiled’.'
9. In the supplementary table it might be good to put what the current protein equivalent is for each AA e.g. 50mg of phe = 1 g protein, 20mg Met etc. We feel that this will depend on this counting system of the individual centre rather than including on each table. 
10. References some need attention e.g. Alex Pinto should be Pinto A etc References are now tidied

 

Reviewer 2 Report

Comments and Suggestions for Authors

The aim of this study is to describe a further analysis of the protein and AA content of fruits, vegetables and starchy roots, specifically focusing on AAs most relevant to IMDs (Phe, Met, Leu, Lys and Tyr). The introduction of the manuscript contains the arguments that support the justification and hypothesis of the study. The objectives are very well defined and associated with the methodology used in the research in question. The methods used in the statistical analysis are in accordance with the study design. The results, discussion and conclusion reflect the hypothesis tested.

Minor revision

In several parts of the manuscript, I noted the separation of IMD and DCU. I consider UCD to be IMD.

The article by Pinto et al. 2023 is restricted to PKU. In this article it is not mentioned that residual enzymatic activity can define the biochemical and clinical phenotype of IMD. There is also a warning in the study by Pinto el al. 2023 that should be considered: “The results of the meta-analysis showed that Phe/natural protein tolerance (expressed as mg or g per day) increases with age, particularly at the beginning of puberty, and reaches its highest level at the end of adolescence. This needs to be interpreted with caution as limited data were available in adult patients. There was also a high degree of heterogeneity between studies due to differences in sample size, the severity of PKU, and target therapeutic levels for blood Phe control”. Furthermore, there are also different degrees of heterogeneity observed in, maple syrup urine disease, homocystinuria, glutaric aciduria type 1 and tyrosinaemias.

The nutritional transition immediately after the industrial revolution presents a culturally differentiated global spectrum. The findings of the present work could be reproduced in other countries, particularly in Latin America, Asia, Oceania and Africa, to validate the content of proteins and amino acids from fruits, dried fruits, cruciferous vegetables, legumes other vegetables or starchy roots, favoring dietary management of patients with IMD from these countries. This approach would be interesting to add to the discussion of the present study. 

Author Response

Many thanks for taking your time to review and provide really valuable feedback on our article.

 

Comment 1: In several parts of the manuscript, I noted the separation of IMD and DCU. I consider UCD to be IMD.

Reply 1: This was used to separate out the Amino acid related disorders (shorthanded as AA related IMDs) to capture PKU, HCU, MSUD, GA1 and Tyrosinanaemia in a shortened manner as these require consideration of specific amino acids versus UCDs which require consideration of overall protein content. In the introduction UCDs are introduced as IMDs

Comment 2: The article by Pinto et al. 2023 is restricted to PKU. In this article it is not mentioned that residual enzymatic activity can define the biochemical and clinical phenotype of IMD. There is also a warning in the study by Pinto el al. 2023 that should be considered: “The results of the meta-analysis showed that Phe/natural protein tolerance (expressed as mg or g per day) increases with age, particularly at the beginning of puberty, and reaches its highest level at the end of adolescence. This needs to be interpreted with caution as limited data were available in adult patients. There was also a high degree of heterogeneity between studies due to differences in sample size, the severity of PKU, and target therapeutic levels for blood Phe control”. Furthermore, there are also different degrees of heterogeneity observed in, maple syrup urine disease, homocystinuria, glutaric aciduria type 1 and tyrosinaemias. Reply 2: Reference changed to references which mention that tolerance will depend on residual enzyme activity. Further discussion on tolerance outside the remit of this paper. 

Comment 3: The nutritional transition immediately after the industrial revolution presents a culturally differentiated global spectrum. The findings of the present work could be reproduced in other countries, particularly in Latin America, Asia, Oceania and Africa, to validate the content of proteins and amino acids from fruits, dried fruits, cruciferous vegetables, legumes other vegetables or starchy roots, favoring dietary management of patients with IMD from these countries. This approach would be interesting to add to the discussion of the present study - Reply 3: interesting point, we have added a line towards the end of the discussion 'We would hope this work could be reproduced in other countries where data on protein and amino acids from fruits, vegetables and starchy roots indigenous to their countries is limited'. 

Reviewer 3 Report

Comments and Suggestions for Authors

The article sent for review entitled: Determination of the protein and amino acid content of the fruit, vegetables and starchy roots for use in inherited metabolic disorders takes into consideration the level of amino acids level in fruits and vegetables.

The article is the systematic analysis of exogenous amino acids: Phe, Met, Leu, Lys, Tyr which must be delivered to the human body.

From the point of different metabolic diseases, this article is important for patients and their minders. Moreover, in the age of vegetarians and vegans, this scientific analysis can be valuable for the above persons and allow the correct nutrition profile arranged.

The language used in the manuscript is correct and makes it readable. Moreover, the selected references were done properly.

I have two comments:

- authors should focus on the level of the exogenous amino acid in the reference protein source as egg

- authors should describe in detail the analysis method (HPLC)

 

In conclusion after answers to my question article is suitable for publication.

 

Author Response

Many thanks for taking the time and expertise to feedback on our article.

In response to the comments the following information has been added to the article

Comment 1: authors should focus on the level of the exogenous amino acid in the reference protein source as egg - protein, Phe, Met, Leu, Lys, Tyr of egg per 100 g = 12.5 g, 800 mg, 450 mg, 1260 mg, 890 mg and 540 mg  (Souci et al.

Average of fruits, veg and starchy roots in this study per 100 g =  g, mg, mg, mg, mg and mg.

In IMDs, this significant difference would be well known. Does the reviewer require mention of this in the body of the text. If so can include in paragraph 2 in the discussion to put into context how low in protein the

• authors should describe in detail the analysis method (HPLC) - We contacted ALS and their response was 
• Chatteris method reference (AM/V/206) and issue(23) HPLC operational requirements such as HPLC setup and chromatographical interpretation as per ALS method AM/V/206 issue 23”