Review Reports - Serotonin, Kynurenine, and Indole Pathways of Tryptophan Metabolism in Humans in Health and Disease

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This article aims to provide a methodical and extensive review of the role of TRP and its metabolites in various physiological and pathological conditions, as well as the use of TRP as a dietary supplement in the therapy of various diseases. The description is methodical as all the sections (some of them partially reiterative) are presented considering the Trp derivatives of the three Trp metabolic routes, Trp_Ser, Trp_Kyn and Trp_Ind. and it throws some light to complexity of Trp derivates. Sections related to the hereditary disorders associated to the 3 routes are very interesting, although some of them are described reiteratively when they are presented in relation to different diseases. Figures summarizing routes, enzymes and main intermediates are smart taking into account the metabolites’ diversity.

In sum. This review is considered positive. The hope of the author is partially satisfied. This methodical and detailed review helps to understand the role of Trp metabolic pathways and what the supplementation with this essential amino acid can and cannot achieve. The complexity of the Trp integral metabolism (host and microbiota) makes difficult a total knowledge of this matter, but it is obvious that this review will lead researchers to further investigation on the effects of this important amino acid in human health.

However, before definitive acceptance, I would like to list a series of points for possible addressing. The authors might improve some controversial aspects about them.

For instance, this section would also consider that at lines 349-352 it is described that serotonin increases production of ROS and secretion of proinflammatory cytokines. The opposite effects (pro/anti-inflammatory) would be discussed, and if possible, in just one section. There are several sections and sentences about antioxidant effects, but also prooxidant (i.e section 6.3.2).

Line 146: About the antiepileptic drug valproate. The mechanism of action of this SFCA is unclear. Proposed mechanisms include affecting GABA levels, blocking voltage-gated sodium channels or inhibiting histone deacetylases, but the link with Trp herein mentioned is not demonstrated. I ask the author reconsidering this point. Anyway, it should be clear that the effect of valproate is not just by changing serum free Trp concentration, as it can be understood from this section.

Section 6.1.1. Please, add some reference for justifying that section. The manuscript is full of excellent references, but some paragraphs are not.

About BBB permeability to Trp and derivates. Reconsider the following sentences extracted from the manuscript: 1-Line 308: Serotonin cannot cross the blood-brain barrier; 2- Line 512: QA, KYNA, and 3-HANA cross the BBB poorly; 3- Line 656: Some indole derivatives, particularly IPA, cross the BBB; 4- Line 1002: Melatonin is both water-and lipid-soluble (‘amphiphilic’) and can freely cross plasma membranes, including the BBB.

In spite that some of those sentences are referenced, a brief reconsideration and discussion about Trp derivates and BBB would clarify this controversial issue. Consider that it is usual TRP administration for boosting serotonin levels in the brain too.

Line 779: About the sentence “KYNA negatively correlated with depression severity and significantly increased after therapy [166]”. Please, check it. It seems a `paradox. According to the first part of the sentence, therapy would decrease the KYNA content.

Lines 836- : Indole-derived uremic toxins act as pathogenic agents, which, via the AHR, induce the transcription of proinflammatory cytokines, apoptosis, and oxidative stress. However, at lines 755 “ the levels of IPA, which exerts benefits on gut homeostasis through AHR and PXR” and line 792 “IPA, which acts as a free radical scavenger and an anti-inflammatory substance, thereby decreasing the production of proinflammatory cytokines [112]. Please, discuss this double-edged sword about indole derivates.

Line 982: 9.8 TRP and Liver Cirrhosis (related to 9.3 TRP and Alcoholism). Even cirrhosis can be caused by other reasons, alcoholism and cirrhosis are related. The analysis of the 3 routes is used so many times throughout the manuscript, and the review becomes a little bit verbose, reiterative and boring. Think about melting 9.3 and 9.8 to shorten the review a little bit avoid reiteration.

Line 1003: Please delete the mention commercial products (Circadin®)

Line 1020: The sentence “serotonin, which is a cancer-promoting factor” seems to be exaggerated in spite of the section about Trp and cancer. Please, re-consider it.

Finally, the abbreviation list found at the end of the text is appropriate, but it should be used. Delete all reiterative clarifications throughout the manuscript. For instance, use BBB in all cases or avoid the mention of branched amino acids after the abbreviation LNAA in some lines, and so on.

Author Response

RESPONSE TO THE COMMENTS OF THE Reviewer #1

Dear Reviewer,

thank you very much for constructive remarks and positive evaluation of the article. Here are the responses to your comments:

Comment

2.1. Antioxidant Properties of TRP. The mechanism for justifying the antioxidant properties would be described or referenced. Mostly, I would like to answer to the following questions: Is the indole ring (or Kyn-related structures) a direct scavenger of ROS? Alternatively, is that action mediated by binding to AHR/PGX?. When binding to those receptors is antioxidant or prooxidant?.For instance, this section would also consider that at lines 349-352 it is described that serotonin increases production of ROS and secretion of proinflammatory cytokines. The opposite effects (pro/anti-inflammatory) would be discussed, and if possible, in just one section. There are several sections and sentences about antioxidant effects, but also prooxidant (i.e section 6.3.2).

Response

The section deals only with chemical properties of indole moiety of TRP and its metabolites to oxidative stress and not by consequences of the TRP metabolism, including effects mediated by its binding to AHR/PXG, which can be both antioxidant and prooxidant. To better understand the meaning of this part of the manuscript, the section has been retitled and edited as follows:

2.1. TRP Sensitivity to Oxidative Stress and Antioxidative Properties of TRP

The indole moiety is sensitive to the influence of a variety of reactive oxygen (ROS) and nitrogen (RNS) species, including hydrogen peroxide, hydroxyl radical, singlet oxygen, nitric oxide, and peroxynitrite anion. Multiple reaction pathways appear to occur in the reaction of ROS and RNS with the indole moiety, and multiple products have been identified, including 3-hydroxytryptophan, dioxindolylalanine, KYN, N-formylkynurenine, and dihydroxypyrroloindole [8]. Because the indole part of TRP can be degraded into multiple toxic products by exposure to light, high or low pH, heat, and oxygen, the storage of TRP-containing supplements, especially in solution form, must be under strict control [8]. A good target of oxidative stress is especially the TRP residues in proteins (Section 5). On the other hand, some TRP metabolites, such as KYNA and melatonin, are recognized as potent antioxidants (Sections 6.3.2 and 7.2.3).

Comment

Response

We do not examine the mechanism of therapeutic effect of valproic acid in therapy of epilepsy. In the revised version of the manuscripts is only stated: “In addition, it has been shown that valproic acid (a drug used for the treatment of epilepsy), can displace protein-bound TRP, increase free TRP level, and affect serotonin synthesis in the brain [28]”.

Comment

Section 6.1.1. Please, add some reference for justifying that section. The manuscript is full of excellent references, but some paragraphs are not.

Response

Done. The following references have been added in the section 6.1.1:

Shih JC, Chen K, Ridd MJ. Role of MAO A and B in neurotransmitter metabolism and behavior. Pol J Pharmacol. 1999;51(1):25-9.
Bortolato M, Chen K, Shih JC. The degradation of serotonin: role of MAO. In: Müller C, Jacobs B, editors. Handbook of Behavioral Neurobiology of Serotonin. Amsterdam, The Netherlands: Elsevier; 2010. pp. 203–218.

Comment

Response

The following sentences have been added into the Section “Summary and Conclusions”:

“For targeting TRP metabolic pathways in the therapy of nervous diseases, the ability of individual TRP metabolites to cross the BBB is important. Melatonin, 5-hydroytryptophan, some derivatives of the TRP-KYN pathway (e.g., KYN, 3-HKYN, and ANA), and the TRP-IND pathway, particularly IPA, can cross the BBB. On the other hand, serotonin cannot cross the BBB, and QA, KYNA, and 3-HANA cross the BBB poorly.”

Comment

Response

Thanks. The misleading sentence “KYNA negatively correlated with depression severity and significantly increased after therapy [166]” has been deleted.

Comment

Response

The paragraph related to TRP-IND pathways in the Section 9.7 has been modified as follows:

“Concentrations of both free and protein-bound indole metabolites, primarily IAA, indoxyl sulfate, and indoxyl-β-D-glucuronide, recognized as uremic toxins, increase in patients with CRI due to impaired gut barrier integrity and their decreased elimination in urine [112,113]. Unlike the positive influence of most indole derivatives in the gut, IAA, indoxyl sulfate, and indoxyl-β-D-glucuronide act in cells of the cardiovascular system as pathogenic agents that, via the AHR, induce the transcription of proinflammatory cytokines, apoptosis, and oxidative stress.”

Comment

Response

In my opinion, merging sections 9.3 and 9.8 is inappropriate because the effects of alcohol on TRP metabolism are different from the changes induced by liver cirrhosis. I think that the examination of changes in the three pathways of TRP metabolism under various physiological and pathological conditions makes the article interesting for a wide range of clinicians and researchers.

Comment

Line 1003: Please delete the mention commercial products (Circadin®)

Response

Done.

Comment

Line 1020: The sentence “serotonin, which is a cancer-promoting factor” seems to be exaggerated in spite of the section about Trp and cancer. Please, re-consider it.

Response

Accepted. The statement has been deleted.

Comment

Response

Done.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

This narrative review addresses a timely and relevant topic within the scope of Nutrients and provides a comprehensive overview of the existing literature. The manuscript is generally well written and informative, and the topic is of clear interest to the readership.

To further strengthen the review, the authors are encouraged to consider the following minor points:

While the review effectively summarizes current evidence, it would benefit from a clearer critical perspective. In particular, briefly commenting on where the literature shows inconsistent results and where evidence is still limited or preliminary (in addition to well-established findings) would improve clarity and balance for the reader.
The manuscript would be strengthened by the inclusion of a concise paragraph outlining key limitations of the existing literature (e.g., heterogeneity of study designs, population differences, confounding factors) and clearly identifying priorities for future research.
To improve clarity and reader engagement, the authors may consider adding a simple schematic figure or conceptual illustration summarizing the main mechanisms and relationships discussed in the review (e.g., a graphical abstract–style overview).

Overall, this is a valuable review that would benefit from these minor revisions, which aim to enhance clarity, critical depth, and accessibility without altering the main conclusions.

Best regards,

Reviewer

Author Response

RESPONSE TO THE COMMENTS OF THE Reviewer #2

Dear Reviewer,

thank you very much for constructive remarks and positive evaluation of the article. Here are the responses to your comments:

Comment

While the review effectively summarizes current evidence, it would benefit from a clearer critical perspective. In particular, briefly commenting on where the literature shows inconsistent results and where evidence is still limited or preliminary (in addition to well-established findings) would improve clarity and balance for the reader.

Response

Please note the following statements in the revised version of the Summary and Conclusion section:

Unfortunately, the understanding and characterization of the mechanism of action of most TRP metabolites are in their early stages, and many of their effects and potential therapeutic uses have not been fully explored. For example, the literature shows very limited knowledge about the effects of cinnabaric and picolinic acid.
Current knowledge is insufficient to provide guidelines for the use of TRP and drugs affecting its metabolic pathways to achieve therapeutic and avoid detrimental effects in most clinical conditions.
In most diseases, we do not know whether the given alterations in TRP metabolism contribute to the disease's pathogenesis or are merely a consequence.

Comment

The manuscript would be strengthened by the inclusion of a concise paragraph outlining key limitations of the existing literature (e.g., heterogeneity of study designs, population differences, confounding factors) and clearly identifying priorities for future research.

Response

The Summary and Conclusion section has been modified to better demonstrate the limited knowledge and gaps in the current literature. For example:

In most diseases, we do not know whether the given alterations in TRP metabolism contribute to the disease's pathogenesis or are merely a consequence. The discrepancies and gaps also exist in the current understanding of the relationships and interactions among the three TRP metabolic pathways. For instance, TRP and most derivatives of its three metabolic pathways act as AHR ligands, and several studies have shown that changes in the flux through the TRP-KYN pathway affect serotonin production in the brain.
Furthermore, the effects and potential therapeutic uses of many TRP metabolites have not been fully explored. For example, the literature shows very limited knowledge about the effects of cinnabaric and picolinic acid.
The goal for clinical research is to explore the options of TRP-targeted therapies and their integration into new therapeutic strategies.
Clinical research should prioritize longitudinal and interventional studies to establish causal links between TRP intake, microbiota-derived metabolites, and host metabolism and neuroimmune responses.
Large randomized clinical trials are needed to define long-term efficacy and clinically relevant outcomes of TRP administration and targeting TRP catabolism pathways.
Rigorous evaluation of the safety and dose–response of TRP supplementation will support the development of personalized nutritional and therapeutic strategies.

Comment

To improve clarity and reader engagement, the authors may consider adding a simple schematic figure or conceptual illustration summarizing the main mechanisms and relationships discussed in the review (e.g., a graphical abstract–style overview).

Response

Accepted. A graphical abstract has been added.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Journal: Nutrients

Manuscript ID

nutrients-4109366

Type

Review

Title

Tryptophan and Its Metabolites in Health and Disease

Authors

Milan Holeček *

Section

Proteins and Amino Acids

OVERVIEW

Based on the statement that tryptophan (TRP) plays a crucial role as an antioxidant and performs important functions in the body, the authors of this article intended to examine sources, nutritional requirements, and TRP catabolism. Alterations of individual TRP catabolism pathways in aging, alcoholism, inflammatory bowel disease, metabolic syndrome, renal insufficiency, liver cirrhosis, cancer, and neurological diseases were investigated, as well as the benefits and adverse effects of its supplementation, The authors concluded that all pathways of TRP catabolism are altered in a broad spectrum of human illnesses, and further investigation is needed for a better understanding of TRP me-tabolism in disease pathogenesis. The goal of clinical research is to explore options for TRP-targeted therapies and their integration into new therapeutic strategies.

TITLE

The title of this manuscript is "Tryptophan and Its Metabolites in Health and Disease".

In my opinion, the title is too broad and does not make the manuscript type clear. I suggest changing it to another one. As an example:

“Regulation of the Serotonin, Kynurenine and Indole Pathways of Tryptophan Metabolism: a review”.

ABSTRACT

In this section we can see that it is divided into 2 main parts (lines 11-26):

“The first parts of the article examine 11 sources, nutritional requirements, and three pathways of TRP catabolism. Physiologically, ~5% of dietary TRP is catabolized through the TRP-serotonin pathway in the brain and enterochromaffin cells of the gut, ~ 85% through the TRP-kynurenine pathway in the liver and immune cells, and ~10% by the TRP-indole pathway in gut microbiota. Alterations of individual TRP catabolism pathways in aging, alcoholism, inflammatory bowel disease, metabolic syndrome, renal insufficiency, liver cirrhosis, cancer, and nervous diseases, e.g., depression, Alzheimer's and Parkinson's diseases, multiple sclerosis, and schizophrenia, are examined in the central section. The final parts are devoted to the benefits and adverse effects of TRP supplementation, therapeutic use of various TRP metabolites, and pharmacological targeting of enzymes, transporters, and receptors involved in TRP catabolism. It 21 is concluded that all pathways of TRP catabolism are altered in a broad spectrum of human illnesses, and further investigation is needed for a better understanding of TRP metabolism in disease pathogenesis. “

I miss the inclusion of the results of the search.
Please check for some minor errors.
I also think it does not explicitly state the criteria for selecting the literature, nor does it clarify the type of review.
The conclusions are generic, and I feel there is a lack of commentary regarding the identification of real gaps and concrete clinical implications.

KEYWORDS

The keywords used by the authors are:

“amino acids; kynurenine; serotonin; indole; melatonin; kynurenic acid; quinolinic acid; metabolic syndrome."

I suggest

“kynurenine; serotonin; melatonin; kynurenic acid; quinolinic acid; neuroinflammation; metabolic disorders".

INTRODUCTION AND DISCUSSION SECTIONS:

In general, the Introduction is fine but long.
I miss the inclusion of Figures in this section.
Figures 3-5 are very nice, but I suggest improving Figure 1.
Still regarding Figures, be sure that all abbreviations used in them are defined in the legends.
Furthermore, I suggest that the authors address the current controversies and the specific gap the review aims to fill.
Another suggestion is that more comparison between studies were included (in my opinion, there is a limited critical synthesis of the literature).
In lines 82-89 (1. Antioxidant Properties of TRP ), we can read that “The indole moiety is sensitive to the influence of a variety of reactive oxygen (ROS) and nitrogen (RNS) species, including hydrogen peroxide, hydroxyl radical, singlet oxygen, nitric oxide, and peroxynitrite anion. Therefore, TRP and some of its metabolites, such as KYNA and melatonin, are potent antioxidants. On the other hand, the storage of TRP-containing supplements, especially in solution form, must be under strict control because the indole part of TRP can be easily degraded into multiple toxic products by exposure to light, high or low pH, heat, and oxygen [8].” I suggest that the authors expand this section. Does this aminoacid have anti-inflammatory properties? Please comment.
I suggest including a separate topic (section) discussing the anti-inflammatory effects of TRP, melatonin, TRP-KYN, and other metabolites (as you did for antioxidant properties). The anti-inflammatory effects are distributed across sections (e.g., lines 384, 398, 459-460, 494, 569, 574), but I believe it would be useful to have a separate section.

Throughout the text, I see that anti-inflammatory aspects were discussed in specific diseases (such as inflammatory bowel disease; line 688: 9.4. TRP and Inflammatory Bowel Disease), but bringing these general aspects together would facilitate understanding.

In the section regarding Nutritional aspects of TRP ( Sources, Requirements, and Transport of Tryptophan in the Blood 116 and Through the Plasma Membrane):

I think the discussion of the superficial nutritional part is superficial. I suggest differentiating modern diets and addressing population variability.

I also suggest a more comprehensive integration with the gut microbiota and pathological states.
Throughout the topics, I feel there is a lack of integration with robust clinical studies, since many claims are based on preclinical studies. Do these studies exist?
I also think there is little discussion of translational aspects and clinical implications.
In the sections 10 (Tryptophan as a Dietary Supplement) and 11 (Therapeutic Possibilities of Targeting Individual Pathways of Tryptophan Metabolism), I would say that the discussion should be a little more cautious because it seems to me that the risk of supplementation is underestimated; it does not discuss in depth what the safe doses are, whether there are adverse effects, and whether there are drug interactions.

LIMITATIONS OF THE STUDY AND FUTURE PERSPECTIVES

I suggest including a topic on the limitations and Future Perspectives for this study.
Regarding Future Perspectives, the authors could mention some points, such as that:

futuere research should prioritize longitudinal and interventional studies to establish causal links between dietary tryptophan intake, microbiota-derived metabolites, and host metabolism and neuroimmune responses.
large randomized clinical trials are needed to define long-term efficacy and clinically relevant outcomes. Integrative multi-omics approaches will be essential to identify molecular signatures and predictive biomarkers of individual responsiveness.
standardization of analytical methods and establishment of reference ranges will improve reproducibility across studies.
investigations using advanced experimental models to elucidate receptor-mediated pathways, particularly those involving AhR and PXR.
finally, rigorous evaluation of the safety and dose–response of tryptophan supplementation will support the development of personalized nutritional and therapeutic strategies.

CONCLUSIONS

In my opinion, in this section, the authors write a relatively generic text. I think there could be a prioritization of knowledge gaps, and they could discuss more strategic points, such as clinical applications.

REFERENCES

This section is fine.

FINAL COMMENT

Dear authors, I wish you good luck with the publication process.

Comments on the Quality of English Language

Some minor errors should be corrected.

Author Response

RESPONSE TO THE COMMENTS OF THE Reviewer #3

Dear Reviewer,

thank you very much for constructive remarks and positive evaluation of the article. Here are the responses to your comments:

TITLE

Comment

The title of this manuscript is "Tryptophan and Its Metabolites in Health and Disease".

In my opinion, the title is too broad and does not make the manuscript type clear. I suggest changing it to another one. As an example: “Regulation of the Serotonin, Kynurenine and Indole Pathways of Tryptophan Metabolism: a review”.

Response

Thank you for the suggestion. I have thought about the title many times. I have changed the title as follows: “Serotonin, Kynurenine, and Indole Pathways of Tryptophan Metabolism in Humans in Health and Disease”.

ABSTRACT

Comment

In this section we can see that it is divided into 2 main parts (lines 11-26):

“The first parts of the article examine 11 sources, nutritional requirements, and three pathways of TRP catabolism. Physiologically, ~5% of dietary TRP is catabolized through the TRP-serotonin pathway in the brain and enterochromaffin cells of the gut, ~ 85% through the TRP-kynurenine pathway in the liver and immune cells, and ~10% by the TRP-indole pathway in gut microbiota. Alterations of individual TRP catabolism pathways in aging, alcoholism, inflammatory bowel disease, metabolic syndrome, renal insufficiency, liver cirrhosis, cancer, and nervous diseases, e.g., depression, Alzheimer's and Parkinson's diseases, multiple sclerosis, and schizophrenia, are examined in the central section. The final parts are devoted to the benefits and adverse effects of TRP supplementation, therapeutic use of various TRP metabolites, and pharmacological targeting of enzymes, transporters, and receptors involved in TRP catabolism. It is concluded that all pathways of TRP catabolism are altered in a broad spectrum of human illnesses, and further investigation is needed for a better understanding of TRP metabolism in disease pathogenesis. “

I miss the inclusion of the results of the search.
Please check for some minor errors.
I also think it does not explicitly state the criteria for selecting the literature, nor does it clarify the type of review.
The conclusions are generic, and I feel there is a lack of commentary regarding the identification of real gaps and concrete clinical implications.

Response

The results of the article are summarized in the statement “It is concluded that all pathways of TRP catabolism are altered in a broad spectrum of human illnesses, and further investigation is needed for a better understanding of TRP metabolism in disease pathogenesis.” A more detailed abstract is not possible due to the limited scope (250 words).
The article's English has been checked by a native speaker and rechecked using the Grammarly software tool.
Literature data were searched using Medline. No pre-defined criteria were used for literature selection. Both original and review articles have been used and referenced in the article.
The section “Summary and Conclusions” has been rewritten and supplemented with comments regarding the gaps in the present knowledge and clinical implications. Please see the revised version of the manuscripts.

KEYWORDS

Comment

The keywords used by the authors are: “amino acids; kynurenine; serotonin; indole; melatonin; kynurenic acid; quinolinic acid; metabolic syndrome." I suggest “kynurenine; serotonin; melatonin; kynurenic acid; quinolinic acid; neuroinflammation; metabolic disorders".

Response

The keywords have been edited as suggested. The words used in the title of the article were not used.

INTRODUCTION AND DISCUSSION SECTIONS:

Comments

In general, the Introduction is fine but long.
I miss the inclusion of Figures in this section.
Figures 3-5 are very nice, but I suggest improving Figure 1.
Still regarding Figures, be sure that all abbreviations used in them are defined in the legends.

Responses

In my opinion, the Introduction describes the main objectives of the article, and its length is K.
The Introduction includes Figure 1 showing the conceptual framework of the review.
Please see the new version of Figure 1.
In the new version of the manuscript, all abbreviations used in figures are defined in the legends.

Comment

Furthermore, I suggest that the authors address the current controversies and the specific gap the review aims to fill.

Response

There are controversial issues both in TRP supplementation and targeting its metabolic pathways. Even

the results of studies evaluating the effects of TRP administration on mood and behavior are controversial, as discussed in Section 10. Please note that some controversies and gaps in the current knowledge are also noted in “Summary and Conclusions”.

Comment

Another suggestion is that more comparison between studies were included (in my opinion, there is a limited critical synthesis of the literature).

Response

In my opinion, comparison between studies and examination of controversies and gaps of several possibilities targeting the pathways of TRP metabolism is beyond the scope of this review. Readers seeking more detailed information should look for articles focused on a specific topic, some of which are referenced.

Comment

In lines 82-89 (1. Antioxidant Properties of TRP ), we can read that “The indole moiety is sensitive to the influence of a variety of reactive oxygen (ROS) and nitrogen (RNS) species, including hydrogen peroxide, hydroxyl radical, singlet oxygen, nitric oxide, and peroxynitrite anion. Therefore, TRP and some of its metabolites, such as KYNA and melatonin, are potent antioxidants. On the other hand, the storage of TRP-containing supplements, especially in solution form, must be under strict control because the indole part of TRP can be easily degraded into multiple toxic products by exposure to light, high or low pH, heat, and oxygen [8].” I suggest that the authors expand this section. Does this aminoacid have anti-inflammatory properties? Please comment.

Response

The section 2.1 Antioxidant properties of TRP has been retitled and rewritten as follows:

2.1. TRP Sensitivity to Oxidative Stress and Antioxidative Properties of TRP

Comment

I suggest including a separate topic (section) discussing the anti-inflammatory effects of TRP, melatonin, TRP-KYN, and other metabolites (as you did for antioxidant properties). The anti-inflammatory effects are distributed across sections (e.g., lines 384, 398, 459-460, 494, 569, 574), but I believe it would be useful to have a separate section.

Response

Thank you for the suggestion. Section 2.2. "Anti-inflammatory Properties of TRP" has been added to the article as follows:

“Anti-inflammatory properties of TRP and many of its derivatives are mediated by the antioxidant properties of the indole ring and affinity to AHR (Section 2.3). Using an in vitro cell-based assay, TRP isolated from human milk exhibited nearly 99-fold higher oxygen radical absorption capacity associated with a decreased response to endotoxin-induced formation of proinflammatory cytokines [9]. In this context, it should be noted that the anti-inflammatory effects of TRP and its derivatives could have both beneficial, e.g., attenuation of inflammation, and detrimental, e.g., weakening the ability to destroy pathogens and tumor cells, effects.”

Comment

In the section regarding Nutritional aspects of TRP ( Sources, Requirements, and Transport of Tryptophan in the Blood and Through the Plasma Membrane):

I think the discussion of the superficial nutritional part is superficial. I suggest differentiating modern diets and addressing population variability.

Response

Accepted. The following paragraph has been added in section 3.2 Nutritional requirements:

“Dietary TRP requirements are higher in subjects consuming vitamin B₃-deficient diets to prevent pellagra (Section 9.1) and during pregnancy and in infants, ~10 mg/kg/day [21]. It has been shown that low TRP levels are associated with increased prevalence of depressive symptoms in pregnant women [22,23]. High TRP content in human milk plays a remarkable role in milk antioxidant potential and mitigates the formation of proinflammatory cytokines [9]. TRP levels in newborns are 2-4 times higher than in adults [24]. Hence, TRP intake influences maternal and fetal health outcomes.”

Comment

I also suggest a more comprehensive integration with the gut microbiota and pathological states.

Response

The following paragraph has been added to section 11.3. “Targeting the TRP-IND Pathway”:

“Optimal composition of the intestinal microbiome is pivotal in maintaining homeostasis and health. The dysbiosis is implicated in aging and mood disorders, and the pathogenesis of various diseases, including metabolic syndrome, IBD, immune and neurological diseases, and cancer [5,260].”

Comment

Throughout the topics, I feel there is a lack of integration with robust clinical studies, since many claims are based on preclinical studies. Do these studies exist?

Response

Larger clinical studies, some referenced in the article, have been published only about the possibilities of targeting the TRP-SER pathway, e.g., effects of TRP, melatonin, TRPH inhibitors, 5-hydroxytryptophan, and SSRI. Unfortunately, although there are several promising preclinical studies devoted to the therapeutic possibilities of influencing TRP-KYN and TRP-IND pathways, as demonstrated in this review, clinical studies are rare. The lack of clinical studies is highlighted in the "Summary and Conclusions".

Thank you for the suggested wording: “Large randomized clinical trials are needed to define long-term efficacy and clinically relevant outcomes”.

Comment

I also think there is little discussion of translational aspects and clinical implications.

Response

Current possibilities for translating knowledge about TRP metabolism into clinical applications have been summarized in Section “Therapeutic Possibilities of Targeting Individual Pathways of Tryptophan Metabolism”. I believe that I have highlighted all the important possibilities.

Comment

In the sections 10 (Tryptophan as a Dietary Supplement) and 11 (Therapeutic Possibilities of Targeting Individual Pathways of Tryptophan Metabolism), I would say that the discussion should be a little more cautious because it seems to me that the risk of supplementation is underestimated; it does not discuss in depth what the safe doses are, whether there are adverse effects, and whether there are drug interactions.

Response

In the revised version of the article is stated: “Administration of up to 5 g of TRP per day for three weeks had no adverse effect in healthy women; based on the 3-HKYN biomarker, a tolerable upper intake level (the highest average daily nutrient intake level that is likely to pose no risk of adverse health effects to most individuals in the general population) was suggested at 4.5 g/d for TRP [96]. Caution should be exercised when supplementing TRP with drugs that affect serotonin metabolism. Life-threatening “serotonin syndrome”, which includes neuromuscular abnormalities and autonomic hyperactivity, can occur under the condition of simultaneous administration of TRP and serotonin reuptake or MAO inhibitors [234]. In this context, it should be noted that the so-called eosinophilia-myalgia syndrome, which emerged at the end of the 1980s following the intake of dietary supplements containing TRP, was caused by bacterial contamination of the product [235].”

Comment

LIMITATIONS OF THE STUDY AND FUTURE PERSPECTIVES

I suggest including a topic on the limitations and Future Perspectives for this study.
Regarding Future Perspectives, the authors could mention some points, such as that:

futuere research should prioritize longitudinal and interventional studies to establish causal links between dietary tryptophan intake, microbiota-derived metabolites, and host metabolism and neuroimmune responses.
large randomized clinical trials are needed to define long-term efficacy and clinically relevant outcomes. Integrative multi-omics approaches will be essential to identify molecular signatures and predictive biomarkers of individual responsiveness.
standardization of analytical methods and establishment of reference ranges will improve reproducibility across studies.
investigations using advanced experimental models to elucidate receptor-mediated pathways, particularly those involving AhR and PXR.
finally, rigorous evaluation of the safety and dose–response of tryptophan supplementation will support the development of personalized nutritional and therapeutic strategie

Response

Thank you very much for your suggestions that have been included in the Summary and Conclusion section as follows:

Regarding the clinical approach, it can be suggested:

Clinical research should prioritize longitudinal and interventional studies to establish causal links between TRP intake, microbiota-derived metabolites, and host metabolism and neuroimmune responses.
Large randomized clinical trials are needed to define long-term efficacy and clinically relevant outcomes.
Rigorous evaluation of the safety and dose–response of TRP supplementation will support the development of personalized nutritional and therapeutic strategies.

Comment

CONCLUSIONS

In my opinion, in this section, the authors write a relatively generic text. I think there could be a prioritization of knowledge gaps, and they could discuss more strategic points, such as clinical applications.

Response

Done. Please see the revised version of the manuscript. Many thanks for your suggestions.

REFERENCES

Comment

This section is fine.

Response

Thank you for checking.

FINAL COMMENT

Dear authors, I wish you good luck with the publication process.

Response

Thank you very much.

Comment

Comments on the Quality of English Language

Some minor errors should be corrected.

Response

We have carefully checked the entire manuscript to improve its style and remove typos. The final version has been checked using the software tool Grammarly.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

This review comprehensively and systematically summarizes the roles of tryptophan (TRP) and its metabolites in health and disease, covering biochemical characteristics, metabolic pathways, alterations under pathological conditions, and therapeutic applications. The structure is clear and the references are abundant, providing a valuable perspective for research in this field. However, there are still some areas that can be improved. The following are the specific review comments:
1. The abstract does not clearly mention the core functional differences of key metabolites in each metabolic pathway. It is suggested to briefly distinguish the core biological effects of the TRP-serotonin, kynurenine, and indole pathways.
2. The downstream signaling pathways activated by AHR and PXR receptors only mention some genes and functions. It is suggested to supplement the cascade reaction mechanism of key signaling molecules.
3. Section 3.2 only provides the dietary requirements of TRP for adults and infants recommended by WHO, without explaining the differences in requirements for special populations (such as pregnant women, the elderly, and patients with chronic diseases).
4. The three metabolic pathways are respectively described throughout the text, but the interaction between the pathways (such as the mutual regulation of metabolic products and the common regulation of physiological functions) is mentioned less.
5. In Section 9.6 on neurological diseases, the specific differences in TRP metabolic disorders in different neurological diseases are not clearly stated. It is suggested to combine the pathological features of specific diseases and refine the targeted mechanisms of changes in metabolic products.
6. In Section 10.1, only a few side effects of TRP supplements are mentioned, and the potential harm to patients with liver and kidney dysfunction from long-term high-dose supplementation is not fully discussed.
7. The references for the clinical research progress of some therapeutic targets need to be updated.
8. It is suggested to supplement the association labels between metabolic products and physiological functions/diseases to enhance the readability and information completeness of the charts.

Author Response

RESPONSE TO THE COMMENTS OF THE Reviewer #4

Dear Reviewer,

thank you very much for constructive remarks and positive evaluation of the article. Here are the responses to your comments:

Comment 1

The abstract does not clearly mention the core functional differences of key metabolites in each metabolic pathway. It is suggested to briefly distinguish the core biological effects of the TRP-serotonin, kynurenine, and indole pathways.

Response

In the new version of the abstract is stated:

“Physiologically, ~5% of dietary TRP is catabolized through the pathway forming serotonin and melatonin in the brain and enterochromaffin cells of the gut, ~85% through the pathway resulting in the formation of nicotinamide nucleotides and kynurenine and its derivatives in the liver and immune cells, and ~10% in gut microbiota to indole derivatives.”

Including more detailed information is not possible because the length of the abstract is limited to 250 words.

Comment 2

The downstream signaling pathways activated by AHR and PXR receptors only mention some genes and functions. It is suggested to supplement the cascade reaction mechanism of key signaling molecules.

Response

The section “ TRP and AHR” has been rewritten. In the revised version is stated:

“The key AHR ligands include several exogenous and endogenous substances with an aryl chemical structure, such as flavonoids, dibenzofurans, benzopyrene, tetrapyrroles, and arachidonic acid metabolites. TRP, and some TRP metabolites, including indole, KYN, KYNA, indole-3-propionic acid (IPA), indole-3-acetic acid (IAA), skatole, and tryptamine, act as agonists of AHR [10-13]. Upon ligand binding, the AHR is translocated into the nucleus and induces transcription of target genes, primarily those encoding enzymes of the cytochrome P450 system (Cyp1a1, Cyp1a2, and Cyp1b1), the AHR repressor, and genes involved in apoptosis, cell proliferation, and differentiation. Hence, AHR plays a remarkable role in immune function, e.g., T-cell differentiation and cytokine formation, maintenance of the mucosal surface in the gut, and carcinogenesis [10,11].”

Comment 3

Section 3.2 only provides the dietary requirements of TRP for adults and infants recommended by WHO, without explaining the differences in requirements for special populations (such as pregnant women, the elderly, and patients with chronic diseases).

Response

The following paragraph has been added in section 3.2.: “Dietary TRP requirements are higher in subjects consuming vitamin B₃-deficient diets to prevent pellagra (Section 9.1) and during pregnancy and in infants, ~10 mg/kg/day [21]. It has been shown that low TRP levels are associated with increased prevalence of depressive symptoms in pregnant women [22,23]. High TRP content in human milk plays a remarkable role in milk antioxidant potential and mitigates the formation of proinflammatory cytokines [9]. TRP levels in newborns are 2-4 times higher than in adults [24]. Hence, TRP intake influences maternal and fetal health outcomes.”

The issue of TRP needs in old age is addressed in section 9.2., TRP as a dietary supplement in illness is discussed in section 10.

Comment 4

The three metabolic pathways are respectively described throughout the text, but the interaction between the pathways (such as the mutual regulation of metabolic products and the common regulation of physiological functions) is mentioned less.

Response

I agree. However, this could be a topic for a separate article. Please note the following statement in the Conclusion section: “The discrepancies and gaps also exist in the current understanding of the relationships and interactions among the three TRP metabolic pathways. For instance, TRP and most derivatives of its three metabolic pathways act as AHR ligands, and several studies have shown that changes in flux through the TRP-KYN pathway affect brain serotonin production.

Comment 5

In Section 9.6 on neurological diseases, the specific differences in TRP metabolic disorders in different neurological diseases are not clearly stated. It is suggested to combine the pathological features of specific diseases and refine the targeted mechanisms of changes in metabolic products.

Response

In the revised manuscript, it is stated: "There are dozens of experimental and clinical studies investigating TRP metabolism in nervous system diseases, which reveal distinct patterns of TRP metabolism dysregulation across various brain regions. Unfortunately, their analysis goes beyond the scope of this article. I will highlight common pathogenic features, particularly the role of neuroinflammation and alterations in the TRP-KYN pathway. Recent articles dedicated to TRP metabolism in specific disorders are cited.”

Comment 6

In Section 10.1, only a few side effects of TRP supplements are mentioned, and the potential harm to patients with liver and kidney dysfunction from long-term high-dose supplementation is not fully discussed.

Response

Accepted. The following explanation has been added in section 10.1:

“TRP supplementation can be detrimental in subjects with liver cirrhosis and renal injury. In liver cirrhosis, TRP supplementation could result in a marked increase in the TRP to BCAA ratio, resulting in anorexia and poor nutritional status due to increased serotonin formation and impaired BCAA transport into the brain and muscles. [29,35,36]. It is a consensus that several TRP metabolites, such as KYN, 5-HIAA, and indoxyl sulfate, exacerbate kidney damage by activating the AHR signaling pathway (Section 9.7). It can therefore be assumed that TRP supplements will increase flux through the TRP-KYN and TRP-IND pathways, increase the production of uremic toxins, and worsen the disease.”

Comment 7

The references for the clinical research progress of some therapeutic targets need to be updated.

Response

Done. Here are examples.

Hui Y, Zhao J, Yu Z, Wang Y, Qin Y, Zhang Y, Xing Y, Han M, Wang A, Guo S, Yuan J, Zhao Y, Ning X, Sun S. The Role of Tryptophan Metabolism in the Occurrence and Progression of Acute and Chronic Kidney Diseases. Mol Nutr Food Res. 2023;67(21):e2300218.
Nayak BN, Buttar HS. Evaluation of the antioxidant properties of tryptophan and its metabolites in vitro assay. J Complement Integ Med 2016; 13: 129-136.
Prescott S, Billeci N, Gotcher M, Patel S, Almon A, Morgan H, Abukhalaf D, Groer M. Tryptophan as a biomarker of pregnancy-related immune expression and modulation: an integrative review. Front Reprod Health. 2025;6:1453714.
Miyake Y, Tanaka K, Okubo H, Sasaki S, Arakawa M. Tryptophan intake is related to a lower prevalence of depressive symptoms during pregnancy in Japan: baseline data from the Kyushu Okinawa Maternal and Child Health Study. Eur J Nutr. 2022;61(8):4215-4222.
Miyamoto K, Sujino T, Kanai T. The tryptophan metabolic pathway of the microbiome and host cells in health and disease. Int Immunol. 2024;36(12):601-16.

Comment 8

It is suggested to supplement the association labels between metabolic products and physiological functions/diseases to enhance the readability and information completeness of the charts.

Response

I have also been thinking about this possibility. Unfortunately, all the images are large and provide a lot of data, and further editing would not be appropriate. I hope that this is partially fulfilled by the graphical abstract that was added to the manuscript.