Review Reports - Essential Trace Elements Zinc, Iron, Copper and Attention-Deficit/Hyperactivity Disorder in Children and Adolescents: A Systematic Review and Meta-Analysis of Case–Control Studies

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,
The manuscript presents several substantial concerns that significantly affect its scientific validity. In particular, there appear to be inconsistencies in the timeline of cited literature (including references to studies published after the reported submission period), as well as a misalignment between the title and the actual focus of the research. Furthermore, there are notable methodological issues, especially regarding the assessment of study quality. Overall, the manuscript would benefit from a thorough revision, as the current structure raises concerns about the robustness of the data and the internal review process.
1. The title indicates an analysis of Zinc (Zn), Selenium (Se), and Copper (Cu). However, Selenium is not reported in the Abstract or Results sections, where it appears to be replaced by Iron (Fe). This inconsistency creates a misalignment between the title and the reported findings, potentially leading to confusion for the reader. Clarification and alignment between the title and the manuscript content are therefore recommended.
2. The Abstract states the inclusion of 43 studies , while the Results section, Figure 1 and Table 1 clearly indicate 45 studies.
3. The authors report the use of the Newcastle–Ottawa Scale (NOS) for cohort studies. However, the inclusion criteria specify that only case–control studies were considered. Applying a cohort-specific version of the NOS to case–control studies may not be methodologically appropriate and could affect the validity of the quality assessment. Clarification on this point, and alignment between the study design and the selected assessment tool, would be advisable. If the cohort-specific version of the NOS had indeed been applied, the quality scores reported in Table 1 would not be technically compatible with case–control studies. This discrepancy raises concerns regarding how the quality assessment was conducted. It would therefore be important for the authors to clarify the methodology used and ensure that the reported scores are consistent with an appropriate and correctly applied assessment tool.
4. Regarding Zinc (Zn) levels, an I² value of 98.8% is reported. Such a high level of heterogeneity indicates that the variability across studies is unlikely to be attributable to chance alone, but rather reflects substantial differences in study populations and/or methodologies. In the presence of extreme heterogeneity, the calculation and interpretation of a pooled effect size (SMD) should be approached with caution, as its clinical interpretability may be limited. Moreover, the manuscript does not appear to include an adequate sensitivity analysis or meta-regression to explore potential sources of heterogeneity. Addressing these aspects would be important to strengthen the robustness and interpretability of the conclusions.
5. In its current form, Table 1 has limited scientific utility. Several formatting issues are present, including overlapping numerical values, which reduce clarity and readability. In addition, the absence of clearly reported standard deviations for key studies (e.g., Viktorinova et al., 2016) limits the ability to properly interpret the data and hinders reproducibility and critical appraisal of the results. A revision of the table to ensure clarity and complete reporting of statistical parameters is therefore recommended.
6. The Discussion section lacks a sufficiently critical interpretation of the observed geographical differences. The manuscript notes lower levels in developing countries but does not adequately explore potential contributing factors, such as dietary patterns, environmental exposures (e.g., heavy metal contamination), or variability in diagnostic practices across settings. As a result, the analysis remains largely descriptive and does not fully develop an interpretative framework that would enhance the scientific value of the findings. A more in-depth contextual discussion would strengthen this section.

Author Response

The title indicates an analysis of Zinc (Zn), Selenium (Se), and Copper (Cu). However, Selenium is not reported in the Abstract or Results sections, where it appears to be replaced by Iron (Fe). This inconsistency creates a misalignment between the title and the reported findings, potentially leading to confusion for the reader. Clarification and alignment between the title and the manuscript content are therefore recommended.
Response:Thank you for your reminder. We have carefully verified and confirmed the inconsistency between the title and content in the original text. We have made the following revisions to the manuscript:

Title Updated: the title has been revised to “Essential Trace Elements Zinc, Iron, Copper and Attention-Deficit/Hyperactivity Disorder in Children and Adolescents: A Systematic Review and Meta-Analysis of Case-Control Studies”.

Full-text Consistency Check: We have carefully reviewed the entire manuscript, including the abstract, results, tables, figures, and keywords, to ensure that all references to the analyzed trace elements (Zn, Fe, Cu) are now fully aligned with the revised title. Any residual mention of Selenium (Se) has been removed.

The Abstract states the inclusion of 43 studies , while the Results section, Figure 1 and Table 1 clearly indicate 45 studies.
Response:Thank you for pointing out the inconsistency between the number of studies reported in the Abstract (43 studies) and the Results section, Figure 1, and Table 1 (45 studies).

Explanation and Correction: We have conducted the latest screening and confirmation. During the final stage of our systematic review process, we identified and included three additional eligible studies through checking the reference lists of relevant articles. Therefore, the total number of studies included in the final analysis is indeed 46. We have taken the following actions to correct this discrepancy: Abstract Updated: The Abstract has been revised, and the statement now correctly reads “46 studies.”. Full-Text Verification: We have thoroughly checked the entire manuscript to ensure that all mentions of the total number of included studies—in the main text, figure captions, and table notes—are now consistently updated to “46 studies,” aligning perfectly with Figure 1 (PRISMA flow diagram) and Table 1 (characteristics of included studies).

The authors report the use of the Newcastle–Ottawa Scale (NOS) for cohort studies. However, the inclusion criteria specify that only case–control studies were considered. Applying a cohort-specific version of the NOS to case–control studies may not be methodologically appropriate and could affect the validity of the quality assessment. Clarification on this point, and alignment between the study design and the selected assessment tool, would be advisable. If the cohort-specific version of the NOS had indeed been applied, the quality scores reported in Table 1 would not be technically compatible with case–control studies. This discrepancy raises concerns regarding how the quality assessment was conducted. It would therefore be important for the authors to clarify the methodology used and ensure that the reported scores are consistent with an appropriate and correctly applied assessment tool.
Response:Thank you for pointing out this issue. As our systematic review included “case-control studies”, applying the Newcastle-Ottawa Scale (NOS) version for cohort studies would be methodologically inappropriate and could compromise the validity of the quality assessment. We have carefully re-examined our methodology. In the revised manuscript, we have clarified and corrected this point in the Methods section to state clearly: “The Newcastle-Ottawa Scale (NOS) adapted for case-control studies was used to assess the quality of the included studies.”. Furthermore, we have re-evaluated all included studies using the correct NOS version for case-control studies.

The quality scores reported in Table 1 have been updated accordingly to reflect this proper application. Also, we have revised the Text as “ All studies received quality scores above 6, with an average score of 6.9. ” in the Results Section.

Regarding Zinc (Zn) levels, an I² value of 98.8% is reported. Such a high level of heterogeneity indicates that the variability across studies is unlikely to be attributable to chance alone, but rather reflects substantial differences in study populations and/or methodologies. In the presence of extreme heterogeneity, the calculation and interpretation of a pooled effect size (SMD) should be approached with caution, as its clinical interpretability may be limited. Moreover, the manuscript does not appear to include an adequate sensitivity analysis or meta-regression to explore potential sources of heterogeneity. Addressing these aspects would be important to strengthen the robustness and interpretability of the conclusions.

Response: Thank you for bringing up this crucial methodological viewpoint, namely the extremely high heterogeneity observed in our meta-analysis of differences in Zn, Fe, and Cu levels. We completely agree with your evaluation. This apparent heterogeneity does indicate substantial differences beyond random opportunities, which may stem from study design (sample size, demographic factors such as age of children and adolescents), laboratory methods (type of testing), or changes in clinical environment (such as dietary characteristics, regional environmental exposure). Therefore, the combined effect size (SMD) should be interpreted with great caution, as its clinical applicability will be limited if the source of heterogeneity is not thoroughly investigated.

In response, we have made the following revisions to the manuscript:

Firstly, we added heterogeneity testing by plotting Galbraith plots to examine heterogeneity patterns. The results showed heterogeneity in the study, but for Zn, 35 out of 37 data points were within the confidence interval; For Cu, 22 data from 24 studies are within the confidence interval; For Fe, 35 out of 32 data items are within the confidence interval. (Figure S3)

Secondly, we explore potential sources of heterogeneity through meta regression based on existing available data, including publication year, sample size, biological sample type, and region. We found that the meta regression results suggest that these factors are not the main causes of high heterogeneity. (Table S4)

Thirdly, we conducted subgroup analysis on important factors, including sample size, economic level, sample size, and age. The research results were consistent with the overall results, indicating the stability of our findings. (Table 2)

Fourth, to assess the robustness of the findings against potential publication bias, a trim-and-fill analysis was conducted. The adjusted results indicated only minor changes in the effect sizes for Zn (SMD = -1.67; 95% CI: -2.17 to -1.16), Fe (SMD = -1.13; 95% CI: -1.77 to -0.48), while ferritin showed no significant change (SMD = -0.54; 95% CI: -1.00 to -0.08), supporting the stability of the pooled estimates.

However, we acknowledge the uncertainty of heterogeneity sources and clarify that the overall differences in this study are preliminary and have limited clinical explanatory significance. We have revised the Conclusions as “In summary, this meta-analysis indicates that children and adolescents with ADHD exhibit significantly lower Zn, Fe, and ferritin levels than healthy control group. This difference is more pronounced among populations in developing countries. When the research subjects are limited to children under 12 years old, the differences in Zn are more pronounced. Reduced serum concentrations of these trace elements may be associated with ADHD. However, considering the relatively high heterogeneity of the pooled results in this study, the clinical value of the research findings needs to be carefully evaluated. Future well‑controlled clinical trials are warranted to evaluate the efficacy of mineral supplementation and to identify potential subgroups of patients who may benefit.”.

In its current form, Table 1 has limited scientific utility. Several formatting issues are present, including overlapping numerical values, which reduce clarity and readability. In addition, the absence of clearly reported standard deviations for key studies (e.g., Viktorinova et al., 2016) limits the ability to properly interpret the data and hinders reproducibility and critical appraisal of the results. A revision of the table to ensure clarity and complete reporting of statistical parameters is therefore recommended.
Response: We have carefully checked and corrected all potential formatting errors in the table to meet the publication requirements of the journal. Please note that due to the lack of reported standard deviations (SD) in some studies, we were unable to include their data in the meta-analysis. This has been clarified in the inclusion and exclusion criteria section. It is worth noting that the direction of effect in one excluded study (Viktorinova et al., 2016)—which compared zinc levels between children with ADHD and controls—is consistent with the overall findings of our meta-analysis. Furthermore, we have acknowledged in the limitations section that our analysis only included studies reporting means and standard deviations, which may have led to the exclusion of otherwise relevant studies that did not provide complete statistical data.
The Discussion section lacks a sufficiently critical interpretation of the observed geographical differences. The manuscript notes lower levels in developing countries but does not adequately explore potential contributing factors, such as dietary patterns, environmental exposures (e.g., heavy metal contamination), or variability in diagnostic practices across settings. As a result, the analysis remains largely descriptive and does not fully develop an interpretative framework that would enhance the scientific value of the findings. A more in-depth contextual discussion would strengthen this section.

Response: Thank you for your valuable feedback. The issue you pointed out about the insufficient interpretation of geographical differences in the discussion section is very relevant, and we fully agree that the current discussion lacks depth. Based on your suggestions, we have significantly expanded and deepened this section in the revised draft, with the following specific modifications:

We have revised the Discussion section as follows: “Subgroup analysis by sample size indicated that larger studies (n > 200) demonstrated more marked differences in Zn and Fe levels, reinforcing the main findings and implying that earlier smaller-scale studies may have underestimated the association between these micronutrients and ADHD due to limited statistical power. Further stratification by economic development level showed that Zn and Fe differences were more pronounced in developing countries, with Fe deficiency reaching statistical significance only in this subgroup. Potential factors contributing to this pattern in developing regions may include less varied diets, inadequate nutrient intake, or environmental exposures[42, 43]. The prevalence of Zn deficiency in low - and middle-income countries is worrying. Globally, Africa has the highest rate of Zn deficiency (23.9%)[44], and accounts for 18 of the top 20 countries most severely affected by multiple micronutrient deficiencies, including Zn and Fe[45]. However, effective strategies to improve Zn nutrition status, such as Zn strengthening programs, micronutrient powder supplementation, dietary diversification, and meat intake promotion, still lack sufficient implementation experience and effectiveness evaluation evidence[46, 47]. Additionally, in many developing countries and regions, heavy metal pollution tends to be more widespread and severe[48]. The combined effects of long-term, low-dose exposure to heavy metals such as Pb and Cd, coupled with insufficient levels of essential nutrients, are progressively causing subclinical neurological damage[49]. This emerging threat is increasingly recognized as a serious public health challenge in developing nations. These findings also suggest that monitoring and timely supplementation of Zn/Fe could represent a cost-effective intervention in resource-limited settings, particularly in developing countries[50].

Admittedly, this study only evaluated differences in trace element levels between children/adolescents with ADHD and controls using a case‑control design, which cannot establish causality. Nonetheless, the results imply that Fe supplementation could be a useful alternative therapy for ADHD patients with Fe deficiency, especially those presenting predominantly inattentive symptoms[51]. A systematic review of randomized controlled trials indicated that dietary supplementation with Zn and Fe was associated with improvement in ADHD severity at the end of treatment compared with placebo, although effect sizes were generally modest and mostly limited to specific symptoms or assessment tools[29].

We also observed lower Cu levels in children with ADHD relative to controls, though the pooled effect size did not attain statistical significance. Cu is an essential trace element that acts as a cofactor for multiple oxidoreductases and is involved in Fe metabolism, antioxidant defense, neuropeptide synthesis, and immune regulation. However, reference ranges for Cu remain uncertain, especially given that excess Cu can promote oxidative stress and cytotoxicity, primarily harming the liver and nervous system, and may disturb the balance of other essential minerals[52]. Subgroup analyses stratified by region and sample type revealed that in developing countries, Cu levels were significantly lower in children with ADHD than in typically developing peers; in blood specimens, the difference in Cu levels appeared more pronounced, yet it still lacked statistical significance.

Sensitivity analyses confirmed the robustness of the main findings. Trim-and-fill analysis further validated the results for Zn and Fe, while revealing a statistically significant reduction in Cu levels among children and adolescents with ADHD compared to controls.”.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors of the meta-analysis titled "Essential Trace Elements Zn, Se, and Cu and Attention-Deficit/Hyperactivity Disorder in Children and Adolescents: A Systematic Review and Meta-Analysis" aimed to summarize existing evidence on serum levels of zinc, iron, and copper in children and adolescents with ADHD.

The manuscript analyzed 43 studies published in English and Chinese and found that zinc and iron levels are significantly lower in children with ADHD, and not significantly lower level in copper. The manuscript is well organized, presenting in the Introduction the issue regarding the trace elements link with ADHD and the need for such a meta-analysis. The results did not establish causality, but they provided a snapshot of the serum levels of these trace elements. More studies are needed to try to find an explanation for the possible role in the ADHD pathogenesis.

The manuscript needs some improvements:

In the title, the authors wringly wrote "Se," from selenium, instead of "iron". Also, they used abbreviated words incorrectly in the title.
In the methods section, the authors mentioned Selenium again, but in the main text, there is no reference to Selenium. If they aimed to include also this trace element, but there were no results, that should be mentioned and discussed. Or maybe it's an error regarding Fe and Se.
Some studies reported ferritin rather than Fe; this should also be discussed and may influence the results.
Table 2 should be placed before the sections presenting results for Zn, Fe, and Cu, as it provides general data on all three trace elements.
Editing must be verified for using the abbreviations

Author Response

Response: Thank you very much for your careful review and valuable feedback on our manuscript. We are pleased to see that you have given a positive evaluation of the overall structure, logical clarity of the research, and our summary of the existing evidence. We have made revisions based on the opinions of three reviewers in order to meet the publication requirements.

The manuscript needs some improvements:

In the title, the authors wringly wrote "Se," from selenium, instead of "iron". Also, they used abbreviated words incorrectly in the title.

Response: Thank you for your valuable feedback. We have noted the issues you pointed out regarding the manuscript title, specifically the incorrect use of “Se” (which refers to selenium) instead of “iron,” as well as the improper use of abbreviations. We have carefully examined the entire text and made corrections to it.

In the methods section, the authors mentioned Selenium again, but in the main text, there is no reference to Selenium. If they aimed to include also this trace element, but there were no results, that should be mentioned and discussed. Or maybe it's an error regarding Fe and Se.

Response: Thank you for pointing this out. You are correct that the mention of selenium (Se) in the Methods section was inconsistent with the content of the manuscript. In our initial design, we had planned to include Se as one of the trace elements of interest. However, after applying the inclusion and exclusion criteria, we found that the number of eligible studies on Se was insufficient for meaningful analysis. Therefore, we ultimately limited our study to the three trace elements: Fe (iron), Cu (copper), and Zn (zinc). We have now revised the manuscript accordingly and removed all references to Se. Thank you for your helpful suggestion.

Some studies reported ferritin rather than Fe; this should also be discussed and may influence the results.

Response: Thank you for your suggestion. We further analyzed the iron indicators (Fe level, ferritin level) separately. The results showed that compared with the healthy control group, children with ADHD had significantly reduced serum iron and ferritin levels (as shown in the figure below).

Meanwhile, the discussion on this point has been added as “In the subgroup analysis of Fe metabolism indicators, we conducted subgroup analysis based on indicator types (serum Fe and ferritin). The results showed that the serum Fe and ferritin levels in children with ADHD were significantly lower than those in healthy control children, suggesting that there may be a common situation of decreased Fe reserves and insufficient circulating Fe supply in ADHD children and adolescents. Fe deficiency may participate in the pathophysiology of ADHD by disrupting dopamine synthesis, transport, and receptor function, as well as affecting neural activity in key brain regions such as the basal ganglia[39, 40].” in the Discussion Section.

Figure 3. Forest plot illustrating the association between Fe levels and ADHD in children and adolescents, presented as the mean difference between case and control groups. Diamonds indicate pooled estimates derived from random-effects meta-analysis. Abbreviation: SMD, standardized mean difference.

Figure 4. Forest plot illustrating the association between ferritin levels and ADHD in children and adolescents, presented as the mean difference between case and control groups. Diamonds indicate pooled estimates derived from random-effects meta-analysis. Abbreviation: SMD, standardized mean difference.

Table 2 should be placed before the sections presenting results for Zn, Fe, and Cu, as it provides general data on all three trace elements.

Response: Thank you for this helpful suggestion. We agree that Table 2, which provides the general data on all three trace elements (Zn, Fe, and Cu), should logically precede the specific result sections for each element. This arrangement will improve the flow and clarity of the manuscript. We have adjusted the order accordingly. Thank you for your attentive review.

Editing must be verified for using the abbreviations.

Response:Thank you for highlighting this point. We have carefully reviewed the manuscript to ensure that all abbreviations (including those for Fe, Cu, and Zn) are properly introduced upon first use and are applied consistently throughout the text. We appreciate your attention to detail in improving the clarity and professionalism of our work.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This is an interesting systematic review and meta-analysis with adequate novelty, which aims to ebaluate the potential associations between zinc (Zn), iron (Fe), copper (Cu), and Attention-Deficit/Hyperactivity Disorder (ADHD) in children and adolescents. However, severa points should be addressed.

Since this systematic review includes only case-control study, this should be mentioned in the title of the paper as "............ A Systematic Review and Meta-Analysis of Case-Control Studies".
A bit more information concerning the background of the research topic of the study should be included in the abstract.
The authors should emphasize in the abstract that this is a systematic review.
In the abstract the sentence "Meta-analysis was performed using Stata 15.0." could be avoided.
The introduction section is quite short. The authors should add more detailed information concerning the topics in the first two paragraphs.
A distinct paragraph reporting the data of randomized clinical trials should be presented in the introduction section.
The authors report in figure 1 that 1,659 records removed after scanning abstracts and titles. To this point, the authors should be more analytic concernign these exclusion criteria due to the large number of studies that they did not take into consideration.
The authors should mention in the results of their study the country of the included studies. A large amount of them has been performed in China. This should also be mention in the limitations of the study at the end of the discussion section.
Most of the included studies has a small amount of cases and controls. This is another limitation that should be reported in the discussion section of the study.
Are ADHD diagnosis criteria the same for each study included in meta-analysis from 1996 to 2025? This issue should also be discussed.
It is reasonable that the age range of the included studies has a high heterogeneity. This is also a crucial issue that should be discussed.
Please be more detailed concerning the statement "Restricting the analysis to participants aged 12 years or younger yielded effect estimates consistent in direction and magnitude with the primary findings, though heterogeneity persisted." reported in the discussion section.
The authors should add a bit more information in the conclusion section.

Comments on the Quality of English Language

English language editing is recommended.

Author Response

Response: Thank you very much for reviewing our manuscript and acknowledging the novelty and value of this research. We highly value the valuable feedback you have provided. You pointed out that there are still several areas in the manuscript that need improvement, which is crucial for enhancing the scientific rigor and depth of the research. We have carefully and comprehensively revised the manuscript based on your and the other two reviewers' specific suggestions.

Since this systematic review includes only case-control study, this should be mentioned in the title of the paper as "............ A Systematic Review and Meta-Analysis of Case-Control Studies".

Response: Thank you for this important clarification. We have revised the title of the manuscript as suggested to accurately reflect the study design, which is limited to case-control studies. The updated title is now:“Essential Trace Elements Zinc, Iron, Copper and Attention-Deficit/Hyperactivity Disorder in Children and Adolescents: A Systematic Review and Meta-Analysis of Case-Control Studies.”

A bit more information concerning the background of the research topic of the study should be included in the abstract.

Response: Thank you for your constructive suggestion to provide more context regarding the research background in the abstract. We have now incorporated additional information to clarify the rationale for examining zinc, iron, and copper in ADHD. The revised abstract section is as follows: “Essential trace elements such as zinc (Zn), iron (Fe), and copper (Cu) play a critical role in neurodevelopment, influencing key processes like neurotransmitter regulation and neuronal signaling. To synthesize the existing evidence, we conducted a systematic review and meta-analysis of case-control studies investigating the associations between these trace elements and Attention-Deficit/Hyperactivity Disorder (ADHD) in children and adolescents. ”.

The authors should emphasize in the abstract that this is a systematic review.

Response: Thank you for your valuable suggestion. We agree that it is important to clearly state the nature of our study in the abstract. As recommended, we have revised the abstract to emphasize at the outset that this is a systematic review and meta-analysis. The updated opening sentences now read: “To synthesize the existing evidence, we conducted a systematic review and meta-analysis of case-control studies investigating the associations between these trace elements and Attention-Deficit/Hyperactivity Disorder (ADHD) in children and adolescents. A comprehensive literature search was performed up to March 2026 across multiple databases, including Web of Science, PubMed, and the Chinese National Knowledge Infrastructure. A total of 46 studies involving 5,515 ADHD cases and 8,166 controls were included. ”.

In the abstract the sentence "Meta-analysis was performed using Stata 15.0." could be avoided.

Response: Thank you for your suggestion. We have removed the sentence “Meta-analysis was performed using Stata 15.0.” from the abstract to keep it more concise and focused on key findings and methodological overview.

The introduction section is quite short. The authors should add more detailed information concerning the topics in the first two paragraphs.

Response: Thank you for your valuable comments. In accordance with your suggestions, we have significantly expanded the first two paragraphs of the Introduction section. The revised paragraphs now incorporate the following key information to provide a more comprehensive background: “Attention-Deficit/Hyperactivity Disorder (ADHD) is a neurodevelopmental disorder primarily characterized by developmentally inappropriate, persistent, and impairing levels of inattention, hyperactivity, and impulsivity, which stem from widespread yet subtle alterations in gene expression across multiple brain regions that collectively disrupt typical neural functioning[1, 2]. It is typically diagnosed in childhood and often persists into adulthood. Recent data report ADHD prevalence rates of 7.6% among children aged 3-12 years and 5.6% among adolescents aged 12-18 years[3]. Between 2016 and 2019, nearly one in ten (9.8%) children in the United States received an ADHD diagnosis based on parent-reported data[4]. The prevalence of ADHD among Chinese children and adolescents in China is estimated to be 6.3%[5]. Children and adolescents with ADHD often experience psychological and social difficulties across home, school, and community settings[6], which may increase their risk of entering a negative developmental trajectory.

There is ample evidence for the heritability of ADHD, but environmental factors, including their direct effects and interactions with genes, are crucial in determining the severity and clinical characteristics of symptoms, which in turn affect diagnosis and treatment pathways[7, 8]. Given the essential role of micronutrients in neurodevelopment, nutritional impairment is closely associated with ADHD[9]. Trace elements such as zinc (Zn), copper (Cu), and iron (Fe) play essential roles in normal brain development, neurotransmitter synthesis and catabolism, cellular metabolism, and dopaminergic pathways[10, 11]. Altered levels of these elements may disrupt the regulation of dopaminergic and noradrenergic systems, potentially contributing to the pathogenesis of ADHD[12].”.

A distinct paragraph reporting the data of randomized clinical trials should be presented in the introduction section.

Response: Thank you for your helpful comment. Based on your suggestion, we have revised the introduction by adding a clear and focused paragraph summarizing the data from randomized clinical trials (RCTs). The updated text in the introduction section now reads as follows: “Considering the various side effects of conventional pharmacotherapy for ADHD[27], researchers have begun to explore novel therapeutic approaches, particularly the investigation of the potential benefits of mineral supplementation [28]. Recent systematic reviews indicated that the specific role of dietary nutrients, including Zn and Fe, in treating ADHD remains controversial, although evidence supporting Zn supplementation appears to be the most robust [29, 30]. Particularly in regions with inadequate diet and nutritional deficiencies, early exposure to micronutrient supplementation may yield more pronounced benefits on children’s behavioral outcomes[31].”. We believe this addition provides a clearer overview of the relevant RCT evidence and strengthens the rationale for our study.

The authors report in figure 1 that 1,659 records removed after scanning abstracts and titles. To this point, the authors should be more analytic concernign these exclusion criteria due to the large number of studies that they did not take into consideration.

Response: Figure 1 shows that after screening through titles and abstracts, 1,659 records were excluded. Given the significant number of studies excluded at this stage, we have indicated the reasons for exclusion in the flowchart: including systematic reviews, clinical guidelines, commentary articles, meta-analyses, case reports, animal studies, studies involving participants who were not children or adolescents, and non-case-control studies.

Figure 1. Flow chart for article selection.

The authors should mention in the results of their study the country of the included studies. A large amount of them has been performed in China. This should also be mention in the limitations of the study at the end of the discussion section.

Response: Thank you for your thoughtful suggestion. We have revised the Limitations Section as follows: “Third, it should be noted that the included studies predominantly involved populations from Asia and Europe, with a significant proportion (19 studies) conducted in China. Additionally, many of these investigations utilized relatively small sample sizes. These factors may limit the statistical power and generalizability of the findings. Therefore, further validation through larger-scale studies and the inclusion of populations from more diverse geographical regions is warranted.”.

Most of the included studies has a small amount of cases and controls. This is another limitation that should be reported in the discussion section of the study.

Response: Thank you for pointing out this important limitation. We acknowledge that the relatively small sample sizes in many of the included studies may impact the statistical power and generalizability of our pooled findings. This limitation has been duly noted and will be included in the discussion section to provide a more balanced interpretation of our results and to underscore the need for larger-scale investigations in the future.

We have revised the Limitations Section as follows: “Additionally, many of these investigations utilized relatively small sample sizes. These factors may limit the statistical power and generalizability of the findings. Therefore, further validation through larger-scale studies and the inclusion of populations from more diverse geographical regions is warranted. ”.

Are ADHD diagnosis criteria the same for each study included in meta-analysis from 1996 to 2025? This issue should also be discussed.

Response: Your point regarding the temporal variation in ADHD diagnostic criteria is a significant consideration in this meta-analysis. Although all included studies relied on clinician diagnosis and primarily applied international standards (e.g., the DSM and ICD series), the evolution of diagnostic criteria from 1996 to 2025—such as the transition from DSM-IV to DSM-5—may have introduced diagnostic heterogeneity across studies. This could potentially affect the comparability of participant populations and the interpretation of pooled results. We will address this as a potential limitation in the discussion section, emphasizing the need for future research to carefully account for the impact of changes in diagnostic criteria when analyzing long-term trends.

We have revised the Limitations Section as follows: “Fourth, another limitation of this study is that the included literature spans a long period (1996–2025), during which the diagnostic criteria for ADHD were not uniform. Notably, major revisions to ADHD diagnostic standards (e.g., from DSM-3 to DSM-5) were implemented in this period, which may have led to heterogeneity in how participants were diagnosed across studies. ”.

It is reasonable that the age range of the included studies has a high heterogeneity. This is also a crucial issue that should be discussed.

Please be more detailed concerning the statement "Restricting the analysis to participants aged 12 years or younger yielded effect estimates consistent in direction and magnitude with the primary findings, though heterogeneity persisted." reported in the discussion section.

Response: We have revised the Discussion section as follows: “Research indicates that the content of Zn and Cu in hair is positively correlated with age[20]. We further conducted an additional meta-analysis of studies including children under 12 years of age. The results showed that, compared with the control group, children with ADHD exhibited a more significant difference in Zn levels (SMD= -1.49), while the differences in Fe (SMD= -0.56) and Cu (SMD= -0.66) levels were relatively moderate. However, only a limited number of studies have directly compared Zn, Fe, and Cu levels between children with ADHD and controls across preschool age, childhood, and adolescence, and the findings have been inconsistent[13, 15, 17]. Most existing studies have not analyzed the groups of children (aged ≤ 12 years) and adolescents (aged > 12 years) independently. Instead, they have included both groups together. Although our subgroup analysis showed that the current main conclusions remained stable and significant in children aged ≤12 years, due to the wide age range of the study population included, the current results still exhibited high heterogeneity.”.

The authors should add a bit more information in the conclusion section.

Response: Thank you for your suggestion. We have revised the conclusion section by adding further information to enhance its comprehensiveness. The updated paragraph is as follows: “In summary, this meta-analysis indicates that children and adolescents with ADHD exhibit significantly lower Zn, Fe, and ferritin levels than healthy control group. This difference is more pronounced among populations in developing countries. When the research subjects are limited to children under 12 years old, the differences in Zn are more pronounced. Reduced serum concentrations of these trace elements may be associated with ADHD. However, considering the relatively high heterogeneity of the pooled results in this study, the clinical value of the research findings needs to be carefully evaluated. Future well‑controlled clinical trials are warranted to evaluate the efficacy of mineral supplementation and to identify potential subgroups of patients who may benefit.”.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have made an effort to address several structural concerns from the previous review round, such as modifying the title and removing Selenium from the text. However, a rigorous evaluation of this revised version reveals critical, numerical inconsistencies in the core data, sample counts, and study selections. Furthermore, despite the authors explicitly stating in their response letter that they thoroughly synchronized all counts across the main text, figure captions, and table notes, several major discrepancies remain unresolved. These flaws compromise the statistical integrity of the meta-analysis and must be thoroughly corrected before any consideration for publication.

Severe Data and Study Count Inconsistencies for Iron (Fe): There is a major mathematical mismatch regarding the exact number of studies included for Iron (Fe) analyses across different sections of the manuscript.

In Section 3.2 (Characteristics of included studies), the text explicitly states that Fe levels were reported in 23 studies.
Conversely, Section 3.3.2 (Meta-analysis of mean Fe levels) opens with the statement that the meta-analysis included a total of 29 studies for Fe.
To add to the confusion, the subgroup analysis table on Page 9 shows completely conflicting totals for the Fe and ADHD subgroups. Stratification by Age sums up to 29 studies (22 + 7), whereas stratification by Biological Sample sums up to 31 studies (27 Blood + 4 Hair), and stratification by Region also sums up to 31 studies (24 Developing + 7 Developed).
The authors must comprehensively audit their dataset, correct these conflicting numbers, and ensure that the main analysis matches the sum of its respective subgroups.

2. Failure to Update Figure 1 (PRISMA Flow Diagram): * In the cover letter, the authors explicitly assured that all mentions of the total number of included studies were updated to "46 studies" to perfectly align with their final screening. However, an inspection of the final submitted Figure 1 reveals that the last box at the bottom still reads: "Number of papers included in this study (n=45)". This direct contradiction between the authors' claims and the actual manuscript content indicates an unacceptable lack of care in their final proofreading process. Figure 1 must be strictly corrected to n=46 to match the Abstract and Results.

3. Inconsistent Literature Search Timeline: The manuscript presents a chronological misalignment regarding the timeline of the literature search. The Abstract states that the comprehensive literature search was performed up to March 2026. On the other hand, Section 2.1 (Methods) explicitly states that the search was conducted up to May 2026. The authors must clarify the exact cut-off date and harmonize this temporal window throughout the text.

4. The Interpretation of High Residual Heterogeneity: The pooled analyses for Zn, Fe, ferritin, and Cu display extreme levels of heterogeneity.

In Section 3.5, the authors note that the meta-regression based on publication year, biological sample, region, and sample size failed to explain the main sources of this high heterogeneity.
Simply reporting the failure of the meta-regression is insufficient for a high-quality systematic review. The authors must significantly expand their Discussion and Limitations sections to qualitatively evaluate other clinical and methodological sources driving this variance, such as differences in ADHD diagnostic criteria (DSM vs. ICD versions), laboratory assay techniques (e.g., ICP-MS vs. AAS), and participant fasting status

Author Response

Comments and Suggestions for Authors

Severe Data and Study Count Inconsistencies for Iron (Fe): There is a major mathematical mismatch regarding the exact number of studies included for Iron (Fe) analyses across different sections of the manuscript.

In Section 3.2 (Characteristics of included studies), the text explicitly states that Fe levels were reported in 23 studies.

Conversely, Section 3.3.2 (Meta-analysis of mean Fe levels) opens with the statement that the meta-analysis included a total of 29 studies for Fe.

To add to the confusion, the subgroup analysis table on Page 9 shows completely conflicting totals for the Fe and ADHD subgroups. Stratification by Age sums up to 29 studies (22 + 7), whereas stratification by Biological Sample sums up to 31 studies (27 Blood + 4 Hair), and stratification by Region also sums up to 31 studies (24 Developing + 7 Developed).

The authors must comprehensively audit their dataset, correct these conflicting numbers, and ensure that the main analysis matches the sum of its respective subgroups.

Response: Thank you for the author’s prompt feedback.. We have carefully reviewed the data and can confirm that the Fe data is accurate, and we have distinctly separated the data for Fe and ferritin, with 23 entries for Fe and 9 entries for ferritin. However, we do not understand why the reviewers received partial content from our previous version instead of the latest revised manuscript. I'm very sorry for any inconvenience caused to your review. Perhaps due to our failure to highlight the modified content, the journal editor did not update. Thank you for your patience. Please review our latest version.

It should be noted that for the subgroup analysis of 37 studies on Zn and ADHD by age, Table 2 explains why the updated total number does not include 37. Because there are 25 items that are ≤ 12 years old; 6-15 years old has 8 items; Four studies also included two age groups (3-13 years old; 0-14 years old; 6-16 years old; 6-18 years old), which did not belong to the first two subgroups, therefore, no analysis was conducted.

Failure to Update Figure 1 (PRISMA Flow Diagram): * In the cover letter, the authors explicitly assured that all mentions of the total number of included studies were updated to "46 studies" to perfectly align with their final screening. However, an inspection of the final submitted Figure 1 reveals that the last box at the bottom still reads: "Number of papers included in this study (n=45)". This direct contradiction between the authors' claims and the actual manuscript content indicates an unacceptable lack of care in their final proofreading process. Figure 1 must be strictly corrected to n=46 to match the Abstract and Results.

Response: Thank you for the reviewer’s comment. We have re-examined all the information in Figure 1 and can confirm that it includes data from 46 studies. We believe the version you currently have access to may not be the latest version we submitted. We have already raised this issue with the editorial office.

Figure 1. Flow chart for article selection.

Inconsistent Literature Search Timeline: The manuscript presents a chronological misalignment regarding the timeline of the literature search. The Abstract states that the comprehensive literature search was performed up to March 2026. On the other hand, Section 2.1 (Methods) explicitly states that the search was conducted up to May 2026. The authors must clarify the exact cut-off date and harmonize this temporal window throughout the text.

Response: Thank you for your valuable comment. In response to the inconsistency noted regarding the timeline of the literature search, we have revised the manuscript to harmonize the information. The cut-off date has been uniformly updated to March 2026 throughout the text, including the Abstract and Section 2.1 (Methods). We apologize for any confusion caused and appreciate your careful review.

The Interpretation of High Residual Heterogeneity: The pooled analyses for Zn, Fe, ferritin, and Cu display extreme levels of heterogeneity.

In Section 3.5, the authors note that the meta-regression based on publication year, biological sample, region, and sample size failed to explain the main sources of this high heterogeneity.

Simply reporting the failure of the meta-regression is insufficient for a high-quality systematic review. The authors must significantly expand their Discussion and Limitations sections to qualitatively evaluate other clinical and methodological sources driving this variance, such as differences in ADHD diagnostic criteria (DSM vs. ICD versions), laboratory assay techniques (e.g., ICP-MS vs. AAS), and participant fasting status

Response: Thank you for your important suggestion. The viewpoint you pointed out is very relevant. Indeed, for meta-analysis results with extremely high heterogeneity, it is not enough to report multiple regression in the methodology section to explain heterogeneity. I have already adopted your core suggestion in the discussion section and conducted a qualitative and in-depth exploration of the clinical and methodological factors that may drive high heterogeneity. The specific modifications are as follows: “The meta-regression indicated that common covariates such as publication year, biological sample type, region, and sample size were insufficient in explaining the primary sources of this high heterogeneity. This suggests that the discrepancies across studies may stem from deeper clinical and methodological factors. These factors include inconsistent diagnostic criteria (e.g., varying versions of DSM or ICD), which may have led to differences in the clinical profiles of enrolled ADHD patients[56]. Another important factor is variability in analytical methods and quality control procedures; for instance, differences in instrumentation (e.g., inductively coupled plasma mass spectroscopy, atomic absorption spectroscopy) and sample pre-treatment across laboratories could impact the comparability of trace element concentrations[57]. Furthermore, insufficient control of key confounders, such as medication use, nutritional status, and inflammatory markers, was often observed, as these were inadequately reported or adjusted for in the original studies[58-60]. Consequently, although the pooled effect size reached statistical significance, its clinical interpretation requires considerable caution. Future research should aim to advance standardization in diagnostic criteria, harmonization of analytical methods, and systematic reporting of confounders to enhance the consistency and reliability of evidence in this field.” in the Discussion Section.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript has significantly be revised and improved.

Author Response

Thank you very much for your positive feedback. We are pleased to hear that you find the revised manuscript significantly improved. We have carefully addressed all the valuable comments and suggestions from you and the other reviewers to enhance the clarity, rigor, and overall quality of the paper. Your guidance has been instrumental in refining this work.

Author Response File: Author Response.pdf