Numerous studies on myocardial ischemia/reperfusion (MI/R) injury have been undertaken in recent years. Hotspots and developmental trends in MI/R research are being rapidly updated. However, there has been no bibliometric analysis that systematically evaluates existing literature on MI/R injury. Our study explores developments in MI/R research over the past decade, and provides a reference for future research.Both experimental and clinical publications on MI/R injury from 2012 to 2021 were retrieved from the Web of Science Core Collection database. The CiteSpace and VOSviewer tools were used to perform a bibliometric analysis.Basic Research in Cardiology had the highest impact factors. The top three co-cited journals were Circulation, Circulation Research, and Cardiovascular Research. According to a co-cited reference analysis, MI/R research can be divided across 10 major subfields of mitophagy, cardioprotection, inflammation, remote ischemic preconditioning, long non-coding RNA, melatonin, postconditioning, mitochondria, microvascular obstruction, and ferroptosis. After 2018, the keywords with strongest citation bursts included extracellular vesicles, long non-coding RNA, cell proliferation, microRNA, mitochondrial quality control, mitophagy, biomarker, and mitochondrial biogenesis.A total of 8419 papers were analyzed. The number of annual publications demonstrated an overall upward trend, rising from 629 publications in 2012 to 1024 publications in 2021. China, the USA, Germany, England, and Italy were the top five contributors to MI/R studies. The Fourth Military Medical University in China contributed the most publications , while the University College London in England cooperated the most with relevant research institutions. Derek J Hausenloy (University College London), Derek M Yellon (University College London), and Gerd Heusch  were the top three most active and influential scholars according to the H-index. Among the top 10 journals with the most publications, The present study reveals the influential authors, cooperating institutions, and main research foci in the field of MI/R injury in the past decade. The latest hotspots are a more in-depth insight into the molecular mechanisms underlying MI/R injury, such as mitochondrial quality control, non-coding RNAs, cell proliferation, and extracellular vesicles.The online version contains supplementary material available at 10.1186/s40001-022-00967-7. Ischemic heart disease has remained a leading cause of morbidity and mortality across the world . RestoraBibliometrics has been widely employed to understand the knowledge base, and to explore developmental trends and research frontiers in various research fields through qualitative and quantitative analyses of the scientific literature . In addiThe present study used the bibliometric tools of CiteSpace and VOSviewer  for the The Web of Science Core Collection (WoSCC) database includes the leading global academic journals, and allows downloading full citation records with new records being updated every day. The WoSCC is the most commonly used database for bibliometric analyses. Previous reports have widely confirmed the validity of bibliometric analyses based solely on the WoSCC database , 13, 15.The search results were screened independently by two investigators to exclude irrelevant, duplicate, and withdrawn articles. Disagreements were resolved by consulting a third investigator.The WoSCC literature analysis report was used to evaluate publication characteristics, such as output in the form of number of annual publications, journals, authors, citation frequency, impact factor (IF), and Hirsch index (H-index). IF reflects a journal's rank or importance by calculating the average number of times that its selected papers are cited in a particular year. The H-index, indicating that the given author/country has published at least H papers which have each been cited at least H times, was used to evaluate the scientific impact of author or country. GraphPad Prism 7.00  was used to analyze publication trends. The growth of publications in the following year was estimated using the polynomial model: Q value) and Mean Silhouette , were used to evaluate the quality of the cluster network formed. If the Q > 0.3, the cluster structure obtained was significant; when the S > 0.7, the clustering was efficient and convincing [CiteSpace, a visualization tool developed by Professor Chaomei Chen, analyzes emerging trends and critical changes in scientific literature . We usednvincing .The VOSviewer  software creates, visualizes, and explores knowledge maps based on network data. We used VOSviewer 1.6.18 to identify co-cited journals, and display the network structure .After scanning, a total of 8419 English records related to MI/R injury were included, of which 6623 were articles (78.7%) and 1796 were reviews (21.3%). The total citation frequency was 214,942, the H-index count was 156, and the average number of citations per item was 25.53. A flowchart of the literature selection is presented in Fig. As presented in Fig. All the studies were distributed among 91 countries and 4847 institutions. As illustrated in Fig. As depicted in Fig. n = 6), China (n = 3), and North America (n = 1). All the top 10 most published authors had total citation counts of over 1000, and their average number of citations per publication was more than 25. In terms of H-index, the top five authors were Derek J Hausenloy, Derek M Yellon, Gerd Heusch, Rainer Schulz, and Erhe Gao.A total of 28,814 authors were involved in MI/R research. As presented in Table The network of author cooperation is presented in Fig. PloS One accounted for the most output, followed by Oxidative Medicine and Cellular Longevity, Molecular Medicine Reports, International Journal of Molecular Sciences, and Journal of Molecular and Cellular Cardiology. Among the top 10 journals with the most publications, Basic Research in Cardiology had the highest IF (17.165), ranking sixth in terms of number of publications. Most of the productive journals were classified as Q1 or Q2 according to the 2020 Journal Citation Reports. In addition, the number of studies on MI/R injury was normalized to the sum of publications in respective journals. These results showed that the proportion of studies on MI/R injury was highest in Basic Research in Cardiology , followed by Journal of Molecular and Cellular Cardiology , Cardiovascular Research , American Journal of Physiology-Heart and Circulatory Physiology , and Oxidative Medicine and Cellular Longevity .The documents were published in 1203 journals, 13 of which had more than 100 publications .A co-citation relationship between two journals arises when they are both cited in the same publication simultaneously. High co-citation counts imply that the journals produce superior academic material and can, therefore, be considered mainstream. As illustrated in Fig. “Ischaemic Accumulation of Succinate Controls Reperfusion Injury Through Mitochondrial ROS” by Edward T Chouchani in 2014 (1235 citations) . The aut“Myocardial Ischemia–Reperfusion Injury: a Neglected Therapeutic Target” by Derek J Hausenloy in 2013 (1231 citations) . This is“Physiological Implications of Hydrogen Sulfide: a Whiff Exploration That Blossomed” by Rui Wang in 2012 (1226 citations) . This coAs presented in Table However, it is important to note that the number of citations does not adequately reflect the knowledge base of a particular field, since older articles are typically cited more frequently than newer ones.Journal of Clinical Investigation was the most frequently co-cited publication [Reference co-citation analysis identifies highly co-cited references that are frequently cited together by other articles, and is, therefore, usually applied for exploring research foci in a given academic field. A reference co-citation network with 685 nodes and 1857 connections is presented in Fig. Journal of the American College of Cardiology by Sean M Davidson in 2019 is the latest of the top 10 co-cited references, and therefore, the time lag between publication and citation might have impacted the burst detection.A paper with strong citation burst can be viewed as representing a major milestone in the knowledge base. Figure Q value of the network of co-cited references was 0.779, indicating that it obtained a good clustering. The average S value of the network was 0.933, suggesting high homogeneity of the references in a cluster. Among the top 10 clusters, the clusters which were most recently active included #4 lncrna (long non-coding RNA) and #9 ferroptosis. The clusters #0 mitophagy, #1 cardioprotection, #2 inflammation, #5 melatonin, and #7 mitochondria emerged as active areas in the period 2013–2018, while activity in the five clusters appeared to be declining. The clusters #3 remote ischemic preconditioning, #6 postconditioning, and #8 microvascular obstruction were formed by documents with a mean publication year of 2010, thereby representing older clusters in the MI/R knowledge base.To further explore characteristics of the temporal evolution of the knowledge base in MI/R research, we performed a cluster analysis using a timeline view tool of CiteSpace Fig. B. The Q Keywords are the focus of attention in a given scientific paper. A total of 25,662 keywords were extracted by CiteSpace, of which 45 appeared more than 100 times. According to their co-occurrence frequency, the top 20 keywords in MI/R research are listed in Fig. To track hotspots and future directions in the field of MI/R research, we further used the burst detection algorithm to analyze the keywords. A keyword with a high burst level indicates that it received greater attention in a specific time period. The top 20 keywords with the strongest appearance bursts are presented in Fig. In the present study, we used bibliometric tools to analyze studies on MI/R injury through the following aspects: (1) the general information was identified and quantified to evaluate individual impact and cooperation information, (2) a co-cited reference analysis was performed to map the knowledge domain, and (3) a keyword analysis was conducted to detect emerging trends. Our findings provide a strong basis for exploring the corpus of knowledge in the MI/R domain.One of the indications of the direction of development in a field is change in annual output. According to the WoSCC database, the 8419 papers were published in 1203 academic journals by 28,814 authors from 4847 institutions across 91 countries/regions. The overall trend between 2012 and 2021 was a slow increase in publications. Among the 10 highest output countries, China was the only one from the developing world. The USA was ahead of other countries/regions in H-index, while England had the highest average number of citations per publication. Moreover, the top six authors with the highest number of published articles were from European countries. This implies that the European countries exert a positive academic influence on MI/R research. The most active collaborations were observed among the USA, France, England, and Italy. Nine of the top 10 most prolific institutions were from China, demonstrating its fast progress in MI/R research over the past decade. However, the cooperation among Chinese institutions was infrequent, and UCL in England was the main collaborating center. Good partnerships and a high degree of collaboration among countries and institutions are important for high-quality research output. Therefore, Chinese researchers are encouraged to cultivate partnerships with institutions across the globe as their research output continues to expand, which will improve their academic influence further.Highlighting the contributions of productive and influential researchers could act as a reference for those scholars and institutions looking for potential collaborators on the basis of research directions . On analBasic Research in Cardiology is a high-quality journal that exerts considerable influence in the field of MI/R injury. However, mainstream journals in specific fields of study need not have high publication capacity. In fact, we found that some journals with high publications did not receive much attention from the academic community. Quality and influence of a journal can also be determined by its co-citation frequency. The top three journals, in terms of co-citation frequency, were Circulation, Circulation Research, and Cardiovascular Research, with IF values greater than 10. This suggests that these three journals influence research foci, and should thus be considered when tracking progress in MI/R research.In terms of a journal’s publications, IF, and ranking, The highly co-cited references can be viewed as the knowledge base in a particular field . ReferenMeanwhile, a cluster analysis was conducted to identify the major subfields in the MI/R research, and a timeline view was used to show the developmental process of these subfields. Over the past decade, there has been considerable progress in the understanding of mechanisms of MI/R injury, and development of strategies to protect the myocardium from ischemia and reperfusion damage , 3, 8. BThe pathophysiological mechanism of MI/R injury is complex, and understood only partially. Besides causing cardiomyocyte death via multiple mechanisms, MI/R also affects other types of cells, such as platelets, fibroblasts, endothelial cells, smooth muscle cells, and immune cells. Cardioprotective strategies that target a single mechanism might be insufficient at a time when clinical situations usually present a number of uncontrolled variables.The majority of preclinical experiments are conducted on young and healthy animals with a virgin coronary circulation . The routinely used animal models do not adequately mimic the clinical scenario of MI/R injury in patients with co-morbidities and co-medications, such as aging, hyperglycemia, hypertension, hyperlipidemia, and the use of P2Y12 antagonists .Translating novel cardioprotective interventions into clinical practice is becoming increasingly challenging due to favorable outcomes after optimal reperfusion therapy. It could be possible that the specific population in a clinical trial did not include those patients who would benefit most from cardioprotective interventions.As for cardioprotective strategies, experimental studies in the early years from 2007 to 2012 had revealed the essential signal transduction pathways underlying local ischemic postconditioning and remote ischemic preconditioning . For exaAlthough all attempts have failed, better adjunct cardioprotection is still a requirement for decreasing the risk of heart failure after acute myocardial infarction . A cliniThe keywords with strongest citation bursts were analyzed to present those research frontiers that have recently attracted close attention from the academic community . The keyMitochondria are abundantly distributed and plentiful in the heart tissue. Despite the canonical role of mitochondria in regulating cellular respiration and oxidative phosphorylation, recent studies have concluded that mitochondria are crucial in sensing and integrating extracellular and intracellular signals . There iIn ongoing research on the regulatory mechanisms of MI/R injury, the focus has gradually shifted from coding RNAs to non-coding RNAs (ncRNAs), particularly microRNAs, lncRNAs, and circular RNAs . microRNLoss of cardiomyocytes is the major contributor to the pathogenesis of MI/R injury . It is wExtracellular vesicles are nanosized vesicles with a double-layer lipid membrane. There is increasing evidence that reveals the vital role of extracellular vesicles as cell-to-cell communication vehicles in the development of MI/R injury . ExosomeThis study found that the MI/R knowledge base has evolved rapidly over the past 10 years. Early MI/R research was initially focused on pharmacological and non-pharmacological approaches to inducing cardioprotection, and on their signal transduction pathways. However, research has gradually shifted focus to insightful molecular mechanisms triggered by MI/R. The findings of this study would act as a reference for researchers looking for research directions, and lay the foundation for further research on MI/R injury. In addition, our results reveal the most productive authors and institutions, which could help researchers decide on collaborations. Journals publishing high-impact studies should be followed by researchers to track the progress of MI/R research.However, the present study does have several limitations. First, studies were not retrieved from databases other than the WoSCC database . Second, only English articles and reviews were included, which could have resulted in a language bias. Third, different H-index values are possible for the same author, because the H-index method is based on the number of publications and citations in a given time period.This study used a bibliometric analysis to provide researchers with a panoptic and holistic perspective on research in the field of MI/R injury. The number of publications on MI/R research has increased over the past decade. Research foci in the MI/R field are the pathophysiology of MI/R injury and development of strategies to protect the myocardium from ischemia and reperfusion damage. Mechanistic research before 2018 had mainly focused on the molecules or pathways involved in cell death, inflammation, and coronary microvascular obstruction, such as mitochondrial permeability transition pore, endoplasmic reticulum stress, oxidative stress, NF-κB, or AMPK pathways. The latest hotspots are more in-depth insights into the molecular mechanisms underlying MI/R injury, such as mitochondrial quality control, ncRNAs, cell proliferation, and extracellular vesicles. With regard to cardioprotective interventions proven to be beneficial in preclinical animal studies, it has been challenging to translate them into clinically beneficial treatments for patients with acute myocardial infarction. Conducting rigorous and systematic in vivo preclinical assessment, such as adopting an animal model with the coexistence of multiple confounding factors, would increase the likelihood of translating novel cardioprotective interventions into clinical practice for patients’ benefit.Additional file 1: Table S1. Top 10 co-cited references for MI/R injury research. Fig. S1. Process of searching, retrieving, and selecting potentially relevant studies.


In patients with anorexia nervosa (AN), gastrointestinal (GI) symptoms are common and usually improve during or after nutritional rehabilitation. It is unclear when exactly GI symptoms change in the timecourse of treatment and to which extent. In this study, we analyzed the timecourse of GI symptoms and their relation to disease-specific, demographic, anthropometric, and psychological factors in inpatients with AN.In weekly intervals, the Gastrointestinal Symptom Rating Scale (GSRS) was completed, and body weight was measured over a mean of 9.5 weeks in inpatients with AN. A total of four self-report questionnaires assessing psychological factors were completed before and after inpatient treatment. Data from 38 inpatients with AN were analyzed using mixed linear models.p = 0.002) and 0.101 (p = 0.004) points per week on the GSRS and were predicted to normalize after 13 (p = 0.002) and 17 (p = 0.004) weeks, respectively. Total GI symptoms tended to normalize after 25 weeks (p = 0.079). Indigestion  was the most severely pathological symptom at admission and did not improve significantly (p = 0.197). Diarrhea and reflux were, on average, not pathological at admission and remained stable during treatment. In addition to treatment time, the strongest predictors were ED pathology at admission for the development of abdominal pain, constipation, reflux, and total GI symptoms; stress for the development of constipation and total GI symptoms; and depression for constipation.Abdominal pain and constipation improved significantly in the timecourse with 0.085 (Informing patients with AN about the course of GI symptoms and their improvement during weight rehabilitation may help support compliance during treatment. Eating disorders (EDs) such as anorexia nervosa (AN) are characterized by aberrant patterns of eating behavior. AN is characterized by a restriction in food intake, which can sometimes be combined with binge-purging behavior, high levels of physical activity, and body image disturbances –3. AsideMany of the GI symptoms found in patients with AN overlap with symptoms typical for functional gastrointestinal disorders (FGIDs) –14, whicOverall, many studies report a change in GI disturbances during treatment—some remain the same, some improve or even disappear, and others newly occur—which appears to be particularly dependent on a specific GI symptom . ExaminiPredictors of the prevalence of GI problems and development during treatment in patients with AN were analyzed by several studies. Psychopathological features in patients with AN were found to be associated with both functional and somatic GI disorders , 32. StaGiving patients a deeper understanding of the development of their GI symptoms in the timecourse of nutritional rehabilitation may support the recovery process by helping to motivate and reassure patients with AN to continue to eat normal amounts of food, despite having severe GI complaints at the beginning of treatment. A more closely monitored longitudinal observation of GI symptoms, which is lacking in the current literature, might contribute to this understanding. The aims of the study were to prospectively analyze (i) the timecourse of various GI symptoms in inpatients with AN and (ii) the relationship of the development of GI symptoms during inpatient treatment with anthropometric, demographic, disease-specific, and psychological factors.Participants with AN were recruited within the context of an inpatient treatment program at the Eating Disorders Unit of the Department of Psychosomatic Medicine and Psychotherapy at the University Medical Hospital in Tübingen, Germany, in the periods from 2016/01 to 2017/01 and 2018/01 to 2019/02. The main inclusion criterion was a diagnosis of AN according to the Diagnostic and Statistical Manual of Mental Disorders 5 (DSM-5) . The participants had to be old enough to be treated in the ED unit for adults, which usually requires the age of 18 years. However, patients who would soon be turning 18 years were also admitted in some cases and were not excluded from analysis for reasons of age. Both sexes were included in order to capture the spectrum of patients with AN. Patients with severe somatic diseases, which are likely to induce GI symptoms like Crohn’s disease, or those with limited knowledge of the German language were excluded from analysis.Within the 1st week after admission, patients with AN with a body mass index (BMI) ≤ 18.5 kg/mAt admission, body height, age, sex, age at the first diagnosis, duration of illness, vomiting behavior, and laxative misuse were assessed. A total of four questionnaires were distributed at admission and at discharge, assessing patients’ mental condition (psychometric variables): the Eating Disorder Inventory 2 (EDI-2), the Generalized Anxiety Disorder 7 (GAD-7), the 9-item depression scale of the Patient Health Questionnaire (PHQ-9), and the Perceived Stress Questionnaire (PSQ). Only the sum scores of these questionnaires were applied, but no subscales. Body weight and the Gastrointestinal Symptom Rating Scale (GSRS) questionnaire were assessed once weekly from the 1st week after admission until discharge. In case of the GSRS, the subscales were also of interest on top of the total score for better differentiation and analysis of GI symptoms. Serum amylase levels were retrieved from the standard laboratory blood measurements. Finally, the length of inpatient stay was recorded.The Gastrointestinal Symptom Rating Scale (GSRS) is a self-report questionnaire evaluating common GI symptoms  examines behaviors and attitudes typical for EDs  is a seven-item anxiety scale of the Patient Health Questionnaire, which was developed and initially validated as a brief self-report screening tool for generalized anxiety disorder (GAD) in clinical practice  is a 9-item depression scale based on the Patient Health Questionnaire (ionnaire . Answersionnaire . AnswersThe Perceived Stress Questionnaire (PSQ) is a 30-item stress questionnaire constructed to measure subjective perception, evaluation, and processing of stress  nested within patients (level 2), a mixed linear model (MLM) approach, also called hierarchical linear model, was considered appropriate to analyze the data. The MLM takes into account that the repeated observations in one patient are dependent. If calculated otherwise, estimators could be biased, and standard errors (SE) could be underestimated. In case of missing data, imputation is not necessary as cases are not excluded listwise, and parameters are estimated with the available data. Within- and between-person predictors were handled following common recommendations . BecauseMLM can be used with different covariance types depending on the structure of the data. The data showed different variance at each timepoint and constant covariance between the measurements; thus, a diagonal covariance structure was applied. Several different models were built in order to analyze the data step by step. (1) A so-called “empty model” without any explanatory variables was calculated to estimate the extent to which the GSRS scores differed between patients. (2) Next, the random intercept model was conducted with the number of treatment weeks, which is the central explanatory predictor at level 1, as the fixed effect. (3) A random slope model was calculated to assess for varying time slopes across patients. The latter two steps were also tested with a quadratic slope by squaring the amount of treatment weeks to test for increasing or decreasing influence of treatment time on the GI development. (4) BMI and amylase, the remaining predictors of level 1 with measures at each timepoint, were applied as fixed effects in separate models. (5) All predictors of level 2 (those which were only measured at the beginning of treatment) were modeled as fixed effects in separate models. Interactions of all predictors with the treatment time in weeks were tested consecutively to examine if the influence of a predictor on GI symptom development changes during treatment time.Model fits calculated as -2 log-likelihood and Akaike information criterion were taken into account in order to compare the fit of different models. MLM was used for the total GSRS score and each subscale separately using repeated measures until week 10. Week 10 was chosen as the last timepoint to be included into analysis as this was the upper limit of the planned treatment time and also matched with the approximate average time of the actual inpatient stay.p values of < 0.005 were considered as statistically significant. Results with p values < 0.05 were, however, also reported and can be considered putative effects.MLM calculations were not adjusted for multiple testing, but only strong 2 according to the DSM-5 criteria, nine as severe (BMI 15.0–15.9 kg/m2), three as moderate (BMI 16.0–16.9 kg/m2), and six as mild AN (BMI > 17.0 kg/m2) ] years, ranging from 17 to 53 years. Only two male patients fulfilled the inclusion criteria (5.3% of patients); 19 patients were categorized as having extreme AN with a BMI of < 15.0 kg/mThe pre- and post-treatment characteristics of the study population are summarized in p < 0.001 in all models calculating random intercepts, irrespective of the GSRS score of the dependent variable, as presented in p < 0.001, SE = 0.132) on the GSRS score for diarrhea  to a highly pathological value of 3.8  for indigestion. These estimates are also similar to the means at admission, which are presented in As the GI development over the timecourse was modeled using the MLM, random intercepts (which are estimates at admission), linear and quadratic fixed and random slopes could be tested in addition to the analysis of weekly development. First of all, models with random intercepts and fixed slopes had good model fits, and random intercepts were significant with p < 0.005 were ED pathology for abdominal pain, constipation, reflux, overall AN-typical GI symptoms, stress for constipation, and AN-typical GI symptoms, as well as depression for constipation. The estimates reported are not effect sizes and not standardized and can therefore not be compared between the different predictors. Instead, they are interpreted in relation with the respective GSRS scores and the average values of the respective predictor. For example, for constipation and ED pathology, this implies that the GSRS score is estimated to be 0.016 GSRS points lower for every full point less on the EDI-2. Given that the average EDI-2 score of our study population is 288.6, substantial differences between the patients in constipation outcomes associated with higher or lower EDI-2 scores appear reasonable.An overview on the assessed predictors of GI symptom improvement is presented in p < 0.05, but not > 0.005, are reported in p < 0.005. Other tested predictors  did not significantly contribute to weekly GI development. Altogether, the most important contributing predictor was treatment time, as reported in the previous section.More factors with GI symptoms are common in patients with AN . In lineAbdominal pain, constipation, and indigestion  are typical GI symptoms in patients with AN and develop differently in the timecourse of treatment. Abdominal pain and constipation, as well as AN-typical symptoms, altogether improved significantly within the observation period. Constipation appeared to improve at a higher rate than abdominal pain, with about 0.1 points on the GSRS score per treatment week. Both symptom complexes did, on average, not normalize during treatment, but due to significant weekly improvement, the normalization of the average GSRS scores of abdominal pain and constipation can be assumed. However, a longer observation period would be required for certainty. These results can be helpful in encouraging patients to continue eating despite GI symptoms troubling the patients particularly at the beginning of treatment.Symptoms of indigestion were very high at the beginning of treatment and neither normalized throughout treatment in more than 80% of the patients nor improved significantly. The persistence of these symptoms is consistent with a previous study . ApparenDiarrhea and reflux symptoms played a minor role in the investigated population with AN as the majority of patients showed normal values from admission onward. In the first week of treatment, diarrhea symptoms appeared to worsen in some patients, but not significantly and not to pathological levels on the GSRS. Reflux symptoms were of particular interest as they might be a consequence of binge-purging behavior, more specifically vomiting. Interestingly, neither serum amylase nor vomiting behavior, factors that can be hypothesized to be connected with reflux symptoms, was found to be associated with the latter for the complete AN group. An analysis within the different AN subgroups—restrictive and binge-purging—would have been necessary but would have resulted in insufficient subgroup sample sizes in this study.A key finding of this study was that disordered ED pathology at admission appeared to be an important predictor for the development of abdominal pain, constipation, reflux, and overall AN-typical GI symptoms. Overall, this suggested that GI symptoms might rather be of functional origin in patients with AN. Although ED pathology appeared to be a predictor for several GI symptoms in this study, the EDI-2 score remained high despite treatment. In line with the high mortality rate of patients with AN due to the difficult course the disease can take, ED pathology was found to be persistent, on average, even after inpatient treatment . SurprisIn addition, the outcome of constipation was also predicted by stress and depression, and AN-typical GI symptoms could be predicted by stress. Anxiety was the only psychological factor that showed no association with any GI symptom complex—a finding which is coherent with an earlier study . Considevia improving depression . Written informed consent to participate in this study was provided by the participants or the participants’ legal guardian/next of kin in case of underage participants.CR was responsible for data analysis and interpretation, drafted the manuscript, and acquired data. IM was responsible for conception, design, and preparation of the study, data interpretation, and manuscript writing. NM was responsible for data interpretation. NS was involved in data management. SZ and PE were responsible for conception and design of the study and data interpretation. KG and AS were involved in data interpretation. All authors revised the manuscript and approved the final version of the manuscript.


The COVID-19 pandemic has led to a worsening of mental health and health behaviors. While physical activity is positively associated mental health, there is limited understanding of how mental health and physical activity evolve throughout the COVID-19 pandemic. This study aimed to examine changes in depression, anxiety and stress and physical activity, and associations between depression, anxiety, and stress with physical activity in Australian adults across three-time points during the COVID-19 pandemic.This study collected both longitudinal and cross-sectional data at three-time points during the COVID-19 pandemic in Australia . Australians aged 18 years and over were invited to complete online surveys hosted on Qualtrics survey platform. Linear mixed models with random subject effect and general linear models were used to analyze the longitudinal and repeated cross-sectional data respectively.d = 1.03, 95% CI = 0.20, 1.85), anxiety , and stress  scores but no significant differences in physical activity across three-time points. On average, participants who met the physical activity guidelines had lower depression , anxiety , and stress  scores compared to those not meeting the guidelines.The number of participants in cross-sectional surveys and longitudinal surveys was 1,877 and 849, respectively. There was an overall reduction between time 2 vs. time 3 in depression (In the context of the ongoing COVID-19 pandemic, both governments and service providers should continue to provide the public with timely mental health support and promote the benefits of physical activity, as a cost-effective strategy to improve mental health and wellbeing. On March 11, 2020, the World Health Organization (WHO) declared the novel coronavirus (COVID-19) a pandemic, due to its rapid global spread and high mortality . Since tStudies exploring the impact of the COVID-19 pandemic on mental health have reported an increased prevalence of mental health problems and psychological distress in both clinical and general populations across the world, including depression, anxiety, stress, and post-traumatic disorder . SystemaLongitudinal studies were also examined changes in mental health over time during the pandemic. Specifically, a UK longitudinal study reported an almost threefold increase in mental health problems in the general population between the pre-pandemic period (2017–2019) and April 2020, with only a slight reduction in the elevated prevalence rate and severity by May/June 2020 . SimilarPrevious studies have also reported significant declines in physical activity following implementation of lockdown measures, as people have been unable to access recreational and gym facilities and engage in incidental physical activities . One of There is evidence demonstrating that an increase of physical activity is associated with lower levels of depression, anxiety, and stress . HoweverGiven the ongoing threats of the pandemic and the importance of mental health and physical activity for our overall health, the present study aimed to examine changes in depression, anxiety and stress, and physical activity, as well as the associations between depression, anxiety and stress with physical activity in Australian adults across three-time points throughout the pandemic. The findings from this study can assist governments and service providers making the decision regarding providing mental health and physical activity support for the public during the COVID-19 pandemic.This study made use of both longitudinal  and repeated cross-sectional  data collected at three-time points during COVID-19 pandemic in Australia. During the first survey, which was conducted from 9th to 19th April 2020, Australian governments had implemented nationwide lockdown measures to reduce the spread of infection including social distancing, stay at home orders, and domestic and international incoming and outgoing travel restrictions. During the second survey, which was conducted from 30th July to 16th August 2020, Victoria was the only Australian state in which a second wave of the COVID-19 pandemic had occurred, and a Victorian state-wide lockdown measure was enforced to protect people and control the rate of infection. In contrast, all other Australian states had reduced their preventive measures due to the low number of infected cases. During the third survey (conducted from 1st to 25th of December 2020), there were no hard lockdowns in place anywhere in Australia.via paid advertisements on Facebook and through social media . Advertisements included a direct link to the surveys hosted on Qualtrics survey platform. A range of images to separately target males and females were used. Central Queensland University internal emails including academic and professional staff were also used for recruitment. Australians aged 18 years and over were invited to complete the surveys. Online informed consent was obtained from all participants after they were provided with the information on the nature of the study and participation. The survey responses were anonymous. Participants were not paid for participation and no information about whether participants were participating in other studies was collected. Participants who completed the first or second survey were invited to participate in subsequent surveys. Those consenting to being re-contacted, provided their e-mail address so they could be advised of subsequent survey rounds. The total number of participants in three cross-sectional surveys were 1887 and in longitudinal surveys (including new participants recruited in survey 2) were 849. The study was approved by the Human Research Ethics Committee of Central Queensland University, Australia .Participants were recruited Depression, anxiety, and stress were assessed using the 21-item Depression, Anxiety and Stress Scale (DASS-21) . The DASPhysical activity was measured using the 8-item Active Australia Survey (AAS) , which a2 or lower), Overweight (BMI of 25.0–29.9 kg/m2), and Obese (BMI of 30.0 kg/m2and above). When assessing for the presence of chronic disease, participants were asked the question “Have you ever been told by a doctor that you have any chronic health problems?” with a “yes” or “no” response.Sociodemographic characteristics included in this study were age, gender, educational attainment, household income, Body Mass Index (BMI), and chronic disease status. Participants’ weekly household income was categorized as: (1) less than AUD $1,000; (2) between AUD $1,000 and 2,000; (3) more than AUD $2,000. BMI was calculated as weight in kilograms divided by height in meters squared and interpreted based on the standard weight status categories: Normal  version 27. Descriptive statistics  were used to describe mental health and physical activity status, and sociodemographic characteristics of the sample at each of the three-time points.To examine changes in depression, anxiety, stress and physical activity, and associations between depression, anxiety, and stress with physical activity across three-time points, linear mixed models with random subject effect were employed for the longitudinal data and general linear models were employed for the repeated cross-sectional data. Robust variance estimator was also used.p-values. Crude estimates and those adjusted for potential confounders were reported with 95% confidence interval. Significance level was set at 0.05 and all p-values were two-tailed.Sociodemographic characteristics were accounted for in multivariable analysis. There were less than 1% missing values for sociodemographic characteristics factors, except for household income which was 16.9% for repeated cross-sectional data, and 12.9% for longitudinal data across three-time points. Therefore, two multivariable models were run with and without household income. As the result between the two models were similar, only the multivariable model with household income is presented here. Given there were three-time points, Bonferroni adjustment method was used to correct inflated Sociodemographic characteristics of the longitudinal sample are shown in d = 1.30, p = 0.003, CI = 0.36, 2.24) and between time 2 and time 3  . There was a significant decrease in stress score between time 1 and time 3  as well as between time 2 and time 3 . There were no significant differences in physical activity between the three-time points.In longitudinal sample, Model 2 shows there was a significant decrease in depression score between time 1 and time 3  . There wd = −2.25, p = 0.001, CI = −3.72, −0.77) and a significant decrease between time 2 and time 3  was found . There was a significant increase in stress score between time 1 and time 2  and significant decrease in stress score between time 2 and time 3 . There were no significant differences in the physical activity level found between the three-time points. The changes in the outcomes were also visualized in In the cross-sectional sample, Model 2 shows a significant increase in depression score between time 1 and time 2 , anxiety  and stress scores  in the longitudinal sample, and also significant lower depression , anxiety  and stress  scores in the cross-sectional sample.The present study examined changes in depression, anxiety, stress and physical activity, as well as associations between depression, anxiety and stress with physical activity in Australian adults across three-time points during the COVID-19 pandemic. The findings showed that depression, anxiety, and stress scores were generally lower at time 3 for both longitudinal and cross-sectional samples. There was, however, one exception to this, the cross-sectional data indicated that from time 1 to time 2, depression, anxiety and stress scores significantly increased and the proportion of participants reporting symptoms of extremely severe depression and severity anxiety, were doubled. This may in part be due to the negative impact of two consecutive lockdowns in Victoria, as there was an increase in the number of participants from Victoria joining the cross-sectional sample from 16.7 to 36.6% at time 2. The negative impact of strict lockdown measures on mental health have been reported in previous studies . SimilarOur findings are also in line with the cross-sectional research outcomes reported by the Australian Bureau of Statistics, where in December 2020, the number of Australian adults feeling negatively impacted by the COVID-19 pandemic in relation to mental health, reduced to its lowest level since the initial peak in April 2020 . Our finIn contrast with studies reporting significant reductions in physical activity during the COVID-19 pandemic , our finThe consistent levels of physical activity found in our samples may be due to several protective factors. Australian governments have actively promoted benefits and practical access to physical activity during the pandemic, including permitting outdoor exercise and recreational activities to be essential reasons for leaving home even during lockdown periods . This waConsistent with previous evidence, the present study also demonstrated that regular participation in physical activity is associated with lower scores for depression, anxiety, and stress . The posThe present study has several strengths, including large sample sizes, broad national inclusion of participants from metropolitan, regional, and remote areas of Australia and application of both longitudinal and cross-sectional study designs. However, it also has limitations, which should be considered when interpreting and generalizing the results to other populations. Firstly, this study does not include pre-pandemic data and it is therefore unknown how mental health and physical activity outcomes changed from before the pandemic to the first time point. Secondly, a large proportion of participants were female, older in age, had higher household incomes, and more years of education than the Australian adult population. Thirdly, this study also relied on voluntary participation and there may have been an over-representation of participants joining in the study who perceived themselves to be affected by the current pandemic. Fourthly, this is an observational study and therefore, it is not possible to control for unknown confounders. Finally, the use of self-reported measures could result in recall bias and further research should consider using objective measures to strengthen the findings.In conclusion, our findings show an improvement in depression, anxiety, and stress and no significant changes in physical activity in Australian adults across three-time points during the COVID-19 pandemic. The significant benefits of engaging in sufficient physical activity on mental health were also demonstrated. These findings have practical implications for physical activity to be utilized as a cost-effective coping method to support people’s mental health, and the need for governments and service providers to continue implement initiatives to promote mental wellbeing and physical activity as the COVID-19 pandemic continues to evolve. The findings from this study may also be applied in similar contexts in which other extraordinary negative events cause large scale psychological distress and changed social conditions . Further research is needed to monitor the long-term impact of the COVID-19 pandemic on mental health and physical activity to inform and guide appropriate public health promotion and intervention in a timely manner.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Human Research Ethics Committee of Central Queensland University, Australia . The patients/participants provided their written informed consent to participate in this study.QT, RS, SK, SW, SA, TT, AF, and CV: conceptualization, methodology, and review and editing. TT: data curation. WZ and QT: data analysis. WZ: manuscript drafting. All authors contributed to the article and approved the submitted version.


Higher nutrition knowledge (p < 0.0001) and higher BMI (p = 0.0370) were correlated with lower nHDI. Higher pHDI characterized people with more favorable attitudes (p < 0.0001) and those using a diet (p = 0.0002). Nutrition knowledge showed an adverse association with nHDI  and no association with pHDI. Thus, declarative nutrition knowledge does not seem to be a good indicator of healthy dietary behavior. Nutrition education programs that concentrate only on knowledge of facts, and neglect the development of favorable attitudes towards food and nutrition, may not be efficient enough to develop adequate dietary behavior of students.The purpose of this study was to examine the relationships between diet quality, dieting, nutrition knowledge and attitudes in a group of Polish young adults. A cross-sectional survey was conducted in 2018 amongst 638 students of food and nutrition-related majors. Based on the frequency of consumption of 24 food groups, the “Pro-Healthy Diet Index” (pHDI) and “Non-Healthy Diet Index” (nHDI) were calculated. To assess the nutrition knowledge, the “GAROTA” test was used. The k-means clustering method was used to identify clusters-attitudes towards food and nutrition. The relationships between pHDI and nHDI indices, dieting, nutrition knowledge (NK), and attitudes towards food and nutrition were verified, using multiple linear regression analysis. The results confirmed some relationships between the variables. Higher nHDI characterized males ( During the transition from adolescence to adulthood, young people are at a greater risk of engaging in inadequate food habits  which, iDuring the transition from adolescence to adulthood, the acquisition of nutrition knowledge may still be related to schooling, where one acquires information from qualified sources without personal interpretation. Simultaneously, the social environment, including social media, can be an important source of nutrition information and attitudes towards food and nutrition . The groMany studies have reported that university students do not consume an adequate diet ,14, whicPrevious studies, both in Europe and other countries, used a variety of tools to measure nutrition knowledge, i.e., the General Nutrition Knowledge Questionnaire (GNKQ)  and the Students majoring in nutrition and medicine are a group of particular interest . The choA cross-sectional survey was conducted between January and March 2018 by either a trained person or an academic teacher. Only first-year students of food and nutrition-related majors participated in the study. The selection of universities and participants was made arbitrarily by the researchers. From the list of Polish universities with food and nutrition-related majors, four universities were selected. Selected universities were characterized by a large number of people studying these majors and represented different parts of Poland, i.e., Northern (University of Warmia and Mazury in Olsztyn), Central (Warsaw University of Life Sciences), and Southern . The study was conducted during lectures and practical classes. All students invited to the study agreed to participate. A paper and pen personal interview (PAPI) technique was used for data collection. In total, 659 students of food and nutrition-related majors gave voluntary consent to participate in the study. Because of missing data in the questionnaires, 21 students were excluded from the sample. The final sample consisted of 638 young adults aged 18–32 years.The frequency of consumption of 24 food groups was assessed with the Beliefs and Eating Habits Questionnaire (KomPAN) , that waTwo indices were calculated. The first “Pro-Healthy Diet Index” (pHDI) comprised 10 food groups with a potentially beneficial influence on health, while the “Non-Healthy Diet Index” (nHDI), included 14 food groups with a potentially negative influence on health .To standardize the range of both indices, the sum of the frequency of food consumption (times/day) and its expression in scale from 0 to 100 points was calculated. The range of 0–33 points for the pHDI/nHDI index indicates a low adherence to a healthy/unhealthy diet, 34–66 indicates a moderate adherence to a healthy/unhealthy diet, and 67–100 indicates a high adherence to a healthy/unhealthy diet .Moreover, students declared whether they followed a diet .The “GAROTA” test was used to measure nutrition knowledge . The tesTrue/false “easy” questions ;True/false and single-choice “moderately difficult” questions ;Multiple-choice and matching “difficult” questions . For this study, the tool contained 42 questions—three questions from each subject block, and each with a varying level of difficulty. The single-choice questions were selected from the “moderately difficult” questions, and the multiple-choice questions from the “difficult” questions. Respondents received one point for each correct answer to all of types of the questions and 0 points for a wrong answer or lack of an answer. The sum of scores ranged between 0 and 42. The higher the score, the more nutrition knowledge the participant had. A high level of nutrition knowledge is attributed to the participant for 80% or more points, a moderate level—60–79%, a low level—40–59%, and an insufficient level—39% or less.To assess attitudes towards food and nutrition, we used seven statements concerning the links between food, nutrition, and health (four items), as well as food, nutrition, and other life aspects  . Items w2); of normal weight (BMI between 18.5 and 24.9 kg/m2); overweight and obese (BMI ≥ 25.0 kg/m2).The sociodemographic profile of the participants was assessed based on questions related to the following variables: gender; age (in years); place of residence . The body mass index (BMI) of the participants was calculated from the self-reported body weight and height. The participants were categorized into three groups, based on their BMI, according to the classification of World Health Organization : underweDescriptive statistics were used to present the sociodemographic characteristics of the study sample. Data were presented as a sample percentage (%) for categorical data or mean and standard deviation (SD) for continuous data. The normality of the distribution of continuous variables was assessed with the normality Kolmogorov–Smirnov test, the Lilliefors test, and normal probability plot. p < 0.001 for all of the variables). Cluster 1 represented “favorable attitudes” towards food and nutrition, while in cluster 2 “unfavorable attitudes” were observed  according to seven statements concerning links between food, nutrition, health, and other life aspects. In the k-means method, the average values for individual clusters obtained using the hierarchical method were used as seeds. Two separate clusters representing different attitudes towards food and nutrition were obtained, which was confirmed by both statistics’ assessment of the selection of clusters, such as cubic clustering criteria–CC = 27.5, pseudo F = 223, and ANOVA  statistics comparing the average values of variables for individual clusters (observed . The ind2). The pHDI and nHDI scores, treated as continuous variables, were introduced into the models separately as a dependent variable. The independent variables included in the models were: using a diet ; nutrition knowledge ; and attitudes towards food and nutrition . Two models were created as follows: model 1—adjusted for the set of confounders for nHDI score; and model 2—adjusted for the same confounders, as in model 1 and pHDI score. Automatic selection of the variables with the forward selection method was applied to the models. Standardized regression coefficient (β), and unstandardized regression coefficient (B) with a 95% confidence interval (CI) were used. Significance was set at p < 0.05 for all of the analyses. Analysis conducted on the Model 1 and Model 2 explained, respectively, 16% and 14% of variance . The Durbin–Watson test was used to check the assumption of no correlation of the random components. The tolerance statistic was applied to assess the assumption of no correlation of the predictors. The Durbin–Watson statistics were 1.98 for Model 1, and 2.01 for Model 2, which means that the assumption was fulfilled. The tolerance statistics were above 0.1 for both models, thus the assumption about a lack of correlation between the predictors was also fulfilled. Significance was confirmed at p < 0.05. The analyses were carried out using IBM SPSS Statistics for Windows, version 26.0 .The associations between the scores of the pHDI and nHDI indices, dieting, nutrition knowledge, and attitudes towards food and nutrition were verified using multiple linear regression analysis, adjusted for gender , age , place of residence , and BMI , which, according to the accepted criteria in the GAROTA test, indicates a low level of nutrition knowledge. The highest score attained (31 points) corresponded to 73.8% of the maximum score, which means a moderate level of nutrition knowledge .In the study group, 153 people (24%) were currently using a diet. In the group declaring the use of a diet, 24.4% were on an elimination diet, 19.3% on a low-calorie diet, 17.6% on a high-protein diet, and 16% on a vegetarian diet. The mean values of both indices describing dietary characteristics, i.e., pHDI and nHDI, were below 33 points. Thus, the study subjects were characterized by a low intensity of both healthy and unhealthy diets . The associations between nutrition knowledge, attitudes, and diet indices are shown in Most people surveyed confirmed the statements that eating healthy foods can improve their appearance and that food choices are determined by concern for their health. In contrast, the respondents were least likely to describe themselves as people who pay attention to the caloric value of the foods they eat .p < 0.05) differences between both clusters were observed in all of the analyzed statements. In general, lower mean values for all of the statements  were observed in cluster 1 compared to cluster 2 (range between “often” and “sometimes”). Therefore, individuals in cluster 1 represented more favorable attitudes towards food and nutrition, compared to those in cluster 2 (p < 0.001).Based on the participants’ opinions about the links between food, nutrition, and health (four items), as well as between food, nutrition, and other aspects of life (three items) two clusters were identified . StatistSociodemographic variables such as gender, age, education, and place of residence were used to profile the clusters. However, no significant differences were found between clusters for these variables, except for a place of residence . More pePeople from cluster 1 had higher nutrition knowledge than the others . They haThe results of the multiple linear regression analysis that examined the relationship between nHDI, dieting, nutrition knowledge, and attitudes towards food and nutrition are shown in The pHDI showed inverse associations with attitudes towards food and nutrition, as well as not using a diet . A higheThe GAROTA test indicated a low level of nutrition knowledge of students entering food and nutrition-related majors, which is not surprising. Such results are, on the one hand, due to the specificity of the test, in which the questions relate to the level of knowledge offered in the programs of technical secondary schools and universities, as well as available in school and academic textbooks on human nutrition . On the The participants were characterized by a low intake of food with both potentially beneficial (pHDI) and adverse influences on health (nHDI). Other studies showed that the diet of young people is characterized by many nutritional irregularities ,46, and In our study, nutrition knowledge correlated negatively with the unhealthy diet index (nHDI), i.e., greater nutrition knowledge was associated with a lower intensity of unhealthy diets (model 1). This is supported by the findings of other studies, indicating that students with greater nutrition knowledge consumed less unhealthy fats and cholesterol , and ateOne of the other explanations for the results obtained may be the fact that the study group consisted of first-year students. Their characteristics may therefore be closer to those of the general secondary school students’ population , and notThe respondents thought that eating healthy foods could improve their appearance, but they also linked their food choices with a concern about their health, which is confirmed by other studies conducted in the group of young people ,64. The Consistent with previous research , nutritiBased on the obtained results, we verified the hypotheses assuming that nutrition knowledge correlates positively with attitudes toward food and nutrition, and that an unhealthy diet shows a relationship with lower nutrition knowledge and unfavorable attitudes towards food and nutrition. The healthy diet index correlated positively with favorable attitudes, whereas the positive correlation between nutrition knowledge and pHDI was not verified either by two-way correlation, or in a linear regression model. The strength of the relationships between the variables was weak. The results confirmed the stronger relationship of favorable attitudes towards food and health with diet quality (lower nHDI and higher pHDI) than nutrition knowledge alone (only correlation with nHDI). These findings could be used in strategies being developed for young people in Poland, but also in other countries, which justifies the high similarity of specificity of this age group, e.g., widespread use of mass communication, social media, dietary mistakes, eating disorders, etc.The limitation of the study was that the study group was not representative of the Polish student population as a whole, but only of the students of food and nutrition-related majors, representing four of the largest universities which offered such courses. Thus, generalizations and extrapolations at a broad level are not permitted. BMI was calculated based on self-reported data. Moreover, it concerned a specific group, i.e., students of nutrition, and did not reflect the available data of the population of young Poles. In 2019, the 20–29 age group of Poles had fewer underweight people (6.3% vs. 10.3 in the study sample), fewer normal weight people (58.7% vs. 76.1%), and more overweight and obese people (35.2% vs. 13.6%) , that caThe study aimed to examine the relationships between nutrition knowledge, attitudes towards food and nutrition, dieting, and the quality of diet of students entering food and nutrition-related majors; these are particularly important because of their role in shaping proper eating habits among people in the future. The results obtained confirmed the relationship between nutrition knowledge and attitudes towards food and nutrition, whereas the association of these constructs with eating behaviors showed a different character. Nutrition knowledge showed an adverse association with nHDI and no association with pHDI, whereas more positive attitudes towards food and nutrition were associated with a higher pHDI and a lower nHDI, i.e., a more adequate diet. Thus, the findings suggested that declarative nutrition knowledge does not seem to be a good indicator of healthy dietary behavior, especially when nutrition knowledge is estimated to be low. Students completing nutrition majors will gain factual nutrition knowledge, however, there is a need to develop their positive attitudes and the skills that can bring about behavioral change. Nutrition education programs that concentrate only on knowledge of the facts, or so-called declarative knowledge, and that neglect the development of favorable attitudes towards food and nutrition, may not be efficient enough to develop adequate eating behaviors of the students, who will provide nutrition counseling in the future and thus shape the correct eating habits in the rest of population.


This improvement of the physiological and biochemical parameters (chlorophyll contents and phenolic compound) of the treated plants reflected the positive impact of AMF, especially those originating from RP. AMF in phosphate mine could be an important source of inoculum to improve plant nutrient efficiency with the direct use of RP as fertilizer.The improvement of plant growth and yield becomes crucial to feed the rising world population, especially in harsh conditions, drought, salt stress, lack of nutrition, and many other challenges. To cope with these stresses, plants developed an adaptation strategy (mycorrhiza), which is an efficient way to reinforce their growth and resistance. For this purpose, we studied the influence of mycorrhizal fungi isolated from a natural rock phosphate mine in the vicinity of some native plants and agricultural soil to assess their capacity in increasing the growth, nutritional profile improvement, and biochemical parameters in the inoculated wheat plants. Results showed a high diversity of isolated arbuscular mycorrhizal fungi (AMF) spores in the agricultural soil, and less diversity in the natural phosphate samples, where three main genera were identified: glomus, gigaspora, and acaulospora. The chlorophyll content increased by 116% in the native inoculum (NM) flowed by Glomus sp2 from agricultural soil (98%) compared to non-mycorrhized plants, which significantly impact the growth and plant biomass . The same rate of change was shown on total phenolic compounds with an increase of 64% in the plants inoculated with Glomus sp2 in the presence of TSP, compared to the non-mycorrhized plants. In conclusion, the inoculation of wheat plants with AMF spores improved plants’ growth  Phosphorus (P) is the second significant element required for plants growth and development . With niMorocco, China, and the United States are the three main producers of rock phosphate in the world. They hold more than 80% of the exploitable deposits. This rock phosphate presents a good opportunity to be applied directly to the soils and considered as a cheaper fertilizer, affordable for low-income countries. However, there is a need for a smart way to convert insoluble rock phosphate to soluble form, easily available for the plants. The use of microorganisms presents one of the innovative and challenging approaches to RP solubilization. Microorganisms produce phosphatase enzymes  and orgaOne of the first benefits of AMF to the plants is the exploitation of water and nutrients, thanks to a very developed root system constituted principally by a huge hyphal network that connects plant interior to surrounding soil. Thus, hyphae are more extensive and more efficient than root hairs in taking up nutrients . HoweverGlomus fasciculatum and Acaulosporalaevis in Sesamum indicum plants were highest in soil with increased concentration of Rock Phosphate (up to 22.5 mg/kg) , disease, and environmental conditions. Under harsh conditions, Mycorrhizal associations help plants to ensure a better balance of nutrition uptake and tolerance to biotic and abiotic stresses . Accordi5 mg/kg) . On othePisum sativum L., Avena sativa L., and Triticum aestivum L. Their results showed that, with the use of fertilizers, root biomass increased in all studied plants. At the opposite, the colonization intensity decreased when superphosphate was introduced into the soil. In unsuitable soil, the Gumi-Omi-Phosphorus could stimulate both partners to form an arbuscular mycorrhizal symbiosis. The unlikable impact of superphosphate is probably due to the presence of readily available phosphorus in greater quantities in the soil. Hence, better and efficient mycorrhization on the plants has been related to the rate of nutrients mobilization from soil to the plants  (The kind of fertilizer used and the concentration of free P in soil significantly impact mycorrhization efficiency. e plants  and helpe plants . Mycorrhe plants . In addisphorus) ; such a sphorus) . BulgareGenerally, these microorganisms (AMF in association with bacteria) release three groups of enzymes able to mineralize P from organic amalgams in the soil environment . The firIn this work, we aimed to study and explore the diversity and the application of AMF isolated from two different environments: Moroccan phosphate mines and agricultural soil. Also, the ability of isolated AMF to improve the growth and nutrition by using different sources of phosphate (RP & TSP), their effects of the growth performance, and the physiological parameters of wheat plants were also investigated.Digitariasanguinalis, Solanum americanum, and Alternanthera caracasana. The plants were picked up from their natural environment (phosphate mine), and their roots were analyzed for the presence of AMF.Three plant species, which grow spontaneously in the phosphate mine of Ben Guerir , mainly in the humid season, were selected. They were identified as The roots were stained according to the method described by Agricultural soil samples were collected from two regions in the vicinity of Settat Town (70 km in the south of Casablanca): the first zone is Douar Boukallou, Latitude: 33.0319; Longitude: −7.6202, and the second zone is Ouled Said, Latitude: 33,019; Longitude: −7.70.A total of five different soil samples belonging to the two different habitats were used for the AMF spores isolation and investigation as presented in –1) and streptomycin (0.2 g L–1)  and the rock phosphate mines (the BenGuerir region) . Each sa2 g L–1)  in orderGlomus sp.1 (G. sp1), Glomus sp.2 (G. sp2), and Acaulosporalongula (A. long). One other morphotype named Morphotype MN (MN) was selected from phosphate mine samples.In this study, four morphotypes were used for inoculum preparation. Three morphotypes were sampled from agricultural soils and named (Hordeum vulgare) were used for AMF multiplication . Barley plants are characterized by very significant ramification of the root system. The latter is fibrous and extensive, which increases the number of potentially colonizable sites but fragile and requires delicacy in the revelation and the realization of the assembly. This cereal has significant compatibility with a wide range of mycorrhizal genera (Glomus intraradices (MG).Barley plants l genera . FragmenTriticum aestivum) were disinfected by ethanol 90° for 30 s to 1 min, and then by a solution of mercury hypochlorite (1%) for 3–4 min. Disinfected seeds were soaked in sterile water and placed in Petri dishes to germinate in the dark at a temperature of 26°C in an incubator. After germination, the seedlings were transplanted into the growth medium (mixture between peat and soil 2/1) and despatched in six categories, non-mycorrhized plants , non-mycorrhized + RP, non-mycorrhized + TSP, mycorrhized , mycorrhized + TSP, and mycorrhized + RP. RP and TSP were used in this study as two different P fertilization regimes, with 45 μg of RP/g of soil and 15-μg TSP/g of the soil, respectively, in order to assess the effect of the mycorrhizal association on the growth and of phosphate uptake.Wheat seeds  (positive control) and without inoculation (negative control) arranged in randomized block design. The pots were then placed in phytotron at 26°C, photoperiod of 16:8 h with an illumination intensity of 240 μmol m–2 s–1. After 8 weeks, the wheat plants were collected for mycorrhizal richness evaluation and for physiological and biochemical parameters determination.The experimental design consisted of five replications in pots, containing the previous mixture  and two controls: the reference inoculum a and b, respectively, using a spectrophotometer , and the concentrations were determined according to the following formula:Leaf fragments (1 g) were grounded in a mortar previously placed in ice with a pinch of magnesium carbonate and 5 g of anhydrous sodium sulfate in 10 ml of 80% acetone. After filtration, the residue was recovered in test tubes. Other extractions were carried out with acetone until a colorless filtrate was obtained (devoid of any traces of chlorophyll pigments). The different filtrates were combined, and the final volume was noted . Opticala = (0.0127 OD663) – (0.00269 OD 645)Chlorophyll b = (0.0229 OD 645) – (0.00468 OD 663)Chlorophyll 645) + (0.00802 OD 663)Total chlorophyll =  were grounded in a mortar, containing 5 ml of ethanol, 50% . Then, the extracts were collected in tubes with lids and numbered. The tubes were then kept at 42CO3 (20%). Then, 0.5 ml of Folin-Ciocalteu reagent was added. After that, the tubes were placed in an oven at 40°C for 30 min. The absorbance was read at 760 nm, and the content of phenolic compounds was calculated. Gallic acid was used for standard curve determination. Results were expressed in milligrams per gram of fresh leaf matter.In the tubes containing the leaf extracts, 0.5 ml of chloroforms was added to 3 ml of extract, and the tubes were vortexed and centrifuged 5 min at 5,000 rpm; the two phases supernatant and pellet were separated . Total pApproximately 0.4 g of each plants were transferred to digestion tubes and the digestion mixture (Sulfuric acid-Selenium) was added to samples. The digestion tubes were transferred to block digester preheated to 300°C. The analysis were carried out using automated Continuous Flow Analyzer (CFA)  according to the manufacturer instructions.2PO4) was prepared in the range of 0.2; 0.4; 0.6; 0.8 and 1 mg P.L−1 . Univariate analysis was used to test significant differences in treatments, accessions, and their interaction for an individual parameter. All statistical analyses were performed with IBM.SPSS statistics, Version 21. The results of each experiment  were repeated three times. The principal component analysis was made by R software.One-way ANOVA was carried out for each parameter studied. Tukey’s –1, and K with average content of 12 mg.g–1  and conductivity of 0.415 mS. Furthermore, for its mineral composition, the P content was around 51 mg/g, the N between 0.42 and 0.46 mg.g2 mg.g–1 . On otheThe sieving and decanting technique revealed the presence of diversity of spore morphotypes in terms of shape, size, color, as well as the presence of conical structures at the external surface of the spores, or of the hyphae in agricultural soil samples .Glomus  were the most abundant in the agricultural soil samples. They were selected for multiplication and, also, for studying their effect on the growth and the nutritional profile of wheat plants.Spores of the morphotype presented in The studied agricultural soil samples showed also characteristic structures , and, sometimes, small spores of unidentified fungi that were observed, which required further studies .Results of relative abundance showed that the morphotype “Brown” spores were the most abundant in agricultural soil, 32% of the total recovered spores, and then the “yellow-conical” and “yellow” spores with 26 and 25%, respectively . FinallyThe presence of mycorrhiza in phosphate mine was very remarkable in this study, since it is an extreme environment, and phosphate inhibits mycorrhization in general. The sieving and decanting technique revealed the presence of less diversity in rock phosphate samples than in agricultural soil, as expected. The isolation of the spores from the phosphate mine samples showed the presence of four morphotypes (depending on the color criterion): brown, yellow, white-yellow, and white, with different sizes , 4. The Spores of the morphotype (white-yellow) were most abundant in phosphate mine with 39%. Then, the brown and yellow spores with 23 and 20%, respectively ; finallyDigitariasanguinalis, Solanum americanum, and Alternanthera caracasana), with the presence of the penetration hyphae, appressoria, and arbuscules  than the plants inoculated with other two morphotypes  , the mycorrhizal intensity (M%), and the richness in arbuscules (A%) showed a high mycorrhization frequency in the plant inoculated with the longula) . SignifiGlomus sp.1, Glomus sp.2, Acaulosporalongula, and morphotype MN. The formation of a hyphopod . All the mycorrhizae phenotypes significantly increased the growth of wheat plant shoots and roots as presented in aradices . In the e medium . Similarhization . Howeveron to RP .Glomus sp2, which reach nearly the same values as the reference inoculum Glomus intraradices  by 98 and 100%, respectively , the content of total chlorophyll (Ch a + Ch b) is significantly higher (10%) in the presence of phosphate rock and TSP in the medium and reched 2.36 mg/g fresh matter (FM) . Howeverectively .Glomus sp2 + TSP, and the positive control inoculum (Glomusintraradices). This improvement reached a value of 158% for MN (spores isolated from phosphate mine) compared to NM-RP, -TSP, and 19% with MN-RP and TSP.The presence of TSP in the medium  increaseGlomus sp1 and the Acaulosporalongula did not show a pronounced effect.This content is slightly reduced in the presence of RP in the medium compared to the TSP; however, it was still much higher to the mycorrhized plant without RP and TSP , and the non-mycorrhized plant. The inocula acted differently according to the presence of RP in the medium . The inoTotal phenolic compounds (TPC) content changed with the presence of RP and TSP and, also, with mycorrhizae inocula . In the Glomus sp2 and positive control inoculum MG, with 64% of increase compared to non-mycorrhized plants with the presence of TSP (NM + TSP), and 35% on mycorrhized plants without TSP. In parallel, some inocula (MN + TSP) showed higher results than others regarding the TPC content , and 100% compared to mycorrhized plants without any source of P. These interesting results recorded in plants were inoculated with the morphotype MN isolated from phosphate mine. In fact, this mycorrhizae helped the plant to use this source of phosphate efficiently. However, the other inocula acted differently but still showed increased levels of TPC.The AMF present one of the preferential microorganisms for supplying the phosphate to the plants . In thisGlomus etunicatum and Glomus intraradices) for phosphate mine land reclamation. The native plants used for the revegetation of phosphate mine lands in Florida were Aronia arbutifolia (L.) Ell. (red chokeberry), Clethra alnifolia L. (sweet pepperbush), Cornusfoemina Mill. (swamp dogwood), Ilex glabra (L.) Gray , and Viburnum nudum L.The deleterious impact of phosphate mine on plant growth and distribution was investigated along a decade . AccordiDigitariasanguinalis, Alternanthera caracasana, and Conyza canadensis that are native in this environment presented mycorrhizal richness. However, this richeness was significantly lower than that reported in agricultural soil. These species could be good candidates for indigenous spore multiplication. In fact, the phosphate mine is a very challenging environment for plant mycorrhization, not just because of the presence of inorganic phosphate but also the existence of the high rate of metals, which could explain the less abundance of wild-plant species (Moricandia arvensis and Diplotaxis harra (Brassicaceae), were able to tolerate high concentrations of Cd, Ni, Zn, and Cr  has a great ability to establish association with mycorrhizae, especially Funneliformismosseae, Rhizoglomusfasciculatum, and Rhizoglomusintraradices . However, sometimes, the association could be ineffective or non-functional, which explains the non-adaptation of other species in the phosphate mine area.One of the native plants investigated in this work , and many Genus, Glomus (10 species), Rhizophagus (2 species), Funneliformis (4 species), Claroideoglomus (1 species), Dentiscutata (2 species), Gigaspora (3 species), Scutellospora (2 species), Racocetra (1 species), Acaulospora (6 species), Entrophospora (1 species), Diversispora (1 species), and Ambispora (1 species). These genera seem to be highly adapted to the host plants of the region but, of course, differ from one site to another one according to the above-cited parameters.In terms of spores diversity, according to Acacia gummifera and Acacia sp., respectively  . Similar results were reported in other species, such as Lactuca sativa L., Lonicera confusa, and Vicia faba L. G. constrictum. According to the same study, the greatest positive effect on growth was recorded after using a mixture of G. constrictum and RP, which increased phosphorus nutrition. Glomus fistulosum had a positive effect on the growth and the nutritional profile of Leucaena plants in the presence of RP; however, the best efficient results were recorded in experiments using combination of G. fistulosum and phosphate-solubilizing fungi (Mortierella sp.). These results were confirmed by Aspergillus niger and Penicillium citrinum).Regarding inoculation assays in wheat plants, we can confirm that the presence of RP and TSP in a medium impacted significantly the plant growth; this source of phosphate nutrition was exploited differently according to the presence of the AMF inoculum or not, and also according to the type of inoculum. We previously reported that the mycorrhized plants presented a high rate of growth in both parts  , which isilicum) . These sG. intraradices (more than 12%). This increase was higher under stress conditions  where the mycorrhization increased chlorophyll content of plants with more than 64%. According to the same authors, the nutritional profile  was increased. The same results were confirmed by in Digitariasanguinalis, showing that inoculation with Funneliformismosseae, Rhizoglomusfasciculatum, and Rhizoglomusintraradices resulted in about 50% more biomass than uninoculated plants.The results of the positive enhancement in morphologic parameters are supported by the physiological essays, especially the photosynthetic activity, which was impacted by the presence of RP and TSP in the medium and, also, the presence of AMF. The results of this study showed that the presence of RP and TSP in the medium increased the chlorophyll content of wheat leaves, especially with the inoculation of AMF spores. Similar effects were previously reported by The mycorrhization leads to better exploitation and absorption of the nutrients, which explained the increase of chlorophyll content. At the same time, it provides to the plant good water exploitation  . The comGlomus sp2 + TSP), with content varies in the average of 3 mg/g FM  plants and positively impacted other physiological parameters. 2 by the stomata). This stimulation could be linked also to an increase in the leaf area and the hydric status on the leaves , in association with Plantago lanceolata. Results showed that both fungi formed more extraradical mycelium at the higher pH, and both fungi formed more arbuscules and vesicles in low pH, with a high rate of colonization of plants by fungi.In the study of The principal components analysis confirmed the previous results of growth and chlorophyll content, in relation with P sources and the AMF inoculation . The mycGlomus sp1 and Acaulosporalongula almost act in the same way, and the positive control Glomusintraradices and Glomus sp2 almost reflected a similar effect .The inoculation of wheat plants with AMF spores improved the growth of the plants The results showed less diversity of AMF in the natural phosphate samples compared to soil samples.Moreover, it is considered that there is an impact on the phosphate solubilization, which explained the positive results obtained in the mycorrhizal plant with rock phosphate (M + RP). Native plants and AMF from phosphate mines could present a reservoir of unexploited microbial diversity that could help in the agronomic efficiency of rock phosphate used as fertilizer.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.ZH, SA, NE, YZ, RD, BB, and IMK participated in designing the study, data processing, spores identification, performed the statistical analysis, and the draft of the manuscript. All authors read and approved the final manuscript.YZ was employed by OCP Group. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Epidemiological evidence of the associations between metal exposure and gout-related outcomes  is scarce. The aim of the study is to investigate the associations of metal exposure with SUA, hyperuricemia and gout in general adults.In this study, the exposure to five blood metals  of general adults was analyzed based on the National Health and Nutrition Examination Survey (NHANES) from 2011 to 2018 . Linear, logistic and weighted quantile sum (WQS) regression models were applied to examine the associations of blood metals with gout-related outcomes. Possible dose-response relationships were analyzed through restricted cubic spline regression.Compared with the lowest quartile of blood metals, mercury (quartile 2 and 4), lead  and selenium (quartile 2 and 4) were found to be positively correlated with SUA and hyperuricemia. Higher levels of mercury and lead were associated with gout, but only those in the fourth quartile had statistical significance (OR [95%CI]: 1.39 [1.10-1.75] and 1.905 [1.41-2.57]) respectively). The WQS index of the blood metals was independently correlated with SUA (β [95%CI]: 0.17 [0.13-0.20]), hyperuricemia (OR [95%CI]: 1.29 [1.16-1.42]) and gout (OR [95%CI]: 1.35 [1.15-1.58]). Among them, lead was the most heavily weighted component . In addition, restricted cubic spline regression models showed a linear association of lead with the prevalence of hyperuricemia and gout.Our results suggested that blood metal mixtures were positively associated with gout-related outcomes, with the greatest effect coming from lead. Uric acid is an end product of the metabolism of purine analogues. If there is an excessive production (an increased endogenous purine catabolism and an excessive exogenous purine intake) or an impaired excretion of uric acid, it may lead to an increase in the level of serum uric acid (SUA) . HyperurScholars have conducted significant research on the factors that influence hyperuricemia, including basic demographic features such as age and gender, lifestyle factors like smoking and alcohol intake, and chronic disorders such as hypertension, diabetes mellitus, and dyslipidemia. In addition, there is a growing interest in the possibility of being exposed to environmental heavy metals . MetalliIn this study, based on the data from the National Health and Nutrition Examination Survey (NHANES) 2011–2018 on general adults, we assessed the relationship of five metals  and their mixtures in blood with gout-related outcomes  through linear, logistic and weighted quantile sum (WQS) regression models. These associations were evaluated to provide epidemiological evidence for further studies on the correlation of metal exposure with hyperuricemia and gout.The NHANES is a nationwide cross-sectional survey conducted periodically by the National Center for Health Statistics (NCHS) of Centers Disease Control and Prevention (CDC), which aims to assess the health and nutritional status of adults and children in the United States. The research protocols were approved by the National Center for Health Statistics (NCHS) Research Ethics Review Board, and all participants provided written informed consent.https://www.cdc.gov/nchs/nhanes), from which we obtained the data over four cycle years . Participants with missing data on five blood metals  were excluded. Moreover, participants aged <18 with missing information on assessment data of gout  or SUA (n=335) and those who were pregnant (n=149) were ruled out. Lastly, a total of 14,871 participants were included in the analysis .All interview and medical examination data can be downloaded from the official website  to analyze the level of metals in participants. We investigated five metals, namely mercury, manganese, lead, cadmium and selenium, meanwhile analyzing their concentration and distribution in blood. The lower limit of detection (LOD) for blood mercury, manganese, lead, cadmium and selenium was 0.28μg/dL, 0.99μg/dL, 0.07μg/dL, 0.10μg/dL and 24.48 μg/dL respectively. The values that were less than the LOD were substituted with a lower LOD divided by the square root of 2.Serum samples were collected from participants and stored at –30°C until they were transported to NCHS/CDC for SUA testing. Hyperuricemia was defined as a level of SUA that was ≥416 μmol/L (7 mg/dL) in men and ≥357 μmol/L (6 mg/dL) in women [9]. Gout was defined as a physician’s self-reported diagnosis, obtained by asking the following question .2), high-density lipoprotein cholesterol , total cholesterol (mg/dL), urinary creatinine (mg/dL), urinary albumin (ug/mL) and estimated glomerular filtration rate . Poverty was assessed using the poverty income ratio (PIR) and defined according to a PIR cutoff that was <1 for a given family. Participants with a serum cotinine value that was >14 ng/mL were defined as smokers. Individuals consuming at least 12 alcoholic drinks in a single calendar year were defined as alcohol users. The intake of energy, nutrients and other food components was calculated by averaging the two values from two 24-hour recalled interviews. We obtained information on the prevalence of hypertension and diabetes among participants using self-reported questionnaires. In addition, the usage of anti-gout medicines, diuretics, and beta-blockers was recorded.Information on baseline data was collected through questionnaires and laboratory tests, including age (years), gender , education level , race/ethnicity , sedentary time , body mass index  or the median (interquartile range), and the categorical variables were presented as n (%). The Kolmogorov-Smirnov statistical test was used to determine if continuous variables were normally distributed. To normalize their distribution, the blood metals were log-transformed. Spearman’s correlation was utilized to determine the exposure correlation coefficients for all metals.th, 35th, 65th, and 95th percentile of each exposure variable was utilized.Using multiple linear regression models, corrected β-coefficients and 95% confidence intervals (CIs) were calculated to determine the level of SUA associated with the blood metals. Multiple logistic regression models were used to calculate adjusted odd ratios (ORs) and 95% CIs to assess the prevalence rate of hyperuricemia and gout associated with the blood metals. The blood metals were divided into quartiles, with the lowest quartile being considered the reference category. To investigate the dose-response curves of exposure variables and the prevalence of hyperuricemia and gout, a restricted cubic spline regression model with knots at the 5The WQS regression model was used to assess the confounding effect of multiple exposure variables on a given dependent variable, each of which was given a weight in the model to indicate the magnitude of its effect on the dependent variable. In the model, 40% of the data was assigned to the training set and 60% to the validation set, meanwhile the training set was bootstrapped 1000 times to maximize the likelihood function of the linear model.Finally, we conducted a stratified analysis in age (>50 years versus ≤ 50 years) and sex . All models were adjusted by age, sex, education level, race, poverty, smoker, alcohol user, energy intake level, sedentary time, BMI, total cholesterol, HDL-C, eGFR, urinary creatinine, urinary albumin, diuretics, beta-blockers, diabetes and hypertension. We performed all statistical analyses using R software (Version 4.2.0).This study comprised 14,871 adults, whose baseline characteristics are presented in , with the exception of a rather minor link between lead and cadmium (r = 0.34).The concentration and detection rate of the blood metals are listed in P < 0.05), as shown in We used linear regression to assess the relationship between blood metals and SUA. The histogram of residuals showed that the residuals were normally distributed  , and those in the 4th quartile of lead showed a statistically significant increase in the prevalence of gout (OR [95% CI]: 1.905 [1.412-2.572]) compared to the lowest referent quartile. Additionally, the restricted cubic spline demonstrated a positive linear correlation between two blood metals (mercury and lead) and the prevalence of hyperuricemia (P for nonlinearity = 0.382 and 0.741)  Figure 2The adverse relationships between metal mixtures and SUA or the prevalence of hyperuricemia and gout were analyzed using WQS regression analysis Table 5.P for interaction = 0.001), lead (P for interaction < 0.001), and selenium (P for interaction = 0.003) in female are statistically different from those in male. In addition, compared to participants aged <50 years, a significantly stronger positive association was observed between cadmium exposure and the prevalence of hyperuricemia in those aged >50 years (P for interaction < 0.001). Further stratified analyses showed that age and sex did not significantly affect the modification of the association between metal exposure and the prevalence of gout .Stratified analysis was performed to explore further whether subgroups in age  and sex  had differential effects on the association of exposure to blood metals with the prevalence of hyperuricemia and gout S3. InteThis study analyzed the cross-sectional data from the 2011–2018 NHANES project to explore the association between five blood metals  and gout-related outcomes  in 14871 general adults. We found a positive association of an increased level of mercury, lead and selenium with gout-related outcomes. The WQS regression model showed that blood metal mixtures were associated with an increased prevalence of hyperuricemia and gout, with the greatest effect coming from lead. In addition, restricted cubic spline regression showed a linear association of blood lead level with the prevalence of hyperuricemia and gout.Metallic mercury, inorganic mercury compounds, methylmercury and other organic compounds are the primary forms of mercury . MercuryMost lead comes from human activities, including burning fossil fuels, mining, smelting and exposure to products containing lead compound . Lead isSelenium is commonly found in organic or inorganic forms in living organisms , which iCadmium is distributed in the environment at a low level, including cadmium fumes and cadmium compounds , the priInconsistent conclusions have been reached for studies on the association of metals with hyperuricemia and gout, which may be related to differences in demographic characteristics and the interference of confounding factors. In addition to exploring the relationship between a single metal and SUA, researchers have increasingly focused on the effect of metal mixtures on it. Ma et al. investigated the combined effect of metal mixtures in urine on SUA and hyperuricemia using three statistical models, through all of which significant roles of arsenic, cadmium and cobalt were found in changes in SUA and hyperuricemia . Using BThe mechanisms underlying the effect of lead level in the human body on SUA are unclear. It is well known that hyperuricemia, gout, and kidney diseases are closely related . StudiesThe main advantage of this study is that it uses the WQS regression to explore the mixture effects of environmental pollutants on gout-related outcomes, which is more in line with the reality of exposure. In terms of study design, most of them involved single metal exposure. However, humans are simultaneously exposed to multiple metals, of which the levels are usually highly correlated. The WQS statistical approach ensures that our results are not confounded by exposure collinearity issues, which allows a specific metal’s statistical significance to be independent of the effect of co-exposure to other metals. Moreover, we identified that lead was the largest contributor to the positive association of metal mixtures with the prevalence of hyperuricemia and gout, which had rarely been assessed in previous studies. Thirdly, since the sample size of this study is large and representative, the conclusions can be generalized to other American adults. Fourthly, the exposure to metals in blood is analyzed in this study, which is different from that in urine . AkerstrThis study has several limitations. First of all, as a cross-sectional study, we were unable to demonstrate a causal association. Secondly, a history of gout based on self-reports might not be completely accurate. Thirdly, blood metals were measured simultaneously without repeated measurements, which might not represent long-term exposure to metals. Finally, the exact mechanism through which metals caused changes in SUA was not known and needed to be confirmed by further research.In this cross-sectional study based on general adults, we found an association between the level of blood mercury, lead and selenium and SUA. Our findings suggested that metal mixtures in blood were associated with an increased prevalence of hyperuricemia and gout, with the greatest effect coming from lead. Additional research is necessary to confirm these findings and clarify the underlying mechanism.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the National Center for Health Statistics Research Ethics Review Board. The patients/participants provided their written informed consent to participate in this study.JX: Conceptualization, Methodology, Formal analysis, Writing-original draft. XZ: Conceptualization, Methodology, Software, Visualization, Writing-original draft. RH: Project administration, Supervision. YX: Project administration, Supervision. SS: Conceptualization, Methodology, Supervision. YZ: Conceptualization, Project administration, Supervision. LZ: Conceptualization, Methodology, Project administration, Writing-review and editing, Supervision. All authors contributed to the article and approved the submitted version.This work was supported by the Science and Technology Talents Support Program of Shaanxi Provincial People’s Hospital .We appreciate the people who contributed to the NHANES data we studied.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.