Alterations in Gut Vitamins and Amino Acids Metabolism are Associated with Symptoms and Neurodevelopment of Children with Autism Spectrum Disorders

Background Accumulated evidence have supported metabolic disturbance may be associated with the pathogenesis of autism spectrum disorders (ASD). Despite abnormalities of some shared metabolic pathways, specic differential compounds are inconsistent in studies, which made a challenge to elucidate the role of metabolism in the mechanism of ASD. Besides, few researches have assessed the correlation between gut metabolites with symptoms of ASD. Objectives The present study aimed to evaluate the gut metabolomic proles of children with ASD and to analyze potential interaction between gut metabolites with symptoms and neurodevelopment of ASD children. Methods In this cross-sectional case-control study, 120 aged 2–6 years ASD children and 60 sex and age matched typically developing (TD) children were included. Autistic symptoms were assessed with the Autism Behavior Checklist (ABC), Childhood Autism Rating Scale (CARS), and the Social Responsiveness Scale (SRS). Neurodevelopment was assessed with the Gesell Developmental Scale (GDS). Fecal samples were analyzed by untargeted liquid chromatography-mass spectrometry (LC-MS) methods, then systematic bioinformatic analyses were performed to characterize the gut metabolomic proles of ASD and TD children. The correlations between metabolites and clinical assessment scores were assessed using Spearman correlation. and amino acids metabolism pathways. Notably, vitamins metabolism abnormalities may play roles in the disturbance of amino acids metabolism. Imbalanced gut metabolites are related to symptoms and neurodevelopment of ASD children. Our ndings provided an improved understanding of perturbations of metabolome networks in ASD.

1. Introduction also observed in the feces of ASD children, which may in uence the balance between excitation and inhibition [6] [10]. Abnormality of tryptophan metabolism and increase of serotonin (5-hydroxytryptamine, 5-HT) in gut of ASD patients has been reported in several studies [11]. Also, isopropanol and phenol substances, including phenol and p-cresol, were found higher in fecal of children with ASD [6] [12]. These ndings suggest that alterations of gut metabolomics may play an important role in the pathogenesis of ASD.
However, speci c compounds are inconsistencies between studies, for multiple potential confounds including ethnicity, age, diets, disease, and medicine can in uence the metabolism status. Besides, methodology of is also a critical factor impact the metabolism ndings. Inconsistent and scattered changes of single metabolites have a limited role in elucidating the pathophysiology of ASD, and thus a comprehensive interpretation of the metabolism pathway network may facilitate exploring the pathogenesis of ASD.
In the present study, we analyzed fecal metabolomic pro les of preschool children with ASD and age, sex, region matched typically developing (TD) children by liquid chromatography-mass spectrometry (LC -MS) methods. We found the differential metabolites between ASD and TD children mainly involved in multiple vitamins and amino acids metabolism pathways. We also investigated the possible link between the gut metabolites with symptoms and neurodevelopment of ASD children, and postulated the interconnection of vitamins and amino acids in the metabolism network of ASD.

Subjects And Methods
2.1. Subjects more serious autistic symptoms. Neurodevelopment in ASD children was assessed with the revised Gesell Developmental Scale (GDS) [16] which is extensively used in China to evaluate cognitional and behavioral development, and the development quotient scores (DQ) re ect the levels of intellectual and behavioral development. DQ <75 indicates developmental delay, and the lower DQ score, the more severe developmental delay.
Additionally, a control group of 60 typically developing (TD) children was recruited and matched to the ASD group by age, gender, and region. The TD children received health examinations at the Department of Child Health in Maternal and Child Care Health Hospital of Hainan Province. They are healthy, and they did not have any signs of developmental disorders or psychiatric diseases, and noticeable gastrointestinal symptoms. Other exclusion criteria were the same as for the ASD group. Fecal samples were collected from each participant and immediately frozen and stored at −80°C until further analysis. The 100 mg of stool for each sample was preserved in sterile tubes for metabolism analysis.

2.2.2.Metabolites extraction
The 100 mg of stool for each sample was separately ground with liquid nitrogen and the homogenate was resuspended in prechilled 80% methanol and 0.1% formic acid by vortexing thoroughly. Samples were incubated on ice for 5 min then centrifuged at 15,000 rpm at 4°C for 5 min. Some supernatants were diluted with LC-MS-grade water to a nal concentration of 60% methanol. Hereafter, samples were transferred into a fresh Eppendorf tube through a 0.22 μm lter then centrifuged at 15,000 g at 4°C for 10 min. Finally, the ltrate was injected into the LC-MS/MS system for analysis.

UHPLC-MS/MS analysis
LC-MS/MS analyses were performed using a Vanquish UHPLC system (Thermo Fisher, USA) and an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher, USA). Brie y, metabolites were rst separated and characterized by using a liquid chromatography system and further detected with a mass spectrometry system. Samples were injected onto an Hyperil Gold column (100 × 2.1 mm, 1.9 μm) at a ow rate of 0.2 mL/min and separated using a 16 min linear gradient. The eluents for positive polarity mode were eluent A (0.1% formic acid in water) and eluent B (methanol). The eluents for negative polarity mode were eluent A (5 mM ammonium acetate, pH 9.0) and eluent B (methanol). The solvent gradient was set as follows: 2% B 1.5 min, 2-100% B 12.0 min, 100% B 14.0 min, 100-2% B 14.1 min, 2% B 16 min.
The Q Exactive HF-X mass spectrometer was operated in positive/negative polarity mode with a spray voltage of 3.2 kV, a sheath gas ow rate of 35 arb, an aux gas ow rate of 10 arb, and capillary temperature of 320°C.

Metabolite analysis
Compound Discoverer 3.0 (CD 3.0, Thermo Fisher) was used to process and normalize the raw data les generated by UHPLC-MS/MS to perform peak alignment, peak selection, and quanti cation for each metabolite. The main parameters were set as follows: retention time tolerance 0.2 min, actual mass tolerance 5 ppm, signal intensity tolerance 30%, signal/noise ratio 3, minimum intensity 100,000. Peak intensities were normalized against the total spectral intensity, and normalized data were used to predict the molecular formula based on additive ions, molecular ion peaks, and fragment ions. Peaks were matched with mzCloud (https://www.mzcloud.org/) and ChemSpider (http://www.chemspider.com/) databases to obtain accurate qualitative and relative quantitative results.
The normalized metabolism data were analyzed by the CentOS (CentOS release 6.6), statistical software R (R version R-3.4.3), and SPSS statistical software (version 19.0, SPSS Inc., USA). With individual metabolite dataset, Partial least squares discriminant analysis (PLS-DA) models were built to visualize the metabolic alteration patterns between ASD and TD children. Furthermore, the cross-validation ANOVA (CV-ANOVA) was calculated to assess the reliability of the models. Differential metabolites between the two groups were selected by combined multivariate and univariate analysis methods. Gut metabolites with fold change >1.5, variable important in projection (VIP) >1, and FDR-corrected P values < 0.05 for student's t-test or Mann-Whitney U test were considered signi cantly differential metabolites between groups. To further demonstrate the biological functions of the associated differential metabolites, the KEGG pathways enrichment analysis was performed (http://www.genome.jp/kegg/), and a hypergeometric test was used to assess the signi cance of KEGG pathway.
The metabolomics analysis was carried according to the standard protocols recommended by Novogene Technology Co., Ltd. (Beijing, China). The raw data were deposited into the MetaboLights database (accession number: MTBLS1946, www.ebi.ac.uk/metabolights).

Statistical Analysis
The demographics and clinical assessment data were analyzed using SPSS statistical software (version 19.0, SPSS Inc., USA). Continuous variables are described as the means with standard deviations or medians (interquartile ranges) when appropriate, and categorical variables are described as percentages.
The two-tailed student's t-test, Mann-Whitney U test, and the chi-square test were used to compare levels between groups. The correlations between metabolites levels with clinical symptoms scores were analyzed by Spearman correlation. P-value < 0.05 was presumed as statistically signi cance.

Characteristics of the subjects
A total of 120 ASD children aged 2-6 years and 60 TD children matched to the ASD group by age, gender and region were selected for this study. Demographic information and clinical features were shown in Table 1. There were no signi cant differences in age-gender composition and z-score of body mass index (BMI) between the two groups. In all of 120 ASD children, 80(66.67%) showed food selectivity, and 58(48.33%) had GI symptoms.

Alterations in gut metabolism pro les of ASD children
To explore the gut metabolic patterns associated with ASD status, a fecal metabolome analysis was performed by LC-MS/MS. A total of 6936 peaks of compounds were obtained, among which 4531 were explored in positive ion mode (ESI+) and 2405 in negative ion mode (ESI-). The supervised PLS-DA showed that the ASD and TD groups were well-clustered with particular metabolic pro les for each(ESI+: R2Y = 0.73 Q2 = 0.61, P <0.001 ; ESI-: R2Y = 0.76 Q2 = 0.65, P <0.001 ) (Figure 1a-b). The permutation test with P <0.001 indicates that the classi cation of global metabolite pro les between ASD and TD are signi cantly different. A total of 96 differential metabolites between the ASD and TD groups were identi ed, 35 of which were signi cantly increased in the ASD group, and 61 metabolites were decreased in the ASD group. Table S1 showed the list of the differential gut metabolites between the ASD and TD groups that achieved statistical signi cance.
To further demonstrate biological functions of the differential metabolites, KEGG pathways enrichment analysis was performed. Twenty-seven KEGG pathways were associated with ASD status (Table S2). Interestingly, the differential metabolites mainly participated in multiple vitamins and amino acids metabolism pathway, with the strongest enrichment identi ed for tryptophan metabolism (P = 0.0006), retinol metabolism(P = 0.009), and cysteine and methionine metabolism (P = 0.008), and vitamin digestion and absorption (P = 0.01) ( Figure 1c). Also, some differential metabolites involved in arachidonic acid, steroid hormone, citrate cycle, and purine metabolism.
As shown in Table 2 and Figure 2, disturbances of various vitamins metabolism were found in children with ASD. In the retinol metabolism pathway, precursors and intermediates of vitamin A showed abnormal levels in fecal of ASD children. Level of 4'-apo-beta-carotenal, b,e-carotene-3,3'-diol, and retinal were increased while retinol was decreased. Concentrations of multiple vitamins B and their derivates were decreased, including thiamine-pyrophosphate (TPP), ribo avin (vitamin B2) and intermediates, vitamin B5, vitamin B6, nicotinate, dihydrofolate (DHF), and 5-methyltetrahydrofolate(5-MTHF). Vitamin C level was also decreased in the feces of children with ASD.
Aberrant amino acids metabolisms were associated with ASD status (Figure 2). In the tryptophan metabolism pathway, concentrations of xanthurenic acid, 5-hydroxy-N-formylkynurenine, 5hydroxytryptophan (5-HTP), serotonin (5-hydroxytryptamine, 5-HT) and N-feruloyl serotonin were signi cantly increased in feces of children with ASD. In contrast, 6-hydroxymelatonin and 5hydroxyindoleacetic acid (5-HIAA) were lower in ASD children. The cysteine-methionine metabolism pathway is closely relevant to folate metabolism. Both pathways were abnormality presents with a lower level of DHF, 5-MTHF, carnitine N-acetylcysteine (NAC), and S-aminoethyl-cysteine, while an excessive accumulation of homocysteine (Hcy) in ASD children. At arginine metabolism, concentrations of polyamines, including agmatine, spermine, and glutathione spermidine, were lower in ASD children. We also nd abnormal glutamate metabolism and glycine metabolism, with decreased glutamine, γaminobutyric acid (GABA), and glycine in children with ASD.
Biologically active metabolites of arachidonic acid showed disturbance, which were crucial regulators in oxidative stress and in ammation. Arachidic acid and 20-hydroxy-leukotriene E4 were increased while leukotriene B4 and 5-trans prostaglandin F2β were decreased in feces of ASD children. Besides, 8hydroxy-deoxyguanosine(8-OHdG), a purine metabolite, is a sensitive marker of oxidative DNA damage [17]. In the present study, 8-OHdG was signi cantly increased with 6.86-fold (P = 0.001) in autistic children compared to TD children.

Correlation between gut metabolites with symptoms and neurodevelopment of ASD children
Spearman correlation were performed to explore the potential links between key fecal metabolites and clinical assessment scores of ASD children ( Figure 3 showed the signi cant correlations). The fecal concentrations of S-aminoethyl-L-cysteine, 5-trans prostaglandin F2β retinol, and ribo avin were positively correlated with neurodevelopment scores, while 8-OHdG, 5-hydroxy-N-formylkynurenine, Hcy, retinal, and serotonin were negatively correlated with neurodevelopment scores. The levels of agmatine, S-aminoethyl-L-cysteine, pyridoxamine, GABA, and 5-trans prostaglandin F2β were negatively correlated with partial subscales or total ABC, SRS or CARS scores. Conversely, concentrations of retinal, Hcy, serotonin, N-feruloyl serotonin, and 5-HIAA in the gut were positively correlated with symptoms of ASD children.

Discussion
This study reports signi cantly different gut metabolomic pro les between young children with ASD and TD children. Interestingly, the differential fecal metabolites are majorly involved in multiple vitamins and amino acids metabolism pathways, with the strongest enrichment identi ed for tryptophan metabolism, retinol metabolism, cysteine and methionine metabolism, and vitamin digestion and absorption. Some of the metabolic perturbations were associated with symptoms and neurodevelopment of ASD children, which may play important roles in the pathogenesis of ASD through the "gut-brain axis".
Vitamin A is an important micronutrient for the systemic development and function of children [18]. Vitamin A de ciency (VAD) is still a public health issue in many developing countries. Studies showed ASD children are more vulnerable to VAD than neurotypical children [19] [20]. In our study, an increased level of 4'-apo-beta-carotenal, b,e-carotene-3,3'-diol and decreased retinol may indicate that ASD children had decreased capacity of absorption and bioconversion of plant-origin precursors of vitamin A. Vitamin A has three active forms, retinal, retinol and retinoic acid (RA) in humans [21]. RA, the main active form of vitamin A, is a crucial signaling molecule that regulate multiple fundamental biological processes [21].
Increased retinal in our study may imply it was suppressed to convert to RA in the gut of ASD children, and excessive retinal may damage the nervous system. We found retinol level was positively correlated with neurodevelopment level while retinal was positively related to the social withdrawal of SRS in ASD children. ALDH1A family are key enzymes to oxidize retinal into RA, and XX Xu et al. [22] found ASD patients with excessive UBE3A (an autism related gene and molecule) may have congenital errors of retinol metabolism, for excessive UBE3A can inhibit the activity of ALDH1A and compromised retinal oxidized to RA. Moreover, gut microbiota can take part in alternative biotransformation of retinal to retinol or RA [23].
Vitamins B are important cofactors implicated in multiple biochemical reactions. TPP, a derivative of thiamine (vitamin B1), is a cofactor of various enzymes in the mitochondria 5. Anwar A et al. [24] found TPP concentration in the plasma of ASD children was signi cantly reduced compared to controls. We also found a lower level of TPP in feces of children with ASD. Decreased TPP can lead to a reduced potential of anti-oxidative and energy produce in mitochondria, and subsequently cellular [25] [26].
Vitamin B2 and B6 also participate in multiple amino acids metabolism process. We also found the level of pyridoxamine, a form of vitamin B6, was slightly negatively related to the ABC and CARS scores of ASD children.
The pathway of cysteine and methionine cycle, folate(vitamin B9) metabolism, and Hcy transsulfuration are interrelated and constitute the folate-related metabolism together [27]. The folate-related pathway has a critical role in cell proliferation, DNA synthesis, immune function, and neural development [28]. Vitamin B6 and B12 are cofactors of these biological processes. Decreased folate and vitamin B6 may lead to an accumulation of homocysteine and decreased methyl production. Much of evidence suggested that folate de cit and excessive Hcy are risk factors of neural tube defects and neurodevelopmental disorders [29]. Several studies showed that children with ASD had decreased folate and elevated levels of Hcy in blood and urine [30] [31]. There was a negative correlation between Hcy levels with neurodevelopment scores in our study, supporting the adverse impact of excessive Hcy on brain development and function.
Moreover, NAC is an antioxidant, and clinical trials showed NAC has potential bene t in treating the irritability of ASD children [32].
Abnormality of tryptophan metabolism in patients with ASD has been reported in multiple studies, which was characterized by decreased concentrations of tryptophan [33] and increased levels of serotonin in blood [11]. In the gut, there are three main tryptophan metabolism pathways leading to serotonin, kynurenine, and indole derivatives[34] [35]. Through the kynurenine pathway, kynurenic acid, xanthurenic acid, and quinolinic acid are generated [35]. In our study, concentrations of xanthurenic acid and 5hydroxy-N-formylkynurenine were signi cantly increased in the feces of children with ASD. Vitamin B6 is a cofactor of kynureninase and kynurenine aminotransferase; therefore, the decrease of B6 may contribute to the increased levels of xanthurenic acid and 5-hydroxy-N-formylkynurenine. In the serotonin pathway, 5-HTP, serotonin, and N-feruloyl serotonin were signi cantly increased in feces of children with ASD, while 6-hydroxymelatonin and 5-HIAA were lower in ASD children. Reproducible evidence suggested impaired serotonin-melatonin pathway in ASD characterized by hyperserotonemia and melatonin de cit in plasma [11][36] [37]. However, few studies reported tryptophan metabolism and serotonin-melatonin levels in the gut of ASD patients. MD Angelis et al. [12] found higher amounts of tryptophan and 3methylindole in the feces of children with ASD. Dan Z et al. [38] also reported abnormal metabolism of tryptophan in the gut of ASD children. An experiment in mice model of autism found a decrease of serotonin in intestine mucosal [39]. However, given 95% of the serotonin in body is generated in the intestine [40], it is likely that blood serotonin levels are correlated with enteric serotonin. Likewise, gastrointestinal tract are also important source of melatonin besides the pineal gland [41]. Melatonin can regulate sleep patterns, immune system, as well as gastrointestinal function [41]. Serotonin can be catabolized to 5-HIAA, and this process depends on ribo avin (vitamin B2) as a cofactor, so ribo avin de ciency may be related to the increase of serotonin. Moreover, dysbiosis of gut microbiota was linked to abnormal tryptophan metabolism [35]. We found a correlation between gut serotonin levels with neurodevelopment scores of ASD children, while serotonin and N-feruloyl serotonin levels were positively correlated with sensory subscales of ABC. Many researches have indicated that the blood levels of serotonin are correlated with autism severity [36]. A Balanced amount of enteric serotonin is bene cial to the functioning of the intestine, nervous system, and gut-brain axis, while excess serotonin may play a harmful role in the progression of ASD.
We found decreased GABA, glutamine, glycine, and polyamines in fecal of children with ASD. GABA were negatively correlated with ABC scores, and agmatine were negatively correlated with SRS scores. These amino derivates are crucial neurotransmitters or neuromodulators in nerve system, and yet are important media of immune and in ammation [42]. GABA and glycine are inhibitory neurotransmitters, and their decrease may impact the excitation-inhibition balance of nervous system [43]. Our ndings were partial supported by previous study of Kang DW et al [6]and Angelis et al. [12], which showed possibly lower GABA concentrations in guts of children with ASD compared with healthy controls. Ford et al. found aberrant glutamate and GABA processes are linked with impaired psychosocial function [44]. Particularly, the synthesis of GABA and glycine depend on vitamin B6 as cofactor [45].
Biologically active metabolites of arachidonic acid showed disturbance, which were key regulators in oxidative stress and in ammation [46]. Besides, 8-OHdG, a purine metabolite, is a sensitive marker of oxidative DNA damage [17]. Studies showed evaluated 8-OHdG levels in cerebellar [47] and urinary excretion [48] of ASD patients. In the present study, 8-OHdG was signi cantly increased with 6.86-fold in autistic children compared to TD children. These results in together indicate that the gastrointestinal tract of ASD children may have higher risk damaged by oxidative stress and in ammation.
Gut microbiota was an important role in the gut metabolism, for micro ora can produce vitamins and participant in the metabolic of numerous substances [49] [23]. Inadequate intake from food could also partly explain the decreased of multiple vitamins and amino acids. Further, the abnormality and de ciency of vitamins may play roles in the disturbance of amino acids metabolism, for vitamins B are implicated in multiple biochemical reactions [45]. Metabolic interventions for ASD patients mainly include supplementation of prebiotics and probiotics, vitamins (e.g., A, C, D, B6, B12, folate), amino acids and derivates (e.g., glycine, N-acetylcysteine) [50]- [53]. Approaches above could sometimes correct the dysbiosis of intestinal ora or nutritional de ciencies in ASD, and partly improve the downstream metabolic consequences. However, these interventions were not always effective [53], for some inborn metabolism errors are hard to be recti ed, and single compound supplementation may be insu cient for extensive abnormalities of metabolic networks in ASD. Besides, metabolism anomaly was only one of many factors related to neurological function and symptoms of ASD.

Limitations
There are limitations in the present study. This cross-sectional study revealed a correlation, but our data do not allow to prove causation of symptoms and gut metabolites outcome. And the correlations were not very strong (correlation coe cient 0.2-0.4), for the metabolic disturbance was just one of many factors related to neurological function and symptoms of ASD. It is unlikely to distinguish whether the metabolites are derived from the host or the gut microbiota. Our participants were preschool children from an island of China with a comparable biology backgrounds; the ndings may not be generalizable to all ASD patients in different regions, races, and ages. ASD children were mostly accompanied by other developmental or behavioral disorders. In our study, 81.7% ASD children had developmental delay, so studies involving different ASD subtypes and other related diseases are needed to evaluate the disease speci city of the metabolomic disturbance. Some metabolic disturbances may be nonspeci c for various neurodevelopmental diseases and have an extensive impact on brain function and neurodevelopment.

Conclusions
In conclusion, ASD children exhibit gut metabolism perturbation mainly associated amino acids and vitamins metabolism, and the imbalance of gut metabolism are related to symptoms and neurodevelopment of children with ASD. Aberrant of gut metabolism pro les may be the result of the interaction of multiple factors, including congenital metabolism errors, decreased intake by abnormal eating pattern, and intestinal micro ora imbalance (Fig. 4). Notably, in the interrelated metabolism networks, vitamins metabolism abnormalities and decreased intake of vitamins may disturb the amino acids metabolism, for vitamins B are essential cofactors implicated in multiple biochemical reactions. The metabolites may affect the brain development and function, and subsequently behavior by nutrients, neurotransmitter, immune-in ammation modulatory, and other pathways. Approach of nutritional supplements and regulating the intestinal ora may be partially bene cial to gut metabolism, nutritional status, and symptoms of ASD. It is essential to formulate detailed evaluation and provide comprehensive and individualized interventions for ASD children. Our ndings provided an extensive understanding of the disturbances of metabolism networks in ASD.

Consent for publication
Not applicable.

Availability of data and materials
All data generated and analyzed in the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.  The two-tailed Student's t test, and the chi-square test were used to analysis. TD = typically developing; ASD = autism spectrum disorders.  Figure 1 Alterations in the gut metabolome of children with ASD compared with typically developing children. (a,

Figures
b) The clustering analyses of partial least-squares discriminant analysis (PLS-DA) of gut metabolome data in positive ion mode (a) and negative ion mode (b). (c) Top 20 KEGG pathways enriched by differential gut metabolites between ASD and TD children. Count, the number of differential metabolites in the pathway. Ratio=the ratio of number of differential metabolites to all detected metabolites in the pathway. P value, P value of hypergeometric test. TD = typically developing; ASD = autism spectrum disorders.

Figure 2
Metabolism pathway networks of the differential metabolites between ASD and TD group Gut metabolites with fold change >1.5, variable important in projection (VIP) >1, and FDR-corrected P values < 0.05 for student's t-test or Mann-Whitney U test were considered signi cantly differential metabolites between groups. Red font (↑), metabolites increased in ASD group; Green font (↓), metabolites decreased Page 23/25 in ASD group; black font, no signi cant difference between ASD and TD groups; grey font, undetected.

Figure 3
Correlations between gut metabolites with symptoms and neurodevelopment of ASD children The correlation coe cient is indicated by a color gradient from green (negative correlation) to red (positive correlation). The * symbol in each lattice represent a signi cant correlation. * P < 0.05, ** P < 0.01.

Figure 4
Hypothesis of interplay between the gut metabolism and the gut-brain axis in ASD Aberrant of gut metabolism pro les may be the result of the interaction of multiple factors, including congenital metabolism errors, decreased intake by abnormal eating pattern, and intestinal micro ora imbalance. In the interrelated metabolism networks, vitamins metabolism abnormalities and decreased intake of vitamins may disturb the amino acids metabolism, for vitamins B are essential cofactors implicated in multiple biochemical reactions. The metabolites may affect the brain development and function, and subsequently behavior by nutrients, neurotransmitters, immune-in ammation modulatory, and other pathways.
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