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Urinary metabolomics of young Italian autistic children supports abnormal tryptophan and purine metabolism

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ABSTRACT

Background: Autism spectrum disorder (ASD) is still diagnosed through behavioral observation, due to a lack of laboratory biomarkers, which could greatly aid clinicians in providing earlier and more reliable diagnoses. Metabolomics on human biofluids provides a sensitive tool to identify metabolite profiles potentially usable as biomarkers for ASD. Initial metabolomic studies, analyzing urines and plasma of ASD and control individuals, suggested that autistic patients may share some metabolic abnormalities, despite several inconsistencies stemming from differences in technology, ethnicity, age range, and definition of “control” status.

Methods: ASD-specific urinary metabolomic patterns were explored at an early age in 30 ASD children and 30 matched controls (age range 2–7, M:F = 22:8) using hydrophilic interaction chromatography (HILIC)-UHPLC and mass spectrometry, a highly sensitive, accurate, and unbiased approach. Metabolites were then subjected to multivariate statistical analysis and grouped by metabolic pathway.

Results: Urinary metabolites displaying the largest differences between young ASD and control children belonged to the tryptophan and purine metabolic pathways. Also, vitamin B6, riboflavin, phenylalanine-tyrosine-tryptophan biosynthesis, pantothenate and CoA, and pyrimidine metabolism differed significantly. ASD children preferentially transform tryptophan into xanthurenic acid and quinolinic acid (two catabolites of the kynurenine pathway), at the expense of kynurenic acid and especially of melatonin. Also, the gut microbiome contributes to altered tryptophan metabolism, yielding increased levels of indolyl 3-acetic acid and indolyl lactate.

Conclusions: The metabolic pathways most distinctive of young Italian autistic children largely overlap with those found in rodent models of ASD following maternal immune activation or genetic manipulations. These results are consistent with the proposal of a purine-driven cell danger response, accompanied by overproduction of epileptogenic and excitotoxic quinolinic acid, large reductions in melatonin synthesis, and gut dysbiosis. These metabolic abnormalities could underlie several comorbidities frequently associated to ASD, such as seizures, sleep disorders, and gastrointestinal symptoms, and could contribute to autism severity. Their diagnostic sensitivity, disease-specificity, and interethnic variability will merit further investigation.

Electronic supplementary material: The online version of this article (doi:10.1186/s13229-016-0109-5) contains supplementary material, which is available to authorized users.

No MeSH data available.


Metabolic pathway analysis plot. Color intensity (white to red) reflects increasing statistical significance, while circle diameter covaries with pathway impact. The graph was obtained plotting on the y-axis the −log of p values from the pathway enrichment analysis and on the x-axis the pathway impact values derived from the pathway topology analysis
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Fig3: Metabolic pathway analysis plot. Color intensity (white to red) reflects increasing statistical significance, while circle diameter covaries with pathway impact. The graph was obtained plotting on the y-axis the −log of p values from the pathway enrichment analysis and on the x-axis the pathway impact values derived from the pathway topology analysis

Mentions: The urinary metabolomes of young autistic and typically developing children are largely distinguishable on the three-dimensional OPLS-DA plot depicting the first three principal components (PC), which together explain 31.4% of the total variance (Fig. 1; accuracy, Q2 and R2 data are shown in Additional file 6). Approximately 10,000 peaks per sample were obtained referring to the KEGG database; among them, 202 metabolites were analyzed more precisely and identified. The top 25 most discriminating metabolites between cases and controls were further defined based on “variable influence on the projection” (VIP) scores >1 (Fig. 2). ROC analysis using this set of 25 metabolites yielded an AUC = 0.893 (95% CI 0.72–0.96), as shown in Additional file 7. The “metabolome overview” obtained through metabolic pathway analysis (MetPA) shows tryptophan metabolism, purine metabolism, vitamin B6 metabolism, and phenylalanine-tyrosine-tryptophan biosynthesis as the four most perturbed metabolic pathways in ASD (Fig. 3).Fig. 1


Urinary metabolomics of young Italian autistic children supports abnormal tryptophan and purine metabolism
Metabolic pathway analysis plot. Color intensity (white to red) reflects increasing statistical significance, while circle diameter covaries with pathway impact. The graph was obtained plotting on the y-axis the −log of p values from the pathway enrichment analysis and on the x-axis the pathway impact values derived from the pathway topology analysis
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5121959&req=5

Fig3: Metabolic pathway analysis plot. Color intensity (white to red) reflects increasing statistical significance, while circle diameter covaries with pathway impact. The graph was obtained plotting on the y-axis the −log of p values from the pathway enrichment analysis and on the x-axis the pathway impact values derived from the pathway topology analysis
Mentions: The urinary metabolomes of young autistic and typically developing children are largely distinguishable on the three-dimensional OPLS-DA plot depicting the first three principal components (PC), which together explain 31.4% of the total variance (Fig. 1; accuracy, Q2 and R2 data are shown in Additional file 6). Approximately 10,000 peaks per sample were obtained referring to the KEGG database; among them, 202 metabolites were analyzed more precisely and identified. The top 25 most discriminating metabolites between cases and controls were further defined based on “variable influence on the projection” (VIP) scores >1 (Fig. 2). ROC analysis using this set of 25 metabolites yielded an AUC = 0.893 (95% CI 0.72–0.96), as shown in Additional file 7. The “metabolome overview” obtained through metabolic pathway analysis (MetPA) shows tryptophan metabolism, purine metabolism, vitamin B6 metabolism, and phenylalanine-tyrosine-tryptophan biosynthesis as the four most perturbed metabolic pathways in ASD (Fig. 3).Fig. 1

View Article: PubMed Central - PubMed

ABSTRACT

Background: Autism spectrum disorder (ASD) is still diagnosed through behavioral observation, due to a lack of laboratory biomarkers, which could greatly aid clinicians in providing earlier and more reliable diagnoses. Metabolomics on human biofluids provides a sensitive tool to identify metabolite profiles potentially usable as biomarkers for ASD. Initial metabolomic studies, analyzing urines and plasma of ASD and control individuals, suggested that autistic patients may share some metabolic abnormalities, despite several inconsistencies stemming from differences in technology, ethnicity, age range, and definition of “control” status.

Methods: ASD-specific urinary metabolomic patterns were explored at an early age in 30 ASD children and 30 matched controls (age range 2–7, M:F = 22:8) using hydrophilic interaction chromatography (HILIC)-UHPLC and mass spectrometry, a highly sensitive, accurate, and unbiased approach. Metabolites were then subjected to multivariate statistical analysis and grouped by metabolic pathway.

Results: Urinary metabolites displaying the largest differences between young ASD and control children belonged to the tryptophan and purine metabolic pathways. Also, vitamin B6, riboflavin, phenylalanine-tyrosine-tryptophan biosynthesis, pantothenate and CoA, and pyrimidine metabolism differed significantly. ASD children preferentially transform tryptophan into xanthurenic acid and quinolinic acid (two catabolites of the kynurenine pathway), at the expense of kynurenic acid and especially of melatonin. Also, the gut microbiome contributes to altered tryptophan metabolism, yielding increased levels of indolyl 3-acetic acid and indolyl lactate.

Conclusions: The metabolic pathways most distinctive of young Italian autistic children largely overlap with those found in rodent models of ASD following maternal immune activation or genetic manipulations. These results are consistent with the proposal of a purine-driven cell danger response, accompanied by overproduction of epileptogenic and excitotoxic quinolinic acid, large reductions in melatonin synthesis, and gut dysbiosis. These metabolic abnormalities could underlie several comorbidities frequently associated to ASD, such as seizures, sleep disorders, and gastrointestinal symptoms, and could contribute to autism severity. Their diagnostic sensitivity, disease-specificity, and interethnic variability will merit further investigation.

Electronic supplementary material: The online version of this article (doi:10.1186/s13229-016-0109-5) contains supplementary material, which is available to authorized users.

No MeSH data available.