Metabolic Perturbance in Autism Spectrum Disorders: A Metabolomics

Oct 29, 2012 - New Jersey Neuroscience Institute, JFK Medical Center, Edison, ... Colgate-Palmolive Technology Center, 909 River Road, Piscataway ... ...
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Metabolic Perturbance in Autism Spectrum Disorders: A Metabolomics Study Xue Ming,*,†,‡ T. Peter Stein,§ Virginia Barnes,∥ Nelson Rhodes,⊥ and Lining Guo⊥ †

Department of Neurosciences and Neurology, UMDNJ-New Jersey Medical School, Newark, New Jersey 07103, United States New Jersey Neuroscience Institute, JFK Medical Center, Edison, New Jersey 08820, United States § Department of Surgery, UMDNJ-School of Osteopathic Medicine, Stratford, New Jersey 08084, United States ∥ Colgate-Palmolive Technology Center, 909 River Road, Piscataway, New Jersey 08855, United States ⊥ Metabolon, Inc., 617 Davis Drive, Suite 400, Durham, North Carolina 27713, United States ‡

S Supporting Information *

ABSTRACT: Autism spectrum disorders (ASD) are a group of biological disorders with associated metabolic derangement. This study aimed to identify a pattern of metabolic perturbance in ASD using metabolomics in urinary specimens from 48 children with ASD and 53 age matched controls. Using a combination of liquid- and gas-chromatography-based mass spectrometry, we detected the levels of 82 metabolites (53 of which were increased) that were significantly altered between the ASD and the control groups using osmolality normalized data. Pattern analysis showed that the levels of several amino acids such as glycine, serine, threonine, alanine, histidine, glutamyl amino acids and the organic acid, taurine were significantly (p ≤ 0.05) lower in ASD children. The levels of antioxidants such as carnosine were also reduced in ASD (p = 0.054). Furthermore, several gut bacterial metabolites were significantly altered in ASD children who had gastrointestinal dysfunction. Overall, this study detected abnormal amino acid metabolism, increased oxidative stress, and altered gut microbiomes in ASD. The relationship of altered gut microbial co-metabolism and the disrupted metabolisms requires further investigation. KEYWORDS: autism spectrum disorders, metabolomics, gut bacteria, metabolic perturbance

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INTRODUCTION Autism spectrum disorders (ASD) have been increasingly recognized as a group of biomedical disorders. Although the diagnosis is made based on behavioral criteria, numerous studies have shown metabolic derangement in individuals with ASD. Abnormal levels of amino acids in the plasma, platelet, urine or cerebral spinal fluid have been documented by many investigators since the 1970s.1−4 Some children with phenylketonuria have phenotypic characteristics consistent with ASD.5 Likewise, ASD were identified in individuals with disorders of purine metabolism.6 Unidentified peaks of organic acids levels were also found in children with autism.1 Significant increases of Krebs cycle analogues and arabinose were found in two brothers with autistic features.7 Altered metabolism in neurotransmitters and hormones such as serotonin, catecholamine/ pterins, melatonin, oxytocin, GABA, endorphins, and so forth has been reported in ASD.8 Reports on disorders of mitochondrial metabolism with abnormal levels of lactate and pyruvate in ASD are increasingly published over the years.9−14 Furthermore, oxidative stress is documented and independently confirmed to be increased in children with ASD by various methods of experimentation.15−18 Biomarkers of vitamin insufficiency, reduced capacity for energy transport, reduced sulfation and detoxification were reported in ASD.18 Likewise, abnormalities of fatty acid metabolism are evident in some of individuals with ASD.19 © 2012 American Chemical Society

In addition to host metabolic perturbance, imbalance in symbiotic gut organisms and its possible association with gastrointestinal dysfunction has gained more recognition in ASD.20 Overgrowth of one or more species of gut bacteria were reported in ASD.21−24 In a study of 13 autistic children with various gastrointestinal symptoms and 8 control children, fecal, gastric and duodenal bacterial species were assessed. Nine species of Clostridium were found in the fecal samples of the autistic children that were not found in the control children, while three different Clostridium species were found in controls but not in the autistic children. In the gastric or duodenal samples, microaerophilic and nonspore forming anaerobic bacteria were identified in four of the five samples from the autistic children, and none from the control specimens.21 Subsequently, the same group of investigators quantified and confirmed significantly higher levels of Clostridium clusters in autistic fecal samples.25 Parracho et al. reported a significantly higher incidence of fecal Clostridium histolyticum group of bacteria in ASD children with gastrointestinal symptoms than their nonautistic siblings or healthy controls.23 Williams et al. reported an association between high levels of intestinal, mucoepithelial-associated Sutterella species and gastrointestinal disturbances in children with autism.24 Oral vancomycin, in an Received: July 9, 2012 Published: October 29, 2012 5856

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Manual of Mental Disorders, Fourth Edition (DSM-IV); 52% of the subjects were further confirmed by Autism Diagnostic Interview-Revise, and/or Autism Diagnostic Observation ScaleGeneric criteria. Control children were screened for medical and developmental disorders during their well-child visits and only those free of any chronic or recurrent medical disorders including gastrointestinal dysfunction were considered healthy and included in this study. Dietary history and vitamin use of all subjects were recorded. Both ASD and control subjects were scored using the six item GI severity scale; the score of 2 SD above the mean of control group was used as a cutoff score for ASD subjects to denote the presence of GI dysfunction. Clinical symptoms of gastrointestinal dysfunction in 29 of the 48 ASD children were reported by care-givers and verified by the clinician using the 6 item gastrointestinal scale.20 This study was approved by the Institutional Review Board of UMDNJ and informed consent was obtained from the parents/guardians of all the subjects.

attempt to reduce the toxin producing deleterious gut Clostridium species, was associated with short-term communication and behavioral improvement.26 Likewise, dietary manipulation and restriction have led to improvement of gastrointestinal symptoms and behaviors in ASD children possibly due to dietary intake that could alter bacterial product profiles. Both host and gut bacterial metabolic disturbances may lead to altered metabolite profiles in body fluid that can be detected by metabolomics. Metabolomics is a method of highthroughput metabolic profiling that could be used to determine perturbance of specific metabolic pathways by comparing a disordered cohort group to controls. Metabolic phenotypes are the products of interactions among additional factors such as dietary, environmental, and genetic.27−30 Recently, Yap et al. reported alterations in nicotinic acid metabolism and gut microbial metabolites in children with autism using 1H NMR spectroscopy and pattern recognition methods,31 suggesting that metabolomic profiling is useful in studying metabolic perturbance in ASD. In this study, we utilized a broad spectrum, untargeted metabolomics platform to confirm and extend the findings of Yap et al. to provide a more comprehensive coverage of metabolites. Our platform could identify 391 detectable compounds in urine samples from children with ASD and healthy controls, aiming to capture multiple metabolic alterations that could aid in deciphering specific biochemical disturbances and pathogenesis of ASD.

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Metabolomic Profiling

The metabolomic profiling was performed by Metabolon (Durham, NC). The detailed descriptions of the protocols were previously described.32,33 Data Normalization, Imputation, and Statistical Analysis

The levels of metabolites were normalized to osmolality and creatinine (Supporting Information Table S1). Osmolality was determined using a Fiske osmometer (Advanced Instruments, Inc., Norwood, MA) with 20 μL of sample. Creatinine values were determined based on UPLC/MS/MS data. For statistical analysis, the missing values for a given metabolite were imputed with the observed minimum detection value based on the assumption that they were below the limits of instrument detection sensitivity. Statistical analysis of the data was performed using JMP (SAS, http://www.jmp.com) and “R” (http://cran.r-project.org/). Welch’s two-sample t tests were performed on the log-transformed data to compare the ASD group and the control group. Multiple comparisons were accounted for with the false discovery (FDR) rate method, and each FDR was estimated using q-values. For the convenience of data visualization, the raw area counts for each biochemical were rescaled by dividing the value for a specific biochemical in each sample by the median value for that specific biochemical. The differences of the metabolites between the ASD and control groups were analyzed by Welch’s t test, with p ≤ 0.05 deemed to be significant. Wilcoxon rank-sum nonparametric analysis was also performed for data that may not be normally distributed (Table S3). Metabolites for all of the biochemicals were evaluated for a gender effect using ANOVA both with and without interaction.

MATERIALS AND METHODS

Subjects and Specimen Collection

Subjects were recruited from Pediatric Neurology and Pediatrics clinical practices at University of Medicine and Dentistry of New Jersey (UMDNJ), New Jersey Medical School. Random spot urine specimens were collected from 48 children with ASD and 53 age-matched healthy controls between 10:00 a.m. and 4:00 p.m. The samples were frozen and stored at −80 °C within 30 min of collection. Demographic characteristics of the subjects are listed in Table 1. All ASD subjects were under the care of the pediatric neurologist (X.M.) and the diagnoses were made by the Diagnostic and Statistical Table 1. Demographics and Characteristics of Subjects Used in This Studya ASDb Subjects (n) Age (years) M/F ASD Diagnosis

GI Severity Scales Diet

Vitamins/Supplements Medication

48 10.7 ± 4.0 36/12 AD: 30 PDD-NOSc: 16 Asperger’s: 2 3.10 ± 2.72 Regular: 44 GCFd: 2 Atkin’s: 1 Low Carbohydrate: 1 25 28

controls 53 10.2 ± 3.8 34/19 NA

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0.17 ± 0.51 Regular: 52 Vegetarian: 1

RESULTS

Metabolomic Profiles

Using an untargeted metabolomic platform consisting of ultrahigh performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) optimized for basic species, UPLC/MS/MS optimized for acidic species, and gas chromatography/mass spectrometry (GC/MS),32,33 a total of 391 metabolites with known chemical structures were identified in the urine samples (Table S1). On the basis of osmolality normalized data, it was found that the levels of 82 metabolites were significantly altered between the ASD and the control groups. The levels of 53 metabolites were increased, while the

27 NA

a

Numbers represent either the number of the subjects in the specific category, year of age, or GI severity scores bASD: autism spectrum disorders. cPDD-NOS: pervasive developmental disorders-not otherwise specified. dGluten and casein free. 5857

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Table 2. Amino Acids and Related Biochemicals ASD/control statistical values biochemical name

fold of change*

glycine N-acetylglycine serine threonine beta-alanine alanine histidine trans-urocanate glutarylcarnitine 3-methylglutarylcarnitine taurine

0.56 0.56 0.66 0.61 0.48 0.70 0.82 1.36 1.22 1.34 0.50

p-value 1.28 × 2.53 × 1.75 × 1.63 × 6.96 × 9.86 × 0.010 2.24 × 7.54 × 6.89 × 6.08 ×

mean values q-value

10−5 10−4 10−4 10−4 10−5 10−4 10−3 10−3 10−3 10−6

1.33 × 6.69 × 6.69 × 6.69 × 4.82 × 0.019 0.060 0.034 0.054 0.054 1.26 ×

10−3 10−3 10−3 10−3 10−3

10−3

control

ASD

1.582 0.945 1.292 1.407 2.212 1.370 1.139 0.989 1.009 1.024 1.582

0.889 0.525 0.857 0.862 1.069 0.954 0.931 1.344 1.229 1.370 0.793

Figure 1. Levels of gamma-glutamyl amino acids in ASD subjects. (A) Box plots of representative gamma-glutamyl amino acids from all ASD subjects are shown. (B) Box plots of these biochemicals with ASD subjects without (GI-) or with (GI+) gastrointestinal dysfunction. Data shown were normalized to osmolality. The box represents the middle 50% of the distribution and upper and lower whiskers represent the entire spread of the data. The solid bar across the box represents the median value of those measured while the + is the mean. Any statistical outliers are represented by a circle. The y axis is the median scaled value (relative level). The p-values are shown.

Figure 2. Box plots of antioxidants and biosynthetic precursors of carnosine. Data shown were normalized to osmolality. The p values are shown.

Amino Acid Metabolism

rest were decreased in ASD specimens. The full statistical table is included in the supplemental table. After mapping the metabolites into general biochemical pathways as illustrated in the Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.genome.jp/kegg/), it was apparent that the most significant differences between the ASD and the control groups were in amino acid metabolism, antioxidant status, and gut bacterial metabolism.

The urinary levels of glycine, serine, threonine, alanine, betaalanine, and histidine, as well as the organic acid taurine, were significantly lower in ASD children compared to controls (p ≤ 0.01). In addition, an intermediate metabolite of the amino acid glycine, N-acetylglycine was also reduced in ASD children (Table 2). Conversely, the levels of urocanate (also referred to as trans-urocanate), a histidine catabolite, were significantly elevated in ASD children (Table 2). Additional perturbations in 5858

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amino acid metabolism can be observed in lysine or tryptophan catabolism and leucine catabolism. The levels of glutaroylcarnitine, a product of lysine and tryptophan catabolism, and 3methylglutaroylcarnitine, derived from leucine catabolism, were significantly increased in the ASD children (Table 2). The urinary level of gamma glutamyl amino acids such as gammaglutamylleucine, gamma-glutamyltyrosine, and gamma-glutamylthreonine were significantly reduced in ASD children (Figure 1A), especially in ASD children with gastrointestinal disorders (Figure 1B). Antioxidants

Carnosine and urate have antioxidant properties. The levels of both biochemicals were reduced in ASD children (Table S1). Carnosine is a dipeptide (β-alanyl- L-histidine) that is particularly abundant in muscles and nervous tissues. The two precursors for carnosine, beta-alanine and histidine, were significantly decreased in the ASD group (Figure 2 and Table 2). Gut Microbiota Metabolism

Various metabolites derived from gut bacteria metabolism of amino acids, carbohydrates, and bile acids were significantly altered in children with ASD. Significantly increased levels of 2(4-hydroxyphenyl)propionate and taurocholenate sulfate were observed in the urine specimens of children with ASD, while the levels of 3-(3-hydroxyphenyl)propionate and 5-aminovalerate were significantly lower in ASD (Figure 3). When analyzing these metabolites in ASD children with and without gastrointestinal dysfunction symptoms separately, the ASD children with gastrointestinal dysfunction symptoms (29 of the 48 ASD subjects) had significantly altered gut microbiome metabolites, whereas ASD children without gastrointestinal dysfunction symptoms exhibited similar gut bacterial metabolite profiles to controls (Table S1).

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DISCUSSION This study revealed a significant perturbation in systemic metabolism in children with ASD. A number of metabolites were significantly increased, while approximately half as many metabolites were reduced in children with ASD. The number of compounds showing significant difference in this study far exceeds random chance of difference in the two groups of test subjects. In addition, the data were normalized to urinary osmolality and creatinine, further excluding the confounding factors of possible renal dysfunction in contributing to the differences. Pattern analysis of the perturbed metabolism showed alterations in several pathways, including amino acid metabolism, increased oxidative stress, and mammalian microbial co-metabolism. A limitation of this study was the reliance on ‘spot’ urines. We tried to limit randomness in the urine collections by collecting urine specimens between 10 a.m. and 4 p.m. Two longitudinal studies collected periodic spot urine specimens and examined temporal variability in the excretion of Bisphenol A and showed that single spot urine specimens were suitable for use as biomarkers in epidemiological studies.34,35 Collection of 24 h urine specimens from children with behavioral problems would be technically difficult and to verify that a true 24 h urine was collected, the data would still have to be controlled for creatinine.36 In addition, we examined the effect of gender, diet, medication, and vitamin/supplements on all the metabolites presented herein and found that none of these variables changed the significance (p ≤ 0.05) between ASD and control children.

Figure 3. Alterations in gut bacterial metabolites in ASD subjects. (A) Box plots of representative bacterially produced biochemicals from all ASD subjects are shown. (B) Box plots of these biochemicals with ASD subjects without (GI-) or with (GI+) gastrointestinal dysfunction. Data shown were normalized to osmolality. The p-values are shown.

Patterns of altered amino acids and their intermediate metabolites do not signal a particular disorder of an inborn error of metabolism. There are several factors affecting urinary amino acid levels, including absorption of amino acids from digested food, biosynthesis, amino acid and protein degradation, secretion and excretion. The significance of the reduced urinary levels glycine, serine, threonine, alanine and histidine in ASD is not apparent, whether there is reduced excretion of these amino acids selectively from proximal renal tubules is not clear. Urocanate is a histidine catabolite, the level of which is modulated by urocanase. Urocanase deficiency leads to urocanic aciduria, an inborn error of metabolism that is associated with neurological disorders.37 The contribution of urocanic aciduria found in this study to symptoms of neurologic 5859

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be under-evaluated. On the other hand, altered gut microbiota may be responsible for the GI dysfunction in ASD children as treatments such as probiotics50 or vacomycin25 improves gastrointestinal symptoms in ASD children. The importance of gut microbiome status to human health has received tremendous attention in the past 10 years. Gut microbiome has been found to be associated with cardiovascular disease,51 obesity,52−54 Crohn’s disease,55 gastrointestinal disorders56,57 and insulin resistance.58 Our result of the altered gut microbial co-metabolism in ASD is consistent with the reports of Yap et al.31 and warrants further in-depth research on this topic.

disorders in ASD children remains to be determined. Abnormal levels of carnitines were reported in ASD children and supplementation of carnitine showed improvement in behaviors in children with ASD.38 Gamma glutamyl amino acids are metabolized by gamma glutamyl transferase. The results of consistently reduced levels of these amino acids in urine specimens from ASD children with gastrointestinal dysfunction may reflect an increase in gamma glutamyl transferase activity. Increased gamma glutamyl transferase activity suggests liver damage, perhaps secondary to medication use. Increased oxidative stress has been consistently reported in ASD. Increased excretion of oxidative stress biomarkers and reduced levels of antioxidants including carnosine were reported in ASD.15,39−41 Carnosine supplementation was found to be beneficial in ASD.40 Furthermore, polymorphism or deletion of genes associated with antioxidants was reported in ASD by independent groups of investigators.15,42−45 Our results of reduced antioxidant levels further reiterate the notion of increased oxidative stress in ASD. Gut microbial co-metabolites are products of metabolism of ingested food by bacteria. These small molecules are then absorbed via intestinal mucosa to systemic circulation, and excreted in urine. Because a number of the subjects in ASD groups had gastrointestinal dysfunction, we performed statistical analysis to assess if the altered gut bacteria metabolites in the ASD group were confounded by gastrointestinal dysfunction. We found that gastrointestinal dysfunction contributed to the differential results. This suggests that gastrointestinal dysfunction in this cohort of ASD children was associated with altered gut bacterial metabolism. Gastrointestinal dysfunction is prevalent in ASD and many investigators reported gut microbial overgrowth in ASD.20−26 Altered gut microbial metabolism could be an inherent part of the ASD pathogenesis, at least in these ASD children with symptoms of gastrointestinal dysfunction. Overgrowth of gut bacterial species such as Clostridium can lead to overproduction of the co-metabolites. Some children with ASD may have increased use of antibiotics for frequent infection, leading to imbalance of resident gut microbiota. Thus, increased levels of microbial derived metabolites in ASD children are in concert with the reported overgrowth of gut bacteria in ASD.21−24 On the other hand, increased absorption of the co-metabolites could also explain the increased urinary levels of the co-metabolites. Increased gut permeability (socalled leaky gut syndrome) has long been debated in ASD, with increasing recognition.46 It is also recognized that an interaction between brain and behaviors and gut microbial imbalance exists.47 It is possible that our results of increased levels of bacterial co-metabolites represent an overproduction of the bacterial metabolites due to bacterial imbalance, increased absorption of the metabolites due to increase intestinal mucosa permeability to small molecules, or both. Our findings of the altered gut microbial co-metabolites in ASD children with symptoms of gastrointestinal dysfunction suggest that this phenomenon is widespread in ASD children. Symptoms of gastrointestinal dysfunction such as constipation, diarrhea, bloating and gas, abdominal pain, and so forth are reported in children with ASD.48,49 However, gastrointestinal symptoms may not be reliably recognized in children with ASD, especially in those who are nonverbal and/or low functioning ones. As such, association of the altered gut microbial cometabolites with gastrointestinal disturbance in this study may

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ASSOCIATED CONTENT

S Supporting Information *

Table S1, metabolites measured in this study. Table S2, statistical comparison of ASD (GI+) versus (GI-) children and percentage of subjects in each group with a detectable level of each biochemical. Table S3, Wilcoxon rank-sum test for ASD/ control osmolality normalized data. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Address: Department of Neurosciences and Neurology, UMDNJ-New Jersey Medical School, 90 Bergen Street, DOC 8100 Newark, NJ 07103. Phone: 973-972-2922. Fax: 973-9729553. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to thank the children and their parents/ guardians who volunteered as participants in this study. We would also like to thank Philip R. Gunst for statistical analysis of the data.

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REFERENCES

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