Comparison of the Metabolic Profiles in the Plasma and Urine Samples between Autistic and Typically Developing Boys: A Preliminary Study
Description
Introduction: Autism spectrum disorder (ASD) is defined as a pervasive developmental disorder which is caused by genetic and environmental risk factors. Besides the core behavioral symptoms, accumulated results indicate children with ASD also share some metabolic abnormalities. Objectives: To analyze the comprehensive metabolic profiles in both of the first-morning urine and plasma samples collected from the same cohort of autistic boys. Methods: In this study, 30 autistic boys and 30 tightly matched healthy control (HC) boys (age range: 2.4 ~ 6.7 years) were recruited. First-morning urine and plasma samples were collected and the liquid chromatography–mass spectrometry (LC-MS) was applied to obtain the untargeted metabolic profiles. The acquired data were processed by multivariate analysis and the screened metabolites were grouped by metabolic pathway. Results: Different discriminating metabolites were found in plasma and urine samples. Notably, taurine and catechol levels were decreased in urine but increased in plasma in the same cohort of ASD children. Enriched pathway analysis revealed that perturbations in taurine and hypotaurine metabolism, phenylalanine metabolism and arginine and proline metabolism could be found in both of the plasma and urine samples. Conclusion: These preliminary results suggest that a series of common metabolic perturbations exist in children with ASD, and confirmed the importance to have a comprehensive analysis of the metabolites in different biological samples to reveal the full picture of the complex metabolic patterns associated with ASD. Further targeted analyses are needed to validate these results in a larger cohort. Note: Other data used in this study are available from the corresponding author upon request. (zhangrong@bjmu.edu.cn)
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Sample Collection Blood samples were collected by trained nurses between 7:00 and 9:30 a.m. Parents were previously informed to have their children fast overnight and allowed only a moderate amount of drinking water to minimize the potential effects of food and water intake. Four milliliters of venous blood was collected into chilled EDTA tubes containing aprotinin (500 KIU/mL blood). Blood samples were centrifuged at 1,600 g for 15 minutes. Plasma was isolated and divided into 500 μL aliquots. The fresh first morning midstream urine was collected in sterile tubes. The samples were placed on ice and immediately frozen into dry ice and transferred to store at -80°C until assay. Sample Extraction For plasma sample extraction, 200μL of methanol was added to 100μL of each thawed sample and vortexed for 60s. The mixture was centrifuged at 12,000 rpm for 10min at 4℃. For urine sample extraction, 100μL of ddH2O was added to 100μL of each thawed sample and vortexed for 5 min. The mixture was centrifuged at 10,000 rpm for 10min at 4℃.The supernatant was then filtered by 0.22 um membrane filtration and prepared for LC-MS analysis. To validate the reproducibility of the LC-MS system, 20 µL from prepared samples was pooled to generate the quality control (QC) samples. Chromatographic Separation and Mass Spectrometry Analysis Chromatographic separation was accomplished in an Acquity UPLC system equipped with an ACQUITY UPLC® BEH C18 (100×2.1mm, 1.7µm, Waters) column maintained at 40℃. The temperature of the autosampler was 4 ℃. Gradient elution of analytes was carried out with 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) at a flow rate of 0.25mL/min. Ten microliter of each sample was injected after equilibration. An increasing linear gradient of solvent B (v/v) was used as follows: 0~1 min, 2% B; 1~9.5min, 2%~50% B; 9.5~14min, 50%~98% B; 14~15 min, 98% B; 15~15.5 min, 98%~2% B; 15.5~17 min, 2% B. The ESI-MSn experiments were executed on the Thermo LTQOrbitrap XL mass spectrometer with the spray voltage of 4.8 kV and -4.5 kV in positive and negative modes. Sheath gas and auxiliary gas were set at 45 and 15 arbitrary units. The capillary temperature was 325℃. The voltages of the capillary and tube were 35V and 50 V, -15 V and -50 V in positive and negative modes. The Orbitrap analyzer scanned over a mass range of m/z 50-1 000 for full scan at a mass resolution of 60 000. Data dependent acquisition (DDA) MS/MS experiments were performed with the CID scan. The normalized collision energy was 30 eV. Dynamic exclusion was implemented with a repeat count of 2, and exclusion duration of 15s. Data Processing and Analysis Urine metabolomic data from UPLC/MS were standardized to eliminate urine volume variability. Multivariate statistics techniques were used to analyze the multiple variables simultaneously. Comprehensive and integrative metabolomic data was mainly analyzed using metaboanalyst 4.0 (https://www.metaboanalyst.ca/).