Analysis Of Biochemical Substances In Noninvasive Biological Samples From Children With Autism And Its Application

Autism spectrum disorders is a representative of extensive developmental disorder,which is a long-term developmental disorder starting in infant period caused by brain dysfunction.It is an important direction that the detection of biochemical index in biological samples to study the occurrence and development,the diagnosis and and treatment research of autism spectrum disorders because there are a lot of complicated relationships between the human multi-system and autism spectrum disorders.Urine is an relatively well samples for noninvasive biological detection,which is easier to be collected and pre-processed than blood,saliva and faeces.The biochemical markers in urine samples can help to better understand the mechanism of ASD and maybe be used to evaluate the health of ASD children.Biochemical markers in urine samples may be a potential diagnostic and intervention indicator in the future.Vitamin B9 and B12 play many indispensable roles in the physiological control of numerous functions of the central nervous system(CNS)and in the formation and metabolism of many important compounds such as the conversion of amino acids and the methylation in the human multi-system.Vitamin B2 is the most popular role in B-vitamins,which participate in a variety of metabolism with other substances.Sulfur-containing amino acids mainly include methionine(Met),homocysteine(Hcy)and cysteine(Cys).Early studies have shown that asulfur-containing amino acid levels has been associated with cognitive impairment.In addition,Histamine(HA)acts as a new type of neurotransmitter,which plays a role in the development of brain and the role of neurotrophic factor.The relationship between histamine and ASD has attracted the attention of researchers because brain neurotransmitter abnormalities in ASD children studies had been found.As the synthesis precursor of HA,Histidine(His),which is a children’s essential amino acids,can not be ignored in ASD research.This study includes the following four aspects:(1)the establishment of analytical method for B-vitamins following a nanofibers based solid phase extraction;(2)determination and data analysis of B-vitamins in urines from children with autism and normal control group children;(3)the establishment of direct-infusion tandem mass spectrometry for analysis of urinary amino acids;(4)determination and data analysis of amino acids in urines from children with autism and normal control group children.The main work of this Paper is as follows:(1)The establishment of analytical method for B-vitamins following a nanofibers based solid phase extractionThough there are many ways to measure the content of B-vitamins,these methods still have shortcomings,whose applicability in large population are not good enough.In this paper,the method of measuring B-vitamins based on fiber pretreatment and high performance liquid chromatography was established.According to the special structure of vitamin B9,B12 and B2 that all contain hydroxyl and amino,polypyrrole nanofibers were successfully prepared for sample enrichment.Diphenylboronic acid 2-aminoethylester(DPBA)was also be used to add in solution to improve sample enrichment efficiency.Then the SPE column was washed with DPBA solution.Other impurities could be removed and the target could be retained on the column.At the same time,the eluent and the ratio of the organic phase to water phase has been optimized.Artificial urine were used for sample preparation,in the appropriate vitamin B9,B12 and B2 concentration level,the correlation coefficient(R squared)was in the range of 0.9925 to 0.9995,the recovery of methods was 84.9%to 111.0%,intraday and interday precision were 1.32%-6.85%and 3.15%-11.7%,respectively,the lowest detection limit(LOD)were 0.04 to 0.096 μg/mL.Results showed that the proposed method is accurate and reliable,providing the simple and accuratee nrichment and detection for B-vitamins in practical samples.(2)Determination and data analysis of B-vitamins in urines from children with autism and normal control group childrenFirst-morning-void urine samples were collected from 45 autism children and 40 normal children,and new methods was used to determine vitamin B9,B12 and B2 in urine sample.Then the results were analyzed by using SPSS 19.0.Compared with control group,the VB12 concentrations in urine of ASD group were significantly higher than that of normal controls,and there was a significant positive correlation between VB9 content and age in ASD children.(3)The establishment of direct-infusion tandem mass spectrometry for analysis of urinary amino acidsThe common analysis and measure methods for amino acid detection mainly include chemical,spectrophotometry,capillary electrophoresis and high performance liquid chromatography,while these methods comsumed longer analysis time and larger reagents consumption.The method of direct injection-type tandem mass spectrometry for the determination of amino acid content is a high-throughput and rapid method and can be used to amino acid detection in human urine.In this paper,the contents of methionine,homocysteine,cysteine,histidine and histamine in urine were measured by direct injection tandem mass spectrometry.Artificial urine were used for sample preparation,in the rang of appropriate concentration level of mixed amino acids,the orrelation coefficient(R squared)was in the range of 0.9933 to 0.9992.The practical application of the method was proved by measuring the concentrations of five target substances in urine samples from 29 obese children and 31 normal control children.The results has been shown that the method is suitable for the detection of amino acids in urine.In addition,the levels of amino acids in obese children and normal children were analyzed.The levels of methionine,homocysteine,cysteine,histidine and histamine in urine were significantly higher than those in normal controls,which may indicate the levels of amino acids in urine are associated with ASD.Accurate measurement of urinary amino acids has clinical meaning to diagnose and treatment.(4)Determination and data analysis of amino acids in urines from children with autism and normal control group childrenFirst-morning-void urine samples were collected from 63 autism children and 45 normal children.The amino acids in urine sample from ASD children and normal children were detected by direct-infusion tandem mass spectrometry.Then the results were analyzed by using SPSS 19.0.Compared with control group,the content of methionine,homocysteine,cysteine,histidine and histamine in urine from ASD group were significantly lower than that of normal children,and the concentrations of homocysteine and cysteine showed significant difference in sex.