KR20230020827A - Composition for diagnosis of autism spectrum disorder using metabolomics profiling and diagnostic kit comprising the same - Google Patents

Abstract

The present invention provides: a composition for the diagnosis of autism spectrum disorder providing a biomarker necessary for diagnosing autism spectrum disorder through a metabolic profiling method, and including metabolites; and a diagnostic kit and a method for providing diagnostic information for the diagnosis of autism spectrum disorder. The composition for the diagnosis of autism spectrum disorder of the present invention can be used for diagnosing autism spectrum disorder by comparing concentrations of metabolites in a patient and a normal control group.

Description

Composition for diagnosis of autism spectrum disorder using metabolomics profiling and diagnostic kit comprising the same}

The present invention provides a biomarker that can be used to diagnose autism spectrum disorder through metabolomic profiling, and provides a diagnostic composition, diagnostic kit, and method for diagnosing autism spectrum disorder.

Autism spectrum disorder (ASD) is known as a neurodevelopmental deteriorating condition characterized by restricted and repetitive patterns of behavior and deficits in social communication and interaction. As ASD is a severe behavioral disorder that develops within the first 3 years of life, early identification and intervention of ASD is the most important factor in improving treatment outcomes for individuals with ASD. However, because there is no method for early diagnosis of ASD, many children miss the optimal time to start treatment. In the United States, the number of children with autism has increased since 1992. A few decades ago, the prevalence of ASD was 1 in 10,000 of the population, but the prevalence is increasing by 11-17% annually. In this regard, the World Health Organization (WHO) selected ASD as a major issue in 2001, and scientific interest in ASD has developed into major areas such as education, psychology, and neurobiological research.

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ana, M.; Gummery, A.; Kneen, R.; Loring, D.W.; Mawer, G.; Meador, K.J.; Shallcross, R.; Clayton-Smith, J.; Liverpool and Manchester Neurodevelopment Group. IQ at 6 Years After in Utero Exposure to Antiepileptic Drugs: A Controlled Cohort Study. Neurology, 2015, 84, 382-90.). 임신 중 VPA에 노출되면 태아 발프로에이트 증후군 (FVS)이 발생하여 비정상적인 발달 지연, 장기 기형, 사회적 기능 또는 의사 소통 장애를 유발한다. 이러한 증후군을 가진 사람들은 자폐성 행동을 보이기 때문에 FVS와 ASD는 서로 관련이있는 것으로 보이지만, FVS는 주로 척추이분증, 독특한 얼굴 특징, 선천성 심장 결함 및 기타 근골격계 이상을 동반한다. VPA에 의해 유발된 ASD를 연구하기 위해 Rodier등은 단일 용량 VPA로 자극된 쥐 배아를 사용하여 VPA 모델을 만들었다(Rodier et al., Embryological origin for autism: Developmental anomalies of the cranial nerve motor nuclei. J. Comp. Neurol. 1996, 370, 247-61.). 또한 Schneider와 Przewlocki는 임신 12.5 일에 VPA를 한 번 주사한 후 쥐의 ASD 행동을 평가하였다. 실험 후, 그들은 VPA에 노출된 수컷 쥐 새끼가 사교성 감소, 반복적 패턴, 통증에 대한 낮은 민감성, 발달 지연과 같은 자폐 적 행동과 이상을 보이는 것을 관찰하였다(Schneider, T.; and Przewlocki, R. Behavioral Alterations in Rats Prenatally Exposed to Valproic Acid: Animal Model of Autism. Neuropsychopharmacology. 2005, 30, 80-9.). Although many causes of ASD have been proposed, the exact causal mechanisms of ASD are unknown. In addition, single gene disorders, congenital metabolic errors, heavy metals and maternal drug exposure are known to increase the risk of ASD. Exposure to drugs such as valproic acid (VPA) has also been suggested as one of the causes of ASD, and clinical studies have shown that consumption of VPA during pregnancy increases the risk of developing neural tube defects, including ASD (Williams et al., Fetal valproate syndrome and autism: Additional evidence of an association. Dev. Med. Child. Neurol. 2001, 43, 202-206. and Christensen et al., Prenatal Valproate Exposure and Risk of Autism Spectrum Disorders and Childhood Autism. JAMA. 2013 , 309, 1696-703). VPA is used to treat epilepsy and bipolar disorder, modulate gamma-amino butyric acid (GABA) neurotransmission, and prevent migraines. A follow-up study in children exposed to VPA during pregnancy showed an approximately 8-fold increase in ASD rates, a reduced number of oculomotor neurons, a shortened caudal region of the facial nucleus, and a reduced number of Purkinje cells ( Baker, GA; Bromley, RL; Briggs, M.; Cheyne, CP; Cohen, MJ; Garc

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ana, M.; Gummery, A.; Kneen, R.; Loring, DW; Mawer, G.; Meador, KJ; Shallcross, R.; Clayton-Smith, J.; Liverpool and Manchester Neurodevelopment Group. IQ at 6 Years After in Utero Exposure to Antiepileptic Drugs: A Controlled Cohort Study. Neurology, 2015, 84, 382-90.). Exposure to VPA during pregnancy causes fetal valproate syndrome (FVS), which causes abnormal developmental delay, organ malformations, and social functioning or communication difficulties. FVS and ASD appear to be related because people with these syndromes exhibit autistic behavior, but FVS is primarily accompanied by spina bifida, distinctive facial features, congenital heart defects, and other musculoskeletal abnormalities. To study VPA-induced ASD, Rodier et al. created a VPA model using rat embryos stimulated with single-dose VPA (Rodier et al., Embryological origin for autism: Developmental anomalies of the cranial nerve motor nuclei. J. Comp. Neurol. 1996, 370, 247-61.). Schneider and Przewlocki also evaluated the ASD behavior of rats after a single injection of VPA on day 12.5 of gestation. After the experiment, they observed that male rat pups exposed to VPA exhibited autistic behaviors and abnormalities such as reduced sociability, repetitive patterns, low sensitivity to pain, and developmental delay (Schneider, T.; and Przewlocki, R. Behavioral). Alterations in Rats Prenatally Exposed to Valproic Acid: Animal Model of Autism. Neuropsychopharmacology. 2005, 30, 80-9.).

The exact cause of most ASD cases is unknown, but it is suggested that they are related to various metabolic abnormalities. Metabolomics has emerged as a promising tool for screening for underlying biochemical abnormalities and managing treatment, as well as a means to investigate possible new biomarkers for disorders. Metabolomics profiling methods are used to monitor metabolic processes in the impaired and control groups to monitor abnormal metabolic pathways. This includes investigating the efficacy or toxicity of drugs or biochemical pathways associated with specific diseases such as humans, microorganisms and plants. In metabolic studies, nuclear magnetic resonance spectroscopy (NMR), high-performance liquid chromatography, and mass spectrometry (MS) are used to identify metabolites, and the results are presented through various data analysis methods including multivariate statistical analysis. Metabolic approaches using NMR spectroscopy are one of the most widely used techniques to identify metabolic changes both in vivo and in vitro. NMR spectroscopy is less sensitive than MS, but NMR spectroscopy allows simultaneous detection of large numbers of compounds, simplifies sample preparation, and provides quantitative and qualitative information on non-redundant metabolites faster, more economically, and with higher reproducibility. there is. In the early diagnosis of ASD using metabolomics approaches, many researchers have reported ASD biomarkers in blood, urine and brain. However, metabolomics approaches to VPA-induced ASD in animal model studies have rarely been conducted.

Therefore, we identified biomarkers of VPA-induced ASD using a metabolomics approach. An object of the present invention is to provide a biomarker for predicting autism spectrum disorders through proton nuclear magnetic resonance ( ¹ H NMR) analysis of brain, serum and urine samples obtained from male offspring of rats exposed to VPA during pregnancy. .

In addition, a composition for diagnosing autism spectrum disorder using the biomarker, a diagnostic kit containing the same as an active ingredient, and a diagnostic information providing method are provided.

However, the problem to be solved by the present invention is not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

According to one embodiment of the present invention, valine, taurine, myoinositol, 3-hydroxybutyrate, 1,3-dihydroxyacetone, glucose, creatine phosphate, lactate, glutamine, threonine, galactose, pimelate, galactonate , A composition for diagnosing autism spectrum disorder is provided, comprising at least one or more metabolites selected from the group consisting of 3-hydroxyisovalerate and valerate.

According to one side, the valine, taurine, myoinositol, 3-hydroxybutyrate and 1,3-dihydroxyacetone may be obtained from the prefrontal cortex of the subject.

According to one aspect, the galactose, 3-hydroxybutyrate, pimelate, galactonate, 3-hydroxyisovalerate and valerate may be obtained from the urine of a subject.

According to one aspect, the glucose, creatine phosphate, lactate, glutamine and threonine may be obtained from the blood of a subject.

According to another embodiment of the present invention, a kit for diagnosing autism spectrum disorder including the composition is provided.

According to one side, the autism spectrum disorder is any one selected from autism disorder, Rett disorder, childhood disintegrative disorder, Asperger's syndrome, pervasive developmental disorder of specific disability, verbal or non-verbal communication disorder, and stereotyped behavioral disorder. can

According to one embodiment of the present invention, providing a sample of the subject; In the above sample, valine, taurine, myoinositol, 3-hydroxybutyrate, 1,3-dihydroxyacetone, glucose, creatine phosphate, lactate, glutamine, threonine, galactose, pimelate, galactonate, 3-hydroxy Detecting the concentration of at least one metabolite from the group consisting of isovalerate and valerate; Confirming the clustering of the metabolite and control through multivariate statistical analysis (PLS-DA); and comparing the concentration of the detected metabolite with a control group; Including, autism spectrum disorder diagnostic information providing method is provided.

According to one aspect, the step of detecting the concentration of the metabolite may be performed by proton nuclear magnetic resonance spectroscopy.

The composition for diagnosing autism spectrum disorder of the present invention uses a biomarker identified by a metabolomics profiling method, and can be used for diagnosing autism spectrum disorder by comparing the concentrations of metabolites in a patient and a normal control group, and can be used for diagnosing autism spectrum disorders early and with high accuracy. Spectrum disorders can be identified.

The effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be inferred from the detailed description of the present invention or the configuration of the invention described in the claims.

Figure 1 shows the behavioral analysis of an animal model in which autism spectrum disorder was induced by administration of valproic acid in terms of sociability index (SI) and social preference index (SPI).
2 shows the results of ¹ H-NMR analysis of brain, serum and urine samples using PCA and PLS-DA.
Figure 3 shows the VIP scores of metabolites by PLS-DA analysis of ¹ H-NMR of brain, serum, and urine samples of the VPA-treated group and the control group.

Hereinafter, embodiments will be described in detail with reference to the accompanying drawings. Like reference numerals in each figure indicate like elements.

Various changes may be made to the embodiments described below. The embodiments described below are not intended to be limiting on the embodiments, and should be understood to include all modifications, equivalents or substitutes thereto.

Terms used in the examples are used only to describe specific examples, and are not intended to limit the examples. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as "include" or "have" are intended to designate that there is a feature, number, step, operation, component, part, or combination thereof described in the specification, but one or more other features It should be understood that the presence or addition of numbers, steps, operations, components, parts, or combinations thereof is not precluded.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiment belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and unless explicitly defined in the present application, they should not be interpreted in an ideal or excessively formal meaning. don't

In addition, in the description with reference to the accompanying drawings, the same reference numerals are given to the same components regardless of reference numerals, and overlapping descriptions thereof will be omitted. In describing the embodiment, if it is determined that a detailed description of a related known technology may unnecessarily obscure the gist of the embodiment, the detailed description will be omitted.

As used herein, the term 'metabolite' is used to refer to any product produced in a metabolic process, and may be produced and influenced by endogenous or exogenous factors.

According to one embodiment of the present invention, valine, taurine, myoinositol, 3-hydroxybutyrate, 1,3-dihydroxyacetone, glucose, creatine phosphate, lactate, glutamine, threonine, galactose, pimelate, galactonate , A composition for diagnosing autism spectrum disorder is provided, comprising at least one or more metabolites selected from the group consisting of 3-hydroxyisovalerate and valerate.

Referring to Example 3, the valine, taurine, myoinositol, 3-hydroxybutyrate, 1,3-dihydroxyacetone, glucose, creatine phosphate, lactate, glutamine, threonine, galactose, pimelate, galacto For nate, 3-hydroxyisovalerate, and valerate, metabolites showing significant differences compared to the control group were selected through metabolomic profiling in the body of ASD-induced rats by VPA treatment during pregnancy.

The composition for diagnosing autism spectrum disorder, the valine, taurine, myoinositol, 3-hydroxybutyrate, 1,3-dihydroxyacetone, glucose, creatine phosphate, lactate, glutamine, threonine, galactose, pimelate, galacto It can be used to diagnose autism spectrum disorder by comparing concentrations with metabolites obtained from a biological sample of a subject, including any one or more of nate, 3-hydroxyisovalerate, and valerate.

According to one side, the 1,3-dihydroxyacetone may be obtained from the prefrontal cortex of the subject. A method of obtaining a sample from the prefrontal cortex of a subject may use any method known in the art, and is not limited to the method performed in the following examples.

According to another embodiment of the present invention, a kit for diagnosing autism spectrum disorder including the composition for diagnosing autism spectrum disorder is provided.

The kit for diagnosing autism spectrum disorder may include, as essential components, a component for analyzing the concentration of the metabolites of a subject and a component for comparing the concentration of the metabolites in a control group, and is not limited to physical specifications such as any size or weight.

According to one side, the autism spectrum disorder is any one selected from autism disorder, Rett disorder, childhood disintegrative disorder, Asperger's syndrome, pervasive developmental disorder of specific disability, verbal or non-verbal communication disorder, and stereotyped behavioral disorder. can

According to another embodiment of the present invention, providing a sample of the subject; In the above sample, valine, taurine, myoinositol, 3-hydroxybutyrate, 1,3-dihydroxyacetone, glucose, creatine phosphate, lactate, glutamine, threonine, galactose, pimelate, galactonate, 3-hydroxy Detecting the concentration of at least one metabolite from the group consisting of isovalerate and valerate; Confirming the clustering of the metabolome and the control group through multivariate statistical analysis; and comparing the concentration of the detected metabolite with a control group; Including, autism spectrum disorder diagnostic information providing method is provided.

The multivariate statistical analysis may preferably use a partial least squares regression analysis method.

According to one aspect, the step of detecting the concentration of the metabolite may be performed by proton nuclear magnetic resonance spectroscopy. The proton nuclear magnetic resonance spectroscopy method may vary depending on the type of sample, and a preferred example is provided in Example 3 below, but the scope of the present invention is not limited thereto.

Hereinafter, the present invention will be described in more detail through examples. The following examples are described for the purpose of illustrating the present invention, but the scope of the present invention is not limited thereto.

Example 1: Provision of VPA-induced ASD expression group

Pregnant Sprague-Dawley rats (n=10) were purchased from OrientBio (Korea). To establish an animal model of VPA-induced ASD, pregnant rats were injected with sodium salt of VPA (purchased from Sigma-Aldrich) dissolved in 0.9% saline at a concentration of 100 mg/mL, pH 7.3. The dose was adjusted according to the weight of the pregnant rat. Pregnant mice received a single subcutaneous injection of 400 mg/kg VPA on day 12 of gestation. Control rats were treated with saline. Lactating dams were housed with their pups in a temperature (22±2°C) and humidity (55±5%) controlled environment with a 12-h light/dark cycle. Only male pups were used in this study. Animal experiments were conducted in accordance with Laboratory Animal Care Principles (NIH Publication No. 85-23, revised in 1985) and Konkuk University Animal Care and Use Guidelines (KU19180).

Example 2: Behavioral analysis of mice expressing ASD

Kim et al. Male-specific alteration in excitatory post-synaptic development and social interaction in pre-natal valproic acid exposure model of autism spectrum disorder. J. Neurochem. As reported in 2013, a social interaction test was performed on 4-week-old pups through a three-chamber method. Behavioral experiments were conducted between 10:00 and 16:00 in a dedicated testing room. In the first session, the amount of time spent with a particular animal confined in a wire cage and in a neutral or empty cage position was investigated. Following the sociability test, a social preference test was performed by placing different strangers in empty cages and measuring the time spent in each location. Movement traces during the experiment were automatically recorded using EthoVision software (Wageningen, Netherlands). Sociability (SI) and Social Preference Index (SPI) were calculated as the ratio of time spent with a specific animal versus time spent in an empty space, and time spent with a novel animal versus time spent with a familiar animal, respectively.

As a result of examining the social interaction behavior of rat pups exposed to VPA during pregnancy using a three-chamber analysis, the VPA-treated group showed a decrease in the dwell time around the same rat and an increase in the dwell time in the empty space compared to the control group. . Therefore, the rate of stay in unfamiliar objects or empty spaces, defined as the sociability index (SI), was significantly lower in the VPA-treated group than in the control group. In addition, rat pups exposed to VPA during pregnancy show impaired social novelty-seeking behavior with lower interest in unfamiliar rats than in familiar rats (social preference index, SPI). These behavioral characteristics are similar to the characteristics of social adjustment disorder and lack of social interaction in children with autistic symptoms, indicating that autism behaviors were successfully reproduced in the VPA-induced ASD animal model (Fig. 1).

Example 3: Metabolomic profiling of brain, serum and urine samples from ASD-expressing mice

After behavioral testing, rats were anesthetized with a Zoletil-Rompun mixture (3:1, v/v), and biological samples including blood, urine, and brain (prefrontal cortex) were collected. Blood samples were collected from the heart via cardiac puncture using a 1 mL syringe. After storage in Eppendorf tubes, the samples were centrifuged at 900 x g for 10 minutes. After centrifugation, only the supernatant was stored in a new Eppendorf tube and stored at -80 °C. Urine samples were collected using a 200 μl pipette during or before whole-brain extraction and stored in a -80 °C freezer. After removing the entire brain from the skull, the prefrontal cortex was carefully removed and rapidly frozen in liquid nitrogen in a 50 ml conical tube. Brain samples were stored at -80 °C until further analysis.

Brain, serum and urine samples from rats treated with or without VPA (400 mg/kg) were analyzed by ¹ H NMR. Brain samples were analyzed on a 400 MHz HR-MAS NMR spectrometer, and serum and urine samples were analyzed on a 600 MHz NMR spectrometer (Agilent Technologies, Santa Clara, CA, USA).

Serum samples were thawed at 4 °C and 350 μL aliquots were added to microcentrifuge tubes containing 350 μL of DO solution with 4 mM TSP-d4. Urine samples were thawed at 4 °C and then centrifuged at 900 x g for 5 min to remove solids. Aliquot 600 μl of urine sample into a microcentrifuge tube containing 70 μL of D2O solution containing 100 mM imidazole and 5 mM DSS (2,2-dimethyl-2-silapentane-5-sulfonic acid), qualitative standards for chemical shift measures for urine. amount was added. Also, 30 μL of 0.42% sodium azide was added. After vortexing, the pH was adjusted to 6.8. Spectra of serum and urine were acquired within 48 hours at 26 °C using a 600 MHz NMR liquid probe. CPMG pulses were used with the following acquisition parameters. s acquisition time and total acquisition time for serum was 13 min 9 sec. In addition, 2D NMR analysis was performed to verify that endogenous metabolites were identified.

NMR data were obtained in FID file format and then spectra were collected by Fourier transform. All acquired spectra were scaled, baseline corrected, and referenced to the TSP-d4 peak (chemical shift of 0 ppm) using VnmrJ 4.2 software (Aligent Technologies, Santa Clara, CA, USA). Identification and quantification of metabolites was performed using Chenomx NMR Suite 8.3 Professional software (Chenomx, Inc., Edmonton, Alberta, Canada) with Chenomx's library database. Internal standards for chemical shift and quantification were TSP-d4 for brain and serum and DSS for urine. For urine samples, concentrations of metabolites were expressed as relative ratio values normalized by creatinine concentration assuming a constant percentage of creatinine excretion.

For pre-processing of NMR spectra, the δ 0.0–10.0 spectral region was divided into regions with a width of 0.04 ppm, and 250 integrated regions were set for each NMR spectrum. The spectral region of water (δ 4.8–5.4) was removed from the analysis for all groups to prevent fluctuations in water inhibition efficiency.

Through pattern analysis in Chenomx NMR software, we identified 42, 36, and 46 metabolites in brain, serum, and urine samples, respectively. Multivariate analysis is used to interpret the collected NMR data and the data are visualized using principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA).

The resulting data were applied to PCA and PLS-DA and the separation of the two groups is shown in Figure 2. Taurine, lactate, myoinositol, glutamate, serine, 4-aminobutyrate, 1,3-dihydroxyacetone, creatine, leucine, valine, 2-oxovalerate, lysine, 3-hydroxybutyrate, glycine, Concentrations of ethylmalonate and butanone were selected as metabolites with a variable importance in projection (VIP) greater than 0.7, and were decreased in the VPA-treated group compared to the control group (Fig. 3(A)). Among them, 3-hydroxybutyrate, 1,3-dihydroxyace, acetoin, myoinositol, and taurine showed significant differences within the 2 groups (p <0.05).

Concentrations of glucose, 3-hydroxybutyrate, lactate, glycine, creatine phosphate, threonine, alanine, glutamine, tyrosine, 3-hydroxyisobutyrate, acetate, and 2-hydroxy-3-methylvalerate in the serum were compared with the VPA-treated group. and the control group were different (VIP> 0.7), and the p-values of glucose, lactate, creatine phosphate, glutamine and 2-hydroxy-3-methylvalerate were less than 0.05 in the t-test (FIG. 3(B)).

In urine, formate, galactose, creatine, 3-hydroxybutyrate, lactate, threonine, myoinositol, succinate, pimelate, arabinitol, galactonate, 3-hydroxyisovalerate, valerate and choline are The VIP score was greater than 0.7, and galactose, 3-hydroxybutyrate, pimelate, galactonate, 3-hydroxyisovalerate and valerate were significantly different between the VPA-treated and control groups (p < 0.05, t-test ) (Fig. 3(C)).

As described above, although the embodiments have been described with limited examples and drawings, those skilled in the art can make various modifications and variations from the above description. For example, even if the described techniques are performed in a different order from the described method, and/or the described components are combined or combined in a different form than the described method, or substituted or replaced by other components or equivalents. Appropriate results can be achieved.

Therefore, other implementations, other embodiments, and equivalents of the claims are within the scope of the following claims.

Claims (8)

Valine, taurine, myoinositol, 3-hydroxybutyrate, 1,3-dihydroxyacetone, glucose, creatine phosphate, lactate, glutamine, threonine, galactose, pimelate, galactonate, 3-hydroxyisovalerate And a composition for diagnosing autism spectrum disorder comprising at least one or more metabolites selected from the group consisting of valerate.
According to claim 1,
The valine, taurine, myoinositol, 3-hydroxybutyrate and 1,3-dihydroxyacetone are obtained from the prefrontal cortex of the subject,
A composition for diagnosing autism spectrum disorder.
According to claim 1,
The galactose, 3-hydroxybutyrate, pimelate, galactonate, 3-hydroxyisovalerate and valerate are obtained from the urine of the subject,
A composition for diagnosing autism spectrum disorder.
According to claim 1,
The glucose, creatine phosphate, lactate, glutamine and threonine are obtained from the blood of the subject,
A composition for diagnosing autism spectrum disorder.
A kit for diagnosing an autism spectrum disorder comprising the composition of any one of claims 1 to 4.
According to any one of claims 1 to 4,
The autism spectrum disorder is any one selected from autism disorder, Rett disorder, childhood disintegration disorder, Asperger's syndrome, pervasive developmental disorder of specific disability, verbal or non-verbal communication disorder, and homologous behavior disorder,
A composition for diagnosing autism spectrum disorder.
providing a sample from the subject;
In the above sample, valine, taurine, myoinositol, 3-hydroxybutyrate, 1,3-dihydroxyacetone, glucose, creatine phosphate, lactate, glutamine, threonine, galactose, pimelate, galactonate, 3-hydroxy Detecting the concentration of at least one metabolite from the group consisting of isovalerate and valerate;
Confirming the clustering of the metabolite and control through multivariate statistical analysis (PLS-DA); and
comparing the concentration of the detected metabolite with that of a control group;
Including, autism spectrum disorder diagnostic information providing method.
According to claim 7,
The step of detecting the concentration of the metabolite is made by proton nuclear magnetic resonance spectroscopy, autism spectrum disorder diagnostic information providing method.

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