KR102351108B1 - Composition or kit for diagnosing autism spectrum disorder and method for diagnosis of autism spectrum disorder using the same - Google Patents

Abstract

A composition and kit for diagnosing autism spectrum disorder (ASD) comprising an agent for measuring the relative concentration ratio of tyrosine metabolites in a biological sample, and a kit for diagnosing autism spectrum disorders using the same A method for calculating the relative concentration ratio is provided. According to this, it can be used to diagnose autism spectrum disorder objectively, simply, with high accuracy and specificity.

Description

Composition or kit for diagnosing autism spectrum disorder and method for diagnosing autism spectrum disorder using the same

A composition and kit for diagnosis of autism spectrum disorder, and a method for detecting a biomarker for diagnosis of autism spectrum disorder using the same.

Autism spectrum disorder (ASD) is a neurological disorder characterized by deficits in social interactions, such as communication, and limited, repetitive behaviors. ASD is called a neurodevelopmental disorder because it usually appears within the first 2 years of life (American Psychiatry Association (APA) 2013).

Globally, the prevalence of ASD is continuously increasing, and as one of the diseases whose pathophysiological causes are not clearly identified, diagnostic and therapeutic drugs have not yet been developed. Because the pathophysiological cause of ASD is not clear, research on it is usually conducted in clinical or experimental animal models with characteristic and diverse characteristics of the disease. Currently, the diagnosis of ASD is made through an interview when there is an impairment in restrictive behavior or social interaction. Since the interview is a subjective diagnosis, it is difficult to determine the degree of complex symptoms and symptoms.

In order to overcome this problem, various biomarker studies that can suggest biological diagnostic criteria are being conducted, but the situation is insufficient (Korean Patent Application Laid-Open No. 10-2011-0114664 and Korean Patent Application Laid-Open No. 10-2018-0039397).

Therefore, there is a need to develop biomarkers with high accuracy and specificity for ASD diagnosis.

A composition for diagnosing autism spectrum disorder is provided.

Provides a kit for diagnosing autism spectrum disorder.

A method for providing information necessary for the diagnosis of autism spectrum disorder is provided.

One aspect provides a composition for diagnosing autism spectrum disorder (ASD), comprising an agent for measuring the relative concentration ratio of tyrosine metabolites in a biological sample.

Tyrosine is one of the aromatic α-amino acids and is a non-essential amino acid. In brain cells, tyrosine can be converted to L-3,4-dihydroxyphenylalanine (L-DOPA) by tyrosine hydroxylase. L-DOPA can be converted into dopamine, a neurotransmitter, and dopamine can be converted into catecholamines such as norepinephrine and epinephrine.

The tyrosine metabolites are L-DOPA, dopamine, 3,4-dihydroxyphenyl acetic acid, 3-methoxytyramine, homovanillic acid, norepinephrine, epinephrine, methanphrine, 3,5-dihydroxyphenylglycine, 3 -Methoxy-4-hydroxyl-phenylene glycol, normethanprine, and may be any one or more selected from the group consisting of vanillylmandelic acid.

The relative concentration ratio is tyrosine, dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid to the sum of the concentrations of tyrosine metabolites in the biological sample. HVA) may be a ratio of one concentration selected from the group consisting of.

The sum of the concentrations of the tyrosine metabolites may be the sum of the concentrations of each of the tyrosine metabolites. The sum of the concentrations of the tyrosine metabolites may be the sum of the concentrations of tyrosine, dopamine, 3-methoxytyramine (3MT), HVA, DOPAC, and norepinephrine (NE).

The agent may be a protein that specifically binds to a tyrosine metabolite, a compound that binds to a tyrosine metabolite, or a combination thereof. The agent may be an antibody or antigen-binding fragment thereof, or an aptamer. The antibody may be a polyclonal antibody or a monoclonal antibody. The term “antibody” may be used interchangeably with the term “immunoglobulin”. The antibody may be a polyclonal antibody or a monoclonal antibody. The antibody may be a full-length antibody. The antigen-binding fragment refers to a polypeptide comprising an antigen-binding site. The antigen-binding fragment may be a single-domain antibody, Fab, Fab', or scFv. The antibody or antigen-binding fragment may be attached to a solid support. The solid support is, for example, a metal chip, a plate, or the surface of a well. The aptamer may be a single-stranded nucleic acid (DNA, RNA or modified nucleic acid) or peptide that specifically binds to a tyrosine metabolite. The protein specifically binding to the tyrosine metabolite may be an enzyme. The enzyme is tyrosine hydroxylase, Dopa decarboxylase, dopamine hydroxylase, phenylethanolamine N-methyltransferase (PNMT), catechol-O-methyltransferase (Catechol- O-methyltransferase: COMT), monoamine oxidase (MAO), or a combination thereof.

The term "autism spectrum disorder (ASD)" is a neurological disorder characterized by a limited and repetitive range of behavioral patterns, interests, and activities, while showing persistent impairment in interactive social communication and social interactions from early childhood onwards. A category of developmental disabilities. The autism spectrum disorder is from the group consisting of Child disintegrative disorder, Autistic Disorder, Asperger's disorder, and Pervasive developmental disorder not otherwise specified. can be selected.

The biological sample may be brain tissue or cerebrospinal fluid. The brain tissue may be a tissue of a corpus striatum.

Another aspect provides a kit for diagnosing autism spectrum disorder, comprising an agent for measuring a relative concentration ratio of a tyrosine metabolite in a biological sample.

The kit may further include a sample required for diagnosis of autism spectrum disorder. The kit may include a solid support, a substrate for immunological detection of an antibody or antigen-binding fragment, a suitable buffer, a chromogenic enzyme, a secondary antibody labeled with a fluorescent material, or a chromogenic substrate.

Another aspect is to measure the concentration of tyrosine metabolites in a subject suspected of having an autism spectrum disorder; measuring the relative concentration ratio of one concentration selected from the group consisting of tyrosine, dopamine, DOPAC, and HVA to the sum of the concentrations of tyrosine metabolites; And it provides a method of calculating the relative concentration ratio of the tyrosine metabolite in order to provide information necessary for the diagnosis of autism spectrum disorder, comprising the step of comparing the measured amount with the relative concentration ratio in the normal control.

Autism spectrum disorder, tyrosine, tyrosine metabolites, the sum of the concentrations of tyrosine metabolites, and the relative concentration ratio are the same as described above.

The method includes determining the concentration of tyrosine metabolites in a subject suspected of having an autism spectrum disorder.

The subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat, or cat. The subject may be suffering from or suspected of having an autism spectrum disorder.

The measuring of the concentration of the tyrosine metabolites may include measuring the concentration of the tyrosine metabolites from a biological sample isolated from the individual or measuring the concentration of the tyrosine metabolites using a nuclear magnetic resonance phenomenon for the individual. Methods for measuring the concentration of tyrosine metabolites from the biological sample isolated from the subject include mass spectrometry (MS), enzyme-linked immunosorbent assay (ELISA), protein chip, immunoprecipitation, and microarray. , electron microscopy, or a combination thereof. A method of measuring the object using a nuclear magnetic resonance phenomenon may be performed by nuclear magnetic resonance imaging (MRI) or magnetic resonance spectroscopy (MRS).

The method includes determining a relative concentration ratio of one concentration selected from the group consisting of tyrosine, dopamine, DOPAC, and HVA to the sum of the concentrations of tyrosine metabolites.

In the step of measuring the relative concentration ratio, the relative concentration ratio may be calculated according to an equation.

The method comprises comparing the measured amount to a relative concentration ratio in a normal control.

The normal control refers to a negative control as an individual without autism spectrum disorder.

The method comprises: for tyrosine, the measured relative concentration ratio is decreased compared to the relative concentration ratio of the normal control; for dopamine, the measured relative concentration ratio increases compared to the relative concentration ratio of the normal control; For DOPAC, the measured relative concentration ratio increases compared to the relative concentration ratio of the normal control; and determining, for HVA, that the measured relative concentration ratio increases compared to the relative concentration ratio of the normal control. Psychological pedagogical treatment or medication may be administered to a diagnosed subject diagnosed with autism spectrum disorder. The drug may be an antidepressant, a stimulant, an anticonvulsant, an antipsychotic, or a combination thereof. Such drugs are, for example, risperidone and aripiprazole.

Another aspect is to measure the concentration of tyrosine metabolites in a subject suspected of having an autism spectrum disorder; measuring the relative concentration ratio of one concentration selected from the group consisting of tyrosine, dopamine, DOPAC, and HVA to the sum of the concentrations of tyrosine metabolites; and comparing the measured amount with a relative concentration ratio in a normal control group.

According to the composition, kit, and method for diagnosing autism spectrum disorder comprising an agent for measuring the relative concentration ratio of tyrosine metabolites in a biological sample, it can be used to objectively, simply, and accurately diagnose autism spectrum disorder with high accuracy and specificity have.

1 is an extracted ion chromatogram of 18 neurochemicals obtained from ultra-high performance liquid chromatography mass spectrometry multiple reaction monitoring mode (1: histamine; 2: GABA; 3: NE; 4: Gln, 5: ACh; 6: Glu; 7: DA; 8: Tyr; 9: 3-MT; 10: KYN; 11: Trp; 12: XA; 13: KA; 14: ILA; 15: IAA; 16: IPA; 17: DOPAC; 18: HVA).
2 is a schematic diagram showing a tyrosine metabolic pathway.
3 is a graph showing the relative concentration ratio of each metabolite to the sum of the tyrosine metabolic pathways in the striatum (*: p<0.05).

Hereinafter, it will be described in more detail through examples. However, these examples are for illustrative purposes of one or more embodiments, and the scope of the present invention is not limited to these examples.

Example 1. Identification of tyrosine metabolites differentially expressed in the striatum of autism spectrum disorder model mice

1. Preparation of Samples from Autism Spectrum Disorder Model Mice

Brain tissue samples from autism spectrum disorder (ASD) valproic acid model mice were collected for each region including the striatum (provided by Konkuk University Medical School). As a negative control, brain tissue samples from normal mice were obtained for each region.

After homogenizing the brain tissue by adding 34 μl of deionized water containing 0.1% (v/v) formic acid to the collected brain tissue, for protein removal, 51 μl of acetonitrile (ACN) An organic solvent was added. This sample was centrifuged at a temperature of 4°C and 13,000xg for 10 minutes to take 30 μl of a supernatant, and 30 μl of an internal standard mixture solution (dopamine-d4, 100 ng/mL; 4-methylumbelliferone (methylumbelliferone) ), 2.5 ng/mL; phenylalanine- ¹³ C ₉ , 25 ng/L) to prepare an assay sample.

The LC insertion vial was placed in the LC vial, and 60 μl of the prepared brain tissue analysis sample was transferred. 5 μl of the assay sample was injected into UPLC-MS/MS for analysis. Brain tissue samples were quantified by the above method, and stored at -80°C until used in experiments.

2. Ultra-High Performance Liquid Chromatography Analysis

Liquid chromatography for the analysis of 18 neurochemicals was performed using an Agilent 1290 infinity II system from Agilent technologies (Palo Alto, CA, USA). As an analytical column for separating analytes, an Acquity UPLC HSS T3 column (2.1 Х 100 mm, 1.8 μm) manufactured by Waters corporation (Milford, MA, USA) was used and maintained at 40°C.

As the mobile phase, water (mobile phase A) to which 0.1% (v/v) formic acid was added and acetonitrile to which 00.1% (v/v) formic acid was added (mobile phase B) were used. A gradient solvent controlling the ratio of mobile phases A and B was used, and gradient solvent conditions were initially maintained for 80% of mobile phase A for 0 minutes to 1.5 minutes. Mobile phase A was changed to 60% for 1.5 minutes to 3 minutes, and mobile phase A to 40% for 3 minutes to 6 minutes. Mobile phase A was held at 5% for 6.5 minutes to 7.5 minutes, and then A was raised to 98% again for 7.5 minutes to 8 minutes. It was allowed to re-equilibrium with mobile phase A 98% for 8 to 12 minutes. The total analysis time is 8 minutes. The temperature of the analytical column was maintained at 40° C., the flow rate was 0.2 mL to 0.5 mL per minute, and the sample injection volume was 5 μl. Table 1 shows the conditions for ultra-high performance liquid chromatography mass spectrometry.

device name Agilent 1290 Infinity II LC system column Waters Acquity UPLC HSS T3 column
(2.1 × 100 mm, 1.8 μm) Mobile phase A: 100% water containing 0.1% (v/v) formic acid
B: 100% ACN containing 0.1% (v/v) formic acid gradient condition hours (minutes) A (percent) B (percent) Flow rate (mL/min) 0 80 20 0.2 1.5 80 20 0.5 3 60 40 0.5 6 40 60 0.5 6.5 5 95 0.5 7.5 5 95 0.5 8 98 2 0.2 12 98 2 0.2 flow rate 0.2 to 0.5 mL/min column temperature 40℃ injection volume 5 μl device name QTRAP 6500+ System ionization mode ESI, positive and negative ion mode scan type Multiple reaction monitoring (MRM) Curtain gas (CUR) 35 psi Ion spray voltage (IS) 5500 V Temperature (TEM) 600℃ Gas pressure 1 (nebulizing gas) 50 psi Gas pressure 2 (heating gas) 50 psi

3. Multiple Reaction Monitoring Analysis

MS/MS analysis of metabolites of glutamate, tryptophan, and tyrosine was performed using the AB SCIEX (Toronto, ON, Canada) QTRAP 6500+ system, which consists of an electrospray ionization (ESI) and triple quadrupole device. . Ionization was analyzed simultaneously in positive and negative mode, and nitrogen gas was used as a nebulizer gas and a heating gas. In order to check the fragment ions of 18 analytes, each analyte was diluted to 100 ng/mL and injected into MS/MS through direct injection. As the analysis mode, multiple reaction monitoring (MRM) mode was used.

The mass spectrometer checks the m/z value and mass fragmentation of the standard material by electrospray ionization. First, MS/MS optimization was performed so that precursor ions and product ions could come out with high sensitivity by injecting a standard material of 10 ng/mL to 100 ng/mL through a syringe. At this time, the values of the first source and gas parameters were used as initial setting values. Each injected standard was checked in Q1 MS scan mode. In the first quadrupole (quadrupole 1, Q1), the precursor is selected in the form of [M+H]+ or [M+H]-, and the declustering potential (DP) value at which the m/z peak of this precursor is generated is determined. became

Then, a collision gas was injected through the second quadrupole to cause fragmentation of the precursor. This fragment was selected as a product ion in the third quadrupole (Q3). The collision energy (CE) value of each fragment is determined, and the last parameter to be optimized is the collision cell exit potential (CXP). In addition, the parameter of the entrance potential (EP) was optimized.

The substances to be analyzed are endogenous substances and have generally low molecular weights between about 100 Da and about 300 Da, and are intended for simultaneous analysis, so care was taken not to overlap in the Q1 MS scan mode. Two ions with good ion peak sensitivity of the analyte materials were selected, and ions with good ion strength were used for quantitative analysis, and the remaining ions were selected for qualitative analysis.

Table 2 shows m/z of Q1 and Q3 of precursor ion and product ion optimized through MS/MS, and DP (declustering potential), CE (Collision energy), and CXP (Collision cell exit potential) values. (†: Quantifier ion). After that, MS source parameters were optimized through FIA (flow injection analysis), an automatic optimization method. Test the values of optimized curtain gas (CUR), ion spray voltage (IS), temperature (TEM), nebulizer gas (GS1), and heating gas (GS2) It allows automatic comparison by setting possible numerical values, and as a result, one optimized setting value can be obtained.

group compound Q1 (m/z) Q3 (m/z) DP (V) CE (V) CXP (V) Glu glutamic acid 147.9 83.8 ^† , 102 2, 40 21,14 10, 12 γ-Aminobutyric acid 104.006 87.0 ^† , 69.0 36 13, 21 10 glutamine 147.003 130 ^† , 84 One 13, 23 18, 12 4-aminobutyric acid-d6 110.085 93 ^† One 15 14 Trp tryptophan 205.047 188.1 ^† , 146.0 31 13, 23 12,18 Indole-3-propionic acid 190 130 ^† , 77 51 19, 61 16, 34 Indole-3-lactic acid 205.988 130.2 ^† , 118.0 61 33,31 14,16 Indole-3-acetic acid 176.046 130.0 ^† , 103.1 46 19, 41 16, 14 kynurenine 209.055 192.1 ^† , 146.1 31 13, 27 12, 18 kynurennic acid 189.986 144.0 ^† , 115.9 116 25, 43 20, 54 xanthurenic acid 205.979 160.1 ^† , 132.0 81 25, 37 10, 22 Tryptophan- ¹³ C ₁₁ 216.088 1991 ^† One 13 12 kynurennic acid-d5 194.933 149.1 ^† , 177.1 One 27,17 14,10 Tyr Tyrosine 182.033 165 ^† , 136 21 13, 17 24, 22 dopamine 154.045 137 ^† , 91 One 13, 31 22, 12 3,4-dihydroxyphenylacetic acid 166.9 122.9 ^† ,95 -5,-10 -10,-26 -19,-41 3-Methoxytyramine 168.101 119 ^† , 151 31 25,13 14,20 Homovanillic acid 180.858 135.9 ^† -30 -12 -13 norepinephrine 152.0 107.1 ^† -30 -12 -13 dopamine-d4 158.073 141.2 ^† One 15 6 Phenylalanine- ¹³ C ₉ 167.1 120.1 ^† ,103 21,16 15,35 14,13 4-methylumbelliferone 174.9 133 ^† -65 -30 -15 histamine 111.968 95.1 ^† , 83.0 One 19, 19 8,14 Acetylcholine 146.146 87.0 ^† , 58.0 One 19, 63 10, 26 Acetylcholine-d4 150.089 91.1 ^† , 43.1 36 19, 45 40,20

4. Check the chromatogram

UPLC-MS/MS was analyzed using the conditions described in Example 1.3 to obtain chromatograms in which 18 neurochemicals were appropriately separated. The obtained chromatogram is shown in Fig. 1 (1: histamine; 2: GABA; 3: NE; 4: Gln, 5: ACh; 6: Glu; 7: DA; 8: Tyr; 9: 3-MT; 10: KYN; 11: Trp; 12: XA; 13: KA; 14: ILA; 15: IAA; 16: IPA; 17: DOPAC; 18: HVA)

*: IS-DA-d4, 100 ng/mL; IS-GABA-d6, IS-Ach-d4, IS-Phe- ¹³ C ₉ , IS-Trp- ¹³ C ₁₁ , IS-KA-d5, 25 ng/mL; IS-4-MUF, 2.5 ng/mL

In addition, the retention time (retention time) of each material is described in Table 3.

compound abbreviation Retention time (minutes) glutamic acid Glu 1.17 γ-Aminobutyric acid GABA 1.14 glutamine Gln 1.16 4-aminobutyric acid-d6 IS-GABA-d6 1.14 tryptophan Trp 1.5 Indole-3-propionic acid IPA 3.88 Indole-3-lactic acid ILA 2.99 Indole-3-acetic acid IAA 3.42 kynurenine KYN 1.37 kynurennic acid KA 1.6 xanthurenic acid XA 1.5 Tryptophan- ¹³ C ₁₁ IS-Trp- ¹³ C ₁₁ 1.5 kynurennic acid-d5 IS-KA-d5 1.6 Tyrosine Tyr 1.23 dopamine DA 1.18 3,4-dihydroxyphenylacetic acid DOPAC 1.63 3-Methoxytyramine 3-MT 1.24 Homovanillic acid HVA 2.12 norepinephrine NE 1.15 dopamine-d4 IS-DA-d4 1.19 Phenylalanine- ¹³ C ₉ IS-Phe- ¹³ C ₉ 1.35 4-methylumbelliferone IS-4-MUF 3.35 histamine Hist 1.01 Acetylcholine Ach 1.17 Acetylcholine-d4 IS-Ach-d4 1.17

5. Calculation of Relative Concentration Ratio of Tyrosine and its Metabolites

From the quantitative analysis results of each tyrosine metabolite, the sum of metabolites corresponding to all tyrosine metabolic pathways (FIG. 2) was obtained, and the relative concentration ratio of each metabolite to the total was calculated according to Equation 1.

Equation 1: Relative Concentration Ratio = Specific Metabolite Concentration / Total Tyrosine Metabolic Pathway Analytes

For example, if Equation 1 is specifically expressed for HVA, it is as Equation 2 below.

Equation 2: HVA Relative Concentration Ratio = HVA Concentration/∑ (Concentrations of Tyr, DA, 3MT, HVA, DOPAC, and NE)

6. Comparison of metabolite relative concentration ratio and fold change in normal control group and autism model group

The ratio of the relative concentration of each metabolite to the sum of tyrosine metabolites for each part of the striatum in brain samples from autistic spectrum disorder valproic acid model mice (n=7) and as a negative control group (n=6) was calculated using the t-test method. Statistical analysis was performed.

The calculated relative concentration ratio of each metabolite is shown in FIG. 3 (*: p<0.05).

As shown in FIG. 3 , the expression of the tyrosine relative concentration ratio decreased to a statistically significant level (0.96 times, p-value=0.002) in the autism model group compared to the normal control group. In addition, the relative concentration ratio of DA, the relative concentration ratio of HVA, and the relative concentration ratio of DOPAC were statistically significant in the autism model group compared to the normal control group (for DA, 1.4 times, p-value = 0.007; for HVA, expression increased by 1.9 fold, p-value=0.003; and for DOPAC, 2.5 fold, p-value=0.002).

Therefore, it was confirmed that the fraction of Tyr, DOPAC, HVA, or DA was significantly changed compared to the total tyrosine metabolic pathway metabolites in the striatum, and the accuracy was excellent as a biomarker for diagnosing autism spectrum disorders.

Claims (12)

A composition for diagnosing autism spectrum disorder (ASD) comprising an agent for measuring the relative concentration ratio of tyrosine metabolites in a biological sample,
The relative concentration ratio is tyrosine, dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid to the sum of the concentrations of tyrosine metabolites in the biological sample. HVA) is the ratio of one concentration selected from the group consisting of,
The biological sample is brain tissue or cerebrospinal fluid,
The sum of the concentrations of the tyrosine metabolites is the sum of the concentrations of tyrosine, dopamine, 3-methoxytyramine (3MT), HVA, DOPAC, and norepinephrine (NE). delete The composition of claim 1, wherein the agent is a protein that specifically binds to a tyrosine metabolite, a compound that binds to a tyrosine metabolite, or a combination thereof. The composition of claim 3, wherein the protein that specifically binds to the tyrosine metabolite is an enzyme. The composition of claim 1 , wherein the autism spectrum disorder is selected from the group consisting of childhood disintegrative disorder, autistic disorder, Asperger's disorder, and pervasive developmental disorder not elsewhere classified. delete A kit for diagnosing autism spectrum disorder comprising an agent for measuring the relative concentration ratio of tyrosine metabolites in a biological sample,
The relative concentration ratio is a ratio of one concentration selected from the group consisting of tyrosine, dopamine, DOPAC, and HVA to the sum of the concentrations of tyrosine metabolites in the biological sample,
The biological sample is brain tissue or cerebrospinal fluid,
The sum of the concentrations of the tyrosine metabolites is the sum of the concentrations of tyrosine, dopamine, 3-methoxytyramine (3MT), HVA, DOPAC, and norepinephrine (NE). measuring concentrations of tyrosine metabolites in a biological sample isolated from a subject suspected of having autism spectrum disorder;
measuring a relative concentration ratio of one concentration selected from the group consisting of tyrosine, dopamine, DOPAC, and HVA to the sum of the concentrations of the tyrosine metabolites; and
Comprising the step of comparing the measured amount with the relative concentration ratio in a normal control,
The biological sample is brain tissue or cerebrospinal fluid,
The sum of the concentrations of the tyrosine metabolites is the sum of the concentrations of tyrosine, dopamine, 3-methoxytyramine (3MT), HVA, DOPAC, and norepinephrine (NE) To provide information necessary for the diagnosis of autism spectrum disorder A method for calculating the relative concentration ratio of tyrosine metabolites. delete The method according to claim 8, wherein the step of measuring the concentration of tyrosine metabolites from the biological sample isolated from the subject is mass spectrometry (MS), enzyme-linked immunosorbent assay (ELISA), a protein chip, Immunoprecipitation, microarray, electron microscopy, or a combination thereof. delete 9. The method of claim 8,
for tyrosine, the measured relative concentration ratio decreases compared to the relative concentration ratio of the normal control;
for dopamine, the measured relative concentration ratio increases compared to the relative concentration ratio of the normal control;
For DOPAC, the measured relative concentration ratio increases compared to the relative concentration ratio of the normal control; and
for HVA, determining at least one of the measured relative concentration ratios increasing as compared to the relative concentration ratios of the normal control.

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