KR102159344B1 - Composition or kit for diagnosing behavioral addiction and method of detecting kynurenine for diagnosis of behavioral addiction using the same - Google Patents

Abstract

The present invention provides a composition or a kit for diagnosing behavioral addiction and a method for detecting kynurenine in order to provide information necessary for the diagnosis of behavioral addiction using the same. According to the present invention, the composition or the kit can be used to objectively and simply diagnose behavioral addiction with high accuracy and specificity.

Description

Composition or kit for diagnosing behavioral addiction and method of detecting kynurenine for diagnosis of behavioral addiction using the same}

It relates to a composition for diagnosis of behavioral addiction, a kit, and a method of detecting a biomarker for diagnosis of behavioral addiction using the same.

Behavioral addiction relates to addiction related to certain behaviors, processes, and activities, such as gambling addiction, sex addiction, pathological Internet use, etc., where brain circuits are involved, tolerance and withdrawal may occur, and impulsiveness develops. It plays an important role in the early stages, and compulsion is key to maintaining behavior. In the modern society, alcohol addiction, drug addiction, Internet addiction, game addiction, and obsessive addiction are prominent.

Biomarkers are known for psychiatric symptoms such as autism, Parkinson's disease, dementia, attention deficit hyperactivity disorder, alcoholism, drug addiction, and obsessive-compulsive disorder (US Publication No. 2005/0084880 A1 (2005.04.21)). , Republic of Korea registration announcement 10-1157526 (2016.06.27), etc.). However, in order to diagnose behavioral addiction, especially internet addiction or game addiction, brain image verification through magnetic resonance imaging (MRI) or self-diagnosis through questionnaire is mainly used, and is an effective biomarker for diagnosis. Is not there yet.

Therefore, it is not necessary to develop a biomarker with high accuracy and specificity of diagnosis, which can diagnose behavioral addiction simply and inexpensively, rather than a self-test method based on expensive and cumbersome brain imaging diagnosis or subjective judgment. There is.

It provides a composition for diagnosis of behavioral addiction.

Provides a kit for diagnosis of behavioral addiction.

Provides a method of providing information necessary for the diagnosis of behavioral addiction.

One aspect provides a composition for diagnosing behavioral addiction, including an agent for measuring the amount of kynurenine.

Kynurenine is a metabolite of the amino acid L-tryptophan and has the formula:

.

.

Kynurenine dilates blood vessels in the inflammatory response and can regulate the immune response.

The amount of kynurenine may be an absolute amount measured from a biological sample, a concentration in a biological sample, or a ratio relative to the amount of kynurenine in a normal control. The biological sample may be blood, plasma, serum, bone marrow fluid, lymph fluid, saliva, urine, tear fluid, mucosal fluid, amniotic fluid, or a combination thereof.

The agent may be an antibody specifically binding to kynurenine, an 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 containing 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 antibody or antigen-binding fragment thereof may be an anti-kynurenine antibody or an antigen-binding fragment thereof. The aptamer may be a single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) or peptide that specifically binds to kynurenine.

The term "behavioral addiction" refers to an addiction to an action, process or activity. Behavioral addiction can lead to tolerance and withdrawal. The behavioral addiction may be selected from the group consisting of internet addiction, smartphone addiction, game addiction, gambling addiction, religious addiction, sex addiction, shopping addiction, exercise addiction, and work addiction. The internet addiction refers to a state of addiction in which physical, mental, or social life is disturbed due to pathological or compulsive internet use. The game addiction refers to pathologically or obsessive immersion in the game, leading to a state of addiction in which physical, mental, or social life is disturbed. The Internet or game includes use by all media such as computers, mobile phones, smart phones, and TVs.

Another aspect provides a kit for diagnosing behavioral addiction, including an agent for measuring the amount of kynurenine.

The kit may further include a sample necessary for diagnosis of behavioral addiction. 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 substance, or a chromogenic substrate.

Another aspect includes measuring the amount of kynurenine in a biological sample isolated from an individual suspected of being addicted to behavior; And it provides a method of detecting kynurenine to provide information necessary for diagnosis of behavioral addiction, comprising the step of comparing the measured amount with the amount of kynurenine of a normal control group.

The method includes measuring the amount of kynurenine in a biological sample isolated from a subject suspected of behavioral addiction.

The subject may be a mammal, for example a human, cow, horse, pig, dog, sheep, goat, or cat. The subject may be a subject suffering from or suspected of having a behavioral addiction.

The biological sample refers to a sample obtained from the individual. The biological sample may be, for example, blood, plasma, serum, bone marrow fluid, lymph fluid, saliva, urine, tear fluid, mucosal fluid, amniotic fluid, or a combination thereof. The biological sample includes a protein sample or a nucleic acid sample obtained from the sample.

The step of measuring the amount of kynurenine may include incubating the biological sample with an antibody specifically binding to kynurenine, an antigen-binding fragment thereof, or an aptamer.

The step of measuring the amount of kynurenine is mass spectrometry (MS), enzyme-linked immunosorbent assay (ELISA), protein chip, immunoprecipitation, microarray, electron microscopy. , Or a combination thereof. The mass spectrometry method may be tandem mass spectrometry (MS/MS). The ELISA may be a direct ELISA, a sandwich ELISA, a competitive ELISA, a reverse ELISA, or a combination thereof.

The step of measuring the amount of kynurenine is not only measuring the amount of kynurenine, but also enzymes related to kynurenine biosynthesis (eg, tryptophan dioxygenase) and indoleamine 2,3-dioxygenase (indoleamine 2, 3-dioxygenase)) or biodegradation-related enzyme gene expression level or protein expression level.

The method includes comparing the measured amount of kynurenine with the amount of kynurenine in a normal control. The method may include checking whether the amount of kynurenine measured is increased compared to the amount of kynurenine in the normal control group. The normal control group is a group that did not suffer from behavioral addiction and means a negative control group. In the above method, when the measured amount of kynurenine is increased compared to the amount of kynurenine in the normal control group, the individual may be diagnosed with behavioral addiction or having a high probability of developing it. Psychosocial therapy or drug therapy can be performed on individuals diagnosed with behavioral addiction. The drugs are, for example, chlordiazepoxide, lorazepam, alprazolam, diazepam, triazolam, flurazepam, and vitamins. , Naltrexone, disulfiram, methadone, acamprosate, bupropion, varenicline, buprenorphine, or a combination thereof have.

According to the composition and kit for diagnosis of behavioral addiction according to an aspect, and a method for detecting kynurenine to provide information necessary for the diagnosis of behavioral addiction using the same, it is possible to diagnose behavioral addiction with objective, simple, high accuracy and specificity. Can be used.

1 is a schematic diagram showing a serum sample pretreatment process.
2 is an extracted ion chromatogram of 28 neurochemicals obtained from ultra-high performance liquid chromatography mass spectrometry multiple reaction monitoring mode.
3 is a graph showing the concentration of kynurenine in a normal control group and behavioral poisoning measured using mass spectrometry.

It will be described in more detail through the following examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1. Identification of differentially expressed neurochemicals among serum components of Internet or game addiction groups

1. Preparation of samples from Internet or game addicts

Serum samples of Internet or game addiction patients (n=35) were collected from Boramae Hospital in Seoul National University Hospital Operation. As a negative control, a serum sample of a normal person (n=35) who was not diagnosed with internet or game addiction was obtained.

To prevent proteolysis or modification of serum samples, cOmplete as a protease inhibitor, Mini EDTA-free protease inhibitor cocktail (Roche), and PhosSTOP (Roche) as a phosphatase inhibitor were added to the serum obtained immediately after the serum was obtained. I did. According to the manufacturer's protocol, one tablet was dissolved in 200 µl of phosphate buffered saline (PBS) to make 50 X, and then added to each sample to a final concentration of 1.5 X. Serum samples to which inhibitors were added were quantified and stored at -80°C until use in experiments.

2. Serum sample preparation

To 10 µl of a serum sample, 30 µl of acetonitrile in which an internal standard mixture was dissolved was added, and protein precipitation using an organic solvent was performed. After taking 30 µl of the supernatant, it was diluted with 30 µl of water to which 0.1% (v/v) formic acid was added. An LC insert vial was added to the LC vial to transfer 60 µl of the diluted serum sample, and 5 µl was injected into UPLC-MS/MS for analysis. The final pretreatment process is shown in FIG. 1.

3. Ultra-high performance liquid chromatography analysis

Liquid chromatography for analyzing 28 neurochemicals was performed using an Agilent 1290 infinity II LC system (Agilent technologies, Inc.). As the analysis column finally selected to separate the analyte, an Acquity UPLC HSS T3 column (Waters corporation, 2.1 Х 100 mm, 1.8 µm) was used.

The mobile phase used for proper separation with the analytical column was water (mobile phase A) with 0.1% (v/v) formic acid added and acetonitrile (mobile phase B) with 0.1% (v/v) formic acid. I did. A gradient solvent that adjusts the ratio of mobile phases A and B was used, and the gradient solvent conditions initially started with 100% mobile phase A and maintained for 1.5 minutes from the start point. Mobile phase A was changed to 40% for 1.5 to 6 minutes, and mobile phase A to 5% for 6 to 7.2 minutes. After maintaining 5% of mobile phase A for 7.2 to 7.5 minutes, A was increased to 100% again for 7.5 to 7.8 minutes. Re-equilibrium time was given to 100% of mobile phase A as initial condition for 7.8 to 9 minutes. The analysis time was a total of 9 minutes, and it is shown in Table 1. The temperature of the analysis column was maintained at 40° C., the flow rate was 0.5 ml/min, and the sample injection volume was 5 μl.

4. Multiple Reaction Monitoring Analysis

MS/MS (mass spectrometry/mass spectrometry or tandem mass spectrometry) analysis of metabolites of glutamate, tryptophan, and tyrosine is performed by the AB SCIEX™ QTRAP 6500 consisting of electro spray ionization (ESI) and triple quadrupole devices. + system(SCIEX) was used. Ionization was simultaneously analyzed in cation mode and anion mode, and nitrogen gas was used as a nebulizing gas and a heating gas. In order to identify fragments of 28 kinds of analytes, each of the analytes was diluted to 100 ng/ml and injected into MS/MS. As the analysis mode, MRM (multiple reaction monitoring) mode was used.

The mass spectrometer confirmed the m/z value and mass fragmentation of the standard material by electrospray ionization. First, a standard substance having a concentration of 10 ng/ml to 100 ng/ml was injected into the mass spectrometer with a syringe, and MS/MS optimization was performed so that precursor ions and product ions can come out with high sensitivity. At this time, the values of the initial source and gas parameters were used as initial set values. Each injected standard was confirmed by 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 m/z peak of this precursor is high, resulting in a declustering potential (DP) ) Value was determined.

After that, a collision gas was injected through the second quadrupole to cause fragmentation of the precursor. This fragment ion was selected as a product ion in the third quadrupole (Q3). The collision energy (CE) value of each fragment ion is determined, and the parameter optimized at the end is CXP (collision cell exit potential), and in addition, parameters of EP (entrance potential) and CEP (collision cell entrance potential) are optimized. I did.

After that, MS raw material parameters were optimized through flow injection analysis (FIA), an automatic optimization method. This is a method of automatically optimizing raw material parameters related to ionization by passing the mixed analyte through an LC system that is not connected to a column. The values of curtain gas (CUR), ion spray voltage (IS), temperature (TEM), nebulizing gas, and heating gas are as described in Table 1 below. Set together.

Device name QTRAP 6500+ System Ionization mode ESI, cation and anion modes Scan type MRM (multiple reaction monitoring) 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

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

The m/z of Q1 and Q3 of the precursor ions and product ions optimized through MS/MS, and DP, CE, and CXP values thereof are shown in Table 2 below.

group compound Q1 (m/z) Q3 (m/z) DP (V) CE (V) CXP (V) Glu Glutamic acid 148.1 83.9 ^† , 130 One 11, 21 6, 4 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-d6 acid 110.085 93 ^† One 15 14 Trp Tryptophan 205.047 188.1 ^† , 146.0 31 13, 21 10, 12 Serotonin 177.071 160.1 ^† , 115.0 16 13, 37 8, 14 6-hydroxy melatonin 249.011 190.1 ^† , 130.0 One 19, 53 10, 10 Indole-3-propionic acid 190 130 ^† , 77 51 19, 61 16, 34 Indole-3-lactic acid 205.988 130.2 ^† , 118.0 61 33 14 Indole-3-acetic acid 176.046 130.0 ^† , 103.1 46 19, 41 16, 14 Tryptamine 161.059 144 ^† , 117.0 One 13, 33 22, 16 Kynurenine 209.055 192.1 ^† , 146.1 31 13, 27 12, 18 Kinurenic acid 189.986 144.0 ^† , 115.9 116 25, 43 20, 54 3-hydroxykynurenine 224.978 110.0 ^† , 162.0 One 21, 27 14, 18 Anthranilic acid 137.99 120 ^† , 92 16 15, 29 16, 12 3-hydroxyanthranylic acid 153.719 136 ^† 150 47 15 Xanthurenic acid 205.979 160.1 ^† , 132.0 81 25, 37 10, 22 Harmane 183.062 115.0 ^† , 168.1 121 39, 43 20, 14 Norharmane 169.059 115.0 ^† , 142.1 131 37, 45 14, 16 ¹³ C-11 Tryptophan 216.18 199.1 ^† One 13 12 Serotonin-d4 181.13 164.1 ^† , 136.1 One 11, 31 8 Kinurenic acid-d5 194.93 149.1 ^† , 177.1 One 17, 27 14 Harman-d3 186.057 115 ^† 131 43 54 Tyr Tyrosine 182.033 165 ^† , 136 21 13, 17 24, 22 Levodopa 197.882 152 ^† , 181 76 13, 19 10, 18 Dopamine 154.045 137 ^† , 91 One 13, 31 22, 12 Metanephrine 198.013 165.1 ^† , 120.1 One 27, 25 14, 8 3-Methoxytyramine 168.101 119 ^† , 151 31 13, 25 20, 14 Dopamine-d4 158.11 141.2 ^† One 15 6 Homovanilic acid 180.858 135.9 ^† -30 -12 -13 Vanillylmandelic acid 196.91 137.8 ^† -40 -16 -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 150.08 91.1 ^† , 43.1 36 19, 45 40,20 Cortisol 362.909 121 ^† , 91.1 One 25, 27 16

(†: Quantifier ion)

5. Check the chromatogram

As described in Example 1.4, UPLC-MS/MS was analyzed to obtain a chromatogram in which neurochemical substances were properly separated.

The obtained chromatogram is shown in FIG. 2, and each retention time is summarized in Table 3 below.

compound Abbreviation Retention time (minutes) Histamine Hist 0.44 Glutamine Gln 0.52 Glutamic acid Glu 0.54 4-aminobutyric-d6 acid IS_GABA-d6 0.54 γ-aminobutyric acid GABA 0.55 Acetylcholine-d4 IS_Ach-d4 0.85 Acetylcholine Ach 0.9 Levodopa L-Dopa 1.22 Dopamine-d4 IS_DA-d4 1.26 Dopamine DA 1.29 3-hydroxykynurenine 3-HK 1.70 Metanephrine MN 1.84 Tyrosine Tyr 1.86 Vanillylmandelic acid VMA 3.03 Serotonin-d4 IS_Ser-d4 3.05 Serotonin Ser 3.05 3-methoxytyramine 3-MT 3.07 Kynurenine KYN 3.12 3-hydroxyanthranylic acid 3-HAA 3.36 Xanthurenic acid XA 3.52 ¹³ C-11 Tryptophan IS_Trp- ¹³ C 3.52 Tryptophan Trp 3.53 Kinurenic acid-d5 IS_KA-d5 3.66 Kinurenic Acid KA 3.66 Tryptamine Trypta 3.70 Norharmann NHM 3.97 Homovanillic acid HVA 4.07 Harman-d3 IS_HM-d3 4.13 Harman HM 4.13 6-hydroxy melatonin 6-HM 4.13 Anthranilic acid AA 4.32 Indole-3-lactic acid ILA 4.55 Indole-3-acetic acid IAA 4.91 Indole-3-propionic acid IPA 5.32 Cortisol corti 5.33

6. Statistical analysis of kynurenine concentration in Internet or game addicts

From the mass spectrometry data, the expression patterns of kynurenine neurochemicals selected as biomarker candidates in 35 control subjects and 35 Internet or game addiction patients were statistically analyzed using the Mann-Whitney U-test, and the results are shown. It is shown in 3.

As shown in Figure 3, it was confirmed that the expression of kynurenine was increased to a statistically significant level in the Internet or game addiction group compared to the normal control group (p value <0.0001). Through this, it was confirmed that kynurenine has excellent accuracy as a biomarker for internet or game addiction.

Claims (10)

As a composition for diagnosing behavioral addiction comprising an agent measuring the amount of kynurenine,
The behavioral addiction is a composition selected from the group consisting of Internet addiction and game addiction. delete The composition of claim 1, wherein the agent is an antibody or antigen-binding fragment thereof that specifically binds to kynurenine, or an aptamer. The composition of claim 3, wherein the antibody is a polyclonal antibody or a monoclonal antibody. As a kit for diagnosing behavioral addiction comprising an agent for measuring the amount of kynurenine,
The behavioral addiction is a kit selected from the group consisting of internet addiction and game addiction. Measuring the amount of kynurenine in a biological sample isolated from an individual suspected of behavioral poisoning;
Comparing the measured amount with the amount of kynurenine in a normal control group; And
Comprising the step of checking whether the amount of kynurenine measured is increased compared to the amount of kynurenine in the normal control,
As a method of detecting kynurenine to provide information necessary for diagnosis of behavioral addiction,
The behavioral addiction is selected from the group consisting of Internet addiction and game addiction. The method of claim 6, wherein the biological sample is blood, plasma, serum, bone marrow fluid, lymph fluid, saliva, urine, tear fluid, mucosal fluid, amniotic fluid, or a combination thereof. The method of claim 6, wherein the measuring of the amount of kynurenine comprises incubating the biological sample with an antibody or antigen-binding fragment thereof, or an aptamer that specifically binds to kynurenine. The method according to claim 6, wherein the measuring the amount of kynurenine comprises mass spectrometry (MS), enzyme-linked immunosorbent assay (ELISA), protein chip, immunoprecipitation, microarray, electron microscope Method (electron microscopy), or a method that is performed by a combination thereof. delete

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