KR101982923B1 - Biomarker composition for diagnosing migraine and diagnostic method of migraine using the same - Google Patents

Abstract

The present invention relates to a biomarker for diagnosis of migraine head and a method for diagnosing migraine head using the biomarker. Specifically, the method of the present invention can diagnose migraine head by measuring the concentration of phenylalanine, which is a specific metabolite in serum samples, and comparing the concentration of phenylalanine with the concentration of phenylalanine in normal subjects. Therefore, the method or the agent capable of specifying the concentration of phenylalanine A kit containing an analytical instrument can be useful for diagnosing migraine headaches.

Description

TECHNICAL FIELD [0001] The present invention relates to a biomarker for diagnosis of migraine head, and a method for diagnosing a migraine head using the biomarker,

The present invention relates to a biomarker for diagnosis of migraine head and a method for diagnosing migraine head using the biomarker.

Migraine is one of the most common neurological disorders, with about 20% of adults with migraine. In general, migraine is a functional disorder of the brain, a sensitive brain state capable of having cerebral cortical dysphoria (CSD) that activate the trigeminal vasculature. However, the mechanism of migraine has not been clearly elucidated because of the complexity of its occurrence.

With regard to the causes of migraine headaches, many fluids are thought to be involved. These include serotonin, histamine, prostaglandins, platelet factors, endorphins and vasoactive neuropeptides.

Prophylactic medication for migraine can only be given to patients who experience two to four or more headaches per month, if the resulting headache is severe enough to interfere with daily life. Beta blockers such as propanolol are most widely used as therapeutic agents. In addition, there are methicillinide maleate, calcium channel blocker, and amitriptylline. All of these treatments have serious side effects such as sedation, loss of energy, dry mouth, weight gain, constipation and gastrointestinal seizures. On the other hand, caffeine-containing ergotamine is mainly used as a popular treatment for migraine.

Migraine is diagnosed as a clinical feature based on the International Headache Classification-3 (ICHD-3) standard, which has two forms that can be clinically distinguished, with or without a preceding symptom. Prefrontal symptoms of migraine include temporary visual impairment or sensory disturbances, which include other symptoms such as intense headache, nausea, increased sensitivity to light and sound. However, there is no objective test to diagnose migraine, so there is limited understanding of the pathophysiology of migraine and the development of a treatment for migraine.

Metabolomics, on the other hand, monitors changes in small molecule metabolites in tissues, blood or organisms to analyze endogenous metabolic profiles in biological samples. Metabolism based on ¹ H-NMR is nondestructive and can be processed in bulk It is an easy quantification technology. Thus, it is used to study toxicology, such as the environmental impacts of pesticide residues and the effects on aquatic organisms due to other harmful substances. In this regard, studies have been reported on the toxicity of lambda-cyhalothrin to goldfish through metabolomic analysis using NMR (Li M. et al . , Aquat Toxicol. , 2014 Jan, 146: 82-92).

Accordingly, the present inventors have made efforts to develop a biomarker that can be used for the diagnosis of migraine. Analysis of ¹ H-NMR spectrum using serum obtained from migraine patients and normal persons revealed that phenylalanine, one of the metabolites, The present inventors have completed the present invention by confirming that they are significantly reduced in migraine patients compared to normal persons.

 Li M. et al., Aquat Toxicol., 2014 Jan, 146: 82-92.

It is an object of the present invention to provide a method for diagnosing migraine head by measuring and comparing the content of a specific metabolite.

Another object of the present invention is to provide a method for accompanying diagnosis of a migraine patient by measuring and comparing the content of a specific metabolite.

It is another object of the present invention to provide a biomarker composition for accompanying diagnosis of a migraine patient comprising an agent for measuring the content of a specific metabolite.

It is still another object of the present invention to provide a biomarker composition and a kit for the diagnosis of migraine, which comprises a preparation or an apparatus for measuring the content of a specific metabolite.

In order to accomplish the above object, the present invention provides a method for measuring the concentration of phenylalanine from a sample; And comparing the phenylalanine concentration with the concentration of the normal subject. The present invention also provides a method for analyzing a metabolite for providing information necessary for diagnosis of migraine headache.

Also, the present invention provides a method for detecting a protein comprising the steps of: 1) measuring the concentration of phenylalanine from a sample; And 2) comparing the phenylalanine concentration of step 1) to the normal individual's concentration.

The present invention also provides a biomarker composition for the diagnosis of migraine head which comprises a preparation for quantification of phenylalanine.

The present invention also provides a biomarker composition for accompanying diagnosis of a migraine patient comprising a preparation for quantifying phenylalanine.

Furthermore, the present invention provides a kit for the diagnosis of migraine, which comprises a preparation for quantitative determination of phenylalanine or a quantitative analysis apparatus.

The method of the present invention can diagnose migraine head by measuring the concentration of phenylalanine, which is a specific metabolite in a serum sample, and comparing the concentration of phenylalanine with the concentration of phenylalanine in a normal individual. Therefore, the method or the analyzer capable of specifying the concentration of phenylalanine The included kit can be useful for diagnosing migraine.

Figure 1 is a graph comparing changes in the expression of metabolites quantified from sera of normal or migraine migraine.
FIG. 2 is a graph showing the results of analysis of metabolites determined from the serum of a normal group, a migraine patient group having a precursor symptom, or a migraine group without a precursor symptom by the PCA method.
FIG. 3 is a graph showing the results of analysis of the metabolites quantified from the serum of the normal group or the migraine patient group by the orthogonal partial least squares discrimination method and comparing the OPLS-DA model (A) of the normal group or the migraine patient group and the S- ).
FIG. 4 is a graph showing the values of the metabolites 2-hydroxybutyrate (A), isocitrate (B), phenylalanine (C), lactate (D) and other EUC values measured from the serum of normal or migraine patients Respectively.
FIG. 5 is a graph showing the AUC values of biomarkers usable in combination among the metabolites quantified from the serum of normal or migraine patients.

Hereinafter, the present invention will be described in detail.

The present invention relates to a method for measuring the concentration of phenylalanine from a sample; And comparing the phenylalanine concentration with the concentration of the normal subject. The present invention also provides a method for analyzing a metabolite for providing information necessary for diagnosis of migraine headache.

The method for analyzing a metabolite according to the present invention provides a step of measuring the concentration of phenylalanine from a sample.

The sample can be any sample whose concentration level of phenylalanine can be changed by migraine. Specifically, the sample may be urine, blood, serum, plasma or cerebrospinal fluid. In one embodiment of the invention, the sample may be serum.

The determination of the concentration in the method according to the present invention can be carried out by any method known to be capable of measuring the concentration of the metabolites, specifically the phenylalanine, contained in the sample. Specifically, the concentration measurement can be performed by a group consisting of nuclear magnetic resonance spectroscopy, chromatography, UV spectroscopy, IR spectroscopy, fluorescence spectroscopy, enzyme-linked immunosorbent assay (ELISA) And may be measured by any one or more devices selected.

In addition, the method for analyzing a metabolite according to the present invention provides a step of comparing the concentration of phenylalanine with that of a normal individual. If the concentration of phenylalanine in the above step is decreased as compared with the normal individuals, it can be determined as migraine.

Furthermore, the method of analyzing a metabolite according to the present invention may further comprise the step of measuring the concentration of at least one selected from the group consisting of 2-hydroxybutyrate and isocitrate. In the above step, if the concentration of 2-hydroxybutyrate or isocitrate is decreased as compared with the normal individuals, it can be judged as migraine.

Specifically, in one embodiment of the present invention, the present inventors obtained serum from a normal person, a migraine patient having a precursor symptom, and a migraine patient without a precursor symptom, and analyzing the concentration of metabolites contained in the obtained serum to determine phenylalanine, 2 -Hydroxybutyrate and isocitrate (see Fig. 1).

In addition, it was confirmed that the above-mentioned three metabolites significantly decreased expression in blood samples isolated from migraine patients (see Table 2). In addition, an analysis of the ROC of the three selected metabolites indicated that each single metabolite showed an AUC value of 0.7 to 0.9 (see Table 3), and the AUC value analyzed for the combination of three metabolites Was found to be 0.899 (see FIG. 5B).

Therefore, the method according to the present invention can be useful for diagnosing migraine head by measuring the concentration of phenylalanine.

Also, the present invention provides a method for detecting a protein comprising the steps of: 1) measuring the concentration of phenylalanine from a sample; And 2) comparing the phenylalanine concentration of step 1) to the normal individual's concentration. The metabolic product analysis method may further include the step of measuring the concentration of at least one selected from the group consisting of 2-hydroxybutyrate and isocitrate.

As used herein, the term " accompanying diagnosis " refers to a diagnostic technique for predicting a patient's response to a specific drug using genetic information (biomarker) such as blood and DNA.

The present invention also provides a biomarker composition for the diagnosis of migraine head which comprises a preparation for quantification of phenylalanine.

The composition may further comprise at least one agent for quantitative determination selected from the group consisting of 2-hydroxybutyrate and isocitrate.

The agent for quantitative determination of phenylalanine may include any substance known to be capable of measuring the concentration of phenylalanine. The preparation for quantification of 2-hydroxybutyrate may include any substance known to be capable of measuring the concentration of 2-hydroxybutyrate. The preparation for quantification of isocitrate may include any substance known to be capable of measuring the concentration of isocitrate.

The biomarker composition may further comprise a detection reagent. The detection reagent may be a conjugate labeled with a detection element such as a chromogenic enzyme, a fluorescent substance, a radioactive isotope or a colloid. The chromogenic enzyme may be a peroxidase, an alkaline phosphatase, or an acid phosphatase, and the fluorescent substance may be a fluorescein carboxylic acid (FCA), a fluorescein isothiocyanate (FITC), a fluorescein thiourea (FTH) 2 ', 7'-dichlorofluorescein-5-yl, 2', 7'-dichlorofluorescein-6 Yl, tetramethylrhodamine-6-yl, 4,4-difluoro-5,7-dimethyl-4-bor- Diaza-s-indacene-3-ethyl or 4,4-difluoro-5,7-diphenyl-4-bora-3a, 4a- diaza- , Cy3, Cy5, polyL-lysine-fluorescein isothiocyanate (FITC), rhodamine-B-isothiocyanate (RITC), PE (Phycoerythrin) or rhodamine.

The detection reagent may further comprise a ligand capable of specifically binding to the detection reagent. The ligand may be a conjugate labeled with a detection element such as a chromogenic enzyme, a fluorescent substance, a radioactive isotope or a colloid, and a ligand treated with streptavidin or avidin.

The composition of the present invention may contain, in addition to the detection reagent as described above, distilled water or a buffer that stably maintains the structure thereof.

Specifically, in one embodiment of the present invention, the present inventors obtained serum from a normal person, a migraine patient having a precursor symptom, and a migraine patient without a precursor symptom, and analyzing the concentration of metabolites contained in the obtained serum to determine phenylalanine, 2 -Hydroxybutyrate and isocitrate (see Fig. 1).

In addition, it was confirmed that the above-mentioned three metabolites significantly decreased expression in blood samples isolated from migraine patients (see Table 2). In addition, an analysis of the ROC of the three selected metabolites indicated that each single metabolite showed an AUC value of 0.7 to 0.9 (see Table 3), and the AUC value analyzed for the combination of three metabolites Was found to be 0.899 (see FIG. 5B).

Therefore, the method according to the present invention can be useful for diagnosing migraine head by measuring the concentration of phenylalanine.

The present invention also provides a biomarker composition for accompanying diagnosis of a migraine patient comprising a preparation for quantifying phenylalanine.

The composition may further comprise at least one agent for quantitative determination selected from the group consisting of 2-hydroxybutyrate and isocitrate.

The agent for quantitative determination of phenylalanine may include any substance known to be capable of measuring the concentration of phenylalanine. The preparation for quantification of 2-hydroxybutyrate may include any substance known to be capable of measuring the concentration of 2-hydroxybutyrate. The preparation for quantification of isocitrate may include any substance known to be capable of measuring the concentration of isocitrate.

The biomarker composition may further comprise a detection reagent. The detection reagent may be a conjugate labeled with a detection element such as a chromogenic enzyme, a fluorescent substance, a radioactive isotope or a colloid. The chromogenic enzyme may be a peroxidase, an alkaline phosphatase, or an acid phosphatase, and the fluorescent substance may be a fluorescein carboxylic acid (FCA), a fluorescein isothiocyanate (FITC), a fluorescein thiourea (FTH) 2 ', 7'-dichlorofluorescein-5-yl, 2', 7'-dichlorofluorescein-6 Yl, tetramethylrhodamine-6-yl, 4,4-difluoro-5,7-dimethyl-4-bor- Diaza-s-indacene-3-ethyl or 4,4-difluoro-5,7-diphenyl-4-bora-3a, 4a- diaza- , Cy3, Cy5, polyL-lysine-fluorescein isothiocyanate (FITC), rhodamine-B-isothiocyanate (RITC), PE (Phycoerythrin) or rhodamine.

The detection reagent may further comprise a ligand capable of specifically binding to the detection reagent. The ligand may be a conjugate labeled with a detection element such as a chromogenic enzyme, a fluorescent substance, a radioactive isotope or a colloid, and a ligand treated with streptavidin or avidin.

The composition of the present invention may contain, in addition to the detection reagent as described above, distilled water or a buffer that stably maintains the structure thereof.

Furthermore, the present invention provides a kit for the diagnosis of migraine, which comprises a preparation for quantitative determination of phenylalanine or a quantitative analysis apparatus.

The kit may further comprise at least one quantitative preparation or quantitative analysis device selected from the group consisting of 2-hydroxybutyrate and isocitrate.

The kit may be used to diagnose migraine headaches on the same principle as the metabolic product analysis method for providing information necessary for the diagnosis of migraine head as described above.

Specifically, the metabolite analysis method can diagnose migraine head by measuring the concentration of phenylalanine from the sample and comparing the measured concentration to the concentration of normal individuals. The method may further comprise measuring the concentration of at least one selected from the group consisting of 2-hydroxybutyrate and isostrate. In the above method, the sample may be urine, blood, serum, plasma or cerebrospinal fluid.

The kit for quantifying phenylalanine contained in the kit may include any substance known to be capable of measuring the concentration of phenylalanine. The preparation for quantification of 2-hydroxybutyrate may include any substance known to be capable of measuring the concentration of 2-hydroxybutyrate. The preparation for quantification of isocitrate may include any substance known to be capable of measuring the concentration of isocitrate. Meanwhile, the quantitative analysis apparatus may be any one selected from the group consisting of nuclear magnetic resonance spectroscopy, chromatography, ultraviolet spectroscopy, infrared spectroscopy, fluorescence spectroscopy, enzyme-linked immunosorbent assay (ELISA) and mass spectrometry.

In the case of diagnosing migraine using the kit according to the present invention, when the concentration of phenylalanine is decreased as compared with the normal individuals, it can be judged as migraine. Further, when the concentration of any one or more selected from the group consisting of 2-hydroxybutyrate and isocitrate is decreased as compared with the normal individuals, it can be determined as migraine.

Specifically, in one embodiment of the present invention, the present inventors obtained serum from a normal person, a migraine patient having a precursor symptom, and a migraine patient without a precursor symptom, and analyzing the concentration of metabolites contained in the obtained serum to determine phenylalanine, 2 -Hydroxybutyrate and isocitrate (see Fig. 1).

In addition, it was confirmed that the above-mentioned three metabolites significantly decreased expression in blood samples isolated from migraine patients (see Table 2). In addition, an analysis of the ROC of the three selected metabolites indicated that each single metabolite showed an AUC value of 0.7 to 0.9 (see Table 3), and the AUC value analyzed for the combination of three metabolites Was found to be 0.899 (see FIG. 5B).

Therefore, the method according to the present invention can be useful for diagnosing migraine head by measuring the concentration of phenylalanine.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are intended to illustrate the present invention, but the present invention is not limited thereto.

Example  1. Preparation of NMR samples from migraine patients

First, NMR samples were prepared in the following manner to identify biomarkers from migraine patients. All experiments were conducted with the approval of the Clinical Research Deliberation Committee of the Gil Medical University Foundation (GBIRB2013-277).

Specifically, we recruited 10 migraine patients with presumptive symptoms, 10 migraine patients without prefrontal symptoms, and 9 normal persons. The classification of patients with migraine according to presence or absence of arousal was based on International Headache Classification-3 (ICHD-3). All patients had no other illnesses and restricted the intake of foods containing biogenic amines (eg, chocolate, dried fruit, cheese, etc.) from one week prior to testing to rule out biochemical interference from diet. In addition, the intake of monoamine oxidase (MAO) activity or changes in the metabolite composition was stopped two weeks before the test. The recruited patients and normal persons were allowed to rest in a lying position for 5 minutes, and then 10 ml of peripheral venous blood was obtained through a venous puncture. The obtained blood was centrifuged at 12,000 x g for 10 minutes to remove the supernatant. Serum was obtained in 30 minutes using a serum-separator tube and stored at -80 DEG C until NMR was performed. The demographic and clinical characteristics of the patients and the normal subjects were summarized in Table 1 below.

Patient ID Age / Sex Features of headache Related symptoms Visual impairment frequency Position / degree /
Deterioration due to exercise Lifespan / Anxiety /
nausea Migraine patients with prefrontal symptoms One F / 35 Left / rupture / + + / + / + + February 2 F / 42 Both sides / rupture / - - / - / + + January 3 F / 47 Both sides / jerking / + + / + / + + January 4 F / 50 Left / jerking / + + / + / + + 1-2 / month 5 F / 39 Left / rupture / + + / + / + + January 6 F / 25 Both sides / jerking / + + / + / + + In May 7 F / 26 Left / rupture / + + / + / + + April 8 F / 20 Both sides / jerking / + + / + / + + 1-2 / week 9 M / 32 Left / Injury / - - / + / + + 3-4 / year 10 F / 24 Both sides / jerking / + + / + / + + December Migraine patients without presymptomatic symptoms One F / 32 Right / Injury / + + / + / + - 2-3 / month 2 F / 52 Right / Injury / + - / - / + - January 3 F / 45 Both sides / jerking / + - / - / + - 2-3 / month 4 F / 43 Left / Injury / - - / - / + - 14 / month 5 F / 31 Right / Rupture / + - / - / + - 1-2 / month 6 M / 36 Both sides / rupture / + - / - / + - January 7 F / 16 Right / Rupture / + - / - / + - June / June 8 F / 40 Right / Injury / - + / - / + - 1-2 / week 9 M / 40 Both sides / jerking / + - / - / + - 2-3 / month 10 F / 32 Both sides / jerking / + - / - / + - 3 years Normal One F / 48 Tension headache 2 M / 45 Tension headache 3 M / 37 Tension headache 4 F / 44 Dizziness before fainting 5 F / 54 Tension headache 6 M / 34 Tension headache 7 F / 22 Tension headache 8 F / 39 Facial spasm 9 F / 61 Tension headache

Example  2. Migraine patients and normal people Metabolite  compare

¹ H-NMR spectral analysis was carried out as follows using the serum of migraine patients and normal persons obtained in Example 1.

Specifically, 400 μl of the serum obtained from the patient and the normal person was dispensed and mixed with 100 μl of NMR buffer containing 550 mM sodium phosphate buffer. NMR final sample is sodium phosphate buffer (pH 7.0) of 100 mM, 2 mM of trimethylsilyl was prepared to include a propanoic acid (TSP), and 10% D ₂ O. To determine the metabolites, the one-dimensional Carr-Purcell-Meiboom-Gill (CPMG) NMR spectrum (cpmgpr1d) was obtained with a Bruker ASCEND Ⅲ 600 spectrometer equipped with a low temperature probe at 298 K. The CPMG pulse sequence was compiled by T2 relaxation time, broad resonance reduction from high molecular weight compounds, and improved observation of low molecular weight metabolites. The water signal was removed by presaturation using a low-power irradiation method on water resonance. The ¹ H-NMR spectrum for each sample consisted of 128 scans with parameters having a spectral width of 12019.2 Hz, a spectral size of 65,536 points, a pulse width (90) of 13.0 μs, and a relaxation delay (RD) of 5.0 s . The free induction decay (FID) was Fourier transformed with LB = 0.3 Hz.

Next, ¹ H-NMR spectra were manually adjusted stepwise using the Bruker Topspin 3.1 software (Bruker GmbH, Germany) and the baseline corrected. At this time, TSP was 0.0 ppm as a reference. Post-treatment baseline corrections were performed with Mnova (Mestrelab Research, Spain). The reference model was constructed on each spectrum using an algorithm of multipoint baseline correction. At this time, the NMR spectral bins of each spectrum are easily affected by subtle changes in pH and ionic strength, and a large bin size composed of one or more resonances leads to difficulties in interpretation of results. Thus, fragment alignment by Mnova and peak alignment using pair-wise peak alignment was applied before binning. On the other hand, a variable bin size in the range of 0.005 to 0.09 ppm was used in spectral analysis to make each single bin possible include a single metabolite. ^{The 1} H-NMR spectrum was divided into spectral regions (bins) of varying sizes between 0.9 and 8.48 ppm, and chemical shift regions containing residual moisture of 4.69 to 5.2 ppm were excluded here. The beans of the result were integrated and used as statistical variables.

The assignment of beans was performed using the Chenomx NMR suite 7.7 (Chenomx Inc., Canada) and evaluated in the ¹ H- ¹³ C HSQC and 2D ¹ H-TOCSY spectra.

As a result, as shown in FIG. 1, phenylalanine, isocitrate and 2-hydroxybutyrate were detected only in patients with migraine, among the metabolites analyzed from migraine patients and normal subjects. (Fig. 1).

Example  3. Migraine patients and normal people Metabolite  Used PCA  analysis

Principal component analysis (PCA) was performed using the NMR results obtained in Example 2 to investigate the differentiation of basic groups.

Specifically, the meta-analysis was performed using MetaboAnalyst (v3.0), a web server-based program, using SPSS version 23 (IBM). The spectrum was classified as normal and migraine patients with or without prefrontal symptoms. The spectral integration (bin) was normalized using a sum average algorithm, which promoted the comparability of the samples. Spectral integrations were scaled using a method called pareto-scaling to provide an adequate balance of high intensity and low intensity signal distributions. Each variable is mean-centered, divided by the square root of the standard deviation. The analysis result was considered extreme when the sample was located outside the 95% confidence ellipsoidal area of the model.

As a result, as shown in Fig. 2, three samples of migraine patients were located close to the boundaries of the 95% confidence ellipse, but they did not find any reason why such results could be regarded as outliers 2). Thus, all samples were used for further statistical analysis.

Example  4. For separation of 2 groups OPLS - DA  Building the Model

In order to characterize the difference between the two groups of migraine patients and normal persons, including all migraine sufferers with and without pre-symptomatic symptoms, a model that maximizes class segregation by eliminating class- (OPLS-DA) was applied to the analysis. We identified important characteristics based on S-plots. The S-plot of the OPLS-DA model is combined with the covariance p for the correlation p (corr) loading profile. This is consistent with the combination of the effects and reliability (modeled correlations) on the model variables with respect to the contribution or size (modeled covariance) of the model variables and the model component scores in relation to the model component scores. The quality of the OPLS-DA model was estimated by R2 (fitness) and Q2 (predictive power) parameters. The quality of the model was verified by a 1,000-random permutation test. As a result, a 2D score plot of OPLS-DA is shown in Fig.

As a result, as shown in Fig. 3, R2 of the OPLS-DA model for comparing normal and migraine patients was 0.983 and Q2 was 0.728 (Fig. 3A). As a result of the S-plot of the OPLS-DA model, a number of isolated beans (metabolites) were identified. In particular, each dotted variable shown in Figure 3B was predicted as an important metabolite contributing to the separation of the group (Figure 3B).

Example  5. Between groups  Contributing to discrimination Metabolite  To verify Univariate  analysis

Univariate analysis was performed to identify metabolites that contribute to discrimination among groups. Standard deviations and uniformity of quantified metabolite concentrations were evaluated using SPSS. The Mann-Whitney test was used because all variables did not satisfy the identity of variance based on the Kolmogorov-Smirnov test-based normality and Levine test - Parameter analysis). In multiple comparisons between groups, p- values were adjusted using the Bonferroni calibration and calculated using the Benjamini-Hochberg method, a multiple test calibration, False Discovery Rate (FDR) Respectively. A final p- value of less than 0.05 was considered significant. As a result, the metabolites showing significant differences between the normal group and the migraine patients are shown in Table 2.

Metabolite Empty range (ppm) Normal people - migraine patients Metabolite change p-value FDR 2-hydroxybutyrate 1.79-1.70 2.4E-4 0.019 ▽ ▽ Isocitrate 2.62-2.58 2.8E-4 0.011 ▽ ▽ Phenylalanine 7.47-7.43 5.7E-4 0.015 ▽ ▽

▽ indicates that the value decreases compared to the normal group.

As shown in Table 2, 2-hydroxybutyrate, isocitrate, and phenylalanine were down-regulated in migraine patients compared to normal individuals.

Example  6. ROC  Migraine through analysis Biomarker  Selection

A ROC curve (AUC), a receiver operating characteristic (ROC) analysis that assesses sensitivity and specificity, was used to select biomarkers that can identify normal and migraine patients. The ROC curve calculation algorithm for multivariate biomarkers was based on the PLS-DA algorithm. Important features were identified by Monte Carlo cross validation (MCCV), which iteratively performs random subsampling. Two-thirds of the samples in each MCCV were used to assess the significance of the variables, and the remaining one-third of the samples were used to construct a validated classification model. The PLS-DA approach with two latent variables was chosen as the classification method. The 95% accuracy of the ROC curve was calculated using the 100 CV method and the performance measurement of the model was evaluated by 1,000 permutation tests. The AUC values of the selected biomarkers are shown in Table 3 below.

Metabolite Empty range (ppm) AUC 2-hydroxybutyrate 1.79-1.70 0.89 Isocitrate 2.62-2.58 0.91 Phenylalanine 7.47-7.43 0.89 Lactate 1.37-1.35
4.15-4.12 0.75
0.73 Other 2.08-2.05 0.73

As shown in Table 3, the metabolites most commonly used for classification were identified as 2-hydroxybutyrate, isocitrate, phenylalanine, and lactate, and as shown in FIG. 4, ≪ / RTI >

On the other hand, as shown in FIG. 5A, when the maximum AUC value obtained by combination of metabolites was combined with three or more metabolites, it could be used to classify normal persons and migraine patients (FIG. 5A). Thus, it can be seen from the above that 2-hydroxybutyrate, isocitrate, and phenylalanine can be used as the biomarkers of migraine.

The results of confirming the AUC value by the combination of the three biomarkers are shown in FIG. 5B. As shown in FIG. 5B, when the three bio-markers were used together, the AUC value was 0.899. As a result of the cross validation analysis of 100 samples, it was possible to accurately diagnose the migraine patient with the accuracy of 0.779.

Claims (15)

1) measuring the concentration of phenylalanine from the sample; And
2) comparing the phenylalanine concentration of step 1) with the normal individual's concentration.
The method according to claim 1, wherein the sample of step 1) is urine, blood, serum, plasma or cerebrospinal fluid.
The method according to claim 1, wherein the concentration of phenylalanine in step 1) is selected from the group consisting of nuclear magnetic resonance spectroscopy, chromatography, ultraviolet spectroscopy, infrared spectroscopy, fluorescence spectroscopy, enzyme-linked immunosorbent assay (ELISA) A method for analyzing metabolites to provide information necessary for the diagnosis of migraine, measured by one or more devices.
The method according to claim 1, wherein the diagnosis of migraine is made when the concentration of phenylalanine is lower than that of a normal subject.
2. The method of claim 1, further comprising the step of measuring the concentration of at least one selected from the group consisting of 2-hydroxybutyrate and isocitrate.
6. The method according to claim 5, wherein, when the concentration of any one or more selected from the group consisting of 2-hydroxybutyrate and isocitrate is decreased as compared with the normal individuals, Metabolism analysis method for.
1) measuring the concentration of phenylalanine from the sample; And
2) comparing the phenylalanine concentration of step 1) with the concentration of the normal individual, to provide information necessary for the accompanying diagnosis of a migraine patient.
8. The method of claim 7, further comprising measuring the concentration of one or more selected from the group consisting of 2-hydroxybutyrate and isocitrate. The metabolic product assay to provide information for the accompanying diagnosis of a migraine patient Way.
A biomarker composition for the diagnosis of migraine headache comprising a preparation for quantification of phenylalanine.
10. The biomarker composition according to claim 9, further comprising at least one quantitative agent selected from the group consisting of 2-hydroxybutyrate and isocitrate.
A biomarker composition for accompanying diagnosis of a migraine patient comprising a preparation for quantifying phenylalanine.
12. The biomarker composition according to claim 11, further comprising at least one quantitative agent selected from the group consisting of 2-hydroxybutyrate and isocitrate.
A kit for the diagnosis of migraine, comprising a preparation for quantification of phenylalanine or a quantitative analysis device.
The diagnostic kit according to claim 13, further comprising at least one quantitative preparation or quantitative analysis device selected from the group consisting of 2-hydroxybutyrate and isocitrate.
The method according to claim 13, wherein the quantitative analysis apparatus is any one or more selected from the group consisting of a nuclear magnetic resonance spectrometer, chromatography, ultraviolet spectroscopy, infrared spectroscopy, fluorescence spectroscopy, ELISA (enzyme-linked immunosorbent assay) Diagnostic kit.

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