KR20170013428A - A Method for diagnosis and progrosis prediction of cardivascular disease using purine metabolomics - Google Patents

Abstract

The present invention relates to a method for diagnosing and predicting the prognosis of a cardiovascular disease by using a purine metabolome. It has been confirmed that employment as a diagnosis marker is possible based on an increase in hypoxanthine and inosine concentrations in angina and myocardial infarction patients and the prognosis of a cardiovascular disease patient can be predicted by the use of the concentration of the purine metabolome through an increase in event likelihood in a case where the concentration of the hypoxanthine increases and the inosine concentration does not differ from those of normal people in the angina and myocardial infarction patients. In addition, it has been confirmed that an early and accurate diagnosis is possible for atrial fibrillation patients based on the measurement of the concentrations of the metabolomes because the concentrations of the purine metabolomes such as hypoxanthine and inosine rapidly decline in the atrial fibrillation patients.

Description

[0001] The present invention relates to a method for diagnosing cardiovascular diseases and prognosis of cardiovascular diseases using purine metabolites,

The present invention relates to the diagnosis of atrial fibrillation, angina pectoris or myocardial infarction, and relates to a purine metabolite analysis method for providing information on atrial fibrillation, angina pectoris or myocardial infarction diagnosis or prognosis prediction using purine metabolism.

Cardiovascular disease is a major cause of death in industrialized countries around the world and has been a leading cause of death in Korea since the 1970s and is one of the fastest growing diseases among the 10 leading causes of death in Koreans. In the case of coronary artery disease, a typical cardiovascular disease, the number increased from 2.2 per 100,000 population in 1983 to 21.9 in 2001, a 10-fold increase in less than 20 years. Today, the incidence of patients with atherosclerosis is almost the same as the number of patients with cancer. The size of the related market is almost several hundred billion won.

Such cardiovascular diseases include myocardial infarction, angina pectoris, atherosclerosis, hypertension, heart failure, aneurysm, stroke, arrhythmia, and the like. Coronary artery disease, which is an important part of cardiovascular disease, is usually caused by clogging or narrowing of the coronary arteries supplying blood to the heart by atherosclerosis. Myocardial infarction is the disease in which the coronary artery is completely blocked by arteriosclerosis and the cardiac muscle tissue is killed, and the coronary artery is narrowed to feel chest tightness or chest pain.

Conventional cardiovascular disease can be diagnosed using a physical method to some extent, and there is a limit to early diagnosis or prediction. In general, cardiovascular disease is diagnosed by X-ray and ultrasound imaging of the inside of the heart and coronary arteries using a contrasting device, which can be diagnosed after the onset.

In order to predict cardiovascular disease, tests for basic lipid-related tests (total cholesterol, low density lipoprotein cholesterol, high density lipoprotein cholesterol, triglycerides) and hsCRP are performed. Such a blood test itself does not directly reflect the atherosclerotic condition of the person, but may also act as a confounding factor if the infected disease or inflammatory vascular disease is accompanied. For this reason, the existing blood markers are very limited in adding radiologic tests including ultrasound, computed tomography, and magnetic resonance imaging, or confirming predictions through invasive examinations .

Because cardiovascular-related illnesses appear suddenly without symptoms, they are closely related to functional limitations in human life and lifestyle. Therefore, it is necessary to predict and diagnose these advanced cardiovascular diseases to save the life of the patient and to improve the quality of life. However, there is a growing need for a biomarker that can be easily diagnosed as a blood sample because the cost of medical examination increases due to various tests, and the screening test is limited to all people.

Biomarkers of known cardiovascular diseases include CRP, IL-6, IL-8, MCP-1 and IP-10. In addition, MCP-1, MCP-2, MCP-3, MCP-4, eotaxin, M-CSF, IL-3, IP-10, TNFa, Ang- (US 2007/0099239) have been filed. IL-5 precursor, IL-6 precursor, CCL2 (MCP-1), CCL5 (RANTES), cathepsin LI precursor (IL-6 precursor) ), Adenylate kinase 1, leukotriene B4 receptor 1, complement factor D, osteopontin, small inducible cytokine AI 7 precursor inducible cytokine AI7 precursor, CXCL10, RANKL / TRANCE, CCL18 precursor, type IV collagenase precursor, neutrophil collagenase precursor, fatty acid binding protein 4 (fatty acid binding protein 4, cathepsin S precursor, IL-13 precursor and sICAM-1 have been patented as markers for cardiovascular disease risk factors.

As described above, there are various blood markers that can predict atherosclerotic vascular disease and the like. However, since the burden of medical expenses is minimized, the markers that can be applied to all persons are limited. In addition, depending on the drugs used, various changes in the markers in the blood may occur, and techniques for appropriately monitoring them are necessary.

Atrial fibrillation is also the most common arrhythmia in the clinic, and it is known that the mechanism of the arrhythmia is associated with structural remodeling and damaged atrial contractility. Atrial fibrillation is a disease with a high risk of stroke, a long hospital stay, poor quality of life, and high mortality, and it is essential to understand the exact mechanism of development for early diagnosis and effective treatment. Atrial fibrillation may be uncomplicated and accurate diagnosis is expected to reduce the risk of complications such as stroke through appropriate treatment of atrial fibrillation.

Purine metabolites are known to act as precursors of uric acid, a well-known marker of heart disease (1). In addition, it is known that ATP is degraded in tissues with hypoxic state, and concentration in blood increases rapidly, and it acts as a main material in cardiovascular diseases (2, 3). However, there is a lack of research on the diagnosis and prognosis of cardiovascular disease using such purine metabolites.

The present inventors performed diagnostic and prognostic prediction of patients suffering from angina / myocardial infarction / atrial fibrillation through quantitative analysis of purine metabolites, and completed the present invention.

(1) Hare JM, Johnson RJ (2003) Uric acid predicted clinical outcomes in heart failure, Insights regarding the role of xanthine oxidase and uric acid in disease pathophysiology. Circulation 107: 1951-1953. (2) Grum CM, Simon RH, Dantzker DR, Fox IH (1985) Evidence for adenosine triphosphate degradation in critically-ill patients. CHEST Journal 88: 763-767. (3) Lewis GD, Wei R, Liu E, Yang E, Shi X, Martinovic M, Farrell L, Asnania, Cyrille M, Ramanathan A, Shaham O, Berriz G, Lowry PA, Palacios IF, Tasan M, Roth FP , Min J, Baumgartner C, Keshishian H, Addona T, Mootha VK, Rosenzweig A, Carr SA Fifer MA, Sabatine MS, Gerszten RE (2008) Metabolite profiling of individuals with myocardial infarction reveals early signs of myocardial injury. J. Clin. Invest. 118: 3503-3512.

It is an object of the present invention to provide a method for analyzing a metabolite for providing information on the diagnosis of atrial fibrillation, angina pectoris or myocardial infarction disease or prediction of prognosis using a purine metabolite.

It is still another object of the present invention to provide a kit capable of diagnosing atrial fibrillation, angina pectoris or myocardial infarction disease or predicting prognosis using a purine metabolite.

In order to accomplish the above object, the present invention provides a pharmaceutical composition comprising hypoxanthine, inosine, xanthine, uric acid, adenosine, adenine, xanthosine, There is provided a kit for the diagnosis or prognosis prediction of atrial fibrillation disease comprising a quantification device for any one or more metabolites selected from the group consisting of guanosine and guanine.

The present invention also provides a kit for diagnosing angina pectoris or myocardial infarction or for predicting prognosis, comprising a quantitative device for hypoxanthine and inosine.

In addition,

i) In a sample isolated from a test sample, a sample containing hypoxanthine, inosine, xanthine, uric acid, adenosine, adenine, xanthosine, guanosine, Measuring the concentration of one or more selected from the group consisting of a metabolite consisting of guanosine and guanine;

ii) comparing the concentration of the metabolite measured in the sample in step i) with the concentration of the metabolite measured in the same type of sample from the normal control, respectively; And

(iii) the amount of hypoxanthine, inosine, xanthine, uric acid, adenosine, adenine, xanthosine, and the like measured in the sample in step ii) , Guanosine and guanine is increased as compared to a normal control group, it is determined that the subject is at risk for atrial fibrillation or atrial fibrillation , A method for analyzing metabolites to provide information on the diagnosis of atrial fibrillation disease or prediction of prognosis.

In addition,

a) measuring the concentration of hypoxanthine and inosine in a sample separated from a subject;

b) comparing the concentration of the metabolite measured in the sample in step a) with the concentration of the metabolite measured in the same type of sample from the normal control, respectively; And

c) when the concentration of hypoxanthine measured in the sample in the step b) is higher than that of a normal control and the concentration of inosine is not different from that of a normal person, The method comprising the steps of: (a) determining whether the patient is suffering from angina pectoris or myocardial infarction.

The present invention relates to a method for diagnosing and prognosing cardiovascular diseases using purine metabolism, which can be used as a diagnostic marker because of increased hypoxanthine and inosine concentrations in patients with angina and myocardial infarction. In patients with myocardial infarction, the prognosis of patients with cardiovascular disease could be predicted using the concentration of purine metabolites by increasing the incidence of events when the concentration of hypoxanthine is higher than that of normal inosine. In addition, since concentrations of purine metabolites such as hypoxanthine and inosine were rapidly decreased in atrial fibrillation patients, it was confirmed that early diagnosis of atrial fibrillation patients was possible by measuring the concentrations of the metabolites.

Figure 1 is a Box-Whisker plot of change in purine metabolite concentration in patients with angina and myocardial infarction (MI);
In this case, the line inside the box represents the standardized median using log, each boundary represents 1 quartile (25 ^th ) and 3 quartiles (75 ^th ), and each boundary of the whisker is 5% % Percentile.
Figure 2 is a Kaplan-Meier curve of the concentration of purine metabolites and the time of event in patients with angina and myocardial infarction (MI);
Here, Hypo: Hypoxanthine;
Ino: inosine;
Group I: Group 1 (hypoxanthine (upper 25%) - inosine (lower 75%)); And
Group II: Group 2 (hypoxanthine (lower 75%) - inosine (upper 25%)).

Hereinafter, the present invention will be described in detail.

The present invention relates to a pharmaceutical composition comprising hypoxanthine, inosine, xanthine, uric acid, adenosine, adenine, xanthosine, guanosine, guanine, (Guanine). The present invention also provides a kit for the diagnosis or prognosis prediction of atrial fibrillation disease comprising at least one metabolite selected from the group consisting of guanine and guanine.

The quantification device is preferably any one selected from the group consisting of nuclear magnetic resonance (NMR), chromatography and mass spectrometry, but is not limited thereto.

In a specific embodiment of the present invention, the purine metabolites were analyzed in patients with atrial fibrillation and analyzed quantitatively using UPLC-Triple Quad-MS. As a result, it was confirmed that the purine metabolites measured in the plasma samples of atrial fibrillation patients were significantly reduced compared with the normal ones. Especially, in the case of hypoxanthine and inosine, in the case of atrial fibrillation, (Table 5). ≪ tb > < TABLE >

Therefore, the results of the present invention confirmed that the above-described verified metabolites can be used to diagnose atrial fibrillation and predict a prognosis.

The present invention also provides a kit for diagnosing angina pectoris or myocardial infarction or for predicting prognosis, comprising a quantitative device for hypoxanthine and inosine.

The quantification device is preferably any one selected from the group consisting of nuclear magnetic resonance (NMR), chromatography and mass spectrometry, but is not limited thereto.

In a specific example of the present invention, the present inventors extracted purine metabolites from serum samples obtained from myocardial infarction, angina pectoris, and normal persons and quantitatively analyzed by UPLC-Triple Quad-MS. As a result, hypoxanthine did not show any significant difference compared to normal subjects, but it was found to increase and was significantly increased in myocardial infarction patients than angina patients (see FIGS. 1A and 3). In addition, inosine was significantly increased in both angina and myocardial infarction patients compared to normal subjects (see FIG. 1B and Table 3).

In addition, the present inventors performed Kaplan-Meier survival analysis and log rank method analysis to confirm whether prediction of angina pectoris and myocardial infarction can be made using hypoxanthine and inosine. As a result, we found that the total frequency of group 1 was 17 and the frequency of events was 5 in the serum samples of 81 patients with angina pectoris (58 patients) and myocardial infarction (23 patients), and the total frequency of group 2 was 64 And the frequency of events was 3 (see Table 4). 2, the Kaplan-Meier curve of Hypoxanthine and inosine concentrations and event time showed that the event rate of group 1 patients was higher than that of group 2, and the survival rate And significantly decreased (p = 0.001, log rank method analysis) (see FIG. 2).

Thus, the results of the present invention have confirmed that the above-described metabolite can be used to diagnose myocardial infarction or angina and to predict a prognosis.

In addition,

i) In a sample isolated from a test sample, a sample containing hypoxanthine, inosine, xanthine, uric acid, adenosine, adenine, xanthosine, guanosine, Measuring the concentration of one or more selected from the group consisting of a metabolite consisting of guanosine and guanine;

ii) comparing the concentration of the metabolite measured in the sample in step i) with the concentration of the metabolite measured in the same type of sample from the normal control, respectively; And

(iii) the amount of hypoxanthine, inosine, xanthine, uric acid, adenosine, adenine, xanthosine, and the like measured in the sample in step ii) , Guanosine and guanine is increased as compared to a normal control group, it is determined that the subject is at risk for atrial fibrillation or atrial fibrillation , A method for analyzing metabolites to provide information on the diagnosis of atrial fibrillation disease or prediction of prognosis.

The separated sample preferably includes at least one selected from the group consisting of urine, blood and plasma, but is not limited thereto.

The concentration of the step i) is preferably measured by any one selected from the group consisting of nuclear magnetic resonance (NMR), chromatography and mass spectrometry, but is not limited thereto.

In addition,

a) measuring the concentration of hypoxanthine and inosine in a sample separated from a subject;

b) comparing the concentration of the metabolite measured in the sample in step a) with the concentration of the metabolite measured in the same type of sample from the normal control, respectively; And

c) when the concentration of hypoxanthine measured in the sample in the step b) is higher than that of a normal control and the concentration of inosine is not different from that of a normal person, The method comprising the steps of: (a) determining whether the patient is suffering from angina pectoris or myocardial infarction.

The separated sample preferably includes at least one selected from the group consisting of urine, blood and plasma, but is not limited thereto.

The concentration of the step a) is preferably measured by any one selected from the group consisting of nuclear magnetic resonance (NMR), chromatography and mass spectrometry, but is not limited thereto.

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

< Example  1> myocardial infarction and angina In patients  Purine Metabolite  analysis

<1-1> Hypoxanthine ( hypoxnathine ) And Inosine  Confirm concentration change

The present inventors extracted purine metabolites from 32 blood samples from 32 patients with myocardial infarction and 32 patients with angina pectoris and 32 healthy controls whose age, sex and body mass index (BMI) matched. The UPLC-Triple Quad-MS .

Specifically, the present inventors performed sample pretreatment as described below for analyzing purine metabolites based on a mass spectrometer.

First, 500 μl of chloroform-methanol (50:50, v / v, 4 ° C) was added to a serum or plasma sample (50 μl) and mixed for 30 seconds. To this, 100 占 퐇 of distilled water was added, and the mixture was stirred for 30 seconds and left at 4 占 폚 for 10 minutes. After centrifugation at 13,000 rpm at 4 ° C for 10 minutes, the supernatant (150 μl) was separated, freeze-dried and stored at -80 ° C until measurement. For mass spectrometer analysis, the sample was redissolved in 250 μl Acetonitile-H ₂ O (75:25, v / v) and xanthine, hypoxanthine, inosine (inosine), and uric acid (uric acid).

Analysis was performed using a hydrophilic interaction chromatography-tandem mass spectrometer (HILIC-MS / MS) for the quantitative analysis of purine metabolites present in serum and plasma polar metabolites. The equipment used was Agilent 1290 UHPLC and Agilent 6490 Triple quadrupole MS. For hydrophilic interaction chromatographic conditions, purine metabolites were separated using gradient elution at 35 ° C using BEH HILIC (2.1 x 100 mm, 1.8 μm, Waters) column. (A) Acetonitile-H ₂ O (95: 5, v / v, ammonium acetate 10 mM, 0.1% formic acid) and Acetonitile-H ₂ O (50:50, v / mM, 0.1% formic acid), and the gradient elution was performed as shown in Table 1 below, with the total analysis time being 15 minutes. 1 μl of the sample was injected, and the tandem mass spectrometer condition (multiple reaction monitoring, MRM) was performed as shown in Table 2 below.

Time (minutes) Mobile A (%) Mobile phase B (%) Flow rate (mL / min) 0.0 99 One 0.4 2.0 99 One 0.4 8.0 45 55 0.4 9.0 One 99 0.4 11.0 One 99 0.4 11.1 99 One 0.4 15.0 99 One 0.4

Compounds m / z Product ion CE ^a Ionized mode ^b Hypoxanthine 137 94 20 + Xanthine 153 94 18/14 + Inosine 269 137 8 + Uric acid 167 124/96 12/16 + Guanine 152 135/110 18/20 + Guanosine 284.1 152.1 20 + Adenine 136.1 119.1 / 94.1 20/20 + Adenosine 268 136.1 18 + Xantosine 283 150.8 20 - IS-Xanthine &lt; / RTI &gt; 155.1 114.1 / 137.1 4/14 + IS-Hypoxanthine 142.1 124.1 / 114.1 24/20 + IS-Inosine (IS-Inosine) 273.1 141/255 14/18 + IS-Uric acid 168.9 125 / 96.9 14/16 - IS-taurine (IS-taurine) 125.9 80 22 -

^a means collision energy.

^b (+) means positive ion mode and (-) means negative ion mode.

The results obtained from the MRM analysis were quantified using Agilent's Qauntitative Analysis Software using a standard curve that was repeated three times. At this time, the concentrations of hypoxanthine, inosine, xanthine and uric acid were corrected using ISs substituted with respective isotopes.

To compare the concentrations of metabolites, standardized concentrations were used for the patients with angina and myocardial infarction. Statistical tests were performed with ANOVA and posttest using Tukey's multiple comparison test.

As a result, as shown in FIG. 1 and Table 3, hypoxanthine did not show a significant difference compared to normal subjects, but it was found to increase, and it was found that it was significantly increased in myocardial infarction patients than in angina patients ( 1a, Table 3). In addition, inosine was significantly increased in both angina pectoris and myocardial infarction patients compared to normal subjects (Fig. 1B, Table 3). The same results were confirmed in serum samples that did not match the age, sex and BMI.

Metabolism type Normal Angina patient Myocardial infarction patient Hypoxanthine 1.14 [0.91, 1.5] ^a 1.07 [0.88, 1.22] 1.33 [1.02, 1.89] Inosine -2.43 [-2.54, 2.24] -1.11 [-1.88, -0.72] -1.38 [-2.14, -0.29]

^a log of the standardized median, the first quartile (25 ^th ), and the third quartile (75 ^th ).

<1-2> Hypoxanthin ( hypoxanthine ) And Inosine ) For prediction of prognosis in patients with angina and myocardial infarction

The present inventors performed Kaplan-Meier survival analysis and log rank method analysis to determine whether prediction of angina pectoris and myocardial infarction can be made using hypoxanthine and inosine.

Specifically, a follow-up study was performed to track the occurrence of angina and myocardial infarction events. Event occurrence was judged to be the patient who underwent surgery or died in the follow-up study. The concentrations of hypoxanthine and inosine were measured in the same manner as in Example <1-1> for serum samples of all disease groups. For comparison with the estimation value of event rate according to the metabolite concentration, The army was divided into two groups.

Group 1: hypoxanthine (upper 25%) - inosine (lower 75%),

Group 2: hypoxanthine (lower 75%) - inosine (upper 25%)

Survival analysis was performed using Kaplan-Meier survival analysis and log-rank method analysis. The significance level was based on p <0.05.

As a result, as shown in Table 4, the total frequency of the group 1 was 17 in the serum samples of 81 angina pectoris (58 patients) and myocardial infarction (23 patients) group, and the event occurrence frequency was 5. The total frequency of group 2 was 64 and the event frequency was 3 (Table 4).

group Total (N) event (N) Survival rate (%) One 17 5 69.7 2 64 3 94.9 all 81 8 -

2, the Kaplan-Meier curve of Hypoxanthine and inosine concentrations and event time showed that the event rate of group 1 patients was higher than that of group 2, and the survival rate And significantly decreased (p = 0.001, log rank method analysis) (Fig. 2).

< Example  2> atrial fibrillation In patients  Purine Metabolite  analysis

To analyze purine metabolites in patients with atrial fibrillation, the present inventors extracted purine metabolites from blood (plasma) samples obtained from 35 patients with atrial fibrillation and 35 patients with normal matched age, sex, and BMI, >, Quantitative analysis was carried out by UPLC-Triple Quad-MS.

Metabolic concentrations were expressed as the median [25th, 3th quartile (75th)] and the Mann-Whitney U test was used to compare the purine metabolites between normal and atrial fibrillation patients .

As a result, as shown in Table 5, it was confirmed that the purine metabolites measured in the plasma samples of the atrial fibrillation patients were significantly decreased as compared with the normal ones. In particular, hypoxanthine and inosine were found to decrease in concentration by a factor of ten in patients with atrial fibrillation compared with normal subjects (Table 5).

Metabolism type Normal subjects (35 subjects, uM) Atrial fibrillation (35 patients, uM) Significance (P value) Hypoxanthine 35.68 [24.16-72.09] 3.83 [2.60-12.03] <0.0001 Inosine 21.36 [3.54-38.31] 0.11 [0.05-1.02] <0.0001 Xanthine 1.72 [0.86-2.29] 0.47 [0.40-0.79] <0.0001 Uric acid 359.98 [157.39-535.46] 179.52 [106.27-537.12] 0.173 Adenosine 0.03 [0.02-0.05] 0.01 [0.01-0.02] <0.0001 Adenine 0.03 [0.02-0.04] 0.01 [0.01-0.02] <0.0001 Xanthosine 0.14 [0.10-0.26] 0.09 [0.06-0.14] 0.0036 Guanosine 0.56 [0.24 - 1.43] 0.00 [0.00-0.04] <0.0001 Guanine 0.76 [0.53-0.17] 0.28 [0.21-0.43] <0.0001

Claims (10)

Inosine, Xanthine, Uric acid, Adenosine, Adenine, Xanthosine, Guanosine and Guanine have been found to be useful in the treatment of cancer, And a quantification device for at least one metabolite selected from the group consisting of: &lt; RTI ID = 0.0 &gt; (a) &lt; / RTI &gt;
The kit according to claim 1, wherein the quantification device is any one selected from the group consisting of nuclear magnetic resonance (NMR), chromatography, and mass spectrometry.
A kit for diagnosing or prognosing myocardial infarction or myocardial infarction, comprising a quantification device for hypoxanthine and inosine.
4. The kit according to claim 3, wherein the quantification device is any one selected from the group consisting of nuclear magnetic resonance (NMR), chromatography, and mass spectrometry.
i) In a sample isolated from a test sample, a sample containing hypoxanthine, inosine, xanthine, uric acid, adenosine, adenine, xanthosine, guanosine, Measuring the concentration of one or more selected from the group consisting of a metabolite consisting of guanosine and guanine;
ii) comparing the concentration of the metabolite measured in the sample in step i) with the concentration of the metabolite measured in the same type of sample from the normal control, respectively; And
(iii) the amount of hypoxanthine, inosine, xanthine, uric acid, adenosine, adenine, xanthosine, and the like measured in the sample in step ii) , Guanosine and guanine is increased as compared to a normal control group, it is determined that the subject is at risk for atrial fibrillation or atrial fibrillation , Atrial fibrillation disease, or prognosis prediction.
[6] The method of claim 5, wherein the separated sample is at least one selected from the group consisting of urine, blood, and plasma.
6. The method according to claim 5, wherein the concentration of step i) is measured by any one selected from the group consisting of nuclear magnetic resonance (NMR), chromatography and mass spectrometry. Method of analysis of metabolites for.
a) measuring the concentration of hypoxanthine and inosine in a sample separated from a subject;
b) comparing the concentration of the metabolite measured in the sample in step a) with the concentration of the metabolite measured in the same type of sample from the normal control, respectively; And
c) when the concentration of hypoxanthine measured in the sample in the step b) is higher than that of a normal control and the concentration of inosine is not different from that of a normal person, Wherein the method comprises the steps of: determining whether the subject is suffering from angina pectoris or myocardial infarction.
9. The method of claim 8, wherein the isolated sample comprises at least one selected from the group consisting of urine, blood, and plasma.
9. The method according to claim 8, wherein the concentration of step a) is measured by any one selected from the group consisting of nuclear magnetic resonance (NMR), chromatography and mass spectrometry. Sieve analysis method.

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