JP2017138141A - Parkinson disease diagnostic indicator - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an easy diagnostic marker that can make a differential diagnosis of Parkinson disease in an early stage.SOLUTION: There is provided a method for measuring the concentration of a component selected from middle-chain to long-chain acylcarnitine and a secondary bile acid in a sample derived from a living body to diagnose Parkinson disease.SELECTED DRAWING: None

Description

  The present invention relates to a method for early diagnosis of Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease in Japan. In addition to progressive movement disorders, non-motor symptoms (cognitive decline, psychiatric symptoms, autonomic symptoms), etc. This will cause serious damage to social assets due to the heavy medical and nursing care burden. In this disease, since about 80% of cases are “spontaneous” cases without a clear genetic background, genetic diagnosis using genomic DNA is not useful in all cases. In addition, changes in disease-specific and high differential diagnostic value have not been identified or established in head MRI examination from the early stage to the end stage of disease, and myocardial MIBG scintigraphy (sympathetic nerve loss occurring in PD is similar to norepinephrine in other nuclear medicine examinations) It is effective for the diagnosis of PD after the middle stage by the method of evaluating by taking down the substance MIBG), DAT (dopamine transporter) scan (to monitor the dopamine nerve terminal decline due to neurodegeneration of nigrostriatal tract, Expensive tests that cost 60,000 yen or more, such as the differentiation of related neurodegenerative diseases, have not been clearly established, but only a certain increase in diagnostic efficiency is recognized.
Further, as a means for diagnosing Parkinson's disease, a diagnostic agent using an α-adrenergic receptor agonist-containing eye drop and a noradrenaline reuptake inhibitor and / or a noradrenaline-releasing agent-containing eye drop (Patent Document 1) is a neurotrophic factor. Methods for measuring the concentration of NRG1 type III in blood (Patent Document 2), methods for measuring the amount of soluble α-synuclein oligomer in cerebrospinal fluid samples (Patent Document 3), benzyl alcohol in skin gas or sweat, etc. A method for measuring the amount (Patent Document 4) has been reported.

JP 2005-162893 A JP2013-181823A JP 2013-504766 gazette Japanese Patent Laying-Open No. 2015-55620

However, conventional methods have problems such as diagnosis that can only be diagnosed after symptom progression, expensive testing is required, and cerebrospinal fluid is used as a sample. A marker (hereinafter referred to as BM) has been desired.
Accordingly, an object of the present invention is to provide a simple diagnostic marker capable of differential diagnosis from an early stage.

  Therefore, the present inventor paid attention to blood components that can be easily measured and conducted a comprehensive analysis of plasma metabolites. As a result, the concentration of a component selected from medium chain to long chain acylcarnitine and secondary bile acids was found to be PD. From the initial stage of the onset, since it changes greatly compared to healthy subjects and Alzheimer's disease patients, it was found that if these components were measured, PD could be diagnosed early, and the present invention was completed.

  That is, the present invention provides the following [1] to [4].

[1] A method of measuring the concentration of a component selected from medium- to long-chain acylcarnitines and secondary bile acids in a biological sample for the purpose of diagnosing Parkinson's disease.
[2] The method according to [1], wherein the medium chain to long chain acylcarnitine is an aliphatic acylcarnitine having 12 to 20 carbon atoms.
[3] The method according to [1] or [2], further comprising a step of comparing the concentration of the component with the concentration of the component of a healthy person.
[4] A diagnostic agent for Parkinson's disease comprising a reagent for measuring a component selected from medium chain to long chain acylcarnitines and secondary bile acids.

  According to the method of the present invention, Parkinson's disease can be diagnosed early and distinguished from other neurodegenerative diseases such as Alzheimer's disease simply by measuring the marker concentration using a sample that can be easily collected such as blood. . Diagnosing before the disease progresses and providing appropriate treatment can stop Parkinson's disease progression.

It is a figure which shows the cluster analysis result of a metabolic compound group. The analysis result of an acylcarnitine (AC) main component is shown. An outline of metabolism of acylcarnitine (AC) and free fatty acid (FA) is shown.

  The present invention is a method for measuring the concentration of a component selected from medium chain to long chain acylcarnitines and secondary bile acids in a biological sample for the purpose of diagnosing Parkinson's disease.

  The biological sample used in the present invention may be a sample containing the above component, but since the component is a component known to exist in blood, blood is preferable, and plasma Is more preferable.

  Examples of the medium chain to long chain acyl carnitine include aliphatic acyl carnitines having 12 to 20 carbon atoms. Here, the aliphatic acyl group includes a saturated or unsaturated aliphatic acyl group. Specific examples include carnitine in which a hydroxy group is acylated with an acyl group derived from a saturated or unsaturated fatty acid having 12 to 20 carbon atoms.

  Secondary bile acids include deoxycholic acid.

  The concentration of medium chain to long chain acylcarnitine and secondary bile acid is measured by, for example, capillary electrophoresis / mass analyzer, liquid chromatography / mass analyzer, mass analyzer / mass analyzer (tandem mass analyzer), etc. be able to.

  As shown in Examples described later, the medium- to long-chain acylcarnitine concentrations in PD patients were significantly lower than those in healthy subjects and Alzheimer's disease patients. On the other hand, the concentration of short-chain acyl (carbon number 4) carnitine in PD patients was significantly higher than that in healthy subjects.

Medium to long chain acylcarnitine concentrations are believed to reflect mitochondrial metabolism in skeletal muscle (UK: CABI; 2014: 157-170). The body mass index, which is an index of obesity, was lower than the control group as shown in the examples below, suggesting a strong tendency to slim in PD patients, but accurately reflecting skeletal muscle mass In the study between the skeletal muscle-derived metabolites, no significant changes were observed for creatinine, carnitine, creatine, and 3-methylhistidine. From the above, it was judged that the decrease in the medium chain to long chain acylcarnitine concentration in the PD group was not considered to be due to changes in skeletal muscle mass.
Furthermore, the fatty acid β oxidation activity index showed a high value only in the PD group. Carnitine / acylcarnitine (16: 0) ratio is inversely correlated with the activity of carnitine palmtoyltransferase I (CPT-I) which converts acyl CoA to acylcarnitine outside the mitochondria (Diabetes care 2012: 35 (3): 605-611) When the index is high, it indicates that CPT-I activity is decreased. The ratio of acylcarnitine (8: 0) / acylcarnitine (16: 0) is inversely correlated with the activity of late fatty acid / oxidation, and the ratio of acylcarnitine (14: 1) / acylcarnitine (16: 0) is the initial fatty acid / oxidation. It is inversely correlated with activity (especially input activity) (Obesity 2010: 18 (9): 1695-1700). Since all three ratios show high values only in the PD group, it indicates that the same activity is reduced in PD. Acylcarnitine close to fatty acid / oxidized end metabolite (4: 0) despite the consistent downward trend of medium- to long-chain acylcarnitine groups indicating a decrease in intermediate materials consumed by fatty acid / oxidation The finding that is elevated is consistent with a decrease in fatty acid and oxidative activity. The raw material of mitochondrial fatty acid / oxidation is blood free fatty acid (FA). Mitochondrial fatty acids and free fatty acids having 22 or less carbon atoms, which are decomposed by oxidation, generally tended to increase. From these, it is considered that the logical interpretation that free fatty acids to be decomposed are stored in mitochondria due to a decrease in mitochondrial fatty acid / oxidation function.
Subsequently, the correlation between medium chain to long chain acylcarnitine and the severity of PD disease was examined. In general, the Hoehn and Yahr classification composed of life function evaluation is used for evaluation of PD severity. The group of 109 cases in the PD group was I grade 26, II grade 52, III grade 21, IV grade 9 and V grade 1. Most importantly, for H & Y I-III degree, acylcarnitine (12: 0), acylcarnitine (13: 1), acylcarnitine (14: 0), acylcarnitine (16: 0), acylcarnitine (16: 1), acylcarnitine (18: 0), acylcarnitine (18: 1), acylcarnitine (18: 2), and acylcarnitine (20: 1) all show a low value with a significant difference compared to the control group. In addition, acyl carnitine (16: 0) and acyl carnitine (18: 0) showed significantly low values in the H & Y I-IV degree. That is, it was revealed that the medium chain to long chain acylcarnitine group decreased from the early stage of the disease although no correlation with disease severity was observed.

  In addition, deoxycholic acid (hereinafter DCA), which is a secondary bile acid in PD group, is increased (also increased in PDD group), and 4 compounds (taurocholic acid (hereinafter referred to as taurocholic acid (hereinafter referred to as “taurocholic acid”)) that are subjected to binding modification by taurine, glycine or the like. TCA), glycoursodeoxycholic acid (hereinafter GUDCA), taurochenodeoxycholic acid (hereinafter TCDCA), and glycochenodeoxycholic acid (hereinafter GCDCA)) were observed to decrease. DCA is a secondary bile acid and is a raw material for TCDCA and GCDCA. Since TCDCA and GCDCA are produced by the enteric bacteria Clostridium perfrigens and Lactobacillus johnsonii, changes in the concentrations of DCA, TCDCA and GCDCA in PD suggest changes in the intestinal bacterial foci, and a PD patient reported in 2015 Consistent with changes in the intestinal flora. This change was not observed in the Alzheimer's disease group, suggesting its usefulness as a differential diagnosis BM from Alzheimer's disease.

  As described above, by comparing the medium chain to long chain acylcarnitine concentration and / or the secondary bile acid concentration in the biological sample of the subject with that of a healthy subject and / or an Alzheimer's disease, PD can be accelerated. Can be diagnosed.

  In the present invention, the medium-long chain acylcarnitine or secondary bile acid concentration can be measured by a liquid chromatography / mass spectrometer, so that the reagent for measuring this is useful as a diagnostic agent for Parkinson's disease.

  EXAMPLES Next, an Example is given and this invention is demonstrated in detail.

Example 1
In the patient groups shown in Table 1 (control group is indicated as control, PD group with dementia is indicated as PDD, and Alzheimer type dementia group is indicated as AD), capillary electrophoresis / mass analyzer, and liquid chromatography / mass analyzer Using these devices, comprehensive analysis of plasma metabolites was performed, and biomarker identification that contributes to early diagnosis of PD was attempted. Since plasma metabolites are susceptible to diet and exercise, fasting (drinking and internal use is possible) from 21:00 on the day before blood collection, blood sampling and plasma extraction are performed at 9-12 o'clock on the day of blood collection, and in liquid nitrogen The analysis was performed within 3 months after extraction.

[About sample adjustment]
1) For capillary electrophoresis / mass spectrometer 50 μL of human plasma was added to 450 mL of methanol solution prepared so that the concentration of the internal standard substance was 10 μM and stirred. To this, 500 μL of chloroform and 200 μL of Milli-Q water were added and stirred, followed by centrifugation (2,300 × g, 4 ° C., 5 minutes). After centrifugation, 400 μL × 1 aqueous layer was transferred to an ultrafiltration tube (Ultra Free MC PLHCC, HMT, centrifugal filter unit 5 kDa). This was centrifuged (9,100 × g, 4 ° C., 120 minutes) and subjected to ultrafiltration treatment. The filtrate was dried and dissolved again in 25 μL of Milli-Q water and used for measurement.

2) For liquid chromatography mass spectrometer 500 mL of human plasma was added to 1,500 mL of 1% formic acid-acetonitrile solution prepared so that the concentration of the internal standard substance was 6 mM and stirred. Thereafter, centrifugation (2,300 × g, 4 ° C., 5 minutes) was performed, the supernatant was recovered, the phospholipid was removed using solid phase extraction, and the filtrate was recovered. This was dried and dissolved in 200 mL of 50% isopropanol solution (v / v) for measurement.
[About measurement]
1) CE-TOFMS measurement In this test, CE-TOFMS measurement in a cation mode and an anion mode was performed under the conditions 1-3) shown below. Judging from the obtained peak intensity and shape, a sample diluted 2 times (5 times for PD69 only) and 5 times for anion mode measurement was used for the measurement in the cation mode.
・ Cationic metabolite (cation mode)
apparatus
Agilent CE-TOFMS system (Agilent Technologies) Unit 6
Capillary: Fused silica capillary id 50 μm × 80 cm
Measurement condition
Run buffer: Cation Buffer Solution (p / n: H3301-1001)
Rinse buffer: Cation Buffer Solution (p / n: H3301-1001)
Sample injection: Pressure injection 50 mbar, 10 sec
CE voltage: Positive, 27 kV
MS ionization: ESI Positive
MS capillary voltage: 4,000 V
MS scan range: m / z 50-1,000
Sheath liquid: HMT Sheath Liquid (p / n: H3301-1020)

・ Anionic metabolites (anion mode)
apparatus
Agilent CE-TOFMS system (Agilent Technologies) Unit 5
Capillary: Fused silica capillary id 50 μm × 80 cm
Measurement condition
Run buffer: Anion Buffer Solution (p / n: I3302-1023)
Rinse buffer: Anion Buffer Solution (p / n: I3302-1023)
Sample injection: Pressure injection 50 mbar, 25 sec
CE voltage: Positive, 30 kV
MS ionization: ESI Negative
MS capillary voltage: 3,500 V
MS scan range: m / z 50-1,000
Sheath liquid: HMT Sheath Liquid (p / n: H3301-1020)

2) LC-TOFMS measurement In this test, positive mode and negative mode LC-TOFMS measurements were performed under the following conditions. Judging from the obtained peak intensity, the injection volume was 1 μL.
Cationic metabolite (positive mode)
apparatus
LC system: Agilent 1200 series RRLC system SL (Agilent Technologies)
Column: ODS column, 2 × 50 mm, 2μm
MS system: Agilent LC / MSD TOF (Agilent Technologies) Unit 4 measurement conditions
Column temp .: 40 ℃
Mobile phase: A: H ₂ O / 0.1% HCOOH
Mobile phase: B: Isopropanol: Acetonitrile: H ₂ O (65: 30: 5) / 0.1% HCOOH, 2mM HCOONH ₄
Flow rate: 0.3 mL / min
Run time: 20 min
Post time: 6 min
Gradient condition: 0-0.5 min: B 1%, 0.5-13.5 min: B 1-100%, 13.5-20 min: B 100%
MS ionization mode: ESI Positive
MS Nebulizer pressure: 40 psi
MS dry gas flow: 10 L / min
MS dry gas temp: 350 ℃
MS capillary voltage: 4,000 V
MS scan range: m / z 100-1,700
Anionic metabolites (negative mode)
apparatus
LC system: Agilent 1200 series RRLC system SL (Agilent Technologies)
Column: ODS column, 2 × 50 mm, 2 μm
MS system: Agilent LC / MSD TOF (Agilent Technologies) Unit 4 measurement conditions
Column temp .: 40 ℃
Mobile phase: A: H ₂ O / 0.1% HCOOH
Mobile phase: B: Isopropanol: Acetonitrile: H2O (65: 30: 5) / 0.1% HCOOH, 2 mM HCOONH ₄
Flow rate: 0.3 mL / min
Run time: 20 min
Post time: 6 min
Gradient condition: 0-0.5 min: B 1%, 0.5-13.5 min: B 1-100%, 13.5-20 min: B 100%
MS ionization mode: ESI Negative
MS Nebulizer pressure: 40 psi
MS dry gas flow: 10 L / min
MS dry gas temp: 350 ℃
MS capillary voltage: 4,000 V
MS scan range: m / z 100-1,700

  FIG. 1 shows a “cluster analysis” result in which each disease group is arranged on the horizontal axis and metabolites are arranged on the vertical axis to search for disease-specific metabolic compound groups. The region indicated by the black dotted line shows a downward trend in the PD group, and the region contained in the region is medium- to long-chain acylcarnitine (hereinafter AC) occupying 15 compounds out of 16 compounds as shown in the right end of the figure. It was. In order to verify this result from various perspectives, a principal component analysis was performed. As a result, an axis (PC3) capable of effectively distinguishing the PD group from the control group was identified, and a compound having a high contribution to the same axis. As shown in the right end of the figure, 14 of 15 compounds were occupied by medium to long chain AC (FIG. 2). All 14 compounds were included in the AC identified by the cluster analysis described above. When the significant difference between PD and the control group was examined for each AC concentration, as shown in Table 2, 14 ACs were significantly decreased in the PD group, and short chain AC (4: 0) was compared with the control group. However, it was significantly increased in the PD group.

  In addition to the acylcarnitine group, this comprehensive analysis revealed that deoxycholic acid (hereinafter DCA), a secondary bile acid, increased in the PD group (also increased in the PDD group), and the secondary bile acid was subjected to binding modification by taurine, glycine, etc. 4 compounds (taurocholic acid (hereinafter TCA), glycoursodeoxycholic acid (hereinafter GUDCA), taurochenotoxicic acid (hereinafter TCDCA), and glycochendodeoxylic acid (hereinafter GCDCA) 3). DCA is a secondary bile acid and is a raw material for TCDCA and GCDCA. Since TCDCA and GCDCA are produced by the enteric bacteria Clostridium perfrigens and Lactobacillus johnsonii, changes in the concentrations of DCA, TCDCA and GCDCA in PD suggest changes in the intestinal bacterial foci, and a PD patient reported in 2015 Consistent with changes in the intestinal flora. This change was not observed in the AD group, suggesting its usefulness as a differential diagnostic biomarker with AD.

  The further examination about medium chain-long chain acyl carnitine group is described. The medium to long chain acylcarnitine group is considered to reflect mitochondrial metabolism in skeletal muscle. As shown in Table 1, the body mass index, which is an index of obesity, was lower in PD / PDD / AD than in the control group, as compared with the control group. However, as shown in Table 4, no significant changes were observed for creatine, carnitine, creatine, and 3-methylhistidine in the study between each group of skeletal muscle-derived metabolites that accurately reflected skeletal muscle mass. From the above, it was determined that the decrease in the medium chain to long chain acylcarnitine group in the PD group was not considered to be due to changes in skeletal muscle mass.

  Furthermore, as shown in Table 5, only in the PD group, the fatty acid / oxidation activity index showed a high value. To understand the change, knowledge of mitochondrial fatty acid and oxidation shown in Fig. 3 is indispensable.

  The carnitine / acylcarnitine (16: 0) ratio is inversely correlated with the activity of carnitine palmtoyltransferase I (CPT-I), which converts acyl CoA to acylcarnitine outside the mitochondria, and when the index is high, CPT-I activity decreases. Indicates that The ratio of acylcarnitine (8: 0) / acylcarnitine (16: 0) is inversely correlated with the activity of late fatty acid / oxidation, and the ratio of acylcarnitine (14: 1) / acylcarnitine (16: 0) is the initial fatty acid / oxidation. Inversely correlated with activity (especially input activity). Since all three ratios show high values only in the PD group, it indicates that the same activity is reduced in PD. Acylcarnitine close to fatty acid / oxidized end metabolite (4: 0) despite the consistent downward trend of medium- to long-chain acylcarnitine groups indicating a decrease in intermediate materials consumed by fatty acid / oxidation The finding that is elevated is consistent with a decrease in fatty acid and oxidative activity. As shown in the schematic diagram of FIG. 3, the raw material for mitochondrial fatty acid / oxidation is blood free fatty acid (hereinafter referred to as free fatty acid). In this comprehensive analysis, as shown in Table 6, mitochondrial fatty acids and free fatty acids having 22 or less carbon atoms decomposed by oxidation (colored cells in Table 6) were generally on an upward trend.

  From these, it is considered that the logical interpretation that free fatty acids to be decomposed are stored in mitochondria due to a decrease in mitochondrial fatty acid / oxidation function.

  Subsequently, the correlation between medium chain to long chain acylcarnitine and the severity of PD disease was examined. As shown in Table 7, in general, Hoehn and Yahr classification composed of life function evaluation is used for evaluation of PD severity.

  As shown in the case group of 109 cases in the PD group, there were 26 people with I degree, 52 people with II degree, 21 people with III degree, 9 people with IV degree, and 1 person with V degree. Most importantly, for H & Y I-III degree, acylcarnitine (12: 0), acylcarnitine (13: 1), acylcarnitine (14: 0), acylcarnitine (16: 0), acylcarnitine (16: 1), acylcarnitine (18: 0), acylcarnitine (18: 1), acylcarnitine (18: 2), and acylcarnitine (20: 1) all show a low value with a significant difference compared to the control group. In addition, acylcarnitine (16: 0) and acylcarnitine (18: 0) showed significantly low values in H & Y I-IV degree. That is, it was revealed that the medium chain to long chain acylcarnitine group decreased from the early stage of the disease although no correlation with the disease severity was observed (Table 8). When the above-mentioned acylcarnitine that was significantly decreased in the PD group compared with the control group was examined by discriminant analysis (using statistical software JMP11, SAS Institute Inc, 2014), AUC = 0.9728, R2 = 0. 5897, indicating that it has excellent diagnostic significance as shown in Table 8 (discriminant analysis) (Table 9).

Claims (4)

  A method of measuring the concentration of a component selected from medium- to long-chain acylcarnitines and secondary bile acids in a biological sample for the purpose of diagnosing Parkinson's disease.   The method according to claim 1, wherein the medium chain to long chain acylcarnitine is an aliphatic acylcarnitine having 12 to 20 carbon atoms.   Furthermore, the method of Claim 1 or 2 including the process of contrasting the density | concentration of the said component and the density | concentration of the said component of a healthy subject.   A diagnostic agent for Parkinson's disease containing a reagent for measuring a component selected from medium- to long-chain acylcarnitines and secondary bile acids.