Methods for measuring higher brain functions

Abstract

To provide an objective, minimally invasive method of evaluating higher brain functions of a subject.SOLUTION: A method of measuring one or more higher brain functions selected from an executive function, an attention function, and cognitive flexibility, the method comprising a step of measuring the level of D-asparagine in a biological sample collected from a subject.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for measuring higher brain functions using D-amino acids. [Background technology]

[0002] Cognitive functions such as executive function, cognitive flexibility, and attention are also known as higher brain functions, and if these functions are impaired by aging, head trauma, stroke, etc., it can become difficult to adapt to daily and social life. Executive functions, which are the highest level among higher brain functions, are also called execution functions and are defined as the ability to maintain an appropriate posture for achieving future goals. They are ranked higher among cognitive functions and are considered essential for all cognitive functions to function normally (Non-Patent Document 1). In addition, cognitive flexibility and attention control, which are among the higher brain functions, are thought to play important roles in promoting new behavioral patterns and optimizing behavior in unconventional situations, supporting human goal-oriented behavior.

[0003] The neural substrate that controls such higher brain functions is generally believed to reside in the prefrontal cortex. Therefore, when higher brain functions are impaired due to insufficient development or damage to the prefrontal cortex, symptoms appear such as unplanned behavior, an inability to prioritize tasks, acting haphazardly, being unable to work efficiently, and being unable to initiate actions without being told to do so.

[0004] Currently, the Trail Making Test (TMT) A and B, which evaluates divided attention and the ability to process multiple tasks, has been established as one of the methods for testing higher brain functions, and is widely used because it can detect higher brain dysfunction early and sensitively. However, it requires regular examinations by dementia specialists and clinical psychologists, and regular hospital visits by patients, and the scores are influenced by the subject's mood, physical condition, and motivation, so there is a demand for the development of simpler and more objective testing methods and indicators. In addition, although cerebrospinal fluid testing is considered useful in diagnosing dementia, its invasiveness is an issue, and it is also necessary to develop testing methods and indicators that can be applied to minimally invasive diagnosis.

[0005] On the other hand, all amino acids except glycine exist in two stereoisomers, D- and L-. L-amino acids are the building blocks of proteins in living organisms, and since the amino acids contained in proteins are in principle L-amino acids, it was thought that L-amino acids were mainly involved in the physiological activities of higher animals. However, with the recent advances in analytical technology, which have improved separation capabilities and sensitivity, the existence and role of D-amino acids in mammals, including humans, has become clear.

[0006] Recently, it has been reported that the amount of D-amino acids and L-amino acids in biological materials of healthy individuals maintain a certain balance, and that in certain diseases, the balance between D-amino acids and L-amino acids is disrupted (Patent Document 1). In this document, the relationship between Alzheimer's disease and D-amino acids in blood is also examined, and it is suggested that the balance of D-serine, D-alanine, D-methionine, D-leucine, D-aspartic acid, D-phenylalanine, or D-allo-isoleucine changes in patients with Alzheimer's disease. However, regarding D-serine, there is a mixed report suggesting that D-serine in cerebrospinal fluid can be a biomarker for early Alzheimer's diagnosis (Non-Patent Document 2), and a report that no correlation is found between D-serine in cerebrospinal fluid and Alzheimer's disease or mild cognitive impairment (Non-Patent Document 3), and there is no unified view on the relationship between brain function and D-amino acids, and there is no report on the relationship with higher brain function. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] JP 2017-223711 A [Non-patent literature]

[0008] [Non-Patent Document 1] Toshiya Fukui, Cognitive Neuroscience (2010) 12: 156-164 [Non-Patent Document 2] C Madeira, MV Lourenco, C Vargas-Lopes, CK Suemoto, CO Brandao, T Reis, REP Leite, J Laks, W Jacob-Filho, CA Pasqualucci, LT Grinberg, ST Ferreira and R Panizzutti., Transl Psychiatry. 2015;5:1-9. doi:10.1038 / tp.2015.52. Epub 2015 May 5. [Non-Patent Document 3] Shorena Samakashvili, Clara Ibanez, Carolina Simo, Francisco J. Gil-Bea, Bengt Winblad, Angel Cedazo-Minguez, Alejandro Cifuentes., Electrophoresis. 2011;32(19):2757-2764. Doi:10.1002 / elps.201100139. Epub 2011 Aug 29. Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention relates to providing an objective, minimally invasive method for assessing higher brain function in a subject. [Means for solving the problem]

[0010] In light of these problems, the inventors conducted research and discovered that D-asparagine is contained in blood, and that the higher the D-asparagine ratio or D-asparagine concentration, the longer it takes to carry out TMT-B. They also found that higher brain function can be measured using the D-asparagine level in a subject's biological sample as an indicator.

[0011] That is, the present invention relates to the following 1). 1) A method for measuring one or more higher brain functions selected from executive function, attention function, and cognitive flexibility, comprising a step of measuring D-asparagine levels in a biological sample collected from a subject. Effect of the Invention

[0012] According to the method of the present invention, the higher brain function of a subject can be examined minimally, objectively and simply, and early changes in the decline of higher brain function can be objectively evaluated. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] In the present invention, "higher brain function" means one or more cognitive functions selected from executive function, attention function, and cognitive flexibility, and preferably includes at least executive function.

[0014] In the present invention, "executive function," also known as executive function, is defined as the ability to maintain an appropriate posture for achieving future goals, and is considered to have the following components: 1) task representation, 2) planning, 3) task completion, 4) inhibition of undesirable responses, and 5) evaluation and correction as necessary (Non-Patent Document 1).

[0015] The "attention function", also called the attention control function, is the function of consciously directing attention to important information from among many pieces of information (Science Co., Ltd., Tadashi Oyama and Yoshiaki Nakajima, eds., Invitation to Experimental Psychology [revised edition], p. 99). The attention function mainly consists of three functions: "selective attention", "attention shifting", and "division of attention" (Waseda University Clinical Psychology Research, Vol. 13, No. 1, p. 33). Here, selective attention is the function of directing attention to a specific stimulus or object from many stimuli or objects, attention shifting is the function of interrupting attention that was directed to a specific stimulus or object as necessary and switching it appropriately to another stimulus or object, and division of attention is the function of allocating attention to multiple objects simultaneously.

[0016] "Cognitive flexibility" refers to the ability to replace maladaptive thoughts and adopt balanced or adaptive thinking (Tokuyoshi Akira and Iwasaki Shoichi (2012) Development and validity of the Japanese version of the Cognitive Flexibility Inventory (CFI) Proceedings of the 76th Annual Meeting of the Japanese Psychological Association, 672).

[0017] In the present invention, higher brain functions (executive function, attention function, and cognitive flexibility) can be evaluated by TMT-B (Trail Making Test B). TMT-B is a method often used to test higher brain functions, and is said to reflect higher attentional functions such as visual attention, visual search, visuomotor coordination, sustained and selective attention, visuomotor coordination, rapid information processing, and short-term memory with interference.

[0018] In the present invention, "measurement of higher brain function" includes the state or degree of higher brain function, and the presence or absence of executive dysfunction, and is preferably measurement of the state of higher brain function. Here, "measurement" can also be replaced with the terms "examination," "determination," "evaluation," or "evaluation support." Note that in this specification, the terms "determination" and "evaluation" do not include determination or evaluation by a doctor.

[0019] The method for measuring higher brain function of the present invention comprises the step of measuring the D-asparagine level in a biological sample collected from a subject.

[0020] In the present invention, the subject is not particularly limited, but examples include middle-aged or older people whose higher brain functions decline with age, people who have subjective symptoms characteristic of higher brain functions, such as executive functions (e.g., being unable to prioritize things, acting haphazardly, being unable to work efficiently, being unable to start an action unless instructed to do so, being unable to modify the content of work as necessary, etc.), and people suspected of a decline in higher brain functions.

[0021] In the present invention, the biological sample mainly includes body fluids such as blood, lymph, cerebrospinal fluid, saliva, and urine, and is preferably a blood sample. Examples of blood samples include blood (whole blood) and blood-derived serum, plasma, and the like, and preferably plasma. Blood can be collected from blood vessels of the systemic circulation (arteries (peripheral arteries), veins (peripheral veins), capillaries) or blood vessels of the pulmonary circulation (pulmonary arteries, pulmonary veins, pulmonary capillaries). From the viewpoint of ease of blood collection, however, it is preferable to collect blood from blood vessels of the systemic circulation, in particular from veins (peripheral veins).

[0022] In the present invention, the D-asparagine level may be the D-asparagine content or composition value, but is preferably the asparagine chiral balance, i.e., the D-asparagine ratio or D-asparagine concentration, shown by the following formula: [D-asparagine concentration / (D-asparagine concentration+L-asparagine concentration)]×100.

[0023] The D-asparagine level in a biological sample can be measured by any method that can separate and measure the D- and L-amino acids in the biological sample. Generally, a separation and quantification method that combines liquid chromatography (LC) and mass spectrometry (MS), such as LC-MS or LC-MS / MS, can be used. Here, examples of methods for separating chiral amino acids include a method in which amino acids are converted into NBD derivatives using 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) reagent, and then separated and quantified by two-dimensional liquid chromatography (LC) using a reversed-phase column (first dimension: molecular species separation) and a chiral column having a stationary phase carrying a chiral identifier (second dimension: chiral separation) (J Chromatogr A. 2010 Feb 12;1217(7):1056-62. doi: 10.1016 / j.chroma.2009.09.002. Epub 2009 Sep 6), and a one-dimensional LC method using one reversed-phase column (ODS column) (Anal Chim Acta. 2015 May 22;875:73-82. doi: 10.1016 / j.aca.2015.02.054. Epub 2015 Feb 23), a method in which amino acids are derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) and separated by one-dimensional LC using a single chiral column (J Pharm Biomed Anal. 2015 Nov 10;115:123-9. doi: 10.1016 / j.jpba.2015.05.024. Epub 2015 Jun 16), and a method in which amino groups are derivatized with AQC and then separated using liquid chromatography connected to a first chiral column having a weak anion exchange stationary phase and a second chiral column having an amphoteric ion exchange stationary phase (JP Patent Publication 2018-100906) are also known. Alternatively, as shown in the examples below, chiral amino acids can be derivatized by reacting the amino group with 2,5-dioxopyrrolidin-1-yl (2-((2-(diethylamino)ethyl)carbamoyl)quinolin-6-yl)carbamate, and then separated using liquid chromatography connected to a chiral column having an amphoteric ion exchange stationary phase (Patent Application No. 2021-042055), and it is preferable to use such a method for separating chiral amino acids. In addition, 2,5-dioxopyrrolidin-1-yl (2-((2-(diethylamino)ethyl)carbamoyl)quinolin-6-yl)carbamate is also called RapiFluor-MS (RFMS) and is commercially available (Waters).

[0024] As shown in the following examples, D-asparagine ratio and D-asparagine concentration have good classification correlation with the execution time of TMT-B, which is widely used as a test of higher brain function.That is, the execution time of TMT-B shows a positive correlation tendency with D-asparagine ratio, and the execution time is positively correlated with D-asparagine concentration.Therefore, by measuring the D-asparagine level in the biological sample of the subject and comparing it with the reference value, it is possible to measure the higher brain function of the subject, specifically one or more selected from executive function, attention function and cognitive flexibility.

[0025] Here, the reference value can be set, for example, as follows based on the correlation between D-asparagine levels and the state of higher brain function. The state of higher brain function is evaluated by TMT-B. Based on the evaluation results, a healthy group consisting of subjects whose higher brain function is judged to be normal and a higher brain dysfunction group consisting of subjects whose higher brain function is judged to be low are created. Separately from this, D-asparagine levels are measured by the above-mentioned method. Then, based on the correlation between the evaluation results of higher brain function and the D-asparagine level, a reference value suitable for evaluating the state of higher brain function is determined. Specifically, based on the results of statistical analysis of the D-asparagine levels of people belonging to each group, a numerical range of the D-asparagine level that characterizes each group is determined. This numerical range is determined by setting a certain range above and below the average value of each group. Here, the "certain range" may be a statistical value such as standard deviation (SD), 1 / 2 SD value, 1 / 3 SD value, etc., or any predetermined numerical value. Alternatively, it may be set to a certain range above and below the median value of each group. Here, the "certain range" may be the first quartile, the third quartile, etc., or any predetermined numerical value. For example, if the D-asparagine level obtained from a subject falls within the range of D-asparagine levels for the higher brain dysfunction group, the subject can be evaluated as having "a decline in higher brain function," "having a higher brain dysfunction," or "highly likely to have a higher brain dysfunction."

[0026] Thus, according to the method of the present invention, the state and degree of higher brain function of a subject can be evaluated simply and accurately in a minimally invasive manner. EXAMPLES

[0027] The present invention will be described more specifically below with reference to examples.

[0028] Test Example: Correlation analysis between higher brain function evaluated by TMT-B and blood D-asparagine ratio and D-asparagine concentration 1. Test Overview (Test Procedure) Twenty men and women aged 65 to 75 years (mean age 71±3 years) were subjected to TMT-B measurement as an evaluation of higher brain function, and a correlation analysis was performed with the blood D-asparagine ratio and D-asparagine concentration. TMT-B was measured with a maximum execution time (seconds) of 300 seconds. In addition, blood was drawn from the forearm vein on the same day as the measurement of higher brain function, and plasma was separated. The plasma was frozen and stored at -80℃, and then the asparagine concentration was measured.

[0029] (Measurement of blood asparagine concentration and calculation of D-asparagine ratio) (1) Preparation of sample solutions and standards 10 μL of human plasma was placed in a 10 mL volumetric flask (product name: Reinforced Hard Screw-cap Test Tube), mixed with 490 μL of a methanol:water (9:1, v / v) solution, and then centrifuged at 3000 rpm for 5 minutes at 4 °C in a centrifuge (manufactured by HITACHI / CF5RE) to remove proteins. The amino acids in the supernatant were collected. Subsequently, in a separate 10 mL volumetric flask, 70 μL of 0.2 mol / L boric acid buffer (pH 8.9), 10 μL of the collected supernatant solution, and 20 μL of a dimethylformamide solution of 2,5-dioxopyrrolidin-1-yl (2-((2-(diethylamino)ethyl)carbamoyl)quinolin-6-yl)carbamate (Waters RapiFluor-MS (RFMS) powder dissolved in ultrapure dimethylformamide at a concentration of 4.5 mg / mL, i.e., 10 mmol / L) were mixed in the order of 70 μL, 10 μL, 20 μL (7:1:2). Immediately after stirring, the sample solution was prepared by heating at 55 °C for 10 minutes. Similarly, 70 μL of 0.2 mol / L boric acid buffer (pH 8.9), 10 μL of a 10 nmol / L D,L-amino acid standard solution (Asn / asparagine, dissolved in 0.2 mol / L boric acid buffer), and 20 μL of the RFMS solution were mixed in the order of 70 μL, 10 μL, 20 μL (7:1:2). Immediately after stirring, the amino acid standard solution was prepared by heating at 55 °C for 10 minutes.

[0030] (2) Chiral LC-MS / MS analysis (1) The sample solution prepared above was subjected to chiral LC-MS / MS analysis under the following conditions to separate, detect, and quantify L-asparagine and D-asparagine. · Instrument Exion LC series (AB SCIEX), mass spectrometer / QTRAP6500 + Linear ion trap type (AB SCIEX)

[0031] · Chromatographic separation Separation chiral column: CHIRALPAK ZWIX(+) <DAICEL> 3.0 mm inner diameter × 150 mm, particle size 3 μm Column temperature: 45 °C Eluent: 0.1% (v / v) formic acid and 55 mM ammonium formate in methanol:water (90:10, v / v) solution Elution method: Isocratic Mobile phase flow rate: 0.25mL / min Injection volume: 5μL

[0032] ·Mass spectrometry Ionization method: Electrospray ionization (ESI) Polarity: Positive ion Curtain Gas (CUR): 30 psi Ionspray voltage(IS):4500V Temperature (TEM): 600℃ Ion Source Gas 1 (GS1): 80 psi Ion Source Gas 1 (GS2): 80 psi Collision Gas(CAD):10

[0033] Detection Mode Selected reaction monitoring (SRM) detection in positive ion mode with protonated ion adduct ([M+H]+m / z=445) as precursor ion (Q1) and RFMS fragment ion (m / z=240) as product ion Calculation of D-asparagine ratio The D-asparagine ratio ([D-asparagine concentration / (D-asparagine concentration+L-asparagine concentration)]×100(%)) was calculated from the measured L-asparagine and D-asparagine concentrations.

[0034] (Correlation analysis) Pearson's product-moment correlation analysis was performed on the measured TMT-B performance time, D-asparagine ratio, and D-asparagine concentration (GraphPad Prism ver 8.0 was used as the statistical analysis software).

[0035] [2. Results] It was revealed that there was a positive correlation between the time to perform TMT-B and the D-asparagine ratio, and that there was a significant positive correlation between the time to perform TMT-B and the D-asparagine concentration, that is, the higher the D-asparagine ratio and D-asparagine concentration, the longer it takes to perform TMT-B. Since TMT-B is a test used as an index of executive function, it was shown that the D-asparagine ratio and D-asparagine concentration can be used as markers for evaluating higher brain function.

[0036] [Table 1]

Claims

1. 2. A method comprising the step of measuring a level of D-asparagine in a blood sample taken from a subject, said level of D-asparagine providing an indication of executive function in said subject.

2. The D-asparagine level is determined according to the following formula: [D-asparagine concentration / (D-asparagine concentration+L-asparagine concentration)]×100 2. The method according to claim 1, wherein the D-asparagine ratio is

3. The method of claim 1, wherein the D-asparagine level is a D-asparagine concentration.

Images