JP2022154081A - Method of measuring higher brain functions - Google Patents

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

The present invention relates to a method for measuring higher brain functions using D-amino acids.

Cognitive functions such as executive function, cognitive flexibility, and attention function are also called higher brain functions, and when these functions are impaired due to aging, head injury, stroke, etc., it is difficult to adapt to daily and social life. may be.
The executive function, which is positioned at the top of the higher brain functions, is also called the executive function, and is defined as the ability to maintain an appropriate posture in order to achieve future goals. It is considered an essential function for normal cognitive function (Non-Patent Document 1). In addition, cognitive flexibility and attention control, which are higher brain functions, play important roles in promoting new behavioral patterns and optimizing behavior in unconventional situations. believed to support such behavior.

It is generally believed that the neural basis for these higher brain functions resides in the prefrontal cortex. Then, symptoms such as unplanned behavior, inability to prioritize things, haphazard behavior, inability to work efficiently, and inability to start behavior without instructions appear.

Currently, "TMT (Trail Making Test) A and B", which evaluate divided attention and ability to process multiple tasks, have been established as one of the test methods for higher brain function. It is widely used because it can be detected sharply. However, it is easier and more objective because it requires regular examinations by dementia specialists and clinical psychologists and regular hospital visits for patients, and the score is affected by the mood, physical condition, motivation, etc. of the subject. Development of inspection methods and indicators is required. Cerebrospinal fluid examination is considered useful in diagnosing dementia, but its invasiveness is a problem. Therefore, it is necessary to develop examination methods and indicators that can be applied to minimally invasive diagnosis.

On the other hand, all amino acids other than glycine have two stereoisomers, D- and L-isomers. Since L-amino acids are constituents of proteins in living organisms, and the amino acids contained in proteins are basically L-amino acids, it was thought that L-amino acids were mainly involved in the physiological activities of higher animals. The presence and role of D-amino acids in mammals, including humans, have been clarified with recent advances in analytical techniques that have improved resolution and sensitivity.

Recently, it has been reported that the amounts of D-amino acids and L-amino acids in biological materials in healthy individuals maintain a certain balance, and that the balance of D-amino acids and L-amino acids is disturbed in certain diseases. (Patent Document 1), and in the document, the relationship between Alzheimer's disease and D-amino acids in blood was also examined, and in Alzheimer's disease patients, D-serine, D-alanine, D-methionine, D-leucine, D - aspartic acid, D-phenylalanine or D-allo-isoleucine balance is suggested to be altered. However, regarding D-serine, there are reports suggesting that D-serine in cerebrospinal fluid can be a biomarker for early diagnosis of Alzheimer's disease (Non-Patent Document 2), D-serine in cerebrospinal fluid and Alzheimer's disease, There is no unified opinion on the relationship between brain function and D-amino acids, such as a mixed report (Non-Patent Document 3) that no correlation with mild cognitive impairment is observed, and the relationship with higher brain function There are also no reports of

JP 2017-223711 A

Toshiya Fukui, Cognitive Neuroscience (2010) 12: 156-164 2015;5 :1-9. doi:10.1038/tp.2015.52. Epub 2015 May 5. 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. 2011 Aug 29.

The present invention relates to providing an objective, minimally invasive method for assessing higher brain function in a subject.

The present inventors have studied in view of such problems, and found that blood contains D-asparagine, and that the higher the D-asparagine ratio or the higher the D-asparagine concentration, the longer it takes to perform TMT-B. and found that higher brain functions can be measured using the D-asparagine level in a biological sample of a subject as an index.

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, attentional function and cognitive flexibility, comprising the step of measuring D-asparagine level in a biological sample collected from a subject.

INDUSTRIAL APPLICABILITY According to the method of the present invention, the higher brain function of a subject can be tested objectively and simply in a minimally invasive manner, and early changes in deterioration of higher brain function can be objectively evaluated.

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.

In the present invention, "executive function" is also referred to as executive function, and is defined as the ability to maintain an appropriate posture to achieve future goals. Specifically, 1) task representation, 2) planning ( planning), 3) execution of tasks, 4) suppression of undesired reactions, and 5) evaluation and correction as necessary (Non-Patent Document 1).

"Attention function" is also known as attention control function, and refers to the function of consciously directing attention to certain important information from a large amount of information (Science Co., Ltd., Tadashi Oyama and Yoshiaki Nakajima, Experimental Psychology). Invitation to School [revised edition], p99). The attention function is mainly composed of three functions of "selective attention", "attention conversion", and "attention division" (Waseda University Clinical Psychology Research, Vol. 13, No. 1, p33). Here, selective attention is the function of directing attention from many stimuli and objects to a specific stimulus or object, and diverting attention is the ability to switch attention from a specific stimulus or object to a specific stimulus or object as needed. It is the ability to pause and switch to other stimuli or objects as appropriate, and splitting attention is the ability to distribute attention to multiple objects simultaneously.

“Cognitive flexibility” refers to the ability to replace maladaptive thoughts and incorporate balanced and adaptive thinking (Yokawa Tokuyoshi and Shoichi Iwasaki (2012) Cognitive Flexibility Scale (CFI) Preparation and Validity of the Japanese Version Proceedings of the 76th Conference of the Japanese Psychological Association, 672).

Higher brain functions (executive function, attention function and cognitive flexibility) in the present invention can be evaluated by TMT-B (Trail Making Test B).
TMT-B is a method that is often used as a test of higher brain functions, and it examines visual attention, visual search, visuomotor coordination, attentional maintenance and selection, visual-motor coordination, speed of information processing, and interference. It is said to reflect higher-order attentional functions such as short-term memory.

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 by the terms "inspection", "judgment", "evaluation" or "evaluation support". In addition, the term "judgment" or "evaluation" used herein does not include judgment or evaluation by a doctor.

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

In the present invention, the subject is not particularly limited. Impossible to prioritize, haphazard behavior, inability to work efficiently, inability to start behavior without instructions, inability to modify work content as needed, etc.), decline in higher brain function. is suspected, and the like.

In the present invention, biological samples mainly include body fluids such as blood, lymph, cerebrospinal fluid, saliva, and urine, preferably blood samples. Examples of blood samples include blood (whole blood) and blood-derived serum, plasma, and the like, 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 point of view, it is preferable to collect blood from blood vessels in the systemic circulation, particularly from veins (peripheral veins).

In the present invention, the D-asparagine level may be the content or composition of D-asparagine. A chiral balance, ie D-asparagine ratio or D-asparagine concentration, is preferred.

Measurement of the D-asparagine level in a biological sample can be performed by separating D-amino acids and L-amino acids in the biological sample, and the means is not limited, but generally liquid chromatography (LC) and mass Separation and quantification methods using LC-MS combined with analysis (MS), LC-MS/MS, or the like can be employed.
Here, as a method for separating chiral amino acids, for example, 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) reagent is used to convert amino acids into NBD derivatives, followed by reverse-phase separation. A method of separating and quantifying by a two-dimensional liquid chromatography (LC) method using a column (first dimension: molecular species separation) and a chiral column (second dimension: chiral separation) having a stationary phase carrying a chiral identifier (J Chromatogr A 2010 Feb 12;1217(7):1056-62. doi: 10.1016/j.chroma.2009.09.002. Epub 2009 Sep 6) and 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 of derivatization and separation by a one-dimensional LC method 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 ), after the amino group is derivatized by AQC, liquid chromatography is used in which a first chiral column having a weak anion-exchange stationary phase and a second chiral column having an amphoteric ion-exchange stationary phase are connected. (JP 2018-100906 A) and the like are known.
Alternatively, a chiral amino acid can be prepared by reacting the amino group with 2,5-dioxopyrrolidin-1-yl (2-((2-(diethylamino)ethyl)carbamoyl)quinolin-6-yl)carbamate, as shown in the Examples below. After derivatization, it can be separated using liquid chromatography connected to a chiral column having an amphoteric ion exchange type stationary phase (Japanese Patent Application No. 2021-042055), and such a method is used for the separation of chiral amino acids. is preferred. Incidentally, 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).

As shown in Examples below, the D-asparagine ratio and D-asparagine concentration were found to have a good classification correlation with the performance time of TMT-B, which is widely used as a test of higher brain function. That is, the performance time of TMT-B shows a trend of positive correlation with the D-asparagine ratio, and the performance time is positively correlated with the D-asparagine concentration. Therefore, by measuring the D-asparagine level in a subject's biological sample and comparing it with a reference value, the subject's higher brain function, specifically executive function, attention function and cognitive flexibility. One or more can be measured.

Here, the reference value can be set as follows, for example, based on the association between the D-asparagine level and the state of higher brain function.
The state of higher brain function is assessed by TMT-B. Based on the evaluation results, create a healthy group consisting of subjects judged to have normal higher brain function and a higher brain dysfunction group consisting of subjects judged to have low higher brain function. do. Separately, D-asparagine levels are measured by the method described above. Based on the correlation between the evaluation result 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 humans belonging to each group, the numerical range of D-asparagine levels that characterize 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 "fixed range" may be a statistical value such as standard deviation (SD), a 1/2 SD value, a 1/3 SD value, or the like, or may be an arbitrary numerical value set in advance. Alternatively, it can be set within a certain range above and below the median value of each group. Here, the “fixed range” may be the first quartile, the third quartile, or the like, or may be an arbitrary numerical value set in advance.
Then, for example, when the D-asparagine level obtained from the subject falls within the range of the D-asparagine level of the higher brain dysfunction group, the subject "has decreased higher brain function", " It can be evaluated as "having higher brain dysfunction" or "high possibility of having higher brain dysfunction".

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 with minimal invasiveness.

EXAMPLES Hereinafter, the present invention will be described more specifically by showing examples.

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)
Targeting 20 males and females aged 65 to 75 (average age 71 ± 3 years), TMT-B was measured as an evaluation of higher brain function, and the blood D-asparagine ratio and D-asparagine concentration were measured. A correlation analysis was performed. TMT-B was measured with a performance time (seconds) as an upper limit of 300 seconds. On the same day as the measurement of higher brain function, blood was collected from the forearm vein and plasma was separated. Plasma was stored frozen at -80°C and then measured for asparagine concentration.

(Measurement of blood asparagine concentration and calculation of D-asparagine ratio)
(1) Preparation of sample solution and standard 10 μL of human plasma is placed in a 10 mL spitch glass (trade name: reinforced hard screw cap test tube), methanol: water (9: 1, v / v) solution 490 μL is mixed, Protein was removed by centrifugation at 3000 rpm for 5 minutes under refrigeration (4° C.) in a centrifuge (manufactured by HITACHI/CF5RE), and amino acids in the supernatant were collected. Then, in a separate 10 mL spitch glass, 0.2 mol / L borate buffer (pH 8.9), collected supernatant solution, 2,5-dioxopyrrolidin-1-yl (2-((2-(diethylamino) Ethyl)carbamoyl)quinolin-6-yl)carbamate in dimethylformamide (Waters RapiFluor-MS (RFMS) powder dissolved in ultra-dehydrated dimethylformamide at a concentration of 4.5 mg/mL, ie, 10 mmol/L) was 70 μL each. , 10 μL, and 20 μL (7:1:2) were mixed in this order, immediately stirred, and then heated at 55° C. for 10 minutes to prepare a sample solution. Similarly, 70 μL each of 0.2 mol/L borate buffer (pH 8.9), 10 nmol/L D, L-amino acid standard solution (Asn/asparagine, dissolved in 0.2 mol/L borate buffer), and RFMS solution. , 10 μL, and 20 μL (7:1:2) were mixed in this order, immediately stirred, and then heated at 55° C. for 10 minutes to prepare an amino acid standard solution.

(2) Chiral LC-MS/MS Analysis The sample solution prepared in (1) was subjected to Chiral LC-MS/MS analysis under the following conditions to separate, detect and quantify L-asparagine and D-asparagine.
・Apparatus Exion LC series (AB SCIEX), mass spectrometer/QTRAP6500 ⁺ linear ion trap type (AB SCIEX)

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

・Mass spectrometry ionization method: electrospray ionization method (ESI)
Polarity: Positive ion Curtain Gas (CUR): 30 psi
Ionspray voltage (IS): 4500V
Temperature (TEM): 600°C
Ion Source Gas 1 (GS1): 80 psi
Ion Source Gas 1 (GS2): 80 psi
Collision gas (CAD): 10

・Detection mode SRM (Selected Reaction Monitoring) in positive ion mode with proton ion adduct molecules ([M+H]+m/z=445) as precursor ions (Q1) and RFMS fragment ions (m/z=240) as product ions Detection/Calculation of D-Asparagine Ratio From the measured L-asparagine and D-asparagine concentrations, the D-asparagine ratio ([D-asparagine concentration/(D-asparagine concentration + L-asparagine concentration)]×100 (%)) was calculated. Calculated.

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

[2. result〕
There is a tendency for a positive correlation between the performance time of TMT-B and the D-asparagine ratio, and there is a significant positive correlation between the performance time of TMT-B and the D-asparagine concentration, that is, the D-asparagine ratio And it was found that the higher the D-asparagine concentration, the longer it takes to perform TMT-B. Since TMT-B is a test used as an indicator of executive function, it was shown that the D-asparagine ratio and D-asparagine concentration serve as markers for evaluating higher brain functions.

Claims (5)

A method for measuring one or more higher brain functions selected from executive function, attention function and cognitive flexibility, comprising the step of measuring D-asparagine level in a biological sample taken from a subject. The D-asparagine level is determined by the formula:
[D-asparagine concentration / (D-asparagine concentration + L-asparagine concentration)] × 100
The method according to claim 1, wherein the D-asparagine ratio is represented by 2. The method of claim 1, wherein the D-asparagine level is D-asparagine concentration. The method according to any one of claims 1 to 3, wherein the biological sample collected from the subject is a blood sample. 5. The method of any one of claims 1-4, wherein the higher brain functions include at least executive functions.