Method for use as an indicator of a disease or condition associated with amyloid beta, and composition and kit for use in said method

Abstract

To provide a method to readily and inexpensively find, in a healthy subject group and the like, a person having a high risk of developing a disease or condition associated with amyloid β, such as MCI.SOLUTION: The inventive method uses an amount of choline esterase in blood isolated from a subject as an index of a disease or condition associated with amyloid β.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for indicating a disease or condition associated with amyloid-β, and to a composition and a kit for use in said method. [Background technology]

[0002] Dementia is a disease whose risk of developing increases as the aging rate increases. The number of dementia patients worldwide was estimated at 47 million in 2017, and is expected to increase to 75 million in 2030 and 131 million in 2050. It has also been pointed out that the increase in dementia could cause medical and nursing care costs to reach 2 trillion dollars worldwide in 2030 and 10.7 trillion yen in Japan in 2025.

[0003] Japan has a dementia prevalence rate of 2.33% of the total population, the highest rate among OECD member states (1.48%). Of dementia in Japan, 50% is Alzheimer's disease (AD), 20% is Lewy body dementia, 15% is vascular dementia, and 15% is other, making treatment and prevention of AD dementia important. The "amyloid cascade hypothesis" supports the idea that AD occurs when amyloid beta (Aβ) accumulates in the brain, causing changes in neurofibrillary fibers and shrinking of the hippocampus, which controls memory.

[0004] There is a condition called mild cognitive impairment (MCI) in which cognitive function can be restored by appropriate prevention / treatment at the pre-dementia stage, and it is known that if this stage can be improved, the onset of dementia can be delayed. It is said that the percentage of people who progress from MCI to dementia is 10% per year, and about 40% in 5 years. In addition, the Japanese Society of Neurology, Dementia Disease Treatment Guidelines 2017, shows that 16-41% of people with MCI will recover if appropriate treatment is given. Therefore, if MCI is detected and evaluated early and preventive measures are taken, the number of dementia patients can be significantly reduced, so there is a strong hope for the development of an early detection method for healthy people at high risk of developing the condition.

[0005] Dementia testing and diagnosis includes memory and language tests, blood tests, brain imaging tests (CT, MRI), cerebral blood flow SPECT (scintigraphy), dopamine transporter scintigraphy (testing for decreased expression levels), and MIBG myocardial scintigraphy (myocardial sympathetic nerve testing).

[0006] Blood tests available for determining the risk of MCI include those that measure apolipoproteins (ApoA1; a protein that excretes Aβ peptides from the brain into the cerebrospinal fluid), complement proteins (C3; a protein that ingests Aβ), and transthyretin (TTR; a protein that binds to Aβ to reduce its toxicity) (MCI screening tests), as well as tests for the APOE gene type (E4 homozygotes have a 15-fold increased risk).

[0007] Research is also being conducted on early detection techniques for dementia, and for example, a method for determining mild cognitive impairment by measuring the expression levels of proteins such as FABP, sTNFR1, TNC, ICAM-1, and cytokines in serum as biomarkers (Patent Document 1), a technique for diagnosing dementia based on the plasmalogen level in red blood cells (Patent Document 2), and a method for diagnosing mild cognitive impairment based on the expression level of miR-206 in olfactory tissue (Patent Document 3) have been proposed. It has also been reported that even if serum magnesium levels are within the standard range of 0.7 to 1.10 mmol / L (1.7 to 2.7 mg / dL), if they are 0.79 mmol / L or less or 1.9 mg / dL or less, the risk of dementia increases by 32%, and if they are 0.90 mmol / L or more or 2.2 mg / dL or more, the risk of dementia increases by 30% (Non-Patent Document 1). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Special Publication No. 2019-522193 [Patent Document 2] International Publication No. 2012 / 090625 [Patent Document 3] Special Publication No. 2018-515145 [Non-patent literature]

[0009] [Non-Patent Document 1] Kieboom et al. Serum magnesium is associated with the risk of dementia.2017, Neurology, 89(16), 1716-1722 Summary of the Invention [Problem to be solved by the invention]

[0010] However, tests for determining the risk of MCI, including blood tests, are generally expensive, and there is a problem that the tests cannot be performed cheaply and safely. Therefore, an object of the present invention is to provide a simple and cheap method for detecting individuals at high risk of developing a disease or condition associated with amyloid β, such as MCI, from a group of healthy individuals, etc. [Means for solving the problem]

[0011] The present inventors have found that the amount of cholinesterase in blood can be correlated with the amount of amyloid beta accumulated in the brain, and have completed the present invention. More specifically, the present invention provides the following.

[0012] [1] A method in which the amount of cholinesterase in blood isolated from a subject is used as an indicator of a disease or condition associated with amyloid beta. [2] The method according to [1] above, wherein the subject is a mammal. [3] The method according to [1] or [2] above, wherein the subject is a human aged 60 or older. [4] The method according to any one of [1] to [3] above, wherein the amount of cholinesterase in the blood, calculated as the amount of cholinesterase in serum obtained from the blood, is 326 IU / L or less, which is an indicator of the possibility of a disease or condition associated with amyloid beta in the subject. [5] The method according to any one of [1] to [4] above, wherein the subject is a male human, and the amount of cholinesterase in the blood, as calculated as the amount of cholinesterase in serum obtained from the blood, is 306 IU / L or less, which is an indication of the possibility of a disease or condition associated with amyloid beta in the subject. [6] The method according to any one of [1] to [5] above, wherein at least one additional test is performed to facilitate the diagnosis of a disease or condition associated with amyloid beta. [7] The method according to [6] above, wherein the additional test comprises using the amount of magnesium in the blood isolated from the subject as an indicator of a disease or condition associated with amyloid beta. [8] The method described in [7] above, wherein the amount of magnesium in the blood, calculated as the amount of magnesium in serum obtained from the blood, is 2.2 mg / dL or more, which is an indicator of the possibility of a disease or condition associated with amyloid beta in the subject. [9] The method according to [7] or [8] above, wherein the subject is a female human and the amount of magnesium in the blood, calculated as the amount of magnesium in serum obtained from the blood, is 2.24 mg / dL or more, which is an indication of the possibility of a disease or condition associated with amyloid beta in the subject.

[10] The method according to [6] above, wherein the additional test is a cognitive function test selected from the group consisting of Mini-cog test, Mini-Mental State Examination (MMSE), ADAS-cog test and Clock Drawing Test.

[11] The method according to any one of [1] to

[10] above, wherein the disease is a neuropathy selected from the group consisting of mild cognitive impairment (MCI), Alzheimer's dementia (AD), Down's syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type), cerebrovascular amyloid angiopathy, and Guam Parkinson's dementia complex, or progressive supranuclear palsy, multiple sclerosis, inclusion body myositis (IBM), Creutzfeldt-Jakob disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), inclusion body myositis (IBM), adult-onset diabetes mellitus (type 2 diabetes), senile cardiac amyloidosis, endocrine tumor, glaucoma, ocular amyloidosis, primary retinal degeneration, macular degeneration, optic nerve drusen, optic neuropathy, optic neuritis, or lattice dystrophy.

[12] The method according to any one of [1] to

[11] above, which is for measuring or detecting the subject's risk of developing a disease or condition associated with amyloid beta, for detecting a disease or condition associated with amyloid beta in the subject, for selecting a subject having a disease or condition associated with amyloid beta, for measuring, evaluating or monitoring the response of the subject to an ingested substance for preventing or treating the disease or condition associated with amyloid beta, or for measuring, evaluating or monitoring the progress of the disease or condition associated with amyloid beta in the subject.

[0013]

[13] A composition for use in the method according to any one of [1] to

[12] above, the composition comprising a reactant for detecting the presence of the cholinesterase and a reagent for quantifying the cholinesterase.

[14] The composition described in

[13] above, wherein the reactant comprises a substrate for the cholinesterase.

[15] A kit for use in the method according to any one of [1] to

[12] above, comprising a reactant for detecting the presence of the cholinesterase and a reagent for quantifying the cholinesterase.

[16] The kit according to

[15] above, wherein the reactant comprises a substrate for the cholinesterase.

[17] A test kit as described in

[15] or

[16] above, which is a kit for use in the method according to any one of claims 7 to 9 and further contains a reagent for quantifying the magnesium. Effect of the Invention

[0014] According to the present invention, it is possible to provide a simple and inexpensive method for identifying individuals at high risk of developing a disease or condition associated with amyloid β, such as MCI, from a group of healthy individuals or the like. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Hereinafter, an embodiment of the present invention will be described, but the present invention is not limited thereto.

[0016] <Method of using as an indicator of amyloid β-related disease or condition> A first aspect of the present invention is a method for using the amount of cholinesterase (ChE) in blood isolated from a subject as an indicator of a disease or condition associated with amyloid β.

[0017] The inventors have discovered that the amount of cholinesterase in blood can be correlated with the amount of amyloid beta accumulated in the brain or a value indicating the amount of amyloid beta accumulated in the brain, such as the Composite Biomarker value described below, and have discovered that the amount of cholinesterase in blood, which can be measured simply and inexpensively in a blood test, can be used as an indicator of a disease or condition associated with amyloid beta.

[0018] Amyloid beta is a partial fragment of the precursor protein APP, which is produced in the brain, and is produced and secreted by successive cleavage by beta-secretase and gamma-secretase. It is composed of about 40 amino acids and there are many peptides, among which Aβ1-40, Aβ1-42 and their precursor APP669-711 are known to be good blood biomarkers for detecting Alzheimer's disease.

[0019] It is believed that amyloid-β-related diseases or conditions are highly likely to be correlated with the amount of amyloid-β accumulated in the brain, and the accumulation of amyloid-β in the brain can be estimated, for example, by measuring the amount of amyloid-β in the blood. The amount of amyloid-β in the blood can be estimated, for example, by a composite biomarker value obtained by scaling and scoring two values, Aβ1-42 and APP669-711, which are particularly related to the onset of amyloid-β-related diseases or conditions such as MCI, Alzheimer's dementia, and other dementias. The first aspect of the present invention may include correlating the amount of amyloid-β accumulated in the brain or the composite biomarker value with the amount (concentration) of cholinesterase in the blood isolated from the subject.

[0020] The subject may be an animal such as a mammal, and may be any animal that can develop amyloid beta-associated disease or condition, and may be selected according to the purpose. For example, rodents such as rats, mice, and guinea pigs, as well as non-human mammals such as rabbits, dogs, cats, pigs, and cows may be included, but humans are preferred. As for humans, humans aged 60 or over are preferred in terms of the correlation, specifically the positive correlation, between the amount of cholinesterase in blood and the amount of amyloid beta-associated brain or the disease or condition associated with amyloid beta, and more preferably male humans, and even more preferably male humans aged 60 or over, in terms of the correlation, specifically the positive correlation, between the amount of cholinesterase in blood and the amount of amyloid beta-associated brain or the disease or condition associated with amyloid beta.

[0021] The amount of cholinesterase in blood may specifically be the amount of cholinesterase in plasma or serum, and is preferably the amount of cholinesterase in serum. The first aspect of the present invention may include obtaining, measuring or determining the amount of cholinesterase in blood.

[0022] The method of obtaining blood, plasma, and serum is not particularly limited, and may be based on a conventionally known method, for example, a method of obtaining (collecting) blood as a clinical test specimen, or a method of separating plasma and serum from blood as a clinical test specimen. For example, blood may be collected in an EDTA tube, heparin tube, or the like, and centrifuged to obtain plasma. Blood may also be collected in a test tube, or the like, and centrifuged to obtain serum.

[0023] The method for measuring the amount of cholinesterase in blood, plasma or serum is not particularly limited as long as it can quantify cholinesterase, and examples thereof include a kit or test composition for quantifying cholinesterase, such as a kit or test composition containing a reactant for detecting the presence of cholinesterase and a reagent for quantifying the cholinesterase. The reactant may contain a substrate for cholinesterase, and examples of the substrate include p-hydroxybenzoylcholine iodide salt (pHBC) described below.

[0024] Methods for measuring the amount of cholinesterase in blood, plasma or serum include, for example, methods employed in general blood tests, specifically, UV method, JSCC standardized method (also called pHBC substrate method), etc. The JSCC standardized method can be used to easily and inexpensively measure the amount of cholinesterase by using, for example, a blood test cholinesterase kit or a test composition such as an in vitro diagnostic drug such as "Serotec" ChE-CL or "Serotec" ChE-ML (manufactured by Serotec). The kit or test composition utilizes the fact that cholinesterase (ChE, nonspecific cholinesterase) in a specimen produces p-hydroxybenzoic acid and choline using p-hydroxybenzoylcholine iodide salt (pHBC) as a substrate, and p-hydroxybenzoic acid hydroxylase (pHBH) acts on the p-hydroxybenzoic acid to produce 3,4-dihydroxybenzoic acid, and at the same time, β-nicotinamide adenine dinucleotide phosphate reduced tetrasodium salt tetrahydrate (β-NADPH) becomes β-nicotinamide adenine dinucleotide phosphate oxidized (β-NADP), and determines the cholinesterase activity value by measuring the rate of decrease in absorbance associated with the decrease in β-NADPH. In addition, protocatechuate-3,4-dioxygenase (PCO) is added to inhibit side reactions, and 3,4-dihydroxybenzoic acid is decomposed to β-carboxymuconic acid. The first reagent containing pHBH, NADPH, and PCO, the second reagent containing pHBC, and serum or the like are mixed and reacted, and the concentration of cholinesterase can be determined based on the rate of decrease in absorbance at 340 nm.

[0025] A blood cholinesterase level of 326 IU / L or less, as calculated as the level of cholinesterase in serum obtained from the blood, can be used as an indicator of the possibility of a disease or condition associated with amyloid beta in a subject. The serum cholinesterase level is preferably that of humans, for example humans of both sexes, specifically humans aged 60 years or older, and particularly humans of both sexes aged 60 years or older.

[0026] In particular, when the subject is a male human, for example a male human aged 60 years or older, an amount of cholinesterase in the blood, expressed as the amount of cholinesterase in serum obtained from the blood, of 306 IU / L or less can be used as an indicator of the possibility of the subject having a disease or condition associated with amyloid beta.

[0027] The above-mentioned value of 326 IU / L or less to 306 IU / L or less has conventionally been considered to be within the normal range, and moreover, although it has been believed that, for example, within the range of 245 to 495 IU / L, which is the reference value for male humans, the higher the value, the more likely it is to correlate with the amount of amyloid beta accumulated in the brain. However, the present inventors have newly discovered that this value may correlate with the amount of amyloid beta accumulated in the brain or a value indicating the amount of amyloid beta accumulated in the brain, for example, a Composite Biomarker value.

[0028] The first aspect of the invention may involve the performance of at least one additional test to facilitate diagnosis of a disease or condition associated with amyloid-β.

[0029] The additional test may include detecting at least one additional biomarker for a disease or condition associated with amyloid β. The additional biomarker may include, for example, magnesium (Mg) in blood isolated from a subject. Specifically, the first aspect of the present invention may include using the amount of magnesium in blood isolated from a subject as an indicator of a disease or condition associated with amyloid β, which is expected to increase the accuracy of the indicator of a disease or condition associated with amyloid β.

[0030] The amount of magnesium in blood may specifically be the amount of magnesium in plasma or serum, and is preferably the amount of magnesium in serum. The method for obtaining blood and serum is not particularly limited, and for example, the same method as that described above for cholinesterase can be applied.

[0031] The method for measuring the amount of magnesium in blood, plasma or serum is not particularly limited as long as it can quantify magnesium, and examples of the method include a method of quantifying magnesium using a kit or test composition for quantifying magnesium, such as a kit or test composition containing a reagent for quantifying magnesium, etc. Examples of the reagent include xylyl azo violet, isocitrate dehydrogenase (ICDH), etc.

[0032] Methods for measuring the amount of magnesium in blood, plasma or serum include, for example, methods employed in general blood tests, specifically, the xylidyl blue method (coloring solution: xylidyl azo violet I), enzyme method, etc. These methods can be used to easily and inexpensively measure the amount of magnesium in blood, plasma or serum by using a test composition such as a blood test kit or an in vitro diagnostic drug. The xylidyl blue method utilizes the fact that magnesium binds with xylylazo violet under alkaline conditions to form a red chelate compound, and involves measuring the red color by absorbance (520 nm, 800 nm).

[0033] In the enzymatic method, isocitrate dehydrogenase (ICDH) produces 2-oxoglutaric acid in the presence of magnesium using isocitrate as a substrate, and simultaneously converts oxidized nicotinamide adenine dinucleotide phosphate (NADP) to reduced nicotinamide adenine dinucleotide phosphate (NADPH), the reaction rate is proportional to the magnesium concentration, and NADPH has an absorption maximum at 340 nm. The magnesium concentration in the sample is determined by measuring the rate of increase in absorbance. For example, D-isocitrate-monopotassium salt and enzyme reagent solution I containing ICDH, enzyme reagent solution II containing NADP are mixed with serum or the like and reacted, and the magnesium concentration can be determined from the rate of increase in absorbance at 340 nm.

[0034] The additional test preferably includes using the amount of magnesium in blood, expressed as the amount of magnesium in serum obtained from the blood, of 2.2 mg / dL or more as an indicator of the possibility of a disease or condition associated with amyloid beta in the subject. The amount of magnesium in the serum is preferably that of a human, for example, a human of both sexes, specifically a human aged 60 years or older, particularly a human of both sexes aged 60 years or older.

[0035] In particular, when the subject is a female human, for example a female human aged 60 years or older, an amount of magnesium in the blood of 2.24 mg / dL or more, calculated as the amount of magnesium in serum obtained from the blood, can be used as an indicator of the possibility of the subject having a disease or condition related to amyloid beta.

[0036] In addition, the at least one additional test for facilitating the diagnosis of a disease or condition associated with amyloid beta may be one or more selected from the group consisting of the Mini-Cog test, the Mini-Mental State Examination (MMSE), the ADAS-Cog test, and the Clock Drawing Test, which are cognitive function tests. These additional tests may be performed instead of or in addition to the additional test including the detection of at least one additional biomarker described above, and can be selected appropriately depending on the cost, simplicity, needs of the subject, etc., but are preferably performed in addition to or in combination with the additional test, as this can increase the accuracy of the possibility of a disease or condition associated with amyloid beta.

[0037] As the additional test, when the subject is a human, the Mini-cog test is preferable. The Mini-cog test is a test that judges the subject's condition by the total score of the "verbal memory" test, in which the subject is asked to memorize three words and is scored 0 to 3 points depending on the number of words that the subject remembers, and the "clock drawing" test, in which the subject is scored 0 or 2 points depending on whether the subject can draw a clock that shows 11:10. The fewer the number of words that the subject remembers in the "verbal memory" test, and the more mistakes are made in both the "verbal memory" and the "clock drawing," the higher the possibility of MCI, Alzheimer's dementia, or other dementias, and this tendency is stronger when the subject is 60 years old or older.

[0038] Examples of diseases or conditions associated with amyloid beta include dementia, Down's syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type), cerebrovascular amyloid angiopathy, and Guam Parkinson's dementia complex, including neuropathy or progressive supranuclear palsy, multiple sclerosis, inclusion body myositis (IBM), Creutzfeldt-Jakob disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), inclusion body myositis (IBM), adult-onset diabetes mellitus (type 2 diabetes), senile cardiac amyloidosis, endocrine tumors, glaucoma, ocular amyloidosis, primary retinal degeneration, macular degeneration, optic nerve drusen, optic neuropathy, optic neuritis, or lattice dystrophy. Examples of the dementia include mild cognitive impairment (MCI), Alzheimer's dementia (Alzheimer's disease; AD), and the like. Among these, mild cognitive impairment (MCI) and dementia such as Alzheimer's disease (Alzheimer's disease; AD) are suitable, with mild cognitive impairment (MCI) being preferable.

[0039] That is, as described above, the first aspect of the present invention is a method in which the amount of cholinesterase (ChE) in blood isolated from a subject is used as an indicator of a disease or condition associated with amyloid beta, and specifically, the method is capable of using the amount of cholinesterase (ChE) as an indicator that a subject has developed or is at risk of developing in the future mild cognitive impairment (MCI), dementia such as Alzheimer's disease (Alzheimer's disease; AD), or other such diseases or conditions.

[0040] The above-mentioned method of the first aspect of the present invention is suitable, for example, for measuring, detecting (including early detection), determining or judging the risk of a subject, such as a healthy individual, for developing a disease or condition associated with amyloid beta; for determining, judging or detecting (including early detection) a disease or condition associated with amyloid beta in a subject; for selecting (or screening) a subject having a disease or condition associated with amyloid beta; for measuring, evaluating or monitoring a subject's response to an ingested substance (e.g., food, supplement, medicine) for preventing or treating a disease or condition associated with amyloid beta (e.g., whether the ingested substance has the effect of preventing or treating the disease or condition); or for measuring, evaluating or monitoring the progress of a disease or condition associated with amyloid beta in a subject (including after the start of treatment, such as medication).

[0041] The method according to the first aspect of the present invention can achieve or support these objectives by performing a blood test that can be performed in vitro, is non-invasive, safe, simple, and inexpensive. The method according to the first aspect of the present invention is also suitable as a method for diagnosing a disease or condition associated with amyloid β, a method for pre-diagnosing the disease or condition, or a method for supporting or assisting in the diagnosis.

[0042] <Composition> The second aspect of the present invention is a composition for use in the method of the first aspect of the present invention, which can measure at least the amount of cholinesterase in blood, plasma or serum. The composition typically comprises a reactant for detecting the presence of cholinesterase and a reagent for quantifying the cholinesterase. Examples of the composition include those described as a test composition for in vitro diagnostic drugs and the like, which can be used to measure the amount of cholinesterase in blood, plasma or serum in the method of the first aspect of the present invention.

[0043] <Kit> The third aspect of the present invention is a kit for use in the method of the first aspect of the present invention, which is capable of measuring at least the amount of cholinesterase in blood, plasma or serum. The kit typically includes a reactant for detecting the presence of cholinesterase and a reagent for quantifying the amount of the cholinesterase. For example, the kit may be one described as a test kit for measuring the amount of cholinesterase in blood, plasma or serum in the method of the first aspect of the present invention.

[0044] The kit may be capable of quantifying magnesium in blood, plasma or serum, for example, when the amount of magnesium in blood isolated from a subject is also used as an index of a disease or condition associated with amyloid β as an additional test for facilitating the diagnosis of a disease or condition associated with amyloid β in the method of the first aspect of the present invention. The kit capable of quantifying magnesium typically includes a reagent for quantifying magnesium, and examples thereof include the kits described as test kits that can be used to measure the amount of magnesium in blood, plasma or serum in the method of the first aspect of the present invention. EXAMPLES

[0045] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0046] 1. Measurement of the amount of amyloid beta An analysis of trace amounts of amyloid beta present in the blood of 474 healthy subjects (humans, who may include patients with mild cognitive impairment (MCI) but have not been diagnosed with dementia) was performed as follows. The plasma of the subjects was treated with magnetic beads bound to anti-amyloid beta monoclonal antibodies, followed by immunoprecipitation (IP), and amyloid beta was eluted from the magnetic beads. Three peptides, Aβ1-40, Aβ1-42, and their precursor APP669-711, were measured by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS), and the biomarker values ​​of APP669-711 / Aβ1-42 and Aβ1-40 / Aβ1-422 species were calculated. Each value was z-scored (values ​​converted so that the values ​​of the data set have a mean value of 0 and a standard deviation of 1), and values ​​with significantly different scales were scaled, and the average value was calculated as the composite biomarker value. Furthermore, the average composite biomarker values ​​for those in their 40s were -0.542 for men and -0.718 for women, for those in their 50s were -0.316 for men and -0.504 for women, for those in their 60s were -0.340 for men and -0.357 for women, and for those in their 70s were -0.195 for men and -0.184 for women.

[0047] 2. Obtaining biomarker values The blood cholinesterase concentration of each subject was measured by the JSCC standardized method, and the blood magnesium concentration of each subject was measured by the enzymatic method.

[0048] 3.Correlation analysis between composite biomarker values ​​and biomarker values To search for biomarkers that have a positive correlation with composite biomarker values, the relationship between composite biomarker values ​​and blood test data was investigated for the above 474 subjects by age group: 40s, 50s, and 60s and above. For the analysis, the average composite biomarker values ​​were calculated for each generation, and a test was performed to see if there was a significant difference in test values ​​between the group of subjects below the average and the group of subjects above the average (t-test).

[0049] As a result of the analysis, when the composite biomarker values ​​were divided into those below the average value and those above the average value, no significant correlation was found, but differences were found in the average values ​​of blood cholinesterase concentration and blood magnesium concentration between the subject group with a composite biomarker value of 0 or above and the subject group with a composite biomarker value below 0. The results are shown in Table 1. In Table 1, a smaller p-value (p-val) indicates a stronger significance, and a p-value of 0.05 or less indicates a particularly statistically significant difference. Furthermore, the composite biomarker value increased with age. The change tended to be greater in women. Furthermore, it is known that the risk of Alzheimer's disease is increased in people with the E4 type of the ApoE gene. Among subjects aged 60 years or older, the proportion of subjects with the E4 type of ApoE was higher among those with composite biomarker values ​​of 0 or higher.

[0050] [Table 1]

[0051] As can be seen from Table 1, serum cholinesterase (ChE) concentrations tend to be low in subjects with high Composite Biomarker values. Specifically, in subjects with Composite Biomarker values ​​of 0 or higher (mixed genders aged 60 years or older), the concentration was 326 IU / L or less. When the subjects were analyzed separately by gender, there was a significant positive correlation in men, with the concentration being 306 IU / L or less. In addition, a positive correlation was observed between the composite biomarker value and serum magnesium concentration from another analysis. Blood magnesium concentration tends to increase with age. In subjects aged 60 years or older, subjects with higher composite biomarker values ​​had significantly higher blood magnesium concentrations. Specifically, serum magnesium concentrations were 2.2 mg / dL or higher in subjects (mixed males and females aged 60 years or older) with composite biomarker values ​​of 0 or higher, and when the subjects were analyzed separately by gender, the positive correlation was significant in women, being 2.24 mg / dL or higher.

[0052] 4. Magnesium intake and its effect on composite biomarker values ​​and blood magnesium levels For each of the 474 subjects, the components and amounts of each meal for an average day's breakfast, lunch, dinner, and snacks were recorded, and dietary magnesium intake was calculated using the Standard Tables of Food Composition in Japan (7th edition). As a result, no relationship was found between dietary magnesium intake and the above-mentioned composite biomarker values ​​or blood magnesium concentrations.

[0053] From the above results, it was found that blood cholinesterase concentration and blood magnesium concentration can be indicators of amyloid beta accumulation.Specifically, in subjects (healthy individuals) aged 60 or over, those with serum cholinesterase of 326 IU / L or less (mixed sex), specifically men with serum cholinesterase of 306 IU / L or less, can be evaluated as having a high risk of developing MCI or dementia, and this evaluation is also correlated with those with serum magnesium of 2.2mg / dL or more (mixed sex), specifically women with serum magnesium of 2.24mg / dL or more.

[0054] 5.Mini-cog test The Mini-cog test was performed on the above subjects. The results are shown in Tables 2 and 3 together with the average amount of amyloid β (average composite biomarker value) as the amount of amyloid β obtained above.

[0055] [Table 2]

[0056] [Table 3]

[0057] As can be seen from a comparison of Tables 2 and 3, there was a tendency for subjects over 60 years old to make mistakes in both the word memory and clock drawing. Subjects who made mistakes in both are presumed to be cases of clear cognitive decline. In addition, because most of the subjects were healthy, no clear overall trend was observed, but for those who answered two questions incorrectly in the Mini-cog test, who are presumed to have a strong tendency toward cognitive decline, although the number was small, a correlation was observed with the Composite Biomarker value (amyloid beta amount). From the above, we were able to determine to some extent the correlation between composite biomarker values ​​(amyloid beta levels) and cognitive function. Therefore, it was found that by measuring blood cholinesterase concentrations, and if necessary, blood magnesium concentrations, primarily in people aged 60 or older, although there are gender differences, it may be possible to detect cognitive decline without having to measure amyloid beta levels or composite biomarker values, which have previously been expensive.

Claims

1. The amount of cholinesterase in blood isolated from the subject is 326 IU / L or less as the amount of cholinesterase in serum obtained from the blood; The amount of magnesium in the blood isolated from the subject is 2.2 mg / dL or more as calculated as the amount of magnesium in serum obtained from the blood; A method for using this as an indicator of mild cognitive impairment (MCI).

2. The method of claim 1 , wherein the subject is a mammal.

3. The method of claim 1 or 2, wherein the subject is a human aged 60 years or older.

4. The method according to any one of claims 1 to 3, wherein the subject is a male human and the amount of cholinesterase in the blood is 306 IU / L or less as the amount of cholinesterase in serum obtained from the blood, which is an indicator of the possibility of mild cognitive impairment (MCI) in the subject.

5. The method according to any one of claims 1 to 4, wherein at least one additional test is performed to facilitate the diagnosis of mild cognitive impairment (MCI).

6. The method according to any one of claims 1 to 3 and 5, wherein the subject is a female human, and the amount of magnesium in the blood, expressed as the amount of magnesium in serum obtained from the blood, is 2.24 mg / dL or more, which is an indicator of the possibility of mild cognitive impairment (MCI) in the subject.

7. The method according to claim 5, wherein the additional test is one or more selected from the group consisting of a Mini-cog test, a Mini-Mental State Examination (MMSE), an ADAS-cog test, and a clock drawing test, which are cognitive function tests.

8. The method according to any one of claims 1 to 7, which is for measuring or detecting the risk of the subject for developing mild cognitive impairment (MCI), for detecting mild cognitive impairment (MCI) in the subject, for selecting a subject having mild cognitive impairment (MCI), for measuring, evaluating or monitoring the response of the subject to an intake for preventing or treating mild cognitive impairment (MCI), or for measuring, evaluating or monitoring the progress of mild cognitive impairment (MCI) in the subject.

9. 9. A composition for use in the method of any one of claims 1 to 8, comprising a reactant for detecting the presence of said cholinesterase, a reagent for quantifying said cholinesterase, and a reagent for quantifying said magnesium.

10. The composition of claim 9 , wherein the reactant comprises a substrate for the cholinesterase.

11. 9. A kit for use in the method according to any one of claims 1 to 8, comprising reactants for detecting the presence of said cholinesterase, reagents for quantifying said cholinesterase, and reagents for quantifying said magnesium.

12. The kit of claim 11 , wherein the reactant comprises a substrate for the cholinesterase.

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