JP7680720B2 - Method for use as an indicator of a disease or condition associated with amyloid beta, and composition and kit for use in said method - Google Patents

Description

The present invention relates to a method for indexing amyloid-β-related diseases or conditions, as well as a composition and a kit for use in said method.

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 predicted 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.

Japan has a dementia prevalence rate of 2.33% of the total population, the highest rate among OECD member states (1.48%). Of all dementia cases 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.

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% over five years. In addition, the Japanese Society of Neurology's Dementia Disease Treatment Guidelines 2017 indicates that 16-41% of people with MCI will recover if they receive appropriate treatment. Therefore, if MCI can be detected and evaluated early and preventive measures can be taken, the number of dementia patients can be significantly reduced, so there is a strong desire to develop a method of early detection in healthy people who are at high risk of developing the disease.

Tests and diagnosis for dementia include 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 test).

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 and processes 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).

Research is also being conducted on early detection techniques for dementia. 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. In addition, it has 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).

Special table 2019-522193 publication International Publication No. 2012/090625 Special table 2018-515145 publication

Kieboom et al. Serum magnesium is associated with the risk of dementia. 2017, Neurology, 89(16), 1716-1722

However, tests to assess the risk of MCI, including blood tests, are generally expensive, and there is a problem that they cannot be performed cheaply and safely. Therefore, the present invention aims to provide a simple and inexpensive method for identifying individuals at high risk of developing amyloid-β-related diseases or conditions, such as MCI, from a group of healthy individuals, etc.

The inventors discovered that the amount of cholinesterase in the blood can be correlated with the amount of amyloid beta accumulated in the brain, and thus completed the present invention. More specifically, the present invention provides the following:

[1] A method for using the amount of cholinesterase in blood isolated from a subject 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, as 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 β in the subject.
[5] The method according to any one of the above [1] to [4], 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 indicator 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 β.
[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 β.
[8] The method according to the above-mentioned [7], wherein the amount of magnesium in the blood is 2.2 mg/dL or more, calculated as the amount of magnesium in serum obtained from the blood, is used as 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 indicator of the possibility of a disease or condition associated with amyloid beta in the subject.
[10] The method according to the above-mentioned [6], 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 tumors, 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 risk of the subject for developing a disease or condition associated with amyloid-β, for detecting the disease or condition associated with amyloid-β in the subject, for selecting a subject having a disease or condition associated with amyloid-β, 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-β, or for measuring, evaluating or monitoring the progress of the disease or condition associated with amyloid-β in the subject.

[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 according to [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 the above-mentioned [15], wherein the reactant comprises a substrate for the cholinesterase.
[17] The test kit according to [15] or [16] above, which is a kit for use in the method according to any one of claims 7 to 9, and further comprises a reagent for quantifying the magnesium.

The present invention provides a simple and inexpensive method for identifying individuals at high risk of developing amyloid-β-related diseases or conditions, such as MCI, from a group of healthy individuals, etc.

The following describes an embodiment of the present invention, but the present invention is not limited to this.

<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.

The inventors have found that the amount of cholinesterase in blood can be correlated with the amount of amyloid β accumulated in the brain or a value indicating the amount of amyloid β accumulated in the brain, for example, the Composite Biomarker value described below, and have found that the amount of cholinesterase in blood, which can be easily and inexpensively measured in a blood test, can be used as an indicator of a disease or condition associated with amyloid β.

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 around 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.

It is believed that a disease or condition associated with amyloid beta is highly likely to correlate with the amount of amyloid beta accumulated in the brain, and the accumulation of amyloid beta in the brain can be estimated, for example, by measuring the amount of amyloid beta in the blood. The amount of amyloid beta 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 diseases or conditions associated with amyloid beta, such as MCI, Alzheimer's dementia, and other dementias. A first aspect of the present invention may include correlating the amount of amyloid beta accumulated in the brain or the Composite Biomarker value with the amount (concentration) of cholinesterase in blood isolated from a subject.

The subject may be an animal such as a mammal, and may be selected according to the purpose without any particular limitation as long as the animal can develop a disease or condition associated with amyloid beta. 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 used, but humans are preferred. As humans, humans aged 60 years or older are preferred in terms of the high correlation, specifically positive correlation, between the amount of amyloid beta accumulated in the brain or a disease or condition associated with amyloid beta, and male humans are more preferred in terms of the high correlation, specifically positive correlation, between the amount of cholinesterase in the blood and the amount of amyloid beta accumulated in the brain or a disease or condition associated with amyloid beta, and male humans aged 60 years or older are even more preferred.

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.

The method of obtaining blood, plasma, and serum is not particularly limited, and may be in accordance with conventionally known methods, such as a method of obtaining (collecting) blood as a clinical test specimen, and a method of separating plasma and serum obtained from blood as a clinical test specimen. For example, plasma can be obtained by collecting blood in an EDTA tube or heparin tube, etc., and centrifuging the collected blood. Serum can also be obtained by collecting blood in a test tube, etc., and centrifuging the collected blood.

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 of the method 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.

Methods for measuring the amount of cholinesterase in blood, plasma or serum include, for example, methods used in general blood tests, specifically, the UV method, the JSCC standardized method (also called the 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, that 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 accompanying 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, and the second reagent containing pHBC are mixed and reacted with serum, etc., and the concentration of cholinesterase can be determined based on the rate of decrease in absorbance at 340 nm.

A blood cholinesterase level of 326 IU/L or less, as determined by serum cholinesterase levels obtained from the blood, can be used as an indicator of the possibility of a disease or condition associated with amyloid-β in the 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.

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

The above-mentioned values of 326 IU/L or less to 306 IU/L or less have traditionally been considered to be within the normal range, and even though there was a view that higher values within the range of 245 to 495 IU/L, which was the reference value for male humans, were more likely to correlate with the amount of amyloid-β accumulated in the brain, the inventors have newly discovered that these values may correlate with the amount of amyloid-β accumulated in the brain or a value indicating the amount of amyloid-β accumulated in the brain, for example, the Composite Biomarker value.

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

The additional test may include detecting at least one additional biomarker for a disease or condition associated with amyloid-β. An example of the additional biomarker is magnesium (Mg) in blood isolated from the subject. Specifically, the first aspect of the present invention may include using the amount of magnesium in blood isolated from the 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.

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.

The method for measuring the amount of magnesium in blood, plasma or serum is not particularly limited as long as it is a method that 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. Examples of the reagent include xylyl azo violet, isocitrate dehydrogenase (ICDH), etc.

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), the enzyme method, etc. These methods can be used easily and inexpensively 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).

In the enzymatic method, isocitrate dehydrogenase (ICDH) produces 2-oxoglutaric acid in the presence of magnesium using isocitrate as a substrate, and at the same time, oxidized nicotinamide adenine dinucleotide phosphate (NADP) is converted 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 rate of increase in absorbance is measured to determine the magnesium concentration in the sample. For example, enzyme reagent solution I containing D-isocitric acid monopotassium salt and ICDH, enzyme reagent solution II containing NADP, and serum or the like are mixed and reacted, and the magnesium concentration can be determined from the rate of increase in absorbance at 340 nm.

The above-mentioned 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 β 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, and particularly a human of both sexes aged 60 years or older.

In particular, when the subject is a female human, for example a female human aged 60 years or older, the amount of magnesium in the blood, expressed as the amount of magnesium in serum obtained from the blood, of 2.24 mg/dL or more can be used as an indicator of the possibility of a disease or condition associated with amyloid-β in the subject.

The at least one additional test to facilitate the diagnosis of a disease or condition associated with amyloid-β 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 may be selected appropriately depending on the cost, convenience, needs of the subject, etc., but are preferably performed in addition to or in combination with the additional test, as this may increase the accuracy of the possibility of a disease or condition associated with amyloid-β.

As the additional test, if the subject is a human, the Mini-cog test is preferable. The Mini-cog test is a test in which the subject is asked to memorize three words and is scored 0 to 3 points depending on the number of words they remember, and a "clock drawing" test is scored 0 or 2 points depending on whether they can draw a clock that shows the time 11:10. The fewer words a subject remembers in the "verbal memory" test, and the more mistakes they make in both the "verbal memory" and "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.

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-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. Typical 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 of the Alzheimer's type (Alzheimer's disease; AD) are preferred, with mild cognitive impairment (MCI) being preferable.

That is, as described above, the 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, and more specifically, the method is a method for using the amount of cholinesterase (ChE) as an indicator of whether 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 disease or condition.

The above-mentioned method of the first aspect of the present invention is suitable for, for example, 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-β, determining, judging or detecting (including early detection) a disease or condition associated with amyloid-β in a subject, selecting (or screening) a subject having a disease or condition associated with amyloid-β, measuring, evaluating or monitoring the subject's response to an ingested substance (e.g., food, supplement, medicine) for preventing or treating a disease or condition associated with amyloid-β (e.g., whether or not the ingested substance has the effect of preventing or treating the disease or condition), or measuring, evaluating or monitoring the progress of a disease or condition associated with amyloid-β in a subject (including after the start of treatment such as medication).

The method of the first aspect of the present invention achieves or supports these objectives by performing a blood test that can be performed in vitro, is non-invasive, safe, simple, and inexpensive. The method of the first aspect of the present invention is also suitable as a method for diagnosing diseases or conditions associated with amyloid β, a method for pre-diagnosing, or a method for supporting or assisting in such diagnoses.

<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.

<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.

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 indicator of a disease or condition associated with amyloid β as an additional test to facilitate diagnosis of the 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 test kit described as being usable for measuring the amount of magnesium in blood, plasma or serum in the method of the first aspect of the present invention.

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

1. Measurement of the amount of amyloid β The amount of trace amounts of amyloid β present in the blood of 474 healthy subjects (humans. Healthy subjects who may include patients with mild cognitive impairment (MCI) but have not been diagnosed with dementia) was analyzed as follows.
The subject's plasma 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 averaged value was calculated as the Composite Biomarker value. The Composite Biomarker values were −0.542 for men and −0.718 for women in their 40s, −0.316 for men and −0.504 for women in their 50s, −0.340 for men and −0.357 for women in their 60s, and −0.195 for men and −0.184 for women in their 70s.

2. Acquisition of biomarker values The blood cholinesterase concentration of each subject was measured by the JSCC standardized method. In addition, the blood magnesium concentration of each subject was measured by the enzymatic method.

3. Correlation analysis between Composite Biomarker value and biomarker value In order to search for a biomarker having a positive correlation with the Composite Biomarker value, the relationship between the Composite Biomarker value and blood test data of the above 474 subjects was investigated for each generation of 40s, 50s, 60s and above. The analysis was carried out by calculating the average value of the Composite Biomarker value for each generation, and testing whether there was a significant difference in the test value between the subject group below the average value and the subject group above the average value (t-test).

As a result of the analysis, when the Composite Biomarker value was divided into those below the average value and those above the average value, the correlation was not significant, but differences were observed in the average values of blood cholinesterase concentration and blood magnesium concentration between the subject group with a Composite Biomarker value of 0 or more and the subject group with a Composite Biomarker value of less than 0. The results are shown in Table 1. In Table 1, a smaller p-value (p-val) indicates a stronger significant difference, and a p-value of 0.05 or less indicates a particularly statistically significant difference.
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 increases in people with the E4 type of ApoE gene. Among subjects aged 60 or older, the proportion of subjects with the E4 type of ApoE was high among those with a Composite Biomarker value of 0 or more.

As can be seen from Table 1, serum cholinesterase (ChE) concentrations tend to be low in subjects with high Composite Biomarker values. Specifically, serum cholinesterase (ChE) concentrations were 326 IU/L or less in subjects with Composite Biomarker values of 0 or more (mixed males and females aged 60 years or older). When the subjects were analyzed separately by gender, a positive correlation was evident in men, with serum ChE concentrations 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 a higher Composite Biomarker value had significantly higher blood magnesium concentration. Specifically, serum magnesium concentration was 2.2 mg/dL or higher in subjects with a Composite Biomarker value of 0 or higher (mixed males and females aged 60 years or older), and when the subjects were analyzed separately by gender, the positive correlation was significant in women, being 2.24 mg/dL or higher.

4. Magnesium intake, Composite Biomarker value, and effect on blood magnesium level For 474 subjects, the components and amounts of each meal for an average day were recorded, and the dietary magnesium intake was calculated according to 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 value or blood magnesium concentration.

These results show that blood cholinesterase and magnesium concentrations can be indicators of amyloid-β accumulation. Specifically, in subjects aged 60 or older (healthy individuals), those with serum cholinesterase levels of 326 IU/L or less (mixed sex), specifically men with serum cholinesterase levels of 306 IU/L or less, can be assessed as being at high risk for developing MCI or dementia, and this assessment was also correlated with those with serum magnesium levels of 2.2 mg/dL or more (mixed sex), specifically women with serum magnesium levels of 2.24 mg/dL or more.

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.

As can be seen from a comparison between Tables 2 and 3, there was a tendency for subjects over 60 years old to make mistakes in both word memory and clock drawing. It is assumed that subjects who make mistakes in both are cases of clear cognitive decline. In addition, since 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 assumed 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, a correlation between the Composite Biomarker value (amyloid β amount) and cognitive function was found to some extent.
Therefore, it was found that by measuring blood cholinesterase concentrations, although there are gender differences, mainly in people aged 60 or older, and also by measuring blood magnesium concentrations if necessary, it may be possible to detect cognitive decline without measuring the amount of amyloid beta or Composite Biomarker values, which are conventionally expensive.

Claims (12)

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 of using this as an indicator of mild cognitive impairment (MCI) . The method of claim 1, wherein the subject is a mammal. The method according to claim 1 or 2, wherein the subject is a human aged 60 years or older. 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, as expressed as the amount of cholinesterase in serum obtained from the blood, is 306 IU/L or less , which is an indicator of the possibility of mild cognitive impairment (MCI) in the subject. 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) . 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. 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. The method according to any one of claims 1 to 7, which is for measuring or detecting the subject's risk of 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 subject's response 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. A composition for use in the method according to 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 . The composition of claim 9 , wherein the reactant comprises a substrate for the cholinesterase. A kit for use in the method according to any one of claims 1 to 8 , comprising a reactant for detecting the presence of the cholinesterase , a reagent for quantifying the cholinesterase , and a reagent for quantifying the magnesium . The kit of claim 11 , wherein the reactant comprises a substrate for the cholinesterase.