JP6478268B2 - Test method of cognitive dysfunction using acrolein metabolite in urine and creatinine as index - Google Patents

Description

  The present invention relates to 3-hydroxypropyl mercapturic acid in urine (hereinafter sometimes abbreviated as 3-HPMA), acrolein-added amino acid (hereinafter sometimes referred to as AC-Acro), and creatinine (hereinafter referred to as The present invention relates to a method for testing dementia and / or mild cognitive impairment, which is referred to as Cre). The present invention also relates to a test method for discriminating Alzheimer's disease dementia with mild cognitive impairment, using 3-HPMA, AC-Acro, and Cre in urine as indexes. More specifically, the present invention uses the ratio of 3-HPMA content to Cre content in urine and / or the ratio of AC-Acro content to Cre content in urine as an indicator. And / or a test method for mild cognitive impairment, and a test method for discriminating Alzheimer's disease from mild cognitive impairment.

  Dementia refers to a syndrome with cognitive dysfunction, in which cognitive function is reduced due to acquired organic brain impairment. Cognitive dysfunction refers to a function realized by information processing in the brain, such as impairment of memory, language, thinking, perception, judgment, etc. Symptoms such as memory impairment, aphasia, apraxia, analgesia, executive dysfunction, etc. Indicates

  Dementia is a neurodegenerative dementia such as Alzheimer type dementia (dementia), Lewy body dementia (dementia with Lewy bodies), and frontotemporal dementia, depending on the disease causing the disease. It is classified into treatable dementias such as multiple cerebral infarction type dementia and vascular dementia such as focal cerebral infarction type dementia and normal pressure hydrocephalus and hypothyroidism.

Alzheimer's disease is a neurodegenerative disease generally referred to as Alzheimer's disease (hereinafter sometimes abbreviated as AD) and is characterized by progressive impairment of cognitive function and behavior. AD is the most common type of dementia, especially in the elderly (non-patent documents 1 and 2). Therefore, there is currently a need for more convenient diagnostic tools, such as readily available biomarkers for the assessment of AD. Recently, the content of amyloid-β (hereinafter abbreviated as Aβ) in plasma has been identified as a promising marker. However, there is a problem that there is an inconsistent report with the result reported for Aβ. In some studies, the content of A [beta] ₄₀ and A [beta] ₄₂ in plasma has been reported that there was no significant difference between the AD subjects and controls (non-patent document 3-6). Meanwhile, in another study, the ratio of A [beta] ₄₂ with respect to A [beta] ₄₂ content or A [beta] ₄₀ in plasma (A [beta] ₄₂ / A [beta] ₄₀ levels) is reported to have significantly reduced in AD subjects (Non-Patent Document 7-10 ).

  Mild cognitive impairment (hereinafter sometimes abbreviated as MCI) refers to a condition in which cognitive function is lower than expected in the normal aging process but is not diagnosed as dementia. It is the pre-stage of dementia, but memory decline is the main symptom rather than cognitive decline. Although subjectively and objectively recognizing memory impairment, general cognitive function and daily living ability are almost maintained. From MCI, there is usually a period of 5 to 10 years, an average of 6 to 7 years, to progress to the extent that is diagnosed as dementia. It is said that 10 to 15% of MCI patients who visit medical institutions shift to dementia annually. Furthermore, it is known that the case with other cognitive disorders together has a much higher risk of progression to dementia than the case with only mild memory impairment. Specifically, the transition rate to dementia after 4 years was 24% in the case of memory impairment alone, and 77% in the case of language, attention, and visual space cognitive impairment.

3-HPMA is a metabolite of a complex of acrolein and glutathione (GSH). Acrolein (CH ₂ CHCHCHO) is known to be formed in the process of metabolism of amino acids and polyamines and in the process of peroxidation of membrane lipids in vivo, and is also mainly produced from polyamines, especially spermine, in tissue injury. (Non-patent documents 11, 12). Spermine is present in cells at concentrations ranging from several mM to several tens of mM, and most of it is present in polyamine-RNA complexes (Non-patent Document 13). When tissues are damaged, spermine is released from RNA and oxidatively degraded by spermine oxidase to generate acrolein and hydrogen peroxide (H ₂ O ₂ ) (Non-patent Document 12). Acrolein is highly reactive and reacts selectively with cysteine, lysine and histidine residues of proteins to inactivate proteins. Acrolein is also highly cytotoxic compared to reactive oxygen species (ROS) such as superoxide anion radical, hydrogen peroxide, and hydroxyl radical, which are considered to be the cause of cell damage (Non-patent Document 14). It is known that it is strong (non-patent documents 15, 16).

Acrolein has been reported to interact much faster with thiol groups (SH groups) than with amino groups (NH ₂ groups) (Non-Patent Document 16), such as GSH with SH groups It is known that the compound of the present invention is bound to a thiol compound of formula I and that its metabolism is performed by an enzyme such as aldehyde dehydrogenase. GSH is known to be the most abundant SH-containing substance in cells, and is present in cells at a concentration of several mM to several tens of mM (Non-patent Documents 17 and 18). That is, acrolein combines with GSH to form an acrolein-GSH conjugate, is metabolized by the enzyme, is converted to 3-HPMA, is detoxified, and is excreted as a urinary metabolite. Therefore, acrolein produced is mostly detoxified by binding to GSH. Decreased GSH levels reduce the metabolism of acrolein, which results in the interaction of acrolein with proteins and nucleic acids, leading to inactivation of these substances.

Recently, the increase in plasma levels of acrolein-modified protein-conjugated acrolein (PC-Acro) and the enzymes involved in the production of polyamine oxidase (spermine oxidase and acetylpolyamine oxidase), as opposed to stroke It is reported that it is a useful biomarker (nonpatent literature 19, patent documents 1-3). Also, the measurement of the acrolein in plasma, acrolein additional amino acids (hereinafter, referred to as ^{AC-Acro) N ε - (} 3- formyl-3,4-dehydro Ropi piperidino) lysine ^([N ε - (3- It is reported that the method can be carried out by measuring the content of (formyl-3,4-dehydropiperidino) lysin (hereinafter referred to as FDP-lysine)) (Patent Document 4).

  In addition, it has been reported that 3-HPMA, which is a major metabolite of acrolein-GSH conjugate, is decreased in the urine of stroke patients (Non-patent Document 20), and the result is that the substance detoxifying acrolein Suggest that 3-HPMA is reduced in stroke. Furthermore, it is disclosed that the level of 3-HPMA / Cre which corrected urine 3-HPMA content or urine 3-HPMA content by urine Cre content serves as a marker for judging stroke and cerebral infarction. (Patent Document 4).

Recently, it has been reported that both PC-Acro content and Aβ ₄₀ / Aβ ₄₂ levels in plasma are significantly higher in MCI and AD subjects as compared to control subjects (Non-patent Document 21). However, it was difficult to clearly distinguish between MCI subjects and AD subjects by these two biomarkers.

In addition, it has been reported that Aβ ₄₀ , Aβ ₄₀ / PC-Acro and Aβ ₄₂ / PC-A in cerebrospinal fluid (CSF) are significantly lower in AD subjects as compared to MCI subjects (Non-patent Document 22) ). However, collecting cerebrospinal fluid is relatively difficult, and the burden on the subject is large.

JP 2005-304476 A International Publication No. WO2008 / 142888 Brochure Japanese Patent Application Publication No. 2005-522669 International Publication No. 2012/173101 pamphlet

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  Biomarkers that can be easily used for the determination of dementia and mild cognitive impairment, which is the first stage, are required, but none have been put into practical use at present. As such biomarkers, it has been reported that Aβ levels in plasma can be used, but there are reports that are not compatible with the reported results. In addition, plasma needs to collect blood from a subject, and there is a problem of invasiveness to a patient.

  The object of the present invention is to find biomarkers for the determination of dementia and mild cognitive impairment which is the previous stage thereof, and to provide a novel test method for these diseases using the biomarkers.

  The present inventors focused on the ratio of 3-HPMA content to Cre content and the ratio of AC-Acro content to Cre content in the urine sample in order to solve the above-mentioned problems, and AD cases and Analysis of the content of 3-HPMA, AC-Acro, and Cre in urine samples of MCI cases was performed. As a result, in the group including both AD cases and MCI cases, 3-HPMA / Cre and AC-Acro / Cre in the urine sample were significantly reduced as compared with healthy persons. Furthermore, in the AD case group, the values of 3-HPMA / Cre and AC-Acro / Cre in the urine sample were significantly reduced as compared to the MCI case group.

  From these findings, by using 3-HPMA / Cre and / or AC-Acro / Cre in urine samples as an index, discrimination between dementia patients and non-dementia patients, and discrimination between AD subjects and MCI subjects And completed a test method for determining dementia and / or mild cognitive impairment, and a test method for discriminating between mild cognitive impairment and Alzheimer's disease.

That is, the present invention is as follows.
“1. A test method for dementia and / or mild cognitive impairment, using 3-HPMA / Cre in a urine sample obtained from a subject and / or AC-Acro / Cre in a urine sample as an index.
2.3. The method of testing for dementia and / or mild cognitive impairment according to the above 1, wherein the index is that HPMA / Cre and / or AC-Acro / Cre are lower than those of healthy individuals.
3. The test method according to the preceding paragraph 2, wherein using an index lower than the healthy person as an index is setting an index lower than a preset cutoff value as an index.
4. The test method according to any one of the above 1 to 3, wherein the dementia is Alzheimer's disease.
5. The test method according to any one of items 1 to 4, further comprising, as an index, that Aβ ₄₀ / Aβ ₄₂ in a blood sample obtained from the subject is higher as a healthy person.
6. A test method for discriminating between mild cognitive impairment and Alzheimer's disease, using 3-HPMA / Cre in a urine sample obtained from a subject and / or AC-Acro / Cre in a urine sample as an index.
7.3. The test method according to the above 6, wherein the subject is determined to have Alzheimer's disease when HPMA / Cre and / or AC-Acro / Cre is low compared to a patient with mild cognitive impairment.
8. The test method of the preceding clause 7 that what is low compared with the said patients with mild cognitive impairment is low compared with the preset cutoff value.
9.3. The test method according to the above 6, wherein the subject is determined to have mild cognitive impairment when HPMA / Cre and / or AC-Acro / Cre are high as compared to a patient with Alzheimer's disease.
10. The test method of the preceding clause 9, wherein being high compared with a patient with Alzheimer type dementia is high compared with a preset cutoff value.
11. A kit for testing dementia and / or mild cognitive impairment, comprising a reagent for measuring 3-HPMA content in a urine sample.
12. The test kit according to the above 11, further comprising a reagent for measuring the AC-Acro content in the urine sample.
13. A test kit for determining Alzheimer's disease dementia with mild cognitive impairment, which comprises a reagent for measuring 3-HPMA content in a urine sample.
14. The test kit according to the above 13, further comprising a reagent for measuring the AC-Acro content in the urine sample. "

  According to the present invention, determination of dementia and / or mild cognitive impairment can be assisted, and discrimination between mild cognitive impairment and Alzheimer's disease can be assisted. Since the test method according to the present invention is a test method using urine as a sample, it has an advantage of being able to be carried out easily and without burdening the subject.

3-HPMA content in urine samples, 3-HPMA / Cre level in a group including both MCI subjects and AD subjects (hereinafter referred to as MCI + AD group) and a control subject group (hereinafter referred to as control group) FIG. 16 shows the results of comparing Cre content, AC-Acro content, AC-Acro / Cre level, and age (Age). Each value is shown as median (horizontal line) ± quartile deviation. The "p" values indicate significance values. Example 1 In the age-matched MCI + AD group and control group, 3-HPMA content in urine sample, 3-HPMA / Cre level, Cre content, AC-Acro content, AC-Acro / Cre level, and age (Age (Age ) Is a diagram showing the results of comparing. Each value is shown as median (horizontal line) ± quartile deviation. The "p" values indicate significance values. Example 1 In the group consisting of MCI subjects only (hereinafter referred to as MCI group) and the group consisting only of AD subjects (hereinafter referred to as AD group), 3-HPMA content in urine sample, 3-HPMA / Cre level, Cre content It is a figure which shows the result of having compared amount, AC-Acro content, AC-Acro / Cre level, and age (Age). Each value is shown as median (horizontal line) ± quartile deviation. The "p" values indicate significance values. Example 1 It is a figure which shows the result of having analyzed the correlation of 3-HPMA content and urine AC-Acro content in the urine sample of MCI + AD group. In the figure, “●” and “Δ” indicate the samples of the MCI subject and the AD subject, respectively. "R _s" indicates the Spearman's rank correlation coefficient. Example 1 It is a figure which shows the result of having analyzed the correlation of the 3-HPMA / Cre level, AC-Acro / Cre level, and Cre content in the urine sample of MCI + AD group, and the result of the mini mental state examination (MMSE). . In the figure, “●” and “Δ” indicate the samples of the MCI subject and the AD subject, respectively. "R _s" indicates the Spearman's rank correlation coefficient. Example 1 Analyze the correlation between 3-HPMA / Cre level, AC-Acro / Cre level, and Cre content in urine samples of MCI + AD group and item sum score (CDR sum of boxes) of clinical dementia rating scale It is a figure which shows the result. In the figure, “●” and “Δ” indicate the samples of the MCI subject and the AD subject, respectively. "R _s" indicates the Spearman's rank correlation coefficient. Example 1 The figure which shows the result of having analyzed the correlation of the 3-HPMA / Cre level, AC-Acro / Cre level, and Cre content in the urine sample of MCI + AD group, and the result of Z-score (Z-score) analysis. It is. In the figure, “●” and “Δ” indicate the samples of the MCI subject and the AD subject, respectively. "R _s" indicates the Spearman's rank correlation coefficient. Example 1 It is a figure which shows the result of having analyzed the correlation of 3-HPMA / Cre level in urine sample of MCI + AD group, AC-Acro / Cre level, and Cre content, and A ( _{beta) 40/42} level in plasma. In the figure, “●” and “Δ” indicate the samples of the MCI subject and the AD subject, respectively. "R _s" indicates the Spearman's rank correlation coefficient. Example 1

  The present invention relates to a method for testing dementia and / or mild cognitive impairment, using acrolein metabolite and Cre in a urine sample of a subject as an index. More specifically, the present invention relates to a method for testing dementia and / or mild cognitive impairment, using 3-HPMA, AC-Acro, and Cre in urine samples of subjects as indicators.

  The present inventors compared 3-HPMA / Cre levels and AC-Acro / Cre levels of urine samples of patients with dementia and / or mild cognitive impairment with those of healthy people as shown in the examples described below. Revealed that it was low. In addition, it was revealed that 3-HPMA / Cre levels and AC-Acro / Cre levels of urine samples of patients with dementia were lower compared to mild cognitive impairment.

  That is, this test method measures the 3-HPMA content and / or the AC-Acro content in the urine sample of the subject and the Cre content, and corrects the measured 3-HPMA content with the Cre content. Dementia and / or mild cognitive impairment based on the 3-HPMA / Cre level obtained and / or the AC-Acro / Cre level obtained by correcting the measured AC-Acro content with the Cre content It may be an inspection method for the determination of This test method uses, as an index, the level of 3-HPMA / Cre in the urine sample of the subject and / or the level of AC-Acro / Cre in the urine sample as compared to those of healthy subjects.

  This test method also measures the 3-HPMA content and / or the AC-Acro content in the urine sample of the subject, and the Cre content, and corrects the measured 3-HPMA content with the Cre content. Mild cognitive impairment and Alzheimer's disease type dementia using the obtained 3-HPMA / Cre level and / or the AC-Acro / Cre level obtained by correcting the measured AC-Acro content with Cre content as an index Test method to determine the In this test method, when the 3-HPMA / Cre level in the urine sample of the subject and / or the AC-Acro / Cre level in the urine sample is low compared to that of patients with mild cognitive impairment, the subject has Alzheimer's disease. It can be determined that In addition, when the 3-HPMA / Cre level in the urine sample of the subject and / or the AC-Acro / Cre level in the urine sample are high compared to those of patients with Alzheimer's disease and low compared to those of healthy people The subject can be determined to have mild cognitive impairment.

(subject)
The "subject" of the present invention refers to a mammal including human. Mammals are intended for any animals classified as mammals, including humans, livestock, non-human primates, athletic animals (horse racing horses), or pet animals such as dogs, horses, cats, cows etc. Is preferably human. Particularly preferred is a human suspected of suffering from mild cognitive impairment or dementia, or a human who needs to determine whether to be suffering from mild cognitive impairment or dementia.

(Healthy person)
In the present specification, a healthy person means a subject without a history of cognitive impairment.

(Dementia and mild cognitive impairment)
"Dementia" refers to a syndrome with cognitive dysfunction that is impaired in cognitive function due to acquired organic brain disorder, and Alzheimer's disease, Lewy body dementia, and frontal head due to its underlying disease Treatable cognition due to neurodegenerative dementia such as temporal dementia, cerebrovascular dementia such as multiple cerebral infarction type dementia and focal cerebral infarction type dementia, and normal pressure hydrocephalus and hypothyroidism Classified as Cognitive dysfunction refers to impairment of functions realized by information processing in the brain, such as memory, language, thinking, perception, and judgment.

  "Mild cognitive impairment" refers to a condition in which cognitive function is lower than expected during normal aging, but is not diagnosed as dementia. In the pre-stage of dementia, memory decline is the main symptom.

  The test method according to the present invention can be used as a method for assisting determination of dementia and / or mild cognitive impairment, preferably dementia, more preferably Alzheimer's disease.

  Further, the test method according to the present invention can be preferably used as a method for assisting the discrimination between Alzheimer's disease and mild cognitive impairment.

(Urine sample)
A urine sample refers to a sample derived from urine, including untreated urine, drug-supplemented urine, and purified urine. Collection and processing of urine from a subject can be carried out by known methods. Urine specimens can, if desired, be stored at -80 ° C until used for testing.

(Indicator of inspection method according to the present invention)
In the present invention, the “index” is the 3-HPMA / Cre level calculated by correcting the 3-HPMA content in the urine sample of the subject with the Cre content in the urine sample, and / or in the urine sample The AC-Acro / Cre level calculated by correcting the AC-Acro content with the Cre content in the urine sample is used. Preferably, 3-HPMA / Cre levels are used as an "index".

  In the present invention, 3-HPMA / Cre levels and / or AC-Acro / Cre levels in the urine sample of the subject are compared with 3-HPMA / Cre levels and / or AC-Acro / Cre levels in the urine sample of healthy subjects When low, preferably statistically significantly low, it can be determined that the subject suffers from or may be suffering from dementia and / or mild cognitive impairment.

  In the present invention, 3-HPMA / Cre level and / or AC-Acro / Cre level in urine sample of subject is 3-HPMA / Cre level and / or AC-Acro / Cre level in urine sample of patients with mild cognitive impairment When the level is low compared to the level, preferably statistically significant, the subject is not mild cognitive impairment but dementia, eg Alzheimer's disease, or possible dementia, eg Alzheimer's disease It can be determined that there is a sex. Conversely, 3-HPMA / Cre levels and / or AC-Acro / Cre levels in the urine sample of the subject, 3-HPMA / Cre levels and / or AC-Acro / Cre levels in the urine sample of Alzheimer's disease patients The subject is mild cognitive impairment when it is high compared to the level, preferably statistically significant and low compared to the healthy subject, preferably statistically significant, or mild cognitive impairment It can be determined that there is a possibility of

  When performing statistical processing, any known statistical processing method generally used for two-group comparison can be used for the execution. Specifically, Wilcoxon rank sum test can be preferably exemplified.

  A subject who is judged to be suffering from or possibly suffering from dementia and / or mild cognitive impairment by the examination method according to the present invention has a head tomographic image taken by MRI, a mini mental state examination (MMSE), and further detailed examination by receiving conventional tests for dementia such as Clinical Dementia Rating Scale Sum of Boxes Scores (CDRsob; Non-patent Literature 23). You can get a rating.

  The test method according to the present invention is preferably performed repeatedly with a fixed period, thereby detecting the change in the 3-HPMA / Cre level and / or the AC-Acro / Cre level in the urine sample during the period. And, thus, the progression of dementia and / or mild cognitive impairment can be assessed early. For example, 3-HPMA / Cre levels and / or AC-Acro / Cre levels in urine samples of subjects diagnosed with mild cognitive impairment are measured at regular intervals during the follow-up period, The subject progressed from mild cognitive impairment to dementia when the value obtained by the measurement decreased compared to the previously measured 3-HPMA / Cre level and / or the AC-Acro / Cre level. Or it can be determined that there is a possibility of progress.

  In the test method according to the present invention, preferably, a cutoff value can be set in advance, and dementia and / or mild cognitive impairment can be determined based on the cutoff value.

  Setting a 3-HPMA / Cre level cutoff value used in testing methods to determine dementia and / or mild cognitive impairment compares subjects with no history of dementia and / or mild cognitive impairment It can be carried out by calculating from the average value of 3-HPMA / Cre levels in the urine sample to be compared. Similarly, the setting of the cutoff value of AC-Acro / Cre level is intended for comparison of subjects without history of dementia and / or mild cognitive impairment, and the AC-Acro / Cre level in the urine sample of the comparison subject is compared It can be implemented by calculating from the average value of Usually, if it is 90% or less, preferably 80% or less, more preferably 70% or less, still more preferably 60% or less, most preferably 50% or less of the standard deviation of the preset cutoff value, dementia and It can be determined that there is a possibility of mild cognitive impairment.

  Setting a cutoff value for 3-HPMA / Cre levels used in testing methods to distinguish between dementia, eg, Alzheimer's disease and mild cognitive impairment, compared subjects who were diagnosed with mild cognitive impairment This can be carried out by calculating from the average value of 3-HPMA / Cre levels in the urine sample to be compared. Similarly, the cutoff value of the AC-Acro / Cre level is calculated from the average value of the AC-Acro / Cre levels in the urine sample of the comparison subject, comparing the subjects diagnosed with mild cognitive impairment. Can be implemented by Usually, it is mild when it is 90% or less, preferably 80% or less, more preferably 70% or less, still more preferably 60% or less, most preferably 50% or less of the standard deviation of the cutoff value set in this way. It can be determined that there is a possibility of dementia, for example, Alzheimer's disease, not cognitive impairment.

  Alternatively, the setting of the 3-HPMA / Cre level cut-off value used in the test method for discriminating between mild cognitive impairment and Alzheimer's disease is used to compare subjects diagnosed with Alzheimer's disease, The cutoff value can be set by calculating from the average value of 3-HPMA / Cre levels in the urine sample to be compared. Similarly, the cutoff value of the AC-Acro / Cre level is set based on the average value of the AC-Acro / Cre levels in the urine sample of the comparison subject, comparing the subjects diagnosed with Alzheimer type dementia. It can carry out by doing. Usually, in the case of 120% or more, preferably 130% or more, more preferably 140% or more, and still more preferably 150% or more of the standard deviation of the cutoff value set in this way, dementia, for example, Alzheimer's disease, etc. Rather, it can be determined that there is a possibility of mild cognitive impairment.

  Another way to set the cutoff value is to create a Receiver Operating Characteristic (ROC) curve based on the 3-HPMA / Cre level or the AC-Acro / Cre level in the urine sample, and the ROC. A method of setting a cutoff value from a curve can be preferably mentioned. The method of creating an ROC curve and the method of setting the cutoff value based on the ROC curve are well known, and when setting the cutoff value from the ROC curve, desired according to the purpose of clinical use such as primary screening The cutoff value can be set in accordance with conditions such as detection sensitivity and specificity.

  For example, a cutoff value for determining dementia and / or mild cognitive impairment using an ROC curve generated based on 3-HPMA / Cre levels (FIG. 1A) in urine samples measured in Examples described later If the desired sensitivity is set to 50%, preferably 60%, more preferably 70%, still more preferably 80%, even more preferably 90%, the cutoff values are 1.54 μmol / g and 1.75 μmol, respectively. It can be set to / g, 2.08 μmol / g, 2.81 μmol / g, 2.99 μmol / g. Desired sensitivity when setting cutoff values to determine dementia and / or mild cognitive impairment using ROC curves generated based on AC-Acro / Cre levels (Figure 1A) in urine samples Is 50%, preferably 60%, more preferably 70%, still more preferably 80%, still more preferably 90%, and the cutoff values are 66.0 μmol / g, 71.4 μmol / g, 92.4 μmol / g, respectively. It can be set to 132.1 μmol / g and 182.2 μmol / g. If the 3-HPMA / Cre level or the AC-Acro / Cre level in the urine sample of the subject is below the cut-off value set in this way, the subject may have dementia and / or mild cognitive impairment It can be determined that Conversely, if the 3-HPMA / Cre level or the AC-Acro / Cre level in the urine sample of the subject is above the cut-off value set in this way, the subject has dementia and / or mild cognitive impairment It can be determined that there is no possibility. The 3-HPMA / Cre level cut-off value and the AC-Acro / Cre level cut-off value can be used alone or in combination.

  In addition, for example, a cut for determining mild cognitive impairment and Alzheimer's disease using ROC curves created based on 3-HPMA / Cre levels (FIG. 2A) in urine samples measured in the examples described later. When setting the off value, when the desired sensitivity is 50%, preferably 60%, more preferably 70%, still more preferably 80%, still more preferably 90%, the cutoff value is 1.29 μmol / g, respectively. It can be set to 1.51 μmol / g, 1.71 μmol / g, 2.31 μmol / g, 2.87 μmol / g. Desired sensitivity when setting cutoff values to determine mild cognitive impairment and Alzheimer's disease using ROC curves generated based on AC-Acro / Cre levels in urine samples (Figure 2A) Is 50%, preferably 60%, more preferably 70%, still more preferably 80%, even more preferably 90%, and the cutoff values are 53.7 μmol / g, 66.0 μmol / g, 69.7 μmol / g, respectively. It can be set to 83.3 μmol / g and 98.8 μmol / g. If the 3-HPMA / Cre level or the AC-Acro / Cre level in the urine sample of the subject is below the cut-off value set in this way, the subject has the possibility of dementia, eg Alzheimer's disease It can be determined that Conversely, the 3-HPMA / Cre level or the AC-Acro / Cre level in the subject's urine sample is above the cut-off value set in this way, and the above-mentioned dementia and / or mild cognitive impairment is judged If the subject is below the cut-off value, the subject can be determined to have a possibility of mild cognitive impairment. The 3-HPMA / Cre level cut-off value and the AC-Acro / Cre level cut-off value can be used alone or in combination.

  In addition, as a method of setting another cutoff value, based on the 3-HPMA / Cre level or AC-Acro / Cre level in the urine sample, a box and whisker plot, which is a statistical graph also called a box plot, is created. , 1st quartile (25% value), 2nd quartile (median; 50% value), and 3rd quartile (75% value) any 1 value is cut off value The method to make can be mentioned. The method of creating box and whisker plots and the method of setting cutoff values based on the first quartile, the second quartile, and the third quartile are well known, and their purpose such as primary screening and scrutiny The cutoff value can be set according to the desired conditions such as detection sensitivity and specificity depending on the purpose of clinical use such as purpose.

  For example, a cut-off to determine dementia and / or mild cognitive impairment using a box and whisker plot created on the basis of 3-HPMA / Cre levels (FIG. 1A) in urine samples measured in Examples described later When setting a value, the third quartile (75% value), preferably the second quartile (median; 50% value), more preferably the first quartile (25% value) The cutoff values can be set to the corresponding values of 2.50 μmol / g, 1.54 μmol / g and 0.78 μmol / g, respectively. When setting a cut-off value to determine dementia and / or mild cognitive impairment using a box and whisker plot created based on AC-Acro / Cre levels (Fig. 1A) in urine samples Quartile (75% value), preferably the second quartile (median; 50% value), more preferably the value corresponding to the first quartile (25% value), 103.9 μmol / l The cutoff values can be set at g, 66.3 μmol / g, and 43.6 μmol / g. If the 3-HPMA / Cre level or the AC-Acro / Cre level in the urine sample of the subject is below the cut-off value set in this way, the subject may have dementia and / or mild cognitive impairment It can be determined that Conversely, if the 3-HPMA / Cre level or the AC-Acro / Cre level in the urine sample of the subject is above the cut-off value set in this way, the subject has dementia and / or mild cognitive impairment It can be determined that there is no possibility. The 3-HPMA / Cre level cut-off value and the AC-Acro / Cre level cut-off value can be used alone or in combination.

  Also, for example, for determining mild cognitive impairment and Alzheimer's disease using a box and whisker plot created based on 3-HPMA / Cre levels (FIG. 2A) in urine samples measured in the examples described later. When setting the cutoff value, the third quartile (75% value), preferably the second quartile (median; 50% value), more preferably the first quartile (25% value) The cut-off values can be set to values corresponding respectively to 2.05 μmol / g, 1.29 μmol / g and 0.68 μmol / g. When setting a cut-off value to determine dementia and / or mild cognitive impairment using a box and whisker plot created based on AC-Acro / Cre levels (Fig. 1A) in urine samples Quartile (75% value), preferably the second quartile (median; 50% value), more preferably the value corresponding to the first quartile (25% value), 72.5 μmol / l The cutoff values can be set to g, 55.9 μmol / g, and 42.4 μmol / g. If the 3-HPMA / Cre level or the AC-Acro / Cre level in the urine sample of the subject is below the cut-off value set in this way, the subject has the possibility of dementia, eg Alzheimer's disease It can be determined that Conversely, the 3-HPMA / Cre level or the AC-Acro / Cre level in the subject's urine sample is above the cut-off value set in this way, and the above-mentioned dementia and / or mild cognitive impairment is judged If the subject is below the cut-off value, the subject can be determined to have a possibility of mild cognitive impairment. The 3-HPMA / Cre level cut-off value and the AC-Acro / Cre level cut-off value can be used alone or in combination.

  The cutoff value of the 3-HPMA / Cre level or the AC-Acro / Cre level in the urine sample of the present invention is not limited to the above-mentioned cutoff value, and can be appropriately changed. That is, methods for determining 3-HPMA, Cre, and AC-Acro / Cre in urine samples, methods for comparing 3-HPMA / Cre levels in urine samples and AC-Acro / Cre levels with subjects, clinical use The cutoff value can be set according to the desired conditions such as detection sensitivity and specificity depending on the purpose.

(Method of measuring Cre in urine sample)
As a method of measuring Cre in a urine sample, a method known per se can be used. Alternatively, it can be carried out using a commercially available creatinine assay kit (Cayman Chemical Co., USA) according to the attached protocol.

(Method of measuring 3-HPMA in urine sample)
As a method for measuring 3-HPMA in a urine sample, a method known per se can be used (Non-patent Document 24). For example, 3-HPMA content in a urine sample can be measured using high performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS / MS) known per se. Preferably, it can be measured by enzyme-linked immunosorbent assay (ELISA) using an antibody specific to 3-HPMA, Western blotting analysis, immunoprecipitation method or the like. The antibody against 3-HPMA used in the measurement may be a monoclonal antibody or a polyclonal antibody. Polyclonal antibodies against 3-HPMA can be obtained from the blood of rabbits, for example, by immunizing rabbits with 3-HPMA using a conventional method for producing peptide antibodies. The fact that the antibody has been produced can be confirmed by collecting blood from the rabbit to which the peptide has been administered and measuring the antibody titer to conduct an assay to determine whether the antibody has reached a sufficient titer.

(Measurement method of AC-Acro in urine sample)
The content of AC-Acro in urine samples, that is, FDP-lysine can be measured according to the attached manual using, for example, ACR-LYSINE ADDUCT ELISA SYSTEM (Nippon Yushi Co., Ltd.).

(Indicators that can be used in addition to 3-HPMA / Cre levels and / or AC-Acro / Cre levels)
In addition to using the 3-HPMA / Cre level and / or the AC-Acro / Cre level in the urine sample as an indicator in the test method according to the present invention, a polyamine metabolite in the blood sample, preferably plasma, is further used. Content and Aβ ₄₀ / Aβ ₄₂ levels can be used as an indicator of the determination of dementia and / or mild cognitive impairment. The more types of biomarkers to be used as indicators, the higher the accuracy of the determination.

  Although acrolein is illustrated as a suitable polyamine metabolite used as a parameter | index in this invention, it is not limited to it. PC-Acro is more preferably used as an indicator, because acrolein is highly reactive and may react with proteins and be present in the form of PC-Acro in the blood.

The PC-Acro content and Aβ ₄₀ / Aβ ₄₂ levels in plasma are higher in subjects with mild cognitive impairment and Alzheimer's disease than in healthy individuals, thereby measuring PC-Acro and / or Aβ ₄₀ / Aβ ₄₂ levels It has been reported to be a useful biochemical marker for subjects with mild cognitive impairment and Alzheimer's disease (Non-patent Document 21). The polyamine is a linear aliphatic hydrocarbon having two or more primary amino groups, and as a biogenic polyamine, putrescine, cadaverine, spermidine, spermine, 1,3-diaminopropane, carzine, homospermidine, 3-amino Propyl cadaverine, norspermine, thermospermine, cardopentamine and the like are known. Preferred polyamines as indicators in the present invention are putrescine, spermidine, spermine. Polyamines are metabolized by oxidation, acetylation, transamination, carbamoylation. As enzymes involved in the oxidation of polyamines, polyamine oxidases (acetylpolyamine oxidase (AcPAO) and spermine oxidase (SMO)) can be preferably mentioned. Polyamine oxidase means an enzyme that oxidizes using diamine or polyamine as a substrate to generate hydrogen peroxide. Polyamines undergo oxidative deamination with polyamine oxidases, resulting in the formation of hydrogen peroxide and aldehydes such as acrolein.

In a test method for the determination of dementia and / or mild cognitive impairment, the acrolein content and Aβ ₄₀ / Aβ ₄₂ level in a blood sample, preferably plasma, can be preferably used as an auxiliary biomarker. Dementia and / or mild cognitive impairment if the acrolein content and Aβ ₄₀ / Aβ ₄₂ levels in the blood sample are high compared to those of subjects without a history of dementia and / or mild cognitive impairment, Alternatively, it can be determined that there is a possibility of dementia and / or mild cognitive impairment.

(Method of measuring acrolein in blood sample)
The acrolein content in blood samples, in particular in plasma, is identified by any method known to the person skilled in the art, for example by measuring the content of the acrolein-added amino acid FDP-lysine (N-formylpiperidino-lysine). can do. The FDP-lysine content can be measured, for example, using ACR-LYSINE ADDUCT ELISA SYSTEM (Nippon Yushi Co., Ltd.) according to the attached manual. The acrolein content can also be measured in the form of derivatives other than FDP-lysine. It is also possible to directly measure the acrolein content, and such a method is described, for example, in the report of Alarcon et al. (Non-patent Document 25). Specifically, 50 μL / well of plasma from a subject and a standard solution are dispensed to an antigen-immobilized plate, and the same amount of primary reaction antibody solution is further added. After standing for 30 minutes at room temperature, remove the solution, wash with a washing solution, and dispense 100 μL / well of the secondary reaction antibody solution. After standing for 1 hour at room temperature, wash with a washing solution, add 100 μl / well of coloring solution and allow to develop color by standing for 15 minutes at room temperature, dispense 50 μl / well of reaction stop solution, and use plate reader Measure the absorbance at 450 nm. The amount of acrolein in plasma is displayed as FDP-lysine content (nmol / mL plasma) per mL of plasma.

(Method of measuring Aβ ₄₀ / Aβ ₄₂ level in blood sample)
Determination of Aβ ₄₀ and Aβ ₄₂ in blood samples such as plasma and calculation of Aβ ₄₀ / Aβ ₄₂ levels can be carried out by known methods described in the previous report (Non-patent Documents 7-10, 21). Also, quantification of A [beta] ₄₀ and A [beta] ₄₂ in the blood sample can be performed using commercially available A [beta] ₄₀ assay kit and A [beta] ₄₂ assay kit. As a commercially available kit for measurement, human β-amyloid (1-40) ELISA kit high sensitive II (Wako Chemical Co.) and human β-amyloid (1-42) ELISA kit high sensitive (Wako Chemical Co.) can be mentioned. .

(Method of screening for therapeutic or prophylactic agent for dementia and / or mild cognitive impairment)
A candidate compound which may be effective for the treatment of dementia and / or mild cognitive impairment is administered to an experimental animal, and the compound is used in the experimental animal in 3-HPMA / Cre level and / or AC-Acro / Cre level Can be provided to provide a method for screening (searching for) a new drug that is effective for the treatment / prevention of dementia and / or mild cognitive impairment.

(Determination kit for dementia and / or mild cognitive impairment)
The present invention provides a test kit for determining dementia and / or mild cognitive impairment, and a test kit for discrimination between dementia, for example, Alzheimer's disease and mild cognitive impairment. The kit contains reagents for measuring 3-HPMA content and / or AC-Acro content in urine samples. Furthermore, as needed, reagents for measuring the content of polyamine metabolites, preferably acrolein, more preferably PC-Acro, in blood samples and urine samples, and the contents of Aβ ₄₀ and Aβ ₄₂ are measured Or both may be included. Furthermore, if necessary, any measuring instrument / device, standard solution, buffer solution and the like known to those skilled in the art can be contained.

EXAMPLES Hereinafter, the present invention will be described in detail by way of examples, but the scope of the present invention is not limited by these examples.
The following examples were conducted in line with the Helsinki Declaration after obtaining informed consent from each participant or their related persons.

(Subject and collection of urine and blood)
In the following example, elderly volunteers were recruited, and 74 control subjects, 22 MCI subjects, and 32 AD subjects were collected. The control subject is a healthy volunteer and lives independently without a clear medical history of MCI and dementia. Diagnosis of MCI and AD subjects was performed as described in previous reports (Non-patent Document 26). Specifically, the diagnosis of AD is based on criteria from the National Institute of Neurological Disorders and Stroke Institute and the Association of Alzheimer's Disease and Related Disorders (NINCDS-ADRDA) (Non-patent Document 27), and the diagnosis of MCI is based on criteria described in the article. It carried out according to (nonpatent literature 28). Plasma PC-Acro has already been reported as a diagnostic marker for stroke and chronic kidney disease (CKD) (Non-patent Documents 12, 13, 28). Thus, all subjects with systemic disease such as stroke, CKD, cardiac dysfunction and other arteriosclerosis have blood and urine tests, cerebral magnetic resonance imaging (cerebral MRI), magnetic resonance angiography, carotid artery Based on the results of carotid ultrasonography and medical history, it was completely excluded from the study. Smokers were excluded from this study due to high urinary 3-HPMA / Cre levels (Non-patent Document 20).

  Evaluation of the item total score (CDRsob) of the Mini Mental State Examination (MMSE) and the Clinical Dementia Rating Scale was performed at the subject's visit. All MCI and AD subjects underwent clinical follow-up over two to three years. Subjects received anticholinesterase drugs as needed.

  Collection of urine samples from the subjects was conducted according to a procedure approved by the Ethics Committee of Chiba University and Higashi Matsudo Hospital. Collected urine was stored at -80 ° C until used for testing.

  Blood was collected from the subject according to a conventional method using heparin. Plasma was obtained by centrifugation of blood containing 3 U / ml heparin at 1500 g for 10 minutes at 4 ° C. The obtained plasma was stored at -80.degree. C. until used for the test.

(Method)
The details of the measurement method of head tomographic image analysis, 3-HPMA, AC-Acro, Cre, Aβ ₄₀ and Aβ ₄₂ and the statistical analysis method are as follows.

(Head image analysis by MRI
All subjects underwent MRI examination. MRI was performed for T1, T2 and fluid decay inversion recovery (FLAIR) sequences using a 1.5 Tesla MR scanner (Toshiba Visart 1.5 T MRI). Three-dimensional volume collection of T1-weighted field echo (FE) sequences has produced a continuous series of thin signal slices. White matter hyperintensities (white matter hyperintensity; WMH) are based on T2 and FLAIR sequences, and periventricular hyperintensity (PVH) and deep cortical white matter hyperintensities (deep and perv) according to Shinohara et al. The evaluation (grade 1 to 4) in both of subcortical WMH; DSWMH) was evaluated with reference to it.

  The degree of gray matter atrophy in the whole brain was evaluated by voxel-based morphometric MRI (VBM-MRI) (Non-patent Document 31). The degree of the region of interest (VOI), which is the bilateral medial temporal lobe including the hippocampal para-rotation region, was determined using the Voxel-based Specific Regional Analysis System for Voxel-based Alzheimer's disease developed by Hirata et al. It evaluated by VSRAD software (Eisai, Tokyo, Japan) (nonpatent literature 32) which is Analysis System for Alzheimer's Disease (VSRAD). For each gray matter image of the subject, a voxel-by-voxel Z-score analysis after voxel normalization to global mean intensities was used to generate a normal database of the same age. A comparison was made with each gray matter image: Z-score = (mean of control-individual volume) / (standard deviation of control) (32). These Z-score maps were displayed superimposed on the tomographic section. VSRAD can automatically analyze 3D T1-weighted MRI data, perform serialization, anatomic standardization, and correction using SPM2 without Matlab programs, and Z-score analysis of VOI Bring Thus, the severity of VOI atrophy in MCI and AD was assessed as a Z-score using VSRAD.

(Measurement of 3-HPMA, AC-Acro, and Cre in urine)
The measurement of 3-HPMA was performed by LC-MS / MS according to the method of Eckert et al. 2 ml of urine sample, 2 ml of ammonium formate buffer (50 mM, pH 2.5), 0.04 ml of formic acid, and 0.02 ml of 10 mg / ml N-acetyl-d ₃ -S- (3 as internal standard) - mixed with hydroxypropyl) cysteine (d ₃ -3-HPMA) dicyclohexylammonium salt (Tronto Chemicals). After centrifugation for 5 minutes at 2000 g, the supernatant is applied to an SPE column (ISOLUTE ENV ⁺ , 100 mg, 3 ml) previously equilibrated with 6 ml of methanol and 6 ml of formic acid (pH 2.5). I let it pass. The cartridge after passage was washed as described in the previous report (Non-patent Document 24), and completely dried by connecting the SPE column to a suction pump. The analyte is eluted with 2.5 ml of formic acid made to 2% (v / v) with methanol, dried, and 5 ml of solvent A (acetonitrile / water (88/12, v / v)). It was dissolved in ammonium acetate, pH 6.5). The extraction rate of 3-HPMA was determined to be 68% under the conditions of this experiment. After centrifugation for 10 minutes at 2000 g, 0.01 ml of the supernatant sample was used for LC-MS / MS analysis.

  LC separation of 3-HPMA is achieved by using hydrophilic interaction chromatography column (XBridge HILIC, particle size 3.5 μm, 2.1 mm × 150 mm, Waters, MA, USA) and corresponding pre-column (HILIC, 2.1 mm × 10 mm) I used to go. The separation of 3-HPMA was performed at a constant flow rate of 0.3 ml / min using ammonium acetate pH 6.5, made 5 mM with acetonitrile / water (88/12, v / v). A triple quadrupole mass spectrometer (amaZon SL, Bruker Daltonics, Billerica, MA, USA) equipped with an electrospray (ESI) interface and a 10-port valve with fractions containing 3-HPMA eluted in 3 to 9 minutes. ) Were injected into the detector. The electrospray needle voltage was set to -4000 V in negative ion mode. The turbo heater was maintained at 475 ° C. Nitrogen was used as atomization gas, turbo heater gas and curtain gas, and the gas pressure was set at 45 psi, 60 psi and 25 psi, respectively. The collision gas (nitrogen) for the MS / MS mode was set to the supply of 3 instrument units. The mass spectrometer was used in multiple reaction monitoring mode (MRM). The retention time of 3-HPMA was 4.8 minutes, and the precursor ion (Q1) and the product ion (Q3) were 220.3 m / z and 91.0 m / z, respectively. The relative standard deviation (RSD) within 1 day of the 1 mol / l 3-HPMA solution and the daily RSD were 3.0% and 4.5%, respectively (n = 6).

A ^{AC-Acro [N ε - (} 3- formyl-3,4-dehydro Ropi piperidino) lysine] - Measurement of ^{([N ε (3-formyl} -3,4-dehydropiperidino) lysine]) is, ACR- Using a lysine adduct enzyme-linked immunosorbent assay system (ACR-LYSINE ADDUCT ELISA SYSTEM; NOF Corporation), the procedure was performed according to the attached protocol.

  The measurement of Cre was performed using a creatinine assay kit (Cayman Chemical CO., Ann Arbor, MI) according to the attached protocol.

(Measurement of Aβ ₄₀ and Aβ ₄₂ )
Measurement of A [beta] ₄₀ and A [beta] ₄₂ in plasma, respectively human β-amyloid (1-40) ELISA kit High Sensitive II (Wako Chemical Co.) and human β-amyloid (1-42) ELISA kit High Sensitive (Wako Chemical Co. The procedure was carried out using the attached procedure. The obtained values were used to calculate Aβ ₄₀ / Aβ ₄₂ levels (hereinafter sometimes abbreviated as Aβ _40/42 ).

(Statistical analysis)
Statistical calculations were performed by GraphPad Prism ^(TM) software ^{(GraphPad Prism (R) Software (} GraphPad Software)). Each group was compared using the Wilcoxon rank sum test. Spearman's rank correlation coefficient also allows statistical correlations between two biochemical markers, as well as statistical correlations between biochemical markers and MMSE, CDRsob, Z-scores or plasma Aβ _40/42. The regression line was drawn by the least squares method.

(Subject baseline sociodemographic variables)
Baseline sociodemographic variables of 74 control subjects, 54 MCI + AD subjects, 22 MCI subjects and 32 AD subjects are shown in Table 1. Each value in Table 1 is shown as median ± quartile deviation.

  As shown in Table 1, median values for MMSE, CDRsob and Z-scores for the MCI + AD, MCI and AD subject groups are 21.0, 24.0 and 18.0; 3.5, 1.5 and 5.0; and 2.04 respectively. , 1.70 and 2.43. The proportions of grades 0, 1-4 and 5-8 of the WMH grades (PVH and DSWMH) are 1.9%, 4.5% and 0% for the MCI + AD, MCI and AD subject groups, respectively; 68.5% 86.4% and 56.2%; and 29.6%, 9.1% and 43.8%. The proportion of female subjects was high in both the MCI and AD subject groups as previously reported (Non-Patent Document 33).

(Results of examination for reduction of 3-HPMA / Cre and AC-Acro / Cre in urine samples of subjects with MCI and AD)
The 3-HPMA content and Cre content in urine samples of subjects of MCI and AD were measured, and compared with the contents of 3-HPMA and Cre in urine samples of control subjects. Also, the ratio of 3-HPMA content to Cre content (3-HPMA / Cre (μmol / g)) was calculated.

  First, without distinction between MCI test subjects and AD test subjects, data analysis was performed on a group including both test subjects (hereinafter referred to as MCI + AD group) in comparison with a control test subject group (hereinafter referred to as a control group). The As shown in FIG. 1-A, 3-HPMA, 3-HPMA / Cre, Cre, AC-Acro, AC-Acro / Cre and median age are for the control subject group and the MCI + AD subject group, respectively. 1.48 μmol / l and 1.42 μmol / l; 2.23 μmol / g and 1.54 μmol / g; 75.1 mg / dl and 91.0 mg / dl; 75.1 nmol / ml and 65.7 nmol / ml; 109 μmol / g and 66.3 μmol / g; And 57.0 and 80.5 years old. As a result, as shown in FIG. 1-A, the level of 3-HPMA / Cre (μmol / g) in the urine sample of the MCI + AD group was compared with the level of 3-HPMA / Cre in the urine sample of the control group Although there was no decrease in 3-HPMA. Thus, AC-Acro was taken as another candidate for acrolein detoxification and its change in the MCI + AD group was tested. As shown in FIG. 1-A, AC-Acro / Cre levels in urine samples of the MCI + AD group were significantly reduced compared to the control group. The results show that in dementia subjects, changes in AC-Acro / Cre levels are the same as changes in 3-HPMA / Cre levels. Similar results were obtained when 3-HPMA / Cre levels and AC-Acro / Cre levels were evaluated in the same age group (22 control subjects aged 63-79 years and 23 MCI + AD subjects) (Fig. 1- B). As shown in FIG. 1-B, 3-HPMA, 3-HPMA / Cre, Cre, AC-Acro, AC-Acro / Cre and median age are for the control subject group and the MCI + AD subject group, respectively. 1.11 μmol / l and 1.45 μmol / l; 2.40 μmol / g and 1.63 μmol / g; 62.3 mg / dl and 90.2 mg / dl; 66.3 nmol / ml and 65.5 nmol / ml; 120 μmol / g and 68.3 μmol / g; And 71.0 and 74.0.

The above results indicate that both 3-HPMA / Cre levels and AC-Acro / Cre levels in urine can be used as urinary biochemical markers for dementia. Because urine collection is non-invasive and generally easier than blood collection, urinary 3-HPMA / Cre levels and AC-Acro / Cre levels are plasma indicators that are indicators of the conventional determination of dementia. Compared to Aβ _40/42 and PC-Acro, it can be a very convenient and useful biomarker for dementia determination.

(Comparison result of 3-HPMA / Cre and AC-Acro / Cre in urine sample between MCI group and AD group)
Next, data analysis is performed by comparing the group consisting of only MCI subjects (hereinafter referred to as MCI group) with the group consisting of only AD subjects (hereinafter referred to as AD group) by distinguishing MCI subjects from AD subjects. The As shown in Figure 2-A, 3-HPMA, 3-HPMA / Cre, Cre, AC-Acro, AC-Acro / Cre and median age are 1.37 μmol for MCI and AD subject groups, respectively. / l and 1.53 μmol / l; 1.89 μmol / g and 1.29 μmol / g; 68.6 mg / dl and 98.1 mg / dl; 58.7 nmol / ml and 66.9 nmol / ml; 92.0 μmol / g and 55.9 μmol / g; Years old and 81.0 years old. As a result, as shown in Fig. 2-A, both 3-HPMA / Cre level and AC-Acro / Cre level of AD group are lower than those of MCI group, and Cre level of AD group is higher than that of MCI group. it was high. In the MCI + AD group, 3-HPMA levels showed a good correlation with AC-Acro levels (FIG. 2-B). 3-HPMA / Cre levels and AC-Acro / Cre levels in urine samples in the AD group are decreased compared to the MCI group, mainly due to the increase in Cre, but in urine samples in AD subjects The mechanism of the increase of Cre is not clear.

  Thus, it was revealed that 3-HPMA / Cre levels and AC-Acro / Cre levels in urine samples of the AD group were lower than those of the MCI group. Because urine collection is non-invasive and generally easier than blood collection, urinary 3-HPMA / Cre levels and AC-Acro / Cre levels can be used to assess the severity of dementia, and AD and MCI subjects It can be used as a very convenient and promising biomarker for the discrimination between

(Correlation between 3-HPMA / Cre, AC-Acro / Cre, and Cre in urine samples and MMSE, CDRsob, image analysis, or Aβ _{40/42 in} plasma)
In previous studies, plasma PC-Acro correlated with MMSE but not with image analysis, ie, Z-score (21). Urinary 3-HPMA / Cre levels and AC-Acro / Cre levels, like PC-Acro in plasma, are biochemical markers of cell injury or tissue damage, so 3-HPMA / Cre levels and AC- The association between Acro / Cre levels and MMSE, CDRsob or Z-scores was examined. In addition, the 3-HPMA / Cre levels and the AC-Acro / Cre levels in urine and the correlation between Cre and plasma Aβ _40/42 were evaluated.

  Decreased levels of 3-HPMA / Cre or AC-Acro / Cre in the MCI + AD group correlated well with decreased MMSE (FIG. 3-A) and increased CDRsob (FIG. 3-B). However, 3-HPMA / Cre levels and AC-Acro / Cre levels did not correlate with the Z score (FIG. 3-C). An increase in Cre levels in the MCI + AD group correlated with both a decrease in MMSE and an increase in CDRsob, but not with the Z-score (FIGS. 3-A, B, and C).

As shown in FIG. 4, 3-HPMA / Cre levels in the MCI + AD group correlated well with Aβ _40/42 . However, AC-Acro / Cre levels and Cre did not correlate with Aβ _40/42 . The result is that, among the three markers in urine: 3-HPMA / Cre levels and AC-Acro / Cre levels and Cre, urinary 3-HPMA / Cre levels are most likely to distinguish between AD and MCI subjects. Indicates a reliable biochemical marker.

  As described above, the levels of 3-HPMA / Cre and AC-Acro / Cre in urine samples of the MCI + AD group are significantly lower than those in the control group, and 3-HPMA in urine samples of the AD group It was found that / Cre levels and AC-Acro / Cre levels were significantly lower compared to the MCI group. Acrolein is mainly detoxified by GSH in vivo. That is, acrolein is metabolized to form acrolein-GSH conjugate. Lack of GSH reduces the metabolism of acrolein, resulting in the interaction of acrolein with proteins and nucleic acids, leading to the inactivation of these substances. Therefore, the 3-HPMA / Cre level is reduced by correcting the urinary sample content of 3-HPMA, which is a metabolite of acrolein-GSH conjugate, with the urinary sample content of Cre, and the inclusion of AC-Acro in the urine sample Any reduction in AC-Acro / Cre levels, corrected for the amount of Cre in the urine sample, is an important biochemical marker of dementia caused by brain tissue damage. Also, both 3-HPMA / Cre levels and AC-Acro / Cre levels in urine samples can be used as reliable biochemical markers to distinguish between AD and MCI groups.

  Thus, 3-HPMA / Cre levels and AC-Acro / Cre levels in urine can be useful biomarkers used in testing methods to aid in the diagnosis of dementia. Since urine collection is much easier than blood collection, a test method using urinary 3-HPMA / Cre levels and / or AC-Acro / Cre levels as markers has the advantage of less invasive to the subject.

In addition, urinary 3-HPMA / Cre levels and AC-Acro / Cre levels are testing methods that aid in the discrimination between MCI and AD, and the progression of dementia from MCI to AD and the severity of dementia It can be a reliable biomarker used in testing methods to assess Recently, it has been reported that AD subjects can be distinguished from MCI subjects by the levels of Aβ ₄₀ , Aβ _40/42 and Aβ ₄₀ / PC-Acro in cerebrospinal fluid (CSF) (Non-patent Document 22). Because urine collection is much easier than cerebrospinal fluid collection, to distinguish AD from MCI, urinary 3-HPMA / Cre levels and / or AC-Acro / Cre levels, or In addition, a test method using PC-Acro and Aβ _40/42 in plasma as a biomarker is considered useful.

  The present invention relates to determination of cognitive impairment and a test method for assisting discrimination between dementia and mild cognitive impairment using 3-HPMA / Cre level and / or AC-Acro / Cre level in urine as an index. It can be used in the clinical laboratory medicine industry, the reagent industry, and the medical device industry.

Claims (10)

  Ratio (3-HPMA / Cre) of 3-hydroxypropyl mercapturic acid (hereinafter referred to as 3-HPMA) content and creatinine (hereinafter referred to as Cre) content in urine samples obtained from subjects And / or dementia and / or mild cognitive impairment based on the ratio (AC-Acro / Cre) of acrolein-added amino acid (hereinafter abbreviated as AC-Acro) content in urine sample to Cre content (AC-Acro / Cre) Inspection method.   The method for testing dementia and / or mild cognitive impairment according to claim 1, wherein 3-HPMA / Cre and / or AC-Acro / Cre are lower than those of healthy individuals.   The dementia and / or mild cognitive impairment according to claim 2, wherein using low as an index compared to the healthy person is using low as an index compared to a preset cutoff value. Inspection method.   The method for testing dementia and / or mild cognitive impairment according to any one of claims 1 to 3, wherein the dementia is Alzheimer's disease. Furthermore, the index (Aβ ₄₀ / Aβ ₄₂ ) of the amyloid β (Aβ) ₄₀ content and the Aβ ₄₂ content in the blood sample obtained from the subject is higher as a healthy person as an index. The inspection method of the dementia and / or mild cognitive impairment as described in any one of -4. Ratio of 3-HPMA content to Cre content (3-HPMA / Cre) in urine sample obtained from the subject, and / or ratio of AC-Acro content to Cre content in urine sample ( A test method that uses AC-Acro / Cre as an index to help distinguish between mild cognitive impairment and Alzheimer's disease. 7. The mild cognitive impairment according to claim 6, wherein the subject is judged to have Alzheimer's disease when 3-HPMA / Cre and / or AC-Acro / Cre is low compared to a patient with mild cognitive impairment And inspection methods to help determine the Lower compared to the mild cognitive impairment patients is lower that in comparison cutoff value set in advance, to assist in distinguishing between mild cognitive impairment and Alzheimer's disease according to claim 7 Inspection method. 7. The mild cognitive impairment according to claim 6, wherein the subject is determined to have mild cognitive impairment when 3-HPMA / Cre and / or AC-Acro / Cre is high compared to a patient with Alzheimer's disease, and Alzheimer's type A test method to help distinguish dementia. Higher as compared to patients Alzheimer type dementia is higher compared cutoff value set in advance, to assist in distinguishing between mild cognitive impairment and Alzheimer's disease according to claim 9 Inspection method.