JP6025568B2 - Test method to determine dementia by blood sample - Google Patents

Description

  The present invention relates to a test method for determining whether a blood sample is derived from a dementia mammal. Moreover, it is related with the method of determining whether a blood sample is derived from a dementia mammal using the result of the said test | inspection method. Moreover, it is related with the method of diagnosing whether a mammal is dementia based on the result of the said determination method. Moreover, it is related with the biomarker for a dementia diagnosis, and use of the said biomarker.

  Diagnosis of dementia mainly consists of interviews and questionnaires, and an objective diagnosis method has not yet been established. The interview method and the questionnaire method have a drawback that there is a difference depending on the diagnostician and lacks quantitativeness. In order to solve such problems, research is being conducted to establish an objective diagnostic method. For example, in AD (Alzheimer's disease), cerebral atrophy is seen as it progresses, so a method of performing image diagnosis using magnetic resonance imaging (MRI) has been devised. In AD, intracellular aggregation (neurofibrillary tangle) of tau protein that is phosphorylated excessively is markedly observed in the brain along with extracellular accumulation of β-amyloid protein (Aβ) (senile plaque). When Aβ accumulates, the microtubules in the nerve cells undergo structural changes and the nerve cells die, and at the same time the nerve cell death occurs, tau protein leaks out of the cells and enters the cerebrospinal fluid. It is thought to accumulate. For this reason, a diagnostic method using tau protein in cerebrospinal fluid as a marker has been devised. However, these methods are not sufficient in terms of convenience, economy, versatility, etc., because large specialized equipment is required.

  In dementia (especially Alzheimer-type dementia, which is said to account for 60% of dementia) (hereinafter referred to as “AD”), abnormalities are observed in the metabolic system of plasmalogen, a myelin lipid. In model animals, it is suggested that the amount of plasmalogens in the brain is reduced. Recently, there has been a report suggesting that plasma AD is reduced not only in the brain but also in serum when AD is affected (Non-patent Document 1). However, since serum contains only a very small amount of plasmalogen from the beginning, it is difficult to easily detect the decrease, and it is convenient, economical and versatile for use as a biomarker for the diagnosis of dementia. It was hard to say that it was sufficient in terms of etc.

J Lipid Res 48, 2485-2498,2007

  An object of the present invention is to provide a test method and a biomarker for easily and safely diagnosing dementia (particularly Alzheimer-type dementia) at low cost.

  The present inventors have surprisingly found that plasmalogen contained in erythrocytes is a useful biomarker for diagnosing dementia (especially AD), and completes the present invention with further improvements. It came to.

That is, the present invention includes, for example, the methods, biomarkers, uses, and kits described in the following sections.
Item 1.
A test method for determining whether a test blood sample is derived from a dementia mammal,
(A) a step of measuring the amount of plasmalogen contained in red blood cells contained in the test blood sample (that is, the amount of plasmalogen in the red blood cell lipid), and (B) (i) contained in red blood cells contained in the test blood sample. And (ii) a plasmalogen amount contained in red blood cells derived from a healthy mammal of the same species as the mammal from which the test blood sample was collected.
Item 2.
Step (A) is a step of measuring the amount of plasmalogen contained in the membrane lipid of erythrocytes contained in the test blood sample by high performance liquid chromatography,
Step (B) comprises (i) the amount of plasmalogen measured in step (A), and (ii) red blood cells derived from a healthy mammal of the same species as the mammal from which the test blood sample was collected, as measured by high performance liquid chromatography Is a step of comparing the amount of plasmalogen contained in the membrane lipid of
Item 1. The inspection method according to Item 1.
Item 3.
Item 3. The inspection method according to Item 2, wherein in step (B), (i) and (ii) are compared by comparing peak areas indicating plasmalogens in chromatograms obtained by high performance liquid chromatography.
Item 4.
Item 4. The inspection method according to Item 3, wherein the peak areas indicating the plasmalogen are compared based on the ratio of the peak area indicating the ethanolamine plasmalogen and the peak area indicating the phosphatidylethanolamine.
Item 5.
Item 5. The test method according to any one of Items 1 to 4, which is a test method for determining that the test blood sample is derived from a dementia mammal when (i) is less than (ii).
Item 6.
A method for determining whether a test blood sample is derived from a dementia mammal, comprising:
The test blood sample contains a dementia feeding when it includes the steps (A) and (B) in the test method according to any one of Items 1 to 4 and (C) the (i) is less than the (ii) A determination method comprising a step of determining that the animal originates.
Item 7.
A biomarker for detecting dementia, comprising a plasmalogen contained in red blood cells.
Item 8.
Use of a plasmalogen contained in erythrocytes as a biomarker for detecting dementia.
Item 9.
A kit for testing a blood sample, determining a blood sample derived from dementia, or diagnosing dementia, comprising a hypotonic solution for erythrocyte hemolysis and / or an organic solvent for lipid extraction.
Item 10.
A method of diagnosing a mammal from which a blood sample has been collected as having dementia,
Including the step (A), the step (B), and the step (C) according to Item 6, and (D) when the test blood sample is determined to be derived from a dementia mammal in the step (C), A diagnostic method comprising a step of diagnosing a mammal from which a test blood sample has been collected as having dementia.
Item 11.
Item 11. The diagnostic method according to Item 10, wherein the mammal is a non-human mammal.
Item 12.
A plasmalogen contained in red blood cells for use in the diagnosis of dementia.

According to the test method of the present invention, data for determining whether a test blood sample is derived from a dementia (particularly, Alzheimer-type dementia) mammal can be obtained simply, safely and at low cost. Moreover, from the result of this test method, it can be determined whether the test blood sample is derived from a dementia mammal, and dementia can be diagnosed from the determination result. Furthermore, with the biomarker or kit for detecting dementia of the present invention, the above-described examination, determination and diagnosis can be performed simply, safely and at low cost.

The chromatogram obtained by analyzing a rat erythrocyte lipid sample by HPLC / ELSD method is shown. The chromatogram obtained by analyzing a rat serum lipid sample by HPLC / ELSD method is shown. Abbreviations for each lipid substance are the same as those in FIG. The chromatogram obtained by analyzing a human erythrocyte lipid sample by HPLC / ELSD method is shown. The chromatogram obtained by analyzing a human serum lipid sample by HPLC / ELSD method is shown. Abbreviations for each lipid substance are the same as those in FIG. The (pl-PE / PE) value is calculated from the peak area of the chromatogram obtained by analyzing erythrocyte lipid samples of normal mice and dementia model mice by HPLC / ELSD method, and the result of statistical processing is shown. Blood samples were collected from each of the patients divided into three groups: normal group, mild dementia (mild AD) group, and advanced dementia (severe AD) group, and erythrocyte lipid samples were prepared and analyzed by HPLC / ELSD method. (Pl-PE / PE) value is calculated from the peak area of the chromatogram obtained in this way, and the result of statistical processing is shown.

  Hereinafter, the present invention will be described in more detail.

  The present invention has been made by finding that the amount of plasmalogen in the erythrocyte membrane decreases when suffering from dementia (particularly AD), and includes an inspection method, a determination method, and a diagnostic method using this feature. To do. Moreover, the kit used for these methods is also included. The present invention also includes a biomarker composed of a plasmalogen contained in erythrocytes and the use of a plasmalogen contained in erythrocytes as a biomarker.

Plasmalogen Plasmalogen generally refers to a glycerophospholipid having a long-chain alkenyl group via a vinyl ether bond at the 1-position of the glycerol skeleton. The general formula of plasmalogen is shown below.

[Wherein R ¹ and R ² represent an aliphatic hydrocarbon group. R ¹ is usually an aliphatic hydrocarbon group having 1 to 20 carbon atoms, and examples thereof include a dodecyl group, a tetradecyl group, a hexadecyl group, an octadecyl group, and an icosanyl group. R ² is usually an aliphatic hydrocarbon group derived from a fatty acid residue. For example, octadecadienoyl group, octadecatrienoyl group, icosatetraenoyl group, icosapentaenoyl group, docosatetraenoyl group, docosa Examples include a pentaenoyl group and a docosahexaenoyl group. In the formula, X represents a polar group. X is preferably —CH ₃ CH ₃ NH ₂ , —CH ₃ CH ₃ N ⁺ (CH ₃ ) ₃ , —CH ₃ CH ₂ (NH ₂ ) COOH, or X is preferably H bound to X Compound (H-X) is inositol or glycerol. ]

In particular, an ethanolamine plasmalogen in which X is an aminoethyl group (—CH ₃ CH ₃ NH ₂ ) and choline in which X is a trimethylaminoethyl group (—CH ₃ CH ₃ N ⁺ (CH ₃ ) ₃ ) Plasmalogens are the main plasmalogens that exist in nature. Red blood cells contain a particularly large amount of ethanolamine plasmalogen as a plasmalogen.

Inspection Method The inspection method of the present invention includes the following steps (A) and (B).
(A) The step of measuring the amount of plasmalogen contained in red blood cells contained in the test blood sample (B) (i) The amount of plasmalogen contained in red blood cells contained in the test blood sample, (ii) Step of comparing the amount of plasmalogen contained in red blood cells derived from healthy mammals of the same species as the collected mammal The test method is used to determine whether the test blood sample is derived from a dementia mammal be able to. In other words, the test method of the present invention can be said to be a test method for determining whether a test blood sample is derived from a dementia mammal (that is, a test method that provides data for determination). In the said determination, when said (i) is less than said (ii), it determines with a test blood sample originating in a dementia mammal.

  In the present specification, examples of mammals include human and non-human mammals. Examples of non-human mammals include pets, laboratory animals, livestock and the like. Specific examples include dogs, cats, monkeys, cows, horses, sheep, goats, pigs, mice, rats, hamsters, rabbits, and the like. In the present specification, dementia is preferably degenerative dementia, specifically, Alzheimer's dementia, Parkinson's disease (with dementia associated with), frontotemporal dementia, Pick's disease, diffuse Examples include Lewy body disease. Alzheimer type dementia and Parkinson's disease (with dementia associated therewith) are preferable, and Alzheimer type dementia is more preferable.

[Process (A)]
As the test blood sample, blood itself collected from a mammal and a sample obtained by subjecting the blood to some kind of processing and containing red blood cells can be used. Examples of the treatment applied to blood (treatment in which red blood cells remain after the treatment) include centrifugation and addition of an anticoagulant. A well-known thing can be used for an anticoagulant, for example, a citric acid, EDTA (ethylenediaminetetraacetic acid) or its metal salt (sodium salt etc.), sodium fluoride, heparin etc. can be illustrated. Although not particularly limited, the test blood sample is preferably blood obtained by a normal blood collection method (for example, syringe blood collection or vacuum blood collection) or a sample obtained by processing the blood.

  For example, HPLC (high performance liquid chromatography; also called high performance liquid chromatography), GC-MS (gas chromatograph mass spectrometer), TLC (thin layer) Chromatography) or the like. Among these, it is preferable to use HPLC.

  Specifically, the measurement using HPLC is performed by centrifuging a test blood sample, removing a supernatant (ie, serum), further removing platelets and white blood cells, preparing a red blood cell sample, and hemolyzing the red blood cell sample. This can be done by extracting the lipid and analyzing it by HPLC.

  Serum, platelets, and leukocytes can be removed, for example, by centrifuging the test blood sample (for example, 1000 × g, 5 minutes) and then removing the supernatant and the upper layer of the blood cell part. It is more preferable to add physiological saline to a sample from which serum, platelets and leukocytes have been removed, mix, and centrifuge (for example, 1000 × g, 5 minutes) to further remove serum components and leukocytes and platelets in the upper layer of blood cells. After removing serum, platelets, and white blood cells, physiological saline can be added to prepare any (eg, about 10%) hematocrit red blood cell suspension (red blood cell sample). The number of red blood cells, hemoglobin value, etc. in this red blood cell suspension can be measured with a blood cell measuring device (Sysmex 800, Sysmex, Kobe, Japan).

  Moreover, in order to hemolyze an erythrocyte sample, a well-known method can be used, for example, it can carry out by adding a solution (hypotonic solution) with a low osmotic pressure to an erythrocyte sample. The hypotonic solution is preferably a buffer, for example, a phosphate buffer. As a method for obtaining a erythrocyte lipid sample by lysing erythrocytes and then extracting lipids, a known lipid extraction method can be used. For example, methanol and chloroform are added to a sample obtained by lysing erythrocytes and mixed. A method for recovering the chloroform layer can be exemplified. More specifically, add 1 to 2 volumes of methanol and chloroform of the same volume to the hemolyzed erythrocyte solution (erythrocyte suspension), mix and leave for 10 minutes to 2 hours, then centrifuge, A method for recovering the chloroform layer can be mentioned. The recovered chloroform layer can be stored at about −20 ° C. to −40 ° C. after drying. The drying can be performed using nitrogen gas.

  For example, by analyzing the erythrocyte lipid sample obtained as described above by HPLC, a chromatogram in which peaks of each lipid component (including plasmalogen) present in the erythrocyte membrane can be obtained. In the analysis by HPLC here, the sample is preferably detected by evaporative light scattering detection (ELSD). That is, it is preferable to perform analysis by HPLC / ELSD method. Specifically, for example, an erythrocyte membrane lipid sample is dissolved in hexane / isopropanol (1: 1), injected into an HPLC column, and detection is performed by ELSD, whereby each lipid component present in the erythrocyte membrane can be analyzed. . For example, hexane / isopropanol / acetic acid (82: 17: 1, volume ratio) can be used for mobile phase A, and isopropanol / water / acetic acid (85: 14: 1, volume ratio) can be used for mobile phase B. The area of each peak in the obtained chromatogram represents the content of each lipid component. For example, the content can be determined by calculating what percentage of the peak area of the entire chromatogram the peak area indicating plasmalogen is. This is because ELSD shows a similar area response if the substance has a similar structure. For example, it is preferable to analyze by charging acidic lipid using acetic acid and triethylamine as a solvent. This is because the same area response can be shown by charging.

  In the measurement of the amount of plasmalogen in the step (A), it is not always necessary to obtain an absolute amount such as a mass value, and in the step (B), it is derived from a healthy mammal of the same species as the mammal from which the test blood sample was collected. It is only necessary to obtain data that can be compared with the amount of plasmalogen contained in red blood cells. For example, (i) the amount of plasmalogen contained in red blood cells contained in the test blood sample (hereinafter also referred to as “test red blood cell PL amount”), and (ii) a healthy mammal of the same species as the mammal from which the test blood sample was collected The amount of plasmalogen contained in erythrocytes derived from blood (hereinafter also referred to as “healthy erythrocyte PL amount”) is measured by the same measurement method, and the obtained data are compared with each other, so that the test erythrocyte PL amount and the healthy erythrocyte PL amount The comparison in the step (B) can be performed without obtaining each absolute amount.

[Process (B)]
In step (B), (i) the amount of plasmalogen contained in the red blood cells contained in the test blood sample (the amount of test red blood cells PL), and (ii) derived from a healthy mammal of the same species as the mammal from which the test blood sample was collected The amount of plasmalogen contained in the red blood cells (the amount of healthy red blood cells PL) is compared. The comparison result (that is, the amount of the test erythrocyte PL amount relative to the healthy erythrocyte PL amount) can be used to determine whether the test blood sample is derived from a dementia mammal.

  As the test erythrocyte PL amount, the plasmalogen amount determined in the above-described step (A) can be used. Hereinafter, red blood cells contained in the test blood sample are also referred to as “test red blood cells”.

  “Red blood cells derived from healthy mammals of the same species as the mammal from which the test blood sample was collected” (hereinafter also referred to as “healthy red blood cells”) are the same species as the mammal from which the test sample was collected and are healthy Red blood cells contained in mammalian blood samples. For example, when the test blood sample is obtained from a human, red blood cells contained in a blood sample collected from a healthy person correspond to healthy red blood cells. In addition, a healthy mammal is a mammal that does not suffer from dementia (preferably healthy).

  The comparison between (i) the test erythrocyte PL amount and (ii) the healthy erythrocyte PL amount may be made by comparing the respective absolute amounts by different or the same measurement method, and the same measurement method is used. Is more preferable. In addition, as described above, (i) and (ii) are not necessarily absolute values as long as (i) and (ii) can be compared, and may be relative values, for example.

  For example, when (i) and (ii) are both obtained as absolute quantity values (for example, mass value or molar value), by aligning the amount of red blood cells used for measurement (ie, the amount of sample used for measurement), (I) and (ii) can be compared. The amount of red blood cells can be aligned by, for example, aligning the mass of red blood cells of the measurement sample, aligning the number of red blood cells of the measurement sample, or aligning the hemoglobin values of the measurement sample. Thus, by comparing the amount of test red blood cells PL present in a certain amount of red blood cells with the amount of red blood cells PL present in the same amount of red blood cells, the amount of test red blood cells PL relative to the amount of healthy red blood cells PL You can decide a little.

  In addition, when (i) and (ii) are obtained as data measured by the same analysis method, these data can be compared.

  For example, by HPLC (detection is preferably ELSD), the membrane lipids of erythrocytes contained in the test blood sample and the membrane lipids of erythrocytes from a healthy mammal of the same species as the mammal from which the test blood sample was collected are analyzed. The areas of the peaks indicating plasmalogen in the respective chromatograms obtained in this manner can be compared with each other. In this case, the amount of the test erythrocyte PL amount relative to the healthy erythrocyte PL amount can be determined by aligning the amount of red blood cells to obtain membrane lipids used for HPLC measurement.

  Furthermore, the ratio of the peak area showing plasmalogen to the peak area showing other specific lipid components is compared with the amount of red blood cells to obtain membrane lipids used for HPLC measurement. It is also possible to determine the amount of the test erythrocyte PL amount relative to the healthy erythrocyte PL amount by comparing the ratios. That is, HPLC analysis is performed on each of the test red blood cells and healthy red blood cells to obtain chromatograms, and in each chromatogram, the ratio (ratio) of the amount of plasmalogen to a specific lipid component amount is obtained, and the magnitude of the ratio is compared. Thus, the amount of plasmalogen can be compared. The larger the ratio, the larger the plasmalogen amount, and the smaller the ratio, the smaller the plasmalogen amount. The ratio can be described as (plasmalogen amount / specific lipid component amount), and can be determined by (peak area showing plasmalogen / peak area showing specific lipid component). In this comparison method, it is preferable to select a specific lipid component that is contained in the test erythrocyte and the healthy erythrocyte and has substantially the same content as the “specific lipid component” used in the denominator of the ratio. Examples of such lipid components include phosphatidylethanolamine, sphingomyelin, cholesterol, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, sphingomyelin, and cholesterol are preferable, and phosphatidylethanolamine is more preferable. In addition, since the amount of erythrocytes for obtaining membrane lipids used for HPLC measurement is aligned and the ratio (ratio) of plasmalogen amount to specific lipid component amount can be obtained, a more accurate comparison result can be obtained. preferable.

  The comparison result of (i) test erythrocyte PL amount and (ii) healthy erythrocyte PL amount obtained by the test method of the present invention is preferable for determining whether or not the test blood sample is derived from a dementia mammal. Used. In the said determination, when said (i) is less than said (ii), it determines with a test blood sample originating in a dementia mammal. That is, the test method of the present invention is a test method for determining that the test blood sample is derived from a dementia mammal when (i) is less than (ii) (that is, obtaining comparative data for determination). Can be preferably used as an inspection method.

  In particular, when the ratio of (plasmalogen amount / specific lipid component amount) is calculated from the chromatogram obtained by the above-mentioned HPLC analysis and the plasmalogen amount is compared using this ratio, (ethanolamine plasmalogen amount / phosphatidyl) It is preferable to compare the ethanolamine amount). The ratio of (ethanolamine plasmalogen amount / phosphatidylethanolamine amount) is about 0.85 to 0.9 in healthy mammals (especially healthy people), whereas it is 0 in demented mammals (especially demented patients). 0.7 or less (about 0.7 to 0.5). It can be said that the (ethanolamine plasmalogen amount / phosphatidylethanolamine amount) ratio value of patients with dementia is 90% or less (preferably 85% or less, more preferably 80% or less) of the ratio value of healthy persons. Therefore, when comparing the ratio value, when the ratio value of the test erythrocyte is 90% or less (preferably 85% or less, more preferably 80% or less) of the ratio of the healthy erythrocyte, the test erythrocyte is dementia. It is thought that it can be determined that it originates from a mammal.

  As described above, the test method of the present invention is useful for determining whether a test blood sample is derived from a dementia mammal. In particular, since the necessary sample is a blood sample and can be carried out by using a general device, it can be said that it is greatly improved in terms of convenience, economy, versatility and the like. The test method of the present invention can be used, for example, for testing a blood sample in a clinical testing company. Further, for example, when a business for shipping dementia model animals is performed, it can be used to check whether the model animals to be shipped suffer from dementia. Or when carrying out the business which ships a normal animal, it can be used in order to check whether the shipped animal does not suffer from dementia.

Determination Method The present invention includes a method for determining whether or not a test blood sample is derived from a dementia mammal. The determination method includes the above steps (A) and (B), and (C) (i) the amount of plasmalogen contained in red blood cells contained in the test blood sample is (ii) collecting the test blood sample A step of determining that the test blood sample is derived from a dementia mammal when the amount of plasmalogen contained in red blood cells derived from a healthy mammal of the same species as that of the mammal is smaller.

  The determination method can also be used in the same manner as described for the inspection method.

Diagnostic Method The present invention also includes a method for diagnosing whether or not a mammal has dementia. This method for diagnosing dementia of the present invention is a method for diagnosing that a mammal from which a test blood sample has been collected has dementia based on the result of the above-described determination method. That is, when the test blood sample is determined to be derived from a dementia mammal by the above-described determination method, the mammal from which the test blood sample is collected is diagnosed as dementia. More specifically, the diagnostic method of the present invention includes steps (A), (B), and (C) in the above-described determination method, and (D) the test blood sample is recognized in step (C). It can be referred to as a diagnostic method including a step of diagnosing a mammal from which the test blood sample has been collected as having dementia when it is determined to be derived from a sick mammal.

  Although not particularly limited, the diagnostic method of the present invention can also include a step of collecting a test blood sample from a mammal to be diagnosed as having dementia before the step (A). As a blood collection method for obtaining a test blood sample, a known method can be used. For example, a syringe blood collection method or a vacuum blood collection method can be exemplified.

Biomarker In this specification, a biomarker for detecting dementia refers to a biomolecule that serves as an index for determining or diagnosing the onset of dementia. It can also be said to be a biomolecule for determining onset of dementia or a diagnostic indicator. The present invention also includes a biomarker for detecting dementia, comprising a plasmalogen contained in erythrocytes. More specifically, a biomarker for detecting dementia, which comprises a plasmalogen contained in the erythrocyte membrane, is also included. Note that plasmalogen contained in erythrocytes isolated from the living body, not plasmalogen present in the living body, is used as the biomarker for detecting dementia of the present invention. Therefore, it can be said that the biomarker of the present invention is a biomarker for detecting dementia composed of plasmalogen extracted from red blood cells (erythrocyte-extracted plasmalogen). It can also be said to be a biomarker for dementia diagnosis.

  The biomarker for detecting dementia of the present invention is characterized in that the amount thereof is reduced in dementia mammals compared to healthy mammals. Therefore, each of the biomarkers is isolated from the test mammal and the healthy mammal, and the amount thereof is measured and compared. If the test mammal is smaller, the test mammal suffers from dementia. Can be diagnosed.

  In addition, as a biomarker for detecting dementia of the present invention, ethanolamine plasmalogen is particularly preferable among plasmalogens. That is, the present invention is a dementia detection biomarker comprising ethanolamine plasmalogen contained in erythrocytes (dementia detection biomarker comprising erythrocyte-extracted ethanolamine plasmalogen, or dementia comprising erythrocyte-extracted ethanolamine plasmalogen. Preferably a diagnostic biomarker).

Kit The present invention also includes a kit (test kit, test kit, or diagnostic kit) used in the above-described test method, determination method, or diagnostic method. More specifically, these kits can be referred to as blood sample test kits, dementia-derived blood sample determination kits, and dementia diagnosis kits.

  The kit of the present invention is provided with a reagent useful for measuring the amount of plasmalogen contained in erythrocytes. Examples of such a reagent include a hypotonic solution for erythrocyte hemolysis and an organic solvent for lipid extraction. Specifically, the hypotonic solution for erythrocyte hemolysis is exemplified by a hypotonic phosphate buffer and the like, and the organic solvent for lipid extraction is exemplified by methanol, chloroform, methanol / chloroform (preferably 1: 1) solution and the like. The kit of the present invention preferably comprises a hypotonic solution for erythrocyte hemolysis and / or an organic solvent for lipid extraction.

  Moreover, the kit of the present invention may further be provided with a blood collecting instrument or reagent. For example, a syringe for blood collection, a tube for blood collection, an anticoagulant and the like can be exemplified. Moreover, the kit of the present invention may further include a solvent for HPLC analysis. Examples of such a solvent include, but are not limited to, hexane / isopropanol / acetic acid (82: 17: 1), isopropanol / water / acetic acid (85: 14: 1), triethylamine, and the like.

  The kit of the present invention can be preferably used in the above-described inspection method, determination method, or diagnostic method.

  Hereinafter, the present invention will be specifically described, but the present invention is not limited to the following examples.

Preparation of measurement sample <blood collection>
Rat blood collection: Blood was collected by cardiac puncture using a syringe. Heparin, an anticoagulant, was added to the collected blood. This is a rat blood sample. The rats used for blood collection were 6 Wistar rats (12 weeks old) raised for 4 weeks on an MF solid diet (Oriental Yeast Co., Ltd.).

  Human blood collection: Venous blood was collected using a syringe. To the collected blood, EDTA-Na (ethylenediaminetetraacetic acid sodium salt) was added as an anticoagulant. This was a human blood sample.

<Preparation of serum and red blood cell samples>
The blood sample obtained as described above was centrifuged at 1000 × g for 5 minutes to remove the supernatant (ie serum). The removed serum was stored (this was used as a serum sample). In addition, platelets and leukocytes were removed. Then, physiological saline was added to the sample from which the platelets and leukocytes had been removed and mixed, followed by centrifugation at 1000 × g for 5 minutes to remove leukocytes and platelets in the upper layer of serum components and blood cells. This operation was further repeated twice. Thereafter, physiological saline was added to prepare a 10% hematocrit erythrocyte suspension. The erythrocyte count and hemoglobin level in the erythrocyte suspension were measured and recorded with a blood cell counter (Sysmex 800, Sysmex, Kobe, Japan).

<Preparation of lipid sample>
2.5 ml of the erythrocyte suspension obtained as described above was placed in a test tube with a stopper and centrifuged (1000 × g, 5 minutes) to remove the supernatant. The erythrocytes thus obtained were hemolyzed by adding 0.25 ml of 5 mM phosphate buffer (pH 8.0), mixed with 4 ml of methanol, and then allowed to stand for 30 minutes. 4 ml of chloroform was added thereto and mixed, and the mixture was allowed to stand at room temperature for 20 minutes to extract lipids. This was centrifuged (1000 × g, 5 minutes), and the supernatant was collected. Then, 4 ml of methanol / chloroform (1: 1) solution was added to the residue to re-extract the lipid. Thereafter, the supernatant was collected by centrifugation (1000 × g, 5 minutes), and the supernatant collected earlier was combined, and 10 ml of 50 mM potassium chloride solution was further added and mixed, followed by centrifugation (1000 × g, 5 minutes). 1 ml of the chloroform layer was collected, dried using nitrogen gas, and stored at −30 ° C. The lipid sample thus obtained was used as an erythrocyte lipid sample. Rat erythrocyte lipid samples and human erythrocyte lipid samples were prepared.

  When extracting lipid from a serum sample, 4 ml of methanol was added to a 0.5 ml serum sample, and then 4 ml of chloroform was added, and then lipid was extracted in the same manner as erythrocytes. The lipid sample thus obtained is used as a serum lipid sample. Rat serum lipid samples and human serum lipid samples were prepared.

Measurement of plasmalogen amount in sample <analysis of erythrocyte lipid sample by HPLC / ELSD method: rat>
Rat erythrocyte lipid samples were dissolved in 0.2 ml hexane / isopropanol (1: 1) and 0.01 ml was injected into the HPLC. The following DIOL column (250 mm × 4 mm, silica gel average particle diameter 5 μm) was used for HPLC. The measurement conditions are described below.

Equipment: Shimadzu LC-10AD (Shimadzu Corporation, Kyoto, Japan)
Column: LiChrospher Diol 100 (250-4, Merck) (no precolumn)
Flow rate: 1.0ml / min
Detection; Evaporative Light Scattering Detector (ELSD) (Shimadzu Corporation, Kyoto, Japan)
Hexane / isopropanol / acetic acid (82: 17: 1, volume ratio) for mobile phase A, isopropanol / water / acetic acid (85: 14: 1, volume ratio) for mobile phase B, (0.08 for both mobile phases A and B) % (V / v) was added triethylamine). Mobile phase A decreases linearly from 95%, remains 63% at 23 minutes, 15% at 27 minutes, 15% from 27 to 28 minutes, returns to the initial conditions in 4 minutes, and continues the initial conditions for 5 minutes. The next sample was injected. Each lipid was detected using an evaporative light scattering detector (ELSD). The results are shown in FIG. 1 (the upper chromatogram in FIG. 1). Peaks indicating various lipids were obtained, and it was found that the peak of ethanolamine plasmalogen (pl-PE), which is a plasmalogen, could be clearly read.

  In addition, since the vinyl ether bond of plasmalogen is more sensitive to acid hydrolysis than acyl bond, plasmalogen is selectively decomposed when the lipid sample is treated with hydrochloric acid. When the rat erythrocyte lipid sample treated with hydrochloric acid was similarly analyzed by HPLC, it was also confirmed that only the peak derived from plasmalogen (pl-PE) disappeared (chromatogram on the lower side of FIG. 1). This also confirmed that the disappeared peak was plasmalogen. In addition, it can be determined whether each peak of a chromatogram is a peak derived from which lipid component by analyzing samples of various lipid components in advance and confirming a peak detection position.

  From the above results, it was confirmed that the plasmalogen contained in the erythrocyte lipid sample can be detected by analysis by the HPLC / ELSD method. Moreover, it was confirmed by HPLC / ELSD method that not only plasmalogens but also other lipid components can be separated and detected at a time.

  Similarly, rat serum lipid samples were analyzed by HPLC / ELSD method. The results are shown in FIG. 2 (the lower chromatogram in FIG. 2 is an enlarged version of the upper chromatogram in FIG. 2). From the results, it was found that there was almost no plasmalogen in the serum (at least difficult to detect under the above-mentioned HPLC analysis conditions).

<Analysis of erythrocyte lipid sample by HPLC / ELSD method: human>
The human erythrocyte lipid sample was analyzed by HPLC / ELSD method in the same manner as the analysis of the rat erythrocyte lipid sample. The results are shown in FIG. 3 (the upper chromatogram of FIG. 3). As in the case of rats, peaks indicating various lipids were obtained, and it was found that the peak of ethanolamine plasmalogen (pl-PE), which is a plasmalogen, could be clearly read.

  Moreover, when the human erythrocyte lipid sample treated with hydrochloric acid was similarly analyzed by the HPLC / ELSD method, it was also confirmed that only the peak derived from plasmalogen (pl-PE) disappeared (chromatogram on the lower side of FIG. 3).

  From the above results, it was confirmed that the plasmalogen contained in the erythrocyte lipid sample can be detected by the analysis by the HPLC / ELSD method. It was also confirmed by HPLC / ELSD that not only plasmalogens but also other lipid components could be separated and detected at a time.

  By analyzing each peak area of the chromatogram on the upper side of FIG. 3, it is possible to determine the ratio of the amount of plasmalogen to the amount of various lipid components, or the ratio of the amount of plasmalogen in the human erythrocyte lipid. The proportion of plasmalogen in human erythrocyte lipid was about 12%.

  In addition, the human serum lipid sample was similarly analyzed by HPLC / ELSD method. The results are shown in FIG. From the results, it was found that there was almost no plasmalogen in the serum (at least difficult to detect under the above-mentioned HPLC analysis conditions).

Comparison between human dementia patients and healthy persons Dementia patients (79-year-old male with Alzheimer's disease, 74-year-old male with Parkinson's disease combined with dementia), and six elderly healthy persons who are the same sex as the patient (68-74 years old: average) 72 years old), blood samples were collected from each of the obtained blood samples as described above, and analyzed by HPLC / ELSD method.

Then, the peak area derived from each lipid component of each chromatogram obtained from each blood was analyzed, and the ratio of the peak area of ethanolamine plasmalogen (pl-PE) to the peak area of phosphatidylethanolamine (PE) ( pl-PE / PE). The result was as follows.
Healthy elderly people: 0.87 ± 0.07
Alzheimer's disease patients 0.58
Parkinson's disease patient 0.63

  From these results, it was confirmed that the amount of plasmalogen in erythrocyte lipid was reduced in patients with dementia. Here, in order to compare the amount of plasmalogen contained in red blood cells of healthy and demented patients, the ratio of the amount of ethanolamine plasmalogen to the amount of phosphatidylethanolamine of one of the lipids contained in the red blood cell membrane was determined. Although calculated and compared (that is, compared with phosphatidylethanolamine as a reference), the ratio of ethanolamine plasmalogen to other lipids can be calculated and compared in the same manner.

Study using dementia model mice
It is known that dementia model mice can be produced by intraperitoneally administering LPS (lipopolysaccharide) to mice (see, for example, Jpn. J. Pharmacol. 87, 195-201 (2001)). Therefore, in the same manner as described in the literature (Lee JW et.al, Neuroinflammation, 2008, Aug 29; 5:37), brain inflammation was caused to produce a dementia model mouse. Specifically, LPS (250 μg / kg, Sigma) was administered intraperitoneally once a day for 7 weeks to 8-12 week old C57BL male mice (Kudo Co.) A dementia model mouse having inflammation was obtained (the mouse group was referred to as LPS group). A control group (Control group) was a group of mice in which physiological saline was administered intraperitoneally for 7 days to 8-12 week old C57BL male mice instead of LPS. The LPS group has n = 5 and the Control group has n = 5.

  24 hours after the final administration of LPS (the final administration of physiological saline in the control group), each mouse was anesthetized by administration of pentobarbital. After laparotomy, blood was collected from the left ventricle to obtain a test blood sample. Then, in the same manner as described above, an erythrocyte lipid sample is prepared from the test blood sample, analyzed by HPLC / ELSD method, and the peak area of ethanolamine plasmalogen (pl-PE) and the peak of phosphatidylethanolamine (PE) are obtained. The ratio to the area (pl-PE / PE) was calculated. Furthermore, a Steel test (nonparametric) test was performed. The results are shown in FIG.

  From the results, it was confirmed that the amount of plasmalogen in the erythrocyte lipid was significantly decreased in the dementia model mouse compared to the normal mouse.

Study revision using Alzheimer's type dementia patients Hasegawa-style simplified intelligence assessment scale evaluates multiple elderly people, out of 30 out of 21 normal (n = 8), 20 to 11 mild dementia ( Mild AD; n = 19) 10 points or less were judged as severe dementia (high AD; n = 14) and divided into groups.

  The revised Hasegawa-style simplified intelligence assessment scale, also referred to as HDS-R, is a method widely used in clinical settings as a method for screening elderly people with dementia from the general elderly (Geriatric Psychiatry Journal) , 2: 1339-1347 (1991), Guide to Intelligent Function Testing for the Elderly, World Planning, (1991) etc.).

  In the same manner as described above, blood samples are collected from each patient to obtain test blood samples, red blood cell lipid samples are prepared from the test blood samples, analyzed by HPLC / ELSD method, and ethanolamine plasmalogen (pl-PE) The ratio (pl-PE / PE) between the peak area and the peak area of phosphatidylethanolamine (PE) was calculated. In addition, a Steel Test (nonparametric) test was performed. The results are shown in FIG. The unit of the vertical axis in FIG. 6 is%.

The results show that plasmalogen levels in erythrocyte lipids decrease in patients with Alzheimer-type dementia, and in particular, plasmalogen levels in erythrocyte lipids are significantly higher in patients with advanced dementia than in non-demented people (normal). I found that it was down.

Claims (8)

A test method for determining whether a test blood sample is derived from a dementia mammal,
(A) a step of measuring the amount of plasmalogen contained in red blood cells contained in the test blood sample, and (B) (i) the amount of plasmalogen contained in red blood cells contained in the test blood sample, and (ii) the test blood A test method comprising a step of comparing the amount of plasmalogen contained in red blood cells derived from a healthy mammal of the same species as the mammal from which the sample was collected. Step (A) is a step of measuring the amount of plasmalogen contained in the membrane lipid of erythrocytes contained in the test blood sample by high performance liquid chromatography,
Step (B) includes (i) the amount of plasmalogen measured in step (A), and (ii) red blood cells derived from a healthy mammal of the same species as the mammal from which the test blood sample was collected, as measured by high performance liquid chromatography 2. The inspection method according to claim 1, which is a step of comparing the amount of plasmalogen contained in the membrane lipid. The method according to claim 2, wherein in step (B), (i) and (ii) are compared by comparing peak areas indicating plasmalogens in a chromatogram obtained by high performance liquid chromatography. . The inspection method according to claim 3, wherein the comparison of the peak areas indicating the plasmalogen is performed based on the ratio of the peak area indicating the ethanolamine plasmalogen and the peak area indicating the phosphatidylethanolamine. The test method according to any one of claims 1 to 4, which is a test method for determining that the test blood sample is derived from a dementia mammal when the (i) is less than the (ii). A method for determining whether a test blood sample is derived from a dementia mammal, comprising:
A test blood sample is a dementia when it includes the steps (A) and (B) in the test method according to any one of claims 1 to 4, and (C) when (i) is less than (ii). A determination method comprising a step of determining that it is derived from a mammal. A biomarker for detecting dementia, comprising a plasmalogen contained in red blood cells. Use of a plasmalogen contained in erythrocytes as a biomarker for detecting dementia.

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