WO2014010667A1 - Method of examining cognitive function and kit therefor - Google Patents

Method of examining cognitive function and kit therefor Download PDF

Info

Publication number
WO2014010667A1
WO2014010667A1 PCT/JP2013/068965 JP2013068965W WO2014010667A1 WO 2014010667 A1 WO2014010667 A1 WO 2014010667A1 JP 2013068965 W JP2013068965 W JP 2013068965W WO 2014010667 A1 WO2014010667 A1 WO 2014010667A1
Authority
WO
WIPO (PCT)
Prior art keywords
ethanolamine
plsetn
serum
plasma
type
Prior art date
Application number
PCT/JP2013/068965
Other languages
French (fr)
Japanese (ja)
Inventor
良太 前場
厚 荒木
佳典 藤原
貴志子 小川
原　博
めぐみ 西向
信一 酒瀬川
松本　英之
優作 松本
大助 杉森
Original Assignee
学校法人帝京大学
地方独立行政法人東京都健康長寿医療センター
国立大学法人北海道大学
旭化成ファーマ株式会社
国立大学法人福島大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 学校法人帝京大学, 地方独立行政法人東京都健康長寿医療センター, 国立大学法人北海道大学, 旭化成ファーマ株式会社, 国立大学法人福島大学 filed Critical 学校法人帝京大学
Priority to JP2014524864A priority Critical patent/JP6185466B2/en
Publication of WO2014010667A1 publication Critical patent/WO2014010667A1/en

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders

Definitions

  • the present invention relates to a test method for identifying a person with dementia or a pre-stage thereof using a biomolecule as an index, a measurement method thereof, a kit used for the test method, an enzyme contained in the kit, and a method for producing the enzyme .
  • Dementia refers to a condition in which intelligence that has developed normally has decreased due to an acquired organic disorder of the brain. Social life becomes difficult due to obstacles such as memory and judgment. In recent years, the number of senile dementia has increased with the aging of the population, and the number is said to be about 2 million, which tends to increase further.
  • Dementia becomes difficult to recover as symptoms progress, and it requires a lot of labor and financial burden for nursing care, but it can be detected in a relatively mild stage and treated with rehabilitation training to prevent onset. A certain degree of recovery is possible. Moreover, since an antidementia drug is a drug for suppressing the progression of symptoms, the prescription is more effective as soon as possible. Therefore, in dementia, it is important to identify the treatment subject at an early stage.
  • a method using a biomolecule (biomarker) as an index has attracted attention.
  • the neuropathological characteristics of Alzheimer's disease one of the typical dementias, are a neurogenic group characterized by the appearance of senile plaques formed by amyloid ⁇ (A ⁇ ) deposition and abnormal phosphorylated tau protein. Fibrous changes and massive neuronal loss.
  • a ⁇ amyloid ⁇
  • a ⁇ induces phosphorylation of tau protein, causing cytoskeletal protein abnormalities and accompanying intracellular transport disturbances, leading to death of nerve cells.
  • a ⁇ is produced by cleaving from amyloid precursor protein with ⁇ -secretase and ⁇ -secretase which do not normally act.
  • a ⁇ 42 consisting mainly of 42 amino acids
  • a ⁇ 40 consisting of 40 amino acids, and the former is considered to be important for the onset of Alzheimer's disease because of the high aggregation ability and neurotoxicity.
  • a decrease in A ⁇ 42 in the cerebrospinal fluid duee to the deposition of A ⁇ 42 in the brain) is considered to be one of the most reliable Alzheimer's disease diagnostic indicators at present.
  • cerebrospinal fluid In Alzheimer's disease, an increase in phosphorylated tau protein in the cerebrospinal fluid has also been confirmed, and it is positioned as a diagnostic index closely related to the disease state.
  • cerebrospinal fluid is different from blood and urine, which are handled in general medical examinations, and it requires special techniques for collection and is a heavy burden on patients, making it unsuitable as a screening test for the elderly. It is.
  • oxidative stress markers In addition, various oxidative stress markers, inflammation markers, apoptosis-related markers, and the like have been examined as test indicators for dementia.
  • Glycated protein glycated by oxidation and 8-hydroxyguanosine with oxidized nucleic acid have been reported to increase in the cerebrospinal fluid of Alzheimer's disease
  • F2-isoprosane an oxidation product of unsaturated fatty acids
  • IL-1 ⁇ , IL-6, TGF- ⁇ 1 and the like are increased in cerebrospinal fluid among inflammation markers
  • TRAIL related to apoptosis is also increased in cerebrospinal fluid.
  • oxidative stress, inflammation, and apoptosis are all closely related to the pathology and pathology of dementia including Alzheimer's disease, they are not necessarily specific, so they are preferable as biomarkers for dementia. It is not a thing.
  • PlsEtn Ethanolamine plasmalogen
  • Pls Plasmalogen
  • Pls is one of the subclasses of glycerophospholipid, accounting for about 20% of phospholipids constituting the human body as a biological membrane component, and is contained in a large amount in the brain and heart. Pls has been studied as a biomarker for lifestyle-related diseases (arteriosclerosis, hyperlipidemia, diabetes, hypertension, central obesity) (Patent Documents 1 and 2).
  • ethanolamine-type plasmalogen (PlsEtn) in the brain is a major component of the myelin membrane that forms the nerve myelin sheath, and is also involved in synapse formation between nerve cells.
  • PlsEtn functions as a membrane modulator and is involved in cell membrane fusion and endocytosis / exocytosis, as well as in the regulation of membrane-bound enzyme activity.
  • Pls is considered to act as an antioxidant in vivo because the vinyl ether bond, which is a structural feature, has radical scavenging ability.
  • PlsEtn has been reported to have a specific decrease in the brain level due to Alzheimer's disease, Down's syndrome, spinal cord injury, multiple sclerosis, and the like (Non-patent Document 1).
  • PlsEtn has attracted attention as an oxidative stress marker for Alzheimer's disease because of the accumulation of biochemical and clinical evidence that supports the importance of oxidative stress in the early stages of Alzheimer's disease ( Non-patent document 2).
  • a decrease in serum or plasma concentration of PlsEtn22: 6, a molecular species of PlsEtn can be an early detection marker for Alzheimer's disease (Non-patent Document 3).
  • Known methods for measuring PlsEtn include the radioactive iodine-high performance liquid chromatography (125I-HPLC) method and LC-MS method (Patent Document 5) described in Patent Document 1.
  • MI Myoinositol
  • MI Myoinositol
  • MI in the living body is biosynthesized from glucose in the body.
  • MI is important as a synthetic material for inositol phospholipids, which are sources of intracellular signal transmitters, and inositol phosphates involved in maintaining calcium ion homeostasis.
  • MI itself is considered to play an important role in maintaining normal function of the central nervous system, as an organic osmotic pressure regulating substance in the brain and possibly in the involvement of myelin formation.
  • the brain MI concentration is about 200 times the blood level, and increases in brain MI have been reported in various pathological conditions such as emotional disorders such as depression and Down's syndrome. An increase in the amount of MI in the brain has also been reported in Alzheimer's disease patients. (Non-patent document 6). A portion of the MI in the brain passes through the brain blood barrier and enters the circulating blood, but most of it is reabsorbed or decomposed by the kidney and is excreted in the urine (hereinafter referred to as “urine MI amount”). There are very few).
  • Patent Document 3 Since the reabsorption process antagonizes blood glucose, the amount of urinary MI increases reflecting the level in hyperglycemia, so it is a focus of attention as a measurement item that can replace blood glucose when detecting borderline diabetes in the glucose tolerance test It is used as a biomarker for the detection of a pre-diabetes group (Patent Document 3) or the detection of mild glucose intolerance and insulin secretion failure (Patent Document 4).
  • JP 2007-33410 A Patent No. 4176749 JP2011-257148A JP 2001-190299 A (Patent No. 3975279) International Publication WO2003 / 083133 (Patent No. 4466912) JP 2011-136926 A
  • biomarkers As described above, early detection is important for preventing or suppressing the progression of dementia, and implementation of large-scale screening using a biomarker that is an objective index is effective. What is required of biomarkers is that they can accurately identify patients with dementia or their predecessors (ie, without being confused with patients with other diseases), and can be performed within the scope of normal health examinations. Such simple measurement (quantification) and determination are possible.
  • the serum and / or plasma PlsEtn amount is promising as a biomarker for dementia testing, particularly for its large-scale screening.
  • the determination accuracy when the serum / plasma PlsEtn amount is used as a single index is not always sufficient. For example, as shown in the examples described later, among subjects whose PlsEtn amount was lower than the average value (58 ⁇ M), the proportion of those who were determined to have dementia in the cognitive function test was less than 60%.
  • An object of the present invention is to provide means for further improving the accuracy of a dementia test using serum and / or plasma PlsEtn levels as biomarkers.
  • an object of the present invention is to provide a convenient method for measuring PlsEtn that can be applied to a general-purpose automatic analyzer.
  • the present inventors improve the accuracy of the dementia test by combining the quantitative value of urinary MI and the quantitative value of serum and / or plasma PlsEtn, which are not related to the cognitive function by the quantification alone. And newly found lysoplasmalogenase (sometimes abbreviated as Lysoplasmalogenase, lyPlsase) and phospholipase (hereinafter sometimes abbreviated as PL) that act specifically on PlsEtn, and PlsEtn in the serum or plasma of the subject
  • the present invention was completed by creating a dementia test reagent kit containing lyPlsase and PL for quantifying the amount of lysine.
  • a method for testing dementia for classifying whether a subject is a dementia patient or a person in the preceding stage, Quantifying myo-inositol (MI) in spontaneously excreted urine of a subject and quantifying ethanolamine plasmalogen (PlsEtn) in serum or plasma of the same subject, A pre-determined threshold of myo-inositol (MI) in spontaneously excreted urine and ethanolamine plasmalogens in serum or plasma, which can distinguish between healthy and demented patients or those in its predecessor
  • a dementia test method comprising a step of comparing a quantitative value of myo-inositol (MI) and a quantitative value of ethanolamine-type plasmalogen (PlsEtn) of the subject with respect to each of previously determined threshold values of (PlsEtn).
  • Hydrolyzing to plasmalogen (lyPlsEtn): (2) Using a hydrolase that can hydrolyze ethanolamine-type lysoplasmalogen (lyPlsEtn) into glycero-3-phosphoethanolamine and an aldehyde and cannot hydrolyze ether-type lysophosphatidylethanolamine, (1) Hydrolyzing ethanolamine-type lysoplasmalogen (lyPlsEtn) produced in step 1 to glycero-3-phosphoethanolamine and aldehyde: and (3-1) hydrolyzing glycero-3-phosphoethanolamine A step of hydrolyzing the glycero-3-phosphoethanolamine obtained in step (2) to ethanolamine with an enzyme that is made to be ethanolamine: (3-2) Step of generating hydrogen peroxide from ethanolamine by an enzyme that oxidizes ethanolamine: (3-3) Step of quantifying hydrogen peroxide by means of hydrogen peroxide quantification: (3-4) Step of calculating the amount of PlsEtn in
  • step of decomposing and substantially eliminating lysophosphatidylethanolamine produced by the step (4) using lipase and / or monoglyceride lipase
  • lyPlsEtn ethanolamine-type lysoplasmalogen
  • the enzyme capable of hydrolyzing the ethanolamine type plasmalogen (PlsEtn) in the step (1) to an ethanolamine type lysoplasmalogen (lyPlsEtn) is one of the following enzymes (c) or (d) [1-2 ] Method.
  • step of decomposing and substantially eliminating lysophosphatidylethanolamine produced by the step (4) using lipase and / or monoglyceride lipase
  • Phospholipase is used as an enzyme that decomposes the phosphatidylethanolamine of the step (4) into lysophosphatidylethanolamine and a fatty acid, and the PL uses the ethanolamine-type plasmalogen (PlsEtn) of the step (1).
  • the step (1) is allowed to proceed without adding new PL, which also serves as an enzyme capable of hydrolyzing ethanolamine-type lysoplasmalogen (lyPlsEtn).
  • the step (1) is completed, and then the steps (2) to (3-4) are performed.
  • Hydrolyzing to plasmalogen (lyPlsEtn): (2) Hydrolyzing enzyme capable of hydrolyzing ethanolamine type lysoplasmalogen (lyPlsEtn) to ethanolamine and plasmenyl phosphatidic acid, hydrolyzing ether type lysophosphatidylethanolamine, and hydrolyzing lysophosphatidylethanolamine Hydrolyzing the ethanolamine-type lysoplasmalogen (lyPlsEtn) produced in the above (1) into ethanolamine and plasmenylphosphatidic acid using: and (3-1) by an enzyme that oxidizes ethanolamine
  • [1-8-1] A method for quantifying ethanolamine-type plasmalogen (PlsEtn) in serum or plasma of the same subject is performed prior to the steps (1) to (3-3) ′ or at least before the step (2) ′.
  • step of decomposing and substantially eliminating lysophosphatidylethanolamine produced by the step (4) using lipase and / or monoglyceride lipase
  • Phospholipase is used as an enzyme that decomposes the phosphatidylethanolamine of the step (4) into lysophosphatidylethanolamine and a fatty acid, and the PL uses the ethanolamine-type plasmalogen (PlsEtn) of the step (1).
  • the step (1) is allowed to proceed without adding new PL, which also serves as an enzyme capable of hydrolyzing ethanolamine-type lysoplasmalogen (lyPlsEtn).
  • the step (1) is completed, and then the steps (2) to (3-3) ′ are carried out [1-8-1].
  • a predetermined threshold value of myo-inositol (MI) in spontaneously excreted urine is a value selected from the range of 21.6 to 63.3 mg / gCr, and ethanolamine-type plasmalogen (PlsEtn) in serum or plasma Is a value selected from the range of 50 to 64 ⁇ M, and the quantitative value of myo-inositol (MI) in the spontaneously excreted urine of the subject exceeds the MI threshold, and the serum of the subject or
  • the method according to [1] wherein when the quantitative value of ethanolamine-type plasmalogen (PlsEtn) in plasma falls below the PlsEtn threshold value, it is distinguished from a dementia patient or a person in the previous stage.
  • the MI amount of the subject is not less than a threshold value set from a range of 21.6 to 55.1 mg / gCr, and the PlsEtn amount is not more than a threshold value set from a range of 50 to 64 ⁇ M.
  • the MI amount of the subject is not less than a threshold value set from a range of 21.6 to 55.1 mg / gCr, and the PlsEtn amount is not more than a threshold value set from a range of 52 to 61 ⁇ M.
  • the MI amount of the subject is not less than a threshold value set from a range of 29.8 to 63.3 mg / gCr, and the PlsEtn amount is not more than a threshold value set from a range of 50 to 64 ⁇ M.
  • the subject's MI amount is not less than a threshold value set from a range of 29.8 to 63.3 mg / gCr, and the PlsEtn amount is not more than a threshold value set from a range of 52 to 61 ⁇ M.
  • a predetermined threshold of myo-inositol (MI) in spontaneously excreted urine, which can distinguish between a healthy person and a person with dementia or in the previous stage, and myo-inositol in the subject's spontaneously excreted urine ( MI) is used for a dementia test for classifying whether a subject is a dementia patient or a person in the previous stage by combining with a step of comparing with a quantitative value of MI, Quantifying ethanolamine-type plasmalogen (PlsEtn) in the serum or plasma of the same subject, during which a healthy person can be distinguished from a patient with dementia or in its predecessor stage,
  • a method for quantifying ethanolamine-type plasmalogen comprising a step of comparing a quantitative value of ethanolamine-type plasmalogen (PlsEtn) of a subject against a predetermined threshold value of ethanolamine-type plasmalogen (PlsEtn).
  • Hydrolyzing to plasmalogen (lyPlsEtn): (2) Using a hydrolase that can hydrolyze ethanolamine-type lysoplasmalogen (lyPlsEtn) into glycero-3-phosphoethanolamine and an aldehyde and cannot hydrolyze ether-type lysophosphatidylethanolamine, (1) Hydrolyzing ethanolamine-type lysoplasmalogen (lyPlsEtn) produced in step 1 to glycero-3-phosphoethanolamine and aldehyde: and (3-1) hydrolyzing glycero-3-phosphoethanolamine A step of hydrolyzing the glycero-3-phosphoethanolamine obtained in step (2) to ethanolamine with an enzyme that is made to be ethanolamine: (3-2) Step of generating hydrogen peroxide from ethanolamine by an enzyme that oxidizes ethanolamine: (3-3) Step of quantifying hydrogen peroxide by means of hydrogen peroxide quantification: (3-4) Step of calculating the amount of PlsEtn in
  • step of decomposing and substantially eliminating lysophosphatidylethanolamine produced by the step (4) using lipase and / or monoglyceride lipase
  • the enzyme capable of hydrolyzing the ethanolamine-type plasmalogen (PlsEtn) in the step (1) to the ethanolamine-type lysoplasmalogen (lyPlsEtn) is one of the following enzymes (c) or (d) [2-2. ] Method.
  • step of decomposing and substantially eliminating lysophosphatidylethanolamine produced by the step (4) using lipase and / or monoglyceride lipase
  • Phospholipase is used as an enzyme that decomposes the phosphatidylethanolamine of the step (4) into lysophosphatidylethanolamine and a fatty acid, and the PL uses the ethanolamine-type plasmalogen (PlsEtn) of the step (1).
  • the step (1) is allowed to proceed without adding new PL, which also serves as an enzyme capable of hydrolyzing ethanolamine-type lysoplasmalogen (lyPlsEtn).
  • the step (1) is completed, and then the steps (2) to (3-4) are performed [2-5].
  • Hydrolyzing to plasmalogen (lyPlsEtn): (2) Hydrolyzing enzyme capable of hydrolyzing ethanolamine type lysoplasmalogen (lyPlsEtn) to ethanolamine and plasmenyl phosphatidic acid, hydrolyzing ether type lysophosphatidylethanolamine, and hydrolyzing lysophosphatidylethanolamine Hydrolyzing the ethanolamine-type lysoplasmalogen (lyPlsEtn) produced in the above (1) into ethanolamine and plasmenylphosphatidic acid using: and (3-1) by an enzyme that oxidizes ethanolamine
  • [2-8] A method for quantifying ethanolamine plasmalogen (PlsEtn) in serum or plasma of the same subject is performed prior to the steps (1) to (3-3) ′ or at least before the step (2).
  • step of decomposing and substantially eliminating lysophosphatidylethanolamine produced by the step (4) using lipase and / or monoglyceride lipase
  • Phospholipase is used as an enzyme that decomposes the phosphatidylethanolamine of the step (4) into lysophosphatidylethanolamine and a fatty acid, and the PL uses the ethanolamine-type plasmalogen (PlsEtn) of the step (1).
  • the step (1) is allowed to proceed without adding new PL, which also serves as an enzyme capable of hydrolyzing ethanolamine-type lysoplasmalogen (lyPlsEtn).
  • the step (1) is completed, and then the steps (2) to (3-3) ′ are performed [2-8].
  • a pre-determined threshold of ethanolamine-type plasmalogen (PlsEtn) in serum or plasma which can distinguish between healthy and demented patients or those in its predecessor, and in the serum or plasma of the subject
  • the subject is used for a dementia test for classifying whether or not the subject is a dementia patient or a person in its previous stage, Quantifying ethanolamine-type plasmalogen (PlsEtn) in the serum or plasma of the same subject, during which a healthy person can be distinguished from a patient with dementia or in its predecessor stage, Quantification of myo-inositol (MI) including the step of comparing the quantitative value of myo-inositol (MI) in spontaneously excreted urine of a subject against a predetermined threshold value of myo-inositol (MI) in spontaneously excreted urine Method.
  • a predetermined threshold value of myo-inositol (MI) in spontaneously excreted urine is a value selected from the range of 21.6 to 63.3 mg / gCr, and ethanolamine-type plasmalogen (PlsEtn) in serum or plasma Is a value selected from the range of 50 to 64 ⁇ M, and the quantitative value of myo-inositol (MI) in the spontaneously excreted urine of the subject exceeds the MI threshold, and the serum of the subject or
  • the method according to [3] wherein when the quantitative value of ethanolamine-type plasmalogen (PlsEtn) in plasma falls below the PlsEtn threshold, it is distinguished from a dementia patient or a person in the previous stage.
  • the subject's MI amount is not less than a threshold value set from the range of 21.6 to 55.1 mg / gCr, and the PlsEtn amount is not more than the threshold value set from the range of 50 to 64 ⁇ M.
  • the subject's MI amount is not less than a threshold value set from a range of 21.6 to 55.1 mg / gCr, and a PlsEtn amount is not more than a threshold value set from a range of 52 to 61 ⁇ M.
  • the subject's MI amount is not less than a threshold value set from a range of 29.8 to 63.3 mg / gCr, and the PlsEtn amount is not more than a threshold value set from a range of 50 to 64 ⁇ M.
  • the subject's MI amount is not less than a threshold value set from a range of 29.8 to 63.3 mg / gCr, and the PlsEtn amount is not more than a threshold value set from a range of 52 to 61 ⁇ M.
  • a method used in a dementia test method for classifying whether a subject is a dementia patient or a person in the previous stage Means for quantifying myo-inositol (MI) in spontaneously excreted urine of a subject, and means for quantifying ethanolamine-type plasmalogen (PlsEtn) in serum or plasma of the same subject, A pre-determined threshold of myo-inositol (MI) in spontaneously excreted urine and ethanolamine plasmalogens in serum or plasma, which can distinguish between healthy and demented patients or those in its predecessor
  • the reagent kit according to [4-1], wherein the reagent kit for quantifying ethanolamine plasmalogen (PlsEtn) in serum or plasma of the same subject comprises the following (1a) to (3a).
  • the hydrolase of (2a) is an enzyme capable of hydrolyzing lysophosphatidylethanolamine
  • the reagent according to [4-1] further includes an enzyme that hydrolyzes lysophosphatidylethanolamine in (1a).
  • the hydrolase of (2a) is an enzyme capable of hydrolyzing lysophosphatidylethanolamine
  • the reagent kit according to [4-2] further includes an enzyme that hydrolyzes lysophosphatidylethanolamine in (1a).
  • [4-3] The reagent kit according to [4-2] or [4-2-1], wherein the hydrolase of (2a) is any of the following enzymes (a) or (b).
  • lyPlsEtn ethanolamine-type lysoplasmalogen
  • the enzyme capable of hydrolyzing the ethanolamine type plasmalogen (PlsEtn) of (1) to an ethanolamine type lysoplasmalogen (lyPlsEtn) is one of the following enzymes (c) or (d) [4-2] Or the reagent kit according to [4-2-1].
  • a predetermined threshold of myo-inositol (MI) in spontaneously excreted urine, which can distinguish between a healthy person and a person with dementia or in the previous stage, and myo-inositol in the subject's spontaneously excreted urine ( MI) is used for a dementia test for classifying whether a subject is a dementia patient or a person in the previous stage by combining with a step of comparing with a quantitative value of MI, Quantifying ethanolamine-type plasmalogen (PlsEtn) in the serum or plasma of the same subject, during which a healthy person can be distinguished from a patient with dementia or in its predecessor stage, Ethanolamine used by comparing the quantitative value of ethanolamine-type plasmalogen (PlsEtn) in serum or plasma of a subject against a predetermined threshold of ethanolamine-type plasmalogen (PlsEtn) in serum or plasma Quantitative means of type plasmalogen.
  • MI myo-inositol
  • PlsEtn
  • the reagent kit according to [5-1], wherein the reagent kit for quantifying ethanolamine plasmalogen (PlsEtn) in serum or plasma of the same subject comprises the following (1a) to (3a).
  • An ethanolamine-type lysoplasmalogen (lyPlsEtn) Hydrolytic enzyme capable of hydrolyzing glycero-3-phosphoethanolamine and aldehyde and not hydrolyzing ether-type lysophosphatidylethanolamine, and additive capable of proceeding the enzymatic reaction (3a) glycero-3-phosphoethanol Enzymes that convert amines to ethanolamine and glycerophosphoric acid, ethanolamines to glycolaldehyde, ammonia, and hydrogen peroxide and additive
  • the reagent kit according to [5-1] further includes an enzyme that hydrolyzes lysophosphatidylethanolamine in (1a).
  • the hydrolase of (2a) is an enzyme capable of hydrolyzing lysophosphatidylethanolamine
  • the reagent kit according to [5-2] further includes an enzyme that hydrolyzes lysophosphatidylethanolamine in (1a).
  • [5-3] The reagent kit according to [5-2] or [5-2-1], wherein the hydrolase of (2a) is any of the following enzymes (a) or (b).
  • lyPlsEtn ethanolamine-type lysoplasmalogen
  • the enzyme capable of hydrolyzing the ethanolamine-type plasmalogen (PlsEtn) of (1a) to an ethanolamine-type lysoplasmogen (lyPlsEtn) is one of the following enzymes (c) or (d) [5-2] Or the reagent kit according to [5-2-1].
  • a pre-determined threshold of ethanolamine-type plasmalogen (PlsEtn) in serum or plasma which can distinguish between healthy and demented patients or those in its predecessor, and in the serum or plasma of the subject
  • the subject is used for a dementia test for classifying whether or not the subject is a dementia patient or a person in its previous stage
  • Quantifying ethanolamine-type plasmalogen (PlsEtn) in the serum or plasma of the same subject during which a healthy person can be distinguished from a patient with dementia or in its predecessor stage
  • Means for quantifying myo-inositol (MI) which is used by comparing the quantitative value of myo-inositol (MI) in spontaneously excreted urine of a subject with a predetermined threshold of myo-inositol (MI) in spontaneously excreted urine .
  • the dementia in the embodiment of the present invention refers to a cognitive disorder including intelligence, memory, and orientation, does not include a phenomenon associated with aging, and refers to a disorder whose ability is reduced morbidly.
  • a “dementia patient” refers to a person (particularly an elderly person) who has difficulty in social life due to a disorder such as memory or judgment. Specifically, it is a person who is determined to have a cognitive impairment level by a cognitive function test (for example, a CDR score of 1.0 or more).
  • a person in the previous stage of dementia is, for example, a person whose decline in memory and judgment is observed in daily life, such as WAIS-R, HDS-R, MMS, MMSE, CDR, MoCA-J.
  • a person having a mildest level that can be detected by a cognitive function testing method such as a CDR score of 0.5 or more and less than 1.0 is preferable.
  • diseases that cause dementia include cerebrovascular disorders, Alzheimer's disease, normal pressure hydrocephalus, metabolic disorders, nutritional disorders, hypothyroidism, and depression, and Alzheimer's disease is an example.
  • Means refers to a general term for a composition, a reagent, an enzyme, or a combination thereof.
  • each component in the composition may be a single composition, but when two or more compositions are separated, the two or more compositions are collectively referred to as a kit.
  • Quantitative means a concept including “measurement”, “detection”, and the like. The following is presented as an invention related to the present invention.
  • a method for quantifying PlsEtn in a sample of a subject comprising the following steps (1) to (3-4): (1) Using an enzyme capable of hydrolyzing ethanolamine-type plasmalogen (PlsEtn) into ethanolamine-type lysoplasmalogen (lyPlsEtn), ethanolamine-type plasmalogen (PlsEtn) in the serum or plasma of the subject is ethanolamine-type lysozyme.
  • step of decomposing and substantially eliminating lysophosphatidylethanolamine produced by the step (4) using lipase and / or monoglyceride lipase
  • the enzyme capable of hydrolyzing the ethanolamine-type plasmalogen (PlsEtn) in the step (1) to an ethanolamine-type lysoplasmalogen (lyPlsEtn) is any one of the following enzymes (c) or (d): [7] The method described.
  • the hydrolase in (2) is an enzyme capable of hydrolyzing lysophosphatidylethanolamine
  • the method for quantifying PlsEtn of a subject sample precedes the steps (1) to (3-4), The method according to [7], wherein the step including the following (4) and (5) is performed at least before the step (2).
  • step of decomposing and substantially eliminating lysophosphatidylethanolamine produced by the step (4) using lipase and / or monoglyceride lipase
  • Phospholipase is used as an enzyme that decomposes the phosphatidylethanolamine of the step (4) into lysophosphatidylethanolamine and a fatty acid, and the PL uses the ethanolamine-type plasmalogen (PlsEtn) of the step (1). Simultaneously with the implementation of the step (5), the step (1) is allowed to proceed without adding new PL, which also serves as an enzyme capable of hydrolyzing ethanolamine-type lysoplasmalogen (lyPlsEtn). Or the method according to [10], wherein the step (1) is completed after the step (5), and the steps (2) to (3-4) are performed thereafter.
  • a reagent kit for quantifying ethanolamine-type plasmalogen (PlsEtn) in serum or plasma of a subject comprising the following (1a) to (3a): (1a) An enzyme capable of hydrolyzing ethanolamine type plasmalogen (PlsEtn) to ethanolamine type lysoplasmalogen (lyPlsEtn) and an additive capable of proceeding with the enzyme reaction. (2a) a hydrolase capable of hydrolyzing ethanolamine-type lysoplasmalogen (lyPlsEtn) into glycero-3-phosphoethanolamine and aldehyde and not hydrolyzing ether-type lysophosphatidylethanolamine, and advancing the enzyme reaction thereof Possible additive.
  • the reagent kit further comprises (1a) an enzyme that hydrolyzes lysophosphatidylethanolamine.
  • the hydrolase of (2a) is an enzyme capable of hydrolyzing lysophosphatidylethanolamine
  • the reagent kit according to [12] further comprising (1a) an enzyme that hydrolyzes lysophosphatidylethanolamine.
  • the enzyme capable of hydrolyzing the ethanolamine type plasmalogen (PlsEtn) of (1a) to an ethanolamine type lysoplasmalogen (lyPlsEtn) is any of the following enzymes (c) or (d) [12] or [ 12-1].
  • a gene comprising the following base sequence (A) or (B).
  • (A) Encodes an enzyme consisting of the amino acid sequence set forth in SEQ ID NO: 1 or 2:
  • B An enzyme comprising an amino acid sequence in which one or a plurality of amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 or 2, and an ethanolamine-type lysoplasmalogen (lyPlsEtn) is hydrolyzed It encodes an enzyme that catalyzes a reaction that decomposes to give glycero-3-phosphoethanolamine and an aldehyde.
  • a method for producing the enzyme comprising: A step of culturing the microorganism having the gene according to [15] or the transformant according to [17] in a medium: Producing and accumulating the enzyme according to [13] in the culture: and collecting the protein from the culture: Including methods.
  • the level of cognitive impairment can be evaluated with high accuracy. Therefore, it is possible to specify not only patients who have already developed dementia, but also those who are in the previous stage of dementia.
  • the correlation diagram which compared the average value of the MI quantitative value in the urinary urine of the test for 3 days, and the fasting second urine MI quantitative value of the test.
  • the correlation diagram which compared the quantitative value of PlsEtn in serum or plasma using 125I-HPLC method and lyPlsase derived from Pseudomonas putida. 125I-HPLC method and Thermocrispum sp.
  • the correlation diagram which compared the quantitative value of PlsEtn in the serum or plasma using 125I-HPLC method and PLD.
  • Thermocrispum sp. SDS-PAGE showing the origin lyPls ase Streptomyces lividans recombinant enzyme (arrow in the figure).
  • Thermocrispum sp. SDS-PAGE showing the derived lyPls as E. coli recombinant enzyme (arrow in the figure).
  • the present invention includes a step of quantifying myo-inositol (MI) in spontaneously excreted urine of a subject and a step of quantifying ethanolamine-type plasmalogen (PlsEtn) in serum or plasma of the same subject. It is a dementia test method for classifying whether a patient or a person in the previous stage.
  • the method for testing dementia includes a step of quantifying ethanolamine-type plasmalogen (PlsEtn) in serum or plasma of the same subject, and comprises a healthy person and a dementia patient or a person in the previous stage.
  • the present invention relates to the method for testing dementia, comprising the step of comparing the quantitative values of the subject's MI and PlsEtn against the predetermined threshold values of MI and PlsEtn, which can be distinguished from each other.
  • the test subject refers to a healthy person or patient who is a subject of urine collection and / or blood collection, and is not limited to age, sex, race, basic disease, and includes non-human laboratory animals. In the case of humans, it may be an individual who participates in clinical trials and receives clinical study drugs or is the subject, ie, clinical trial monitor, pharmaceutical monitor, pharmaceutical volunteer, drug discovery volunteer, etc., and WAIS-R, HDS- It may be a patient or a healthy person suspected of having a dementia in a cognitive function test such as R, MMS, MMSE, CDR, or MoCA-J, or a patient or a healthy person having a consciousness and / or objective symptom of dementia.
  • the subject does not need to have a dietary restriction, but usually has a dietary restriction in the case of a medical examination.
  • the urine in the embodiment of the present invention is spontaneously excreted urine that is not forcibly excreted by drugs or the like.
  • This spontaneously excreted urine may be anytime urine excreted when feeling urinary, but is preferably early morning fasting urine excreted before breakfast in the average life cycle, more preferably early morning fasting Second fasting urine excreted on an empty stomach after urine.
  • These urines are quantitatively measured every day or every other day (within one month) for 3 days. If it is difficult to collect urine for 3 days, it may be 2 days or 1 day, but the urine in that case is preferably fasting second urine. When it is difficult to collect fasting second urine, early morning fasting urine may be used, but in that case, it is preferable to collect urine for three days or more.
  • the present invention includes a step of quantifying MI in a subject's spontaneous excretion urine.
  • the MI in the embodiment of the present invention includes a known MI described in the background art. MI is quantified by GC-MASS (Toshimitsu Niwa, J. Chromatography, 227 (1983), 25-39), immunoassay (Japanese Patent Laid-Open No. 8-21835), enzyme method [Rosita Dolohofer, O. H. Wieland. , J .; Clin. Chem. Clin. Biochem.
  • the urine in the embodiment of the present invention is preferably stored refrigerated until it is fixed (stable for one year or more). There is no problem even if it is left at room temperature within a few days. Addition of 0.05% sodium azide or 0.1% procrine as a preservative does not affect the determination.
  • MI may be performed by a method of detecting the presence of MI by a qualitative reaction, but is preferably quantified.
  • MI is quantified by detecting MI in a known method for urine of a subject that may contain MI and a calibration sample containing MI at a known concentration.
  • concentration of MI in the urine of a subject who may be subject to the detection amount for the urine of a subject who may contain MI and the detection amount for a calibration sample containing MI at a known concentration Is calculated by comparing.
  • the amount of urine MI is displayed as a value (mg) standardized with the creatinine 1 g (gCr) value measured simultaneously, and is usually preferably calculated as an average value of a plurality of samples, and other quantitative values among three or more samples If there is a specimen showing a value that is significantly different from the quantitative value, it is preferable to exclude this. Further, when only two samples are quantified, if the quantified values of both are greatly different, re-quantification may be performed.
  • the present invention includes the step of quantifying PlsEtn in the serum or plasma (serum and / or plasma) of the same subject to quantify MI.
  • the same subject for quantifying MI includes subjects whose MI has been quantified, and the order of quantification of MI and PlsEtn is arbitrary.
  • Blood is collected from the subject as usual, centrifuged as soon as possible, and the collected serum or plasma is immediately refrigerated (4 ° C.) or frozen ( ⁇ 20 ° C. to ⁇ 80 ° C.). In the case of temporary storage in the state of blood, it is preferable to store in a refrigerated state and to perform treatment up to serum or plasma separation at least on the day of blood collection.
  • a separating agent or antiplasmin agent is not particularly limited, and anticoagulants and antiglycolytic agents such as EDTA, sodium fluoride, sodium citrate, heparin sodium, monoiodoacetic acid, etc. Whether or not is used is not particularly limited.
  • the “specimen” or “sample” in the embodiment of the present invention is a sample expected to contain PlsEtn, and may include urine, serum and plasma of the above-mentioned subject, and may be a solution, serum and plasma to which PlsEtn is added.
  • the “specimen” or “sample” in the embodiment of the present invention is not limited as long as it is a sample expected to contain PlsEtn, but is preferably urine, serum, and / or plasma of a subject. Examples of other samples include urine derived from laboratory animals, serum, plasma, plant tissue, seawater, natural water, fruit juice, beverages, waste liquid, and the like.
  • PlsEtn is an alkenyl acyl glycerophospholipid (alkenylacyl ether phospholipid) in which a fatty acid is vinyl ether-bonded to the C1 (sn-1) position of glycerophospholipid, and the base is ethanolamine. and thrombosis, Vol. 14, No. 1, 2007, pp. 12-18 and J. Am. Psychiatry Neurosci. 2010, Vol. 35, No. 1, pp. 59-62, etc., which is a known PlsEtn, and PlsEtn of the following formula. :
  • PlsEtn in the embodiment of the present invention may include a PlsEtn lyso form (lyPlsEtn) of the following formula.
  • the present invention comprises a step of quantifying MI in spontaneously excreted urine of a subject and a step of quantifying PlsEtn in the serum or plasma of the same subject.
  • a dementia test method for classifying MI and PlsEtn which can discriminate between a healthy person and a dementia patient or a person in the previous stage, at a predetermined threshold value.
  • inspection method including the process of comparing the quantitative value of MI and PlsEtn of a test subject is also included.
  • the urinary MI amount criterion for classifying a subject as dementia or a person in the previous stage can distinguish a healthy person from a dementia patient or a person in the previous stage.
  • the quantitative values of the subject's MI and PlsEtn are compared with the predetermined threshold values of MI and PlsEtn.
  • the threshold value of urine MI may be set from the range from the average value of normal adults ⁇ the upper limit of standard deviation to the average value of healthy elderly people ⁇ the upper limit of standard deviation.
  • the urinary MI amount standard for classifying a subject as dementia or a person in the previous stage is 21.6 to 55.1 mg / gCr in the case of fasting second urine. It is good also as more than the threshold set up.
  • the detection accuracy of dementia is improved by the combined use with the quantification of the serum and / or plasma PlsEtn amount. It is also preferable to adopt 25 mg / gCr as the threshold of the MI amount.
  • the detection accuracy of dementia is improved by the combined use with the quantification of the serum and / or plasma PlsEtn amount. It is also preferable to adopt 36.6 mg / gCr as the threshold value for the urine MI amount.
  • the amount of ad libitum urine MI is on average increased by 8.2 mg / gCr from the fasting second urine, so it is set within the range of 29.8 to 63.3 mg / gCr. It may be greater than the threshold, and in the case of occasional urine, it is also preferable to employ 33.2 or 44.8 mg / gCr as the threshold.
  • the lower limit value of the PlsEtn threshold value in the serum or plasma of the subject may be set 1 to 2 ⁇ M lower than the average value of the normal adult ⁇ the lower limit value of the standard deviation and the average value of the healthy elderly person ⁇ the lower limit value of the standard deviation.
  • the upper limit of the threshold value of PlsEtn in the serum or plasma of the test subject is the correlation curve equation of CDR and PlsEtn created for all elderly people (healthy + cognitive impairment) and older subjects with average urinary MI levels or higher.
  • Y 0.5 (CDR, suspected cognitive impairment) may be used as x.
  • the step of quantifying PlsEtn in a subject's serum or plasma is a threshold value of the serum and / or plasma PlsEtn amount when the amount of serum and / or plasma PlsEtn described below is quantified using a 125I-HPLC method.
  • the step of quantifying PlsEtn in the serum or plasma of the subject comprises the upper limit of the threshold value of the serum and / or plasma PlsEtn amount when the amount of serum and / or plasma PlsEtn described below is quantified using the 125I-HPLC method.
  • the serum and / or plasma PlsEtn amount standard for classifying a subject as dementia or a person in the previous stage is the step of quantifying PlsEtn in the subject's serum or plasma.
  • the serum and / or plasma PlsEtn amount to be quantified using the 125I-HPLC method may be equal to or lower than a threshold value set from a range of 52 to 61 ⁇ M.
  • the average value of the measured values is from the average value of ⁇ 2 ⁇ M to +3 ⁇ M in the case of quantification using the 125I-HPLC method. Range. Therefore, in the embodiment of the present invention, the serum and / or plasma PlsEtn amount standard for classifying a subject as dementia or a person in the previous stage is the step of quantifying PlsEtn in the serum or plasma of the subject. In the case of quantification using the enzyme, the serum and / or plasma PlsEtn amount may be set to a threshold value or less set from the range of 50 to 64 ⁇ M.
  • the fasting second urine MI amount threshold is 36.6 mg / gCr
  • urinary urine, and serum and / or plasma PlsEtn amount is quantified by 125I-HPLC method
  • the threshold value of serum and / or plasma PlsEtn amount is about 58 ⁇ M as described later
  • the accuracy of dementia detection is optimal, and it is particularly preferable to adopt these threshold values.
  • the step of quantifying PlsEtn in the serum or plasma of a subject is quantification using an enzyme described later, it is particularly preferable to employ a threshold value of about 56 or 61 ⁇ M for the plasma PlsEtn amount.
  • the urine MI amount and the serum and / or plasma PlsEtn amount can be quantified for all subjects, and classification can be determined based on the above-mentioned criteria.
  • the urinary MI amount may be quantified for a subject whose serum and / or plasma PlsEtn amount is a threshold or less, or the serum and / or plasma PlsEtn amount may be quantified for a subject whose urine MI amount is a threshold value or more.
  • the amount of urine MI whose quantification procedure is simpler can be quantified for all subjects, and the amount of serum and / or plasma PlsEtn can be quantified based on the results, but is not limited thereto.
  • quantifying the amount of serum and / or plasma PlsEtn means that serum or plasma of a subject who may contain PlsEtn and a calibration sample containing PlsEtn at a known concentration, respectively.
  • PlsEtn alone is detected by known methods and / or the methods of the present invention, and the concentration of PlsEtn in the serum or plasma of a subject that may contain PlsEtn may be subject to PsEtn Is calculated by comparing the detected amount of serum or plasma with the detected amount of a calibration sample containing PlsEtn at a known concentration.
  • glycero-3-phosphoethanolamine or ethanolamine since 1 mol of glycero-3-phosphoethanolamine or ethanolamine is usually obtained as a product from 1 mol of PlsEtn, glycero-3-phosphoethanolamine or ethanolamine can be quantified. PlsEtn amount can be calculated. In the embodiment of the present invention, since 1 mol of hydrogen peroxide is usually obtained as a product from 1 mol of PlsEtn, the amount of PlsEtn can be calculated by quantifying hydrogen peroxide.
  • the amount of serum and / or plasma PlsEtn can be quantified by, for example, a radioactive iodine-high performance liquid chromatography (125I-HPLC) method described in Patent Document 1.
  • PlsEtn is quantified by the 125I-HPLC method
  • serum and / or plasma is stored in a refrigerator or rapidly frozen and thawed, and a fixed amount (usually 200 ⁇ L) of serum and / or plasma is used, and lipid components are extracted by a prescribed method.
  • the sample for measurement is preferably used for the rapid quantification of PlsEtn. However, if it is temporarily stored until measurement, avoid methanol and diethyl ether and store frozen in chloroform (-20 ° C to -80 ° C). C.).
  • the step of quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention may be quantification using an enzyme.
  • the step of quantifying the amount of serum and / or plasma PlsEtn may include any one or more of the following steps (1) to (3-4), and further includes (1) to All of (3-4) may be included.
  • Hydrolyzing to plasmalogen (lyPlsEtn): (2) Using a hydrolase that can hydrolyze ethanolamine-type lysoplasmalogen (lyPlsEtn) into glycero-3-phosphoethanolamine and aldehyde, but cannot hydrolyze ether-type lysophosphatidylethanolamine, (1) The step of hydrolyzing the ethanolamine-type lysoplasmalogen (lyPlsEtn) produced into glycero-3-phosphoethanolamine and an aldehyde: and (3-1) hydrolysis of glycero-3-phosphoethanolamine to ethanol A step of hydrolyzing the glycero-3-phosphoethanolamine obtained in the step (2) to an ethanolamine with an enzyme that is converted into an amine: (3-2) Step of generating hydrogen peroxide from ethanolamine by an enzyme that oxidizes ethanolamine: (3-3) Step of quantifying hydrogen peroxide by means of hydrogen peroxide quantification: (3-4) Step of calculating the amount of
  • Step (1) is a step of hydrolyzing PlsEtn to lyPlsEtn by the action of PL.
  • an enzyme that hydrolyzes lysophosphatidylethanolamine preferably a step of hydrolyzing lysophosphatidylethanolamine using lysophospholipase (may be abbreviated as LYPL) may be included.
  • Step (2) is a step of hydrolyzing lyPlsEtn by the action of a hydrolase.
  • Step (3-1) is a step in which the product obtained in step (2) is converted to hydrogen peroxide (H 2 O 2 ) by the action of glycephophosphocholine phosphodiesterase (GPCP) and ethanolamine oxidase (EAO). Hydrogen peroxide in the step (3-2) may be quantified by a known method described later.
  • GPCP glycephophosphocholine phosphodiesterase
  • EAO ethanolamine oxidase
  • a preferred example of the step (1) shown in FIG. 7 is a step of hydrolyzing PlsEtn into lyPlsEtn and a fatty acid by the action of PL.
  • a preferred example of the above step (2) shown in FIG. 7 is to hydrolyze lyPlsEtn into aldehyde corresponding to glycero-3-phosphoethanolamine by the action of lyPls ase. It is a process.
  • a preferred example of the above step (3-1) shown in FIG. 7 is that glycero-3-phosphoethanolamine obtained in step (2) is glycero-3-phosphoethanolamine (GPCP) by the action of glycerophosphophosphoesterase (GPCP).
  • Ethanolamine is hydrolyzed to ethanolamine and glycerophosphoric acid (sn-glycerol 3-phosphate), and ethanolamine is converted into hydrogen peroxide (H 2 O 2 ), ammonia (NH 3 , ammonium salt by the action of ethanolamine oxidase. And oxidation to glycolaldehyde (glycolaldehyde).
  • PL used in step (1) acts to hydrolyze PlsEtn to lyPlsEtn.
  • the enzyme is not limited as long as it catalyzes.
  • a preferred example of such an enzyme is PL having the amino acid sequence of SEQ ID NO: 5.
  • Examples of the amino acid sequence shown in SEQ ID NO: 5 include an enzyme consisting of an amino acid sequence in which one or a plurality of amino acids are deleted, substituted or added, and catalyzing the action of hydrolyzing Pls to lyPls. Is the same as in the case of lyPlase of the present invention described later.
  • the PL is a novel phospholipase A 1 that can be produced by the production method described in International Publication Number: WO2012 / 105565 A1 using NA297 (deposited as NITE BP-1014) classified as Streptomyces albidoflavus.
  • step (1) is phospholipase A 2 (phospholipase A 2 and PLA 2 ), phospholipase A 1 (sometimes abbreviated as phospholipase A 1 and PLA 1 ), and phospholipase B (PLB).
  • phospholipase A 2 phospholipase A 2 and PLA 2
  • phospholipase A 1 sometimes abbreviated as phospholipase A 1 and PLA 1
  • PLB phospholipase B
  • PLA 2 contains an enzyme classified as EC 3.1.1.4. Suitable examples of PLA 2 include PLA2 derived from Streptomyces violaceoruber (PLA 2 (Asahi Kasei Pharma Corporation (T-31)) and PLA 2 Nagase (Nagase ChemteX Corp.)), Aspergillus niger derived PLA 2 (Maxa Pearl A 2 And cakezyme (both DSM Japan Co., Ltd.), porcine pancreas-derived PLA 2 (Lipomod 699L (Genencore Kyowa Co., Ltd.)), Aspergillus-derived phospholipase (Recitase (Novozymes Japan Co., Ltd.)), porcine pancreas Origin PLA 2 (SIGMA (P223)).
  • PLA 1 contains an enzyme classified as EC 3.1.1.32.
  • a preferable example of PLA 1 is derived from Aspergillus oryzae (Mitsubishi Chemical Foods Co., Ltd.).
  • PLB combines the actions of PLA 1 and PLA 2 equally or unevenly.
  • PLB include the enzyme disclosed in Japanese Patent No. 5060666, the enzyme disclosed in WO2007-010892, LPBP (Asahi Kasei Pharma Corporation (T-63)) and CEBP (Asahi Kasei Pharma Corporation (T-66)). )).
  • the degrading enzyme may be an enzyme including an enzyme that hydrolyzes lyPlsEtn to glycero-3-phosphoethanolamine and a corresponding aldehyde (may be simply referred to as aldehyde), but is not limited thereto.
  • the hydrolase is an enzyme that does not hydrolyze at least ether type lysophosphatidylethanolamine. Pseudomonas putida-derived, rat-derived, and Thermocrispum sp. The enzyme that hydrolyzes lyPlsEtn of the derived lyPlsEtn into glycero-3-phosphoethanolamine and aldehyde does not hydrolyze ether type lysophosphatidylethanolamine.
  • the hydrolase used in step (2) is EC 3.3. Includes lyPls as classified as 2.2 or EC 3.3.2.5. Examples of such enzymes include THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 28, pp. Examples include rat-derived lyPlsase disclosed in 24916-24930, and lyPlsase of the present invention described later is preferable.
  • GPCP in the method for quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention including the above steps (1) to (3-4), in step (3-1), glycerophosphocholine phosphodiesterase, GPCP May be abbreviated).
  • the GPCP includes an enzyme classified as EC 3.1.4.2, and is not limited as long as it is an enzyme that catalyzes the action of hydrolyzing glycero-3-phosphoethanolamine with ethanolamine and glycerophosphate.
  • a preferred example of GPCP is GPCP derived from Glicadium roseum (Asahi Kasei Pharma Corporation (T-33)).
  • ethanolamine oxidase can be used in step (3-2).
  • the ethanolamine oxidase is classified as monoethanolamine oxidase (EC 1.4.3.8), monoamine oxidase (EC 1.4.3.4), and primary amine oxidase (EC 1.4.3.21).
  • the enzyme is not limited as long as it is an enzyme that catalyzes the action of oxidizing ethanolamine to H 2 O 2 , NH 3 and glycol aldehyde.
  • ethanolamine oxidase an enzyme derived from Arthrobacter genus (Narrod SA and Jakoby WB, J. Biol. Chem., 239, 2189-2193, 1964), derived from Pharmacia regina Enzyme (Kulkarni AP and Hodgson E., Comp. Biochem. Physiol., B44, 407-422, 1973), Arthrobacter sp. (FERM P-06240 and BP-0421) derived from primary amine oxidase (Ota H. et al., Biosci. Biotechnol. Biochem., 72, 2732-2738, 2008), which has substrate specificity for ethanolamine.
  • step of decomposing and substantially eliminating lysophosphatidylethanolamine produced by the step (4) using lipase and / or monoglyceride lipase
  • hydrolase of (2) is an enzyme capable of hydrolyzing lysophosphatidylethanolamine
  • the subject is in a dementia patient or in the previous stage in the quantitative value of PlsEtn of this embodiment.
  • lysophosphatidylethanolamine is hydrolyzed to an extent that affects the purpose of classifying it as a person.
  • the degree of hydrolysis of lysophosphatidylethanolamine is 15% or less, further 12% or less, and may be 10% or less with respect to lyPlsEtn.
  • an enzyme that hydrolyzes lyPlsEtn derived from Pseudomonas putida and rat described later to glycero-3-phosphoethanolamine and an aldehyde is an enzyme that does not hydrolyze lysophosphatidylethanolamine.
  • Thermocrispum sp.
  • the enzyme that hydrolyzes derived lyPlsEtn into glycero-3-phosphoethanolamine and aldehyde hydrolyzes lysophosphatidylethanolamine because it hydrolyzes lysophosphatidylethanolamine to lyPlsEtn by 12%.
  • the method for quantifying the amount of serum and / or plasma PlsEtn comprises converting the phosphatidylethanolamine of the step (4) into lysophosphatidylethanolamine and a fatty acid.
  • PL is used as an enzyme for decomposing, and the PL serves as an enzyme capable of hydrolyzing PlsEtn in the step (1) into lyPlsEtn, and the step of the step (1) without adding new PL.
  • the step (1) is completed simultaneously with the implementation of the step (5) or after the implementation of the step (5), and then the steps (2) to (3-4) are carried out. good.
  • the serum or plasma of the subject does not contain phosphatidylethanolamine and / or lysophosphatidylethanolamine, or lyPlsEtn in the above step (2) is glycero-3-phosphoethanol. If the enzyme that hydrolyzes amines and aldehydes does not hydrolyze lysophosphatidylethanolamine, it may not be performed, but phosphatidylethanolamine and / or lysophosphatidylethanolamine may be present in the serum or plasma of the subject.
  • the enzyme that hydrolyzes lyPlsEtn in the step (2) into glycero-3-phosphoethanolamine and aldehyde has the action of hydrolyzing lysophosphatidylethanolamine.
  • “Erase” in “substantially erase” means that the object to be erased (for example, by the action of an enzyme) is decomposed (converted) into a substance that is not involved in the determination of PlsEtn in the present embodiment.
  • Substances not involved in quantification vary depending on the reactivity of the enzyme used, reagents, etc., but for example, the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention including the above steps (1) to (3-4) is quantified.
  • examples of the substance not involved in the measurement include glycerophosphatidic acid, glycol aldehyde, ammonia, water, oxygen and the like.
  • the above-mentioned “erasing” includes converting the erasure target into a colorless substance and excluding the detection target. .
  • “Substantially eliminate” means that the object to be erased does not affect the purpose of classifying whether the subject is a dementia patient or a person in the previous stage in the quantitative value of PlsEtn of the present embodiment.
  • Means to erase For example, when the measurement method of the present embodiment is used as a dementia inspection method described later, this means that the erasure target is erased to an extent that does not affect the accuracy of the inspection method. More specifically, although it may vary depending on the disease, for example, when the erasure target is phosphatidylethanolamine and / or lysophosphatidylethanolamine, the total amount of phosphatidylethanolamine after the erasure process is eliminated. It is preferably 10% or less before performing the step, more preferably 9% or less, and most preferably 8% or less. In the steps (4) and (5), the ether type ethanolamine phospholipid containing PlsEtn is not eliminated.
  • the PL used in the above step (4) is the same as that in the above step (1), but PLA 2 is preferable from the viewpoint of “substantially erasing” in the above steps (4) and (5). From the viewpoint of quick reactivity, Streptomyces violaceoruber-derived PLA 2 is more preferable.
  • the enzyme used in the above step (5) is not limited as long as it has an action of hydrolyzing lysophosphatidylethanolamine into glycero-3-phosphoethanolamine and a fatty acid, but may be abbreviated as lysophospholipase (LYPL). And / or monoglyceride lipase (may be abbreviated as MGLP).
  • LYPL lysophospholipase
  • MGLP monoglyceride lipase
  • the LYPL contains an enzyme classified as EC 3.1.1.5, and a preferred example is Vibrio genus-derived LYPL (Asahi Kasei Pharma Corporation (T-32)).
  • the MGLP contains an enzyme classified as EC 3.1.1.23, and preferable examples include Bacillus genus MGLP (Asahi Kasei Pharma Corporation (T-59)).
  • the step of quantifying the amount of serum and / or plasma PlsEtn is a quantification using an enzyme
  • the quantification of hydrogen peroxide is performed according to peroxidase (EC 1.11.1.1.7).
  • a dye can be produced by oxidative condensation between a chromogen such as a Trinder reagent and a coupler, and colorimetric analysis can be performed.
  • a chromogen of the Trinder type reagent phenol derivatives, aniline derivatives, toluidine derivatives and the like can be used.
  • N-dimethylaniline N, N-diethylaniline, 2,4-dichlorophenol
  • N -Ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline DAOS
  • N-ethyl-N-sulfopropyl-3,5-dimethylaniline MAOS
  • MAOS N-ethyl-N -(2-hydroxy-3-sulfopropyl) -3,5-dimethylaniline
  • MAOS N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine
  • TOOS N-ethyl- N-sulfopropyl-m-anisidine
  • ADPS N-ethyl-N-sulfopropylaniline
  • DAPS N-ethyl-N Sulfopropyl-3,5-dimethoxyaniline
  • Hydrogen peroxide can also be colored by using a leuco reagent in the presence of POD.
  • this reagent include O-dianisidine, O-tolidine, 3,3-diaminobenzidine, 3,3,5,5-tetramethylbenzidine (above, Doujin Chemical Laboratory Co., Ltd.), N- (carboxymethylamino) And carbonyl) -4,4-bis (dimethylamino) biphenylamine (DA64), 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine (DA67), and the like.
  • the quantification of hydrogen peroxide uses an absorption method, KMnO 4 or the like in addition to the above method.
  • the oxidation / reduction method, the fluorescence method, the luminescence method, or the electrode method can be used.
  • a compound that emits fluorescence by oxidation such as homovanillic acid, 4-hydroxyphenylacetic acid, tyramine, paracresol, a diacetylfluorescin derivative, or the like can be used.
  • luminol, lucigenin, isoluminol, pyrogallol, or the like can be used as a catalyst.
  • the electrode used for the electrode method is not particularly limited as long as it is a material that can exchange electrons with hydrogen peroxide. Examples thereof include platinum, gold, silver, and the like. , Known methods such as potentiometry and coulometry can be used.
  • an electron carrier may be interposed in the reaction between the oxidase or substrate and the electrode, and the resulting oxidation, reduction current, or electric quantity thereof may be measured.
  • the electron carrier any substance having an electron transfer function can be used, and examples thereof include substances such as ferrocene derivatives and quinone derivatives.
  • an electron carrier may be interposed between hydrogen peroxide generated by the oxidase reaction and the electrode, and the resulting oxidation, reduction current, or electric quantity thereof may be measured.
  • the step of quantifying the amount of serum and / or plasma PlsEtn may include one or more of the following steps (1) to (3-3) ′, and further includes (1) to ( 3-3) all of 'may be included.
  • a hydrolase capable of hydrolyzing ethanolamine-type lysoplasmalogen (lyPlsEtn) into ethanolamine and plasmenylphosphatidic acid and hydrolyzing ether-type lysophosphatidylethanolamine The step of hydrolyzing the produced ethanolamine-type
  • FIG. 8 schematically shows another example of the method for quantifying the serum and / or plasma PlsEtn amount of the embodiment of the present invention including the steps (1) to (3-3) ′.
  • Step (1) is a step of hydrolyzing PlsEtn to lyPlsEtn by the action of PL.
  • Step (2) ′ is a step of hydrolyzing lyPlsEtn by the action of a hydrolase.
  • Step (3-1) ′ is a step in which ethanolamine, which is the product obtained in step (2) ′, is converted into hydrogen peroxide by the action of EAO. What is necessary is just to quantify hydrogen peroxide by the well-known method mentioned later.
  • the PL used in the step (1) is the same as that in the above step (1). Same as the case.
  • the hydrolase includes an enzyme that hydrolyzes lyPlsEtn into plasmenyl phosphatidic acid and ethanolamine, and phospholipase D (sometimes abbreviated as phospholipase D, PLD) is preferable.
  • PLD phospholipase D
  • Examples of such PLD are derived from the genus Streptomyces (Asahi Kasei Pharma Co., Ltd.
  • T-07 Streptomyces chromofuscus (Asahi Kasei Pharma Co., Ltd. (T-39)), derived from cabbage (SIGMA (P8398)), and from the genus Actinomadura. (Meisei Sangyo Co., Ltd.) PLD. From the viewpoint of high substrate specificity for lyPlsEtn, Streptomyces chromofuscus-derived PLD is preferred.
  • Steps (1) to (3-3) ′ above In the method for quantifying the amount of serum and / or plasma PlsEtn according to an embodiment of the present invention including EAO, EAO used in step (3-1) ′ is the same as in step (3-2) described above.
  • the phosphatidylethanolamine in the above step (4) is converted into lysophosphatidylethanolamine and a fatty acid.
  • PL is used as an enzyme that decomposes into, and the PL functions as an enzyme capable of hydrolyzing PlsEtn in the step (1) to lyPlsEtn without adding new PL.
  • the step (1) is completed simultaneously with the implementation of the step (5) or after the execution of the step (5), and then the steps (2) ′ to (3-3) ′ are performed. The method to do is also good.
  • the serum or plasma of the subject does not contain phosphatidylethanolamine and / or lysophosphatidylethanolamine, or the lyPlsEtn of the above step (2) ′ is changed to ethanolamine and plasmenyl phosphatidine. If a hydrolase that can hydrolyze to acid does not hydrolyze lysophosphatidylethanolamine, it may not be carried out, but phosphatidylethanolamine and / or lysophosphatidylethanolamine may be present in the serum or plasma of a subject.
  • hydrolase that is included and can hydrolyze the lyPlsEtn of the step (2) ′ into ethanolamine and plasmenyl phosphatidic acid has an action of hydrolyzing lysophosphatidylethanolamine. Good.
  • “erase” of “substantially erase” is the same as described above, and substances that are not involved in quantification vary depending on the reactivity of the enzyme used, reagents, etc., but for example, PlsEtn of this embodiment
  • substances not involved in the measurement include glycerophosphoethanolamine (however, , Monoacylglycerol, diacylglycerol, phosphorylethanolamine, fatty acid, ceramide, glycerol-3-phosphate, ammonia, glycolaldehyde, water, oxygen and the like.
  • the PlsEtn quantification according to the present embodiment is performed by quantification using colorimetric analysis
  • the above-mentioned “erasing” includes converting the erasure target into a colorless substance and excluding the detection target. .
  • “Substantially erase” is the same as described above. More specifically, it may vary depending on the disease, but when the object to be erased is, for example, phosphatidylethanolamine and / or lysophosphatidylethanolamine, The total amount in the case of phosphatidylethanolamine and / or lysophosphatidylethanolamine after performing these erasing steps is preferably 10% or less, more preferably 9% or less before performing the erasing step. Most preferably, it is 8% or less.
  • the step of quantifying the serum and / or plasma PlsEtn amount using an enzyme according to the embodiment of the present invention may be a method described in JP2012-210179A.
  • any enzyme that can be used in the quantification using the enzyme in the step of quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention its mutants are similarly used. Included in each enzyme range. For example, even an enzyme having an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of each enzyme is included in the range of each enzyme as long as the desired activity is maintained. Also, when each enzyme is obtained by translation of a base sequence, the base sequence to be used is not particularly limited as long as the obtained enzyme has a desired activity.
  • the secondary structure, tertiary structure and quaternary structure, properties, purity, origin, trade name and price of each enzyme are not particularly limited.
  • ascorbate oxidase is used to eliminate ascorbate contained in the sample.
  • oxidase and ASOM may be abbreviated).
  • ASOM preferably has an action of substantially eliminating ascorbic acid, and more preferably an enzyme classified as EC 1.10.3.3.
  • an ASOM derived from a Cucurbitaceae plant can be used.
  • Preferred examples are ascorbate oxidase (Asahi Kasei Pharma Co., Ltd.
  • T-53 derived from the genus Acremonium from the viewpoint of high enzyme stability, and Amano enzyme from the viewpoint of not being inhibited by sodium azide.
  • Ascorbic acid oxidase (trade name: ASO-3) manufactured by KK
  • the amount of the sample concentration is 300 ⁇ M or less and the sample / reagent ratio (sample amount: total amount of reagent) is 1:10
  • the lower limit is 0.1 U / mL or more, preferably 1 U / mL or more, more preferably 5 U / mL or more.
  • the upper limit is not particularly provided, it is 100 U / mL or less, preferably 50 U / mL or less, more preferably 30 U / mL or less.
  • the amount of enzyme used is preferably higher from the viewpoint of reagent stability, and lower from the economical viewpoint. In particular, when performing a rate assay, the amount of enzyme used is preferably low, and the lower limit is 0.01 U / mL.
  • catalase may be used to eliminate hydrogen peroxide. is there.
  • the catalase preferably has an action of substantially eliminating hydrogen peroxide, more preferably an enzyme classified as EC 1.11.1.6, but is not limited thereto.
  • the amount of catalase used is not limited, it is usually used in the range of 10 to 5000 U / mL.
  • Preferable examples of catalase include catalase derived from Arthrobacter genus (Asahi Kasei Pharma Co., Ltd.) from the viewpoint of high purity and animal origin (SIGMA (C1345 etc.)) from the viewpoint of being inexpensive and easily available.
  • the step of quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention is quantification using an enzyme, from the viewpoint of maintaining and / or enhancing the reactivity and / or stability of the enzyme used. It is preferable to use a pH buffer as appropriate.
  • the pH buffer is not limited as long as the target pH can be maintained, but Good's pH buffer (MES, Bis-Tris, ADA, PIPES, ACES, BES, MOPS, TES, HEPES, DIPSO, TAPSO, POPSO, HEPPSO , EPPS, Tricine, Bicine, TAPS, CHES, CAPS, etc.), Tris buffer, diethanolamine buffer, carbonate buffer, glycine buffer, borate buffer, phosphate buffer, glycylglycine buffer, acetate buffer, Examples include citrate buffer, succinate buffer, maleate buffer, trisethanolamine buffer, imidazole buffer, and the like. These buffers can be used by adjusting to a pH range that can be used as a buffer using a strong acid such as hydrochloric acid or a strong alkali such as NaOH.
  • the pH varies depending on the enzyme used, it is preferably a weakly alkaline pH, and the lower limit is exemplified by pH 7.0 or higher, preferably pH 7.3 or higher, more preferably pH 7.5 or higher, and the upper limit is exemplified.
  • the pH is 9.0 or less, preferably pH 8.4 or less, more preferably pH 8.0 or less.
  • the pH is exemplified by pH 7.0 or more as the lower limit, and pH 9.5 or less, preferably as the upper limit
  • the pH is 9 or less, more preferably 8.5 or less.
  • the concentration of the pH buffer is not particularly limited as long as the target pH can be maintained, but the lower limit is 3 mM or more, preferably 5 mM or more, more preferably 10 mM or more, and the upper limit is 500 mM or less, preferably 200 mM. Hereinafter, more preferably 100 mM or less.
  • a surfactant can be present in the step.
  • the type of surfactant is not limited, but polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene glycerin fatty acid esters, polyoxyethylene sorbit fatty acid esters, polyoxyethylene alkylphenyl formaldehyde condensates, Polyoxyethylene castor oil, polyoxyethylene sterols, polyoxyethylene polyoxypropylene alkyl ethers, polyoxyethylene lanolins, polyoxyethylene alkylamine / fatty acid amides, polyoxyethylene alkyl ether phosphates / phosphates, Polyoxyethylene alkyl ether sulfates, polyglycerol fatty acid esters, glycerol fatty acid esters, propylene glycol fatty acid esters, sol Tan fatty acid esters, N
  • an ether type or an ester type of a nonionic surfactant is used, and more preferable examples include polyoxyethylene derivatives, polyoxyethylene alkyl ethers, and polyoxyethylene alkyl amines having an HLB value of 13 to 15.
  • Nonionic surfactants selected from the group consisting of: polyoxyethylene sodium lauryl sulfate, polyoxyethylene alkyl (12-14) ether, polyoxyethylene octylphenyl ether, polyoxyethylene nonylphenyl Ether. From the viewpoint of the solubility of the surfactant, a polyoxyethylene alkylphenyl ether-based nonionic surfactant is preferable.
  • Trade names include Triton X-100 (TX-100), Tween 20, Emar 20C (Kao Corporation), NIKKOL BT-9 (Nikko Chemicals Corporation), Nonion HS-208, HS-210, HS-208, HS- 208.5, NS210 (NOF Corporation), etc. are illustrated.
  • n-Dodecyl- ⁇ -D-maltoside DDM
  • n-Octyl- ⁇ -D-glucoside n-Nonyl- ⁇ -D-thiomaltoside
  • Nonionic surfactants having a sugar chain in the hydrophilic portion such as -thioglucoside and 3-Oxatridecyl- ⁇ -D-mannoside
  • a plurality of surfactants may be used as a mixture.
  • polar surfactants are known to have a denaturation effect on proteins such as enzymes
  • neutral (nonpolar) surfactants are used to dissolve lipids when using lipid-based enzymes.
  • the present invention is not limited to this.
  • a surfactant when a surfactant is used, its concentration is not particularly limited as long as the desired action of the enzyme used is obtained.
  • the step of quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention is quantification using an enzyme, and the surfactant is lyPls as and / or PL of the present invention.
  • the upper limit of the concentration is 0.25 (w / v)% or less, preferably 0.1 (w / v)% or less, more preferably 0.05 (w / v)% or less.
  • the surfactant may not be used.
  • the concentration of the surfactant is usually 0 as the lower limit.
  • a salt such as NaCl, KCl, ammonium sulfate or NH 3 Cl can be present in the step,
  • the amount of the lower limit is 0.1 mM or more, preferably 5 mM or more, more preferably 50 mM or more, and the upper limit is not particularly limited, but is preferably 200 mM or less, more preferably 150 mM or less, particularly preferably 120 mM or less. Is exemplified.
  • These salts can function as an enzyme stabilizer for a dementia test reagent kit described later.
  • the step of quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention is quantification using an enzyme
  • sugar can be present in the step, and the concentration thereof is within a soluble range.
  • the lower limit value is 0.05 (w / v)% or more, preferably 0.1 (w / v)% or more, more preferably 0.3 (%) of the dementia test reagent kit described later.
  • the upper limit is 30 (w / v)% or less, preferably 10 (w / v)% or less, and more preferably 5 (w / v)% or less.
  • the lower limit is 0.05 (w / v)% or more, preferably 0.1 (w / v)% or more, more preferably 0.3 (w / v)% or more
  • the upper limit is 3 (w / v)% or less, preferably 2 (w / v)% or less, and more preferably 2 (w / v)% or less.
  • examples of other sugars include trehalose and cyclodextrin. These sugars can function as an enzyme for a dementia test reagent kit described later and a stabilizer for the kit contents themselves, and as a freeze-drying excipient when the kit contents are freeze-dried.
  • a preservative can be present in the step, and the type and concentration thereof are not limited.
  • the lower limit is 0.005 (w / v)% or more, preferably 0.01 (w / v)% or more, more preferably 0.03 (w) of the dementia test reagent kit described later. / V)% or more
  • the upper limit is 1 (w / v)% or less, preferably 0.5 (w / v)% or less, more preferably 0.1 (w / v)% or less.
  • the lower limit is 5 ⁇ g / mL or more, preferably 10 ⁇ g / mL or more, more preferably 30 ⁇ g / mL or more, and the upper limit is 100 ⁇ g / mL or less, preferably 75 ⁇ g / mL or less, more preferably 60 ⁇ g. / ML or less.
  • the step of quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention is quantification using an enzyme
  • the step can contain a chelating agent such as EDTA, EGTA, NAT, etc.
  • concentration is not limited.
  • EDTA it is usually in the range of 0.05 mM to 10 mM.
  • the chelating agent can function as a stabilizer for the enzyme in the kit by the action of inhibiting the activity when a protease that utilizes a metal for activity expression is mixed in the kit contents.
  • the step of quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention is quantification using an enzyme
  • the step has a catalytic action such as bovine albumin, ovalbumin, human albumin, or crystallin.
  • bovine albumin there are no proteins, and their types and concentrations are not limited.
  • bovine albumin when bovine albumin is contained, the content is usually in the range of 0.01 (w / v)% to 5 (w / v)%. Since these proteins are protease substrates, they may be enzyme stabilizers. Moreover, it can become a freeze-drying excipient during freeze-drying.
  • the additive capable of proceeding with the enzyme reaction includes an additive for the purpose of maintaining and / or enhancing the reactivity and / or stability of the enzyme as described above, and examples thereof include a pH buffer, a surfactant, Examples thereof include salts, sugars, preservatives, chelating agents, and proteins having no catalytic action.
  • the reaction time specifically measures the amount of PlsEtn in the serum and / or plasma.
  • the lower limit of the reaction time is 15 seconds or more, preferably 1 minute or longer, more preferably 3 minutes or longer.
  • the lower limit of the reaction time is 15 seconds or more, preferably 1 minute or longer, more preferably 3 minutes or longer.
  • the lower limit of the reaction time is 15 seconds or more, preferably 1 minute or longer, more preferably 3 minutes or longer.
  • the lower limit of the reaction time using the contents of each container is 15 seconds. As mentioned above, Preferably it is 1 minute or more, More preferably, it is 3 minutes or more. There is no particular upper limit, but it is preferably 30 minutes or less, more preferably 15 minutes or less, and particularly preferably 10 minutes or less.
  • the reaction time using the contents of each container may not be the same.
  • the reaction time of the method for quantifying serum and / or plasma PlsEtn amount using the action of lyPlsase and / or PL of the embodiment of the present invention is preferably within 30 minutes, more preferably within 15 minutes. Particularly preferably within 10 minutes.
  • the temperature at which each step is performed specifically determines the amount of PlsEtn in the serum and / or plasma.
  • the temperature is not limited as long as it can be measured, and the temperature of each step may not be the same.
  • the lower limit is 15 ° C. or higher, preferably 20 ° C. or higher, more preferably 25 ° C. or higher
  • the upper limit is 70 ° C. or lower, preferably 50 ° C. or lower, more preferably 40 ° C. or lower, preferably 37 It is around °C.
  • PlsEtn For the amount of serum and / or plasma PlsEtn, PlsEtn can be quantified for each molecular species by the LC-MS / MS method described in Patent Document 2, for example.
  • the 125I-HPLC method is inferior in versatility because it uses expensive equipment and radioactive iodine. It is preferable because there is a lot of data.
  • the step of quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention is quantification using an enzyme, and a method using an enzyme further comprising the steps (1) to (3-4) ( FIG. 7) is preferable because it can be applied to a general-purpose automatic analyzer and is convenient.
  • the method for quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention further comprises the steps (1) to (3-3) ′ described above (FIG. 8).
  • the amount of the enzyme used is inferior to that of the method using the enzyme comprising steps (3-4), but it is preferable because it can be applied to a general-purpose automatic analyzer and is convenient.
  • the LC-MS / MS method is inferior in versatility because it uses an expensive apparatus, but is preferable because it can be quantified for each molecular species. That is, the method for quantifying the amount of serum and / or plasma PlsEtn of the present invention is not limited to 125I-HPLC method, LC-MS / MS method, enzyme method, etc. For example, measurement sensitivity, specificity, reproducibility, economical Those skilled in the art can appropriately change the reason according to the reason, safety purpose, applicable laws and the like.
  • the lyPls as of the present invention includes Pseudomonas putida (KT2440) or Thermocrispum sp. LyPlsase having the amino acid sequence of SEQ ID NO: 1 or 2 derived from (NITE BP-01628). Further, lyPlsase of the present invention that catalyzes the reaction of hydrolyzing lyPlsEtn in the presence of water to obtain the corresponding aldehyde and glycero-3-phosphoethanolamine can also be mentioned.
  • the microorganism can be any microorganism that produces an enzyme that catalyzes the reaction of hydrolyzing lyPlsEtn to obtain the corresponding aldehyde and glycero-3-phosphoethanolamine.
  • microorganisms belonging to the genus Pseudomonas or Thermocrispum preferably Pseudomonas putida or Thermocrispum sp. And most preferably Pseudomonas putida (KT2440) or Thermocrispum sp. (NITE BP-01628).
  • Pseudomonas putida (KT2440) is a DSM no. 6125 can be purchased from Leibniz-Institut DSMZ (Deutsche Sammlung von Microorganismen und Zellkulturen GmbH).
  • a strain isolated from soil, lakes, seas, organism surfaces, body cavities, etc. is a microorganism belonging to the genus Pseudomonas or Thermocrispum, for example, “Bergey's Manual 2nd edition (2001)”, “Microorganisms” Classification / identification experiment method-focusing on molecular genetics and molecular biological methods (Springer Lab Manual) Springer Fairlark Tokyo, September 2001 ", etc., commercial identification test products (for example, BIOMERIEUX For example, “Techno Suruga Lab Co., Ltd. (Shizuoka City)” or the like.
  • strains are Pseudomonas putida or Thermocrispum sp. Is determined according to the method described in “Stackackt E., Ebers J .: Taxonomic parameters revised: tumbled gold standards, Microbiology today, nov, p. 152-155, etc.”. That is, if DNA-DNA hybridization has 70% or more homology, or 16s rRNA is 98.5% or more identical, it can be judged as the same genera. Preferably, if the DNA-DNA hybridization has a homology of 70% or more, it can be judged as the same genera.
  • Separation of natural microorganisms can be carried out using techniques known to those skilled in the art. For example, referring to the microorganism separation method described in the Biotechnology Experiments edited by the Japanese Society for Biotechnology (2002 revised edition, Baifukan) It can be carried out.
  • the lyPlsase of the present invention includes: the amino acid sequence of SEQ ID NO: 1 or 2, and lyPlsase catalyzing the reaction of hydrolyzing lyPlsEtn to obtain glycero-3-phosphoethanolamine and aldehyde: SEQ ID NO: 1 or 2
  • Examples include lyPlsase, which consists of an amino acid sequence in which various amino acid residues are added to the amino acid sequence of No.
  • lyPlsase by adding a functional protein such as a thioredoxin protein or other amino acid sequence to the N-terminal side and / or C-terminal side of the lyPlsase of the present invention.
  • a portion called a tag that can be purified or confirmed by the added portion may be fused, and in some cases, all or part of the tag portion may be deleted after the fusion.
  • signal peptides for transporting the lyPlsase of the present invention to the outside of the cell or to the periplasm, or 5 to 10 His for efficient purification may be added. May be added.
  • protease-recognizing amino acid sequences may be arranged between these amino acid sequences and added. Similar to the above addition examples, deletions or substitutions can be made. For example, in the amino acid sequence of SEQ ID NO: 1 or 2, lyPlsEtn is hydrolyzed to give glycero-3-phosphoethanolamine and an aldehyde.
  • deletions can be combined. Deletions, substitutions or additions may be combined as appropriate. Specifically, even if the signal peptide consisting of 27 amino acids of SEQ ID NO: 6 in the amino acid sequence of SEQ ID NO: 2 is deleted, it is lyPlsEtn of the present invention. Even if the signal peptide consisting of 33 amino acids of SEQ ID NO: 7 in the amino acid sequence of SEQ ID NO: 5 is deleted, it is a PL of the present invention. Also, Botechnol Lett. In reference to the method disclosed in 2011, 33, 727-731, a promoter, signal peptide, and terminator sequence of phospholipase D derived from Streptoverclum cinnamoneum can also be used.
  • the lyPlsase of the present invention is a protein encoded by a base sequence complementary to the base sequence shown in SEQ ID NO: 3 or 4 and a base sequence that hybridizes under stringent conditions described later, and hydrolyzes lyPlsEtn. Alternatively, it may be lyPlsase which catalyzes the reaction for obtaining glycero-3-phosphoethanolamine and aldehyde.
  • the present invention includes: a gene comprising a base sequence encoding lyPlsase consisting of the amino acid sequence of SEQ ID NO: 1 or 2; and the amino acid sequence of SEQ ID NO: 1 or 2, wherein one or more amino acids are deleted, substituted or substituted
  • the present invention also relates to a gene comprising a base sequence encoding a protein comprising an added amino acid sequence and hydrolyzing lyPlsEtn to catalyze a reaction for obtaining glycero-3-phosphoethanolamine and an aldehyde.
  • Examples of such a gene include, but are not limited to, for example: a gene containing the nucleotide sequence set forth in SEQ ID NO: 3 or 4, and one or more bases deleted in the nucleotide sequence set forth in SEQ ID NO: 3 or 4.
  • a nucleotide sequence consisting of a substituted or added nucleotide sequence, which encodes a protein that catalyzes the reaction of hydrolyzing lyPlsEtn to give glycero-3-phosphoethanolamine and an aldehyde specifically: SEQ ID NO: In the amino acid sequence of SEQ ID NO: 1 or 2, wherein lyPlsEtn is hydrolyzed to glycero-3-phos Deletion of one or more amino acids not involved in the action of catalyzing the reaction of obtaining foethanolamine and aldehyde, Gene comprising a nucleotide sequence encoding a substituted or added in the amino acid sequence is exemplified.
  • the gene of the present invention is a base sequence that hybridizes with a base sequence complementary to the base sequence shown in SEQ ID NO: 3 or 4 under stringent conditions, and hydrolyzes lyPlsEtn to give glycero-3-phospho It may be a gene containing a base sequence encoding a protein that catalyzes a reaction for obtaining a tanolamine and an aldehyde.
  • the above stringent conditions are usually about 5 ° C. to about 30 ° C., preferably about 10 ° C. to about 25 ° C. below the melting temperature (Tm) of the complete hybrid, and a specific hybrid is formed.
  • Tm melting temperature
  • J. Examples include the conditions described in Sambrook et al., Molecular Cloning, Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989).
  • the condition may be such that DNAs having 90% or more homology hybridize and DNAs having lower homology do not hybridize.
  • composition of SSC solution of 1 ⁇ concentration is 150 mM
  • Sodium chloride, 15 mM sodium citrate preferably the conditions for hybridization at a salt concentration corresponding to 0.1 ⁇ SSC.
  • the vector into which the gene of the present invention is incorporated is not particularly limited, but a phage or plasmid constructed for gene recombination among phages or plasmids that can grow autonomously in a host microorganism is suitable.
  • phage vectors include E. coli. ⁇ gt ⁇ ⁇ C, ⁇ gt ⁇ ⁇ B, etc. can be used when a microorganism belonging to the above group is used as a host.
  • the plasmid vector for example, when Escherichia coli is used as a host, Novagen's pET vector, or pBR322, pBR325, pACYC184, pUC12, pUC13, pUC18, pUC19, pUC118, pIN I, Bluescript KS +, etc. PW1520, pUB110, pKH300PLK, etc., when using actinomycetes as the host, pIJ680, pIJ702, etc., and when using yeast, particularly Saccharomyces cerevisiae as the host, YRp7, pYC1, YEp13, etc. can be used.
  • the recombinant vector of the present invention From the viewpoint that the recombinant vector of the present invention has been confirmed to be safe, a ministerial ordinance that stipulates anti-diffusion measures to be taken for industrial use, etc., among the second type use of genetically modified organisms, etc.
  • a recombinant vector into which the gene of the present invention is inserted is preferable.
  • the promoter is not particularly limited as long as it can be expressed in the host.
  • the recombinant vector of the present invention can be prepared by a technique known to those skilled in the art using, for example, the gene of the present invention and the above vector.
  • the transformant containing the above recombinant vector of the present invention is not limited as long as it is a transformant transformed with the above recombinant vector.
  • Examples of the host include Escherichia coli, Bacillus subtilis, Streptomyces and Rhodococcus. Examples include actinomycetes belonging to the genus, Saccharomyces cerevisiae, Pichia pastoris, and fungi. From the viewpoint that the transformant of the present invention has been confirmed to be safe, a ministerial ordinance that establishes measures to prevent diffusion in industrial use, etc. among second-class use of genetically modified organisms, etc.
  • the transformant of the present invention can be prepared by a technique known to those skilled in the art using, for example, the recombinant vector of the present invention and the above host.
  • the present invention is also characterized in that a microorganism that produces the lyPlsase of the present invention is cultured in a medium, the lyPlsase of the present invention is produced and accumulated in the culture, and the lyPlsase of the present invention is collected from the culture.
  • the present invention also relates to a method for producing lyPlsase of the present invention.
  • Culture conditions in the microorganism cultivation process and the production and accumulation process may be appropriately selected in view of the nutritional physiological properties, and are usually performed by liquid culture, but industrially, it is advantageous to perform deep aeration and agitation culture. .
  • a nutrient source of the medium those usually used for culturing microorganisms such as LB, PDA, MA, OA or LcA medium can be widely used.
  • the culture temperature can be appropriately changed within the range in which the lyPlsase of the present invention is produced. In the case of natural microorganisms, the lower limit is 5 ° C. or higher, preferably 15 ° C. or higher, more preferably 20 ° C. or higher, and the upper limit is thermophilic.
  • the temperature is about 100 ° C. in the case of archaebacteria and bacteria, but it is usually 55 ° C. or lower, preferably 45 ° C. or lower, more preferably 40 ° C. or lower. Particularly in the case of fungi, the lower limit is 4 ° C or higher, preferably 10 ° C or higher, more preferably 20 ° C or higher, and the upper limit is 50 ° C or lower, preferably 42 ° C or lower, more preferably 37 ° C or lower.
  • the culture time may vary depending on the culture conditions, but the culture may be terminated at an appropriate time in anticipation of the time when the production of the lyPlsase of the present invention reaches the maximum amount.
  • the lower limit is 17 hours or more, preferably 20 It is not less than time, more preferably not less than 24 hours, and the upper limit is not more than 80 hours, preferably not more than 72 hours, more preferably not more than 48 hours.
  • the lower limit is 1 day or more, preferably 2 days or more, and the upper limit is about 10 days, preferably 4 days or less, more preferably 3 days or less.
  • the medium pH can be appropriately changed within the range in which microorganisms grow and produce the lyPlsase of the present invention, but the lower limit is preferably pH 4 or higher, more preferably pH 5 or higher, and the upper limit is preferably pH 8.5 or lower, more preferably pH 7 .5 or less. Especially in the case of fungi, a low pH is preferred.
  • the present invention is also characterized in that the above-described transformant of the present invention is cultured in a medium, the lyPlsase of the present invention is produced and accumulated in the culture, and the lyPlsase is collected from the culture. It also relates to a method for producing the lyPlsase of the invention.
  • the culture conditions in the transformant culturing step and the production and accumulation step are the same as in the case of the microorganism, and can be appropriately selected according to the type of the transformant.
  • the lower limit of the culture temperature is 10 ° C or higher, preferably 20 ° C or higher, more preferably 25 ° C or higher, and the upper limit is 45 ° C or lower, preferably 42 ° C or lower, more preferably 37 ° C. It is as follows.
  • the lower limit is 4 ° C or higher, preferably 10 ° C or higher, more preferably 20 ° C or higher, and the upper limit is 50 ° C or lower, preferably 42 ° C or lower, more preferably 37 ° C or lower.
  • the culture time may vary depending on the conditions, but the culture may be terminated at an appropriate time in anticipation of the time when the production of the lyPlsase of the present invention reaches the maximum amount. 10 hours or more, preferably 12 hours or more, more preferably 17 hours or more, and the upper limit is 60 hours or less, preferably 48 hours or less, more preferably 30 hours or less.
  • the lower limit is usually 17 hours or longer, preferably 20 hours or longer, more preferably 24 hours or longer, and the upper limit is 80 hours or shorter, preferably 72 hours or shorter, more preferably 48 hours or shorter.
  • the pH of the medium can be appropriately changed within the range in which the transformant develops and produces the lyPlsase of the present invention, but in the case of Escherichia coli and actinomycetes, the lower limit is preferably pH 5.8 or more, more preferably pH 6.2 or more.
  • the upper limit is preferably pH 8.5 or less, more preferably pH 7.5 or less.
  • the method for collecting the lyPlsase of the present invention produced and accumulated in the culture is not particularly limited, but for convenience, the lyPlsase of the present invention is collected as it is, including cells containing cells, regardless of sterilization or non-sterilization. May be. It is also preferable to collect the lyPlsase of the present invention while lightly removing the culture impurities and cell debris and leaving the impurities remaining. Depending on the purpose and application, it is also preferable to collect the lyPlsase of the present invention which does not substantially contain impurities.
  • lyPlsase of the present invention is collected with a purity of 50% or more, 70% or more, or 95% or more.
  • the purity can be confirmed using a known method such as SDS-PAGE or HPLC.
  • lyPlsase of the present invention A method for purifying lyPlsase of the present invention will be described below.
  • the microbial cells are collected from the obtained culture by means such as filtration or centrifugation after completion of the culture.
  • this bacterial cell is destroyed by a mechanical method or an enzymatic method such as lysozyme, and EDTA and / or an appropriate surfactant is added as necessary to concentrate the lyPlsase, and then acetone, methanol,
  • the lyPlsase of the present invention can be precipitated and recovered by applying a fractional precipitation method using an organic solvent such as ethanol, a salting out method using ammonium sulfate, sodium chloride, or the like. This precipitate is subjected to dialysis and isoelectric precipitation, if necessary, and then purified by adsorption using gel filtration, affinity chromatography, etc., ion exchange chromatography, or hydrophobic chromatography.
  • Inventive lyPls as can be obtained.
  • the above methods can be combined as appropriate.
  • the microbial cells are removed from the culture by means of filtration or centrifugation to obtain a culture solution, and then formed in the microbial cells.
  • Purified lyPlsase of the present invention can be obtained by carrying out the same treatment as described above.
  • the lyPlsase obtained by the above-described method for producing lyPlsase of the present invention is added with various salts, saccharides, proteins, lipids, surfactants, etc. as stabilizers as necessary, and concentrated by ultrafiltration and freeze-dried. Etc., it can be made liquid or solid. When freeze-drying, about 0.5 to 10% of sucrose, mannitol, sodium chloride, albumin, ammonium sulfate, etc. may be added as a stabilizer.
  • the lyPlsase of the present invention can be distinguished from the conventionally known lyPlsase in amino acid sequence.
  • a known amino acid sequence having homology with the amino acid sequence described in SEQ ID NO: 1 is obtained from NCBI BLAST (Basic Local Alignment Search Tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi)).
  • NCBI BLAST Basic Local Alignment Search Tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi)).
  • Protein BLAST search the amino acid sequence derived from Pseudomonas putida KT2440 strain can be searched as of June 29, 2013, but the definition (definition) is a hypothetical protein and is a protein of unknown function.
  • Tmem86b (inventor ⁇ : lyPlsase gene) has only been identified inverteds, inclusive humans, mice, rats, cows, dogs, and iszef”. That is, conventionally, lyPlsase of vertebrates including humans, mice, rats, cows, dogs, and zebrafish has been known.
  • the lyPlsase of the present invention is derived from microorganisms as described above, and is a lyPlsase derived from microorganisms newly discovered by the present inventors.
  • the step of quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention is quantification using an enzyme, and the enzyme is lyPlsase and / or PL of the present invention.
  • the enzyme can be used as an enzyme that catalyzes the reaction of hydrolyzing lyPlsEtn to obtain glycero-3-phosphoethanolamine and an aldehyde.
  • the present invention also includes a means for quantifying MI in spontaneously excreted urine of a subject and a means for quantifying PlsEtn in the serum or plasma of the same subject.
  • a dementia test means for classification which is capable of distinguishing between a healthy person and a dementia patient or a person in the previous stage, with respect to respective predetermined threshold values of MI and PlsEtn
  • the present invention relates to the above-mentioned dementia test means, which uses the subject's MI and PlsEtn quantitative values in comparison.
  • the means include, but are not limited to, a composition, a test reagent, an enzyme kit, and the like.
  • the present invention also includes an enzyme for quantifying MI in spontaneously excreted urine of a subject, and an enzyme for quantifying PlsEtn in serum or plasma of the same subject.
  • an enzyme for quantifying MI in spontaneously excreted urine of a subject and an enzyme for quantifying PlsEtn in serum or plasma of the same subject.
  • MI and PlsEtn each of which can distinguish between a healthy person and a dementia patient or a person in its previous stage
  • the present invention relates to the above-described test reagent kit, which uses the subject's MI and PlsEtn quantitative values in comparison with a predetermined threshold value.
  • a reagent kit for quantifying PlsEtn is a method for quantifying PlsEtn by a 125I-HPLC method, and a method for quantifying the amount of serum and / or plasma PlsEtn according to an embodiment of the present invention including the steps (1) to (3-4) above.
  • a reagent kit for carrying out a method for quantification by the LC-MS / MS method, etc., and a reagent kit for quantification using the action of lyPlsase and / or PL of the present invention is preferred.
  • Dementia test reagent kit (the step of quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention is quantification using an enzyme, and the enzyme further comprises lyPlsase and / or Alternatively, the step of quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention and the dementia test reagent kit that is PL further comprises the steps (1) to (3-4) or (1) to (3- 3)
  • a dementia test reagent kit, which is a quantification using the method for quantifying the amount of serum and / or plasma PlsEtn according to an embodiment of the present invention including ') includes various enzymes, PL, and lyPls as described herein.
  • GPCP ethanolamine oxidase
  • PLA 1 PLA 2, PLB , LYPL, MGLP, peroxidase, catalase, the ASOM like, dementia test method described above
  • Preferred examples thereof are the same as those in the method in which the step of quantifying the amount of serum and / or plasma PlsEtn according to the above-described embodiment of the present invention is quantification using an enzyme.
  • the dementia test reagent kit of this embodiment is used for the purpose of improving the quality of measurement sensitivity, accuracy, reproducibility, kit stability, etc., salt, sugar, preservative, chelating agent, protein, surface activity
  • the type and amount of the components contained can be changed as appropriate according to the purpose of use and the measurement application of the dementia test reagent kit. For example, measurement sensitivity, specificity, reproducibility, economic reason, safety purpose Those skilled in the art can appropriately change them according to applicable laws and regulations. Preferred examples thereof are the same as those in the method in which the step of quantifying the amount of serum and / or plasma PlsEtn according to the above-described embodiment of the present invention is quantification using an enzyme.
  • the present invention is an enzyme reagent kit comprising an enzyme for quantifying MI in spontaneously excreted urine of a subject and an enzyme for quantifying PlsEtn in the serum or plasma of the same subject, wherein the subject is a dementia patient or a preceding stage thereof
  • the present invention also relates to a dementia test reagent kit characterized in that it is classified as a person in the above. It is preferable that an enzyme reagent kit containing an enzyme for quantifying MI in spontaneously excreted urine of a subject contains at least myo-inositol dehydrogenase.
  • the dementia test reagent kit according to the embodiment of the present invention provides a subject with a dementia patient or a pre-stage thereof by setting a threshold value for each quantified value of MI in spontaneously excreted urine and PlsEtn in serum and / or plasma. It is also preferable to classify as a person who is in the category, and such a threshold is the same as described above. Further, in the subject's spontaneously excreted urine, serum and / or plasma as determined by the dementia test reagent kit of the embodiment of the present invention, the same as described above.
  • the reagent kit for dementia testing according to the embodiment of the present invention uses the step of quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention, and further includes the steps (1) to (3-4) or ( In the case of a kit using the method for quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention including 1) to (3-3) ′, each component of the contents may be used as a single container kit. Usually, it is preferable to separate into two or more containers.
  • the dementia test reagent kit of the present embodiment is divided into an erasing container 1 for erasing substances other than PlsEtn in the serum or plasma of a subject and a container 2 for measuring PlsEtn. .
  • the dementia test reagent kit of the embodiment of the present invention includes the following (1a) to (3a), wherein (1a) to (3a) are for erasing substances other than PlsEtn in the serum or plasma of a subject
  • the container can be divided into an erasing container 1 and a container 2 for measuring PlsEtn.
  • the hydrolase of (2a) is an enzyme capable of hydrolyzing lysophosphatidylethanolamine
  • the reagent kit further comprises (1a) an enzyme that hydrolyzes lysophosphatidylethanolamine.
  • a preferred example of such a reagent kit is a dementia test reagent kit including a container 1 containing at least (1a) and (3a) and a container 2 containing at least (2a).
  • the serum and / or plasma of the subject contains phosphatidylethanolamine, and an enzyme that catalyzes the action of hydrolyzing lyPlsEtn into glycero-3-phosphoethanolamine and an aldehyde has the effect of hydrolyzing lysophosphatidylethanolamine.
  • the container 1 should just be made into the container 1 which contains the enzyme which has the effect
  • the dementia test reagent kit of the embodiment of the present invention includes the following (1a) to (3a) ′, wherein (1a) to (3a) ′ erase substances other than PlsEtn in the serum or plasma of the subject It can be divided into an erasing container 1 for measuring and a container 2 for measuring PlsEtn.
  • a preferred example of such a reagent kit is a dementia test reagent kit including a container 1 containing at least (1a) and (3a) ′ and a container 2 containing at least (2a) ′.
  • the container 1 may be a container 1 that further contains an enzyme having the action of decomposing and substantially eliminating lysophosphatidylethanolamine as a content.
  • the dementia test reagent kit of the present embodiment may be used together with a calibration reagent containing at least a known amount of PlsEtn.
  • the calibration reagent is, for example, a reagent containing at least a known amount of PlsEtn and / or lyPls, and preferably contains a pH buffer, a preservative such as sodium azide or antibiotics, and a stabilizer such as sugar.
  • the conditions such as the type and concentration can be appropriately determined by those skilled in the art with reference to the conditions described for the dementia test reagent kit.
  • As the calibration method a single inspection quantity, a multi-inspection quantity (a broken line or a spline), a linear regression of the multi-inspection quantity, and the like can be selected.
  • the known amount of PlsEtn in the calibration reagent is not particularly limited, and may be appropriately selected so that PlsEtn in the serum or plasma of the subject can be accurately measured.
  • the lower limit value of the known amount of PlsEtn is 10 ⁇ M or more, preferably 50 ⁇ M or more, more preferably 80 ⁇ M or more, and the upper limit value is 500 ⁇ M or less, preferably 300 ⁇ M. In the following, it can be more preferably 250 ⁇ M or less.
  • the dementia test reagent kit of the present embodiment and the calibration reagent described above are liquid products, frozen products of liquid products, freeze-dried products of liquid products, or dried products of liquid products (heat-dried and / or air-dried and / or reduced pressure). For example, by drying). Also, for example, in the case of use in a capillary of a point-of-care device or use as an enzyme sensor, the concentration of each component is preferably higher than usual. For example, it is fixed, soaked in paper or a film, gel -It is preferable to use it as a sol-like dementia test reagent kit.
  • the subject obtained informed consent with the approval of the ethics committee of the Tokyo Metropolitan Health and Longevity Medical Center “Forgetfulness” outpatient and elderly healthy “Brain Imaging” volunteers. Blood and urine were collected.
  • biomarkers WBC, RBC, Hb, Ht, MCV, MCH, MCHC, PLT, TP, Alb, T-Bil, other than serum plasmalogens (choline type plasmalogen: PlsCho, ethanolamine type plasmalogen: PlsEtn) Cre, UA, ⁇ 2-ub, AMY, GOT, GPT, LDH, Na, Cl, K, TSH, FreeT3, FreeT4, Glu, HbA1c, CRP, TNF- ⁇ , IL-6, TG, TC, HDL-C, LDL-C, PL, VB9, VB12, ⁇ -Toc, ⁇ -Toc, ⁇ -Toc, A ⁇ 1-40 and A ⁇ 1-42) are available at SRL, Inc. (2-1-1 Nishi-Shinjuku, Shinjuku-ku, Tokyo) It was measured.
  • Spontaneously excreted urine MI (urine MI) was measured with a myo-inositol measurement reagent (Lucica MI, Asahi Kasei Pharma Co., Ltd., 1-chome, Kanda Jimbocho, Chiyoda-ku, Tokyo), and the average value for one or three days was standardized with Cre. It was expressed as a value (mg / gCr).
  • GDS Geriatric Depression Rating Scale
  • reagents used in the present examples unless otherwise specified, Wako Pure Chemical Industries, Ltd., Sigma-Aldrich, etc., which are commercially available and can be easily obtained, reagent manufacturer, purity, price These are not particularly limited, and can be appropriately selected and used by those skilled in the art as needed.
  • Human pooled serum was purchased from Yamakyu Kasei Co., Ltd. (Kyoyodai 4-2C-1007, Inagi City, Tokyo).
  • the techniques used in the present examples include, for example, the prior art document described in this specification, the method of Maniatis et al. (Maniatis, T.
  • the enzyme activity of lyPlsase was measured as follows.
  • Reaction reagent mixture 50 mM BES / NaOH buffer pH 7.5 5U / mL GPCP (Asahi Kasei Pharma Corporation (T-33) 5U / mL POD (SIGMA (P8250) 0.02% TODB (Dojindo Laboratories (OC22)) 0.03% 4-AA (012-14471) 8 mM lyPlsEtn (Avanti Polar Lipids, Inc.
  • the following [Hitachi 7080 automatic analyzer parameters] used in this example and the like are easily set by those skilled in the art with reference to the instruction manual of the Hitachi 7080 automatic analyzer. Can be used. Taking the “Hitachi 7080 automatic analyzer parameter 1” as an example, the method of using the analyzer will be briefly described below.
  • the analysis method colorimetric analysis
  • the measurement wavelength has a dominant wavelength of 660 nm and a sub wavelength of 750 nm.
  • the sample volume (serum and / or plasma volume) is 12 ⁇ L
  • the test reagent kit 1 for the first step is used as 160 ⁇ L as R1
  • the test reagent kit 2 for the next step is used as 40 ⁇ L as R3.
  • the blank ([]) in the parameter means to leave it blank.
  • Example 1 Dementia test by urine MI quantitative value and serum and / or plasma PlsEtn quantitative value
  • the amount of serum and / or plasma PlsEtn was measured by a radioactive iodine-high performance liquid chromatography method (125I-HPLC method) described in Patent Document 1.
  • MI in fasting second urine was measured.
  • Urinary MI amount Based on generational / gender measurement data of urinary MI amount, a significant difference test was performed between groups, and it was confirmed that the urinary MI amount increased significantly in the elderly group for both men and women. The amount of urinary MI in the elderly group was extremely different among individuals compared to the young and middle-aged groups. In addition, there was no significant difference in urinary MI between healthy individuals and dementia patients among the elderly (FIG. 1, Table 4).
  • the urine MI amount and the HbA1c amount were divided into 4 groups with the average value of all measured values as a boundary for each of the healthy group and the “forgetful” group, and the PlsEtn amount in each group was compared. In the healthy elderly, there was no significant difference between the four groups in the PlsEtn amount.
  • the serum PlsEtn (EP) level (low level) and urinary MI level (high level) group are all compared to the other three groups.
  • Significant cognitive decline was observed ( Figure 3, Table 6). From the above analysis, it was confirmed that dementia patients can be classified with high accuracy by combining the measurement of serum PlsEtn amount and urine MI amount.
  • the correlation between the ability to evaluate the cognitive function of the serum PlsEtn amount and the face-to-face cognitive function test method is as follows. Examination of the three groups of the elderly group showed that the correlation between the serum PlsEtn amount and the cognitive function test results was increased as the urinary MI level was increased (FIG. 6, Table). 7). From the above results, combining measurement of serum PlsEtn and measurement of urinary MI makes it possible to classify patients with dementia with high accuracy, and further enhances cognitive function evaluation ability using serum PlsEtn as an index. Was confirmed.
  • Example 2 Comparison of MI quantitative values in urine from time to time and fasting second urine.
  • the quantitative values of MI in ad hoc urine and fasting second urine were compared, and in the dementia test method according to the embodiment of the present invention, it was confirmed that the occasional urine could be replaced with fasting second urine.
  • MI MI quantification value
  • y-axis MI was quantified by collecting urine from fasting second urine for three days every day or every other suitable day (within one month) from the subject, and the average value was determined as the MI quantification value (x axis) of fasting second urine.
  • the results are shown in FIG.
  • the slopes of the calibration curves of the quantitative values of PlsEtn in serum and PlsEtn in 0.9% saline were the same and the correlation coefficient was close to 1.
  • the intercept of the calibration value of PlsEtn in serum is the effect of PlsEtn originally contained in the serum, and the intercept of the calibration curve of PlsEtn in 0.9% saline is the effect of the reagent blank. . Moreover, the manufacturing method of lyPlsase derived from Pseudomonas putida will be described later.
  • composition 1-1b 50 mM BES-NaOH pH 7.5 10U / mL Peroxidase 50U / mL Ethanolamine oxidase 5U / mL GPCP 1U / mL International publication number: PL of WO2012 / 105565 A1
  • Composition 1-1b 50 mM BES-NaOH pH 7.5 4.2 U / mL rat-derived lyPls as 12 ⁇ M DA67
  • rat-derived lyPlsase was produced as a recombinant Escherichia coli from rats by the method described in The journal of biochemical chemistry, 2011, 286, 24916-24930.
  • Example 4 Comparison of quantified value of PlsEtn in serum or plasma using 125I-HPLC method and enzyme 1] 47 serum specimens were measured in two ways: (I) 125I-HPLC method, (Ii) Enzymatic method using lyPlsase derived from Pseudomonas putida ([Composition 1-1a] and [Composition 2-1a]).
  • Each test reagent kit was measured as R1 and R2 using a Hitachi 7080 automatic analyzer with [Hitachi 7080 automatic analyzer parameter 2].
  • the correlation diagram of each quantitative value is shown in FIG.
  • Example 5 Comparison 2 of quantitative value of PlsEtn in serum or plasma using 125I-HPLC method and enzyme
  • 47 serum specimens were measured in two ways: (I) 125I-HPLC method, (Iii) Thermocrispum sp. Enzymatic method using [Composition 1-2c] and [Composition 2-2c].
  • Each test reagent kit was measured as R1 and R2 using a Hitachi 7080 automatic analyzer with [Hitachi 7080 automatic analyzer parameter 2].
  • composition 1-2c 20 mM BES / NaOH buffer pH 7.5 0.05 mM ALPS (Dojindo Laboratories (OC04)) 2 mM CaCl 2 70 mM sucrose 120 mM Mannitol 5U / mL GPCP (Asahi Kasei Pharma Corporation (T-33)) 40 U / mL TOD of JP-A-2005-52034 5U / mL International publication number: PL of WO2012 / 105565 A1 10U / mL POD (SIGMA (P8250)) 5U / mL ASOM (Asahi Kasei Pharma Corporation (T-53)) 5U / mL LYPL (Asahi Kasei Pharma Corporation (T-59)) [Composition 2-2c] 20 mM BES / NaOH buffer pH 7.5 1 mM Na 2 SO 4 0.12 mM DA-67 (Wako Pure Chemical Industries, Ltd.
  • Thermocrispum sp. Origin lyPls as 2% ⁇ -cyclodextrin
  • the correlation diagram of each quantitative value is shown in FIG.
  • PlsEtn in the specimen could be measured by an enzymatic method using the derived lyPlsase.
  • Each test reagent kit was measured as R1 and R2 using a Hitachi 7080 automatic analyzer with [Hitachi 7080 automatic analyzer parameter 2].
  • composition 1-3 20 mM Tris / HCl buffer pH 8.5 0.05 mM ALPS (Dojindo Laboratories (OC04)) 2 mM CaCl 2 2 mM MgCl 2 2U / mL GPCP (Asahi Kasei Pharma Corporation (T-33)) 40U / mL TOD (Asahi Kasei Pharma Corporation (T-25)) 5U / mL PLA 2 (Asahi Kasei Pharma Corporation (T-31)) 30U / mL LYPL (Asahi Kasei Pharma Corporation (T-59)) 10U / mL POD (SIGMA (P8250)) 5U / mL ASOM (Asahi Kasei Pharma Corporation (T-53)) 0.1% TX-100 [Composition 2-3] 20 mM HEPES / NaOH buffer pH 7.5 10 mM CaCl 2 0.12 mM DA-67 (Wako Pure Chemical Industries
  • Example 7 Comparison 4 of quantified value of PlsEtn in serum or plasma using 125I-HPLC method and enzyme
  • 47 serum specimens were measured by the following four methods: (I) 125I-HPLC method, (Ii) Enzymatic method using lyPlsase derived from Pseudomonas putida ([Composition 1-1a] and [Composition 2-1a]), (Iii) Thermocrispum sp. Enzymatic method using (composition 1-2c and composition 2-2c), (Iv) Enzymatic method using PLD ([Composition 1-3] and [Composition Key 2-3]).
  • Each test reagent kit was measured as R1 and R2 using a Hitachi 7080 automatic analyzer with [Hitachi 7080 automatic analyzer parameter 2].
  • Table 8 shows the average value of the measured values by each method.
  • the enzyme method using lyPlsase derived from Pseudomonas putida was about 2 ⁇ M lower than the quantified value of PlsEtn by 125I-HPLC method, and (iii) Thermocrispum sp. It has been found that the average value of about 3 ⁇ M may be high in the enzyme method using (iv) and the enzyme method using (iv) PLD.
  • Example 8 Production method of lyPlsase recombinant enzyme derived from Pseudomonas putida] Pseudomonas putida (KT2440) chromosomal DNA was extracted by the phenol / chloroform method. Using KOD FX (Toyobo Co., Ltd., Code No. KFX-101) using chromosomal DNA as a template, using the nucleotide sequence set forth in SEQ ID NO: 8 as a sense primer and the nucleotide sequence set forth in SEQ ID NO: 9 as an antisense primer, PCR was performed to obtain PCR product 1.
  • KOD FX Toyobo Co., Ltd., Code No. KFX-101
  • the resulting PCR product 1 is cloned into pCR-Blunt using Zero Blunt PCR Cloning Kit (Invitrogen, product number K2700-20), and a recombinant vector comprising a polynucleotide comprising a base sequence encoding the protein of the present invention PP_lyPlaase / pCR-Blunt was obtained.
  • the pET-21a (+) vector (Novagen, Cat. No 69740-3) was cleaved with NdeI and EcoRI to purify about 5.4 kbp of DNA.
  • PP_lyPlaase / pCR-Blunt was cleaved with restriction enzymes NdeI and EcoR I to purify about 0.7 kbp of DNA. Each of the obtained purified DNAs was transferred to DNA Ligation Kit Ver. 2.1 (Takara Bio Inc., product code 6022) was ligated to obtain a recombinant vector PP_lyPlaase / pET-21a (+) containing a polynucleotide comprising a base sequence encoding the protein of the present invention. PP_lyPlaase / pET-21a (+) was replaced with One Shot BL21 (DE3) Chemically Competent E.I.
  • transformant PP_lyPlaase / pET-21a (+) / BL21 () having a recombinant vector comprising a polynucleotide comprising a nucleotide sequence encoding the protein of the present invention after being transformed into E. coli (Invitrogen, product number C6000-03) DE3) was obtained.
  • PP_lyPlaase / pET-21a (+) / BL21 (DE3) was inoculated into 1.6L (2L jar) Overnight Express Instant Medium (Merck, order number 71757-5) containing 50 ⁇ g / ml ampicillin. . Cultivate at 34 ° C.
  • Example 9 Thermocrispum sp. Method for producing derived lyPls as] Thermocrispum sp. (Deposited with NITE BP-01628, National Institute of Technology and Evaluation, Patent Microorganism Depositary Center) was inoculated into 2.4 L of IPS2 medium (Nippon Becton Dickinson Co., Ltd., catalog number 277010) according to a conventional method. Cultured with shaking at 180 ° C. for 84 h. The lyPlsase activity of the culture supernatant was about 0.058 U / mL (total amount 104 U).
  • the culture supernatant obtained by sterilization by centrifugation was filtered through a 0.45 ⁇ m filter, and the protein was concentrated with 85% saturated ammonium sulfate.
  • the concentrate obtained by dialysis and desalting against 20 mM Tris / HCl buffer (pH 9.0) was partially purified with TOYOPEARL GigaCup Q-650M (TOSOH BIOSCIENCE) under the following conditions: 20 mM Tris containing 1.0 M NaCl. Chromatography using a linear gradient of 20 CV (column volume) using an / HCl buffer solution (pH 9.0) and a 20 mM Tris / HCl buffer solution (pH 9.0).
  • Ammonium sulfate was added to the active fraction so as to be 1.5 M, and partially purified by TOYOPEARL PPG-600M (TOSOH BIOSCIENCE) under the following conditions: 20 mM Tris / HCl containing 1.5 M (NH 4 ) 2 SO 4 Column chromatography by linear gradient of 20 CV (column volume) using buffer solution (pH 8.0) and 20 mM Tris / HCl buffer solution (pH 8.0). The active fraction was partially purified by Resource Q (GE Healthcare Bioscience) under the following conditions: 20 mM Tris / HCl buffer (pH 8.0) containing 0.5 M NaCl and 20 mM Tris / HCl buffer (pH 8.
  • Example 10 Thermocrispum sp. Method 1 for producing derived lyPlsase recombinant enzyme (Streptomyces lividans recombinant)] Thermocrispum sp.
  • the chromosomal DNA was extracted by the phenol / chloroform method.
  • PCR was performed using KOD FX using the nucleotide sequence set forth in SEQ ID NO: 10 as the sense primer and the nucleotide sequence set forth in SEQ ID NO: 11 as the antisense primer to obtain a PCR product 2 of about 900 bp.
  • the PCR product 2 was digested with NheI and BglII and inserted into the NheI-BglII site of the actinomycete plasmid as an expression vector to obtain a recombinant plasmid lyPlaase / Ex.
  • lyPlaase / Ex is transformed into a protoplast actinomycete Streptomyces vividans) 1326 / S. lividans was obtained.
  • lyPlaase / Ex / S. lividans was cultured for 48 hours at 28 ° C.
  • the crude enzyme solution was buffer-exchanged to 20 mM Tris / HCl buffer (pH 9.0) by dialysis, and partially purified with TOYOPEARL GigaCap Q-650M (TOSOH BIOSCIENCE) under the following conditions: 20 mM Tris / HCl buffer containing 1 M NaCl.
  • 20 mM Tris / HCl buffer containing 1 M NaCl 20 mM Tris / HCl buffer containing 1 M NaCl.
  • Ammonium sulfate was added to the active fraction so as to be 1.5 M, and partially purified with TOYOPEARL PPG-600M (TOSOH BIOSSCENCE) under the following conditions: 20 mM Tris / HCl buffer (pH 8.0 containing 1.5 M ammonium sulfate). And 10 CV (column volume) linear gradient using 20 mM Tris / HCl buffer (pH 8.0).
  • the active fraction was buffer exchanged to 20 mM Tris / HCl buffer (pH 9.0) by dialysis and purified by Mono Q (GE Healthcare Bioscience) under the following conditions: 20 mM Tris / HCl containing 0.5 M NaCl.
  • Example 11 Thermocrispum sp. Method 2 for producing derived lyPlsase recombinant enzyme (E. coli recombinant)] Thermocrispum sp.
  • the chromosomal DNA was extracted by the phenol / chloroform method.
  • PCR was performed using KOD FX with the nucleotide sequence set forth in SEQ ID NO: 12 as the sense primer and the nucleotide sequence set forth in SEQ ID NO: 13 as the antisense primer to obtain a PCR product 3 of about 900 bp.
  • T_lyPlaase / pET-21a (+) was replaced with One Shot BL21 (DE3) Chemically Competent E.I.
  • a transformant T_lyPlaase / pET-21a (+) / BL21 (DE3) having a recombinant vector containing a polynucleotide comprising a nucleotide sequence encoding the protein of the present invention was obtained by transforming into E. coli.
  • T_lyPlaase / pET-21a (+) / BL21 (DE3) was inoculated into 50 mL (500 mL Erlenmeyer flask) Overnight Express Instant TB Medium containing 50 ⁇ g / ml ampicillin.
  • the culture solution is centrifuged at 37 ° C. and pH 6.8 for 24 hours, and the culture solution is collected by centrifugation, suspended in 20 mM Tris / HCl buffer (pH 8.5), sonicated and centrifuged. The obtained supernatant was used as a crude protein solution. Thermocrispum sp. In the crude protein solution.
  • the derived lyPlsase recombinant enzyme was 20 U, and SDS-PAGE of the crude protein solution is shown in FIG. lyPlsase is indicated by an arrow.
  • Example 12 Substrate specificity of lyPlsase of the present invention
  • Table 10 shows the results of measuring the action of the lyPlsase of the present invention on various substrates in accordance with the above activity measurement method.
  • the results were expressed as relative activities, with the measured value when lyPlsEtn was used as a substrate being 100.

Abstract

There are provided: a method of examination for identifying persons suffering from dementia or persons in the preliminary stage thereof, using a biomolecule as an index; a kit for use in this method of examination; an enzyme contained in this kit; and a method of manufacturing this enzyme. The method of inspection includes a step of assaying myoinositol (MI) in naturally excreted urine of the patient and a step of assaying ethanolamine plasmalogen (PlsEtn) in serum or plasma of the same patient, and classifying patients whose MI content is at least a set threshold value and whose PlsEtn content is no more than a set threshold value as persons suffering from dementia or persons in the preliminary stage thereof.

Description

認知機能検査法、及びそのキットCognitive function testing method and kit
本発明は、生体分子を指標として認知症またはその前段階にある者を特定する検査方法、とその測定方法、該検査方法に用いるキット、該キットに含有させる酵素、及び該酵素の製造方法に関する。 The present invention relates to a test method for identifying a person with dementia or a pre-stage thereof using a biomolecule as an index, a measurement method thereof, a kit used for the test method, an enzyme contained in the kit, and a method for producing the enzyme .
 
１．認知症
認知症は後天的な脳の器質的障害により、正常に発達した知能が低下した状態をいう。記憶、判断力などの障害により社会生活が困難となる。近年では高齢化に伴い老人性認知症者が増加しており、その数は約２００万人といわれ、さらに増加する傾向にある。 1. Dementia Dementia refers to a condition in which intelligence that has developed normally has decreased due to an acquired organic disorder of the brain. Social life becomes difficult due to obstacles such as memory and judgment. In recent years, the number of senile dementia has increased with the aging of the population, and the number is said to be about 2 million, which tends to increase further.
 
認知症は症状が進行すると回復が困難になり、介護には多くの労力と金銭的負担が必要となるが、比較的軽度の段階で発見してリハビリ訓練等の治療を施すことにより発症予防やある程度の症状の回復が可能である。また抗認知症薬は症状の進行を抑制するための薬剤であることから、その処方は早ければ早いほど有効である。従って、認知症においては早期に治療対象者を特定することが重要となる。
Dementia becomes difficult to recover as symptoms progress, and it requires a lot of labor and financial burden for nursing care, but it can be detected in a relatively mild stage and treated with rehabilitation training to prevent onset. A certain degree of recovery is possible. Moreover, since an antidementia drug is a drug for suppressing the progression of symptoms, the prescription is more effective as soon as possible. Therefore, in dementia, it is important to identify the treatment subject at an early stage.
 
２．認知症検査
従来、認知症の判定には、ウェクスラー成人知能検査（ＷＡＩＳ－Ｒ）、改訂長谷川式簡易知能評価スケール（ＨＤＳ－Ｒ）、ミニメンタルステート検査（ＭＭＳ、ＭＭＳＥ）、クリニカルデメンチアレーティング検査（ＣＤＲ）、モントリオールコグニティブアセスメント検査日本語版（ＭｏＣＡ－Ｊ）等の認知機能検査が利用されている。これらの検査は、専門の検査官が被験者と対面して行うため、大規模なスクリーニングには不向きである。また被験者の意識状態、気分、意欲などが成績に影響を及ぼすことや、うつ病やせん妄、あるいは難聴との識別も別途に必要となるなどの問題を有している。
2. Dementia test Conventionally, for the determination of dementia, Wexler adult intelligence test (WAIS-R), revised Hasegawa simplified intelligence evaluation scale (HDS-R), mini mental state test (MMS, MMSE), clinical dementia rating Cognitive function tests such as the test (CDR) and the Montreal Cognitive Assessment Test Japanese version (MoCA-J) are used. These tests are not suitable for large-scale screening because specialized inspectors face the subject. In addition, the subject's consciousness state, mood, motivation, etc. have an effect on the results, and there is a problem that separate identification from depression, delirium, or hearing loss is necessary.
 
そこで、より客観的かつ簡便な認知症の検査方法として、生体分子（バイオマーカー）を指標とする方法が注目を集めている。例えば、代表的な認知症の一つであるアルツハイマー病の神経病理学的特徴は、アミロイドβ（Ａβ）の沈着により形成される老人班と、異常リン酸化タウ蛋白の出現に特徴付けられる神経原線維変化、そして大量の神経細胞脱落である。今日、アルツハイマー病の病態過程はＡβの産生により惹起されるというアミロイド仮説が提唱されている。これは、Ａβの産生に伴い神経細胞に酸化ストレスやカルシウム制御異常が誘導され、神経細胞死を引き起こす。また同時に、Ａβはタウ蛋白のリン酸化を誘導し、細胞骨格蛋白異常とそれに伴う細胞内輸送の障害を引き起こし、神経細胞を死に至らしめるというものである。Ａβはアミロイド前駆体蛋白から通常では作用しないβセクレターゼおよびγセクレターゼにより切断されて生成する。Ａβには主に４２個のアミノ酸から構成されるＡβ４２と４０個のアミノ酸から成るＡβ４０があり、前者が凝集能や神経毒性が高い点から、アルツハイマー病の発症により重要であると考えられている。脳脊髄液中のＡβ４２の減少（脳内でＡβ４２の沈着に起因）は、現状では最も信頼のできるアルツハイマー病診断指標の一つとされている。また、アルツハイマー病では脳脊髄液中のリン酸化タウ蛋白の増加も確認されており、病態と密接に関連した診断指標として位置づけられている。しかし、脳脊髄液は一般的な健康診断で扱う血液や尿とは異なり、その採取には特殊な技術を要し、また患者への負担も大きいことから、高齢者等のスクリーニング検査としては不向きである。
Therefore, as a more objective and simple test method for dementia, a method using a biomolecule (biomarker) as an index has attracted attention. For example, the neuropathological characteristics of Alzheimer's disease, one of the typical dementias, are a neurogenic group characterized by the appearance of senile plaques formed by amyloid β (Aβ) deposition and abnormal phosphorylated tau protein. Fibrous changes and massive neuronal loss. Today, the amyloid hypothesis has been proposed that the pathological process of Alzheimer's disease is triggered by Aβ production. As a result of the production of Aβ, oxidative stress and calcium regulation abnormalities are induced in nerve cells, causing nerve cell death. At the same time, Aβ induces phosphorylation of tau protein, causing cytoskeletal protein abnormalities and accompanying intracellular transport disturbances, leading to death of nerve cells. Aβ is produced by cleaving from amyloid precursor protein with β-secretase and γ-secretase which do not normally act. There are Aβ42 consisting mainly of 42 amino acids and Aβ40 consisting of 40 amino acids, and the former is considered to be important for the onset of Alzheimer's disease because of the high aggregation ability and neurotoxicity. . A decrease in Aβ42 in the cerebrospinal fluid (due to the deposition of Aβ42 in the brain) is considered to be one of the most reliable Alzheimer's disease diagnostic indicators at present. In Alzheimer's disease, an increase in phosphorylated tau protein in the cerebrospinal fluid has also been confirmed, and it is positioned as a diagnostic index closely related to the disease state. However, cerebrospinal fluid is different from blood and urine, which are handled in general medical examinations, and it requires special techniques for collection and is a heavy burden on patients, making it unsuitable as a screening test for the elderly. It is.
 
その他にもさまざまな酸化ストレスマーカーや炎症マーカー、アポトーシス関連マーカーなどが認知症の検査指標として検討されている。酸化により糖化修飾されたｇｌｙｃａｔｅｄ　ｐｒｏｔｅｉｎや核酸が酸化された８－ｈｙｄｒｏｘｙｇｕａｎｏｓｉｎｅはアルツハイマー病の脳脊髄液中で増加することが報告されており、不飽和脂肪酸の酸化生成物であるＦ２－ｉｓｏｐｒｏｓｔａｎｅは脳脊髄液、血液、尿中で増加すると報告されている。また、炎症マーカーではＩＬ－１βやＩＬ－６、ＴＧＦ－β１などが脳脊髄液中で増加、アポトーシスに関連するＴＲＡＩＬも脳脊髄液中での増加が報告されている。しかし、酸化ストレスや炎症、アポトーシスは、いずれもアルツハイマー病を含む認知症の病理・病態に深く関係する現象ではあるが、必ずしも特異的であるとは言えないことから、認知症のバイオマーカーとして好ましいものではない。
In addition, various oxidative stress markers, inflammation markers, apoptosis-related markers, and the like have been examined as test indicators for dementia. Glycated protein glycated by oxidation and 8-hydroxyguanosine with oxidized nucleic acid have been reported to increase in the cerebrospinal fluid of Alzheimer's disease, and F2-isoprosane, an oxidation product of unsaturated fatty acids, has been reported It is reported to increase in fluid, blood and urine. In addition, IL-1β, IL-6, TGF-β1 and the like are increased in cerebrospinal fluid among inflammation markers, and TRAIL related to apoptosis is also increased in cerebrospinal fluid. However, although oxidative stress, inflammation, and apoptosis are all closely related to the pathology and pathology of dementia including Alzheimer's disease, they are not necessarily specific, so they are preferable as biomarkers for dementia. It is not a thing.
 
３．エタノールアミン型プラスマローゲン（以下ＰｌｓＥｔｎと略す。）
プラスマローゲン（以下Ｐｌｓと略す。）はグリセロリン脂質のサブクラスの一つで、生体膜成分として人体を構成するリン脂質の約２割を占め、脳や心臓などに多く含まれている。このＰｌｓは、生活習慣病（動脈硬化症、高脂血症、糖尿病、高血圧症、中心性肥満症）のバイオマーカーとして検討されている（特許文献１、２）。また、特に脳内のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）は、神経髄鞘を成すミエリン膜の主要構成成分であり、また神経細胞間でのシナプス形成などに関わるとされる。ＰｌｓＥｔｎは膜モデュレーターとして機能し、細胞膜融合やエンドサイトーシス／エキソサイトーシスに関与するのに加え、膜結合酵素の活性調節などにも関わっている。また、Ｐｌｓはその構造上の特徴を成すビニルエーテル結合にラジカル消去能があることから、生体内で抗酸化物質として作用すると考えられている。
3. Ethanolamine plasmalogen (hereinafter abbreviated as PlsEtn)
Plasmalogen (hereinafter abbreviated as Pls) is one of the subclasses of glycerophospholipid, accounting for about 20% of phospholipids constituting the human body as a biological membrane component, and is contained in a large amount in the brain and heart. Pls has been studied as a biomarker for lifestyle-related diseases (arteriosclerosis, hyperlipidemia, diabetes, hypertension, central obesity) (Patent Documents 1 and 2). In particular, ethanolamine-type plasmalogen (PlsEtn) in the brain is a major component of the myelin membrane that forms the nerve myelin sheath, and is also involved in synapse formation between nerve cells. PlsEtn functions as a membrane modulator and is involved in cell membrane fusion and endocytosis / exocytosis, as well as in the regulation of membrane-bound enzyme activity. In addition, Pls is considered to act as an antioxidant in vivo because the vinyl ether bond, which is a structural feature, has radical scavenging ability.
 
ＰｌｓＥｔｎはアルツハイマー病やダウン症候群、脊髄損傷、多発性硬化症などで脳内レベルの特異的な減少が報告されている（非特許文献１）。特に、アルツハイマー病では酸化ストレスがその発症の初期段階で重要であることを支持する生化学的・臨床的証拠が集積していることから、ＰｌｓＥｔｎはアルツハイマー病の酸化ストレスマーカーとして注目されている（非特許文献２）。例えば、ＰｌｓＥｔｎの一分子種であるＰｌｓＥｔｎ２２：６の血清または血漿中濃度の低下がアルツハイマー病の早期発見マーカーとなり得ることが報告されている（非特許文献３）。ただし、ＰｌｓＥｔｎ２２：６の測定（定量）には液体クロマトグラフィー質量分析（ＬＣ－ＭＳ／ＭＳ）などの高価な分析機器が必要であり、また定量のための内部標準物質の調整等も煩雑であることから、通常の健康診断に付随させることのできる大規模スクリーニングには不向きである。一方、本発明者らは、特許文献１等に記載の簡便な方法で健康高齢者および認知障害者の血清または血漿中のＰｌｓＥｔｎ量（以下、「血清／血漿ＰｌｓＥｔｎ量」と記載することがある）を測定（定量）し、認知機能検査（ＣＤＲ）の結果別に比較したところ、ＰｌｓＥｔｎ量は認知機能の低下を良く反映することを見出している（非特許文献４）。
PlsEtn has been reported to have a specific decrease in the brain level due to Alzheimer's disease, Down's syndrome, spinal cord injury, multiple sclerosis, and the like (Non-patent Document 1). In particular, PlsEtn has attracted attention as an oxidative stress marker for Alzheimer's disease because of the accumulation of biochemical and clinical evidence that supports the importance of oxidative stress in the early stages of Alzheimer's disease ( Non-patent document 2). For example, it has been reported that a decrease in serum or plasma concentration of PlsEtn22: 6, a molecular species of PlsEtn, can be an early detection marker for Alzheimer's disease (Non-patent Document 3). However, measurement (quantification) of PlsEtn22: 6 requires expensive analytical instruments such as liquid chromatography mass spectrometry (LC-MS / MS), and adjustment of internal standard substances for quantification is complicated. Therefore, it is not suitable for large-scale screening that can be accompanied by a normal health examination. On the other hand, the present inventors may describe the amount of PlsEtn in serum or plasma of healthy elderly people and cognitively impaired persons (hereinafter referred to as “serum / plasma PlsEtn amount”) by a simple method described in Patent Document 1 and the like. ) Was measured (quantitatively) and compared according to cognitive function test (CDR) results, it was found that the amount of PlsEtn well reflects the decline in cognitive function (Non-patent Document 4).
 
ＰｌｓＥｔｎの測定方法としては、特許文献１に記載された放射性ヨウ素－高速液体クロマトグラフィー（１２５Ｉ－ＨＰＬＣ）法、ＬＣ－ＭＳ法（特許文献５）が公知である。
Known methods for measuring PlsEtn include the radioactive iodine-high performance liquid chromatography (125I-HPLC) method and LC-MS method (Patent Document 5) described in Patent Document 1.
 
４．ミオイノシトール（以下ＭＩと略す場合がある。）
ＭＩは９種類あるイノシトールの異性体の一つで、人体を含む天然に最も豊富に存在する。生体中のＭＩは食餌からの摂取に加え、体内でグルコースからも生合成される。ＭＩは細胞内情報伝達物質のソースとなるイノシトールリン脂質や、カルシウムイオンの恒常性維持に関わるイノシトールリン酸の合成材料として重要である。さらに、ＭＩ自身も脳内の有機性浸透圧調製物質として、また、おそらくミエリン形成への関与の可能性といった点から、中枢神経系の正常な機能維持に重要な役割をもつと考えられる。
4). Myoinositol (hereinafter sometimes abbreviated as MI)
MI is one of nine isomers of inositol and is most abundant in nature, including the human body. In addition to intake from diet, MI in the living body is biosynthesized from glucose in the body. MI is important as a synthetic material for inositol phospholipids, which are sources of intracellular signal transmitters, and inositol phosphates involved in maintaining calcium ion homeostasis. Furthermore, MI itself is considered to play an important role in maintaining normal function of the central nervous system, as an organic osmotic pressure regulating substance in the brain and possibly in the involvement of myelin formation.
 
脳内ＭＩ濃度は血中レベルの２００倍程度もあり、うつ病などの感情障害やダウン症候群など様々な病態で脳内ＭＩの増加が報告されている。またアルツハイマー病患者においても脳内ＭＩ量の増加が報告されている。（非特許文献６）。脳内ＭＩの一部は、脳血液関門を通過して循環血液に入るが、ほとんどが腎臓で再吸収されるか分解され、尿に排泄されるＭＩ量（以下、「尿ＭＩ量」と記載することがある）はごくわずかである。再吸収過程が血糖と拮抗することから、高血糖状態ではそのレベルを反映して尿ＭＩ量が増加するため、糖負荷試験による境界型糖尿病の検出の際に血糖値に代替できる測定項目として注目されており（非特許文献５）、糖尿病予備群の検査（特許文献３）あるは軽症耐糖能異常やインスリン分泌不全の検出（特許文献４）のためのバイオマーカーとして使用されている。
The brain MI concentration is about 200 times the blood level, and increases in brain MI have been reported in various pathological conditions such as emotional disorders such as depression and Down's syndrome. An increase in the amount of MI in the brain has also been reported in Alzheimer's disease patients. (Non-patent document 6). A portion of the MI in the brain passes through the brain blood barrier and enters the circulating blood, but most of it is reabsorbed or decomposed by the kidney and is excreted in the urine (hereinafter referred to as “urine MI amount”). There are very few). Since the reabsorption process antagonizes blood glucose, the amount of urinary MI increases reflecting the level in hyperglycemia, so it is a focus of attention as a measurement item that can replace blood glucose when detecting borderline diabetes in the glucose tolerance test It is used as a biomarker for the detection of a pre-diabetes group (Patent Document 3) or the detection of mild glucose intolerance and insulin secretion failure (Patent Document 4).
　
５．ＰｌｓとＭＩの関係
ＭＩは体内（特に脳内）でのＰｌｓ合成に於いて重要な役割を担うと考えられ、ＭＩの体外ロス（長期の持続的な尿中排泄の増加）はＰｌｓの体内（特に脳内）合成の低下を招くと推測される。ＭＩの尿中排泄はグルコースと拮抗することから、高血糖状態はＭＩの尿中排泄量を増加させる一因と思われる。したがって、特に、糖尿病などの高血糖状態を有する者では、高血糖状態　→　尿ＭＩ排泄の増加　→　脳内ＰｌｓＥｔｎの減少　→　認知症発症という仮説が提唱できる。また、高血糖に限らず、何らかの原因で尿ＭＩ排泄の増加がみられる場合も、脳内ＰｌｓＥｔｎの減少　→　認知症発症に至るリスクが高いと考えられる。ただし、尿ＭＩ量に関しては高齢健常者と認知症患者とで有意差は認められず、また認知機能検査の結果と尿ＭＩ量にも有意な相関が得られないことが報告されている（非特許文献７）。
5. Relationship between Pls and MI MI is considered to play an important role in the synthesis of Pls in the body (especially in the brain), and MI extracorporeal loss (long-term sustained increase in urinary excretion) occurs in the body of Pls ( It is speculated that this will lead to a decrease in synthesis, particularly in the brain. Since urinary excretion of MI antagonizes glucose, a hyperglycemic state seems to be a cause of increasing the urinary excretion of MI. Therefore, in particular, in a person with a hyperglycemic state such as diabetes, the hypothesis that hyperglycemic state → increased urinary MI excretion → decrease in brain PlsEtn → onset of dementia can be proposed. Further, not only hyperglycemia but also increase in urinary MI excretion for some reason, it is considered that there is a high risk of reducing PlsEtn in the brain → dementia. However, regarding urinary MI levels, no significant difference was found between elderly healthy subjects and patients with dementia, and it was reported that there was no significant correlation between cognitive function test results and urinary MI levels. Patent Document 7).
 
特開２００７－３３４１０号公報（特許第４１７６７４９号）JP 2007-33410 A (Patent No. 4176749) 特開２０１１－２５７１４８号公報JP2011-257148A 特開２００１－１９０２９９号公報（特許第３９７５２７９号）JP 2001-190299 A (Patent No. 3975279) 国際公開ＷＯ２００３／０８３１３３（特許第４４６６９１２号）International Publication WO2003 / 083133 (Patent No. 4466912) 特開２０１１－１３６９２６号公報JP 2011-136926 A
前記のとおり、認知症を予防し、あるいはその進行を抑制するためには早期の発見が重要であり、客観的な指標であるバイオマーカーを用いた大規模なスクリーニングの実施が有効である。そしてバイオマーカーに求められることは、認知症患者またはその前段階にある者を的確に（すなわち他の疾患患者と混同することなく）特定できることであり、また通常の健康診断の範囲内で実施できるような簡便な測定（定量）と判定が可能なことである。 As described above, early detection is important for preventing or suppressing the progression of dementia, and implementation of large-scale screening using a biomarker that is an objective index is effective. What is required of biomarkers is that they can accurately identify patients with dementia or their predecessors (ie, without being confused with patients with other diseases), and can be performed within the scope of normal health examinations. Such simple measurement (quantification) and determination are possible.
 
このような観点から、血清及び／又は血漿ＰｌｓＥｔｎ量は認知症検査、特にその大規模スクリーニングのためのバイオマーカーとして有望視されている。しかしながら、血清／血漿ＰｌｓＥｔｎ量を単独指標とした場合の判定精度は必ずしも十分なものではない。たとえば後記の実施例に示したように、ＰｌｓＥｔｎ量がその平均的な値（５８μＭ）を下回った被験者のうち、認知機能検査で認知症と判定された者の割合は６０％未満である。
From this point of view, the serum and / or plasma PlsEtn amount is promising as a biomarker for dementia testing, particularly for its large-scale screening. However, the determination accuracy when the serum / plasma PlsEtn amount is used as a single index is not always sufficient. For example, as shown in the examples described later, among subjects whose PlsEtn amount was lower than the average value (58 μM), the proportion of those who were determined to have dementia in the cognitive function test was less than 60%.
 
本発明は、血清及び／又は血漿ＰｌｓＥｔｎ量をバイオマーカーとする認知症検査の精度をさらに向上させる手段を提供することを課題としている。
An object of the present invention is to provide means for further improving the accuracy of a dementia test using serum and / or plasma PlsEtn levels as biomarkers.
 
また前記のとおり従来のＰｌｓＥｔｎ測定法の１２５Ｉ－ＨＰＬＣ法は高額な装置や放射性ヨウ素を使用するので汎用性が劣る。ＬＣ－ＭＳ／ＭＳ法は高額な装置を使用するので汎用性が劣る。以上のような背景のもと本発明は、汎用自動分析機に応用でき、利便性高いＰｌｓＥｔｎの測定方法を提供することを目的とする。
Further, as described above, the conventional PlsEtn measurement method 125I-HPLC is inferior in versatility because it uses expensive equipment and radioactive iodine. The LC-MS / MS method is inferior in versatility because an expensive apparatus is used. Under the background as described above, an object of the present invention is to provide a convenient method for measuring PlsEtn that can be applied to a general-purpose automatic analyzer.
 
本発明者らは、それ単独の定量では認知機能との関連性が存在しない尿ＭＩ量の定量値と血清及び／又は血漿ＰｌｓＥｔｎの定量値を組み合わせることで、認知症検査の精度が向上することを見出し、ＰｌｓＥｔｎに特異的に作用するリゾプラスマローゲナーゼ（Ｌｙｓｏｐｌａｓｍａｌｏｇｅｎａｓｅ、ｌｙＰｌｓ　ａｓｅと略す場合がある）とホスホリパーゼ（以下ＰＬと略す場合がある。）を新規に見出し、被験者の血清または血漿中のＰｌｓＥｔｎを定量するためのｌｙＰｌｓ　ａｓｅとＰＬを含有する認知症検査試薬キットを創出して本発明を完成させた。 The present inventors improve the accuracy of the dementia test by combining the quantitative value of urinary MI and the quantitative value of serum and / or plasma PlsEtn, which are not related to the cognitive function by the quantification alone. And newly found lysoplasmalogenase (sometimes abbreviated as Lysoplasmalogenase, lyPlsase) and phospholipase (hereinafter sometimes abbreviated as PL) that act specifically on PlsEtn, and PlsEtn in the serum or plasma of the subject The present invention was completed by creating a dementia test reagent kit containing lyPlsase and PL for quantifying the amount of lysine.
 
すなわち、本出願は前記の課題を解決する発明として以下を提供する。
That is, this application provides the following as invention which solves the said subject.
 
［１］
被験者を認知症患者またはその前段階にある者か否かを分類するための認知症検査方法であって、
　被験者の自然排泄尿中のミオイノシトール（ＭＩ）を定量する工程と、同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する工程とを含み、
健常者と、認知症患者またはその前段階にある者とを区別することができる、自然排泄尿中のミオイノシトール（ＭＩ）の予め求められた閾値と、血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）の予め求められた閾値のそれぞれに対して、前記被験者のミオイノシトール（ＭＩ）の定量値とエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）の定量値を比較する工程を含む認知症検査方法。
[1]
A method for testing dementia for classifying whether a subject is a dementia patient or a person in the preceding stage,
Quantifying myo-inositol (MI) in spontaneously excreted urine of a subject and quantifying ethanolamine plasmalogen (PlsEtn) in serum or plasma of the same subject,
A pre-determined threshold of myo-inositol (MI) in spontaneously excreted urine and ethanolamine plasmalogens in serum or plasma, which can distinguish between healthy and demented patients or those in its predecessor A dementia test method comprising a step of comparing a quantitative value of myo-inositol (MI) and a quantitative value of ethanolamine-type plasmalogen (PlsEtn) of the subject with respect to each of previously determined threshold values of (PlsEtn).
 
［１－１］
同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する工程が、酵素を利用する反応により、最終的に過酸化水素が生成され、該エタノールアミン型プラスマローゲンの存在量に対応して発生する過酸化水素の量を検出して、血清または血漿中のＰｌｓＥｔｎ量を算出する工程を含む［１］の方法。
[1-1]
The process of quantifying ethanolamine plasmalogen (PlsEtn) in the serum or plasma of the same subject finally generates hydrogen peroxide by the reaction using an enzyme, and corresponds to the abundance of the ethanolamine plasmalogen. The method according to [1], further comprising the step of calculating the amount of PlsEtn in serum or plasma by detecting the amount of hydrogen peroxide generated.
 
［１－２］
同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する方法が、以下の（１）～（３－４）の工程を含む［１］に記載の方法。
（１）エタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解できる酵素を用いて、被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解する工程：
（２）エタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）をグリセロ－３－ホスフォエタノールアミンとアルデヒドに加水分解でき、かつエーテル型リゾホスファチジルエタノールアミンを加水分解できない加水分解酵素を用いて、前記（１）にて生成されたエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）をグリセロ－３－ホフォエタノールアミンとアルデヒドに加水分解する工程：及び
（３－１）グリセロ－３－ホスフォエタノールアミンを加水分解してエタノールアミンにせしめる酵素にて、工程（２）で得られたグリセロ－３－ホスフォエタノールアミンを加水分解してエタノールアミンにする工程：
（３－２）エタノールアミンを酸化する酵素により、エタノールアミンから過酸化水素を発生せしめる工程：
（３－３）過酸化水素を過酸化水素定量手段により定量する工程：及び
（３－４）前記被験者の血清または血漿中のＰｌｓＥｔｎ量を算出する工程：
または前記（２）の加水分解酵素が、リゾホスファチジルエタノールアミンを加水分解できる酵素である場合、同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する方法が、前記（１）～（３－４）の工程に先立つか、少なくとも前記（２）工程の前までに、下記（４）及び（５）を含む工程を行うことを特徴とする方法。
（４）ホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する酵素を用いて、血清または血漿中に混在するホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する工程：及び
（５）リゾホスフォリパーゼ及び／又はモノグリセリドリパーゼを用いて、前記（４）工程により生成されるリゾホスファチジルエタノールアミンを分解し、実質的に消去する工程。
[1-2]
The method according to [1], wherein the method for quantifying ethanolamine-type plasmalogen (PlsEtn) in serum or plasma of the same subject comprises the following steps (1) to (3-4).
(1) Using an enzyme capable of hydrolyzing ethanolamine-type plasmalogen (PlsEtn) into ethanolamine-type lysoplasmalogen (lyPlsEtn), ethanolamine-type plasmalogen (PlsEtn) in the serum or plasma of the subject is ethanolamine-type lysozyme. Hydrolyzing to plasmalogen (lyPlsEtn):
(2) Using a hydrolase that can hydrolyze ethanolamine-type lysoplasmalogen (lyPlsEtn) into glycero-3-phosphoethanolamine and an aldehyde and cannot hydrolyze ether-type lysophosphatidylethanolamine, (1) Hydrolyzing ethanolamine-type lysoplasmalogen (lyPlsEtn) produced in step 1 to glycero-3-phosphoethanolamine and aldehyde: and (3-1) hydrolyzing glycero-3-phosphoethanolamine A step of hydrolyzing the glycero-3-phosphoethanolamine obtained in step (2) to ethanolamine with an enzyme that is made to be ethanolamine:
(3-2) Step of generating hydrogen peroxide from ethanolamine by an enzyme that oxidizes ethanolamine:
(3-3) Step of quantifying hydrogen peroxide by means of hydrogen peroxide quantification: (3-4) Step of calculating the amount of PlsEtn in the serum or plasma of the subject:
Alternatively, when the hydrolase of (2) is an enzyme capable of hydrolyzing lysophosphatidylethanolamine, the method for quantifying ethanolamine-type plasmalogen (PlsEtn) in the serum or plasma of the same subject is described in (1) A method comprising performing the steps (4) and (5) below prior to the steps (3-4) or at least before the step (2).
(4) Step of decomposing phosphatidylethanolamine mixed in serum or plasma into lysophosphatidylethanolamine and fatty acid using an enzyme that decomposes phosphatidylethanolamine into lysophosphatidylethanolamine and fatty acid: and (5) lysophospho A step of decomposing and substantially eliminating lysophosphatidylethanolamine produced by the step (4) using lipase and / or monoglyceride lipase.
 
［１－３］
前記工程（２）の加水分解酵素が、下記（ａ）または（ｂ）の酵素のいずれかである［１－２］に記載の方法。
（ａ）配列番号１または２に記載のアミノ酸配列：
（ｂ）配列番号１または２に記載のアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列：
からなる酵素であって、エタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）を加水分解し、グリセロ－３－ホスフォエタノールアミンとアルデヒドとを得る反応を触媒する。
[1-3]
The method according to [1-2], wherein the hydrolase in the step (2) is any of the following enzymes (a) and (b).
(A) the amino acid sequence set forth in SEQ ID NO: 1 or 2:
(B) Amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 or 2:
Which catalyzes the reaction of hydrolyzing ethanolamine-type lysoplasmalogen (lyPlsEtn) to give glycero-3-phosphoethanolamine and an aldehyde.
 
［１－４］
前記工程（１）のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解できる酵素、が下記（ｃ）または（ｄ）のいずれかの酵素である［１－２］に記載の方法。
（ｃ）配列番号５に記載のアミノ酸配列：
（ｄ）配列番号５に記載のアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列であり、エタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解する作用を触媒する。 
[1-4]
The enzyme capable of hydrolyzing the ethanolamine type plasmalogen (PlsEtn) in the step (1) to an ethanolamine type lysoplasmalogen (lyPlsEtn) is one of the following enzymes (c) or (d) [1-2 ] Method.
(C) the amino acid sequence set forth in SEQ ID NO: 5:
(D) An amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 5, and ethanolamine type plasmalogen (PlsEtn) is changed to ethanolamine type lysoplasmalogen (lyPlsEtn). To catalyze the action of hydrolysis.
 
［１－５］
前記（２）の加水分解酵素が、リゾホスファチジルエタノールアミンを加水分解できる酵素である場合、同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する方法が、前記（１）～（３－４）の工程に先立つか、少なくとも前記（２）工程の前までに、下記（４）及び（５）を含む工程を行うことを特徴とする［１－１］～［１－４］に記載の方法。
（４）ホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する酵素を用いて、血清または血漿中に混在するホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する工程：及び
（５）リゾホスフォリパーゼ及び／又はモノグリセリドリパーゼを用いて、前記（４）工程により生成されるリゾホスファチジルエタノールアミンを分解し、実質的に消去する工程。
[1-5]
When the hydrolase of (2) is an enzyme capable of hydrolyzing lysophosphatidylethanolamine, the method for quantifying ethanolamine-type plasmalogen (PlsEtn) in serum or plasma of the same subject is described in (1) to [1-1] to [1-4] characterized in that the steps including the following (4) and (5) are performed prior to the step (3-4) or at least before the step (2). ] Method.
(4) Step of decomposing phosphatidylethanolamine mixed in serum or plasma into lysophosphatidylethanolamine and fatty acid using an enzyme that decomposes phosphatidylethanolamine into lysophosphatidylethanolamine and fatty acid: and (5) lysophospho A step of decomposing and substantially eliminating lysophosphatidylethanolamine produced by the step (4) using lipase and / or monoglyceride lipase.
 
［１－６］
前記工程（４）のホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する酵素としてホスフォリパーゼ（ＰＬ）を使用し、該ＰＬは、前記工程（１）のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解できる酵素としての作用を兼ねさせて新たな該ＰＬを添加することなく前記工程（１）の工程を進行せしめ、前記工程（５）を実施と同時、または前記工程（５）の実施後に、前記工程（１）が完了し、以後前記工程（２）～（３－４）を実施する［１－５］に記載の方法。
[1-6]
Phospholipase (PL) is used as an enzyme that decomposes the phosphatidylethanolamine of the step (4) into lysophosphatidylethanolamine and a fatty acid, and the PL uses the ethanolamine-type plasmalogen (PlsEtn) of the step (1). Simultaneously with the implementation of the step (5), the step (1) is allowed to proceed without adding new PL, which also serves as an enzyme capable of hydrolyzing ethanolamine-type lysoplasmalogen (lyPlsEtn). Alternatively, after the step (5) is performed, the step (1) is completed, and then the steps (2) to (3-4) are performed.
 
［１－７］
同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する方法が、以下の（１）～（３－３）´の工程を含む［１－１］に記載の方法。
（１）エタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解できる酵素を用いて、被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解する工程：
（２）´エタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）をエタノールアミンとプラズメニルホスファチジン酸に加水分解でき、かつエーテル型リゾホスファチジルエタノールアミンを加水分解でき、かつリゾホスファチジルエタノールアミンを加水分解できる加水分解酵素を用いて、前記（１）にて生成されたエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）をエタノールアミンとプラズメニルホスファチジン酸に加水分解する工程：及び
（３－１）´エタノールアミンを酸化する酵素により、工程（２）´で得られたエタノールアミンから過酸化水素を発生せしめる工程：
（３－２）´過酸化水素を過酸化水素定量手段により定量する工程：及び
（３－３）´前記被験者の血清または血漿中のＰｌｓＥｔｎ量を算出する工程。
[1-7]
The method according to [1-1], wherein the method for quantifying ethanolamine plasmalogen (PlsEtn) in serum or plasma of the same subject comprises the following steps (1) to (3-3) ′.
(1) Using an enzyme capable of hydrolyzing ethanolamine-type plasmalogen (PlsEtn) into ethanolamine-type lysoplasmalogen (lyPlsEtn), ethanolamine-type plasmalogen (PlsEtn) in the serum or plasma of the subject is ethanolamine-type lysozyme. Hydrolyzing to plasmalogen (lyPlsEtn):
(2) Hydrolyzing enzyme capable of hydrolyzing ethanolamine type lysoplasmalogen (lyPlsEtn) to ethanolamine and plasmenyl phosphatidic acid, hydrolyzing ether type lysophosphatidylethanolamine, and hydrolyzing lysophosphatidylethanolamine Hydrolyzing the ethanolamine-type lysoplasmalogen (lyPlsEtn) produced in the above (1) into ethanolamine and plasmenylphosphatidic acid using: and (3-1) by an enzyme that oxidizes ethanolamine The step of generating hydrogen peroxide from the ethanolamine obtained in step (2) ′:
(3-2) 'Step of quantifying hydrogen peroxide by means of hydrogen peroxide determination: and (3-3)' Step of calculating the amount of PlsEtn in the serum or plasma of the subject.
 
［１－８－１］
同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する方法が、前記（１）～（３－３）´の工程に先立つか、少なくとも前記（２）´工程の前までに、下記（４）及び（５）を含む工程を行うことを特徴とする［１－７］に記載の方法。
（４）ホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する酵素を用いて、血清または血漿中に混在するホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する工程：及び
（５）リゾホスフォリパーゼ及び／又はモノグリセリドリパーゼを用いて、前記（４）工程により生成されるリゾホスファチジルエタノールアミンを分解し、実質的に消去する工程。
[1-8-1]
A method for quantifying ethanolamine-type plasmalogen (PlsEtn) in serum or plasma of the same subject is performed prior to the steps (1) to (3-3) ′ or at least before the step (2) ′. The method according to [1-7], wherein a process comprising the following (4) and (5) is performed.
(4) Step of decomposing phosphatidylethanolamine mixed in serum or plasma into lysophosphatidylethanolamine and fatty acid using an enzyme that decomposes phosphatidylethanolamine into lysophosphatidylethanolamine and fatty acid: and (5) lysophospho A step of decomposing and substantially eliminating lysophosphatidylethanolamine produced by the step (4) using lipase and / or monoglyceride lipase.
 
［１－８－２］
前記工程（４）のホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する酵素としてホスフォリパーゼ（ＰＬ）を使用し、該ＰＬは、前記工程（１）のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解できる酵素としての作用を兼ねさせて新たな該ＰＬを添加することなく前記工程（１）の工程を進行せしめ、前記工程（５）を実施と同時、または前記工程（５）の実施後に、前記工程（１）が完了し、以後前記工程（２）～（３－３）´を実施する［１－８－１］に記載の方法。
[1-8-2]
Phospholipase (PL) is used as an enzyme that decomposes the phosphatidylethanolamine of the step (4) into lysophosphatidylethanolamine and a fatty acid, and the PL uses the ethanolamine-type plasmalogen (PlsEtn) of the step (1). Simultaneously with the implementation of the step (5), the step (1) is allowed to proceed without adding new PL, which also serves as an enzyme capable of hydrolyzing ethanolamine-type lysoplasmalogen (lyPlsEtn). Alternatively, after the step (5) is carried out, the step (1) is completed, and then the steps (2) to (3-3) ′ are carried out [1-8-1].
 
［１－９］
自然排泄尿中のミオイノシトール（ＭＩ）の予め求められた閾値が、２１．６～６３．３ｍｇ／ｇＣｒの範囲から選択される値であり、血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）の予め求められた閾値が、５０～６４μＭの範囲から選択される値であって、被験者の自然排泄尿中のミオイノシトール（ＭＩ）の定量値が、前記ＭＩ閾値を越え、かつ被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）の定量値が、前記ＰｌｓＥｔｎ閾値を下回った場合に、認知症患者またはその前段階にある者と区分する［１］に記載の方法。
[1-9]
A predetermined threshold value of myo-inositol (MI) in spontaneously excreted urine is a value selected from the range of 21.6 to 63.3 mg / gCr, and ethanolamine-type plasmalogen (PlsEtn) in serum or plasma Is a value selected from the range of 50 to 64 μM, and the quantitative value of myo-inositol (MI) in the spontaneously excreted urine of the subject exceeds the MI threshold, and the serum of the subject or The method according to [1], wherein when the quantitative value of ethanolamine-type plasmalogen (PlsEtn) in plasma falls below the PlsEtn threshold value, it is distinguished from a dementia patient or a person in the previous stage.
 
［１－１０］ 
前記ＭＩ量の閾値が２５ｍｇ／ｇＣｒである［１－９］に記載の方法。
[1-10]
The method according to [1-9], wherein the MI amount threshold is 25 mg / gCr.
 
［１－１１］
前記ＭＩ量の閾値が３３．２ｍｇ／ｇＣｒである［１－９］に記載の方法。
[1-11]
The method according to [1-9], wherein the MI amount threshold is 33.2 mg / gCr.
 
［１－１２］ 
前記ＭＩ閾値が３６．６ｍｇ／ｇＣｒである［１－９］に記載の方法。
[1-12]
The method according to [1-9], wherein the MI threshold is 36.6 mg / gCr.
 
［１－１３］
前記ＭＩ量の閾値が４４．８ｍｇ／ｇＣｒである［１－９］に記載の方法。
[1-13]
The method according to [1-9], wherein the MI amount threshold is 44.8 mg / gCr.
 
［１－１４］
前記エタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）閾値が、５８μＭである［１－９］に記載の方法。
[1-14]
The method according to [1-9], wherein the ethanolamine type plasmalogen (PlsEtn) threshold value is 58 μM.
 
［１－１５］
前記エタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）閾値が、６１μＭである［１－９］に記載の方法。
[1-15]
The method according to [1-9], wherein the ethanolamine type plasmalogen (PlsEtn) threshold is 61 μM.
 
［１－１６］
前記エタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）閾値が、５６μＭである［１－９］に記載の方法。
[1-16]
The method according to [1-9], wherein the ethanolamine type plasmalogen (PlsEtn) threshold value is 56 μM.
 
［１－１７］
前記被験者のＭＩ量が２１．６～５５．１ｍｇ／ｇＣｒの範囲から設定される閾値以上であり、ＰｌｓＥｔｎ量が５０～６４μＭの範囲から設定される閾値以下である［１－９］に記載の方法。
[1-17]
[1-9] The MI amount of the subject is not less than a threshold value set from a range of 21.6 to 55.1 mg / gCr, and the PlsEtn amount is not more than a threshold value set from a range of 50 to 64 μM. Method.
 
［１－１８］
前記被験者のＭＩ量が２１．６～５５．１ｍｇ／ｇＣｒの範囲から設定される閾値以上であり、ＰｌｓＥｔｎ量が５２～６１μＭの範囲から設定される閾値以下である［１－９］に記載の方法。
[1-18]
[1-9] The MI amount of the subject is not less than a threshold value set from a range of 21.6 to 55.1 mg / gCr, and the PlsEtn amount is not more than a threshold value set from a range of 52 to 61 μM. Method.
 
［１－１９］
前記被験者のＭＩ量が２９．８～６３．３ｍｇ／ｇＣｒの範囲から設定される閾値以上であり、ＰｌｓＥｔｎ量が５０～６４μＭの範囲から設定される閾値以下である［１―９］に記載の方法。
[1-19]
[1-9] The MI amount of the subject is not less than a threshold value set from a range of 29.8 to 63.3 mg / gCr, and the PlsEtn amount is not more than a threshold value set from a range of 50 to 64 μM. Method.
 
［１－２０］
前記被験者のＭＩ量が２９．８～６３．３ｍｇ／ｇＣｒの範囲から設定される閾値以上であり、ＰｌｓＥｔｎ量が５２～６１μＭの範囲から設定される閾値以下である［１―９］に記載の方法。
[1-20]
The subject's MI amount is not less than a threshold value set from a range of 29.8 to 63.3 mg / gCr, and the PlsEtn amount is not more than a threshold value set from a range of 52 to 61 μM. Method.
 
［１－２１］
前記ＭＩ量の閾値が３６．６ｍｇ／ｇＣｒであり、ＰｌｓＥｔｎ量の閾値が５８μＭである［１－９］記載の方法。
[1-21]
[1-9] The method according to [1-9], wherein the MI amount threshold is 36.6 mg / gCr, and the PlsEtn amount threshold is 58 μM.
 
［１－２２］
前記ＭＩ量の閾値が３６．６ｍｇ／ｇＣｒであり、ＰｌｓＥｔｎ量の閾値が５６μＭ又は６１μＭである［１－９］に記載の方法。
[1-22]
[1-9] The method according to [1-9], wherein the MI amount threshold value is 36.6 mg / gCr, and the PlsEtn amount threshold value is 56 μM or 61 μM.
 
［１－２３］
前記ＭＩを定量する尿が、特に被験者の空腹時随意尿である［１－９］に記載の方法。
[1-23]
[1-9] The method according to [1-9], wherein the urine for quantifying the MI is particularly a fasting voluntary urine of the subject.
 
［２］
健常者と、認知症患者またはその前段階にある者とを区別することができる、自然排泄尿中のミオイノシトール（ＭＩ）の予め求められた閾値と、被験者の自然排泄尿中のミオイノシトール（ＭＩ）の定量値とを比較する工程と組み合わせることにより、被験者を認知症患者またはその前段階にある者か否かを分類する認知症検査に用いるものであって、
同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する工程を含み、該工程中に、健常者と、認知症患者またはその前段階にある者とを区別することができる、エタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）の、予め求められた閾値に対して、被験者のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）の定量値を比較する工程を含む、エタノールアミン型プラスマローゲンの定量方法。
[2]
A predetermined threshold of myo-inositol (MI) in spontaneously excreted urine, which can distinguish between a healthy person and a person with dementia or in the previous stage, and myo-inositol in the subject's spontaneously excreted urine ( MI) is used for a dementia test for classifying whether a subject is a dementia patient or a person in the previous stage by combining with a step of comparing with a quantitative value of MI,
Quantifying ethanolamine-type plasmalogen (PlsEtn) in the serum or plasma of the same subject, during which a healthy person can be distinguished from a patient with dementia or in its predecessor stage, A method for quantifying ethanolamine-type plasmalogen, comprising a step of comparing a quantitative value of ethanolamine-type plasmalogen (PlsEtn) of a subject against a predetermined threshold value of ethanolamine-type plasmalogen (PlsEtn).
 
［２－１］
同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する工程が、酵素を利用する反応により、最終的に過酸化水素が生成され、該エタノールアミン型プラスマローゲンの存在量に対応して発生する過酸化水素の量を検出して、血清または血漿中のＰｌｓＥｔｎ量を算出する工程を含む［２］の方法。
[2-1]
The process of quantifying ethanolamine plasmalogen (PlsEtn) in the serum or plasma of the same subject finally generates hydrogen peroxide by the reaction using an enzyme, and corresponds to the abundance of the ethanolamine plasmalogen. The method according to [2], further comprising the step of calculating the amount of PlsEtn in serum or plasma by detecting the amount of hydrogen peroxide generated.
 
［２－２］
同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する方法が、以下の（１）～（３－４）の工程を含む［２］に記載の方法。
（１）エタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解できる酵素を用いて、被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解する工程：
（２）エタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）をグリセロ－３－ホスフォエタノールアミンとアルデヒドに加水分解でき、かつエーテル型リゾホスファチジルエタノールアミンを加水分解できない加水分解酵素を用いて、前記（１）にて生成されたエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）をグリセロ－３－ホフォエタノールアミンとアルデヒドに加水分解する工程：及び
（３－１）グリセロ－３－ホスフォエタノールアミンを加水分解してエタノールアミンにせしめる酵素にて、工程（２）で得られたグリセロ－３－ホスフォエタノールアミンを加水分解してエタノールアミンにする工程：
（３－２）エタノールアミンを酸化する酵素により、エタノールアミンから過酸化水素を発生せしめる工程：
（３－３）過酸化水素を過酸化水素定量手段により定量する工程：及び
（３－４）前記被験者の血清または血漿中のＰｌｓＥｔｎ量を算出する工程：
または前記（２）の加水分解酵素が、リゾホスファチジルエタノールアミンを加水分解できる酵素である場合、同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する方法が、前記（１）～（３－４）の工程に先立つか、少なくとも前記（２）工程の前までに、下記（４）及び（５）を含む工程を行うことを特徴とする方法。
（４）ホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する酵素を用いて、血清または血漿中に混在するホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する工程：及び
（５）リゾホスフォリパーゼ及び／又はモノグリセリドリパーゼを用いて、前記（４）工程により生成されるリゾホスファチジルエタノールアミンを分解し、実質的に消去する工程。
[2-2]
The method according to [2], wherein the method for quantifying ethanolamine plasmalogen (PlsEtn) in serum or plasma of the same subject comprises the following steps (1) to (3-4).
(1) Using an enzyme capable of hydrolyzing ethanolamine-type plasmalogen (PlsEtn) into ethanolamine-type lysoplasmalogen (lyPlsEtn), ethanolamine-type plasmalogen (PlsEtn) in the serum or plasma of the subject is ethanolamine-type lysozyme. Hydrolyzing to plasmalogen (lyPlsEtn):
(2) Using a hydrolase that can hydrolyze ethanolamine-type lysoplasmalogen (lyPlsEtn) into glycero-3-phosphoethanolamine and an aldehyde and cannot hydrolyze ether-type lysophosphatidylethanolamine, (1) Hydrolyzing ethanolamine-type lysoplasmalogen (lyPlsEtn) produced in step 1 to glycero-3-phosphoethanolamine and aldehyde: and (3-1) hydrolyzing glycero-3-phosphoethanolamine A step of hydrolyzing the glycero-3-phosphoethanolamine obtained in step (2) to ethanolamine with an enzyme that is made to be ethanolamine:
(3-2) Step of generating hydrogen peroxide from ethanolamine by an enzyme that oxidizes ethanolamine:
(3-3) Step of quantifying hydrogen peroxide by means of hydrogen peroxide quantification: (3-4) Step of calculating the amount of PlsEtn in the serum or plasma of the subject:
Alternatively, when the hydrolase of (2) is an enzyme capable of hydrolyzing lysophosphatidylethanolamine, the method for quantifying ethanolamine-type plasmalogen (PlsEtn) in the serum or plasma of the same subject is described in (1) A method comprising performing the steps (4) and (5) below prior to the steps (3-4) or at least before the step (2).
(4) Step of decomposing phosphatidylethanolamine mixed in serum or plasma into lysophosphatidylethanolamine and fatty acid using an enzyme that decomposes phosphatidylethanolamine into lysophosphatidylethanolamine and fatty acid: and (5) lysophospho A step of decomposing and substantially eliminating lysophosphatidylethanolamine produced by the step (4) using lipase and / or monoglyceride lipase.
 
［２－３］
前記工程（２）の加水分解酵素が、下記（ａ）または（ｂ）の酵素のいずれかである［２－２］に記載の方法。
（ａ）配列番号１または２に記載のアミノ酸配列：
（ｂ）配列番号１または２に記載のアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列：
からなる酵素であって、エタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）を加水分解し、グリセロ－３－ホスフォエタノールアミンとアルデヒドとを得る反応を触媒する。
[2-3]
The method according to [2-2], wherein the hydrolase in the step (2) is any of the following enzymes (a) and (b).
(A) the amino acid sequence set forth in SEQ ID NO: 1 or 2:
(B) Amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 or 2:
Which catalyzes the reaction of hydrolyzing ethanolamine-type lysoplasmalogen (lyPlsEtn) to give glycero-3-phosphoethanolamine and an aldehyde.
 
［２－４］
前記工程（１）のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解できる酵素が、下記（ｃ）または（ｄ）のいずれかの酵素である［２－２］に記載の方法。
（ｃ）配列番号５に記載のアミノ酸配列：
（ｄ）配列番号５に記載のアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列であり、エタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解する作用を触媒する。 
[2-4]
The enzyme capable of hydrolyzing the ethanolamine-type plasmalogen (PlsEtn) in the step (1) to the ethanolamine-type lysoplasmalogen (lyPlsEtn) is one of the following enzymes (c) or (d) [2-2. ] Method.
(C) the amino acid sequence set forth in SEQ ID NO: 5:
(D) An amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 5, and ethanolamine type plasmalogen (PlsEtn) is changed to ethanolamine type lysoplasmalogen (lyPlsEtn). To catalyze the action of hydrolysis.
 
［２－５］
前記（２）の加水分解酵素が、リゾホスファチジルエタノールアミンを加水分解できる酵素である場合、同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する方法が、前記（１）～（３－４）の工程に先立つか、少なくとも前記（２）工程の前までに、下記（４）及び（５）を含む工程を行うことを特徴とする［２－２］～［２－４］に記載の方法。
（４）ホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する酵素を用いて、血清または血漿中に混在するホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する工程：及び
（５）リゾホスフォリパーゼ及び／又はモノグリセリドリパーゼを用いて、前記（４）工程により生成されるリゾホスファチジルエタノールアミンを分解し、実質的に消去する工程。
[2-5]
When the hydrolase of (2) is an enzyme capable of hydrolyzing lysophosphatidylethanolamine, the method for quantifying ethanolamine-type plasmalogen (PlsEtn) in serum or plasma of the same subject is described in (1) to Prior to the step (3-4) or at least before the step (2), the steps including the following (4) and (5) are performed [2-2] to [2-4] ] Method.
(4) Step of decomposing phosphatidylethanolamine mixed in serum or plasma into lysophosphatidylethanolamine and fatty acid using an enzyme that decomposes phosphatidylethanolamine into lysophosphatidylethanolamine and fatty acid: and (5) lysophospho A step of decomposing and substantially eliminating lysophosphatidylethanolamine produced by the step (4) using lipase and / or monoglyceride lipase.
 
［２－６］
前記工程（４）のホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する酵素としてホスフォリパーゼ（ＰＬ）を使用し、該ＰＬは、前記工程（１）のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解できる酵素としての作用を兼ねさせて新たな該ＰＬを添加することなく前記工程（１）の工程を進行せしめ、前記工程（５）を実施と同時、または前記工程（５）の実施後に、前記工程（１）が完了し、以後前記工程（２）～（３－４）を実施する［２－５］に記載の方法。
[2-6]
Phospholipase (PL) is used as an enzyme that decomposes the phosphatidylethanolamine of the step (4) into lysophosphatidylethanolamine and a fatty acid, and the PL uses the ethanolamine-type plasmalogen (PlsEtn) of the step (1). Simultaneously with the implementation of the step (5), the step (1) is allowed to proceed without adding new PL, which also serves as an enzyme capable of hydrolyzing ethanolamine-type lysoplasmalogen (lyPlsEtn). Alternatively, after the step (5), the step (1) is completed, and then the steps (2) to (3-4) are performed [2-5].
 
［２－７］
同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する方法が、以下の（１）～（３－３）´の工程を含む［２－１］に記載の方法。
（１）エタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解できる酵素を用いて、被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解する工程：
（２）´エタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）をエタノールアミンとプラズメニルホスファチジン酸に加水分解でき、かつエーテル型リゾホスファチジルエタノールアミンを加水分解でき、かつリゾホスファチジルエタノールアミンを加水分解できる加水分解酵素を用いて、前記（１）にて生成されたエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）をエタノールアミンとプラズメニルホスファチジン酸に加水分解する工程：及び
（３－１）´エタノールアミンを酸化する酵素により、工程（２）´で得られたエタノールアミンから過酸化水素を発生せしめる工程：
（３－２）´過酸化水素を過酸化水素定量手段により定量する工程：及び
（３－３）´前記被験者の血清または血漿中のＰｌｓＥｔｎ量を算出する工程。
[2-7]
The method according to [2-1], wherein the method for quantifying ethanolamine plasmalogen (PlsEtn) in serum or plasma of the same subject comprises the following steps (1) to (3-3) ′.
(1) Using an enzyme capable of hydrolyzing ethanolamine-type plasmalogen (PlsEtn) into ethanolamine-type lysoplasmalogen (lyPlsEtn), ethanolamine-type plasmalogen (PlsEtn) in the serum or plasma of the subject is ethanolamine-type lysozyme. Hydrolyzing to plasmalogen (lyPlsEtn):
(2) Hydrolyzing enzyme capable of hydrolyzing ethanolamine type lysoplasmalogen (lyPlsEtn) to ethanolamine and plasmenyl phosphatidic acid, hydrolyzing ether type lysophosphatidylethanolamine, and hydrolyzing lysophosphatidylethanolamine Hydrolyzing the ethanolamine-type lysoplasmalogen (lyPlsEtn) produced in the above (1) into ethanolamine and plasmenylphosphatidic acid using: and (3-1) by an enzyme that oxidizes ethanolamine The step of generating hydrogen peroxide from the ethanolamine obtained in step (2) ′:
(3-2) 'Step of quantifying hydrogen peroxide by means of hydrogen peroxide determination: and (3-3)' Step of calculating the amount of PlsEtn in the serum or plasma of the subject.
 
［２－８］
同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する方法が、前記（１）～（３－３）´の工程に先立つか、少なくとも前記（２）工程の前までに、下記（４）及び（５）を含む工程を行うことを特徴とする［２－７］に記載の方法。
（４）ホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する酵素を用いて、血清または血漿中に混在するホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する工程：及び
（５）リゾホスフォリパーゼ及び／又はモノグリセリドリパーゼを用いて、前記（４）工程により生成されるリゾホスファチジルエタノールアミンを分解し、実質的に消去する工程。
[2-8]
A method for quantifying ethanolamine plasmalogen (PlsEtn) in serum or plasma of the same subject is performed prior to the steps (1) to (3-3) ′ or at least before the step (2). The method according to [2-7], wherein a process comprising the following (4) and (5) is performed.
(4) Step of decomposing phosphatidylethanolamine mixed in serum or plasma into lysophosphatidylethanolamine and fatty acid using an enzyme that decomposes phosphatidylethanolamine into lysophosphatidylethanolamine and fatty acid: and (5) lysophospho A step of decomposing and substantially eliminating lysophosphatidylethanolamine produced by the step (4) using lipase and / or monoglyceride lipase.
 
［２－９］
前記工程（４）のホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する酵素としてホスフォリパーゼ（ＰＬ）を使用し、該ＰＬは、前記工程（１）のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解できる酵素としての作用を兼ねさせて新たな該ＰＬを添加することなく前記工程（１）の工程を進行せしめ、前記工程（５）を実施と同時、または前記工程（５）の実施後に、前記工程（１）が完了し、以後前記工程（２）～（３－３）´を実施する［２－８］に記載の方法。
[2-9]
Phospholipase (PL) is used as an enzyme that decomposes the phosphatidylethanolamine of the step (4) into lysophosphatidylethanolamine and a fatty acid, and the PL uses the ethanolamine-type plasmalogen (PlsEtn) of the step (1). Simultaneously with the implementation of the step (5), the step (1) is allowed to proceed without adding new PL, which also serves as an enzyme capable of hydrolyzing ethanolamine-type lysoplasmalogen (lyPlsEtn). Alternatively, after the step (5) is performed, the step (1) is completed, and then the steps (2) to (3-3) ′ are performed [2-8].
 
［３］
健常者と、認知症患者またはその前段階にある者とを区別することができる、血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）の予め求められた閾値と、被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）の定量値とを比較する工程と組み合わせることにより、被験者を認知症患者またはその前段階にある者か否かを分類する認知症検査に用いるものであって、
同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する工程を含み、該工程中に、健常者と、認知症患者またはその前段階にある者とを区別することができる、自然排泄尿中のミオイノシトール（ＭＩ）の、予め求められた閾値に対して、被験者の自然排泄尿中のミオイノシトール（ＭＩ）の定量値を比較する工程を含む、ミオイノシトール（ＭＩ）の定量方法。
[3]
A pre-determined threshold of ethanolamine-type plasmalogen (PlsEtn) in serum or plasma, which can distinguish between healthy and demented patients or those in its predecessor, and in the serum or plasma of the subject In combination with the step of comparing the ethanolamine-type plasmalogen (PlsEtn) with a quantitative value, the subject is used for a dementia test for classifying whether or not the subject is a dementia patient or a person in its previous stage,
Quantifying ethanolamine-type plasmalogen (PlsEtn) in the serum or plasma of the same subject, during which a healthy person can be distinguished from a patient with dementia or in its predecessor stage, Quantification of myo-inositol (MI) including the step of comparing the quantitative value of myo-inositol (MI) in spontaneously excreted urine of a subject against a predetermined threshold value of myo-inositol (MI) in spontaneously excreted urine Method.
 
［３－１］
自然排泄尿中のミオイノシトール（ＭＩ）の予め求められた閾値が、２１．６～６３．３ｍｇ／ｇＣｒの範囲から選択される値であり、血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）の予め求められた閾値が、５０～６４μＭの範囲から選択される値であって、被験者の自然排泄尿中のミオイノシトール（ＭＩ）の定量値が、前記ＭＩ閾値を越え、かつ被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）の定量値が、前記ＰｌｓＥｔｎ閾値を下回った場合に、認知症患者またはその前段階にある者と区分する［３］に記載の方法。
[3-1]
A predetermined threshold value of myo-inositol (MI) in spontaneously excreted urine is a value selected from the range of 21.6 to 63.3 mg / gCr, and ethanolamine-type plasmalogen (PlsEtn) in serum or plasma Is a value selected from the range of 50 to 64 μM, and the quantitative value of myo-inositol (MI) in the spontaneously excreted urine of the subject exceeds the MI threshold, and the serum of the subject or The method according to [3], wherein when the quantitative value of ethanolamine-type plasmalogen (PlsEtn) in plasma falls below the PlsEtn threshold, it is distinguished from a dementia patient or a person in the previous stage.
 
［３－２］ 
前記ＭＩ量の閾値が２５ｍｇ／ｇＣｒである［３－１］に記載の方法。
[3-2]
The method according to [3-1], wherein the MI amount threshold is 25 mg / gCr.
 
［３－３］
前記ＭＩ量の閾値が３３．２ｍｇ／ｇＣｒである［３－１］ に記載の方法。
[3-3]
The method according to [3-1], wherein the threshold value of the MI amount is 33.2 mg / gCr.
 
［３－４］ 
前記ＭＩ閾値が３６．６ｍｇ／ｇＣｒである［３－１］ に記載の方法。
[3-4]
The method according to [3-1], wherein the MI threshold is 36.6 mg / gCr.
 
［３－５］ 
前記ＭＩ量の閾値が４４．８ｍｇ／ｇＣｒである［３－１］ に記載の方法。
[3-5]
The method according to [3-1], wherein the threshold value of the MI amount is 44.8 mg / gCr.
 
［３－６］ 
前記エタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）閾値が、５８μＭである［３－１］ に記載の方法。
[3-6]
The method according to [3-1], wherein the ethanolamine type plasmalogen (PlsEtn) threshold value is 58 μM.
 
［３－７］
前記エタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）閾値が、６１μＭである［３－１］ に記載の方法。
[3-7]
The method according to [3-1], wherein the ethanolamine type plasmalogen (PlsEtn) threshold is 61 μM.
 
［３－８］
前記エタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）閾値が、５６μＭである［３－１］ に記載の方法。
[3-8]
The method according to [3-1], wherein the ethanolamine type plasmalogen (PlsEtn) threshold is 56 μM.
 
［３－９］
前記被験者のＭＩ量が２１．６～５５．１ｍｇ／ｇＣｒの範囲から設定される閾値以上であり、ＰｌｓＥｔｎ量が５０～６４μＭの範囲から設定される閾値以下である［３－１］に記載の方法。
[3-9]
The subject's MI amount is not less than a threshold value set from the range of 21.6 to 55.1 mg / gCr, and the PlsEtn amount is not more than the threshold value set from the range of 50 to 64 μM. Method.
 
［３－１０］
前記被験者のＭＩ量が２１．６～５５．１ｍｇ／ｇＣｒの範囲から設定される閾値以上であり、ＰｌｓＥｔｎ量が５２～６１μＭの範囲から設定される閾値以下である［３－１］に記載の方法。
[3-10]
The subject's MI amount is not less than a threshold value set from a range of 21.6 to 55.1 mg / gCr, and a PlsEtn amount is not more than a threshold value set from a range of 52 to 61 μM. Method.
 
［３－１１］
前記被験者のＭＩ量が２９．８～６３．３ｍｇ／ｇＣｒの範囲から設定される閾値以上であり、ＰｌｓＥｔｎ量が５０～６４μＭの範囲から設定される閾値以下である［３－１］に記載の方法。
[3-11]
The subject's MI amount is not less than a threshold value set from a range of 29.8 to 63.3 mg / gCr, and the PlsEtn amount is not more than a threshold value set from a range of 50 to 64 μM. Method.
 
［３－１２］
前記被験者のＭＩ量が２９．８～６３．３ｍｇ／ｇＣｒの範囲から設定される閾値以上であり、ＰｌｓＥｔｎ量が５２～６１μＭの範囲から設定される閾値以下である［３－１］に記載の方法。
[3-12]
The subject's MI amount is not less than a threshold value set from a range of 29.8 to 63.3 mg / gCr, and the PlsEtn amount is not more than a threshold value set from a range of 52 to 61 μM. Method.
 
［３－１３］
前記ＭＩ量の閾値が３６．６ｍｇ／ｇＣｒであり、ＰｌｓＥｔｎ量の閾値が５８μＭである［３－１］ 記載の方法。
[3-13]
[3-1] The method according to [3-1], wherein the threshold value for the MI amount is 36.6 mg / gCr, and the threshold value for the PlsEtn amount is 58 μM.
 
［３－１４］
前記ＭＩ量の閾値が３６．６ｍｇ／ｇＣｒであり、ＰｌｓＥｔｎ量の閾値が５６μＭ又は６１μＭである［３－１］ に記載の方法。
[3-14]
The method according to [3-1], wherein the threshold value for the amount of MI is 36.6 mg / gCr, and the threshold value for the amount of PlsEtn is 56 μM or 61 μM.
 
［３－１５］
前記ＭＩを定量する尿が、特に被験者の空腹時随意尿である［３－１］ に記載の方法。
[3-15]
The method according to [3-1], wherein the urine for quantifying the MI is particularly a fasting voluntary urine of the subject.
 
［４］
被験者を認知症患者またはその前段階にある者か否かを分類するための認知症検査方法に用いられる手段であって、
　被験者の自然排泄尿中のミオイノシトール（ＭＩ）を定量する手段と、同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する手段とを含み、
健常者と、認知症患者またはその前段階にある者とを区別することができる、自然排泄尿中のミオイノシトール（ＭＩ）の予め求められた閾値と、血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）の予め求められた閾値のそれぞれに対して、前記被験者のミオイノシトール（ＭＩ）の定量値とエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）の定量値を比較して用いる認知症検査手段。
[4]
A method used in a dementia test method for classifying whether a subject is a dementia patient or a person in the previous stage,
Means for quantifying myo-inositol (MI) in spontaneously excreted urine of a subject, and means for quantifying ethanolamine-type plasmalogen (PlsEtn) in serum or plasma of the same subject,
A pre-determined threshold of myo-inositol (MI) in spontaneously excreted urine and ethanolamine plasmalogens in serum or plasma, which can distinguish between healthy and demented patients or those in its predecessor The dementia test | inspection means which compares and uses the quantitative value of myo-inositol (MI) of said test subject, and the quantitative value of ethanolamine type plasmalogen (PlsEtn) with respect to each of the threshold value calculated | required in advance of (PlsEtn).
 
［４－１］
前記手段が試薬キットである［４］に記載の認知症検査手段。
[4-1]
The dementia test means according to [4], wherein the means is a reagent kit.
 
［４－２］
同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する試薬キットが、以下の（１ａ）～（３ａ）を含む［４－１］に記載の試薬キット。
（１ａ）エタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解できる酵素およびその酵素反応を進めることのできる添加物。
（２ａ）エタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）をグリセロ－３－ホスフォエタノールアミンとアルデヒドに加水分解でき、かつエーテル型リゾホスファチジルエタノールアミンを加水分解できない加水分解酵素およびその酵素反応を進めることのできる添加物。
（３ａ）グリセロ－３－ホスフォエタノールアミンをエタノールアミンとグリセロリン酸に変換する酵素、エタノールアミンをグリコールアルデヒド、アンモニア、及び過酸化水素に変換する酵素とその酵素反応を進めるにことのできる添加物、さらに必要により過酸化水素を定量するための過酸化水素定量手段。
または前記（２ａ）の加水分解酵素がリゾホスファチジルエタノールアミンを加水分解できる酵素である場合、前記（１ａ）に、さらにリゾホスファチジルエタノールアミンを加水分解する酵素を含む［４－１］に記載の試薬キット。
[4-2]
The reagent kit according to [4-1], wherein the reagent kit for quantifying ethanolamine plasmalogen (PlsEtn) in serum or plasma of the same subject comprises the following (1a) to (3a).
(1a) An enzyme capable of hydrolyzing ethanolamine type plasmalogen (PlsEtn) to ethanolamine type lysoplasmalogen (lyPlsEtn) and an additive capable of proceeding with the enzyme reaction.
(2a) a hydrolase capable of hydrolyzing ethanolamine-type lysoplasmalogen (lyPlsEtn) into glycero-3-phosphoethanolamine and aldehyde and not hydrolyzing ether-type lysophosphatidylethanolamine, and advancing the enzyme reaction thereof Possible additive.
(3a) An enzyme that converts glycero-3-phosphoethanolamine into ethanolamine and glycerophosphoric acid, an enzyme that converts ethanolamine into glycolaldehyde, ammonia, and hydrogen peroxide, and an additive capable of advancing the enzymatic reaction Further, hydrogen peroxide determination means for determining hydrogen peroxide if necessary.
Alternatively, when the hydrolase of (2a) is an enzyme capable of hydrolyzing lysophosphatidylethanolamine, the reagent according to [4-1] further includes an enzyme that hydrolyzes lysophosphatidylethanolamine in (1a). kit.
 
［４－２－１］
前記（２ａ）の加水分解酵素がリゾホスファチジルエタノールアミンを加水分解できる酵素である場合、前記（１ａ）に、さらにリゾホスファチジルエタノールアミンを加水分解する酵素を含む［４－２］に記載の試薬キット。
[4-2-1]
When the hydrolase of (2a) is an enzyme capable of hydrolyzing lysophosphatidylethanolamine, the reagent kit according to [4-2] further includes an enzyme that hydrolyzes lysophosphatidylethanolamine in (1a). .
 
［４－３］
前記（２ａ）の加水分解酵素が、下記（ａ）または（ｂ）の酵素のいずれかである［４－２］または［４－２－１］に記載の試薬キット。
（ａ）配列番号１または２に記載のアミノ酸配列：
（ｂ）配列番号１または２に記載のアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列：
からなる酵素であって、エタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）を加水分解し、グリセロ－３－ホスフォエタノールアミンとアルデヒドとを得る反応を触媒する。
[4-3]
The reagent kit according to [4-2] or [4-2-1], wherein the hydrolase of (2a) is any of the following enzymes (a) or (b).
(A) the amino acid sequence set forth in SEQ ID NO: 1 or 2:
(B) Amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 or 2:
Which catalyzes the reaction of hydrolyzing ethanolamine-type lysoplasmalogen (lyPlsEtn) to give glycero-3-phosphoethanolamine and an aldehyde.
 
［４－４］
前記（１）のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解できる酵素、が下記（ｃ）または（ｄ）のいずれかの酵素である［４－２］または［４－２－１］に記載の試薬キット。
（ｃ）配列番号５に記載のアミノ酸配列：
（ｄ）配列番号５に記載のアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列であり、エタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解する作用を触媒する。 
[4-4]
The enzyme capable of hydrolyzing the ethanolamine type plasmalogen (PlsEtn) of (1) to an ethanolamine type lysoplasmalogen (lyPlsEtn) is one of the following enzymes (c) or (d) [4-2] Or the reagent kit according to [4-2-1].
(C) the amino acid sequence set forth in SEQ ID NO: 5:
(D) An amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 5, and ethanolamine type plasmalogen (PlsEtn) is changed to ethanolamine type lysoplasmalogen (lyPlsEtn). To catalyze the action of hydrolysis.
 
［５］
健常者と、認知症患者またはその前段階にある者とを区別することができる、自然排泄尿中のミオイノシトール（ＭＩ）の予め求められた閾値と、被験者の自然排泄尿中のミオイノシトール（ＭＩ）の定量値とを比較する工程と組み合わせることにより、被験者を認知症患者またはその前段階にある者か否かを分類する認知症検査に用いるものであって、
同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する工程を含み、該工程中に、健常者と、認知症患者またはその前段階にある者とを区別することができる、血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）の、予め求められた閾値に対して、被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）の定量値を比較して用いる、エタノールアミン型プラスマローゲンの定量手段。
[5]
A predetermined threshold of myo-inositol (MI) in spontaneously excreted urine, which can distinguish between a healthy person and a person with dementia or in the previous stage, and myo-inositol in the subject's spontaneously excreted urine ( MI) is used for a dementia test for classifying whether a subject is a dementia patient or a person in the previous stage by combining with a step of comparing with a quantitative value of MI,
Quantifying ethanolamine-type plasmalogen (PlsEtn) in the serum or plasma of the same subject, during which a healthy person can be distinguished from a patient with dementia or in its predecessor stage, Ethanolamine used by comparing the quantitative value of ethanolamine-type plasmalogen (PlsEtn) in serum or plasma of a subject against a predetermined threshold of ethanolamine-type plasmalogen (PlsEtn) in serum or plasma Quantitative means of type plasmalogen.
 
［５－１］
前記手段が試薬キットである［５］に記載の定量手段。
[5-1]
The quantitative means according to [5], wherein the means is a reagent kit.
 
［５－２］
同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する試薬キットが、以下の（１ａ）～（３ａ）を含む［５－１］に記載の試薬キット。
（１ａ）エタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解できる酵素およびその酵素反応を進めることのできる添加物
（２ａ）エタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）をグリセロ－３－ホスフォエタノールアミンとアルデヒドに加水分解でき、かつエーテル型リゾホスファチジルエタノールアミンを加水分解できない加水分解酵素およびその酵素反応を進めることのできる添加物
（３ａ）グリセロ－３－ホスフォエタノールアミンをエタノールアミンとグリセロリン酸に変換し、エタノールアミンをグリコールアルデヒド、アンモニア、及び過酸化水素に変換する酵素とその酵素反応を進めるにことのできる添加物、さらに必要により過酸化水素を定量するための過酸化水素定量手段。
または前記（２ａ）の加水分解酵素がリゾホスファチジルエタノールアミンを加水分解できる酵素である場合、前記（１ａ）に、さらにリゾホスファチジルエタノールアミンを加水分解する酵素を含む［５－１］記載の試薬キット。
[5-2]
The reagent kit according to [5-1], wherein the reagent kit for quantifying ethanolamine plasmalogen (PlsEtn) in serum or plasma of the same subject comprises the following (1a) to (3a).
(1a) An enzyme capable of hydrolyzing ethanolamine-type plasmalogen (PlsEtn) into ethanolamine-type lysoplasmalogen (lyPlsEtn) and an additive capable of proceeding with the enzyme reaction (2a) An ethanolamine-type lysoplasmalogen (lyPlsEtn) Hydrolytic enzyme capable of hydrolyzing glycero-3-phosphoethanolamine and aldehyde and not hydrolyzing ether-type lysophosphatidylethanolamine, and additive capable of proceeding the enzymatic reaction (3a) glycero-3-phosphoethanol Enzymes that convert amines to ethanolamine and glycerophosphoric acid, ethanolamines to glycolaldehyde, ammonia, and hydrogen peroxide and additives that can drive the enzyme reaction, and if necessary Determination of hydrogen peroxide means for quantifying the hydrogen peroxide.
Alternatively, when the hydrolase of (2a) is an enzyme capable of hydrolyzing lysophosphatidylethanolamine, the reagent kit according to [5-1] further includes an enzyme that hydrolyzes lysophosphatidylethanolamine in (1a). .
 
［５－２－１］
前記（２ａ）の加水分解酵素がリゾホスファチジルエタノールアミンを加水分解できる酵素である場合、前記（１ａ）に、さらにリゾホスファチジルエタノールアミンを加水分解する酵素を含む［５－２］に記載の試薬キット。
[5-2-1]
When the hydrolase of (2a) is an enzyme capable of hydrolyzing lysophosphatidylethanolamine, the reagent kit according to [5-2] further includes an enzyme that hydrolyzes lysophosphatidylethanolamine in (1a). .
 
［５－３］
前記（２ａ）の加水分解酵素が、下記（ａ）または（ｂ）の酵素のいずれかである［５－２］または［５－２－１］に記載の試薬キット。
（ａ）配列番号１または２に記載のアミノ酸配列：
（ｂ）配列番号１または２に記載のアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列：
からなる酵素であって、エタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）を加水分解し、グリセロ－３－ホスフォエタノールアミンとアルデヒドとを得る反応を触媒する。
[5-3]
The reagent kit according to [5-2] or [5-2-1], wherein the hydrolase of (2a) is any of the following enzymes (a) or (b).
(A) the amino acid sequence set forth in SEQ ID NO: 1 or 2:
(B) Amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 or 2:
Which catalyzes the reaction of hydrolyzing ethanolamine-type lysoplasmalogen (lyPlsEtn) to give glycero-3-phosphoethanolamine and an aldehyde.
 
［５－４］
前記（１ａ）のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解できる酵素、が下記（ｃ）または（ｄ）のいずれかの酵素である［５－２］または［５－２－１］に記載の試薬キット。
（ｃ）配列番号５に記載のアミノ酸配列：
（ｄ）配列番号５に記載のアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列であり、エタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解する作用を触媒する。
[5-4]
The enzyme capable of hydrolyzing the ethanolamine-type plasmalogen (PlsEtn) of (1a) to an ethanolamine-type lysoplasmogen (lyPlsEtn) is one of the following enzymes (c) or (d) [5-2] Or the reagent kit according to [5-2-1].
(C) the amino acid sequence set forth in SEQ ID NO: 5:
(D) An amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 5, and ethanolamine type plasmalogen (PlsEtn) is changed to ethanolamine type lysoplasmalogen (lyPlsEtn). To catalyze the action of hydrolysis.
 
［６］
健常者と、認知症患者またはその前段階にある者とを区別することができる、血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）の予め求められた閾値と、被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）の定量値とを比較する工程と組み合わせることにより、被験者を認知症患者またはその前段階にある者か否かを分類する認知症検査に用いるものであって、
同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する工程を含み、該工程中に、健常者と、認知症患者またはその前段階にある者とを区別することができる、自然排泄尿中のミオイノシトール（ＭＩ）の、予め求められた閾値に対して、被験者の自然排泄尿中のミオイノシトール（ＭＩ）の定量値を比較して用いる、ミオイノシトール（ＭＩ）の定量手段。
[6]
A pre-determined threshold of ethanolamine-type plasmalogen (PlsEtn) in serum or plasma, which can distinguish between healthy and demented patients or those in its predecessor, and in the serum or plasma of the subject In combination with the step of comparing the ethanolamine-type plasmalogen (PlsEtn) with a quantitative value, the subject is used for a dementia test for classifying whether or not the subject is a dementia patient or a person in its previous stage,
Quantifying ethanolamine-type plasmalogen (PlsEtn) in the serum or plasma of the same subject, during which a healthy person can be distinguished from a patient with dementia or in its predecessor stage, Means for quantifying myo-inositol (MI), which is used by comparing the quantitative value of myo-inositol (MI) in spontaneously excreted urine of a subject with a predetermined threshold of myo-inositol (MI) in spontaneously excreted urine .
 
［６－１］
前記手段が試薬キットである［６］に記載の定量手段。
[6-1]
The quantitative means according to [6], wherein the means is a reagent kit.
 
なお、本発明の実施の形態における認知症とは、知能、記憶、見当識含む認知の障害を指し、加齢に伴う現象は含まず、病的に能力が低下する障害を指す。「認知症患者」とは、記憶、判断力などの障害により社会生活が困難な者（特に高齢者）をいう。具体的には、認知機能検査によって認知障害を有するレベルと判定される者（例えば、ＣＤＲスコアが１．０以上）である。また「認知症の前段階にある者」とは、例えば日常生活において記憶、判断力に衰えが観察される者などであり、ＷＡＩＳ－Ｒ、ＨＤＳ－Ｒ、ＭＭＳ、ＭＭＳＥ、ＣＤＲ、ＭｏＣＡ－Ｊ等の認知機能検査方法で検出できる最も軽度な段階を含み、例えばＣＤＲスコアが０．５以上、１．０未満のような者が好ましい。認知症の原因となる疾患は、例えば脳血管障害、アルツハイマー病、正常圧水頭症、代謝障害、栄養障害、甲状腺機能低下、鬱病などがあるが、一例としてアルツハイマー病が挙げられる。
In addition, the dementia in the embodiment of the present invention refers to a cognitive disorder including intelligence, memory, and orientation, does not include a phenomenon associated with aging, and refers to a disorder whose ability is reduced morbidly. A “dementia patient” refers to a person (particularly an elderly person) who has difficulty in social life due to a disorder such as memory or judgment. Specifically, it is a person who is determined to have a cognitive impairment level by a cognitive function test (for example, a CDR score of 1.0 or more). In addition, “a person in the previous stage of dementia” is, for example, a person whose decline in memory and judgment is observed in daily life, such as WAIS-R, HDS-R, MMS, MMSE, CDR, MoCA-J. A person having a mildest level that can be detected by a cognitive function testing method such as a CDR score of 0.5 or more and less than 1.0 is preferable. Examples of diseases that cause dementia include cerebrovascular disorders, Alzheimer's disease, normal pressure hydrocephalus, metabolic disorders, nutritional disorders, hypothyroidism, and depression, and Alzheimer's disease is an example.
 
「手段」とは組成物、試薬、酵素あるいはそれらの組み合わせの総称を言う。組成物は組成物中の各成分を単一の組成物としてもよいが、２以上の組成物に分離する場合、このような２以上の組成物を合わせてキットという。
「定量」とは「測定」、「検出」等を含む概念をいう。
又本発明に関連する発明として以下が提示される。
“Means” refers to a general term for a composition, a reagent, an enzyme, or a combination thereof. In the composition, each component in the composition may be a single composition, but when two or more compositions are separated, the two or more compositions are collectively referred to as a kit.
“Quantitative” means a concept including “measurement”, “detection”, and the like.
The following is presented as an invention related to the present invention.
 
［７］
被験者の試料のＰｌｓＥｔｎを定量する方法であって、以下の（１）～（３－４）の工程を含む方法。
（１）エタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解できる酵素を用いて、被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解する工程：
（２）エタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）をグリセロ－３－ホスフォエタノールアミンとアルデヒドに加水分解でき、エーテル型リゾホスファチジルエタノールアミンを加水分解できない加水分解酵素を用いて、前記（１）にて生成されたエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）をグリセロ－３－ホスフォエタノールアミンとアルデヒドに加水分解する工程：及び
（３－１）グリセロ－３－ホスフォエタノールアミンを加水分解してエタノールアミンにせしめる酵素にて、工程（２）で得られたグリセロ－３－ホスフォエタノールアミンを加水分解してエタノールアミンにする工程：
（３－２）エタノールアミンを酸化する酵素により、エタノールアミンから過酸化水素を発生せしめる工程：
（３－３）過酸化水素を過酸化水素定量手段により定量する工程：及び
（３－４）前記被験者の血清または血漿中のＰｌｓＥｔｎ量を算出する工程：
または前記（２）の加水分解酵素が、リゾホスファチジルエタノールアミンを加水分解できる酵素である場合、同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する方法が、前記（１）～（３－４）の工程に先立つか、少なくとも前記（２）工程の前までに、下記（４）及び（５）を含む工程を行うことを特徴とする方法。
（４）ホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する酵素を用いて、血清または血漿中に混在するホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する工程：及び
（５）リゾホスフォリパーゼ及び／又はモノグリセリドリパーゼを用いて、前記（４）工程により生成されるリゾホスファチジルエタノールアミンを分解し、実質的に消去する工程。
[7]
A method for quantifying PlsEtn in a sample of a subject, comprising the following steps (1) to (3-4):
(1) Using an enzyme capable of hydrolyzing ethanolamine-type plasmalogen (PlsEtn) into ethanolamine-type lysoplasmalogen (lyPlsEtn), ethanolamine-type plasmalogen (PlsEtn) in the serum or plasma of the subject is ethanolamine-type lysozyme. Hydrolyzing to plasmalogen (lyPlsEtn):
(2) Using a hydrolase that can hydrolyze ethanolamine-type lysoplasmalogen (lyPlsEtn) into glycero-3-phosphoethanolamine and aldehyde, but cannot hydrolyze ether-type lysophosphatidylethanolamine, (1) Hydrolyzing ethanolamine-type lysoplasmalogen (lyPlsEtn) produced into glycero-3-phosphoethanolamine and aldehyde: and (3-1) hydrolyzing glycero-3-phosphoethanolamine to ethanol A step of hydrolyzing the glycero-3-phosphoethanolamine obtained in the step (2) to an ethanolamine with an enzyme that is converted into an amine:
(3-2) Step of generating hydrogen peroxide from ethanolamine by an enzyme that oxidizes ethanolamine:
(3-3) Step of quantifying hydrogen peroxide by means of hydrogen peroxide quantification: (3-4) Step of calculating the amount of PlsEtn in the serum or plasma of the subject:
Alternatively, when the hydrolase of (2) is an enzyme capable of hydrolyzing lysophosphatidylethanolamine, the method for quantifying ethanolamine-type plasmalogen (PlsEtn) in the serum or plasma of the same subject is described in (1) A method comprising performing the steps (4) and (5) below prior to the steps (3-4) or at least before the step (2).
(4) Step of decomposing phosphatidylethanolamine mixed in serum or plasma into lysophosphatidylethanolamine and fatty acid using an enzyme that decomposes phosphatidylethanolamine into lysophosphatidylethanolamine and fatty acid: and (5) lysophospho A step of decomposing and substantially eliminating lysophosphatidylethanolamine produced by the step (4) using lipase and / or monoglyceride lipase.
 
［８］
前記工程（２）の加水分解酵素が、下記（ａ）または（ｂ）の酵素のいずれかである［７］に記載の方法。
（ａ）配列番号１または２に記載のアミノ酸配列：
（ｂ）配列番号１または２に記載のアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列：
からなる酵素であって、エタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）を加水分解し、グリセロ－３－ホスフォエタノールアミンとアルデヒドとを得る反応を触媒する。
[8]
[7] The method according to [7], wherein the hydrolase in the step (2) is one of the following enzymes (a) and (b).
(A) the amino acid sequence set forth in SEQ ID NO: 1 or 2:
(B) Amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 or 2:
Which catalyzes the reaction of hydrolyzing ethanolamine-type lysoplasmalogen (lyPlsEtn) to give glycero-3-phosphoethanolamine and an aldehyde.
 
［９］
前記工程（１）のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解できる酵素、が下記（ｃ）または（ｄ）のいずれかの酵素である［７］に記載の方法。
（ｃ）配列番号５に記載のアミノ酸配列：
（ｄ）配列番号５に記載のアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列であり、エタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解する作用を触媒する。
[9]
The enzyme capable of hydrolyzing the ethanolamine-type plasmalogen (PlsEtn) in the step (1) to an ethanolamine-type lysoplasmalogen (lyPlsEtn) is any one of the following enzymes (c) or (d): [7] The method described.
(C) the amino acid sequence set forth in SEQ ID NO: 5:
(D) An amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 5, and ethanolamine type plasmalogen (PlsEtn) is changed to ethanolamine type lysoplasmalogen (lyPlsEtn). To catalyze the action of hydrolysis.
 
［１０］
前記（２）に加水分解酵素が、リゾホスファチジルエタノールアミンを加水分解できる酵素である場合、被験者の試料のＰｌｓＥｔｎを定量する方法が、前記（１）～（３－４）の工程に先立つか、少なくとも前記（２）工程の前までに、下記（４）及び（５）を含む工程を行うことを特徴とする［７］に記載の方法。
（４）ホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する酵素を用いて、血清または血漿中に混在するホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する工程：及び
（５）リゾホスフォリパーゼ及び／又はモノグリセリドリパーゼを用いて、前記（４）工程により生成されるリゾホスファチジルエタノールアミンを分解し、実質的に消去する工程。
[10]
When the hydrolase in (2) is an enzyme capable of hydrolyzing lysophosphatidylethanolamine, the method for quantifying PlsEtn of a subject sample precedes the steps (1) to (3-4), The method according to [7], wherein the step including the following (4) and (5) is performed at least before the step (2).
(4) Step of decomposing phosphatidylethanolamine mixed in serum or plasma into lysophosphatidylethanolamine and fatty acid using an enzyme that decomposes phosphatidylethanolamine into lysophosphatidylethanolamine and fatty acid: and (5) lysophospho A step of decomposing and substantially eliminating lysophosphatidylethanolamine produced by the step (4) using lipase and / or monoglyceride lipase.
 
［１１］
前記工程（４）のホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する酵素としてホスフォリパーゼ（ＰＬ）を使用し、該ＰＬは、前記工程（１）のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解できる酵素としての作用を兼ねさせて新たな該ＰＬを添加することなく前記工程（１）の工程を進行せしめ、前記工程（５）を実施と同時、または前記工程（５）の実施後に、前記工程（１）が完了し、以後前記工程を（２）～（３－４）実施する［１０］に記載の方法。
[11]
Phospholipase (PL) is used as an enzyme that decomposes the phosphatidylethanolamine of the step (4) into lysophosphatidylethanolamine and a fatty acid, and the PL uses the ethanolamine-type plasmalogen (PlsEtn) of the step (1). Simultaneously with the implementation of the step (5), the step (1) is allowed to proceed without adding new PL, which also serves as an enzyme capable of hydrolyzing ethanolamine-type lysoplasmalogen (lyPlsEtn). Or the method according to [10], wherein the step (1) is completed after the step (5), and the steps (2) to (3-4) are performed thereafter.
 
［１２］
被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する試薬キットであって、以下の（１ａ）～（３ａ）を含む試薬キット。
（１ａ）エタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解できる酵素およびその酵素反応を進めることのできる添加物。
（２ａ）エタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）をグリセロ－３－ホスフォエタノールアミンとアルデヒドに加水分解でき、かつエーテル型リゾホスファチジルエタノールアミンを加水分解できない加水分解酵素およびその酵素反応を進めることのできる添加物。
（３ａ）グリセロ－３－ホスフォエタノールアミンをエタノールアミンとグリセロリン酸に変換する酵素、エタノールアミンをグリコールアルデヒド、アンモニア、及び過酸化水素に変換する酵素とその酵素反応を進めるにことのできる添加物、さらに必要により過酸化水素を定量するための過酸化水素定量手段。
[12]
A reagent kit for quantifying ethanolamine-type plasmalogen (PlsEtn) in serum or plasma of a subject, comprising the following (1a) to (3a):
(1a) An enzyme capable of hydrolyzing ethanolamine type plasmalogen (PlsEtn) to ethanolamine type lysoplasmalogen (lyPlsEtn) and an additive capable of proceeding with the enzyme reaction.
(2a) a hydrolase capable of hydrolyzing ethanolamine-type lysoplasmalogen (lyPlsEtn) into glycero-3-phosphoethanolamine and aldehyde and not hydrolyzing ether-type lysophosphatidylethanolamine, and advancing the enzyme reaction thereof Possible additive.
(3a) An enzyme that converts glycero-3-phosphoethanolamine into ethanolamine and glycerophosphoric acid, an enzyme that converts ethanolamine into glycolaldehyde, ammonia, and hydrogen peroxide, and an additive capable of advancing the enzymatic reaction Further, hydrogen peroxide determination means for determining hydrogen peroxide if necessary.
　または前記（２ａ）の加水分解酵素がリゾホスファチジルエタノールアミンを加水分解できる酵素である場合、前記（１ａ）に、さらにリゾホスファチジルエタノールアミンを加水分解する酵素を含む試薬キット。 Alternatively, when the hydrolase of (2a) is an enzyme capable of hydrolyzing lysophosphatidylethanolamine, the reagent kit further comprises (1a) an enzyme that hydrolyzes lysophosphatidylethanolamine.
 
［１２－１］
前記（２ａ）の加水分解酵素がリゾホスファチジルエタノールアミンを加水分解できる酵素である場合、前記（１ａ）に、さらにリゾホスファチジルエタノールアミンを加水分解する酵素を含む［１２］に記載の試薬キット。
[12-1]
When the hydrolase of (2a) is an enzyme capable of hydrolyzing lysophosphatidylethanolamine, the reagent kit according to [12], further comprising (1a) an enzyme that hydrolyzes lysophosphatidylethanolamine.
 
［１３］
前記（２ａ）の加水分解酵素が、下記（ａ）または（ｂ）の酵素のいずれかである［１２］または［１２－１］に記載の試薬キット。
（ａ）配列番号１または２に記載のアミノ酸配列：
（ｂ）配列番号１または２に記載のアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列：
からなる酵素であって、エタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）を加水分解し、グリセロ－３－ホスフォエタノールアミンとアルデヒドとを得る反応を触媒する。
[13]
The reagent kit according to [12] or [12-1], wherein the hydrolase of (2a) is any of the following enzymes (a) or (b).
(A) the amino acid sequence set forth in SEQ ID NO: 1 or 2:
(B) Amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 or 2:
Which catalyzes the reaction of hydrolyzing ethanolamine-type lysoplasmalogen (lyPlsEtn) to give glycero-3-phosphoethanolamine and an aldehyde.
 
［１４］
前記（１ａ）のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解できる酵素が、下記（ｃ）または（ｄ）のいずれかの酵素である［１２］または［１２－１］に記載の試薬キット。
（ｃ）配列番号５に記載のアミノ酸配列：
（ｄ）配列番号５に記載のアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列であり、エタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解する作用を触媒する。
[14]
The enzyme capable of hydrolyzing the ethanolamine type plasmalogen (PlsEtn) of (1a) to an ethanolamine type lysoplasmalogen (lyPlsEtn) is any of the following enzymes (c) or (d) [12] or [ 12-1].
(C) the amino acid sequence set forth in SEQ ID NO: 5:
(D) An amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 5, and ethanolamine type plasmalogen (PlsEtn) is changed to ethanolamine type lysoplasmalogen (lyPlsEtn). To catalyze the action of hydrolysis.
 
［１５］
　下記（Ａ）または（Ｂ）の塩基配列を含む遺伝子。
（Ａ）配列番号１または２に記載のアミノ酸配列からなる酵素をコードする：　
（Ｂ）配列番号１または２に記載のアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなる酵素であって、エタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）を加水分解し、グリセロ－３－ホスフォエタノールアミンとアルデヒドとを得る反応を触媒する酵素をコードする。
[15]
A gene comprising the following base sequence (A) or (B).
(A) Encodes an enzyme consisting of the amino acid sequence set forth in SEQ ID NO: 1 or 2:
(B) An enzyme comprising an amino acid sequence in which one or a plurality of amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 or 2, and an ethanolamine-type lysoplasmalogen (lyPlsEtn) is hydrolyzed It encodes an enzyme that catalyzes a reaction that decomposes to give glycero-3-phosphoethanolamine and an aldehyde.
 
［１６］
［１５］に記載の遺伝子を含む組換えベクター。
[16]
A recombinant vector comprising the gene according to [15].
 
［１７］
［１６］に記載の組換えベクターを含む形質転換体。
[17]
A transformant comprising the recombinant vector according to [16].
 
［１８］
前記酵素の製造方法であって：
［１５］に記載の遺伝子をもつ微生物、または［１７］に記載の形質転換体を培地で培養する工程：
培養物中に［１３］に記載の酵素を生成蓄積させる工程：及び
該培養物から該蛋白質を採取する工程：
を含む方法。
[18]
A method for producing the enzyme comprising:
A step of culturing the microorganism having the gene according to [15] or the transformant according to [17] in a medium:
Producing and accumulating the enzyme according to [13] in the culture: and collecting the protein from the culture:
Including methods.
 
［１９］
前記ＰｌｓＥｔｎを定量する試薬キットに含有させる下記（ｃ）または（ｄ）の酵素：
（ｃ）配列番号５に記載のアミノ酸配列：
（ｄ）配列番号５に記載のアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列であり、エタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解する作用を触媒する。
[19]
The following enzyme (c) or (d) contained in the reagent kit for quantifying PlsEtn:
(C) the amino acid sequence set forth in SEQ ID NO: 5:
(D) An amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 5, and ethanolamine type plasmalogen (PlsEtn) is changed to ethanolamine type lysoplasmalogen (lyPlsEtn). To catalyze the action of hydrolysis.
 
微生物の寄託Deposit of microorganisms
本発明に係る以下の微生物をブダペスト条約に基づき国際寄託している。
［ＮＡ２９７］
寄託機関：独立行政法人製品評価技術基盤機構特許微生物寄託センター
住所：〒２９２－０８１８　日本国千葉県木更津市かずさ鎌足２－５－８
寄託日：２０１０年１２月7日
受託番号：ＮＩＴＥ　ＢＰ－１０１４
［Ｔｈｅｒｍｏｃｒｉｓｐｕｍ　ｓｐ．］
寄託機関：独立行政法人製品評価技術基盤機構特許微生物寄託センター
住所：〒２９２－０８１８　日本国千葉県木更津市かずさ鎌足２－５－８　１２２号
寄託日：２０１３年５月１７日
受託番号：ＮＩＴＥ　ＢＰ－０１６２８
  The following microorganisms according to the present invention are deposited internationally based on the Budapest Treaty.
[NA297]
Depositary Institution: National Institute of Technology and Evaluation Patent Microorganisms Depositary Center Address: 2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture, Japan 292-0818
Deposit date: December 7, 2010 Deposit number: NITE BP-1014
[Thermocrispum sp. ]
Depositary institution: National Institute for Product Evaluation Technology Patent Microorganism Depositary Center Address: 2-5-8 122 Kazusa Kamashi, Kisarazu, Chiba Prefecture, Japan 292-0818 Japan Date of deposit: May 17, 2013 Deposit number: NITE BP-01628
検査の実施形態および被験者への負担を実施的に増大させることなく、血清及び／又は血漿中ＰｌｓＥｔｎ量の定量による認知検査の精度を向上させることが可能となる。 It is possible to improve the accuracy of the cognitive test by quantitatively determining the amount of PlsEtn in serum and / or plasma without effectively increasing the test embodiment and the burden on the subject.
 
尿ＭＩ量の定量と血清及び／又は血漿ＰｌｓＥｔｎ量の定量を併用することで、認知機能の障害のレベルを高い確度で評価することもできる。従って、すでに認知症を発症している患者のみらならず、認知症の前段階にある者をも特定することが可能となる。
By combining the determination of the urine MI amount and the determination of the serum and / or plasma PlsEtn amount, the level of cognitive impairment can be evaluated with high accuracy. Therefore, it is possible to specify not only patients who have already developed dementia, but also those who are in the previous stage of dementia.
 
被験者の血清または血漿中のＰｌｓＥｔｎを定量するためのｌｙＰｌｓ　ａｓｅとＰＬを含有する認知症検査試薬キットを提供することが可能となる。
It is possible to provide a dementia test reagent kit containing lyPlsase and PL for quantifying PlsEtn in serum or plasma of a subject.
 
実施例において定量した全被験者を群別した場合の尿ＭＩ量である。実測値は表５に示した。It is the amount of urine MI when all subjects quantified in the examples are grouped. The measured values are shown in Table 5. 実施例において、健常者群と「もの忘れ」群のそれぞれについて尿ＭＩ量およびＨｂＡ１ｃ量がＰｌｓＥｔｎ量に及ぼす影響を検討した結果である。In an Example, it is the result of having examined the influence which the amount of urine MI and the amount of HbA1c have on PlsEtn amount about each of a healthy subject group and a "forgetfulness" group. 実施例において、全高齢者（健常＋「もの忘れ」群）を対象に血清ＰｌｓＥｔｎ（ＥＰ）量と尿ＭＩ量の全定量値の平均値を境に４群に分割して、各群での認知機能検査結果を比較した結果である。実測値は表６に示した。In the examples, for all elderly people (healthy + “forgetfulness” group), the average value of the total quantified values of serum PlsEtn (EP) amount and urine MI amount was divided into 4 groups, and each group was divided into 4 groups. It is the result of comparing cognitive function test results. The measured values are shown in Table 6. 血清ＰｌｓＥｔｎ量が５８μＭ未満、尿ＭＩ量が２５ｍｇ／ｇＣｒ以上をそれぞれの基準とした場合の認知症患者（ＣＤＲ≧０．５）の検出確度を検討した結果である。It is the result of examining the detection accuracy of patients with dementia (CDR ≧ 0.5) when the serum PlsEtn amount is less than 58 μM and the urinary MI amount is 25 mg / gCr or more. 全高齢者、尿ＭＩ量＞２５ｍｇ／ｇＣｒ高齢者群および尿ＭＩ量＞３６．６ｍｇ／ｇＣｒ高齢者群の３群について、血清ＰｌｓＥｔｎ量の認知機能正常（ＣＤＲ値：０）と認知機能非正常（ＣＤＲ値：０．５以上）を分別する精度について、Ｒｅｃｅｉｖｅｒ　Ｏｐｅｒａｔｉｎｇ　Ｃｈａｒａｃｔｅｒｉｓｔｉｃ（ＲＯＣ）解析によるＲＯＣ曲線下の面積（Ａｒｅａ　ｕｎｄｅｒ　ｔｈｅ　ｃｕｒｖｅ：ＡＵＣ）を求めることによって調べた結果である。Normal cognitive function (CDR value: 0) and abnormal cognitive function for serum PlsEtn levels in all elderly groups, urinary MI level> 25 mg / gCr elderly group and urinary MI level> 36.6 mg / gCr elderly group This is the result of examining the area under the ROC curve (Area under the curve: AUC) by Receiver Operating Characteristic (ROC) analysis for the accuracy of sorting (CDR value: 0.5 or more). 血清ＰｌｓＥｔｎ量の認知機能評価能と既存の認知機能検査法との相関性を、対象者を全高齢者、尿ＭＩ量＞２５ｍｇ／ｇＣｒ高齢者群および尿ＭＩ量＞３６．６ｍｇ／ｇＣｒ高齢者群の３群について検討した結果である。実測値は表７に示した。Correlation between the cognitive function evaluation ability of serum PlsEtn amount and the existing cognitive function test method, the subjects were all elderly people, urine MI amount> 25 mg / gCr elderly group and urine MI amount> 36.6 mg / gCr elderly It is the result of having examined about 3 groups. The measured values are shown in Table 7. 本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を、ＰＬ及び加水分解酵素を用いて定量する方法の一例を示す概略図。Schematic which shows an example of the method of quantifying the amount of serum and / or plasma PlsEtn of embodiment of this invention using PL and a hydrolase. 本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を、ＰＬ及び加水分解酵素を用いて定量する方法の、図７と別異な一例を示す概略図。Schematic which shows an example different from FIG. 7 of the method of quantifying the amount of serum and / or plasma PlsEtn of embodiment of this invention using PL and a hydrolase. ホスファチジルエタノールアミンを実質的に消去する工程を示す概略図。Schematic which shows the process of substantially eliminating phosphatidylethanolamine. 被験の、３日分の随時尿中ＭＩ定量値と空腹時二番尿中ＭＩ定量値の平均値を比較した相関図。The correlation diagram which compared the average value of the MI quantitative value in the urinary urine of the test for 3 days, and the fasting second urine MI quantitative value of the test. プール血清中ＰｌｓＥｔｎ（図中白丸）と０．９％食塩水中ＰｌｓＥｔｎ（図中黒丸）を、酵素を利用して定量した検量線。A calibration curve in which PlsEtn (white circle in the figure) in pooled serum and PlsEtn (black circle in the figure) in 0.9% saline were quantified using an enzyme. １２５Ｉ－ＨＰＬＣ法とＰｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ由来ｌｙＰｌｓ　ａｓｅを利用した血清または血漿中のＰｌｓＥｔｎの定量値を比較した相関図。The correlation diagram which compared the quantitative value of PlsEtn in serum or plasma using 125I-HPLC method and lyPlsase derived from Pseudomonas putida. １２５Ｉ－ＨＰＬＣ法とＴｈｅｒｍｏｃｒｉｓｐｕｍ　ｓｐ．由来ｌｙＰｌｓ　ａｓｅを利用した血清または血漿中のＰｌｓＥｔｎの定量値を比較した相関図。125I-HPLC method and Thermocrispum sp. The correlation diagram which compared the quantitative value of PlsEtn in serum or plasma using origin lyPlsase. １２５Ｉ－ＨＰＬＣ法とＰＬＤを利用した血清または血漿中のＰｌｓＥｔｎの定量値を比較した相関図。The correlation diagram which compared the quantitative value of PlsEtn in the serum or plasma using 125I-HPLC method and PLD. Ｔｈｅｒｍｏｃｒｉｓｐｕｍ　ｓｐ．由来ｌｙＰｌｓ　ａｓｅＳｔｒｅｐｔｏｍｙｃｅｓ　ｌｉｖｉｄａｎｓ組換体酵素を示す（図中矢印）ＳＤＳ－ＰＡＧＥ。Thermocrispum sp. SDS-PAGE showing the origin lyPls ase Streptomyces lividans recombinant enzyme (arrow in the figure). 粗蛋白質液中Ｔｈｅｒｍｏｃｒｉｓｐｕｍ　ｓｐ．由来ｌｙＰｌｓ　ａｓｅ大腸菌組換体酵素を示す（図中矢印）ＳＤＳ－ＰＡＧＥ。In a crude protein solution, Thermocrispum sp. SDS-PAGE showing the derived lyPls as E. coli recombinant enzyme (arrow in the figure).
 
本発明は、被験者の自然排泄尿中のミオイノシトール（ＭＩ）を定量する工程と、同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する工程とを含み、被験者を認知症患者またはその前段階にある者か否かを分類するための認知症検査方法である。該認知症検査方法は、同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する工程を含むものであって、健常者と、認知症患者またはその前段階にある者とを区別することができる、ＭＩとＰｌｓＥｔｎとのそれぞれの、予め求められた閾値に対して、被験者のＭＩとＰｌｓＥｔｎの定量値を比較する工程を含む、前記の認知症検査方法に関する。 The present invention includes a step of quantifying myo-inositol (MI) in spontaneously excreted urine of a subject and a step of quantifying ethanolamine-type plasmalogen (PlsEtn) in serum or plasma of the same subject. It is a dementia test method for classifying whether a patient or a person in the previous stage. The method for testing dementia includes a step of quantifying ethanolamine-type plasmalogen (PlsEtn) in serum or plasma of the same subject, and comprises a healthy person and a dementia patient or a person in the previous stage. The present invention relates to the method for testing dementia, comprising the step of comparing the quantitative values of the subject's MI and PlsEtn against the predetermined threshold values of MI and PlsEtn, which can be distinguished from each other.
 
被験者とは採尿及び／又は採血の対象となる健常人や患者を指し、年齢、性別、人種、基礎疾患などは限定せず、ヒト以外の実験動物なども含む。ヒトの場合、治験及び臨床試験に参加し治験薬の投与を受けるか又はその対象となる個人、すなわち治験モニター、医薬モニター、医薬ボランティア、創薬ボランティアなどの場合でも良く、ＷＡＩＳ－Ｒ、ＨＤＳ－Ｒ、ＭＭＳ、ＭＭＳＥ、ＣＤＲ、ＭｏＣＡ－Ｊ等の認知機能検査で認知症が疑われた患者又は健常人、認知症の自覚及び／又は他覚症状がある患者又は健常人でも良い。被験者には、食事制限などは設けなくても良いが、健康診断の場合は通常食事制限がある。
The test subject refers to a healthy person or patient who is a subject of urine collection and / or blood collection, and is not limited to age, sex, race, basic disease, and includes non-human laboratory animals. In the case of humans, it may be an individual who participates in clinical trials and receives clinical study drugs or is the subject, ie, clinical trial monitor, pharmaceutical monitor, pharmaceutical volunteer, drug discovery volunteer, etc., and WAIS-R, HDS- It may be a patient or a healthy person suspected of having a dementia in a cognitive function test such as R, MMS, MMSE, CDR, or MoCA-J, or a patient or a healthy person having a consciousness and / or objective symptom of dementia. The subject does not need to have a dietary restriction, but usually has a dietary restriction in the case of a medical examination.
 
本発明の実施の形態における尿は、薬剤等により強制的に排泄されたものではない自然排泄尿である。この自然排泄尿は、尿意を感じた際に排泄された随時尿であってもよいが、好ましくは平均的な生活サイクルにおいて朝食時前に排泄される早朝空腹時尿、さらに好ましくは早朝空腹時尿の後の空腹時に排泄される空腹時二番尿である。これらの尿は、連日あるいは適当な隔日（１ヶ月以内）の３日分を定量対象とする。３日分の採尿が困難な場合は、２日分あるいは１日分でもよいが、その場合の尿は空腹時二番尿であることが好ましい。また空腹時二番尿の採取が困難な場合は、早朝空腹時尿でよいが、その場合は３日分以上の尿の採取が好ましい。
The urine in the embodiment of the present invention is spontaneously excreted urine that is not forcibly excreted by drugs or the like. This spontaneously excreted urine may be anytime urine excreted when feeling urinary, but is preferably early morning fasting urine excreted before breakfast in the average life cycle, more preferably early morning fasting Second fasting urine excreted on an empty stomach after urine. These urines are quantitatively measured every day or every other day (within one month) for 3 days. If it is difficult to collect urine for 3 days, it may be 2 days or 1 day, but the urine in that case is preferably fasting second urine. When it is difficult to collect fasting second urine, early morning fasting urine may be used, but in that case, it is preferable to collect urine for three days or more.
 
本発明は被験者の自然排泄尿中のＭＩを定量する工程を含む。本発明の実施の形態におけるＭＩは背景技術に記載した公知のＭＩを含む。ＭＩはＧＣ－ＭＡＳＳで定量する方法（Ｔｏｓｈｉｍｉｔｓｕ　Ｎｉｗａ，Ｊ．Ｃｈｒｏｍａｔｏｇｒａｐｈｙ，２２７（１９８３），２５－３９）、免疫測定法（特開平８－２１８３５号公報）、酵素法［Ｒｏｓｗｉｔｈａ　Ｄｏｌｈｏｆｅｒ，Ｏ．Ｈ．Ｗｉｅｌａｎｄ．，Ｊ．Ｃｌｉｎ．Ｃｈｅｍ．Ｃｌｉｎ．Ｂｉｏｃｈｅｍ．，２５，７３３－７３６（１９８７）］、液体クロマトグラフィー質量分析（ＬＣ－ＭＳ）法やＨＰＬＣ法等の公知の方法で定量すれば良く、好ましくはミオイノシトールデヒドロゲナーゼ（ＥＣ　１．１．１．１８）を用いる方法（特許文献３、４）であり、さらに好ましくはミオイノシトール測定試薬ルシカＭＩ（旭化成ファーマ株式会社）を用いて定量する。
The present invention includes a step of quantifying MI in a subject's spontaneous excretion urine. The MI in the embodiment of the present invention includes a known MI described in the background art. MI is quantified by GC-MASS (Toshimitsu Niwa, J. Chromatography, 227 (1983), 25-39), immunoassay (Japanese Patent Laid-Open No. 8-21835), enzyme method [Rosita Dolohofer, O. H. Wieland. , J .; Clin. Chem. Clin. Biochem. , 25, 733-736 (1987)], known methods such as liquid chromatography mass spectrometry (LC-MS) method and HPLC method, preferably myo-inositol dehydrogenase (EC 1.1.1.18). ) (Patent Documents 3 and 4), and more preferably quantification using a myo-inositol measuring reagent Lucica MI (Asahi Kasei Pharma Co., Ltd.).
 
本発明の実施の形態における尿は定量まで冷蔵保存することが好ましい（１年以上安定）。室温放置でも数日以内であれば問題はない。また、防腐剤として０．０５％アジ化ナトリウムあるいは０．１％プロクリンを添加しても定量に影響しない。
The urine in the embodiment of the present invention is preferably stored refrigerated until it is fixed (stable for one year or more). There is no problem even if it is left at room temperature within a few days. Addition of 0.05% sodium azide or 0.1% procrine as a preservative does not affect the determination.
 
ＭＩは定性反応によりＭＩの存在を検出する方法により行ってもよいが、定量することが好ましい。ＭＩを定量するとは、ＭＩが含まれている可能性のある被験者の尿と、ＭＩを既知の濃度で含む校正用の試料を、それぞれ単独にＭＩを公知の方法で検出し、ＭＩが含まれている可能性のある被験者の尿中のＭＩの濃度は、ＭＩが含まれている可能性のある被験者の尿についての検出量と、ＭＩを既知の濃度で含む校正用の試料についての検出量とを比較することにより算出することである。尿ＭＩ量は、同時に測定するクレアチニン１ｇ（ｇＣｒ）値で標準化した値（ｍｇ）として表示し、通常、複数検体の平均値として算出することが好ましく、３検体以上の定量値の中で他の定量値と大きく異なる値を示す検体がある場合は、これを除外することが好ましい。また、２検体しか定量されていない場合で、両者の定量値が大きく異なる場合は、再定量すれば良い。
MI may be performed by a method of detecting the presence of MI by a qualitative reaction, but is preferably quantified. MI is quantified by detecting MI in a known method for urine of a subject that may contain MI and a calibration sample containing MI at a known concentration. The concentration of MI in the urine of a subject who may be subject to the detection amount for the urine of a subject who may contain MI and the detection amount for a calibration sample containing MI at a known concentration Is calculated by comparing. The amount of urine MI is displayed as a value (mg) standardized with the creatinine 1 g (gCr) value measured simultaneously, and is usually preferably calculated as an average value of a plurality of samples, and other quantitative values among three or more samples If there is a specimen showing a value that is significantly different from the quantitative value, it is preferable to exclude this. Further, when only two samples are quantified, if the quantified values of both are greatly different, re-quantification may be performed.
 
本発明はＭＩを定量する同一被験者の血清又は血漿中（血清及び／又は血漿）のＰｌｓＥｔｎを定量する工程を含む。ＭＩを定量する同一被験者にはＭＩを定量された被験者を含み、ＭＩとＰｌｓＥｔｎの定量の順序は任意である。被験者から血液を通常通り採血し、できるだけ早期に遠心分離し、採取した血清または血漿は速やかに冷蔵（４℃）あるいは凍結保存（－２０℃～－８０℃）する。血液の状態で一時保管する場合は冷蔵保存し、少なくとも採血日当日に血清あるいは血漿分離までの処理を施すことが好ましい。血漿を取得する場合には、分離剤や抗プラスミン剤等の使用の有無は特に限定されず、ＥＤＴＡ、フッ化ナトリウム、クエン酸ナトリウム、ヘパリンナトリウム、モノヨード酢酸等の抗凝固剤や解糖阻止剤の使用の有無も特に限定されない。
The present invention includes the step of quantifying PlsEtn in the serum or plasma (serum and / or plasma) of the same subject to quantify MI. The same subject for quantifying MI includes subjects whose MI has been quantified, and the order of quantification of MI and PlsEtn is arbitrary. Blood is collected from the subject as usual, centrifuged as soon as possible, and the collected serum or plasma is immediately refrigerated (4 ° C.) or frozen (−20 ° C. to −80 ° C.). In the case of temporary storage in the state of blood, it is preferable to store in a refrigerated state and to perform treatment up to serum or plasma separation at least on the day of blood collection. In the case of obtaining plasma, the presence or absence of the use of a separating agent or antiplasmin agent is not particularly limited, and anticoagulants and antiglycolytic agents such as EDTA, sodium fluoride, sodium citrate, heparin sodium, monoiodoacetic acid, etc. Whether or not is used is not particularly limited.
 
本発明の実施の形態における「検体」または「試料」はＰｌｓＥｔｎを含むと予想される試料であり、上記の被験者の尿、血清及び血漿を含み、ＰｌｓＥｔｎを添加した溶液、血清及び血漿でもよい。本発明の実施の形態における「検体」または「試料」はＰｌｓＥｔｎを含むと予想される試料であれば限定されないが、被験者の尿、血清、及び/または血漿であれば好ましい。その他の試料の例としては、実験動物由来の尿、血清、血漿や植物組織、海水、天然水、果汁、飲料、廃液等が挙げられる。
The “specimen” or “sample” in the embodiment of the present invention is a sample expected to contain PlsEtn, and may include urine, serum and plasma of the above-mentioned subject, and may be a solution, serum and plasma to which PlsEtn is added. The “specimen” or “sample” in the embodiment of the present invention is not limited as long as it is a sample expected to contain PlsEtn, but is preferably urine, serum, and / or plasma of a subject. Examples of other samples include urine derived from laboratory animals, serum, plasma, plant tissue, seawater, natural water, fruit juice, beverages, waste liquid, and the like.
 
本発明の実施の形態におけるＰｌｓＥｔｎは、グリセロリン脂質のＣ１（ｓｎ－１）位に脂肪酸がビニルエーテル結合したアルケニルアシル型グリセロリン脂質（アルケニルアシル型エーテルリン脂質）で塩基がエタノールアミンである、Ｊｏｕｒａｎａｌ　ｏｆ　ａｔｈｅｒｏｓｃｌｅｒｏｓｉｓ　ａｎｄ　ｔｈｒｏｍｂｏｓｉｓ、１４巻、１号、２００７年、１２－１８頁やＪ．Ｐｓｙｃｈｉａｔｒｙ　Ｎｅｕｒｏｓｃｉ．、２０１０年、３５巻、１号、５９－６２頁等で開示された公知のＰｌｓＥｔｎであり、以下の式のＰｌｓＥｔｎである。：
In the embodiment of the present invention, PlsEtn is an alkenyl acyl glycerophospholipid (alkenylacyl ether phospholipid) in which a fatty acid is vinyl ether-bonded to the C1 (sn-1) position of glycerophospholipid, and the base is ethanolamine. and thrombosis, Vol. 14, No. 1, 2007, pp. 12-18 and J. Am. Psychiatry Neurosci. 2010, Vol. 35, No. 1, pp. 59-62, etc., which is a known PlsEtn, and PlsEtn of the following formula. :
Figure JPOXMLDOC01-appb-C000001
Figure JPOXMLDOC01-appb-C000001
（式中、Ｒ１及びＲ２は、それぞれ独立して、低級若しくは高級アルキル基又はアルケニル基である）
 
本発明の実施の形態におけるＰｌｓＥｔｎには以下の式のＰｌｓＥｔｎのリゾ体（ｌｙＰｌｓＥｔｎ）を含む場合もある。 (Wherein, R 1 and R 2 are each independently a lower or higher alkyl group or an alkenyl group)

PlsEtn in the embodiment of the present invention may include a PlsEtn lyso form (lyPlsEtn) of the following formula.
Figure JPOXMLDOC01-appb-C000002
Figure JPOXMLDOC01-appb-C000002
 （式中、Ｒ３は、低級若しくは高級アルキル基又はアルケニル基である）
 
本発明は、被験者の自然排泄尿中のＭＩを定量する工程と、同一被験者の血清または血漿中のＰｌｓＥｔｎを定量する工程とを含む、該被験者を認知症患者またはその前段階にある者か否かを分類するための認知症検査方法であって、健常者と、認知症患者またはその前段階にある者とを区別することができる、ＭＩとＰｌｓＥｔｎとのそれぞれの、予め求められた閾値に対して、被験者のＭＩとＰｌｓＥｔｎの定量値を比較する工程を含む、前記の認知症検査方法も含む。 (Wherein R 3 is a lower or higher alkyl group or an alkenyl group)

The present invention comprises a step of quantifying MI in spontaneously excreted urine of a subject and a step of quantifying PlsEtn in the serum or plasma of the same subject. A dementia test method for classifying MI and PlsEtn, which can discriminate between a healthy person and a dementia patient or a person in the previous stage, at a predetermined threshold value. On the other hand, the said dementia test | inspection method including the process of comparing the quantitative value of MI and PlsEtn of a test subject is also included.
 
本発明の実施の形態において被験者を認知症またはその前段階にある者と分類する尿ＭＩ量の基準は、健常者と、認知症患者またはその前段階にある者とを区別することができる、ＭＩとＰｌｓＥｔｎとのそれぞれの、予め求められた閾値に対して、被験者のＭＩとＰｌｓＥｔｎの定量値を比較する。尿ＭＩの閾値は、健常成人の平均値±標準偏差の上限値から、健常高齢者の平均値±標準偏差の上限値までの範囲から設定すれば良い。
In the embodiment of the present invention, the urinary MI amount criterion for classifying a subject as dementia or a person in the previous stage can distinguish a healthy person from a dementia patient or a person in the previous stage. The quantitative values of the subject's MI and PlsEtn are compared with the predetermined threshold values of MI and PlsEtn. The threshold value of urine MI may be set from the range from the average value of normal adults ± the upper limit of standard deviation to the average value of healthy elderly people ± the upper limit of standard deviation.
 
一例として、尿ＭＩの閾値は、健常成人の平均値±標準偏差（１３．１±８．５ｍｇ／ｇＣｒ、ｎ＝４４４、平均年齢３９．６±１０．６歳）の上限値である２１．６ｍｇ／ｇＣｒから、健常高齢者の平均値±標準偏差（３３．０±２２．１ｍｇ／ｇＣｒ、ｎ＝７６、平均年齢７２．７±５．６歳）の上限値である５５．１ｍｇ／ｇＣｒまでの範囲から設定できる。
As an example, the threshold value of urine MI is an upper limit value of the average value ± standard deviation (13.1 ± 8.5 mg / gCr, n = 444, average age 39.6 ± 10.6 years old) of healthy adults. From 6 mg / gCr, 55.1 mg / gCr which is the upper limit of the average value ± standard deviation (33.0 ± 22.1 mg / gCr, n = 76, average age 72.7 ± 5.6 years old) of healthy elderly people It can be set from the range up to.
 
従って、本発明の実施の形態において被験者を認知症またはその前段階にある者と分類する尿ＭＩ量の基準は、空腹時二番尿の場合、２１．６～５５．１ｍｇ／ｇＣｒの範囲から設定される閾値以上としても良い。また、下記の実施例に示したとおり、２５ｍｇ／ｇＣｒを尿ＭＩ量の閾値とすることにより、血清及び／又は血漿ＰｌｓＥｔｎ量の定量との併用による認知症の検出確度が向上することから、尿ＭＩ量の閾値として２５ｍｇ／ｇＣｒを採用することも好ましい。さらに、下記の実施例に示したとおり、３６．６ｍｇ／ｇＣｒを尿ＭＩ量の閾値とすることにより、血清及び／又は血漿ＰｌｓＥｔｎ量の定量との併用による認知症の検出確度が向上することから、尿ＭＩ量の閾値として３６．６ｍｇ／ｇＣｒを採用することも好ましい。下記の実施例に示したとおり、随時尿ＭＩ量は平均的に空腹時二番尿より８．２ｍｇ／ｇＣｒ増加していることから、２９．８～６３．３ｍｇ／ｇＣｒの範囲から設定される閾値以上としても良く、随時尿の場合３３．２又は４４．８ｍｇ／ｇＣｒを閾値として採用することも好ましい。
Therefore, in the embodiment of the present invention, the urinary MI amount standard for classifying a subject as dementia or a person in the previous stage is 21.6 to 55.1 mg / gCr in the case of fasting second urine. It is good also as more than the threshold set up. In addition, as shown in the following examples, by setting 25 mg / gCr as a threshold value for the urine MI amount, the detection accuracy of dementia is improved by the combined use with the quantification of the serum and / or plasma PlsEtn amount. It is also preferable to adopt 25 mg / gCr as the threshold of the MI amount. Furthermore, as shown in the examples below, by setting 36.6 mg / gCr as the threshold for the urine MI amount, the detection accuracy of dementia is improved by the combined use with the quantification of the serum and / or plasma PlsEtn amount. It is also preferable to adopt 36.6 mg / gCr as the threshold value for the urine MI amount. As shown in the Examples below, the amount of ad libitum urine MI is on average increased by 8.2 mg / gCr from the fasting second urine, so it is set within the range of 29.8 to 63.3 mg / gCr. It may be greater than the threshold, and in the case of occasional urine, it is also preferable to employ 33.2 or 44.8 mg / gCr as the threshold.
 
被験者の血清または血漿中のＰｌｓＥｔｎの閾値の下限値は、健常成人の平均値±標準偏差の下限値および健常高齢者の平均値±標準偏差の下限値より１～２μＭ低く設定すれば良い。被験者の血清または血漿中のＰｌｓＥｔｎの閾値の上限値は、全高齢者（健常＋認知障害）における尿ＭＩ量の平均値以上の高齢者を対象に作出されたＣＤＲとＰｌｓＥｔｎの相関近似曲線式に、ｙ＝０．５（ＣＤＲ、認知障害疑い）を代入して得られるｘとすれば良い。
The lower limit value of the PlsEtn threshold value in the serum or plasma of the subject may be set 1 to 2 μM lower than the average value of the normal adult ± the lower limit value of the standard deviation and the average value of the healthy elderly person ± the lower limit value of the standard deviation. The upper limit of the threshold value of PlsEtn in the serum or plasma of the test subject is the correlation curve equation of CDR and PlsEtn created for all elderly people (healthy + cognitive impairment) and older subjects with average urinary MI levels or higher. , Y = 0.5 (CDR, suspected cognitive impairment) may be used as x.
 
一例として、被験者の血清または血漿中のＰｌｓＥｔｎを定量する工程が、後述する血清及び／又は血漿ＰｌｓＥｔｎ量を１２５Ｉ－ＨＰＬＣ法を利用して定量したる場合の血清及び／又は血漿ＰｌｓＥｔｎ量の閾値の下限値は、健常成人の平均値±標準偏差（７３．０±１９．５μＭ、ｎ＝４３９）の下限値は５３．５であり、健常高齢者の平均値±標準偏差（７３．３±２０．５μＭ、ｎ＝９７）の下限値は５２．８μＭなので、５２μＭとすることもできる。また、被験者の血清または血漿中のＰｌｓＥｔｎを定量する工程が、後述する血清及び／又は血漿ＰｌｓＥｔｎ量を１２５Ｉ－ＨＰＬＣ法を利用して定量したる場合の血清及び／又は血漿ＰｌｓＥｔｎ量の閾値の上限値は、全高齢者（健常＋認知障害）における尿ＭＩ量の平均値である３６．６　ｍｇ／ｇＣｒ以上の高齢者を対象に作出されたＣＤＲとＰｌｓＥｔｎの相関近似曲線式（ｙ＝－０．０１９７ｘ＋１．６９０）に、ｙ＝０．５（ＣＤＲ、認知障害疑い）を代入して得られるｘ（ＰｌｓＥｔｎ）の値６０．４μＭと概算することもできる。
As an example, the step of quantifying PlsEtn in a subject's serum or plasma is a threshold value of the serum and / or plasma PlsEtn amount when the amount of serum and / or plasma PlsEtn described below is quantified using a 125I-HPLC method. The lower limit is 53.5 for the average value ± standard deviation (73.0 ± 19.5 μM, n = 439) for healthy adults, and the average value ± standard deviation (73.3 ± 20 for healthy elderly people). The lower limit value of .5 μM, n = 97) is 52.8 μM, so it can be set to 52 μM. In addition, the step of quantifying PlsEtn in the serum or plasma of the subject comprises the upper limit of the threshold value of the serum and / or plasma PlsEtn amount when the amount of serum and / or plasma PlsEtn described below is quantified using the 125I-HPLC method. The value is an approximate curve equation of correlation between CDR and PlsEtn (y = −0) created for an elderly person with an average urinary MI amount of 36.6 mg / gCr or higher in all elderly persons (healthy + cognitive impairment). .0197x + 1.690) and y = 0.5 (CDR, suspected cognitive impairment) can be approximated to a value of 60.4 μM for x (PlsEtn).
 
従って、本発明の実施の形態において、被験者を認知症またはその前段階にある者と分類する血清及び／又は血漿ＰｌｓＥｔｎ量の基準は、被験者の血清または血漿中のＰｌｓＥｔｎを定量する工程が、後述する血清及び／又は血漿ＰｌｓＥｔｎ量を１２５Ｉ－ＨＰＬＣ法を利用した定量である場合、血清及び／又は血漿ＰｌｓＥｔｎ量が５２～６１μＭの範囲から設定される閾値以下としても良い。
Therefore, in the embodiment of the present invention, the serum and / or plasma PlsEtn amount standard for classifying a subject as dementia or a person in the previous stage is the step of quantifying PlsEtn in the subject's serum or plasma. When the serum and / or plasma PlsEtn amount to be quantified using the 125I-HPLC method, the serum and / or plasma PlsEtn amount may be equal to or lower than a threshold value set from a range of 52 to 61 μM.
 
被験者の血清または血漿中のＰｌｓＥｔｎを定量する工程が、後述する酵素を利用した定量である場合、測定値の平均値は、１２５Ｉ－ＨＰＬＣ法を利用した定量である場合の平均値－２μＭから＋３μＭの範囲である。したがって、本発明の実施の形態において、被験者を認知症またはその前段階にある者と分類する血清及び／又は血漿ＰｌｓＥｔｎ量の基準は、被験者の血清または血漿中のＰｌｓＥｔｎを定量する工程が、後述する酵素を利用した定量である場合、血清及び／又は血漿ＰｌｓＥｔｎ量が５０～６４μＭの範囲から設定される閾値以下としても良い。
When the step of quantifying PlsEtn in the serum or plasma of the subject is quantification using the enzyme described later, the average value of the measured values is from the average value of −2 μM to +3 μM in the case of quantification using the 125I-HPLC method. Range. Therefore, in the embodiment of the present invention, the serum and / or plasma PlsEtn amount standard for classifying a subject as dementia or a person in the previous stage is the step of quantifying PlsEtn in the serum or plasma of the subject. In the case of quantification using the enzyme, the serum and / or plasma PlsEtn amount may be set to a threshold value or less set from the range of 50 to 64 μM.
 
さらに、下記の実施例に示したとおり、空腹時二番尿ＭＩ量の閾値が３６．６ｍｇ／ｇＣｒ、随時尿中のであり、血清及び／又は血漿ＰｌｓＥｔｎ量を１２５Ｉ－ＨＰＬＣ法により定量する場合、血清及び／又は血漿ＰｌｓＥｔｎ量の閾値が後述するように約５８μＭである場合に認知症検出の確度が最適となっており、これらの閾値を採用することが特に好ましい。また、被験者の血清または血漿中のＰｌｓＥｔｎを定量する工程が、後述する酵素を利用した定量である場合、血漿ＰｌｓＥｔｎ量の閾値が約５６または６１μＭの閾値を採用することが特に好ましい。
Furthermore, as shown in the Examples below, when the fasting second urine MI amount threshold is 36.6 mg / gCr, urinary urine, and serum and / or plasma PlsEtn amount is quantified by 125I-HPLC method, When the threshold value of serum and / or plasma PlsEtn amount is about 58 μM as described later, the accuracy of dementia detection is optimal, and it is particularly preferable to adopt these threshold values. In addition, when the step of quantifying PlsEtn in the serum or plasma of a subject is quantification using an enzyme described later, it is particularly preferable to employ a threshold value of about 56 or 61 μM for the plasma PlsEtn amount.
 
なお、実際の検査においては、尿ＭＩ量と血清及び／又は血漿ＰｌｓＥｔｎ量は全ての被験者について定量し、前記の基準に基づいて分類判定を行うことができる。あるいは、血清及び／又は血漿ＰｌｓＥｔｎ量が閾値以下の被験者について尿ＭＩ量を定量するか、または尿ＭＩ量が閾値以上の被験者について血清及び／又は血漿ＰｌｓＥｔｎ量を定量してもよい。好ましくは、定量の手順がより簡便である尿ＭＩ量を全被験者について定量し、その結果に基づいて血清及び／又は血漿ＰｌｓＥｔｎ量の定量を行うことができるがこれに限定されない。
In an actual test, the urine MI amount and the serum and / or plasma PlsEtn amount can be quantified for all subjects, and classification can be determined based on the above-mentioned criteria. Alternatively, the urinary MI amount may be quantified for a subject whose serum and / or plasma PlsEtn amount is a threshold or less, or the serum and / or plasma PlsEtn amount may be quantified for a subject whose urine MI amount is a threshold value or more. Preferably, the amount of urine MI whose quantification procedure is simpler can be quantified for all subjects, and the amount of serum and / or plasma PlsEtn can be quantified based on the results, but is not limited thereto.
 
本発明の実施の形態において血清及び／又は血漿ＰｌｓＥｔｎ量を定量するとは、ＰｌｓＥｔｎが含まれている可能性のある被験者の血清又は血漿と、ＰｌｓＥｔｎを既知の濃度で含む校正用の試料を、それぞれ単独にＰｌｓＥｔｎを公知の方法及び/または本発明の方法で検出し、ＰｌｓＥｔｎが含まれている可能性のある被験者の血清又は血漿のＰｌｓＥｔｎの濃度は、ＰｌｓＥｔｎが含まれている可能性のある被験者の血清又は血漿についての検出量と、ＰｌｓＥｔｎを既知の濃度で含む校正用の試料についての検出量とを比較することにより算出することである。本発明の実施の形態においては、通常１モルのＰｌｓＥｔｎから1モルのグリセロ－３－ホスフォエタノールアミン又はエタノールアミンが生成物として得られるのでグリセロ－３－ホスフォエタノールアミン又はエタノールアミンを定量すればＰｌｓＥｔｎ量を算出できる。本発明の実施の形態においては、通常１モルのＰｌｓＥｔｎから1モルの過酸化水素が生成物として得られるので過酸化水素を定量すればＰｌｓＥｔｎ量を算出できる。
In the embodiment of the present invention, quantifying the amount of serum and / or plasma PlsEtn means that serum or plasma of a subject who may contain PlsEtn and a calibration sample containing PlsEtn at a known concentration, respectively. PlsEtn alone is detected by known methods and / or the methods of the present invention, and the concentration of PlsEtn in the serum or plasma of a subject that may contain PlsEtn may be subject to PsEtn Is calculated by comparing the detected amount of serum or plasma with the detected amount of a calibration sample containing PlsEtn at a known concentration. In the embodiment of the present invention, since 1 mol of glycero-3-phosphoethanolamine or ethanolamine is usually obtained as a product from 1 mol of PlsEtn, glycero-3-phosphoethanolamine or ethanolamine can be quantified. PlsEtn amount can be calculated. In the embodiment of the present invention, since 1 mol of hydrogen peroxide is usually obtained as a product from 1 mol of PlsEtn, the amount of PlsEtn can be calculated by quantifying hydrogen peroxide.
 
本発明の実施の形態において血清及び／又は血漿ＰｌｓＥｔｎ量は、例えば特許文献１に記載された放射性ヨウ素－高速液体クロマトグラフィー（１２５Ｉ－ＨＰＬＣ）法により、定量できる。ＰｌｓＥｔｎを１２５Ｉ－ＨＰＬＣ法で定量する場合、血清及び／又は血漿は冷蔵保存あるいは速やかに凍結融解した血清及び／又は血漿を一定量（通常、２００μＬ）使用し、脂質成分を規定の方法で抽出する。測定用試料（抽出脂質成分）は速やかにＰｌｓＥｔｎの定量に供することが好ましいが、測定まで一時的に保管する場合はメタノールやジエチルエーテル中は避け、クロロホルム中で冷凍保存（－２０℃～－８０℃）することが好ましい。
In the embodiment of the present invention, the amount of serum and / or plasma PlsEtn can be quantified by, for example, a radioactive iodine-high performance liquid chromatography (125I-HPLC) method described in Patent Document 1. When PlsEtn is quantified by the 125I-HPLC method, serum and / or plasma is stored in a refrigerator or rapidly frozen and thawed, and a fixed amount (usually 200 μL) of serum and / or plasma is used, and lipid components are extracted by a prescribed method. . The sample for measurement (extracted lipid component) is preferably used for the rapid quantification of PlsEtn. However, if it is temporarily stored until measurement, avoid methanol and diethyl ether and store frozen in chloroform (-20 ° C to -80 ° C). C.).
 
本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する工程は、酵素を利用した定量であっても良い。
The step of quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention may be quantification using an enzyme.
 
本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する工程は、以下の（１）～（３－４）の工程のいずれかまたは複数を含んでもよく、また、さらに（１）～（３－４）の全てを含んでもよい。
（１）エタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解できる酵素を用いて、被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解する工程：
（２）エタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）をグリセロ－３－ホスフォエタノールアミンとアルデヒドに加水分解でき、エーテル型リゾホスファチジルエタノールアミンを加水分解できない加水分解酵素を用いて、前記（１）にて生成されたエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）をグリセロ－３－ホフォエタノールアミンとアルデヒドに加水分解する工程：及び
（３－１）グリセロ－３－ホスフォエタノールアミンを加水分解してエタノールアミンにせしめる酵素にて、工程（２）で得られたグリセロ－３－ホスフォエタノールアミンを加水分解してエタノールアミンにする工程：
（３－２）エタノールアミンを酸化する酵素により、エタノールアミンから過酸化水素を発生せしめる工程：
（３－３）過酸化水素を過酸化水素定量手段により定量する工程：及び
（３－４）前記被験者の血清または血漿中のＰｌｓＥｔｎ量を算出する工程：
または前記（２）の加水分解酵素が、リゾホスファチジルエタノールアミンを加水分解できる酵素である場合、同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する方法が、前記（１）～（３－４）の工程に先立つか、少なくとも前記（２）工程の前までに、下記（４）及び（５）を含む工程を行うことを特徴とする方法。
The step of quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention may include any one or more of the following steps (1) to (3-4), and further includes (1) to All of (3-4) may be included.
(1) Using an enzyme capable of hydrolyzing ethanolamine-type plasmalogen (PlsEtn) into ethanolamine-type lysoplasmalogen (lyPlsEtn), ethanolamine-type plasmalogen (PlsEtn) in the serum or plasma of the subject is ethanolamine-type lysozyme. Hydrolyzing to plasmalogen (lyPlsEtn):
(2) Using a hydrolase that can hydrolyze ethanolamine-type lysoplasmalogen (lyPlsEtn) into glycero-3-phosphoethanolamine and aldehyde, but cannot hydrolyze ether-type lysophosphatidylethanolamine, (1) The step of hydrolyzing the ethanolamine-type lysoplasmalogen (lyPlsEtn) produced into glycero-3-phosphoethanolamine and an aldehyde: and (3-1) hydrolysis of glycero-3-phosphoethanolamine to ethanol A step of hydrolyzing the glycero-3-phosphoethanolamine obtained in the step (2) to an ethanolamine with an enzyme that is converted into an amine:
(3-2) Step of generating hydrogen peroxide from ethanolamine by an enzyme that oxidizes ethanolamine:
(3-3) Step of quantifying hydrogen peroxide by means of hydrogen peroxide quantification: (3-4) Step of calculating the amount of PlsEtn in the serum or plasma of the subject:
Alternatively, when the hydrolase of (2) is an enzyme capable of hydrolyzing lysophosphatidylethanolamine, the method for quantifying ethanolamine-type plasmalogen (PlsEtn) in the serum or plasma of the same subject is described in (1) A method comprising performing the steps (4) and (5) below prior to the steps (3-4) or at least before the step (2).
 
上記工程（１）～（３－４）を含む本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する方法の一例を概略して図７に示した。工程（１）はＰＬの作用で、ＰｌｓＥｔｎをｌｙＰｌｓＥｔｎに加水分解する工程である。さらに必要によりリゾホスファチジルエタノールアミンを加水分解する酵素、好ましくはリゾホスフォリパーゼ（ｌｙｓｏｐｈｏｓｐｈｏｌｉｐａｓｅ、ＬＹＰＬと略す場合がある）を用いてリゾホスファチジルエタノールアミンを加水分解する工程を含む場合があるがこれに限定されない。工程（２）は加水分解酵素の作用で、ｌｙＰｌｓＥｔｎを加水分解する工程である。工程（３－１）は工程（２）で得られた生成物をＧｌｙｃｅｒｏｐｈｏｓｐｈｏｃｈｏｌｉｎｅ　ｐｈｏｓｐｈｏｄｉｅｓｔｅｒａｓｅ（ＧＰＣＰ）とエタノールアミンオキシダーゼ（ＥＡＯ）の作用で過酸化水素（Ｈ２Ｏ２）とする工程である。工程（３－２）の過酸化水素は後述する公知の方法で定量すれば良い。
An example of a method for quantifying the serum and / or plasma PlsEtn amount of the embodiment of the present invention including the above steps (1) to (3-4) is schematically shown in FIG. Step (1) is a step of hydrolyzing PlsEtn to lyPlsEtn by the action of PL. If necessary, an enzyme that hydrolyzes lysophosphatidylethanolamine, preferably a step of hydrolyzing lysophosphatidylethanolamine using lysophospholipase (may be abbreviated as LYPL) may be included. Not. Step (2) is a step of hydrolyzing lyPlsEtn by the action of a hydrolase. Step (3-1) is a step in which the product obtained in step (2) is converted to hydrogen peroxide (H 2 O 2 ) by the action of glycephophosphocholine phosphodiesterase (GPCP) and ethanolamine oxidase (EAO). Hydrogen peroxide in the step (3-2) may be quantified by a known method described later.
 
図７に示した上記工程（１）の好適な例は、ＰＬの作用により、ＰｌｓＥｔｎをｌｙＰｌｓＥｔｎと脂肪酸に加水分解する工程である。図７に示した上記工程（２）の好適な例は、ｌｙＰｌｓ　ａｓｅの作用により、ｌｙＰｌｓＥｔｎをグリセロ－３－ホスフォエタノールアミン（ｇｌｙｃｅｒｏ－３－ｐｈｏｓｐｈｏｅｔｈａｎｏｌａｍｉｎｅ）と対応するアルデヒド（ａｌｄｅｈｙｄｅ）に加水分解する工程である。図７に示した上記工程（３－１）の好適な例は、工程（２）で得られたグリセロ－３－ホスフォエタノールアミンをＧｌｙｃｅｒｏｐｈｏｓｐｈｏｃｈｏｌｉｎｅ　ｐｈｏｓｐｈｏｄｉｅｓｔｅｒａｓｅ（ＧＰＣＰ）の作用により、グリセロ－３－ホスフォエタノールアミンをエタノールアミン（ｅｔｈａｎｏｌａｍｉｎｅ）とグリセロリン酸（ｓｎ－ｇｌｙｃｅｒｏｌ　３－ｐｈｏｓｐｈａｔｅ）に加水分解し、エタノールアミンオキシダーゼの作用でエタノールアミンを過酸化水素（Ｈ２Ｏ２）、アンモニア（ＮＨ３、アンモニウム塩も含む）とグリコールアルデヒド（ｇｌｙｃｏｌａｌｄｅｈｙｄｅ）に酸化する工程である。
A preferred example of the step (1) shown in FIG. 7 is a step of hydrolyzing PlsEtn into lyPlsEtn and a fatty acid by the action of PL. A preferred example of the above step (2) shown in FIG. 7 is to hydrolyze lyPlsEtn into aldehyde corresponding to glycero-3-phosphoethanolamine by the action of lyPls ase. It is a process. A preferred example of the above step (3-1) shown in FIG. 7 is that glycero-3-phosphoethanolamine obtained in step (2) is glycero-3-phosphoethanolamine (GPCP) by the action of glycerophosphophosphoesterase (GPCP). Ethanolamine is hydrolyzed to ethanolamine and glycerophosphoric acid (sn-glycerol 3-phosphate), and ethanolamine is converted into hydrogen peroxide (H 2 O 2 ), ammonia (NH 3 , ammonium salt by the action of ethanolamine oxidase. And oxidation to glycolaldehyde (glycolaldehyde).
 
上記工程（１）～（３－４）を含む本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する方法において、被験者の血清及び／又は血漿中にＰｌｓＥｔｎのみ、又はＰｌｓＥｔｎとｌｙＰｌｓＥｔｎが混在する場合は、後述する本発明のｌｙＰｌｓ　ａｓｅ及びＰＬの作用を利用して血清及び／又は血漿ＰｌｓＥｔｎ量を定量することが好ましいがこれに限定されない。被験者の血清及び／又は血漿中にｌｙＰｌｓＥｔｎのみが存在する場合は本発明のｌｙＰｌｓ　ａｓｅの作用を利用して血清及び／又は血漿ＰｌｓＥｔｎ量を定量することが好ましいがこれに限定されない。
In the method for quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention including the above steps (1) to (3-4), only PlsEtn, or PlsEtn and lyPlsEtn are contained in the serum and / or plasma of a subject. In the case of coexistence, it is preferable to quantify the amount of serum and / or plasma PlsEtn using the action of lyPlsase and PL of the present invention described later, but it is not limited thereto. When only lyPlsEtn is present in the serum and / or plasma of a subject, it is preferable to quantify the amount of serum and / or plasma PlsEtn using the action of lyPlsase of the present invention, but the present invention is not limited thereto.
 
上記工程（１）～（３－４）を含む本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する方法において、工程（１）で用いるＰＬは、ＰｌｓＥｔｎをｌｙＰｌｓＥｔｎに加水分解する作用を触媒する酵素であれば限定されない。この様な酵素の好ましい例として配列番号５のアミノ酸配列を有するＰＬが挙げられる。また、配列番号５に記載のアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、ＰｌｓをｌｙＰｌｓに加水分解する作用を触媒する酵素も挙げられ、その例は後述する本発明のｌｙＰｌｓ　ａｓｅの場合と同様である。該ＰＬはＳｔｒｅｐｔｏｍｙｃｅｓ　ａｌｂｉｄｏｆｌａｖｕｓに分類されるＮＡ２９７（ＮＩＴＥ　ＢＰ－１０１４として寄託済み）を用いて国際公開番号：ＷＯ２０１２／１０５５６５　Ａ１に記載の製造方法で製造できる新規なホスホリパーゼＡ１である。
In the method for quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention including the above steps (1) to (3-4), PL used in step (1) acts to hydrolyze PlsEtn to lyPlsEtn. The enzyme is not limited as long as it catalyzes. A preferred example of such an enzyme is PL having the amino acid sequence of SEQ ID NO: 5. Examples of the amino acid sequence shown in SEQ ID NO: 5 include an enzyme consisting of an amino acid sequence in which one or a plurality of amino acids are deleted, substituted or added, and catalyzing the action of hydrolyzing Pls to lyPls. Is the same as in the case of lyPlase of the present invention described later. The PL is a novel phospholipase A 1 that can be produced by the production method described in International Publication Number: WO2012 / 105565 A1 using NA297 (deposited as NITE BP-1014) classified as Streptomyces albidoflavus.
 
上記工程（１）～（３－４）を含む本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する方法において、工程（１）で用いるその他のＰＬとしてはホスホリパーゼＡ２（ｐｈｏｓｐｈｏｌｉｐａｓｅ　Ａ２、ＰＬＡ２と略す場合がある）、ホスホリパーゼＡ１（ｐｈｏｓｐｈｏｌｉｐａｓｅ　Ａ１、ＰＬＡ１と略す場合がある）、及びホスホリパーゼＢ（ＰＬＢ）が挙げられる。
In the method for quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention including the above steps (1) to (3-4), other PL used in step (1) is phospholipase A 2 (phospholipase A 2 and PLA 2 ), phospholipase A 1 (sometimes abbreviated as phospholipase A 1 and PLA 1 ), and phospholipase B (PLB).
 
ＰＬＡ２は、ＥＣ　３．１．１．４と分類される酵素を含む。ＰＬＡ２の好適な例としては、Ｓｔｒｅｐｔｏｍｙｃｅｓ　ｖｉｏｌａｃｅｏｒｕｂｅｒ由来ＰＬＡ２（ＰＬＡ２（旭化成ファーマ株式会社（Ｔ－３１））及びＰＬＡ２ナガセ（ナガセケムテックス（株）））、Ａｓｐｅｒｇｉｌｌｕｓ　ｎｉｇｅｒ由来ＰＬＡ２（マキサパールＡ２及びケーキザイム（ともにディー・エス・エム　ジャパン（株）））、豚の膵臓由来ＰＬＡ２（リポモッド６９９Ｌ（ジェネンコア協和（株）））、Ａｓｐｅｒｇｉｌｌｕｓ由来ホスホリパーゼ（レシターゼ（ノボザイムズ　ジャパン（株））、ｐｏｒｃｉｎｅ　ｐａｎｃｒｅａｓ由来ＰＬＡ２（ＳＩＧＭＡ（Ｐ２２３））が挙げられる。
PLA 2 contains an enzyme classified as EC 3.1.1.4. Suitable examples of PLA 2 include PLA2 derived from Streptomyces violaceoruber (PLA 2 (Asahi Kasei Pharma Corporation (T-31)) and PLA 2 Nagase (Nagase ChemteX Corp.)), Aspergillus niger derived PLA 2 (Maxa Pearl A 2 And cakezyme (both DSM Japan Co., Ltd.), porcine pancreas-derived PLA 2 (Lipomod 699L (Genencore Kyowa Co., Ltd.)), Aspergillus-derived phospholipase (Recitase (Novozymes Japan Co., Ltd.)), porcine pancreas Origin PLA 2 (SIGMA (P223)).
 
ＰＬＡ１は、ＥＣ　３．１．１．３２と分類される酵素を含む。ＰＬＡ１の好適な例としてはＡｓｐｅｒｇｉｌｌｕｓ　ｏｒｙｚａｅ由来（三菱化学フーズ（株））が挙げられる。
PLA 1 contains an enzyme classified as EC 3.1.1.32. A preferable example of PLA 1 is derived from Aspergillus oryzae (Mitsubishi Chemical Foods Co., Ltd.).
 
ＰＬＢはＰＬＡ１及びＰＬＡ２の作用を均等に又は不均等に併せ持つ。ＰＬＢの好適な例としては、特許第５０６０６６６号で開示された酵素、ＷＯ２００７－０１０８９２で開示された酵素、ＬＰＢＰ（旭化成ファーマ株式会社（Ｔ－６３））及びＣＥＢＰ（旭化成ファーマ株式会社（Ｔ－６６））が挙げられる。
PLB combines the actions of PLA 1 and PLA 2 equally or unevenly. Preferable examples of PLB include the enzyme disclosed in Japanese Patent No. 5060666, the enzyme disclosed in WO2007-010892, LPBP (Asahi Kasei Pharma Corporation (T-63)) and CEBP (Asahi Kasei Pharma Corporation (T-66)). )).
 
上記工程（１）～（３－４）を含む本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する方法において、工程（２）でｌｙＰｌｓＥｔｎを加水分解する酵素を用いる場合、該加水分解酵素はｌｙＰｌｓＥｔｎをグリセロ－３－ホスフォエタノールアミンと対応するアルデヒド（単にアルデヒドと記載する場合がある）に加水分解する酵素を含む酵素であれば良いがこれに限定されない。
In the method for quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention including the above steps (1) to (3-4), when an enzyme that hydrolyzes lyPlsEtn is used in step (2), The degrading enzyme may be an enzyme including an enzyme that hydrolyzes lyPlsEtn to glycero-3-phosphoethanolamine and a corresponding aldehyde (may be simply referred to as aldehyde), but is not limited thereto.
 
 
また、工程（２）でｌｙＰｌｓＥｔｎを加水分解する酵素を用いる場合、該加水分解酵素は、少なくともエーテル型リゾホスファチジルエタノールアミンを加水分解しない酵素であればさらに好ましい。後述のＰｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ由来、ラット由来、及びＴｈｅｒｍｏｃｒｉｓｐｕｍ　ｓｐ．由来のｌｙＰｌｓＥｔｎのｌｙＰｌｓＥｔｎをグリセロ－３－ホスフォエタノールアミンとアルデヒドに加水分解する酵素はエーテル型リゾホスファチジルエタノールアミンを加水分解しない。

In addition, when an enzyme that hydrolyzes lyPlsEtn is used in step (2), it is more preferable that the hydrolase is an enzyme that does not hydrolyze at least ether type lysophosphatidylethanolamine. Pseudomonas putida-derived, rat-derived, and Thermocrispum sp. The enzyme that hydrolyzes lyPlsEtn of the derived lyPlsEtn into glycero-3-phosphoethanolamine and aldehyde does not hydrolyze ether type lysophosphatidylethanolamine.
 
上記工程（１）～（３－４）を含む本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する方法において、工程（２）で用いる加水分解酵素には、ＥＣ　３．３．２．２またはＥＣ　３．３．２．５と分類されるｌｙＰｌｓ　ａｓｅを含む。この様な酵素の例としてはＴＨＥ　ＪＯＵＲＮＡＬ　ＯＦ　ＢＩＯＬＯＧＩＣＡＬ　ＣＨＥＭＩＳＴＲＹ　ＶＯＬ．２８６，ＮＯ．２８，ｐｐ．２４９１６－２４９３０で開示されたラット由来のｌｙＰｌｓ　ａｓｅが挙げられ、好ましくは後述する本発明のｌｙＰｌｓ　ａｓｅである。
In the method for quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention including the above steps (1) to (3-4), the hydrolase used in step (2) is EC 3.3. Includes lyPls as classified as 2.2 or EC 3.3.2.5. Examples of such enzymes include THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 28, pp. Examples include rat-derived lyPlsase disclosed in 24916-24930, and lyPlsase of the present invention described later is preferable.
 
上記工程（１）～（３－４）を含む本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する方法において、工程（３－１）では、グリセロホスホコリンホスホジエステラーゼ（ｇｌｙｃｅｒｏｐｈｏｓｐｈｏｒｙｌｃｈｏｌｉｎｅ　ｐｈｏｓｐｈｏｄｉｅｓｔｅｒａｓｅ、ＧＰＣＰと略す場合がある）を利用することができる。該ＧＰＣＰはＥＣ　３．１．４．２と分類される酵素を含み、グリセロ－３－ホスフォエタノールアミンをエタノールアミンとグリセロリン酸加水分解する作用を触媒する酵素であれば限定されない。ＧＰＣＰの好適な例としてはＧｌｉｃｌａｄｉｕｍ　ｒｏｓｅｕｍ由来ＧＰＣＰ（旭化成ファーマ株式会社（Ｔ－３３））が挙げられる。
In the method for quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention including the above steps (1) to (3-4), in step (3-1), glycerophosphocholine phosphodiesterase, GPCP May be abbreviated). The GPCP includes an enzyme classified as EC 3.1.4.2, and is not limited as long as it is an enzyme that catalyzes the action of hydrolyzing glycero-3-phosphoethanolamine with ethanolamine and glycerophosphate. A preferred example of GPCP is GPCP derived from Glicadium roseum (Asahi Kasei Pharma Corporation (T-33)).
 
上記工程（１）～（３－４）を含む本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する方法において、工程（３－２）ではエタノールアミンオキシダーゼを利用することができる。該エタノールアミンオキシダーゼは、モノエタノールアミンオキシダーゼ（ＥＣ　１．４．３．８）、モノアミンオキシダーゼ（ＥＣ　１．４．３．４）、及び一級アミンオキシダーゼ（ＥＣ　１．４．３．２１）と分類される酵素を含み、エタノールアミンをＨ２Ｏ２、ＮＨ３とグリコールアルデヒドに酸化する作用を触媒する酵素であれば限定されない。エタノールアミンオキシダーゼの好適な例としてはＡｒｔｈｒｏｂａｃｔｅｒ属由来の酵素（Ｎａｒｒｏｄ　Ｓ．Ａ．及びＪａｋｏｂｙ　Ｗ．Ｂ．、Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．、２３９巻、２１８９－２１９３頁、１９６４年）、Ｐｈｏｒｍｉａ　ｒｅｇｉｎａ由来の酵素（Ｋｕｌｋａｒｎｉ　Ａ．Ｐ．及びＨｏｄｇｓｏｎ　Ｅ．、Ｃｏｍｐ．Ｂｉｏｃｈｅｍ．Ｐｈｙｓｉｏｌ．、Ｂ４４巻、４０７－４２２頁、１９７３年）、Ａｒｔｈｒｏｂａｃｔｅｒ　ｓｐ．（ＦＥＲＭ　Ｐ－０６２４０及びＢＰ－０４２１）由来の一級アミンオキシダーゼ（Ｏｔａ　Ｈ．ら、Ｂｉｏｓｃｉ．Ｂｉｏｔｅｃｈｎｏｌ．Ｂｉｏｃｈｅｍ．、７２巻、２７３２－２７３８頁、２００８年）が挙げられ、エタノールアミンに対する基質特異性が高いという観点からは特開２００５－５２０３４号公報で開示された分析方法に用いられるモノアミンオキシダーゼ（チラミンオキシダーゼ）が特に好ましい。
上記工程（１）～（３－４）を含む本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する方法は、前記（２）の加水分解酵素が、リゾホスファチジルエタノールアミンを加水分解できる酵素である場合、上記（１）～（３－４）の工程に先立つか、少なくとも前記（２）工程の前までに、下記（４）及び（５）を含む工程を行う方法であれば好ましいがこれに限定されない。工程（４）及び（５）は図９に概略して示した。
（４）ホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する酵素を用いて、血清または血漿中に混在するホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する工程：及び
（５）リゾホスフォリパーゼ及び／又はモノグリセリドリパーゼを用いて、前記（４）工程により生成されるリゾホスファチジルエタノールアミンを分解し、実質的に消去する工程。
In the method for quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention including the above steps (1) to (3-4), ethanolamine oxidase can be used in step (3-2). The ethanolamine oxidase is classified as monoethanolamine oxidase (EC 1.4.3.8), monoamine oxidase (EC 1.4.3.4), and primary amine oxidase (EC 1.4.3.21). The enzyme is not limited as long as it is an enzyme that catalyzes the action of oxidizing ethanolamine to H 2 O 2 , NH 3 and glycol aldehyde. As a suitable example of ethanolamine oxidase, an enzyme derived from Arthrobacter genus (Narrod SA and Jakoby WB, J. Biol. Chem., 239, 2189-2193, 1964), derived from Pharmacia regina Enzyme (Kulkarni AP and Hodgson E., Comp. Biochem. Physiol., B44, 407-422, 1973), Arthrobacter sp. (FERM P-06240 and BP-0421) derived from primary amine oxidase (Ota H. et al., Biosci. Biotechnol. Biochem., 72, 2732-2738, 2008), which has substrate specificity for ethanolamine. From the viewpoint of high, monoamine oxidase (tyramine oxidase) used in the analysis method disclosed in JP-A-2005-52034 is particularly preferable.
In the method for quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention including the above steps (1) to (3-4), the hydrolase of (2) described above hydrolyzes lysophosphatidylethanolamine. In the case of an enzyme that can be used, any method can be used that includes the steps (4) and (5) below prior to the steps (1) to (3-4) or at least before the step (2). Although preferable, it is not limited to this. Steps (4) and (5) are shown schematically in FIG.
(4) Step of decomposing phosphatidylethanolamine mixed in serum or plasma into lysophosphatidylethanolamine and fatty acid using an enzyme that decomposes phosphatidylethanolamine into lysophosphatidylethanolamine and fatty acid: and (5) lysophospho A step of decomposing and substantially eliminating lysophosphatidylethanolamine produced by the step (4) using lipase and / or monoglyceride lipase.
 
ここで「前記（２）の加水分解酵素が、リゾホスファチジルエタノールアミンを加水分解できる酵素である場合」とは本実施の形態のＰｌｓＥｔｎの定量値において、被験者を認知症患者またはその前段階にある者か否かを分類する目的に影響を与える程度にリゾホスファチジルエタノールアミンを加水分解することを意味する。例えば、リゾホスファチジルエタノールアミンを加水分解する程度がｌｙＰｌｓＥｔｎに対して１５％以下、更には１２％以下であり、１０％以下の場合があるがこれに限定しない。
Here, “when the hydrolase of (2) is an enzyme capable of hydrolyzing lysophosphatidylethanolamine” means that the subject is in a dementia patient or in the previous stage in the quantitative value of PlsEtn of this embodiment. This means that lysophosphatidylethanolamine is hydrolyzed to an extent that affects the purpose of classifying it as a person. For example, the degree of hydrolysis of lysophosphatidylethanolamine is 15% or less, further 12% or less, and may be 10% or less with respect to lyPlsEtn.
 
例えば、後述のＰｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ由来とラット由来のｌｙＰｌｓＥｔｎをグリセロ－３－ホスフォエタノールアミンとアルデヒドに加水分解する酵素はリゾホスファチジルエタノールアミンを加水分解しない酵素である。また、後述のＴｈｅｒｍｏｃｒｉｓｐｕｍ　ｓｐ．由来のｌｙＰｌｓＥｔｎをグリセロ－３－ホスフォエタノールアミンとアルデヒドに加水分解する酵素は、リゾホスファチジルエタノールアミンをｌｙＰｌｓＥｔｎに対して１２％加水分解するので、リゾホスファチジルエタノールアミンを加水分解する酵素である。
For example, an enzyme that hydrolyzes lyPlsEtn derived from Pseudomonas putida and rat described later to glycero-3-phosphoethanolamine and an aldehyde is an enzyme that does not hydrolyze lysophosphatidylethanolamine. In addition, Thermocrispum sp. The enzyme that hydrolyzes derived lyPlsEtn into glycero-3-phosphoethanolamine and aldehyde hydrolyzes lysophosphatidylethanolamine because it hydrolyzes lysophosphatidylethanolamine to lyPlsEtn by 12%.
 
　上記工程（１）～（３－４）を含む本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する方法は、前記工程（４）のホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する酵素としてＰＬを使用し、該ＰＬは、前記工程（１）のＰｌｓＥｔｎをｌｙＰｌｓＥｔｎに加水分解できる酵素としての作用を兼ねさせて新たな該ＰＬを添加することなく前記工程（１）の工程を進行せしめ、前記工程（５）を実施と同時、または前記工程（５）の実施後に、前記工程（１）が完了し、以後前記工程（２）～（３－４）を実施する方法でも良い。
The method for quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention including the steps (1) to (3-4) described above comprises converting the phosphatidylethanolamine of the step (4) into lysophosphatidylethanolamine and a fatty acid. PL is used as an enzyme for decomposing, and the PL serves as an enzyme capable of hydrolyzing PlsEtn in the step (1) into lyPlsEtn, and the step of the step (1) without adding new PL. In the method in which the step (1) is completed simultaneously with the implementation of the step (5) or after the implementation of the step (5), and then the steps (2) to (3-4) are carried out. good.
 
上記工程（４）及び（５）は、被験者の血清または血漿中にホスファチジルエタノールアミン及び／又はリゾホスファチジルエタノールアミンが含まれない、または、前記工程（２）のｌｙＰｌｓＥｔｎをグリセロ－３－ホスフォエタノールアミンとアルデヒドに加水分解する酵素がリゾホスファチジルエタノールアミンを加水分解しない場合には、実施しなくても良い場合があるが、被験者の血清または血漿中にホスファチジルエタノールアミン及び／又はリゾホスファチジルエタノールアミンが含まれ、かつ、前記工程（２）のｌｙＰｌｓＥｔｎをグリセロ－３－ホスフォエタノールアミンとアルデヒドに加水分解する酵素がリゾホスファチジルエタノールアミンを加水分解する作用を有する場合には、実施することが好ましい。
In the above steps (4) and (5), the serum or plasma of the subject does not contain phosphatidylethanolamine and / or lysophosphatidylethanolamine, or lyPlsEtn in the above step (2) is glycero-3-phosphoethanol. If the enzyme that hydrolyzes amines and aldehydes does not hydrolyze lysophosphatidylethanolamine, it may not be performed, but phosphatidylethanolamine and / or lysophosphatidylethanolamine may be present in the serum or plasma of the subject. It is preferably carried out if it is contained and the enzyme that hydrolyzes lyPlsEtn in the step (2) into glycero-3-phosphoethanolamine and aldehyde has the action of hydrolyzing lysophosphatidylethanolamine.
 
「実質的に消去する」の「消去する」とは消去対象を（例えば酵素の作用により）本実施の形態のＰｌｓＥｔｎの定量に関与しない物質へ分解（変換）することをいう。定量に関与しない物質は、用いる酵素の反応性や試薬等によっても異なるが、例えば上記工程（１）～（３－４）を含む本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する方法の場合、測定に関与しない物質の例としては、グリセロホスファチジン酸、グリコールアルデヒド、アンモニア、水、酸素などが挙げられる。例えば比色分析を用いた定量によって本実施の形態のＰｌｓＥｔｎの定量を行う場合には、消去対象を無色の物質に変換し、検出の対象外とすることも上記の「消去する」に含まれる。
“Erase” in “substantially erase” means that the object to be erased (for example, by the action of an enzyme) is decomposed (converted) into a substance that is not involved in the determination of PlsEtn in the present embodiment. Substances not involved in quantification vary depending on the reactivity of the enzyme used, reagents, etc., but for example, the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention including the above steps (1) to (3-4) is quantified. In the case of this method, examples of the substance not involved in the measurement include glycerophosphatidic acid, glycol aldehyde, ammonia, water, oxygen and the like. For example, when the PlsEtn quantification according to the present embodiment is performed by quantification using colorimetric analysis, the above-mentioned “erasing” includes converting the erasure target into a colorless substance and excluding the detection target. .
 
「実質的に消去する」とは、消去対象を、本実施の形態のＰｌｓＥｔｎの定量値において、被験者を認知症患者またはその前段階にある者か否かを分類する目的に影響を与えない程度に消去することを意味する。例えば、本実施の形態の測定方法を、後述する認知症検査方法として用いる場合、消去対象が、検査方法の正確性に影響を与えない程度まで消去されることを意味する。より具体的には、疾患によっても変動し得るが、消去対象が例えばホスファチジルエタノールアミン及び／又はリゾホスファチジルエタノールアミンの場合、これらを消去する工程を行った後のホスファチジルエタノールアミンの総量が、消去する工程を行う前の１０％以下となることが好ましく、更に好ましくは９％以下であり、最も好ましくは８％以下である。なお、上記の工程（４）及び（５）ではＰｌｓＥｔｎを含むエーテル型のエタノールアミン型リン脂質は消去しない。
“Substantially eliminate” means that the object to be erased does not affect the purpose of classifying whether the subject is a dementia patient or a person in the previous stage in the quantitative value of PlsEtn of the present embodiment. Means to erase. For example, when the measurement method of the present embodiment is used as a dementia inspection method described later, this means that the erasure target is erased to an extent that does not affect the accuracy of the inspection method. More specifically, although it may vary depending on the disease, for example, when the erasure target is phosphatidylethanolamine and / or lysophosphatidylethanolamine, the total amount of phosphatidylethanolamine after the erasure process is eliminated. It is preferably 10% or less before performing the step, more preferably 9% or less, and most preferably 8% or less. In the steps (4) and (5), the ether type ethanolamine phospholipid containing PlsEtn is not eliminated.
 
上記工程（４）で用いるＰＬは、上述の工程（１）の場合と同様であるが、上記の工程（４）及び（５）の「実質的に消去する」という観点からはＰＬＡ２が好ましく、反応性が早いという観点からＳｔｒｅｐｔｏｍｙｃｅｓ　ｖｉｏｌａｃｅｏｒｕｂｅｒ由来ＰＬＡ２がさらに好ましい。
The PL used in the above step (4) is the same as that in the above step (1), but PLA 2 is preferable from the viewpoint of “substantially erasing” in the above steps (4) and (5). From the viewpoint of quick reactivity, Streptomyces violaceoruber-derived PLA 2 is more preferable.
 
上記工程（５）で用いる酵素はリゾホスファチジルエタノールアミンをグリセロ－３－ホスフォエタノールアミンと脂肪酸に加水分解する作用があれば限定されないが、リゾホスフォリパーゼ（ｌｙｓｏｐｈｏｓｐｈｏｌｉｐａｓｅ、ＬＹＰＬと略す場合がある）、及び／又はモノグリセリドリパーゼ（ｍｏｎｏｇｌｙｃｅｒｉｄｅ　ｌｐａｓｅ、ＭＧＬＰと略す場合がある）が好ましい。該ＬＹＰＬはＥＣ　３．１．１．５と分類される酵素を含み、好適な例としてはＶｉｂｒｉｏ属由来ＬＹＰＬ（旭化成ファーマ株式会社（Ｔ－３２））が挙げられる。該ＭＧＬＰはＥＣ　３．１．１．２３と分類される酵素を含み、好適な例としてはＢａｃｉｌｌｕｓ属由来ＭＧＬＰ（旭化成ファーマ株式会社（Ｔ－５９））が挙げられる。
The enzyme used in the above step (5) is not limited as long as it has an action of hydrolyzing lysophosphatidylethanolamine into glycero-3-phosphoethanolamine and a fatty acid, but may be abbreviated as lysophospholipase (LYPL). And / or monoglyceride lipase (may be abbreviated as MGLP). The LYPL contains an enzyme classified as EC 3.1.1.5, and a preferred example is Vibrio genus-derived LYPL (Asahi Kasei Pharma Corporation (T-32)). The MGLP contains an enzyme classified as EC 3.1.1.23, and preferable examples include Bacillus genus MGLP (Asahi Kasei Pharma Corporation (T-59)).
 
本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する工程が、酵素を利用した定量である場合、過酸化水素の定量は、ペルオキシダーゼ（ｐｅｒｏｘｉｄａｓｅ、ＥＣ　１．１１．１．７）の作用を利用して、例えばトリンダー試薬等の色原体とカップラーとの酸化縮合により色素を生成させて比色分析により行うことができる。トリンダー型試薬の色原体としては、フェノール誘導体、アニリン誘導体、トルイジン誘導体等が使用可能であり、具体例としてＮ，Ｎ－ジメチルアニリン、Ｎ，Ｎ－ジエチルアニリン、２，４－ジクロロフェノール、Ｎ－エチル－Ｎ－（２－ヒドロキシ－３－スルホプロピル）－３，５－ジメトキシアニリン（ＤＡＯＳ）、Ｎ－エチル－Ｎ－スルホプロピル－３，５－ジメチルアニリン（ＭＡＰＳ）、Ｎ－エチル－Ｎ－（２－ヒドロキシ－３－スルホプロピル）－３，５－ジメチルアニリン（ＭＡＯＳ）、Ｎ－エチル－Ｎ－（２－ヒドロキシ－３－スルホプロピル）－ｍ－トルイジン（ＴＯＯＳ）、Ｎ－エチル－Ｎ－スルホプロピル－ｍ－アニシジン（ＡＤＰＳ）、Ｎ－エチル－Ｎ－スルホプロピルアニリン（ＡＬＰＳ）、Ｎ－エチル－Ｎ－スルホプロピル－３，５－ジメトキシアニリン（ＤＡＰＳ）、Ｎ－スルホプロピル－３，５－ジメトキシアニリン（ＨＤＡＰＳ）、Ｎ－エチル－Ｎ－スルホプロピル－ｍ－トルイジン（ＴＯＰＳ）、Ｎ－エチル－Ｎ－（２－ヒドロキシ－３－スルホプロピル）－３－ｍ－アニシジン（ＡＤＯＳ）、Ｎ－エチル－Ｎ－（２－ヒドロキシ－３－スルホプロピル）アニリン（ＡＬＯＳ）、Ｎ－（２－ヒドロキシ－３－スルホプロピル）－３，５－ジメトキシアニリン（ＨＤＡＯＳ）、Ｎ－スルホプロピル－アニリン（ＨＡＬＰＳ）、Ｎ，Ｎ－ビス－（４－スルホブチル）－３－メチルアニリン（ＴＯＤＢ）、Ｎ－エチル－Ｎ－（２－サクシニルアミノエチル）－ｍ－トルイジン（ＥＳＥＴ）等（以上株式会社同人化学研究所）が挙げられる。なかでもＴＯＯＳとＴＯＤＢは吸光係数が大きいため好ましい。また過酸化水素はＰＯＤの存在下、ロイコ型試薬を用いても発色させることができる。この試薬の具体例としては、Ｏ－ジアニシジン、Ｏ－トリジン、３，３－ジアミノベンジジン、３，３，５，５－テトラメチルベンジジン（以上株式会社同人化学研究所）、Ｎ－（カルボキシメチルアミノカルボニル）－４，４－ビス（ジメチルアミノ）ビフェニルアミン（ＤＡ６４）、１０－（カルボキシメチルアミノカルボニル）－３，７－ビス（ジメチルアミノ）フェノチアジン（ＤＡ６７）等が挙げられる。ＤＡ－６４やＤＡ－６７など、不安定な試薬を用いる場合、公知の方法により、亜硫酸チオ硫酸を添加したり、紫外線や可視光線を吸収する効果のある色素を共存させたりすることで安定化させることができる。カップラーの例としては、４－アミノアンチピリン（４－ＡＡ）、３－メチル－２－ベンゾチアゾリノンヒドラゾン（ＭＢＴＨ）が挙げられる。
When the step of quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention is a quantification using an enzyme, the quantification of hydrogen peroxide is performed according to peroxidase (EC 1.11.1.1.7). By utilizing the action, for example, a dye can be produced by oxidative condensation between a chromogen such as a Trinder reagent and a coupler, and colorimetric analysis can be performed. As the chromogen of the Trinder type reagent, phenol derivatives, aniline derivatives, toluidine derivatives and the like can be used. Specific examples include N, N-dimethylaniline, N, N-diethylaniline, 2,4-dichlorophenol, N -Ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (DAOS), N-ethyl-N-sulfopropyl-3,5-dimethylaniline (MAPS), N-ethyl-N -(2-hydroxy-3-sulfopropyl) -3,5-dimethylaniline (MAOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine (TOOS), N-ethyl- N-sulfopropyl-m-anisidine (ADPS), N-ethyl-N-sulfopropylaniline (ALPS), N-ethyl-N Sulfopropyl-3,5-dimethoxyaniline (DAPS), N-sulfopropyl-3,5-dimethoxyaniline (HDAPS), N-ethyl-N-sulfopropyl-m-toluidine (TOPS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-m-anisidine (ADOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) aniline (ALOS), N- (2-hydroxy-3- Sulfopropyl) -3,5-dimethoxyaniline (HDAOS), N-sulfopropyl-aniline (HALPS), N, N-bis- (4-sulfobutyl) -3-methylaniline (TODB), N-ethyl-N- (2-succinylaminoethyl) -m-toluidine (ESET) and the like (above Doujin Chemical Laboratory). Of these, TOOS and TODB are preferable because of their large extinction coefficient. Hydrogen peroxide can also be colored by using a leuco reagent in the presence of POD. Specific examples of this reagent include O-dianisidine, O-tolidine, 3,3-diaminobenzidine, 3,3,5,5-tetramethylbenzidine (above, Doujin Chemical Laboratory Co., Ltd.), N- (carboxymethylamino) And carbonyl) -4,4-bis (dimethylamino) biphenylamine (DA64), 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine (DA67), and the like. When unstable reagents such as DA-64 and DA-67 are used, stabilization is achieved by adding thiosulfurous acid sulfuric acid or coexisting with a dye that has the effect of absorbing ultraviolet rays or visible light by known methods. Can be made. Examples of couplers include 4-aminoantipyrine (4-AA) and 3-methyl-2-benzothiazolinone hydrazone (MBTH).
 
本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する工程が、酵素を利用した定量である場合、過酸化水素の定量は、上記の方法のほか、吸光法、ＫＭｎＯ４等を利用する酸化還元法、蛍光法、発光法又は電極法等の公知の方法を用いて行うことができる。蛍光法には、酸化によって蛍光を発する化合物、例えばホモバニリン酸、４－ヒドロキシフェニル酢酸、チラミン、パラクレゾール、ジアセチルフルオレスシン誘導体等を用いることができる。発光法には、触媒としてルミノール、ルシゲニン、イソルミノール、ピロガロール等を用いることが出来る。電極法に用いる電極は、過酸化水素との間で電子を授受することの出来る材料である限り特に制限されないが、例えば白金、金、銀等が挙げられ、電極測定方法としては、アンペロメトリー、ポテンショメトリー、クーロメトリー等の公知の方法を用いることが出来る。更にオキシダーゼ又は基質と電極との間の反応に電子伝達体を介在させ、得られる酸化、還元電流又はその電気量を測定してもよい。電子伝達体としては、電子伝達機能を有する任意の物質が使用可能であり、例えばフェロセン誘導体、キノン誘導体等の物質が挙げられる。またオキシダーゼ反応により生成する過酸化水素と電極の間に電子伝達体を介在させ、得られる酸化、還元電流又はその電気量を測定してもよい。
When the step of quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention is quantification using an enzyme, the quantification of hydrogen peroxide uses an absorption method, KMnO 4 or the like in addition to the above method. The oxidation / reduction method, the fluorescence method, the luminescence method, or the electrode method can be used. In the fluorescence method, a compound that emits fluorescence by oxidation, such as homovanillic acid, 4-hydroxyphenylacetic acid, tyramine, paracresol, a diacetylfluorescin derivative, or the like can be used. In the luminescence method, luminol, lucigenin, isoluminol, pyrogallol, or the like can be used as a catalyst. The electrode used for the electrode method is not particularly limited as long as it is a material that can exchange electrons with hydrogen peroxide. Examples thereof include platinum, gold, silver, and the like. , Known methods such as potentiometry and coulometry can be used. Further, an electron carrier may be interposed in the reaction between the oxidase or substrate and the electrode, and the resulting oxidation, reduction current, or electric quantity thereof may be measured. As the electron carrier, any substance having an electron transfer function can be used, and examples thereof include substances such as ferrocene derivatives and quinone derivatives. Further, an electron carrier may be interposed between hydrogen peroxide generated by the oxidase reaction and the electrode, and the resulting oxidation, reduction current, or electric quantity thereof may be measured.
 
本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する工程は、以下の（１）～（３－３）´の工程のいずれかまたは複数を含んでもよく、さらに（１）～（３－３）´の全てを含んでもよい。
（１）エタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解でき、かつリゾホスファチジルエタノールアミンを加水分解できる酵素を用いて、被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解する工程：
（２）´エタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）をエタノールアミンとプラズメニルホスファチジン酸に加水分解でき、かつエーテル型リゾホスファチジルエタノールアミンを加水分解できる加水分解酵素を用いて、前記（１）にて生成されたエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）をエタノールアミンとプラズメニルホスファチジン酸に加水分解する工程： 
（３－１）´エタノールアミンを酸化する酵素により、工程（２）´で得られたエタノールアミンから過酸化水素を発生せしめる工程：
（３－２）´過酸化水素を過酸化水素定量手段により定量する工程：及び
（３－３）´前記被験者の血清または血漿中のＰｌｓＥｔｎ量を算出する工程。
The step of quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention may include one or more of the following steps (1) to (3-3) ′, and further includes (1) to ( 3-3) all of 'may be included.
(1) Ethanolamine type in serum or plasma of a subject using an enzyme capable of hydrolyzing ethanolamine type plasmalogen (PlsEtn) to ethanolamine type lysoplasmalogen (lyPlsEtn) and hydrolyzing lysophosphatidylethanolamine Hydrolyzing plasmalogen (PlsEtn) to ethanolamine-type lysoplasmalogen (lyPlsEtn):
(2) In the above (1), using a hydrolase capable of hydrolyzing ethanolamine-type lysoplasmalogen (lyPlsEtn) into ethanolamine and plasmenylphosphatidic acid and hydrolyzing ether-type lysophosphatidylethanolamine The step of hydrolyzing the produced ethanolamine-type lysoplasmalogen (lyPlsEtn) to ethanolamine and plasmenyl phosphatidic acid:
(3-1) Step of generating hydrogen peroxide from the ethanolamine obtained in step (2) ′ by an enzyme that oxidizes “ethanolamine”:
(3-2) 'Step of quantifying hydrogen peroxide by means of hydrogen peroxide determination: and (3-3)' Step of calculating the amount of PlsEtn in the serum or plasma of the subject.
 
上記工程（１）～（３－３）´を含む本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する方法の別異な一例を概略して図８に示した。工程（１）はＰＬの作用で、ＰｌｓＥｔｎをｌｙＰｌｓＥｔｎに加水分解する工程である。工程（２）´は加水分解酵素の作用で、ｌｙＰｌｓＥｔｎを加水分解する工程である。工程（３－１）´は工程（２）´で得られた生成物であるエタノールアミンをＥＡＯの作用で過酸化水素とする工程である。過酸化水素は後述する公知の方法で定量すれば良い。
FIG. 8 schematically shows another example of the method for quantifying the serum and / or plasma PlsEtn amount of the embodiment of the present invention including the steps (1) to (3-3) ′. Step (1) is a step of hydrolyzing PlsEtn to lyPlsEtn by the action of PL. Step (2) ′ is a step of hydrolyzing lyPlsEtn by the action of a hydrolase. Step (3-1) ′ is a step in which ethanolamine, which is the product obtained in step (2) ′, is converted into hydrogen peroxide by the action of EAO. What is necessary is just to quantify hydrogen peroxide by the well-known method mentioned later.
 
上記工程（１）～（３－３）´を含む本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する方法において、工程（１）で用いるＰＬは、上述の工程（１）の場合と同様である。
In the method for quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention including the steps (1) to (3-3) ′, the PL used in the step (1) is the same as that in the above step (1). Same as the case.
 
上記工程（１）～（３－３）´を含む本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する方法において、工程（２）´でｌｙＰｌｓＥｔｎを加水分解する酵素を用いる場合、該加水分解酵素はｌｙＰｌｓＥｔｎをプラズメニルホスファチジン酸とエタノールアミンに加水分解する酵素を含み、ホスホリパーゼＤ（ｐｈｏｓｐｈｏｌｉｐａｓｅ　Ｄ、ＰＬＤと略す場合がある）が好ましい。そのようなＰＬＤの例としてはＳｔｒｅｐｔｏｍｙｃｅｓ属由来（旭化成ファーマ株式会社（Ｔ－０７））、Ｓｔｒｅｐｔｏｍｙｃｅｓ　ｃｈｒｏｍｏｆｕｓｃｕｓ由来（旭化成ファーマ株式会社（Ｔ－３９））、キャベツ由来（ＳＩＧＭＡ（Ｐ８３９８））、Ａｃｔｉｎｏｍａｄｕｒａ属由来（名糖産業（株））ＰＬＤが挙げられる。ｌｙＰｌｓＥｔｎに対する基質特異性が高いという観点からは、Ｓｔｒｅｐｔｏｍｙｃｅｓ　ｃｈｒｏｍｏｆｕｓｃｕｓ由来ＰＬＤが好ましい。
In the method for quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention including the above steps (1) to (3-3) ′, when using an enzyme that hydrolyzes lyPlsEtn in step (2) ′, The hydrolase includes an enzyme that hydrolyzes lyPlsEtn into plasmenyl phosphatidic acid and ethanolamine, and phospholipase D (sometimes abbreviated as phospholipase D, PLD) is preferable. Examples of such PLD are derived from the genus Streptomyces (Asahi Kasei Pharma Co., Ltd. (T-07)), derived from Streptomyces chromofuscus (Asahi Kasei Pharma Co., Ltd. (T-39)), derived from cabbage (SIGMA (P8398)), and from the genus Actinomadura. (Meisei Sangyo Co., Ltd.) PLD. From the viewpoint of high substrate specificity for lyPlsEtn, Streptomyces chromofuscus-derived PLD is preferred.
 
上記工程（１）～（３－３）´
を含む本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する方法において、工程（３－１）´で用いるＥＡＯは、上述の工程（３－２）の場合と同様である。
Steps (1) to (3-3) ′ above
In the method for quantifying the amount of serum and / or plasma PlsEtn according to an embodiment of the present invention including EAO, EAO used in step (3-1) ′ is the same as in step (3-2) described above.
 
上記工程（１）～（３－３）´を含む本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する方法は、上記（１）～（３－３）´の工程に先立つか、少なくとも前記（２）´工程の前までに、上記の（４）及び（５）を含む工程を行う方法であれば好ましいがこれに限定されない。
Is the method for quantifying the amount of serum and / or plasma PlsEtn of the embodiment including the steps (1) to (3-3) ′ prior to the steps (1) to (3-3) ′ described above? Although it is preferable to use the method including the steps (4) and (5) at least before the step (2) ′, the method is not limited to this.
 
上記工程（１）～（３－３）´を含む本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する方法は、前記工程（４）のホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する酵素としてＰＬを使用し、該ＰＬは、前記工程（１）のＰｌｓＥｔｎをｌｙＰｌｓＥｔｎに加水分解できる酵素としての作用を兼ねさせて新たな該ＰＬを添加することなく前記工程（１）の工程を進行せしめ、前記工程（５）を実施と同時、または前記工程（５）の実施後に、前記工程（１）が完了し、以後前記工程（２）´～（３－３）´を実施する方法でも良い。
In the method for quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention including the above steps (1) to (3-3) ′, the phosphatidylethanolamine in the above step (4) is converted into lysophosphatidylethanolamine and a fatty acid. PL is used as an enzyme that decomposes into, and the PL functions as an enzyme capable of hydrolyzing PlsEtn in the step (1) to lyPlsEtn without adding new PL. The step (1) is completed simultaneously with the implementation of the step (5) or after the execution of the step (5), and then the steps (2) ′ to (3-3) ′ are performed. The method to do is also good.
 
上記工程（４）及び（５）は、被験者の血清または血漿中にホスファチジルエタノールアミン及び／又はリゾホスファチジルエタノールアミンが含まれない、または、前記工程（２）´のｌｙＰｌｓＥｔｎをエタノールアミンとプラズメニルホスファチジン酸に加水分解できる加水分解酵素がリゾホスファチジルエタノールアミンを加水分解しない場合には、実施しなくても良い場合があるが、被験者の血清または血漿中にホスファチジルエタノールアミン及び／又はリゾホスファチジルエタノールアミンが含まれ、かつ、前記工程（２）´のｌｙＰｌｓＥｔｎをエタノールアミンとプラズメニルホスファチジン酸に加水分解できる加水分解酵素がリゾホスファチジルエタノールアミンを加水分解する作用を有する場合には、実施することが好ましい。
In the above steps (4) and (5), the serum or plasma of the subject does not contain phosphatidylethanolamine and / or lysophosphatidylethanolamine, or the lyPlsEtn of the above step (2) ′ is changed to ethanolamine and plasmenyl phosphatidine. If a hydrolase that can hydrolyze to acid does not hydrolyze lysophosphatidylethanolamine, it may not be carried out, but phosphatidylethanolamine and / or lysophosphatidylethanolamine may be present in the serum or plasma of a subject. It is preferably carried out when the hydrolase that is included and can hydrolyze the lyPlsEtn of the step (2) ′ into ethanolamine and plasmenyl phosphatidic acid has an action of hydrolyzing lysophosphatidylethanolamine. Good.
 
ここで、「実質的に消去する」の「消去する」とは上述と同様であり、定量に関与しない物質は、用いる酵素の反応性や試薬等によっても異なるが、例えば本実施の形態のＰｌｓＥｔｎの測定を、ＰＬＤで分解し、得られたエタノールアミンをさらに酸化して得られる過酸化水素を用いて定量する方法により行う場合、測定に関与しない物質の例としては、グリセロホスホエタノールアミン（ただし、用いるＰＬＤがグリセロホスホエタノールアミンに作用しない場合）、モノアシルグリセロール、ジアシルグリセロール、ホスホリルエタノールアミン、脂肪酸、セラミド、グリセロール－３－リン酸、アンモニア、グリコールアルデヒド、水、酸素などが挙げられる。例えば比色分析を用いた定量によって本実施の形態のＰｌｓＥｔｎの定量を行う場合には、消去対象を無色の物質に変換し、検出の対象外とすることも上記の「消去する」に含まれる。
Here, “erase” of “substantially erase” is the same as described above, and substances that are not involved in quantification vary depending on the reactivity of the enzyme used, reagents, etc., but for example, PlsEtn of this embodiment In the case of measuring by the method of determining by using hydrogen peroxide obtained by further decomposing with PLD and further oxidizing the obtained ethanolamine, examples of substances not involved in the measurement include glycerophosphoethanolamine (however, , Monoacylglycerol, diacylglycerol, phosphorylethanolamine, fatty acid, ceramide, glycerol-3-phosphate, ammonia, glycolaldehyde, water, oxygen and the like. For example, when the PlsEtn quantification according to the present embodiment is performed by quantification using colorimetric analysis, the above-mentioned “erasing” includes converting the erasure target into a colorless substance and excluding the detection target. .
 
「実質的に消去する」とは、上述と同様であり、より具体的には、疾患によっても変動し得るが、消去対象が例えばホスファチジルエタノールアミン及び／又はリゾホスファチジルエタノールアミンの場合である場合、これらを消去する工程を行った後のホスファチジルエタノールアミン及び／又はリゾホスファチジルエタノールアミンの場合の総量が、消去する工程を行う前の１０％以下となることが好ましく、更に好ましくは９％以下であり、最も好ましくは８％以下である。
“Substantially erase” is the same as described above. More specifically, it may vary depending on the disease, but when the object to be erased is, for example, phosphatidylethanolamine and / or lysophosphatidylethanolamine, The total amount in the case of phosphatidylethanolamine and / or lysophosphatidylethanolamine after performing these erasing steps is preferably 10% or less, more preferably 9% or less before performing the erasing step. Most preferably, it is 8% or less.
 
本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量の酵素を利用した定量する工程は、特開２０１２－２１０１７９号に記載の方法でも良い。
The step of quantifying the serum and / or plasma PlsEtn amount using an enzyme according to the embodiment of the present invention may be a method described in JP2012-210179A.
 
本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する工程が、酵素を利用した定量で使用し得るいずれの酵素についても、所望の活性が維持される限り、その変異体も同様に各酵素の範囲に含まれる。例えば、各酵素のアミノ酸配列において１又は複数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列を有する酵素であっても、所望の活性が維持される限り、各酵素の範囲に含まれる。また、各酵素を塩基配列の翻訳によって得る場合にも、用いる塩基配列は、得られる酵素が所望の活性を有する限り特に限定されない。各酵素の二次構造、三次構造及び四次構造、性質、純度、由来、商品名並びに価格についても特に限定されない。
As long as the desired activity is maintained for any enzyme that can be used in the quantification using the enzyme in the step of quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention, its mutants are similarly used. Included in each enzyme range. For example, even an enzyme having an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of each enzyme is included in the range of each enzyme as long as the desired activity is maintained. Also, when each enzyme is obtained by translation of a base sequence, the base sequence to be used is not particularly limited as long as the obtained enzyme has a desired activity. The secondary structure, tertiary structure and quaternary structure, properties, purity, origin, trade name and price of each enzyme are not particularly limited.
 
本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する工程が、酵素を利用した定量である場合、一態様において、試料中に含まれるアスコルビン酸を消去するためにアスコルビン酸オキシダーゼ（ａｓｃｏｒｂａｔｅ　ｏｘｉｄａｓｅ、ＡＳＯＭと略す場合がある）を使用することができる。ＡＳＯＭは、アスコルビン酸を実質的に消去する作用を有するものが好ましく、ＥＣ　１．１０．３．３と分類される酵素がより好ましい。例えば、ウリ科植物由来のＡＳＯＭを使用することができる。好適な例としては、酵素の安定性が高いという観点からはＡｃｒｅｍｏｎｉｕｍ属由来のアスコルビン酸オキシダーゼ（旭化成ファーマ株式会社（Ｔ－５３））、アジ化ナトリウムによる阻害を受けないという観点からは天野エンザイム（株）社のアスコルビン酸オキシダーゼ（商品名ＡＳＯ－３）が挙げられる。
When the step of quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention is quantification using an enzyme, in one aspect, ascorbate oxidase (ascorbate) is used to eliminate ascorbate contained in the sample. oxidase and ASOM may be abbreviated). ASOM preferably has an action of substantially eliminating ascorbic acid, and more preferably an enzyme classified as EC 1.10.3.3. For example, an ASOM derived from a Cucurbitaceae plant can be used. Preferred examples are ascorbate oxidase (Asahi Kasei Pharma Co., Ltd. (T-53)) derived from the genus Acremonium from the viewpoint of high enzyme stability, and Amano enzyme from the viewpoint of not being inhibited by sodium azide. Ascorbic acid oxidase (trade name: ASO-3) manufactured by KK
 
本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する工程が、酵素を利用した定量である場合、その量は下記のカタラーゼの量を除き、例えば、被験者の血清または血漿中のＰｌｓＥｔｎの濃度が３００μＭ以下で、検体試薬比（試料の量：試薬の全量）が１：１０の場合、下限値が０．１Ｕ／ｍＬ以上、好ましくは１Ｕ／ｍＬ以上、更に好ましくは５Ｕ／ｍＬ以上であり、上限は特に設けないが、１００Ｕ／ｍＬ以下、好ましくは５０Ｕ／ｍＬ以下、更に好ましくは３０Ｕ／ｍＬ以下である。使用する酵素の量は、試薬の安定性という観点からは高い方が好ましく、経済性の観点からは低い方が好ましい。特にレートアッセイを行う場合には使用する酵素の量は低い方が好ましく、下限は０．０１Ｕ／ｍＬである。
When the step of quantifying the amount of serum and / or plasma PlsEtn according to an embodiment of the present invention is a quantification using an enzyme, the amount excludes the amount of catalase described below, for example, PlsEtn in the serum or plasma of a subject. When the sample concentration is 300 μM or less and the sample / reagent ratio (sample amount: total amount of reagent) is 1:10, the lower limit is 0.1 U / mL or more, preferably 1 U / mL or more, more preferably 5 U / mL or more. Although the upper limit is not particularly provided, it is 100 U / mL or less, preferably 50 U / mL or less, more preferably 30 U / mL or less. The amount of enzyme used is preferably higher from the viewpoint of reagent stability, and lower from the economical viewpoint. In particular, when performing a rate assay, the amount of enzyme used is preferably low, and the lower limit is 0.01 U / mL.
 
本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する工程が、酵素を利用した定量である場合、一態様において、過酸化水素を消去するためにカタラーゼ（ｃａｔａｌａｓｅ）を使用する場合がある。カタラーゼは、過酸化水素を実質的に消去する作用を有するものが好ましく、ＥＣ　１．１１．１．６と分類される酵素がより好ましいがこれに限定されない。カタラーゼの使用量は限定されないが、通常１０～５０００Ｕ／ｍＬの範囲で使用する。カタラーゼの好適な例としては、純度が高いという観点からはＡｒｔｈｒｏｂａｃｔｅｒ属由来（旭化成ファーマ株式会社）、安価で入手が容易である観点からは動物由来（ＳＩＧＭＡ（Ｃ１３４５等））のカタラーゼが挙げられる。
When the step of quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention is quantification using an enzyme, in one embodiment, catalase may be used to eliminate hydrogen peroxide. is there. The catalase preferably has an action of substantially eliminating hydrogen peroxide, more preferably an enzyme classified as EC 1.11.1.6, but is not limited thereto. Although the amount of catalase used is not limited, it is usually used in the range of 10 to 5000 U / mL. Preferable examples of catalase include catalase derived from Arthrobacter genus (Asahi Kasei Pharma Co., Ltd.) from the viewpoint of high purity and animal origin (SIGMA (C1345 etc.)) from the viewpoint of being inexpensive and easily available.
 
本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する工程が、酵素を利用した定量である場合、使用する酵素の反応性及び／又は安定性を維持及び／又は高めるという観点から、適宜ｐＨ緩衝剤を用いることが好ましい。ｐＨ緩衝剤は、目的のｐＨを保つことができれば限定されないが、グッドのｐＨ緩衝液（ＭＥＳ、Ｂｉｓ－Ｔｒｉｓ、ＡＤＡ、ＰＩＰＥＳ、ＡＣＥＳ、ＢＥＳ、ＭＯＰＳ、ＴＥＳ、ＨＥＰＥＳ、ＤＩＰＳＯ、ＴＡＰＳＯ、ＰＯＰＳＯ、ＨＥＰＰＳＯ、ＥＰＰＳ、Ｔｒｉｃｉｎｅ、Ｂｉｃｉｎｅ、ＴＡＰＳ、ＣＨＥＳ、ＣＡＰＳ等）、Ｔｒｉｓ緩衝液、ジエタノールアミン緩衝液、炭酸緩衝液、グリシン緩衝液、硼酸緩衝液、リン酸緩衝液、グリシルグリシン緩衝液、酢酸緩衝液、クエン酸緩衝液、コハク酸緩衝液、マレイン酸緩衝液、トリスエタノールアミン緩衝液、イミダゾール緩衝液等が例示できる。これらの緩衝液は、塩酸等の強酸やＮａＯＨ等の強アルカリを用いて、緩衝剤として使用可能なｐＨ範囲に調整して使用することができる。
When the step of quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention is quantification using an enzyme, from the viewpoint of maintaining and / or enhancing the reactivity and / or stability of the enzyme used. It is preferable to use a pH buffer as appropriate. The pH buffer is not limited as long as the target pH can be maintained, but Good's pH buffer (MES, Bis-Tris, ADA, PIPES, ACES, BES, MOPS, TES, HEPES, DIPSO, TAPSO, POPSO, HEPPSO , EPPS, Tricine, Bicine, TAPS, CHES, CAPS, etc.), Tris buffer, diethanolamine buffer, carbonate buffer, glycine buffer, borate buffer, phosphate buffer, glycylglycine buffer, acetate buffer, Examples include citrate buffer, succinate buffer, maleate buffer, trisethanolamine buffer, imidazole buffer, and the like. These buffers can be used by adjusting to a pH range that can be used as a buffer using a strong acid such as hydrochloric acid or a strong alkali such as NaOH.
 
ｐＨは、用いる酵素によっても異なるが、弱アルカリ性のｐＨであることが好ましく、下限値としてｐＨ７．０以上、好ましくはｐＨ７．３以上、更に好ましくはｐＨ７．５以上が例示され、上限値としてはｐＨ９．０以下、好ましくはｐＨ８．４以下、更に好ましくはｐＨ８．０以下が例示される。例えば、ｌｙＰｌｓ　ａｓｅ、ＰＬ、ＬＹＰＬ、ＧＰＣＰを安定化及び／又は活性化するという観点からは、そのｐＨは、下限値としてｐＨ７．０以上が例示され、上限値としてはｐＨ９．５以下、好ましくはｐＨ９以下、更に好ましくはｐＨ８．５以下が例示される。
Although the pH varies depending on the enzyme used, it is preferably a weakly alkaline pH, and the lower limit is exemplified by pH 7.0 or higher, preferably pH 7.3 or higher, more preferably pH 7.5 or higher, and the upper limit is exemplified. The pH is 9.0 or less, preferably pH 8.4 or less, more preferably pH 8.0 or less. For example, from the viewpoint of stabilizing and / or activating lyPlsase, PL, LYPL, GPCP, the pH is exemplified by pH 7.0 or more as the lower limit, and pH 9.5 or less, preferably as the upper limit The pH is 9 or less, more preferably 8.5 or less.
 
ｐＨ緩衝剤の濃度は目的のｐＨを保つことができる限り特に限定されないが、下限値として３ｍＭ以上、好ましくは５ｍＭ以上、更に好ましくは１０ｍＭ以上が例示され、上限値としては５００ｍＭ以下、好ましくは２００ｍＭ以下、更に好ましくは１００ｍＭ以下が例示される。
The concentration of the pH buffer is not particularly limited as long as the target pH can be maintained, but the lower limit is 3 mM or more, preferably 5 mM or more, more preferably 10 mM or more, and the upper limit is 500 mM or less, preferably 200 mM. Hereinafter, more preferably 100 mM or less.
 
本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する工程が、酵素を利用した定量である場合、該工程には界面活性剤を存在させられる。界面活性剤の種類は限定されないが、ポリオキシエチレンアルキルエーテル類、ポリオキシエチレンソルビタン脂肪酸エステル類、ポリオキシエチレングリセリン脂肪酸エステル類、ポリオキシエチレンソルビット脂肪酸エステル類、ポリオキシエチレンアルキルフェニルホルムアルデヒド縮合物、ポリオキシエチレンヒマシ油、ポリオキシエチレンステロール類、ポリオキシエチレンポリオキシプロピレンアルキルエーテル類、ポリオキシエチレンラノリン類、ポリオキシエチレンアルキルアミン・脂肪酸アミド類、ポリオキシエチレンアルキルエーテルリン酸・リン酸塩類、ポリオキシエチレンアルキルエーテル硫酸塩類、ポリグリセリン脂肪酸エステル類、グリセリン脂肪酸エステル類、プロピレングリコール脂肪酸エステル類、ソルビタン脂肪酸エステル類、Ｎアシルアミノ酸塩類、アルキルエーテルカルボン酸塩類、アルキルリン酸塩、Ｎアシルタウリン酸塩、スルホン酸塩、アルキル硫酸、酢酸ベタイン型両性界面活性剤、イミダゾリン型両性界面活性剤、レシチン誘導体、ポリエチレングリコール類、ポリエチレングリコールラウリルエーテル、ポリエチレングリコールイソオクチルフェニルエーテル、ポリプロピレングリコール、ポリビニルアルコール等が例示される。好ましくは、非イオン性界面活性剤のエーテル型又はエステル型が挙げられ、更に好ましい例としては、ＨＬＢ値が１３以上１５以下のポリオキシエチレン誘導体、ポリオキシエチレンアルキルエーテル、及びポリオキシエチレンアルキルアミンからなる群から選択される非イオン界面活性剤が挙げられ、更に好ましくは、ポリオキシエチレンラウリル硫酸ナトリウム、ポリオキシエチレンアルキル（１２－１４）エーテル、ポリオキシエチレンオクチルフェニルエーテル、ポリオキシエチレンノニルフェニルエーテルが挙げられる。界面活性剤の溶解性の観点からは、ポリオキシエチレンアルキルフェニルエーテル系非イオン性界面活性剤が好ましい。商品名としてはトリトンＸ－１００（ＴＸ－１００）、Ｔｗｅｅｎ２０、エマール２０Ｃ（花王株式会社）、ＮＩＫＫＯＬ　ＢＴ－９（日光ケミカルズ株式会社）、ノニオンＨＳ－２０８、ＨＳ－２１０、ＨＳ－２０８、ＨＳ－２０８．５、ＮＳ２１０（日油株式会社）等が例示される。また、膜酵素安定化の観点からはｎ－Ｄｏｄｅｃｙｌ－β－Ｄ－ｍａｌｔｏｓｉｄｅ（ＤＤＭ）、ｎ－Ｏｃｔｙｌ－β－Ｄ－ｇｌｕｃｏｓｉｄｅ、ｎ－Ｎｏｎｙｌ－β－Ｄ－ｔｈｉｏｍａｌｔｏｓｉｄｅ、ｎ－Ｏｃｔｙｌ－β－Ｄ－ｔｈｉｏｇｌｕｃｏｓｉｄｅ、３－Ｏｘａｔｒｉｄｅｃｙｌ－α－Ｄ－ｍａｎｎｏｓｉｄｅのような親水部に糖鎖を持った非イオン性界面活性剤を使用する事もできる。界面活性剤は複数種混合して使用してもよい。
When the step of quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention is quantification using an enzyme, a surfactant can be present in the step. The type of surfactant is not limited, but polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene glycerin fatty acid esters, polyoxyethylene sorbit fatty acid esters, polyoxyethylene alkylphenyl formaldehyde condensates, Polyoxyethylene castor oil, polyoxyethylene sterols, polyoxyethylene polyoxypropylene alkyl ethers, polyoxyethylene lanolins, polyoxyethylene alkylamine / fatty acid amides, polyoxyethylene alkyl ether phosphates / phosphates, Polyoxyethylene alkyl ether sulfates, polyglycerol fatty acid esters, glycerol fatty acid esters, propylene glycol fatty acid esters, sol Tan fatty acid esters, N acylamino acid salts, alkyl ether carboxylates, alkyl phosphates, N acyl taurates, sulfonates, alkyl sulfates, betaine acetate amphoteric surfactants, imidazoline amphoteric surfactants, lecithin Examples include derivatives, polyethylene glycols, polyethylene glycol lauryl ether, polyethylene glycol isooctyl phenyl ether, polypropylene glycol, polyvinyl alcohol, and the like. Preferably, an ether type or an ester type of a nonionic surfactant is used, and more preferable examples include polyoxyethylene derivatives, polyoxyethylene alkyl ethers, and polyoxyethylene alkyl amines having an HLB value of 13 to 15. Nonionic surfactants selected from the group consisting of: polyoxyethylene sodium lauryl sulfate, polyoxyethylene alkyl (12-14) ether, polyoxyethylene octylphenyl ether, polyoxyethylene nonylphenyl Ether. From the viewpoint of the solubility of the surfactant, a polyoxyethylene alkylphenyl ether-based nonionic surfactant is preferable. Trade names include Triton X-100 (TX-100), Tween 20, Emar 20C (Kao Corporation), NIKKOL BT-9 (Nikko Chemicals Corporation), Nonion HS-208, HS-210, HS-208, HS- 208.5, NS210 (NOF Corporation), etc. are illustrated. From the viewpoint of membrane enzyme stabilization, n-Dodecyl-β-D-maltoside (DDM), n-Octyl-β-D-glucoside, n-Nonyl-β-D-thiomaltoside, n-Octyl-β-D Nonionic surfactants having a sugar chain in the hydrophilic portion such as -thioglucoside and 3-Oxatridecyl-α-D-mannoside can also be used. A plurality of surfactants may be used as a mixture.
 
極性をもつ界面活性剤は酵素などのタンパク質の変性作用を有することが知られているため、脂質を基質とする酵素を用いる場合、脂質を溶解するために中性（非極性）の界面活性剤存在下で実施することが好ましいがこれに限定されない。本実施の形態において、界面活性剤を用いる場合、その濃度は用いる酵素の所望の作用が得られる限り特に限定されない。例えば本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する工程が、酵素を利用した定量であり、さらに該酵素が本発明のｌｙＰｌｓ　ａｓｅ及び／またはＰＬである場合の界面活性剤の濃度は、上限値としては０．２５（ｗ／ｖ）％以下、好ましくは０．１（ｗ／ｖ）％以下、更に好ましくは０．０５（ｗ／ｖ）％以下が例示され、酵素の所望の作用が得られる限り、界面活性剤を用いなくてもよい。界面活性剤により酵素を安定化及び／又は活性化する、基質を分散させる、再現性等の酵素の作用を向上するという観点からは、通常、界面活性剤の濃度は、下限値としては０．００１（ｗ／ｖ）％以上、好ましくは０．００５（ｗ／ｖ）％以上、更に好ましくは０．０１（ｗ／ｖ）％以上が例示され、上限値としては１（ｗ／ｖ）％以下、好ましくは０．５（ｗ／ｖ）％以下が例示される。
Since polar surfactants are known to have a denaturation effect on proteins such as enzymes, neutral (nonpolar) surfactants are used to dissolve lipids when using lipid-based enzymes. However, the present invention is not limited to this. In the present embodiment, when a surfactant is used, its concentration is not particularly limited as long as the desired action of the enzyme used is obtained. For example, the step of quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention is quantification using an enzyme, and the surfactant is lyPls as and / or PL of the present invention. The upper limit of the concentration is 0.25 (w / v)% or less, preferably 0.1 (w / v)% or less, more preferably 0.05 (w / v)% or less. As long as the desired action is obtained, the surfactant may not be used. From the viewpoint of stabilizing and / or activating the enzyme with the surfactant, dispersing the substrate, and improving the action of the enzyme, such as reproducibility, the concentration of the surfactant is usually 0 as the lower limit. 001 (w / v)% or more, preferably 0.005 (w / v)% or more, more preferably 0.01 (w / v)% or more, and the upper limit is 1 (w / v)% Hereinafter, preferably 0.5 (w / v)% or less is exemplified.
 
本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する工程が、酵素を利用した定量である場合、該工程にはＮａＣｌ、ＫＣｌ、硫安やＮＨ３Ｃｌ等の塩を存在させられ、その量は、下限値として０．１ｍＭ以上、好ましくは５ｍＭ以上、更に好ましくは５０ｍＭ以上が例示され、上限値は特に制限されないが、好ましくは２００ｍＭ以下、更に好ましくは１５０ｍＭ以下、特に好ましくは１２０ｍＭ以下が例示される。これらの塩は後述する認知症検査試薬キットの酵素の安定化剤として機能し得る。
When the step of quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention is a quantification using an enzyme, a salt such as NaCl, KCl, ammonium sulfate or NH 3 Cl can be present in the step, The amount of the lower limit is 0.1 mM or more, preferably 5 mM or more, more preferably 50 mM or more, and the upper limit is not particularly limited, but is preferably 200 mM or less, more preferably 150 mM or less, particularly preferably 120 mM or less. Is exemplified. These salts can function as an enzyme stabilizer for a dementia test reagent kit described later.
 
本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する工程が、酵素を利用した定量である場合、該工程には糖を存在させることができ、その濃度は溶解可能な範囲内であれば限定されない。例えばシュークロースを含有する場合、下限値は後述する認知症検査試薬キットの０．０５（ｗ／ｖ）％以上、好ましくは０．１（ｗ／ｖ）％以上、更に好ましくは０．３（ｗ／ｖ）％以上であり、上限値は３０（ｗ／ｖ）％以下、好ましくは１０（ｗ／ｖ）％以下、更に好ましくは５（ｗ／ｖ）％以下である。例えばマンニトールを含有する場合、下限値は０．０５（ｗ／ｖ）％以上、好ましくは０．１（ｗ／ｖ）％以上、更に好ましくは０．３（ｗ／ｖ）％以上であり、上限値は３（ｗ／ｖ）％以下、好ましくは２（ｗ／ｖ）％以下、更に好ましくは２（ｗ／ｖ）％以下である。その他の糖としてはトレハロースやシクロデキストリン等が挙げられる。これらの糖は、後述する認知症検査試薬キットの酵素やキット内容物自体の安定化剤として、また、キット内容物を凍結乾燥する場合は凍結乾燥賦型剤として機能し得る。
When the step of quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention is quantification using an enzyme, sugar can be present in the step, and the concentration thereof is within a soluble range. There is no limitation as long as there is. For example, when sucrose is contained, the lower limit value is 0.05 (w / v)% or more, preferably 0.1 (w / v)% or more, more preferably 0.3 (%) of the dementia test reagent kit described later. The upper limit is 30 (w / v)% or less, preferably 10 (w / v)% or less, and more preferably 5 (w / v)% or less. For example, when mannitol is contained, the lower limit is 0.05 (w / v)% or more, preferably 0.1 (w / v)% or more, more preferably 0.3 (w / v)% or more, The upper limit is 3 (w / v)% or less, preferably 2 (w / v)% or less, and more preferably 2 (w / v)% or less. Examples of other sugars include trehalose and cyclodextrin. These sugars can function as an enzyme for a dementia test reagent kit described later and a stabilizer for the kit contents themselves, and as a freeze-drying excipient when the kit contents are freeze-dried.
 
本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する工程が、酵素を利用した定量である場合、該工程には防腐剤を存在させられ、その種類や濃度は限定されない。例えばアジ化ナトリウムの場合、下限値は後述する認知症検査試薬キットの０．００５（ｗ／ｖ）％以上、好ましくは０．０１（ｗ／ｖ）％以上、更に好ましくは０．０３（ｗ／ｖ）％以上であり、上限値は１（ｗ／ｖ）％以下、好ましくは０．５（ｗ／ｖ）％以下、更に好ましくは０．１（ｗ／ｖ）％以下である。例えば抗生物質の場合、下限値は５μｇ／ｍＬ以上、好ましくは１０μｇ／ｍＬ以上、更に好ましくは３０μｇ／ｍＬ以上であり、上限値は１００μｇ／ｍＬ以下、好ましくは７５μｇ／ｍＬ以下、更に好ましくは６０μｇ／ｍＬ以下である。
When the step of quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention is quantification using an enzyme, a preservative can be present in the step, and the type and concentration thereof are not limited. For example, in the case of sodium azide, the lower limit is 0.005 (w / v)% or more, preferably 0.01 (w / v)% or more, more preferably 0.03 (w) of the dementia test reagent kit described later. / V)% or more, and the upper limit is 1 (w / v)% or less, preferably 0.5 (w / v)% or less, more preferably 0.1 (w / v)% or less. For example, in the case of antibiotics, the lower limit is 5 μg / mL or more, preferably 10 μg / mL or more, more preferably 30 μg / mL or more, and the upper limit is 100 μg / mL or less, preferably 75 μg / mL or less, more preferably 60 μg. / ML or less.
 
本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する工程が、酵素を利用した定量である場合、該工程にはＥＤＴＡ、ＥＧＴＡ、ＮＡＴ等のキレート剤を含有させられ、その種類や濃度は限定されない。例えばＥＤＴＡを含有する場合、通常は０．０５ｍＭ以上１０ｍＭ以下の範囲である。キレート剤は、金属を活性発現に利用するプロテアーゼがキット内容物中に混在する場合、該活性を阻害する作用により、キット中の酵素の安定化剤として機能し得る。
When the step of quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention is quantification using an enzyme, the step can contain a chelating agent such as EDTA, EGTA, NAT, etc. The concentration is not limited. For example, when it contains EDTA, it is usually in the range of 0.05 mM to 10 mM. The chelating agent can function as a stabilizer for the enzyme in the kit by the action of inhibiting the activity when a protease that utilizes a metal for activity expression is mixed in the kit contents.
 
本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する工程が、酵素を利用した定量である場合、該工程には牛アルブミン、卵アルブミン、ヒトアルブミン、クリスタリン等の触媒作用をもたないタンパク質を存在させられ、その種類や濃度は限定されない。例えば牛アルブミンを含有する場合、通常その含有量は０．０１（ｗ／ｖ）％以上５（ｗ／ｖ）％以下の範囲である。これらのタンパク質はプロテアーゼの基質となるため、酵素の安定化剤となる場合がある。また、凍結乾燥に際しては凍結乾燥賦型剤となり得る。
When the step of quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention is quantification using an enzyme, the step has a catalytic action such as bovine albumin, ovalbumin, human albumin, or crystallin. There are no proteins, and their types and concentrations are not limited. For example, when bovine albumin is contained, the content is usually in the range of 0.01 (w / v)% to 5 (w / v)%. Since these proteins are protease substrates, they may be enzyme stabilizers. Moreover, it can become a freeze-drying excipient during freeze-drying.
 
酵素反応を進めることのできる添加物とは、上記のような酵素の反応性及び／又は安定性を維持及び／又は高める目的の添加物を含み、その例としてはｐＨ緩衝剤、界面活性剤、塩、糖、防腐剤、キレート剤、触媒作用をもたないタンパク質等が挙げられる。
The additive capable of proceeding with the enzyme reaction includes an additive for the purpose of maintaining and / or enhancing the reactivity and / or stability of the enzyme as described above, and examples thereof include a pH buffer, a surfactant, Examples thereof include salts, sugars, preservatives, chelating agents, and proteins having no catalytic action.
 
本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する工程が、酵素を利用した定量である場合、その反応時間は、血清及び／又は血漿中のＰｌｓＥｔｎ量を特異的に測定することができる限り限定されないが、例えば後述する本発明の実施の形態の認知症検査試薬キット内容物の各成分を単一の容器のキットとして用いる場合、反応時間の下限値は１５秒以上、好ましくは１分以上、更に好ましくは３分以上である。上限値は特に設けないが、好ましくは３０分以下、更に好ましくは１５分以下、特に好ましくは１０分以下である。例えば例えば後述する本発明の実施の形態の認知症検査試薬キット内容物の各成分を２以上の容器に分離して用いる場合、それぞれの容器の内容物を使用する反応時間の下限値は１５秒以上、好ましくは１分以上、更に好ましくは３分以上である。上限値は特に設けないが、好ましくは３０分以下、更に好ましくは１５分以下、特に好ましくは１０分以下である。それぞれの容器の内容物を使用する反応時間は同一でなくてもよい。一態様において、本発明の実施の形態のｌｙＰｌｓ　ａｓｅ及び／またはＰＬの作用を利用する血清及び／又は血漿ＰｌｓＥｔｎ量を定量する方法の反応時間は、好ましくは３０分以内、更に好ましくは１５分以内、特に好ましくは１０分以内に完了する。
When the step of quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention is a quantification using an enzyme, the reaction time specifically measures the amount of PlsEtn in the serum and / or plasma. Is not limited as much as possible, for example, when each component of the dementia test reagent kit contents of the embodiment of the present invention described later is used as a single container kit, the lower limit of the reaction time is 15 seconds or more, preferably 1 minute or longer, more preferably 3 minutes or longer. There is no particular upper limit, but it is preferably 30 minutes or less, more preferably 15 minutes or less, and particularly preferably 10 minutes or less. For example, when each component of the contents of the dementia test reagent kit according to the embodiment of the present invention described later is used separately in two or more containers, the lower limit of the reaction time using the contents of each container is 15 seconds. As mentioned above, Preferably it is 1 minute or more, More preferably, it is 3 minutes or more. There is no particular upper limit, but it is preferably 30 minutes or less, more preferably 15 minutes or less, and particularly preferably 10 minutes or less. The reaction time using the contents of each container may not be the same. In one aspect, the reaction time of the method for quantifying serum and / or plasma PlsEtn amount using the action of lyPlsase and / or PL of the embodiment of the present invention is preferably within 30 minutes, more preferably within 15 minutes. Particularly preferably within 10 minutes.
 
本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する工程が、酵素を利用した定量である場合、各工程を実施する温度は、血清及び／又は血漿中のＰｌｓＥｔｎ量を特異的に測定することができる温度であれば限定されず、各工程の温度は同一でなくてもよい。各工程に酵素を用いる場合、用いる酵素の作用温度の範囲内で行うことが好ましい。例えば、下限値は１５℃以上、好ましくは２０℃以上、更に好ましくは２５℃以上であり、上限値は７０℃以下、好ましくは５０℃以下、更に好ましくは４０℃以下であり、好適には３７℃付近である。
When the step of quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention is a quantification using an enzyme, the temperature at which each step is performed specifically determines the amount of PlsEtn in the serum and / or plasma. The temperature is not limited as long as it can be measured, and the temperature of each step may not be the same. When using an enzyme for each process, it is preferable to carry out within the range of the working temperature of the enzyme used. For example, the lower limit is 15 ° C. or higher, preferably 20 ° C. or higher, more preferably 25 ° C. or higher, and the upper limit is 70 ° C. or lower, preferably 50 ° C. or lower, more preferably 40 ° C. or lower, preferably 37 It is around ℃.
 
血清及び／又は血漿ＰｌｓＥｔｎ量は、例えば特許文献２に記載されたＬＣ－ＭＳ／ＭＳ法によってＰｌｓＥｔｎを各分子種ごとに定量することもできる。
For the amount of serum and / or plasma PlsEtn, PlsEtn can be quantified for each molecular species by the LC-MS / MS method described in Patent Document 2, for example.
 
本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する方法において、１２５Ｉ－ＨＰＬＣ法は高額な装置や放射性ヨウ素を使用するので汎用性が劣るが、従来多くの測定実績があり比較対象データが多いので好ましい。本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する工程が、酵素を利用した定量であり、さらに上記の（１）～（３－４）の工程を含む酵素を利用する方法（図７）は、汎用自動分析機に応用でき、利便性が良いので好ましい。本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する工程が、さらに上記の（１）～（３－３）´の工程を含む酵素を利用する方法（図８）は、（１）～（３－４）の工程を含む酵素を利用する方法に比べて使用する酵素が多く経済性に劣るが、汎用自動分析機に応用でき、利便性が良いので好ましい。ＬＣ－ＭＳ／ＭＳ法は高額な装置を使用するので汎用性が劣るが、各分子種ごとに定量できるので好ましい。すなわち本発明の血清及び／又は血漿ＰｌｓＥｔｎ量の定量方法は、１２５Ｉ－ＨＰＬＣ法、ＬＣ－ＭＳ／ＭＳ法、酵素法等に限定されず、例えば、測定感度、特異度、再現性、経済的な理由、安全性目的、適用法令等に応じて当業者であれば適宜変更し得る。
In the method for quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention, the 125I-HPLC method is inferior in versatility because it uses expensive equipment and radioactive iodine. It is preferable because there is a lot of data. The step of quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention is quantification using an enzyme, and a method using an enzyme further comprising the steps (1) to (3-4) ( FIG. 7) is preferable because it can be applied to a general-purpose automatic analyzer and is convenient. The method for quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention further comprises the steps (1) to (3-3) ′ described above (FIG. 8). ) To (3-4), the amount of the enzyme used is inferior to that of the method using the enzyme comprising steps (3-4), but it is preferable because it can be applied to a general-purpose automatic analyzer and is convenient. The LC-MS / MS method is inferior in versatility because it uses an expensive apparatus, but is preferable because it can be quantified for each molecular species. That is, the method for quantifying the amount of serum and / or plasma PlsEtn of the present invention is not limited to 125I-HPLC method, LC-MS / MS method, enzyme method, etc. For example, measurement sensitivity, specificity, reproducibility, economical Those skilled in the art can appropriately change the reason according to the reason, safety purpose, applicable laws and the like.
 
本発明のｌｙＰｌｓ　ａｓｅとしては、Ｐｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ（ＫＴ２４４０）又はＴｈｅｒｍｏｃｒｉｓｐｕｍ　ｓｐ．（ＮＩＴＥ　ＢＰ－０１６２８）由来の配列番号１または２のアミノ酸配列を有するｌｙＰｌｓ　ａｓｅが挙げられる。また、水の存在下でｌｙＰｌｓＥｔｎを加水分解し、対応するアルデヒドとグリセロ－３－ホスフォエタノールアミンを得る反応を触媒する本発明のｌｙＰｌｓ　ａｓｅも挙げられる。
The lyPls as of the present invention includes Pseudomonas putida (KT2440) or Thermocrispum sp. LyPlsase having the amino acid sequence of SEQ ID NO: 1 or 2 derived from (NITE BP-01628). Further, lyPlsase of the present invention that catalyzes the reaction of hydrolyzing lyPlsEtn in the presence of water to obtain the corresponding aldehyde and glycero-3-phosphoethanolamine can also be mentioned.
 
本発明のｌｙＰｌｓ　ａｓｅが天然の微生物由来である場合、該微生物は、ｌｙＰｌｓＥｔｎを加水分解し、対応するアルデヒドとグリセロ－３－ホスフォエタノールアミンを得る反応を触媒する酵素を生産する微生物であれば特に限定されないが、例えばＰｓｅｕｄｏｍｏｎａｓ属又はＴｈｅｒｍｏｃｒｉｓｐｕｍ属に属する微生物であり、好ましくは、Ｐｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ又はＴｈｅｒｍｏｃｒｉｓｐｕｍ　ｓｐ．に属する微生物であり、最も好ましくはＰｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ（ＫＴ２４４０）又はＴｈｅｒｍｏｃｒｉｓｐｕｍ　ｓｐ．（ＮＩＴＥ　ＢＰ－０１６２８）である。Ｐｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ（ＫＴ２４４０）はＤＳＭ　Ｎｏ．６１２５としてＬｅｉｂｎｉｚ－Ｉｎｓｔｉｔｕｔ　ＤＳＭＺ（Ｄｅｕｔｓｃｈｅ　Ｓａｍｍｌｕｎｇ　ｖｏｎ　Ｍｉｋｒｏｏｒｇａｎｉｓｍｅｎ　ｕｎｄ　Ｚｅｌｌｋｕｌｔｕｒｅｎ　ＧｍｂＨ）で購入できる。
When the lyPlsase of the present invention is derived from a natural microorganism, the microorganism can be any microorganism that produces an enzyme that catalyzes the reaction of hydrolyzing lyPlsEtn to obtain the corresponding aldehyde and glycero-3-phosphoethanolamine. Although not particularly limited, for example, microorganisms belonging to the genus Pseudomonas or Thermocrispum, preferably Pseudomonas putida or Thermocrispum sp. And most preferably Pseudomonas putida (KT2440) or Thermocrispum sp. (NITE BP-01628). Pseudomonas putida (KT2440) is a DSM no. 6125 can be purchased from Leibniz-Institut DSMZ (Deutsche Sammlung von Microorganismen und Zellkulturen GmbH).
 
土壌、湖沼、海、生物の表面や体腔内等から分離した菌株が、Ｐｓｅｕｄｏｍｏｎａｓ属又はＴｈｅｒｍｏｃｒｉｓｐｕｍ属に属する微生物であるかどうかは、例えば「Ｂｅｒｇｅｙ’ｓ　Ｍａｎｕａｌ　第２版（２００１年）」、「微生物の分類・同定実験法―分子遺伝学・分子生物学的手法を中心に（Ｓｐｒｉｎｇｅｒ　Ｌａｂ　Ｍａｎｕａｌ）シュプリンガー・フェアラーク東京、２００１年９月」等に記載の方法、市販の同定検査用製品（例えばＢＩＯＭＥＲＩＥＵＸ社）を使用する方法、「株式会社テクノスルガ・ラボ（静岡県静岡市）」等に委託する方法等により確認すればよい。さらにそれらの菌株が、Ｐｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ又はＴｈｅｒｍｏｃｒｉｓｐｕｍ　ｓｐ．であるかどうかは、「Ｓｔａｃｋｅｂｒａｎｄｔ　Ｅ．、Ｅｂｅｒｓ　Ｊ．：　Ｔａｘｏｎｏｍｉｃ　ｐａｒａｍｅｔｅｒｓ　ｒｅｖｉｓｉｔｅｄ：　ｔａｒｎｉｓｈｅｄ　ｇｏｌｄ　ｓｔａｎｄａｒｄｓ，　Ｍｉｃｒｏｂｉｏｌｏｇｙ　ｔｏｄａｙ，　ｎｏｖ，　１５２－１５５頁、２００６年」に記載の方法等により判断すればよい。すなわち、ＤＮＡ－ＤＮＡハイブリダイゼーションで７０％以上の相同性がある、又は１６ｓ　ｒＲＮＡが９８．５％以上同一であれば同属同種と判断できる。好ましくはＤＮＡ－ＤＮＡハイブリダイゼーションで７０％以上の相同性があれば同属同種と判断することができる。２８Ｓ　ｒＤＮＡ－Ｄ１／Ｄ２及び／又はＩＴＳ－５．８Ｓ　ｒＤＮＡ塩基配列によりＰｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ又はＴｈｅｒｍｏｃｒｉｓｐｕｍ　ｓｐ．であるかどうかを判断する場合も同様である。
Whether a strain isolated from soil, lakes, seas, organism surfaces, body cavities, etc. is a microorganism belonging to the genus Pseudomonas or Thermocrispum, for example, “Bergey's Manual 2nd edition (2001)”, “Microorganisms” Classification / identification experiment method-focusing on molecular genetics and molecular biological methods (Springer Lab Manual) Springer Fairlark Tokyo, September 2001 ", etc., commercial identification test products (for example, BIOMERIEUX For example, “Techno Suruga Lab Co., Ltd. (Shizuoka City)” or the like. Furthermore, those strains are Pseudomonas putida or Thermocrispum sp. Is determined according to the method described in “Stackackt E., Ebers J .: Taxonomic parameters revised: tumbled gold standards, Microbiology today, nov, p. 152-155, etc.”. That is, if DNA-DNA hybridization has 70% or more homology, or 16s rRNA is 98.5% or more identical, it can be judged as the same genera. Preferably, if the DNA-DNA hybridization has a homology of 70% or more, it can be judged as the same genera. According to 28S rDNA-D1 / D2 and / or ITS-5.8S rDNA base sequence, Pseudomonas putida or Thermocrispum sp. The same applies to the determination of whether or not.
 
天然の微生物の分離は、当業者に公知の手法を用いて行うことができ、例えば、日本生物工学会編生物工学実験書（２００２年改訂版、培風館）に記載の微生物分離方法を参考にして行うことができる。
Separation of natural microorganisms can be carried out using techniques known to those skilled in the art. For example, referring to the microorganism separation method described in the Biotechnology Experiments edited by the Japanese Society for Biotechnology (2002 revised edition, Baifukan) It can be carried out.
 
本発明のｌｙＰｌｓ　ａｓｅとしては：配列番号１または２のアミノ酸配列を含み、ｌｙＰｌｓＥｔｎを加水分解し、グリセロ－３－ホスフォエタノールアミンとアルデヒドとを得る反応を触媒するｌｙＰｌｓ　ａｓｅ：配列番号１または２に記載のアミノ酸配列において、ｌｙＰｌｓＥｔｎを加水分解し、グリセロ－３－ホスフォエタノールアミンとアルデヒドとを得る反応の触媒作用に関与しない一部のアミノ酸を変異させたアミノ酸配列からなるｌｙＰｌｓ　ａｓｅ：及び配列番号１または２のアミノ酸配列に各種のアミノ酸残基が付加されたアミノ酸配列からなり、ｌｙＰｌｓＥｔｎを加水分解し、グリセロ－３－ホスフォエタノールアミンとアルデヒドとを得る反応を触媒するｌｙＰｌｓ　ａｓｅが挙げられる。本発明のｌｙＰｌｓ　ａｓｅのＮ末端側及び／又はＣ末端側にチオレドキシン蛋白質等機能性蛋白質やその他のアミノ酸配列からなる部分を付加する等により融合ｌｙＰｌｓ　ａｓｅとすることも好ましい。また、該付加する部分により精製や確認等をせしめることのできるタグと呼ばれる部分を融合させてもよく、場合によっては、融合後、そのタグ部分の全部又は一部を削除してよい。例えば、本発明のｌｙＰｌｓ　ａｓｅを菌体外やペリプラズムへ輸送する為の約２０個のシグナルペプチドや、効率的な精製を行う為の５～１０個のＨｉｓを付加してもよく、それらを直列して付加してもよい。それらのアミノ酸配列の間等に数個のプロテアーゼ認識アミノ酸配列を配置して付加してもよい。上述の付加の例と同様に、欠失、又は置換を行うことができ、例えば、配列番号１または２のアミノ酸配列において、ｌｙＰｌｓＥｔｎを加水分解し、グリセロ－３－ホスフォエタノールアミンとアルデヒドとを得る反応を触媒する作用とは無関係の、数個のアミノ酸からなるドメインや、複数個のアミノ酸からなるギャップが存在する場合、それらの欠失を組み合わせることもできる。欠失、置換又は付加を適宜組み合わせてもよい。具体的には配列番号２のアミノ酸配列において配列番号６の２７アミノ酸からなるシグナルペプチドを欠失しても本発明のｌｙＰｌｓＥｔｎである。配列番号５のアミノ酸配列において配列番号７の３３アミノ酸からなるシグナルペプチドを欠失しても本発明のＰＬである。また、Ｂｏｔｅｃｈｎｏｌ　Ｌｅｔｔ．２０１１年、３３巻、７２７－７３１頁で開示された方法を参考に、Ｓｔｒｅｐｔｏｖｅｒｔｃｌｌｕｍ　ｃiｎｎａｍｏｎｅｕｍ由来のｐｈｏｓｐｈｏｌｉｐａｓｅ　Ｄのプロモーター、シグナルペプチド、ターミネーター配列も使用できる。
The lyPlsase of the present invention includes: the amino acid sequence of SEQ ID NO: 1 or 2, and lyPlsase catalyzing the reaction of hydrolyzing lyPlsEtn to obtain glycero-3-phosphoethanolamine and aldehyde: SEQ ID NO: 1 or 2 The lyPls as: comprising the amino acid sequence obtained by hydrolyzing lyPlsEtn and mutating some amino acids not involved in the catalytic action of the reaction for obtaining glycero-3-phosphoethanolamine and aldehyde Examples include lyPlsase, which consists of an amino acid sequence in which various amino acid residues are added to the amino acid sequence of No. 1 or 2, and catalyzes a reaction of hydrolyzing lyPlsEtn to obtain glycero-3-phosphoethanolamine and an aldehyde. . It is also preferable to obtain a fused lyPlsase by adding a functional protein such as a thioredoxin protein or other amino acid sequence to the N-terminal side and / or C-terminal side of the lyPlsase of the present invention. In addition, a portion called a tag that can be purified or confirmed by the added portion may be fused, and in some cases, all or part of the tag portion may be deleted after the fusion. For example, about 20 signal peptides for transporting the lyPlsase of the present invention to the outside of the cell or to the periplasm, or 5 to 10 His for efficient purification may be added. May be added. Several protease-recognizing amino acid sequences may be arranged between these amino acid sequences and added. Similar to the above addition examples, deletions or substitutions can be made. For example, in the amino acid sequence of SEQ ID NO: 1 or 2, lyPlsEtn is hydrolyzed to give glycero-3-phosphoethanolamine and an aldehyde. When there is a domain consisting of several amino acids or a gap consisting of a plurality of amino acids irrespective of the action of catalyzing the reaction to be obtained, these deletions can be combined. Deletions, substitutions or additions may be combined as appropriate. Specifically, even if the signal peptide consisting of 27 amino acids of SEQ ID NO: 6 in the amino acid sequence of SEQ ID NO: 2 is deleted, it is lyPlsEtn of the present invention. Even if the signal peptide consisting of 33 amino acids of SEQ ID NO: 7 in the amino acid sequence of SEQ ID NO: 5 is deleted, it is a PL of the present invention. Also, Botechnol Lett. In reference to the method disclosed in 2011, 33, 727-731, a promoter, signal peptide, and terminator sequence of phospholipase D derived from Streptoverclum cinnamoneum can also be used.
 
本発明のｌｙＰｌｓ　ａｓｅは、配列番号３または４に記載の塩基配列に相補的な塩基配列と後述するストリンジェントな条件下でハイブリダイズする塩基配列によりコードされる蛋白質であって、ｌｙＰｌｓＥｔｎを加水分解し、グリセロ－３－ホスフォエタノールアミンとアルデヒドとを得る反応を触媒するｌｙＰｌｓ　ａｓｅであってもよい。
The lyPlsase of the present invention is a protein encoded by a base sequence complementary to the base sequence shown in SEQ ID NO: 3 or 4 and a base sequence that hybridizes under stringent conditions described later, and hydrolyzes lyPlsEtn. Alternatively, it may be lyPlsase which catalyzes the reaction for obtaining glycero-3-phosphoethanolamine and aldehyde.
 
本発明は：配列番号１または２のアミノ酸配列からなるｌｙＰｌｓ　ａｓｅをコードする塩基配列を含む遺伝子：及び配列番号１または２に記載のアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、ｌｙＰｌｓＥｔｎを加水分解し、グリセロ－３－ホスフォエタノールアミンとアルデヒドとを得る反応を触媒する蛋白質をコードする塩基配列を含む遺伝子にも関する。
The present invention includes: a gene comprising a base sequence encoding lyPlsase consisting of the amino acid sequence of SEQ ID NO: 1 or 2; and the amino acid sequence of SEQ ID NO: 1 or 2, wherein one or more amino acids are deleted, substituted or substituted The present invention also relates to a gene comprising a base sequence encoding a protein comprising an added amino acid sequence and hydrolyzing lyPlsEtn to catalyze a reaction for obtaining glycero-3-phosphoethanolamine and an aldehyde.
 
そのような遺伝子としては、特に限定されないが、例えば：配列番号３または４に記載の塩基配列を含む遺伝子：及び配列番号３または４に記載の塩基配列において１又は複数個の塩基が欠失、置換若しくは付加された塩基配列からなり、ｌｙＰｌｓＥｔｎを加水分解し、グリセロ－３－ホスフォエタノールアミンとアルデヒドとを得る反応を触媒する蛋白質をコードする塩基配列が挙げられ、具体的には：配列番号３または４の塩基配列を大腸菌や放線菌等宿主のコドン使用頻度に合わせて変更した塩基配列を含む遺伝子：及び配列番号１または２のアミノ酸配列において、ｌｙＰｌｓＥｔｎを加水分解し、グリセロ－３－ホスフォエタノールアミンとアルデヒドとを得る反応を触媒する作用に関与しない１又は複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列をコードする塩基配列を含む遺伝子が例示される。
Examples of such a gene include, but are not limited to, for example: a gene containing the nucleotide sequence set forth in SEQ ID NO: 3 or 4, and one or more bases deleted in the nucleotide sequence set forth in SEQ ID NO: 3 or 4. A nucleotide sequence consisting of a substituted or added nucleotide sequence, which encodes a protein that catalyzes the reaction of hydrolyzing lyPlsEtn to give glycero-3-phosphoethanolamine and an aldehyde, specifically: SEQ ID NO: In the amino acid sequence of SEQ ID NO: 1 or 2, wherein lyPlsEtn is hydrolyzed to glycero-3-phos Deletion of one or more amino acids not involved in the action of catalyzing the reaction of obtaining foethanolamine and aldehyde, Gene comprising a nucleotide sequence encoding a substituted or added in the amino acid sequence is exemplified.
 
本発明の遺伝子は、配列番号３または４に記載の塩基配列に相補的な塩基配列とストリンジェントな条件下でハイブリダイズする塩基配列であって、ｌｙＰｌｓＥｔｎを加水分解し、グリセロ－３－ホスフォタノールアミンとアルデヒドとを得る反応を触媒する蛋白質をコードする塩基配列を含む遺伝子であってもよい。
The gene of the present invention is a base sequence that hybridizes with a base sequence complementary to the base sequence shown in SEQ ID NO: 3 or 4 under stringent conditions, and hydrolyzes lyPlsEtn to give glycero-3-phospho It may be a gene containing a base sequence encoding a protein that catalyzes a reaction for obtaining a tanolamine and an aldehyde.
 
上記のストリンジェントな条件は、通常、完全ハイブリッドの融解温度（Ｔｍ）より約５℃～約３０℃、好ましくは約１０℃～約２５℃低い温度であって、特異的なハイブリッドが形成される条件であり、例えばＪ．Ｓａｍｂｒｏｏｋら，Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ，ＡＬａｂｏｒａｔｏｒｙ　Ｍａｎｎｕａｌ，Ｓｅｃｏｎｄ　Ｅｄｉｔｉｏｎ，Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓ（１９８９）に記載されている条件が挙げられる。また、例えば、９０％以上の相同性を有するＤＮＡ同士がハイブリダイズし、それより相同性が低いＤＮＡ同士がハイブリダイズしない条件であってもよい。具体的には、例えば、完全ハイブリッドのＴｍ～（Ｔｍ－３０）℃、好ましくはＴｍ～（Ｔｍ－２０）℃の温度範囲で、かつ１×ＳＳＣ（１倍濃度のＳＳＣ溶液の組成は、１５０ｍＭ塩化ナトリウム、１５ｍＭクエン酸ナトリウム）、好ましくは０．１×ＳＳＣに相当する塩濃度でハイブリダイズを行う条件が挙げられる。
The above stringent conditions are usually about 5 ° C. to about 30 ° C., preferably about 10 ° C. to about 25 ° C. below the melting temperature (Tm) of the complete hybrid, and a specific hybrid is formed. For example, J. Examples include the conditions described in Sambrook et al., Molecular Cloning, Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989). Further, for example, the condition may be such that DNAs having 90% or more homology hybridize and DNAs having lower homology do not hybridize. Specifically, for example, the temperature range of Tm to (Tm-30) ° C., preferably Tm to (Tm-20) ° C. of the complete hybrid, and 1 × SSC (composition of SSC solution of 1 × concentration is 150 mM) Sodium chloride, 15 mM sodium citrate), preferably the conditions for hybridization at a salt concentration corresponding to 0.1 × SSC.
 
本発明の遺伝子を組み込むベクターは特に限定されないが、宿主微生物体内で自律的に増殖しうるファージ又はプラスミドのうち遺伝子組換用として構築されたものが適しており、ファージベクターとしては、例えば、大腸菌に属する微生物を宿主とする場合にはλｇｔ・λＣ、λｇｔ・λＢ等が使用できる。プラスミドベクターとしては、例えば、大腸菌を宿主とする場合には、Ｎｏｖａｇｅｎ社のｐＥＴベクター、又はｐＢＲ３２２、ｐＢＲ３２５、ｐＡＣＹＣ１８４、ｐＵＣ１２、ｐＵＣ１３、ｐＵＣ１８、ｐＵＣ１９、ｐＵＣ１１８、ｐＩＮ　Ｉ、ＢｌｕｅｓｃｒｉｐｔＫＳ＋等、バチラス・サチリスを宿主とする場合にはｐＷＨ１５２０、ｐＵＢ１１０、ｐＫＨ３００ＰＬＫ等、放線菌を宿主とする場合にはｐＩＪ６８０、ｐＩＪ７０２等、酵母、特にサッカロマイセス・セレビジアエを宿主とする場合にはＹＲｐ７、ｐＹＣ１、ＹＥｐ１３等が使用できる。本発明の組換えベクターは、安全性が確認されているという観点から、遺伝子組換え生物等の第二種使用等のうち産業上の使用等に当たって執るべき拡散防止措置等を定める省令（平成十六年財務省、厚生労働省、農林水産省、経済産業省、環境省令第一号）別表第一号の規定に基づき経済産業大臣が定めるＧＩＬＳＰ遺伝子組換え微生物の、別表第一に掲げられたベクターに、上記の本発明の遺伝子が挿入された組換えベクターが好ましい。プロモーターは宿主中で発現できるものであれば特に限定されない。本発明の組換えベクターは、例えば本発明の遺伝子及び上記のベクターを用いて、当業者に公知の手法で作成することができる。
The vector into which the gene of the present invention is incorporated is not particularly limited, but a phage or plasmid constructed for gene recombination among phages or plasmids that can grow autonomously in a host microorganism is suitable. Examples of phage vectors include E. coli. Λgt · λC, λgt · λB, etc. can be used when a microorganism belonging to the above group is used as a host. As the plasmid vector, for example, when Escherichia coli is used as a host, Novagen's pET vector, or pBR322, pBR325, pACYC184, pUC12, pUC13, pUC18, pUC19, pUC118, pIN I, Bluescript KS +, etc. PW1520, pUB110, pKH300PLK, etc., when using actinomycetes as the host, pIJ680, pIJ702, etc., and when using yeast, particularly Saccharomyces cerevisiae as the host, YRp7, pYC1, YEp13, etc. can be used. From the viewpoint that the recombinant vector of the present invention has been confirmed to be safe, a ministerial ordinance that stipulates anti-diffusion measures to be taken for industrial use, etc., among the second type use of genetically modified organisms, etc. Six years Ministry of Finance, Ministry of Health, Labor and Welfare, Ministry of Agriculture, Forestry and Fisheries, Ministry of Economy, Trade and Industry, Ministry of the Environment Ordinance No.1) Furthermore, a recombinant vector into which the gene of the present invention is inserted is preferable. The promoter is not particularly limited as long as it can be expressed in the host. The recombinant vector of the present invention can be prepared by a technique known to those skilled in the art using, for example, the gene of the present invention and the above vector.
 
本発明の上記の組換えベクターを含む形質転換体は、上記組換えベクターで形質転換された形質転換体であれば限定されず、宿主としては、大腸菌、バチラス・サチリス、ストレプトマイセス属やロドコッカス属に属する放線菌、サッカロマイセス・セレビジアエ、ピキア・パストリス、麹カビ等が挙げられる。本発明の形質転換体は、安全性が確認されているという観点から、遺伝子組換え生物等の第二種使用等のうち産業上の使用等に当たって執るべき拡散防止措置等を定める省令（平成十六年財務省、厚生労働省、農林水産省、経済産業省、環境省令第一号）別表第一号の規定に基づき経済産業大臣が定めるＧＩＬＳＰ遺伝子組換え微生物の、別表第一に掲げられた宿主に形質転換したものが好ましい。本発明の形質転換体は、例えば本発明の組換えベクター及び上記の宿主を用いて、当業者に公知の手法で作成することができる。
The transformant containing the above recombinant vector of the present invention is not limited as long as it is a transformant transformed with the above recombinant vector. Examples of the host include Escherichia coli, Bacillus subtilis, Streptomyces and Rhodococcus. Examples include actinomycetes belonging to the genus, Saccharomyces cerevisiae, Pichia pastoris, and fungi. From the viewpoint that the transformant of the present invention has been confirmed to be safe, a ministerial ordinance that establishes measures to prevent diffusion in industrial use, etc. among second-class use of genetically modified organisms, etc. Six years Ministry of Finance, Ministry of Health, Labor and Welfare, Ministry of Agriculture, Forestry and Fisheries, Ministry of Economy, Trade and Industry, Ministry of the Environment Ordinance No. 1) Hosts listed in Appendix 1 of GILSP genetically modified microorganisms established by the Minister of Economy, Trade and Industry based on the provisions of Schedule No. 1 Those transformed into are preferred. The transformant of the present invention can be prepared by a technique known to those skilled in the art using, for example, the recombinant vector of the present invention and the above host.
 
本発明はまた、本発明のｌｙＰｌｓ　ａｓｅを生成する微生物を培地で培養し、培養物中に本発明のｌｙＰｌｓ　ａｓｅを生成蓄積させ、該培養物から本発明のｌｙＰｌｓ　ａｓｅを採取することを特徴とする、本発明のｌｙＰｌｓ　ａｓｅの製造方法にも関連する。
The present invention is also characterized in that a microorganism that produces the lyPlsase of the present invention is cultured in a medium, the lyPlsase of the present invention is produced and accumulated in the culture, and the lyPlsase of the present invention is collected from the culture. The present invention also relates to a method for producing lyPlsase of the present invention.
 
微生物の培養工程及び生成蓄積工程における培養条件はその栄養生理的性質を考慮して適宜選択すればよく、通常液体培養で行うが、工業的には深部通気撹拌培養を行うのが有利であり得る。培地の栄養源としては、ＬＢ、ＰＤＡ、ＭＡ、ＯＡ又はＬｃＡ培地等微生物の培養に通常用いられるものが広く使用され得る。培養温度は本発明のｌｙＰｌｓ　ａｓｅが生成される範囲で適宜変更し得るが、天然の微生物の場合、下限が５℃以上、好ましくは１５℃以上、更に好ましくは２０℃以上、上限は、好熱性の古細菌やバクテリアの場合の約１００℃となるが、通常は５５℃以下、好ましくは４５℃以下、更に好ましくは４０℃以下である。特に真菌の場合は、下限が４℃以上、好ましくは１０℃以上、更に好ましくは２０℃以上、上限が５０℃以下、好ましくは４２℃以下、更に好ましくは３７℃以下である。培養時間は、培養条件によって変動し得るが、本発明のｌｙＰｌｓ　ａｓｅの生成が最高量に達する時期を見計らって適当な時期に培養を終了すればよく、通常は下限が１７時間以上、好ましくは２０時間以上、更に好ましくは２４時間以上、上限が８０時間以下、好ましくは７２時間以下、更に好ましくは４８時間以下である。特に真菌の場合は、下限が１日以上、好ましくは２日以上、上限は約１０日、好ましくは４日以下、更に好ましくは３日以下である。培地ｐＨは微生物が発育し、本発明のｌｙＰｌｓ　ａｓｅを生成する範囲で適宜変更し得るが、下限が好ましくはｐＨ４以上、更に好ましくはｐＨ５以上、上限が好ましくはｐＨ８．５以下、より好ましくはｐＨ７．５以下である。特に真菌の場合は、低いｐＨが好ましい。
Culture conditions in the microorganism cultivation process and the production and accumulation process may be appropriately selected in view of the nutritional physiological properties, and are usually performed by liquid culture, but industrially, it is advantageous to perform deep aeration and agitation culture. . As a nutrient source of the medium, those usually used for culturing microorganisms such as LB, PDA, MA, OA or LcA medium can be widely used. The culture temperature can be appropriately changed within the range in which the lyPlsase of the present invention is produced. In the case of natural microorganisms, the lower limit is 5 ° C. or higher, preferably 15 ° C. or higher, more preferably 20 ° C. or higher, and the upper limit is thermophilic. The temperature is about 100 ° C. in the case of archaebacteria and bacteria, but it is usually 55 ° C. or lower, preferably 45 ° C. or lower, more preferably 40 ° C. or lower. Particularly in the case of fungi, the lower limit is 4 ° C or higher, preferably 10 ° C or higher, more preferably 20 ° C or higher, and the upper limit is 50 ° C or lower, preferably 42 ° C or lower, more preferably 37 ° C or lower. The culture time may vary depending on the culture conditions, but the culture may be terminated at an appropriate time in anticipation of the time when the production of the lyPlsase of the present invention reaches the maximum amount. Usually, the lower limit is 17 hours or more, preferably 20 It is not less than time, more preferably not less than 24 hours, and the upper limit is not more than 80 hours, preferably not more than 72 hours, more preferably not more than 48 hours. In particular, in the case of fungi, the lower limit is 1 day or more, preferably 2 days or more, and the upper limit is about 10 days, preferably 4 days or less, more preferably 3 days or less. The medium pH can be appropriately changed within the range in which microorganisms grow and produce the lyPlsase of the present invention, but the lower limit is preferably pH 4 or higher, more preferably pH 5 or higher, and the upper limit is preferably pH 8.5 or lower, more preferably pH 7 .5 or less. Especially in the case of fungi, a low pH is preferred.
 
本発明はまた、上記の本発明の形質転換体を培地で培養し、培養物中に本発明のｌｙＰｌｓ　ａｓｅを生成蓄積させ、該培養物から該ｌｙＰｌｓ　ａｓｅを採取することを特徴とする、本発明のｌｙＰｌｓ　ａｓｅの製造方法にも関連する。
The present invention is also characterized in that the above-described transformant of the present invention is cultured in a medium, the lyPlsase of the present invention is produced and accumulated in the culture, and the lyPlsase is collected from the culture. It also relates to a method for producing the lyPlsase of the invention.
 
形質転換体の培養工程及び生成蓄積工程における培養条件は、上記微生物の場合と同様であり、形質転換体の種類に応じて適宜選択することができる。例えば、形質転換体が大腸菌の場合、培養温度は、下限が１０℃以上、好ましくは２０℃以上、更に好ましくは２５℃以上、上限が４５℃以下、好ましくは４２℃以下、更に好ましくは３７℃以下である。形質転換体が放線菌の場合、下限が４℃以上、好ましくは１０℃以上、更に好ましくは２０℃以上、上限が５０℃以下、好ましくは４２℃以下、更に好ましくは３７℃以下である。培養時間は、条件によって変動し得るが、本発明のｌｙＰｌｓ　ａｓｅの生成が最高量に達する時期を見計らって適当な時期に培養を終了すればよく、形質転換体が大腸菌の場合、通常は下限が１０時間以上、好ましくは１２時間以上、更に好ましくは１７時間以上、上限が６０時間以下、好ましくは４８時間以下、更に好ましくは３０時間以下である。形質転換体が放線菌の場合、通常は下限が１７時間以上、好ましくは２０時間以上、更に好ましくは２４時間以上、上限が８０時間以下、好ましくは７２時間以下、更に好ましくは４８時間以下である。培地ｐＨは、形質転換体が発育し、本発明のｌｙＰｌｓ　ａｓｅを生成する範囲で適宜変更し得るが、大腸菌や放線菌の場合、下限が好ましくはｐＨ５．８以上、更に好ましくはｐＨ６．２以上、上限が好ましくはｐＨ８．５以下、更に好ましくはｐＨ７．５以下である。
The culture conditions in the transformant culturing step and the production and accumulation step are the same as in the case of the microorganism, and can be appropriately selected according to the type of the transformant. For example, when the transformant is E. coli, the lower limit of the culture temperature is 10 ° C or higher, preferably 20 ° C or higher, more preferably 25 ° C or higher, and the upper limit is 45 ° C or lower, preferably 42 ° C or lower, more preferably 37 ° C. It is as follows. When the transformant is actinomycetes, the lower limit is 4 ° C or higher, preferably 10 ° C or higher, more preferably 20 ° C or higher, and the upper limit is 50 ° C or lower, preferably 42 ° C or lower, more preferably 37 ° C or lower. The culture time may vary depending on the conditions, but the culture may be terminated at an appropriate time in anticipation of the time when the production of the lyPlsase of the present invention reaches the maximum amount. 10 hours or more, preferably 12 hours or more, more preferably 17 hours or more, and the upper limit is 60 hours or less, preferably 48 hours or less, more preferably 30 hours or less. When the transformant is actinomycetes, the lower limit is usually 17 hours or longer, preferably 20 hours or longer, more preferably 24 hours or longer, and the upper limit is 80 hours or shorter, preferably 72 hours or shorter, more preferably 48 hours or shorter. . The pH of the medium can be appropriately changed within the range in which the transformant develops and produces the lyPlsase of the present invention, but in the case of Escherichia coli and actinomycetes, the lower limit is preferably pH 5.8 or more, more preferably pH 6.2 or more. The upper limit is preferably pH 8.5 or less, more preferably pH 7.5 or less.
 
上記の培養物中に生成蓄積された本発明のｌｙＰｌｓ　ａｓｅを採取する方法は特に限定されないが、簡便には殺菌、非殺菌を問わず菌体を含む細胞等のまま本発明のｌｙＰｌｓ　ａｓｅを採取してもよい。培養不純物や細胞破砕物等を軽く除き、不純物が残存したまま、本発明のｌｙＰｌｓ　ａｓｅを採取することも好ましい。目的や用途等によっては実質的に不純物を包含しない本発明のｌｙＰｌｓ　ａｓｅを採取することも好ましい。例えば５０％以上、７０％以上又は９５％以上の純度で本発明のｌｙＰｌｓ　ａｓｅを採取することが例示される。純度はＳＤＳ－ＰＡＧＥやＨＰＬＣ等の公知の方法を用いて確認することができる。
The method for collecting the lyPlsase of the present invention produced and accumulated in the culture is not particularly limited, but for convenience, the lyPlsase of the present invention is collected as it is, including cells containing cells, regardless of sterilization or non-sterilization. May be. It is also preferable to collect the lyPlsase of the present invention while lightly removing the culture impurities and cell debris and leaving the impurities remaining. Depending on the purpose and application, it is also preferable to collect the lyPlsase of the present invention which does not substantially contain impurities. For example, it is exemplified that lyPlsase of the present invention is collected with a purity of 50% or more, 70% or more, or 95% or more. The purity can be confirmed using a known method such as SDS-PAGE or HPLC.
 
本発明のｌｙＰｌｓ　ａｓｅを精製する方法を以下に説明する。本発明のｌｙＰｌｓ　ａｓｅが、菌体内に形成される場合には、培養終了後、得られた培養物から、濾過又は遠心分離等の手段により菌体を採集する。次いで、この菌体を機械的方法又はリゾチーム等の酵素的方法で破壊し、必要に応じてＥＤＴＡ、及び／又は適当な界面活性剤等を添加して該ｌｙＰｌｓ　ａｓｅを濃縮し、アセトン、メタノール、エタノール等の有機溶媒による分別沈殿法、硫酸アンモニウム、食塩等による塩析法等を適用して本発明のｌｙＰｌｓ　ａｓｅを沈殿させ回収することができる。この沈殿物について、必要に応じて透析、等電点沈殿を行った後、ゲル濾過、アフィニティークロマトグラフィー等の吸着クロマトグラフィー、イオン交換クロマトグラフィーや疎水的クロマトグラフィーにより処理して、精製された本発明のｌｙＰｌｓ　ａｓｅを得ることができる。上記の方法は適宜組み合わせて行うことができる。また、本発明のｌｙＰｌｓ　ａｓｅが培養液中に形成される場合には、培養物から濾過又は遠心分離等の手段により菌体を除去して培養液を得て、前記菌体内に形成される場合と同様の処理を行うことにより、精製された本発明のｌｙＰｌｓ　ａｓｅを得ることができる。
A method for purifying lyPlsase of the present invention will be described below. When the lyPlsase of the present invention is formed in the microbial cells, the microbial cells are collected from the obtained culture by means such as filtration or centrifugation after completion of the culture. Next, this bacterial cell is destroyed by a mechanical method or an enzymatic method such as lysozyme, and EDTA and / or an appropriate surfactant is added as necessary to concentrate the lyPlsase, and then acetone, methanol, The lyPlsase of the present invention can be precipitated and recovered by applying a fractional precipitation method using an organic solvent such as ethanol, a salting out method using ammonium sulfate, sodium chloride, or the like. This precipitate is subjected to dialysis and isoelectric precipitation, if necessary, and then purified by adsorption using gel filtration, affinity chromatography, etc., ion exchange chromatography, or hydrophobic chromatography. Inventive lyPls as can be obtained. The above methods can be combined as appropriate. In addition, when the lyPlsase of the present invention is formed in a culture solution, the microbial cells are removed from the culture by means of filtration or centrifugation to obtain a culture solution, and then formed in the microbial cells. Purified lyPlsase of the present invention can be obtained by carrying out the same treatment as described above.
 
上記の本発明のｌｙＰｌｓ　ａｓｅの製造方法によって得られるｌｙＰｌｓ　ａｓｅは、必要に応じて安定化剤として、各種の塩類、糖類、蛋白質、脂質、界面活性剤等を加え、限外濾過濃縮、凍結乾燥等の方法により、液状又は固形とすることができる。凍結乾燥を行う場合、安定化剤としてサッカロース、マンニトール、食塩、アルブミン、硫安等を０．５～１０％程度添加してもよい。
The lyPlsase obtained by the above-described method for producing lyPlsase of the present invention is added with various salts, saccharides, proteins, lipids, surfactants, etc. as stabilizers as necessary, and concentrated by ultrafiltration and freeze-dried. Etc., it can be made liquid or solid. When freeze-drying, about 0.5 to 10% of sucrose, mannitol, sodium chloride, albumin, ammonium sulfate, etc. may be added as a stabilizer.
 
本発明のｌｙＰｌｓ　ａｓｅは、従来公知のｌｙＰｌｓ　ａｓｅとはアミノ酸配列において区別できる。例えば、配列番号１に記載のアミノ酸配列と相同性を有する公知のアミノ酸配列を、ＮＣＢＩ　ＢＬＡＳＴ（Ｂａｓｉｃ　Ｌｏｃａｌ　Ａｌｉｇｎｍｅｎｔ　Ｓｅａｒｃｈ　Ｔｏｏｌ（ｈｔｔｐ：／／ｂｌａｓｔ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／Ｂｌａｓｔ．ｃｇｉ））でＰｒｏｔｅｉｎ　ＢＬＡＳＴ検索すると、平成２５年６月２９日時点で、Ｐｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ　ＫＴ２４４０株由来のアミノ酸配列が検索できるものの、その定義（Ｄｅｆｉｎｉｔｉｏｎ）はｈｙｐｏｔｈｅｔｉｃａｌ　ｐｒｏｔｅｉｎであり、機能未知の蛋白質ででる。同様に配列番号２に記載のアミノ酸配列と相同性を有する公知のアミノ酸配列をＮＣＢＩ　ＢＬＡＳＴでＰｒｏｔｅｉｎ　ＢＬＡＳＴ検索すると、平成２５年６月２９日時点で、Ｓｔａｃｋｅｂｒａｎｄｔｉａ　ｎａｓｓａｕｅｎｓｉｓ由来のアミノ酸配列が検索できるものの、その定義（Ｄｅｆｉｎｉｔｉｏｎ）はｇｌｙｃｅｒｏｐｈｏｓｐｈｏｒｙｌ　ｄｉｅｓｔｅｒ　ｐｈｏｓｐｈｏｄｉｅｓｔｅｒａｓｅであり、全く異なることが明らかである。さらに従来公知のラット由来のｌｙＰｌｓ　ａｓｅを開示したＴＨＥ　ＪＯＵＲＮＡＬ　ＯＦ　ＢＩＯＬＯＧＩＣＡＬ　ＣＨＥＭＩＳＴＲＹ　ＶＯＬ．２８６，ＮＯ．２８，の２４９２３頁右３行目には「Ｔｍｅｍ８６ｂ　（発明者註：ｌｙＰｌｓ　ａｓｅ遺伝子）　ｈａｓ　ｏｎｌｙ　ｂｅｅｎ　ｉｄｅｎｔｉｆｉｅｄ　ｉｎ　ｖｅｒｔｅｂｒａｔｅｓ，ｉｎｃｌｕｄｉｎｇ　ｈｕｍａｎｓ，ｍｉｃｅ，ｒａｔｓ，ｃｏｗｓ，ｄｏｇｓ，ａｎｄ　ｚｅｂｒａｆｉｓｈ．」と記載されている。すなわち、従来、ヒト、マウス、ラット、ウシ、イヌ、及びゼブラフィッシュを含む脊椎動物のｌｙＰｌｓ　ａｓｅが公知であった。本発明のｌｙＰｌｓ　ａｓｅは、上記のように微生物由来であり、本発明者らにより新規に発見された微生物由来のｌｙＰｌｓ　ａｓｅである。
The lyPlsase of the present invention can be distinguished from the conventionally known lyPlsase in amino acid sequence. For example, a known amino acid sequence having homology with the amino acid sequence described in SEQ ID NO: 1 is obtained from NCBI BLAST (Basic Local Alignment Search Tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi)). When Protein BLAST search is performed, the amino acid sequence derived from Pseudomonas putida KT2440 strain can be searched as of June 29, 2013, but the definition (definition) is a hypothetical protein and is a protein of unknown function. Similarly, when a protein BLAST search with NCBI BLAST is performed for a known amino acid sequence having homology with the amino acid sequence shown in SEQ ID NO: 2, as of June 29, 2013, an amino acid sequence derived from Stackeblandia nassauensis can be searched. It is clear that the definition is glycerphophoryl disperse phosphoresterase and is quite different. Furthermore, THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. Which disclosed a conventionally known rat-derived lyPlsase. 286, NO. 28, page 24923, the third line on the right, “Tmem86b (inventor 註: lyPlsase gene) has only been identified inverteds, inclusive humans, mice, rats, cows, dogs, and iszef”. That is, conventionally, lyPlsase of vertebrates including humans, mice, rats, cows, dogs, and zebrafish has been known. The lyPlsase of the present invention is derived from microorganisms as described above, and is a lyPlsase derived from microorganisms newly discovered by the present inventors.
 
この様なｌｙＰｌｓ　ａｓｅは、本本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する工程が、酵素を利用した定量であり、さらに該酵素が本発明のｌｙＰｌｓ　ａｓｅ及び／またはＰＬである場合、ｌｙＰｌｓＥｔｎを加水分解し、グリセロ－３－ホスフォエタノールアミンとアルデヒドとを得る反応を触媒する酵素として利用できる。
For such lyPlsase, the step of quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention is quantification using an enzyme, and the enzyme is lyPlsase and / or PL of the present invention. In this case, it can be used as an enzyme that catalyzes the reaction of hydrolyzing lyPlsEtn to obtain glycero-3-phosphoethanolamine and an aldehyde.
 
本発明はまた被験者の自然排泄尿中のＭＩの定量手段と、同一被験者の血清または血漿中のＰｌｓＥｔｎの定量手段とを含む、該被験者を認知症患者またはその前段階にある者か否かを分類するための認知症検査手段であって、健常者と、認知症患者またはその前段階にある者とを区別することができる、ＭＩとＰｌｓＥｔｎとのそれぞれの、予め求められた閾値に対して、被験者のＭＩとＰｌｓＥｔｎの定量値を比較して用いる、前記の認知症検査手段に関わる。前記手段としては、組成物、検査試薬、酵素キット等が挙げられるがこれに限定されない。
The present invention also includes a means for quantifying MI in spontaneously excreted urine of a subject and a means for quantifying PlsEtn in the serum or plasma of the same subject. A dementia test means for classification, which is capable of distinguishing between a healthy person and a dementia patient or a person in the previous stage, with respect to respective predetermined threshold values of MI and PlsEtn In addition, the present invention relates to the above-mentioned dementia test means, which uses the subject's MI and PlsEtn quantitative values in comparison. Examples of the means include, but are not limited to, a composition, a test reagent, an enzyme kit, and the like.
 
本発明はまた、被験者の自然排泄尿中のＭＩを定量する為の酵素と、同一被験者の血清または血漿中のＰｌｓＥｔｎを定量する為の酵素とを含む、該被験者を認知症患者またはその前段階にある者か否かを分類するための認知症検査試薬キットであって、健常者と、認知症患者またはその前段階にある者とを区別することができる、ＭＩとＰｌｓＥｔｎとのそれぞれの、予め求められた閾値に対して、被験者のＭＩとＰｌｓＥｔｎの定量値を比較して用いる、前記の検査試薬キットに関わる。
The present invention also includes an enzyme for quantifying MI in spontaneously excreted urine of a subject, and an enzyme for quantifying PlsEtn in serum or plasma of the same subject. Each of MI and PlsEtn, each of which can distinguish between a healthy person and a dementia patient or a person in its previous stage, The present invention relates to the above-described test reagent kit, which uses the subject's MI and PlsEtn quantitative values in comparison with a predetermined threshold value.
 
ＰｌｓＥｔｎを定量する試薬キットは、ＰｌｓＥｔｎを１２５Ｉ－ＨＰＬＣ法で定量する方法、上記工程（１）～（３－４）を含む本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する方法、ＬＣ－ＭＳ／ＭＳ法により定量する方法などを実施するための試薬キットであり、本発明のｌｙＰｌｓ　ａｓｅ及び／またはＰＬの作用を利用して定量するための試薬キットが好ましい。
A reagent kit for quantifying PlsEtn is a method for quantifying PlsEtn by a 125I-HPLC method, and a method for quantifying the amount of serum and / or plasma PlsEtn according to an embodiment of the present invention including the steps (1) to (3-4) above. A reagent kit for carrying out a method for quantification by the LC-MS / MS method, etc., and a reagent kit for quantification using the action of lyPlsase and / or PL of the present invention is preferred.
 
本実施の形態の認知症検査試薬キット（本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する工程が、酵素を利用した定量であり、さらに該酵素が本発明のｌｙＰｌｓ　ａｓｅ及び／またはＰＬである認知症検査試薬キット及び本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する工程が、さらに上記工程（１）～（３－４）又は（１）～（３－３）´を含む本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する方法を利用した定量である認知症検査試薬キット）は、本明細書に記載の各種酵素、ＰＬ、ｌｙＰｌｓ　ａｓｅ、ＧＰＣＰ、エタノールアミンオキシダーゼ、ＰＬＡ１、ＰＬＡ２、ＰＬＢ、ＬＹＰＬ、ＭＧＬＰ、ペルオキシダーゼ、カタラーゼ、ＡＳＯＭ等を、上述の認知症検査方法の記載を参照して、適宜組み合わせて１以上含むことができる。それらの好ましい例は上述の本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する工程が、酵素を利用した定量である方法の場合と同様である。
Dementia test reagent kit according to the present embodiment (the step of quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention is quantification using an enzyme, and the enzyme further comprises lyPlsase and / or Alternatively, the step of quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention and the dementia test reagent kit that is PL further comprises the steps (1) to (3-4) or (1) to (3- 3) A dementia test reagent kit, which is a quantification using the method for quantifying the amount of serum and / or plasma PlsEtn according to an embodiment of the present invention including '), includes various enzymes, PL, and lyPls as described herein. , GPCP, ethanolamine oxidase, PLA 1, PLA 2, PLB , LYPL, MGLP, peroxidase, catalase, the ASOM like, dementia test method described above With reference to the description can comprise one or more in combination. Preferred examples thereof are the same as those in the method in which the step of quantifying the amount of serum and / or plasma PlsEtn according to the above-described embodiment of the present invention is quantification using an enzyme.
 
本実施の形態の認知症検査試薬キットは、測定の感度、正確性、再現性、キットの安定性等の品質を向上する目的等で、塩、糖、防腐剤、キレート剤、タンパク質、界面活性剤等他の成分を含有してもよい。含有される成分の種類及び量は、認知症検査試薬キットの使用目的や測定用途に応じて適宜変更することができ、例えば、測定感度、特異度、再現性、経済的な理由、安全性目的、適用法令等に応じて当業者であれば適宜変更し得る。それらの好ましい例は上述の本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する工程が、酵素を利用した定量である方法の場合と同様である。
The dementia test reagent kit of this embodiment is used for the purpose of improving the quality of measurement sensitivity, accuracy, reproducibility, kit stability, etc., salt, sugar, preservative, chelating agent, protein, surface activity You may contain other components, such as an agent. The type and amount of the components contained can be changed as appropriate according to the purpose of use and the measurement application of the dementia test reagent kit. For example, measurement sensitivity, specificity, reproducibility, economic reason, safety purpose Those skilled in the art can appropriately change them according to applicable laws and regulations. Preferred examples thereof are the same as those in the method in which the step of quantifying the amount of serum and / or plasma PlsEtn according to the above-described embodiment of the present invention is quantification using an enzyme.
 
本発明は、被験者の自然排泄尿中のＭＩを定量する酵素と、同一被験者の血清または血漿中のＰｌｓＥｔｎを定量する酵素とを含む酵素試薬キットであって、被験者を認知症患者またはその前段階にある者として分類することを特徴とする認知症検査試薬キットにも関する。被験者の自然排泄尿中のＭＩを定量する酵素を含む酵素試薬キットには少なくともミオイノシトールデヒドロゲナーゼを含むことが好ましい。
The present invention is an enzyme reagent kit comprising an enzyme for quantifying MI in spontaneously excreted urine of a subject and an enzyme for quantifying PlsEtn in the serum or plasma of the same subject, wherein the subject is a dementia patient or a preceding stage thereof The present invention also relates to a dementia test reagent kit characterized in that it is classified as a person in the above. It is preferable that an enzyme reagent kit containing an enzyme for quantifying MI in spontaneously excreted urine of a subject contains at least myo-inositol dehydrogenase.
 
本発明の実施の形態の認知症検査試薬キットは、自然排泄尿中のＭＩと血清及び／又は血漿中のＰｌｓＥｔｎ、それぞれの定量値に閾値を設定することによって被験者を認知症患者またはその前段階にある者として分類することも好ましく、そのような閾値は上述と同様である。また、本発明の実施の形態の認知症検査試薬キットで定量する被験者の自然排泄尿、血清及び／又は血漿中については上述と同様である。
The dementia test reagent kit according to the embodiment of the present invention provides a subject with a dementia patient or a pre-stage thereof by setting a threshold value for each quantified value of MI in spontaneously excreted urine and PlsEtn in serum and / or plasma. It is also preferable to classify as a person who is in the category, and such a threshold is the same as described above. Further, in the subject's spontaneously excreted urine, serum and / or plasma as determined by the dementia test reagent kit of the embodiment of the present invention, the same as described above.
 
本発明の実施の形態の認知症検査試薬キットが、本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する工程を利用し、さらに上記工程（１）～（３－４）又は（１）～（３－３）´を含む本発明の実施の形態の血清及び／又は血漿ＰｌｓＥｔｎ量を定量する方法を利用したキットの場合、内容物の各成分を単一の容器のキットとしてもよいが、通常は２以上の容器に分離することが好ましい。一態様において、本実施の形態の認知症検査試薬キットは、被験者の血清または血漿中のＰｌｓＥｔｎ以外の物質を消去するための消去用容器１と、ＰｌｓＥｔｎを測定するための容器２とに分けられる。
The reagent kit for dementia testing according to the embodiment of the present invention uses the step of quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention, and further includes the steps (1) to (3-4) or ( In the case of a kit using the method for quantifying the amount of serum and / or plasma PlsEtn according to the embodiment of the present invention including 1) to (3-3) ′, each component of the contents may be used as a single container kit. Usually, it is preferable to separate into two or more containers. In one aspect, the dementia test reagent kit of the present embodiment is divided into an erasing container 1 for erasing substances other than PlsEtn in the serum or plasma of a subject and a container 2 for measuring PlsEtn. .
 
本発明の実施の形態の認知症検査試薬キットは、以下の（１ａ）～（３ａ）を含み、（１ａ）～（３ａ）は被験者の血清または血漿中のＰｌｓＥｔｎ以外の物質を消去するための消去用容器１と、ＰｌｓＥｔｎを測定するための容器２とに分けることができる。
The dementia test reagent kit of the embodiment of the present invention includes the following (1a) to (3a), wherein (1a) to (3a) are for erasing substances other than PlsEtn in the serum or plasma of a subject The container can be divided into an erasing container 1 and a container 2 for measuring PlsEtn.
 
（１ａ）ＰｌｓＥｔｎをｌｙＰｌｓＥｔｎに加水分解できる酵素およびその酵素反応を進めることのできる添加物。
（２ａ）ｌｙＰｌｓＥｔｎをグリセロ－３－ホスフォエタノールアミンとアルデヒドに加水分解でき、かつエーテル型リゾホスファチジルエタノールアミンを加水分解できない加水分解酵素およびその酵素反応を進めることのできる添加物。
（３ａ）グリセロ－３－ホスフォエタノールアミンをエタノールアミンとグリセロリン酸に変換する酵素、エタノールアミンをグリコールアルデヒド、アンモニア、及び過酸化水素に変換する酵素とその酵素反応を進めるにことのできる添加物、さらに必要により過酸化水素を定量するための過酸化水素定量手段。
または前記（２ａ）の加水分解酵素がリゾホスファチジルエタノールアミンを加水分解できる酵素である場合、前記（１ａ）に、さらにリゾホスファチジルエタノールアミンを加水分解する酵素を含む試薬キット。
(1a) An enzyme capable of hydrolyzing PlsEtn to lyPlsEtn and an additive capable of proceeding with the enzyme reaction.
(2a) A hydrolase capable of hydrolyzing lyPlsEtn into glycero-3-phosphoethanolamine and an aldehyde and not hydrolyzing ether-type lysophosphatidylethanolamine and an additive capable of proceeding with the enzyme reaction.
(3a) An enzyme that converts glycero-3-phosphoethanolamine into ethanolamine and glycerophosphoric acid, an enzyme that converts ethanolamine into glycolaldehyde, ammonia, and hydrogen peroxide, and an additive capable of advancing the enzymatic reaction Further, hydrogen peroxide determination means for determining hydrogen peroxide if necessary.
Alternatively, when the hydrolase of (2a) is an enzyme capable of hydrolyzing lysophosphatidylethanolamine, the reagent kit further comprises (1a) an enzyme that hydrolyzes lysophosphatidylethanolamine.
 
そのような試薬キットの好ましい例は、少なくとも上記（１ａ）及び（３ａ）を含有する容器１：並びに少なくとも（２ａ）を含有する容器２を含む、認知症検査試薬キットである。
A preferred example of such a reagent kit is a dementia test reagent kit including a container 1 containing at least (1a) and (3a) and a container 2 containing at least (2a).
 
被験者の血清及び／又は血漿中にホスファチジルエタノールアミンが含まれ、かつ、ｌｙＰｌｓＥｔｎをグリセロ－３－ホスフォエタノールアミンとアルデヒドに加水分解する作用を触媒する酵素がリゾホスファチジルエタノールアミンを加水分解する作用を有する場合は、容器１にさらにリゾホスファチジルエタノールアミンを分解し、実質的に消去する作用を有する酵素を内容物として含有する容器１とすれば良い。
The serum and / or plasma of the subject contains phosphatidylethanolamine, and an enzyme that catalyzes the action of hydrolyzing lyPlsEtn into glycero-3-phosphoethanolamine and an aldehyde has the effect of hydrolyzing lysophosphatidylethanolamine. When it has, the container 1 should just be made into the container 1 which contains the enzyme which has the effect | action which decomposes | disassembles lysophosphatidylethanolamine further and eliminates it as a content.
 
本発明の実施の形態の認知症検査試薬キットは、以下の（１ａ）～（３ａ）´を含み、（１ａ）～（３ａ）´は被験者の血清または血漿中のＰｌｓＥｔｎ以外の物質を消去するための消去用容器１と、ＰｌｓＥｔｎを測定するための容器２とに分けることができる。
The dementia test reagent kit of the embodiment of the present invention includes the following (1a) to (3a) ′, wherein (1a) to (3a) ′ erase substances other than PlsEtn in the serum or plasma of the subject It can be divided into an erasing container 1 for measuring and a container 2 for measuring PlsEtn.
 
（１ａ）ＰｌｓＥｔｎをｌｙＰｌｓＥｔｎに加水分解できる酵素およびその酵素反応を進めることのできる添加物。
（２ａ）´ｌｙＰｌｓＥｔｎをプラズメニルホスファチジン酸とエタノールアミンに分解でき、かつエーテル型リゾホスファチジルエタノールアミンを加水分解でき、かつリゾホスファチジルエタノールアミンを加水分解できる加水分解酵素およびその酵素反応を進めることのできる添加物。
（３ａ）´エタノールアミンをグリコールアルデヒド、アンモニア、及び過酸化水素に変換する酵素とその酵素反応を進めるにことのできる添加物、さらに必要により過酸化水素を定量するための過酸化水素定量手段。
(1a) An enzyme capable of hydrolyzing PlsEtn to lyPlsEtn and an additive capable of proceeding with the enzyme reaction.
(2a) Hydrolyzing enzyme capable of decomposing 'lyPlsEtn into plasmenylphosphatidic acid and ethanolamine, hydrolyzing ether-type lysophosphatidylethanolamine, and hydrolyzing lysophosphatidylethanolamine, and the enzymatic reaction thereof Additive.
(3a) 'An enzyme that converts ethanolamine into glycolaldehyde, ammonia, and hydrogen peroxide, an additive that can promote the enzyme reaction, and a hydrogen peroxide quantification means for quantifying hydrogen peroxide if necessary.
 
そのような試薬キットの好ましい例は、少なくとも上記（１ａ）及び（３ａ）´を含有する容器１：並びに少なくとも（２ａ）´を含有する容器２を含む、認知症検査試薬キットである。
A preferred example of such a reagent kit is a dementia test reagent kit including a container 1 containing at least (1a) and (3a) ′ and a container 2 containing at least (2a) ′.
 
被験者の血清及び／又は血漿中にホスファチジルエタノールアミンが含まれ、かつ、ｌｙＰｌｓＥｔｎをプラズメニルホスファチジン酸とエタノールアミンに加水分解する作用を触媒する酵素がリゾホスファチジルエタノールアミンを加水分解する作用を有する場合は、容器１にさらにリゾホスファチジルエタノールアミンを分解し、実質的に消去する作用を有する酵素を内容物として含有する容器１とすれば良い。
When phosphatidylethanolamine is contained in the serum and / or plasma of a subject and the enzyme that catalyzes the action of hydrolyzing lyPlsEtn to plasmenyl phosphatidic acid and ethanolamine has the action of hydrolyzing lysophosphatidylethanolamine Further, the container 1 may be a container 1 that further contains an enzyme having the action of decomposing and substantially eliminating lysophosphatidylethanolamine as a content.
 
本実施の形態の認知症検査試薬キットは、少なくとも既知量のＰｌｓＥｔｎを含むキャリブレーション試薬とともに用いてもよい。上記のキャリブレーション試薬は、例えば、少なくとも既知量のＰｌｓＥｔｎ及び／又はｌｙＰｌｓを含む試薬であり、好ましくはｐＨ緩衝剤、アジ化ナトリウムや抗生物質等の防腐剤、糖等の安定化剤を含む。これらの種類や濃度等の条件等は上記認知症検査試薬キットに関して記載した条件を参照して当業者であれば適宜決定することができる。キャリブレーション方法は、一点検量の他、多点検量（折れ線やスプライン）や多点検量の直線回帰などが選択できる。
The dementia test reagent kit of the present embodiment may be used together with a calibration reagent containing at least a known amount of PlsEtn. The calibration reagent is, for example, a reagent containing at least a known amount of PlsEtn and / or lyPls, and preferably contains a pH buffer, a preservative such as sodium azide or antibiotics, and a stabilizer such as sugar. The conditions such as the type and concentration can be appropriately determined by those skilled in the art with reference to the conditions described for the dementia test reagent kit. As the calibration method, a single inspection quantity, a multi-inspection quantity (a broken line or a spline), a linear regression of the multi-inspection quantity, and the like can be selected.
 
キャリブレーション試薬中のＰｌｓＥｔｎの既知量は特に限定されず、被験者の血清または血漿中のＰｌｓＥｔｎを正確に測定することができるよう、適宜選択すればよい。
The known amount of PlsEtn in the calibration reagent is not particularly limited, and may be appropriately selected so that PlsEtn in the serum or plasma of the subject can be accurately measured.
 
また、本実施の形態の認知症検査試薬キットのキャリブレーション試薬の場合、ＰｌｓＥｔｎの既知量の下限値は１０μＭ以上、好ましくは５０μＭ以上、更に好ましくは８０μＭ以上、上限値は５００μＭ以下、好ましくは３００μＭ以下、更に好ましくは２５０μＭ以下とすることができる。
In the case of the calibration reagent of the dementia test reagent kit of the present embodiment, the lower limit value of the known amount of PlsEtn is 10 μM or more, preferably 50 μM or more, more preferably 80 μM or more, and the upper limit value is 500 μM or less, preferably 300 μM. In the following, it can be more preferably 250 μM or less.
 
本実施の形態の認知症検査試薬キット及び上述のキャリブレーション試薬は、液状品、液状品の凍結物、液状品の凍結乾燥品又は液状品の乾燥品（加熱乾燥及び／又は風乾及び／又は減圧乾燥等による）等の形態で提供することができる。また、例えば、ポイントオブケア装置のキャピラリーへの使用又は酵素センサーとしての使用の場合、各成分の濃度は通常よりも高い濃度が好ましく、例えば、固定化したり、紙や膜に染み込ませたり、ゲル・ゾル状認知症検査試薬キットとしたりして使用することが好ましい。
The dementia test reagent kit of the present embodiment and the calibration reagent described above are liquid products, frozen products of liquid products, freeze-dried products of liquid products, or dried products of liquid products (heat-dried and / or air-dried and / or reduced pressure). For example, by drying). Also, for example, in the case of use in a capillary of a point-of-care device or use as an enzyme sensor, the concentration of each component is preferably higher than usual. For example, it is fixed, soaked in paper or a film, gel -It is preferable to use it as a sol-like dementia test reagent kit.
 
以下、本発明を参考例及び実施例（実施例等）に基づいて説明するが、本発明の範囲は以下の実施例等に限定して解釈されるものではない。
EXAMPLES Hereinafter, although this invention is demonstrated based on a reference example and an Example (Example etc.), the range of this invention is limited to a following example etc. and is not interpreted.
 
本実施例等において、被験者は、東京都健康長寿医療センター「もの忘れ」外来来院者および高齢健常者「脳画像診断」ボランティアの中で、同センター倫理委員会の承認下、インフォームドコンセントが得て血液および尿を採取した。
In this example, the subject obtained informed consent with the approval of the ethics committee of the Tokyo Metropolitan Health and Longevity Medical Center “Forgetfulness” outpatient and elderly healthy “Brain Imaging” volunteers. Blood and urine were collected.
 
血清プラスマローゲン（コリン型プラスマローゲン：ＰｌｓＣｈｏ、エタノールアミン型プラスマローゲン：ＰｌｓＥｔｎ）以外の他のバイオマーカー（ＷＢＣ、ＲＢＣ、Ｈｂ、Ｈｔ、ＭＣＶ、ＭＣＨ、ＭＣＨＣ、ＰＬＴ、ＴＰ、Ａｌｂ、Ｔ－Ｂｉｌ、Ｃｒｅ、ＵＡ、β２－ｕｂ、ＡＭＹ、ＧＯＴ、ＧＰＴ、ＬＤＨ、Ｎａ、Ｃｌ、Ｋ、ＴＳＨ、ＦｒｅｅＴ３、ＦｒｅｅＴ４、Ｇｌｕ、ＨｂＡ１ｃ、ＣＲＰ、ＴＮＦ－α、ＩＬ－６、ＴＧ、ＴＣ、ＨＤＬ－Ｃ、ＬＤＬ－Ｃ、ＰＬ、ＶＢ９、ＶＢ１２、α－Ｔｏｃ、β－Ｔｏｃ、γ－Ｔｏｃ、Ａβ１－４０およびＡβ１－４２）は、株式会社エスアールエル（東京都新宿区西新宿２－１－１）にて測定した。
Other biomarkers (WBC, RBC, Hb, Ht, MCV, MCH, MCHC, PLT, TP, Alb, T-Bil, other than serum plasmalogens (choline type plasmalogen: PlsCho, ethanolamine type plasmalogen: PlsEtn) Cre, UA, β2-ub, AMY, GOT, GPT, LDH, Na, Cl, K, TSH, FreeT3, FreeT4, Glu, HbA1c, CRP, TNF-α, IL-6, TG, TC, HDL-C, LDL-C, PL, VB9, VB12, α-Toc, β-Toc, γ-Toc, Aβ1-40 and Aβ1-42) are available at SRL, Inc. (2-1-1 Nishi-Shinjuku, Shinjuku-ku, Tokyo) It was measured.
 
自然排泄尿ＭＩ（尿ＭＩ）はミオイノシトール測定試薬（ルシカＭＩ、旭化成ファーマ株式会社、東京都千代田区神田神保町１丁目１０５）で測定し、１日あるいは３日分の平均値をＣｒｅで標準化した値（ｍｇ／ｇＣｒ）で示した。
Spontaneously excreted urine MI (urine MI) was measured with a myo-inositol measurement reagent (Lucica MI, Asahi Kasei Pharma Co., Ltd., 1-chome, Kanda Jimbocho, Chiyoda-ku, Tokyo), and the average value for one or three days was standardized with Cre. It was expressed as a value (mg / gCr).
 
認知機能検査はＣＤＲ、ＨＤＳ－Ｒ、ＭＭＳＥおよびＭｏＣＡ－Ｊを行ない、うつ状態の検査として「老年期うつ病評価尺度（ＧＤＳ）」による検査を実施した。
For the cognitive function test, CDR, HDS-R, MMSE and MoCA-J were performed, and a test based on the “Geriatric Depression Rating Scale (GDS)” was performed as a test for depression.
 
本実施例等で用いた試薬類としては、特に断らない限り、和光純薬工業株式会社、シグマアルドリッチ社等市販され容易に入手することができる任意のものを使用し、試薬メーカー、純度、価格等は特に限定されず、必要に応じて当業者であれば適宜選択して用いることができる。ヒトプール血清は、有限会社山久化成（東京都稲城市向陽台４－２－Ｃ－１００７）から購入したものを使用した。本実施例等に使用した技術は、例えば本明細書に記載の先行技術文献、マニアティスらの方法（Ｍａｎｉａｔｉｓ，Ｔ．ら、Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ、Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ、１９８２年及び１９８９年）、蛋白質・酵素の基礎実験法（改訂第２版、堀尾武一、１９９４年南光堂参照）及び市販の各種酵素又はキット類に添付された手順に従って、当業者であれば実施することができるものである。
As reagents used in the present examples, unless otherwise specified, Wako Pure Chemical Industries, Ltd., Sigma-Aldrich, etc., which are commercially available and can be easily obtained, reagent manufacturer, purity, price These are not particularly limited, and can be appropriately selected and used by those skilled in the art as needed. Human pooled serum was purchased from Yamakyu Kasei Co., Ltd. (Kyoyodai 4-2C-1007, Inagi City, Tokyo). The techniques used in the present examples include, for example, the prior art document described in this specification, the method of Maniatis et al. (Maniatis, T. et al., Molecular Cloning, Cold Spring Harbor Laboratory, 1982 and 1989), Those skilled in the art can carry out the basic experiment method of the enzyme (Ref. 2nd edition, Takeichi Horio, 1994 Nankodo) and procedures attached to various commercially available enzymes or kits.
 
ｌｙＰｌｓ　ａｓｅの酵素活性は以下の様にして測定した。
The enzyme activity of lyPlsase was measured as follows.
 
石英製の１．０ｃｍキュベットに反応試薬混合液を１ｍＬとり、３７℃で５分間予備加温する。１２０ｍＭ　マンニトール及び７０ｍＭシュークロースを含む２０ｍＭ　Ｔｒｉｓ／ＨＣｌ緩衝液（ｐＨ７．５）で適当な濃度に希釈した本発明のｌｙＰｌｓ　ａｓｅ　２０μＬを反応試薬混合液に混和して反応を開始する。５４６ｎｍにおける吸光度を１０分間測定し、求められた吸光変化をＡｓ／ｍｉｎ、本発明の蛋白質溶液の代わりに精製水を用いた盲検をＡｂ／ｍｉｎとして、１分間に１μｍｏｌのＰｌｓＥｔｎを酸化する酵素量を１Ｕとし、酵素活性（Ｕ／ｍＬ）を下記［式］により算出する。
Take 1 mL of the reaction reagent mixture in a 1.0 cm cuvette made of quartz and preheat at 37 ° C. for 5 minutes. 20 μL of lyPlsase of the present invention diluted to an appropriate concentration with 20 mM Tris / HCl buffer (pH 7.5) containing 120 mM mannitol and 70 mM sucrose is mixed with the reaction reagent mixture to start the reaction. An enzyme that oxidizes 1 μmol of PlsEtn per minute by measuring absorbance at 546 nm for 10 minutes, assuming the change in absorbance to be As / min, and blinding using purified water instead of the protein solution of the present invention as Ab / min. The amount is 1 U, and the enzyme activity (U / mL) is calculated by the following [formula].
　酵素活性（Ｕ／ｍＬ）
＝｛（Ａｓ／ｍｉｎ－Ａｂ／ｍｉｎ）／１８｝×１．０２／０．０２×希釈倍数・・［式］
 
［反応試薬混合液］
５０ｍＭ      ＢＥＳ／ＮａＯＨ緩衝液　ｐＨ７．５
５Ｕ／ｍＬ    ＧＰＣＰ（旭化成ファーマ株式会社（Ｔ－３３）
５Ｕ／ｍＬ    ＰＯＤ　（ＳＩＧＭＡ（Ｐ８２５０）
０．０２％    ＴＯＤＢ（同仁化学研究所（ＯＣ２２））
０．０３％    ４－ＡＡ（０１２－１４４７１）
８ｍＭ        ｌｙＰｌｓＥｔｎ
　　　　（Ａｖａｎｔｉ　Ｐｏｌａｒ　Ｌｉｐｉｄｓ，Ｉｎｃ．（８５００９５））
 
本実施例等で用いた下記の［日立７０８０形自動分析機パラメーター］は、日立７０８０形自動分析機の取扱説明書等を参考にして、当業者であれば容易に設定し、該分析機を使用することができる。［日立７０８０形自動分析機パラメーター１］を例に、以下に簡単に分析機の使用方法を説明する。分析法（比色分析）は、１０分反応の２ポイントエンド法で、反応開始後約１０分後と５分後の吸光度の差を出力する（セルブランク、水ブランク、試薬量等の補正後に出力される）。測定波長は、主波長が６６０ｎｍであり、副波長は７５０ｎｍである。サンプル量（血清及び／又は血漿の量）は１２μＬ、最初の工程のための検査試薬キット１はＲ１として１６０μＬ使用し、次の工程のための検査試薬キット２はＲ３として４０μＬ使用する。なお、パラメーター中の空欄（［　］）は、空欄にしておくという意味である。 Enzyme activity (U / mL)
= {(As / min−Ab / min) / 18} × 1.02 / 0.02 × dilution factor ··· [formula]

[Reaction reagent mixture]
50 mM BES / NaOH buffer pH 7.5
5U / mL GPCP (Asahi Kasei Pharma Corporation (T-33)
5U / mL POD (SIGMA (P8250)
0.02% TODB (Dojindo Laboratories (OC22))
0.03% 4-AA (012-14471)
8 mM lyPlsEtn
(Avanti Polar Lipids, Inc. (850095))

The following [Hitachi 7080 automatic analyzer parameters] used in this example and the like are easily set by those skilled in the art with reference to the instruction manual of the Hitachi 7080 automatic analyzer. Can be used. Taking the “Hitachi 7080 automatic analyzer parameter 1” as an example, the method of using the analyzer will be briefly described below. The analysis method (colorimetric analysis) is a two-point end method of 10-minute reaction, and outputs the difference in absorbance after about 10 minutes and 5 minutes after the start of the reaction (after correction of cell blank, water blank, reagent amount, etc.) Output). The measurement wavelength has a dominant wavelength of 660 nm and a sub wavelength of 750 nm. The sample volume (serum and / or plasma volume) is 12 μL, the test reagent kit 1 for the first step is used as 160 μL as R1, and the test reagent kit 2 for the next step is used as 40 μL as R3. Note that the blank ([]) in the parameter means to leave it blank.
 
［日立７０８０形自動分析機パラメーター１］
分析法：　　　　　　　［２ポイントエンド］［１０］［１６］［３１］［０］［０］
波長（副波長／主波長）：［７５０］／［６６０］
検体量：　　　　　　　　種別１
標準：　　　　　　　　［１２．０］［０．０］［０］
試薬分注量
　Ｒ１：　　　　　　　［１６０］［０］［　］［９９］
　Ｒ２：　　　　　　　［０］［０］［　］［９９］
　Ｒ３：　　　　　　　［４０］［０］［　］［９９］
　Ｒ４：　　　　　　　［０］［０］［　］［９９］
 
［日立７０８０形自動分析機パラメーター２］
分析法：　　　　　　　［２ポイントエンド］［１０］［１６］［３１］［０］［０］
波長（副波長／主波長）：［７５０］／［６６０］
検体量：　　　　　　　　種別１
標準：　　　　　　　　［６．０］［０．０］［０］
試薬分注量
　Ｒ１：　　　　　　　［１６０］［０］［　］［９９］
　Ｒ２：　　　　　　　［０］［０］［　］［９９］
　Ｒ３：　　　　　　　［４０］［０］［　］［９９］
　Ｒ４：　　　　　　　［０］［０］［　］［９９］
 
以下に示した測定値等は、様々な条件、例えば、試薬の純度、測定の条件、使用機器の精度等、使用機器の置かれた温度や気圧等の雰囲気により変化し得るが、同条件で測定した場合には以下の実施例等で得られたものと同じ傾向を示す結果が得られることは当業者であれば理解できるであろう。
[Hitachi 7080 automatic analyzer parameter 1]
Analysis method: [2 point end] [10] [16] [31] [0] [0]
Wavelength (subwavelength / main wavelength): [750] / [660]
Sample volume: Type 1
Standard: [12.0] [0.0] [0]
Reagent dispensing amount R1: [160] [0] [] [99]
R2: [0] [0] [] [99]
R3: [40] [0] [] [99]
R4: [0] [0] [] [99]

[Hitachi 7080 automatic analyzer parameter 2]
Analysis method: [2 point end] [10] [16] [31] [0] [0]
Wavelength (subwavelength / main wavelength): [750] / [660]
Sample volume: Type 1
Standard: [6.0] [0.0] [0]
Reagent dispensing amount R1: [160] [0] [] [99]
R2: [0] [0] [] [99]
R3: [40] [0] [] [99]
R4: [0] [0] [] [99]

The measurement values shown below may vary depending on various conditions, for example, the purity of the reagent, the measurement conditions, the accuracy of the equipment used, and the atmosphere such as the temperature and pressure of the equipment used. Those skilled in the art will understand that when measured, results showing the same tendency as those obtained in the following Examples and the like can be obtained.
 
［実施例１：尿ＭＩ定量値と血清及び／又は血漿ＰｌｓＥｔｎ定量値による認知症検査］
本実施例では、血清及び／又は血漿ＰｌｓＥｔｎ量は特許文献１に記載された放射性ヨウ素－高速液体クロマトグラフィー法（１２５Ｉ－ＨＰＬＣ法）で測定した。本実施例では、空腹時二番尿中のＭＩを測定した。 [Example 1: Dementia test by urine MI quantitative value and serum and / or plasma PlsEtn quantitative value]
In this example, the amount of serum and / or plasma PlsEtn was measured by a radioactive iodine-high performance liquid chromatography method (125I-HPLC method) described in Patent Document 1. In this example, MI in fasting second urine was measured.
 
１．血清ＰｌｓＥｔｎ量
全測定データ（表１－１、１－２）を基に、認知機能と相関性を示す項目を検索したところ、年齢、平均赤血球ヘモグロビン濃度（ＭＣＨＣ）、遊離トリヨードサイロニン（ＦｒｅｅＴ３）、アルブミン（Ａｌｂ）、グルコース（Ｇｌｕ）、インターロイキン６（ＩＬ－６）に加えプラスマローゲン関連（ＰｌｓＥｔｎ、ＰｌｓＣｈｏ／ＰｌｓＥｔｎ比、全リン脂質中のＰｌｓＥｔｎ：ＰｌｓＥｔｎ／ＰＬ）項目で有意な相関を示すことが確認された（表２）。これらの項目についてＣＤＲ別に有意差検定を行なったところ、ＣＤＲ３群（０、０．５、１≦）すべてで有意差がみられた項目は認知機能検査（ＨＤＳ－Ｒ、ＭＭＳＥ、ＭｏＣＡ－Ｊ）を除いてはＰｌｓＥｔｎとＰｌｓＥｔｎ／ＰＬのみであった（表３）。以上の解析から、血清ＰｌｓＥｔｎは認知症を分類するバイオマーカーとして機能することが確認された。
1. Based on the serum PlsEtn total measurement data (Tables 1-1 and 1-2), items showing a correlation with cognitive function were searched, and age, mean erythrocyte hemoglobin concentration (MCHC), free triiodothyronine (FreeT3) ), Albumin (Alb), glucose (Glu), interleukin 6 (IL-6) plus plasmalogen-related (PlsEtn, PlsCho / PlsEtn ratio, PlsEtn: PlsEtn / PL in all phospholipids) It was confirmed to show (Table 2). When significant difference test was performed for each of these items by CDR, items that showed significant differences in all CDR3 groups (0, 0.5, 1 ≦) were cognitive function tests (HDS-R, MMSE, MoCA-J). Except for PlsEtn and PlsEtn / PL (Table 3). From the above analysis, it was confirmed that serum PlsEtn functions as a biomarker for classifying dementia.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000006
  
２．尿ＭＩ量
尿ＭＩ量の世代別・性別測定データを基に、群間での有意差検定を行なったところ、男女とも高齢者群で尿ＭＩ量が有意に増加することが確認された。高齢者群の尿ＭＩ量は、若年および中年者群と比べて個人差がきわめて大きかった。また、高齢者の中の健常者と認知症患者の間では尿ＭＩ量に有意差を認めなかった（図１、表４）。
Figure JPOXMLDOC01-appb-T000006
2. Urinary MI amount Based on generational / gender measurement data of urinary MI amount, a significant difference test was performed between groups, and it was confirmed that the urinary MI amount increased significantly in the elderly group for both men and women. The amount of urinary MI in the elderly group was extremely different among individuals compared to the young and middle-aged groups. In addition, there was no significant difference in urinary MI between healthy individuals and dementia patients among the elderly (FIG. 1, Table 4).
Figure JPOXMLDOC01-appb-T000007
  
全測定データ（表１）を基に、尿ＭＩ量と相関性を示す項目を検索したところ、「もの忘れ」群のみでプラスマローゲン関連（ＰｌｓＥｔｎ、ＰｌｓＣｈｏ＋ＰｌｓＥｔｎ、ＰｌｓＣｈｏ／ＰｌｓＥｔｎ比）項目とヘモグロビンＡ１ｃ（ＨｂＡ１ｃ）および腫瘍壊死因子α（ＴＮＦ－α）で尿ＭＩと有意な相関を示すことが確認された（表５－１、５－２）。
Figure JPOXMLDOC01-appb-T000007
Based on the entire measurement data (Table 1), the items showing the correlation with the urinary MI amount were searched. It was confirmed that HbA1c) and tumor necrosis factor α (TNF-α) showed a significant correlation with urine MI (Tables 5-1 and 5-2).
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000009
  
ＰｌｓＥｔｎ量、尿ＭＩ量およびＨｂＡ１ｃ量の３者の関係に注目して、尿ＭＩ量およびＨｂＡ１ｃ量がＰｌｓＥｔｎ量に及ぼす影響を検討した。健常者群と「もの忘れ」群ごとに、尿ＭＩ量およびＨｂＡ１ｃ量を全測定値の平均値を境に４群に分割して、各群でのＰｌｓＥｔｎ量を比較した。健常高齢者ではＰｌｓＥｔｎ量に４群間で有意差を認めなかった。一方、「もの忘れ」群では、高ＨｂＡ１ｃ者（＞５．５　％）において高尿ＭＩ者（＞３６．６ｍｇ／ｇＣｒ）と低尿ＭＩ者間でＰｌｓＥｔｎ量に有意差を認めた。また、高尿ＭＩ者では健常者と「もの忘れ」群間でＰｌｓＥｔｎ量に有意差を認めた（図２）。以上の解析により、尿ＭＩ量を考慮する（高ＭＩ者を選択する）ことで、血清及び／又は血漿ＰｌｓＥｔｎ量の定量による認知症患者の検出確度（そして認知機能の評価能）が高まることが推測された。
Figure JPOXMLDOC01-appb-T000009
Paying attention to the relationship between the PlsEtn amount, the urine MI amount, and the HbA1c amount, the effects of the urine MI amount and the HbA1c amount on the PlsEtn amount were examined. The urine MI amount and the HbA1c amount were divided into 4 groups with the average value of all measured values as a boundary for each of the healthy group and the “forgetful” group, and the PlsEtn amount in each group was compared. In the healthy elderly, there was no significant difference between the four groups in the PlsEtn amount. On the other hand, in the “forgetful” group, there was a significant difference in PlsEtn amount between high urine MI (> 36.6 mg / gCr) and low urine MI in high HbA1c (> 5.5%). Moreover, in the high urine MI person, a significant difference was observed in the PlsEtn amount between the healthy person and the “forgetful” group (FIG. 2). Based on the above analysis, considering the urinary MI amount (selecting a high MI person), the detection accuracy (and the ability to evaluate cognitive function) of patients with dementia by quantification of the serum and / or plasma PlsEtn amount can be increased. Was guessed.
 
３．尿ＭＩ量＋血清及び／又は血漿ＰｌｓＥｔｎ量
全高齢者（健常＋「もの忘れ」群）を対象に、血清ＰｌｓＥｔｎ（ＥＰ）量と尿ＭＩ量の全測定値の平均値を境に４群に分割して、各群での認知機能検査結果を比較した。認知機能検査（ＣＤＲ、ＨＤＳ－Ｒ、ＭＭＳＥ）の結果に対応させた場合、血清ＰｌｓＥｔｎ（ＥＰ）量（低レベル）かつ尿ＭＩ量（高レベル）群は他の３群と比較していずれも有意な認知機能低下を示した（図３、表６）。以上の解析から、血清ＰｌｓＥｔｎ量と尿ＭＩ量の測定を組み合わせることで、高精度で認知症患者を分類できることが確認された。
3. Urinary MI level + serum and / or plasma PlsEtn amount for all elderly (healthy + “Forgetfulness” group), 4 groups with the average of all measured values of serum PlsEtn (EP) amount and urinary MI amount as the boundary Divided and compared cognitive function test results in each group. When matched to the results of cognitive function tests (CDR, HDS-R, MMSE), the serum PlsEtn (EP) level (low level) and urinary MI level (high level) group are all compared to the other three groups. Significant cognitive decline was observed (Figure 3, Table 6). From the above analysis, it was confirmed that dementia patients can be classified with high accuracy by combining the measurement of serum PlsEtn amount and urine MI amount.
Figure JPOXMLDOC01-appb-T000010
  
さらに、血清ＰｌｓＥｔｎ量が５８μＭ未満、尿ＭＩ量が２５ｍｇ／ｇＣｒ以上をそれぞれの基準とした場合の検出確度を調べたところ、７２．２％（８３／１１５）の確度で認知症患者を検出することができた（図４）。
Figure JPOXMLDOC01-appb-T000010
Furthermore, when the detection accuracy when the serum PlsEtn amount was less than 58 μM and the urine MI amount was 25 mg / gCr or more was used as the standard, a patient with dementia was detected with an accuracy of 72.2% (83/115). (Fig. 4).
 
また、全高齢者、尿ＭＩ量＞２５ｍｇ／ｇＣｒ高齢者群および尿ＭＩ量＞３６．６ｍｇ／ｇＣｒ高齢者群の３群について、血清ＰｌｓＥｔｎの認知機能正常（ＣＤＲ値：０）と認知機能非正常（ＣＤＲ値：０．５以上）を分別する精度について、Ｒｅｃｅｉｖｅｒ　Ｏｐｅｒａｔｉｎｇ　Ｃｈａｒａｃｔｅｒｉｓｔｉｃ（ＲＯＣ）解析によるＲＯＣ曲線下の面積（Ａｒｅａ　ｕｎｄｅｒ　ｔｈｅ　ｃｕｒｖｅ：ＡＵＣ）を求めることによって調べた。その結果、尿ＭＩ量が高レベルの高齢者を対象にするほど血清ＰｌｓＥｔｎの分別能が高くなることが示された。すなわち、尿ＭＩ量＞３６．６ｍｇ／ｇＣｒの高齢者を対象とした場合の血清ＰｌｓＥｔｎ量の分別能（ＡＵＣ）は０．８３８と、対面式認知機能検査（ＨＤＳ－Ｒ、ＭＭＳＥ、ＭｏＣＡ－Ｊ）のＡＵＣ（０．８７６～０．８９４）に匹敵した（図５）。
Moreover, cognitive function normal (CDR value: 0) of serum PlsEtn and non-cognitive function were observed in all three groups, the elderly group of urinary MI> 25 mg / gCr and the group of urinary MI> 36.6 mg / gCr. The accuracy of fractionation of normal (CDR value: 0.5 or more) was examined by determining the area under the ROC curve (Area under the curve: AUC) by Receiver Operating Characteristic (ROC) analysis. As a result, it was shown that the fractionation ability of serum PlsEtn increases as the target of elderly people with high levels of urine MI. In other words, the serum PlsEtn fractionation ability (AUC) for elderly people with urinary MI amount> 36.6 mg / gCr was 0.838, and the face-to-face cognitive function test (HDS-R, MMSE, MoCA-J) ) AUC (0.876-0.894) (Fig. 5).
 
さらに、血清ＰｌｓＥｔｎ量の認知機能評価能と対面式認知機能検査法との相関性を、対象者を全高齢者、尿ＭＩ量＞２５ｍｇ／ｇＣｒ高齢者群および尿ＭＩ量＞３６．６ｍｇ／ｇＣｒ高齢者群の３群について検討したところ、尿ＭＩ量が高レベルの高齢者を対象にするほど血清ＰｌｓＥｔｎ量と認知機能検査結果との相関性が高くなることが示された（図６、表７）。以上の結果から、血清ＰｌｓＥｔｎ量の測定と尿ＭＩ量の測定を組み合わせることで、高確度で認知症患者を分類できるようになり、さらには血清ＰｌｓＥｔｎ量を指標とした認知機能評価能が高まることが確認された。
Furthermore, the correlation between the ability to evaluate the cognitive function of the serum PlsEtn amount and the face-to-face cognitive function test method is as follows. Examination of the three groups of the elderly group showed that the correlation between the serum PlsEtn amount and the cognitive function test results was increased as the urinary MI level was increased (FIG. 6, Table). 7). From the above results, combining measurement of serum PlsEtn and measurement of urinary MI makes it possible to classify patients with dementia with high accuracy, and further enhances cognitive function evaluation ability using serum PlsEtn as an index. Was confirmed.
Figure JPOXMLDOC01-appb-T000011
  
［実施例２：随時尿中と空腹時二番尿中のＭＩ定量値の比較］
本実施例では、随時尿中と空腹時二番尿中のＭＩ定量値を比較し、本発明の実施の形態の認知症検査方法において随時尿が空腹時二番尿に代替できることを確認した。
Figure JPOXMLDOC01-appb-T000011
[Example 2: Comparison of MI quantitative values in urine from time to time and fasting second urine]
In this example, the quantitative values of MI in ad hoc urine and fasting second urine were compared, and in the dementia test method according to the embodiment of the present invention, it was confirmed that the occasional urine could be replaced with fasting second urine.
 
５０人の被験者から、連日あるいは適当な隔日（１ヶ月以内）の３日分の随時尿を採尿してＭＩを定量し平均値を随時尿中のＭＩ定量値（ｙ軸）とした。被験者から、連日あるいは適当な隔日（１ヶ月以内）の３日分の空腹時二番尿を採尿してＭＩを定量し平均値を空腹時二番尿のＭＩ定量値（ｘ軸）とした。それぞれの定量値の相関図を図１０に示した。相関式はｙ　＝１．０５ｘ＋６．６４、相関係数ｒ＝０．８４５と高い相関があったので、随時尿が空腹時二番尿に代替できることが確認できた。
From 50 subjects, urine was collected every day or every other day (within 1 month) for 3 days, and the MI was quantified, and the average value was taken as the urine MI quantification value (y-axis). MI was quantified by collecting urine from fasting second urine for three days every day or every other suitable day (within one month) from the subject, and the average value was determined as the MI quantification value (x axis) of fasting second urine. The correlation diagram of each quantitative value is shown in FIG. Since the correlation equation was highly correlated with y = 1.05x + 6.64 and correlation coefficient r = 0.845, it was confirmed that urine from time to time could be replaced with fasting second urine.
 
また、本実施例に用いた５０人の被験者の随時尿中ＭＩ量と空腹時二番尿中ＭＩ量の平均はそれぞれ３２．３ｍｇ／ｇＣｒと４０．５ｍｇ／ｇＣｒであり、空腹時二番尿中ＭＩ量＋７．８＝随時尿中ＭＩ量の関係になる場合があることが分かった。
In addition, the average of the urinary MI amount and the fasting second urine MI amount of the 50 subjects used in this example were 32.3 mg / gCr and 40.5 mg / gCr, respectively. It was found that the relationship between medium MI amount + 7.8 = urinary MI amount was sometimes obtained.
 
［実施例３：酵素を利用した血清、血漿等中のＰｌｓＥｔｎの定量］
［組成物１－１ａ］
２０ｍＭ      ＢＥＳ／ＮａＯＨ緩衝液　ｐＨ７．５
０．０５ｍＭ  ＡＬＰＳ（同仁化学研究所（ＯＣ０４））
２ｍＭ　      ＣａＣｌ２
７０ｍＭ      Ｓｕｃｒｏｓｅ
１２０ｍＭ    Ｍａｎｎｉｔｏｌ
５Ｕ／ｍＬ    ＧＰＣＰ（旭化成ファーマ株式会社（Ｔ－３３））
４０Ｕ／ｍＬ  特開２００５－５２０３４号公報のＴＯＤ
５Ｕ／ｍＬ    国際公開番号：ＷＯ２０１２／１０５５６５　Ａ１のＰＬ
１０Ｕ／ｍＬ  ＰＯＤ　（ＳＩＧＭＡ（Ｐ８２５０））
５Ｕ／ｍＬ    ＡＳＯＭ（旭化成ファーマ株式会社（Ｔ－５３））
 
［組成物２－１ａ］
２０ｍＭ      ＢＥＳ／ＮａＯＨ緩衝液　ｐＨ７．５
１ｍＭ        Ｎａ２ＳＯ４
０．１２ｍＭ  ＤＡ－６７（和光純薬工業株式会社（０４６－２２３４１））
７０ｍＭ      Ｓｕｃｒｏｓｅ
１２０ｍＭ    Ｍａｎｎｉｔｏｌ
５Ｕ／ｍＬ    Ｐｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ由来ｌｙＰｌｓ　ａｓｅ
２％          β－ｃｙｃｌｏｄｅｘｔｒｉｎ
０．１％      ＤＤＭ（同仁化学研究所（Ｄ３８２））
 
上記の［組成物１－１ａ］と［組成物２－１ａ］を調製し、それぞれＲ１、Ｒ２として、［日立７０８０形自動分析機パラメーター１］で日立７０８０形自動分析機を用いて、０、２５、５０、・・・、１２５、１５０μＭの各濃度になるように、ＰｌｓＥｔｎ（Ａｖａｎｔｉ　Ｐｏｌａｒ　Ｌｉｐｉｄｓ，Ｉｎｃ．Ａｌａｂａｍａ、ＵＳＡ、８５２８０４Ｃ）をプール血清と０．９％食塩水に添加した検体を測定した。
[Example 3: Determination of PlsEtn in serum, plasma, etc. using enzyme]
[Composition 1-1a]
20 mM BES / NaOH buffer pH 7.5
0.05 mM ALPS (Dojindo Laboratories (OC04))
2 mM CaCl 2
70 mM sucrose
120 mM Mannitol
5U / mL GPCP (Asahi Kasei Pharma Corporation (T-33))
40 U / mL TOD of JP-A-2005-52034
5U / mL International publication number: PL of WO2012 / 105565 A1
10U / mL POD (SIGMA (P8250))
5U / mL ASOM (Asahi Kasei Pharma Corporation (T-53))

[Composition 2-1a]
20 mM BES / NaOH buffer pH 7.5
1 mM Na 2 SO 4
0.12 mM DA-67 (Wako Pure Chemical Industries, Ltd. (046-22231))
70 mM sucrose
120 mM Mannitol
5U / mL Pseudomonas putida derived lyPls as
2% β-cyclodextrin
0.1% DDM (Dojindo Laboratories (D382))

[Composition 1-1a] and [Composition 2-1a] were prepared, and R1 and R2, respectively, were set to 0, using a Hitachi 7080 automatic analyzer in [Hitachi 7080 automatic analyzer parameter 1]. Samples obtained by adding PlsEtn (Avanti Polar Lipids, Inc. Alabama, USA, 852804C) to pooled serum and 0.9% saline so as to have respective concentrations of 25, 50,..., 125, 150 μM were measured. .
 
結果を図１１に示した。図中白丸はＰｌｓＥｔｎをプール血清に添加した検体の定量値の検量線であり、ｙ＝２．１０ｘ＋１６４、相関係数ｒ＝０．９９７で酵素を利用した血清、血漿中のＰｌｓＥｔｎの定量ができた。黒丸はＰｌｓＥｔｎを０．９％食塩水に添加した検体の定量値の検量線であり、ｙ＝２．２８ｘ－５４、相関係数ｒ＝０．９９８で試料のＰｌｓＥｔｎの定量ができた。血清中ＰｌｓＥｔｎと０．９％食塩水中ＰｌｓＥｔｎの定量値の検量線の傾きが同じでかつ相関係数が１に近かった。
The results are shown in FIG. The white circle in the figure is a calibration curve of the quantitative value of the sample in which PlsEtn was added to the pooled serum, and it was possible to quantify PlsEtn in serum and plasma using enzyme with y = 2.10x + 164 and correlation coefficient r = 0.997. It was. A black circle is a calibration curve of a quantitative value of a sample obtained by adding PlsEtn to 0.9% saline, and PlsEtn of the sample was quantified with y = 2.28x−54 and a correlation coefficient r = 0.998. The slopes of the calibration curves of the quantitative values of PlsEtn in serum and PlsEtn in 0.9% saline were the same and the correlation coefficient was close to 1.
 
なお、血清中ＰｌｓＥｔｎの検量線の定量値の、切片は元々血清に含まれていたＰｌｓＥｔｎの影響であり、０．９％食塩水中ＰｌｓＥｔｎの定量値の検量線の切片は試薬ブランクの影響である。また、Ｐｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ由来ｌｙＰｌｓ　ａｓｅの製造方法は後述する。
The intercept of the calibration value of PlsEtn in serum is the effect of PlsEtn originally contained in the serum, and the intercept of the calibration curve of PlsEtn in 0.9% saline is the effect of the reagent blank. . Moreover, the manufacturing method of lyPlsase derived from Pseudomonas putida will be described later.
 
［組成物１－１ｂ］
５０ｍＭ             ＢＥＳ－ＮａＯＨ　ｐＨ７．５
１０Ｕ／ｍＬ         Ｐｅｒｏｘｉｄａｓｅ
５０Ｕ／ｍＬ         Ｅｔｈａｎｏｌａｍｉｎｅ　ｏｘｉｄａｓｅ
５Ｕ／ｍＬ           ＧＰＣＰ
１Ｕ／ｍＬ           国際公開番号：ＷＯ２０１２／１０５５６５　Ａ１のＰＬ
 
［組成物１－１ｂ］
５０ｍＭ             ＢＥＳ－ＮａＯＨ　ｐＨ７．５
４．２Ｕ／ｍＬ              ラット由来のｌｙＰｌｓ　ａｓｅ
１２μＭ             ＤＡ６７
 
ここで、ラット由来のｌｙＰｌｓ　ａｓｅはＴｈｅ　ｊｏｕｒｎａｌ　ｏｆ　ｂｉｏｌｏｇｉｃａｌ　ｃｈｅｍｉｓｔｒｙ、２０１１年、２８６巻、２４９１６～２４９３０頁に記載の方法でラット由来のｌｙＰｌｓ　ａｓｅを大腸菌組換体として製造した。
[Composition 1-1b]
50 mM BES-NaOH pH 7.5
10U / mL Peroxidase
50U / mL Ethanolamine oxidase
5U / mL GPCP
1U / mL International publication number: PL of WO2012 / 105565 A1

[Composition 1-1b]
50 mM BES-NaOH pH 7.5
4.2 U / mL rat-derived lyPls as
12 μM DA67

Here, rat-derived lyPlsase was produced as a recombinant Escherichia coli from rats by the method described in The journal of biochemical chemistry, 2011, 286, 24916-24930.
 
１０％　ＤＤＭにＰｌｓＥｔｎを０～２００μＭになるように添加して検体とした。検量線は相関式ｙ＝２．２７ｘ－５３で表され、相関係数ｒ＝０．９９８であった。なお、切片が－５３となった理由は、上記と同様である。
PlsEtn was added to 10% DDM to a concentration of 0 to 200 μM to prepare a specimen. The calibration curve was expressed by the correlation equation y = 2.27x-53, and the correlation coefficient r = 0.998. The reason why the intercept is −53 is the same as described above.
 
本実施例により酵素を利用した血清、血漿等中のＰｌｓＥｔｎが正確に定量できることが示された。
This example showed that PlsEtn in serum, plasma, etc. using enzymes can be accurately quantified.
 
［実施例４：１２５Ｉ－ＨＰＬＣ法と酵素を利用した血清または血漿中のＰｌｓＥｔｎの定量値の比較１］
４７の血清検体を、次の２種類の方法で測定した：
（ｉ）１２５Ｉ－ＨＰＬＣ法、
（ｉｉ）Ｐｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ由来ｌｙＰｌｓ　ａｓｅを使用した酵素法（［組成物１－１ａ］と［組成物２－１ａ］）。
[Example 4: Comparison of quantified value of PlsEtn in serum or plasma using 125I-HPLC method and enzyme 1]
47 serum specimens were measured in two ways:
(I) 125I-HPLC method,
(Ii) Enzymatic method using lyPlsase derived from Pseudomonas putida ([Composition 1-1a] and [Composition 2-1a]).
 
それぞれの検査試薬キットをＲ１、Ｒ２として、［日立７０８０形自動分析機パラメーター２］で日立７０８０形自動分析機を使用して測定した。
Each test reagent kit was measured as R1 and R2 using a Hitachi 7080 automatic analyzer with [Hitachi 7080 automatic analyzer parameter 2].
 
それぞれの定量値の相関図を図１２に示した。相関式はｙ　＝０．５５ｘ＋２６．９、相関係数ｒ＝０．８８０と高い相関があり、Ｐｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉ１ｄａ由来ｌｙＰｌｓ　ａｓｅを使用した酵素法で検体中のＰｌｓＥｔｎが測定できた。
The correlation diagram of each quantitative value is shown in FIG. The correlation equation is highly correlated with y = 0.55x + 26.9 and the correlation coefficient r = 0.880, and PlsEtn in the sample could be measured by the enzyme method using lyPlsase derived from Pseudomonas puti1da.
 
［実施例５：１２５Ｉ－ＨＰＬＣ法と酵素を利用した血清または血漿中のＰｌｓＥｔｎの定量値の比較２］
４７の血清検体を、次の２種類の方法で測定した：
（ｉ）１２５Ｉ－ＨＰＬＣ法、
（ｉｉｉ）Ｔｈｅｒｍｏｃｒｉｓｐｕｍ　ｓｐ．を使用した酵素法（［組成物１－２ｃ］と［組成物２－２ｃ］）。
[Example 5: Comparison 2 of quantitative value of PlsEtn in serum or plasma using 125I-HPLC method and enzyme]
47 serum specimens were measured in two ways:
(I) 125I-HPLC method,
(Iii) Thermocrispum sp. Enzymatic method using [Composition 1-2c] and [Composition 2-2c].
 
それぞれの検査試薬キットをＲ１、Ｒ２として、［日立７０８０形自動分析機パラメーター２］で日立７０８０形自動分析機を使用して測定した。
Each test reagent kit was measured as R1 and R2 using a Hitachi 7080 automatic analyzer with [Hitachi 7080 automatic analyzer parameter 2].
 
［組成物１－２ｃ］
２０ｍＭ      ＢＥＳ／ＮａＯＨ緩衝液　ｐＨ７．５
０．０５ｍＭ  ＡＬＰＳ（同仁化学研究所（ＯＣ０４））
２ｍＭ　      ＣａＣｌ２
７０ｍＭ      Ｓｕｃｒｏｓｅ
１２０ｍＭ    Ｍａｎｎｉｔｏｌ
５Ｕ／ｍＬ    ＧＰＣＰ（旭化成ファーマ株式会社（Ｔ－３３））
４０Ｕ／ｍＬ  特開２００５－５２０３４号公報のＴＯＤ
５Ｕ／ｍＬ    国際公開番号：ＷＯ２０１２／１０５５６５　Ａ１のＰＬ
１０Ｕ／ｍＬ  ＰＯＤ　（ＳＩＧＭＡ（Ｐ８２５０））
５Ｕ／ｍＬ    ＡＳＯＭ（旭化成ファーマ株式会社（Ｔ－５３））
５Ｕ／ｍＬ    ＬＹＰＬ（旭化成ファーマ株式会社（Ｔ－５９））
 
［組成物２－２ｃ］
２０ｍＭ      ＢＥＳ／ＮａＯＨ緩衝液　ｐＨ７．５
１ｍＭ        Ｎａ２ＳＯ４
０．１２ｍＭ  ＤＡ－６７（和光純薬工業株式会社（０４６－２２３４１））
７０ｍＭ      Ｓｕｃｒｏｓｅ
１２０ｍＭ    Ｍａｎｎｉｔｏｌ
５Ｕ／ｍＬ    Ｔｈｅｒｍｏｃｒｉｓｐｕｍ　ｓｐ．由来ｌｙＰｌｓ　ａｓｅ
２％          β－ｃｙｃｌｏｄｅｘｔｒｉｎ
 
それぞれの定量値の相関図を図１３に示した。相関式はｙ　＝０．７８ｘ＋１６．８、相関係数ｒ＝０．８５８と高い相関があり、Ｔｈｅｒｍｏｃｒｉｓｐｕｍ　ｓｐ．由来ｌｙＰｌｓ　ａｓｅを使用した酵素法で検体中のＰｌｓＥｔｎが測定できた。
[Composition 1-2c]
20 mM BES / NaOH buffer pH 7.5
0.05 mM ALPS (Dojindo Laboratories (OC04))
2 mM CaCl 2
70 mM sucrose
120 mM Mannitol
5U / mL GPCP (Asahi Kasei Pharma Corporation (T-33))
40 U / mL TOD of JP-A-2005-52034
5U / mL International publication number: PL of WO2012 / 105565 A1
10U / mL POD (SIGMA (P8250))
5U / mL ASOM (Asahi Kasei Pharma Corporation (T-53))
5U / mL LYPL (Asahi Kasei Pharma Corporation (T-59))

[Composition 2-2c]
20 mM BES / NaOH buffer pH 7.5
1 mM Na 2 SO 4
0.12 mM DA-67 (Wako Pure Chemical Industries, Ltd. (046-22231))
70 mM sucrose
120 mM Mannitol
5 U / mL Thermocrispum sp. Origin lyPls as
2% β-cyclodextrin

The correlation diagram of each quantitative value is shown in FIG. The correlation equation is highly correlated with y = 0.78x + 16.8 and a correlation coefficient r = 0.858, and Thermocrispum sp. PlsEtn in the specimen could be measured by an enzymatic method using the derived lyPlsase.
 
［実施例６：１２５Ｉ－ＨＰＬＣ法と酵素を利用した血清または血漿中のＰｌｓＥｔｎの定量値の比較３］
４７の血清検体を、次の２種類の方法で測定した：
（ｉ）１２５Ｉ－ＨＰＬＣ法、
（ｉｖ）ＰＬＤを使用した酵素法（［組成物１－３］と［組成物キ２－３］）。
[Example 6: Comparison 3 of quantitative value of PlsEtn in serum or plasma using 125I-HPLC method and enzyme]
47 serum specimens were measured in two ways:
(I) 125I-HPLC method,
(Iv) Enzymatic method using PLD ([Composition 1-3] and [Composition Key 2-3]).
 
それぞれの検査試薬キットをＲ１、Ｒ２として、［日立７０８０形自動分析機パラメーター２］で日立７０８０形自動分析機を使用して測定した。
Each test reagent kit was measured as R1 and R2 using a Hitachi 7080 automatic analyzer with [Hitachi 7080 automatic analyzer parameter 2].
 
［組成物１－３］
２０ｍＭ      Ｔｒｉｓ／ＨＣｌ緩衝液　ｐＨ８．５
０．０５ｍＭ  ＡＬＰＳ（同仁化学研究所（ＯＣ０４））
２ｍＭ　      ＣａＣｌ２
２ｍＭ　      ＭｇＣｌ２
２Ｕ／ｍＬ    ＧＰＣＰ（旭化成ファーマ株式会社（Ｔ－３３））
４０Ｕ／ｍＬ  ＴＯＤ（旭化成ファーマ株式会社（Ｔ－２５））
５Ｕ／ｍＬ    ＰＬＡ２（旭化成ファーマ株式会社（Ｔ－３１））
３０Ｕ／ｍＬ  ＬＹＰＬ（旭化成ファーマ株式会社（Ｔ－５９））
１０Ｕ／ｍＬ  ＰＯＤ　（ＳＩＧＭＡ（Ｐ８２５０））
５Ｕ／ｍＬ    ＡＳＯＭ（旭化成ファーマ株式会社（Ｔ－５３））
０．１％      ＴＸ－１００
 
［組成物２－３］
２０ｍＭ      ＨＥＰＥＳ／ＮａＯＨ緩衝液　ｐＨ７．５
１０ｍＭ　    ＣａＣｌ２
０．１２ｍＭ  ＤＡ－６７（和光純薬工業株式会社（０４６－２２３４１））
１０Ｕ／ｍＬ  ＰＬＤ（旭化成ファーマ株式会社（Ｔ－３９））
２％          β－ｃｙｃｌｏｄｅｘｔｒｉｎ
１％          ＴＸ－１００
 
それぞれの定量値の相関図を図１４に示した。相関式はｙ＝０．７４ｘ＋１９．８、相関係数ｒ＝０．８２５と高い相関があり、ＰＬＤを使用した酵素法で検体中のＰｌｓＥｔｎが測定できた。
［実施例７：１２５Ｉ－ＨＰＬＣ法と酵素を利用した血清または血漿中のＰｌｓＥｔｎの定量値の比較４］
 
４７の血清検体を、次の４種類の方法で測定した：   
（ｉ）１２５Ｉ－ＨＰＬＣ法、
（ｉｉ）Ｐｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ由来ｌｙＰｌｓ　ａｓｅを使用した酵素法（［組成物１－１ａ］と［組成物２－１ａ］）、
（ｉｉｉ）Ｔｈｅｒｍｏｃｒｉｓｐｕｍ　ｓｐ．を使用した酵素法（［組成物１－２ｃ］と［組成物２－２ｃ］）、
（ｉｖ）ＰＬＤを使用した酵素法（［組成物１－３］と［組成物キ２－３］）。
[Composition 1-3]
20 mM Tris / HCl buffer pH 8.5
0.05 mM ALPS (Dojindo Laboratories (OC04))
2 mM CaCl 2
2 mM MgCl 2
2U / mL GPCP (Asahi Kasei Pharma Corporation (T-33))
40U / mL TOD (Asahi Kasei Pharma Corporation (T-25))
5U / mL PLA 2 (Asahi Kasei Pharma Corporation (T-31))
30U / mL LYPL (Asahi Kasei Pharma Corporation (T-59))
10U / mL POD (SIGMA (P8250))
5U / mL ASOM (Asahi Kasei Pharma Corporation (T-53))
0.1% TX-100

[Composition 2-3]
20 mM HEPES / NaOH buffer pH 7.5
10 mM CaCl 2
0.12 mM DA-67 (Wako Pure Chemical Industries, Ltd. (046-22231))
10U / mL PLD (Asahi Kasei Pharma Corporation (T-39))
2% β-cyclodextrin
1% TX-100

The correlation diagram of each quantitative value is shown in FIG. The correlation equation was highly correlated with y = 0.74x + 19.8 and the correlation coefficient r = 0.825, and PlsEtn in the sample could be measured by an enzymatic method using PLD.
[Example 7: Comparison 4 of quantified value of PlsEtn in serum or plasma using 125I-HPLC method and enzyme]

47 serum specimens were measured by the following four methods:
(I) 125I-HPLC method,
(Ii) Enzymatic method using lyPlsase derived from Pseudomonas putida ([Composition 1-1a] and [Composition 2-1a]),
(Iii) Thermocrispum sp. Enzymatic method using (composition 1-2c and composition 2-2c),
(Iv) Enzymatic method using PLD ([Composition 1-3] and [Composition Key 2-3]).
 
それぞれの検査試薬キットをＲ１、Ｒ２として、［日立７０８０形自動分析機パラメーター２］で日立７０８０形自動分析機を使用して測定した。
Each test reagent kit was measured as R1 and R2 using a Hitachi 7080 automatic analyzer with [Hitachi 7080 automatic analyzer parameter 2].
 
各方法による測定値の平均値を表８に示した。
Table 8 shows the average value of the measured values by each method.
Figure JPOXMLDOC01-appb-T000012
  
１２５Ｉ－ＨＰＬＣ法によるＰｌｓＥｔｎ定量値に比べて、（ｉｉ）Ｐｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ由来ｌｙＰｌｓ　ａｓｅを使用した酵素法は約２μＭ低くなり、（ｉｉｉ）Ｔｈｅｒｍｏｃｒｉｓｐｕｍ　ｓｐ．を使用した酵素法と（ｉｖ）ＰＬＤを使用した酵素法は約３μＭ平均値が高くなる場合があることが分かった。
Figure JPOXMLDOC01-appb-T000012
The enzyme method using lyPlsase derived from Pseudomonas putida was about 2 μM lower than the quantified value of PlsEtn by 125I-HPLC method, and (iii) Thermocrispum sp. It has been found that the average value of about 3 μM may be high in the enzyme method using (iv) and the enzyme method using (iv) PLD.
 
［実施例８：Ｐｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ由来ｌｙＰｌｓ　ａｓｅ組換体酵素の製造方法］
Ｐｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ（ＫＴ２４４０）の染色体ＤＮＡを、フェノール・クロロホルム法にて抽出した。染色体ＤＮＡをテンプレートにし、配列番号８に記載の塩基配列をセンスプライマー、配列番号９に記載の塩基配列をアンチセンスプライマーとして、ＫＯＤ　ＦＸ（東洋紡績株式会社、Ｃｏｄｅ　Ｎｏ．ＫＦＸ－１０１）を用いてＰＣＲし、ＰＣＲ産物１を得た。得られたＰＣＲ産物１をＺｅｒｏ　Ｂｌｕｎｔ　ＰＣＲ　Ｃｌｏｎｉｎｇ　Ｋｉｔ（Ｉｎｖｉｔｒｏｇｅｎ、製品番号Ｋ２７００－２０）を用いてｐＣＲ－Ｂｌｕｎｔにクローニングして、本発明の蛋白質をコードする塩基配列からなるポリヌクレオチドを含む組換えベクターＰＰ＿ｌｙＰｌａ　ａｓｅ／ｐＣＲ－Ｂｌｕｎｔを得た。ｐＥＴ－２１ａ（＋）ベクター（Ｎｏｖａｇｅｎ、Ｃａｔ．Ｎｏ　６９７４０－３）をＮｄｅＩとＥｃｏＲ　Ｉで切断して約５．４ｋｂｐのＤＮＡを精製した。ＰＰ＿ｌｙＰｌａ　ａｓｅ／ｐＣＲ－Ｂｌｕｎｔを制限酵素ＮｄｅＩとＥｃｏＲ　Ｉで切断し、約０．７ｋｂｐのＤＮＡを精製した。得られたそれぞれの精製ＤＮＡを、ＤＮＡ　Ｌｉｇａｔｉｏｎ　Ｋｉｔ　Ｖｅｒ．２．１（タカラバイオ株式会社、製品コード６０２２）でライゲーションして本発明の蛋白質をコードする塩基配列からなるポリヌクレオチドを含む組換えベクターＰＰ＿ｌｙＰｌａ　ａｓｅ／ｐＥＴ－２１ａ（＋）を得た。ＰＰ＿ｌｙＰｌａ　ａｓｅ／ｐＥＴ－２１ａ（＋）を、Ｏｎｅ　Ｓｈｏｔ　ＢＬ２１（ＤＥ３）Ｃｈｅｍｉｃａｌｌｙ　Ｃｏｍｐｅｔｅｎｔ　Ｅ．ｃｏｌｉ（Ｉｎｖｉｔｒｏｇｅｎ、製品番号Ｃ６０００－０３）に形質転換して本発明の蛋白質をコードする塩基配列からなるポリヌクレオチドを含む組換えベクターを有する形質転換体ＰＰ＿ｌｙＰｌａ　ａｓｅ／ｐＥＴ－２１ａ（＋）／ＢＬ２１（ＤＥ３）を得た。ＰＰ＿ｌｙＰｌａ　ａｓｅ／ｐＥＴ－２１ａ（＋）／ＢＬ２１（ＤＥ３）を、５０μｇ／ｍｌのアンピシリンを含む、１．６Ｌ（２Ｌジャー）のＯｖｅｒｎｉｇｈｔ　Ｅｘｐｒｅｓｓ　Ｉｎｓｔａｎｔ　ＴＢ　Ｍｅｄｉｕｍ（メルク、注文番号７１７５７－５）に植菌した。３４℃、ｐＨ６．８で３０時間培養して培養液を遠心分離して集菌し、７０ｍＭ　Ｓｕｃｒｏｓｅ、１２０ｍＭ　Ｍａｎｎｉｔｏｌ、及び１．２％　ＤＤＭを含む２０ｍＭ　Ｔｒｉｓ／ＨＣｌ緩衝液（ｐＨ７．５）で４℃１時間懸濁した。懸濁液を遠心分離し、上清の粗Ｐｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ由来ｌｙＰｌｓ　ａｓｅを約１７０Ｕを得た。本実施例で得たＰｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ由来ｌｙＰｌｓ　ａｓｅは、適宜濃縮して実施例３、４、７、１２で使用した。
[Example 8: Production method of lyPlsase recombinant enzyme derived from Pseudomonas putida]
Pseudomonas putida (KT2440) chromosomal DNA was extracted by the phenol / chloroform method. Using KOD FX (Toyobo Co., Ltd., Code No. KFX-101) using chromosomal DNA as a template, using the nucleotide sequence set forth in SEQ ID NO: 8 as a sense primer and the nucleotide sequence set forth in SEQ ID NO: 9 as an antisense primer, PCR was performed to obtain PCR product 1. The resulting PCR product 1 is cloned into pCR-Blunt using Zero Blunt PCR Cloning Kit (Invitrogen, product number K2700-20), and a recombinant vector comprising a polynucleotide comprising a base sequence encoding the protein of the present invention PP_lyPlaase / pCR-Blunt was obtained. The pET-21a (+) vector (Novagen, Cat. No 69740-3) was cleaved with NdeI and EcoRI to purify about 5.4 kbp of DNA. PP_lyPlaase / pCR-Blunt was cleaved with restriction enzymes NdeI and EcoR I to purify about 0.7 kbp of DNA. Each of the obtained purified DNAs was transferred to DNA Ligation Kit Ver. 2.1 (Takara Bio Inc., product code 6022) was ligated to obtain a recombinant vector PP_lyPlaase / pET-21a (+) containing a polynucleotide comprising a base sequence encoding the protein of the present invention. PP_lyPlaase / pET-21a (+) was replaced with One Shot BL21 (DE3) Chemically Competent E.I. transformant PP_lyPlaase / pET-21a (+) / BL21 () having a recombinant vector comprising a polynucleotide comprising a nucleotide sequence encoding the protein of the present invention after being transformed into E. coli (Invitrogen, product number C6000-03) DE3) was obtained. PP_lyPlaase / pET-21a (+) / BL21 (DE3) was inoculated into 1.6L (2L jar) Overnight Express Instant Medium (Merck, order number 71757-5) containing 50 μg / ml ampicillin. . Cultivate at 34 ° C. for 30 hours at pH 6.8, collect the culture by centrifugation, collect 4 in 20 mM Tris / HCl buffer (pH 7.5) containing 70 mM Sucrose, 120 mM Mannitol, and 1.2% DDM. Suspended at 1 ° C for 1 hour. The suspension was centrifuged to obtain about 170 U of a crude crude Pseudomonas putida-derived lyPlsase. The lyPlsase derived from Pseudomonas putida obtained in this example was appropriately concentrated and used in Examples 3, 4, 7, and 12.
 
［実施例９：Ｔｈｅｒｍｏｃｒｉｓｐｕｍ　ｓｐ．由来ｌｙＰｌｓ　ａｓｅの製造方法］
Ｔｈｅｒｍｏｃｒｉｓｐｕｍ　ｓｐ．（ＮＩＴＥ　ＢＰ－０１６２８として独立行政法人製品評価技術基盤機構、特許微生物寄託センターに寄託）を２．４ＬのＩＰＳ２培地（日本ベクトン・ディッキンソン株式会社、カタログ番号２７７０１０）へ常法に従い植菌し、４５℃、１８０ｒｐｍで８４ｈ振とう培養した。培養上清のｌｙＰｌｓ　ａｓｅ活性は約０．０５８Ｕ／ｍＬ（総量１０４Ｕ）であった。遠心分離にて除菌して得た培養上清を０．４５μmのフィルターで濾過し、８５％飽和量硫安でタンパク質を濃縮した。２０ｍＭ　Ｔｒｉｓ／ＨＣｌ緩衝液（ｐＨ９．０）に対して透析脱塩を行った濃縮液はＴＯＹＯＰＥＡＲＬ ＧｉｇａＣｕｐ Ｑ‐６５０Ｍ（ＴＯＳＯＨ　ＢＩＯＳＣＩＥＮＣＥ）にて次の条件で部分精製した：１．０Ｍ　ＮａＣｌを含む２０ｍＭ　Ｔｒｉｓ／ＨＣｌ緩衝液（ｐＨ９．０）と２０ｍＭ　Ｔｒｉｓ／ＨＣｌ緩衝液（ｐＨ９．０）を用いた２０ＣＶ（ｃｏｌｕｍｎ　ｖｏｌｕｍｅ）のリニアグラジェントによるカラムクロマトグラフィー。活性画分に１．５Ｍとなるように硫酸アンモニウムを添加し、ＴＯＹＯＰＥＡＲＬ　ＰＰＧ‐６００Ｍ（ＴＯＳＯＨ　ＢＩＯＳＣＩＥＮＣＥ）にて次の条件で部分精製した：１．５Ｍ　（ＮＨ４）２ＳＯ４を含む２０ｍＭ　Ｔｒｉｓ／ＨＣｌ緩衝液（ｐＨ８．０）と２０ｍＭ　Ｔｒｉｓ／ＨＣｌ緩衝液（ｐＨ８．０）を用いた２０ＣＶ（ｃｏｌｕｍｎ　ｖｏｌｕｍｅ）のリニアグラジェントによるカラムクロマトグラフィー。活性画分はＲｅｓｏｕｒｃｅ　Ｑ（ＧＥヘルスケアバイオサイエンス）にて次の条件で部分精製した：０．５Ｍ　ＮａＣｌを含む２０ｍＭ　Ｔｒｉｓ／ＨＣｌ緩衝液（ｐＨ８．０）と２０ｍＭ　Ｔｒｉｓ／ＨＣｌ緩衝液（ｐＨ８．０）を用いた４０ＣＶ（ｃｏｌｕｍｎ　ｖｏｌｕｍｅ）のリニアグラジェントによるカラムクロマトグラフィー。活性画分に０．１５Ｍとなるように塩化ナトリウムを添加し、Ｓｕｐｅｒｄｅｘ　２００（ＧＥヘルスケアバイオサイエンス）でさらに部分精製した：２０　ｍＭ　Ｔｒｉｓ／ＨＣｌ緩衝液（ｐＨ　８．０）＋０．１５　Ｍ　ＮａＣｌ。活性画分は１．６５　Ｍ　となるように硫酸アンモニウムを添加し、Ｒｅｓｏｕｒｃｅ　Ｉｓｏ（ＧＥヘルスケアバイオサイエンス）にて次の条件でさらに部分精製した：１．６５Ｍ　（ＮＨ４）２ＳＯ４を含む２０ｍＭ　Ｔｒｉｓ／ＨＣｌ緩衝液（ｐＨ８．０）と２０ｍＭ　Ｔｒｉｓ／ＨＣｌ緩衝液（ｐＨ８．０）を用いた４０ＣＶ（ｃｏｌｕｍｎ　ｖｏｌｕｍｅ）のリニアグラジェントによるカラムクロマトグラフィー。活性画分はＭｏｎｏ　Ｑ（ＧＥヘルスケアバイオサイエンス）にて次の条件でさらに部分精製した：０．４Ｍ　ＮａＣｌを含む２０ｍＭ　Ｔｒｉｓ／ＨＣｌ緩衝液（ｐＨ８．０）と２０ｍＭ　Ｔｒｉｓ／ＨＣｌ緩衝液（ｐＨ８．０）を用いた４０ＣＶのリニアグラジェントによるカラムクロマトグラフィー。活性画分は培養上清から約２８５倍精製した部分精製ｌｙＰｌｓ　ａｓｅを１．５５Ｕ（比活性５０．６Ｕ ｍｇ-１）得た。
[Example 9: Thermocrispum sp. Method for producing derived lyPls as]
Thermocrispum sp. (Deposited with NITE BP-01628, National Institute of Technology and Evaluation, Patent Microorganism Depositary Center) was inoculated into 2.4 L of IPS2 medium (Nippon Becton Dickinson Co., Ltd., catalog number 277010) according to a conventional method. Cultured with shaking at 180 ° C. for 84 h. The lyPlsase activity of the culture supernatant was about 0.058 U / mL (total amount 104 U). The culture supernatant obtained by sterilization by centrifugation was filtered through a 0.45 μm filter, and the protein was concentrated with 85% saturated ammonium sulfate. The concentrate obtained by dialysis and desalting against 20 mM Tris / HCl buffer (pH 9.0) was partially purified with TOYOPEARL GigaCup Q-650M (TOSOH BIOSCIENCE) under the following conditions: 20 mM Tris containing 1.0 M NaCl. Chromatography using a linear gradient of 20 CV (column volume) using an / HCl buffer solution (pH 9.0) and a 20 mM Tris / HCl buffer solution (pH 9.0). Ammonium sulfate was added to the active fraction so as to be 1.5 M, and partially purified by TOYOPEARL PPG-600M (TOSOH BIOSCIENCE) under the following conditions: 20 mM Tris / HCl containing 1.5 M (NH 4 ) 2 SO 4 Column chromatography by linear gradient of 20 CV (column volume) using buffer solution (pH 8.0) and 20 mM Tris / HCl buffer solution (pH 8.0). The active fraction was partially purified by Resource Q (GE Healthcare Bioscience) under the following conditions: 20 mM Tris / HCl buffer (pH 8.0) containing 0.5 M NaCl and 20 mM Tris / HCl buffer (pH 8. Column chromatography with a linear gradient of 40 CV (column volume) using 0). Sodium chloride was added to the active fraction to a concentration of 0.15 M, and further partially purified with Superdex 200 (GE Healthcare Bioscience): 20 mM Tris / HCl buffer (pH 8.0) +0.15 M NaCl . Ammonium sulfate was added so that the active fraction was 1.65 M, and further partial purification was performed under the following conditions at Resource Iso (GE Healthcare Bioscience): 20 mM containing 1.65 M (NH 4 ) 2 SO 4. Column chromatography by linear gradient of 40 CV (column volume) using Tris / HCl buffer (pH 8.0) and 20 mM Tris / HCl buffer (pH 8.0). The active fraction was further partially purified with Mono Q (GE Healthcare Bioscience) under the following conditions: 20 mM Tris / HCl buffer (pH 8.0) containing 0.4 M NaCl and 20 mM Tris / HCl buffer (pH 8). Column) with a linear gradient of 40 CV using. As the active fraction, 1.55 U (specific activity 50.6 U mg- 1 ) of partially purified lyPlsase purified about 285 times from the culture supernatant was obtained.
 
［実施例１０：Ｔｈｅｒｍｏｃｒｉｓｐｕｍ　ｓｐ．由来ｌｙＰｌｓ　ａｓｅ組換体酵素の製造方法１（Ｓｔｒｅｐｔｏｍｙｃｅｓ　ｌｉｖｉｄａｎｓ組み換え体）］
Ｔｈｅｒｍｏｃｒｉｓｐｕｍ　ｓｐ．の染色体ＤＮＡを、フェノール・クロロホルム法にて抽出した。配列番号１０に記載の塩基配列をセンスプライマー、配列番号１１に記載の塩基配列をアンチセンスプライマーとして、ＫＯＤ　ＦＸを用いてＰＣＲし、約９００ｂｐのＰＣＲ産物２を得た。ＰＣＲ産物２をＮｈｅＩとＢｇｌＩＩで消化し、発現ベクターである放線菌プラスミドのＮｈｅＩ－ＢｇｌＩＩ部位に挿入して、組換えプラスミドｌｙＰｌａ　ａｓｅ／Ｅｘを得た。ｌｙＰｌａ　ａｓｅ／Ｅｘを「ＰＲＡＣＴＩＣＡＬ　ＳＴＲＥＰＴＯＭＹＣＥＳ　ＧＥＮＥＴＩＣＳ（Ｋｉｅｓｅｒら、Ｊｏｈｎ　Ｉｎｎｅｓ　Ｆｏｕｎｄａｔｉｏｎ、２０００年）」に記載の方法に従い、プロトプラスト化された放線菌Ｓｔｒｅｐｔｏｍｙｃｅｓ　ｌｉｖｉｄａｎｓ）１３２６を形質転換し、組換え放線菌ｌｙＰｌａ　ａｓｅ／Ｅｘ／Ｓ．　ｌｉｖｉｄａｎｓを得た。ｌｙＰｌａ　ａｓｅ／Ｅｘ／Ｓ．　ｌｉｖｉｄａｎｓを、５μｇ／ｍＬのチオストレプトン、１％　グルコース及び１％　トリプトンを含む０．８Ｌトリプチックソイ培地（日本ベクトン・ディッキンソン株式会社）で２８℃、１８０回転で４８時間培養した。培養後、遠心分離にて除菌して得た培養上清に、８５％飽和量となるように硫酸アンモニウムを添加し、生じた沈殿を遠心分離（１０,０００ｒｐｍ、１０分、４℃）により回収した。この沈殿を可溶化し、２０ｍＭ　Ｔｒｉｓ／ＨＣｌ緩衝液（ｐＨ９．０）で透析し、粗酵素液を得た。粗酵素液は透析で２０ｍＭ　Ｔｒｉｓ／ＨＣｌ緩衝液（ｐＨ９．０）にｂｕｆｆｅｒ交換し、ＴＯＹＯＰＥＡＲＬ　ＧｉｇａＣａｐ　Ｑ－６５０Ｍ（ＴＯＳＯＨ　ＢＩＯＳＣＩＥＮＣＥ）にて次の条件で部分精製した：１Ｍ　ＮａＣｌを含む２０ｍＭ　Ｔｒｉｓ／ＨＣｌ緩衝液（ｐＨ９．０）と２０ｍＭ　Ｔｒｉｓ／ＨＣｌ緩衝液（ｐＨ９．０）を用いた１５ＣＶ（ｃｏｌｕｍｎ　ｖｏｌｕｍｅ）のリニアグラジェントによるカラムクロマトグラフィー。活性画分に１．５Ｍとなるように硫酸アンモニウムを添加し、ＴＯＹＯＰＥＡＲＬ　ＰＰＧ－６００Ｍ（ＴＯＳＯＨ　ＢＩＯＳＣＩＥＮＣＥ）にて次の条件で部分精製した：１．５Ｍ　硫酸アンモニウムを含む２０ｍＭ　Ｔｒｉｓ／ＨＣｌ緩衝液（ｐＨ８．０）と２０ｍＭ　Ｔｒｉｓ／ＨＣｌ緩衝液（ｐＨ８．０）を用いた１０ＣＶ（ｃｏｌｕｍｎ　ｖｏｌｕｍｅ）のリニアグラジェントによるカラムクロマトグラフィー。活性画分は透析で２０ｍＭ　Ｔｒｉｓ／ＨＣｌ緩衝液（ｐＨ９．０）にｂｕｆｆｅｒ交換し、Ｍｏｎｏ　Ｑ（ＧＥヘルスケアバイオサイエンス）にて次の条件で精製した：０．５Ｍ　ＮａＣｌを含む２０ｍＭ　Ｔｒｉｓ／ＨＣｌ緩衝液（ｐＨ９．０）と２０ｍＭ　Ｔｒｉｓ／ＨＣｌ緩衝液（ｐＨ９．０）を用いた４０ＣＶ（ｃｏｌｕｍｎ　ｖｏｌｕｍｅ）のリニアグラジェントによるカラムクロマトグラフィー。活性画分は透析で２５ｍＭ　Ｂｉｓ‐Ｔｒｉｓ／ＮａＯＨ緩衝液（ｐＨ６．３）にｂｕｆｆｅｒ交換し、Ｍｏｎｏ　Ｐ（ＧＥヘルスケアバイオサイエンス）にて次の条件で精製した：開始ｂｕｆｆｅｒ２５ｍＭ　Ｂｉｓ‐Ｔｒｉｓ／ＮａＯＨ緩衝液（ｐＨ６．３）、溶出Ｂｕｆｆｅｒ　ポリバッファー７４（１０倍希釈、ｐＨ４．０）。本実施例による精製結果を表９に、精製酵素のＳＤＳ－ＰＡＧＥ（図中矢印）を図１５に示した。本実施例で得たＴｈｅｒｍｏｃｒｉｓｐｕｍ　ｓｐ．由来ｌｙＰｌｓ　ａｓｅは、適宜濃縮して実施例５、７、１２で使用した。
[Example 10: Thermocrispum sp. Method 1 for producing derived lyPlsase recombinant enzyme (Streptomyces lividans recombinant)]
Thermocrispum sp. The chromosomal DNA was extracted by the phenol / chloroform method. PCR was performed using KOD FX using the nucleotide sequence set forth in SEQ ID NO: 10 as the sense primer and the nucleotide sequence set forth in SEQ ID NO: 11 as the antisense primer to obtain a PCR product 2 of about 900 bp. The PCR product 2 was digested with NheI and BglII and inserted into the NheI-BglII site of the actinomycete plasmid as an expression vector to obtain a recombinant plasmid lyPlaase / Ex. In accordance with the method described in “PRACTICAL STREPTOMYCES GENETICS (Kieser et al., John Inns Foundation, 2000)”, lyPlaase / Ex is transformed into a protoplast actinomycete Streptomyces vividans) 1326 / S. lividans was obtained. lyPlaase / Ex / S. lividans was cultured for 48 hours at 28 ° C. and 180 rpm in 0.8 L tryptic soy medium (Nihon Becton Dickinson) containing 5 μg / mL thiostrepton, 1% glucose and 1% tryptone. After culturing, ammonium sulfate is added to the culture supernatant obtained by sterilization by centrifugation so that the saturation amount is 85%, and the resulting precipitate is recovered by centrifugation (10,000 rpm, 10 minutes, 4 ° C.). did. This precipitate was solubilized and dialyzed against 20 mM Tris / HCl buffer (pH 9.0) to obtain a crude enzyme solution. The crude enzyme solution was buffer-exchanged to 20 mM Tris / HCl buffer (pH 9.0) by dialysis, and partially purified with TOYOPEARL GigaCap Q-650M (TOSOH BIOSCIENCE) under the following conditions: 20 mM Tris / HCl buffer containing 1 M NaCl. Column chromatography with a linear gradient of 15 CV (column volume) using a liquid (pH 9.0) and a 20 mM Tris / HCl buffer (pH 9.0). Ammonium sulfate was added to the active fraction so as to be 1.5 M, and partially purified with TOYOPEARL PPG-600M (TOSOH BIOSSCENCE) under the following conditions: 20 mM Tris / HCl buffer (pH 8.0 containing 1.5 M ammonium sulfate). And 10 CV (column volume) linear gradient using 20 mM Tris / HCl buffer (pH 8.0). The active fraction was buffer exchanged to 20 mM Tris / HCl buffer (pH 9.0) by dialysis and purified by Mono Q (GE Healthcare Bioscience) under the following conditions: 20 mM Tris / HCl containing 0.5 M NaCl. Column chromatography by linear gradient of 40 CV (column volume) using a buffer solution (pH 9.0) and a 20 mM Tris / HCl buffer solution (pH 9.0). The active fraction was buffer-exchanged to 25 mM Bis-Tris / NaOH buffer (pH 6.3) by dialysis and purified with Mono P (GE Healthcare Bioscience) under the following conditions: Starting buffer 25 mM Bis-Tris / NaOH buffer Solution (pH 6.3), elution Buffer polybuffer 74 (10-fold dilution, pH 4.0). The purification results of this example are shown in Table 9, and SDS-PAGE (arrow in the figure) of the purified enzyme is shown in FIG. Thermocrispum sp. Obtained in this example. The derived lyPlsase was appropriately concentrated and used in Examples 5, 7, and 12.
Figure JPOXMLDOC01-appb-T000013
 ［実施例１１：Ｔｈｅｒｍｏｃｒｉｓｐｕｍ　ｓｐ．由来ｌｙＰｌｓ　ａｓｅ組換体酵素の製造方法２（大腸菌組換え体）］
Ｔｈｅｒｍｏｃｒｉｓｐｕｍ　ｓｐ．の染色体ＤＮＡを、フェノール・クロロホルム法にて抽出した。配列番号１２に記載の塩基配列をセンスプライマー、配列番号１３に記載の塩基配列をアンチセンスプライマーとして、ＫＯＤ　ＦＸを用いてＰＣＲし、約９００ｂｐのＰＣＲ産物３を得た。ＰＣＲ産物３をＮｄｅＩとＥｃｏＲＩで消化し、発現ベクターであるｐＥＴ－２１ａ（＋）ベクターのＮｄｅＩ－ＥｃｏＲＩ部位に挿入して、Ｔ＿ｌｙＰｌａ　ａｓｅ／ｐＥＴ－２１ａ（＋）を得た。Ｔ＿ｌｙＰｌａ　ａｓｅ／ｐＥＴ－２１ａ（＋）を、Ｏｎｅ　Ｓｈｏｔ　ＢＬ２１（ＤＥ３）　Ｃｈｅｍｉｃａｌｌｙ　Ｃｏｍｐｅｔｅｎｔ　Ｅ．ｃｏｌｉに形質転換して本発明の蛋白質をコードする塩基配列からなるポリヌクレオチドを含む組換えベクターを有する形質転換体Ｔ＿ｌｙＰｌａ　ａｓｅ／ｐＥＴ－２１ａ（＋）／ＢＬ２１（ＤＥ３）を得た。Ｔ＿ｌｙＰｌａ　ａｓｅ／ｐＥＴ－２１ａ（＋）／ＢＬ２１（ＤＥ３）を、５０μｇ／ｍｌのアンピシリンを含む、５０ｍＬ（５００ｍＬ三角フラスコ）のＯｖｅｒｎｉｇｈｔ　Ｅｘｐｒｅｓｓ　Ｉｎｓｔａｎｔ　ＴＢ　Ｍｅｄｉｕｍに植菌した。３７℃、ｐＨ６．８で２４時間振とう培養して培養液を遠心分離して集菌し、２０ｍＭ　Ｔｒｉｓ／ＨＣｌ緩衝液（ｐＨ８．５）に懸濁し超音波破砕して、遠心分離し、得られた上清を粗蛋白質液とした。粗蛋白質液中のＴｈｅｒｍｏｃｒｉｓｐｕｍ　ｓｐ．由来ｌｙＰｌｓ　ａｓｅ組換体酵素は２０Ｕで、粗蛋白質液のＳＤＳ－ＰＡＧＥを図１６に示した。ｌｙＰｌｓ　ａｓｅは矢印で示した。
Figure JPOXMLDOC01-appb-T000013
 [Example 11: Thermocrispum sp. Method 2 for producing derived lyPlsase recombinant enzyme (E. coli recombinant)]
Thermocrispum sp. The chromosomal DNA was extracted by the phenol / chloroform method. PCR was performed using KOD FX with the nucleotide sequence set forth in SEQ ID NO: 12 as the sense primer and the nucleotide sequence set forth in SEQ ID NO: 13 as the antisense primer to obtain a PCR product 3 of about 900 bp. PCR product 3 was digested with NdeI and EcoRI and inserted into the NdeI-EcoRI site of the expression vector pET-21a (+) to obtain T_lyPlaase / pET-21a (+). T_lyPlaase / pET-21a (+) was replaced with One Shot BL21 (DE3) Chemically Competent E.I. A transformant T_lyPlaase / pET-21a (+) / BL21 (DE3) having a recombinant vector containing a polynucleotide comprising a nucleotide sequence encoding the protein of the present invention was obtained by transforming into E. coli. T_lyPlaase / pET-21a (+) / BL21 (DE3) was inoculated into 50 mL (500 mL Erlenmeyer flask) Overnight Express Instant TB Medium containing 50 μg / ml ampicillin. The culture solution is centrifuged at 37 ° C. and pH 6.8 for 24 hours, and the culture solution is collected by centrifugation, suspended in 20 mM Tris / HCl buffer (pH 8.5), sonicated and centrifuged. The obtained supernatant was used as a crude protein solution. Thermocrispum sp. In the crude protein solution. The derived lyPlsase recombinant enzyme was 20 U, and SDS-PAGE of the crude protein solution is shown in FIG. lyPlsase is indicated by an arrow.
 
［実施例１２：本発明のｌｙＰｌｓ　ａｓｅの基質特異性］
表１０に、各種基質に対する本発明のｌｙＰｌｓ　ａｓｅの作用を、上記の活性測定法に準じて測定した結果を示す。
[Example 12: Substrate specificity of lyPlsase of the present invention]
Table 10 shows the results of measuring the action of the lyPlsase of the present invention on various substrates in accordance with the above activity measurement method.
 
結果は、ｌｙＰｌｓＥｔｎを基質とした場合の測定値を１００として、相対活性で示した。
The results were expressed as relative activities, with the measured value when lyPlsEtn was used as a substrate being 100.
Figure JPOXMLDOC01-appb-T000014
 表２から、本発明の蛋白質は、Ｌ－α－ｌｙｓｏｐｈｏｓｐｈａｔｉｄｙｌｅｔｈａｎｏｌａｍｉｎｅ　ｐｌａｓｍａｌｏｇｅｎ（ｌｙＰｌｓＥｔｎ）に対して高い活性を有することが明らかになった。
Figure JPOXMLDOC01-appb-T000014
 From Table 2, it was revealed that the protein of the present invention has high activity against L-α-lysophosphatidylethanolamine plasma (lyPlsEtn).
 
血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）量をバイオマーカーとする、大規模スクリーニングに提起した認知症検査に利用できる。 It can be used for a dementia test proposed for large-scale screening using the amount of ethanolamine plasmalogen (PlsEtn) in serum or plasma as a biomarker.

Claims (34)

  1. 被験者を認知症患者またはその前段階にある者か否かを分類するための認知症検査方法であって、
    　被験者の自然排泄尿中のミオイノシトール（ＭＩ）を定量する工程と、同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する工程とを含み、
    健常者と、認知症患者またはその前段階にある者とを区別することができる、自然排泄尿中のミオイノシトール（ＭＩ）の予め求められた閾値と、血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）の予め求められた閾値のそれぞれに対して、前記被験者のミオイノシトール（ＭＩ）の定量値とエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）の定量値を比較する工程を含む認知症検査方法。     A method for testing dementia for classifying whether a subject is a dementia patient or a person in the preceding stage,
    Quantifying myo-inositol (MI) in spontaneously excreted urine of a subject and quantifying ethanolamine plasmalogen (PlsEtn) in serum or plasma of the same subject,
    A pre-determined threshold of myo-inositol (MI) in spontaneously excreted urine and ethanolamine plasmalogens in serum or plasma, which can distinguish between healthy and demented patients or those in its predecessor A dementia test method comprising a step of comparing a quantitative value of myo-inositol (MI) and a quantitative value of ethanolamine-type plasmalogen (PlsEtn) of the subject with respect to each of previously determined threshold values of (PlsEtn).
  2. 同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する工程が、酵素を利用する反応により、最終的に過酸化水素が生成され、該エタノールアミン型プラスマローゲンの存在量に対応して発生する過酸化水素の量を検出して、血清または血漿中のＰｌｓＥｔｎ量を算出する工程を含む請求項１の方法。 The process of quantifying ethanolamine plasmalogen (PlsEtn) in the serum or plasma of the same subject finally generates hydrogen peroxide by the reaction using an enzyme, and corresponds to the abundance of the ethanolamine plasmalogen. The method according to claim 1, further comprising the step of calculating the amount of PlsEtn in serum or plasma by detecting the amount of hydrogen peroxide generated.
  3. 同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する方法が、以下の（１）～（３－４）（枝番号を付したものも全て含む。以下同様）の工程を含む請求項２に記載の方法。
    （１）エタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解できる酵素を用いて、被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解する工程：
    （２）エタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）をグリセロ－３－ホスフォエタノールアミンとアルデヒドに加水分解でき、かつエーテル型リゾホスファチジルエタノールアミンを加水分解できない加水分解酵素を用いて、前記（１）にて生成されたエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）をグリセロ－３－ホフォエタノールアミンとアルデヒドに加水分解する工程：及び
    （３－１）グリセロ－３－ホスフォエタノールアミンを加水分解してエタノールアミンにせしめる酵素にて、工程（２）で得られたグリセロ－３－ホスフォエタノールアミンを加水分解してエタノールアミンにする工程：
    （３－２）エタノールアミンを酸化する酵素により、エタノールアミンから過酸化水素を発生せしめる工程：
    （３－３）過酸化水素を過酸化水素定量手段により定量する工程：及び
    （３－４）前記被験者の血清または血漿中のＰｌｓＥｔｎ量を算出する工程：
    または前記（２）の加水分解酵素が、リゾホスファチジルエタノールアミンを加水分解できる酵素である場合、同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する方法が、前記（１）～（３－４）の工程に先立つか、少なくとも前記（２）工程の前までに、下記（４）及び（５）を含む工程を行うことを特徴とする方法。
    （４）ホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する酵素を用いて、血清または血漿中に混在するホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する工程：及び
    （５）リゾホスフォリパーゼ及び／又はモノグリセリドリパーゼを用いて、前記（４）工程により生成されるリゾホスファチジルエタノールアミンを分解し、実質的に消去する工程：     A method for quantifying ethanolamine-type plasmalogen (PlsEtn) in serum or plasma of the same subject comprises the following steps (1) to (3-4) (including all those with branch numbers, the same applies hereinafter). The method of claim 2 comprising.
    (1) Using an enzyme capable of hydrolyzing ethanolamine-type plasmalogen (PlsEtn) into ethanolamine-type lysoplasmalogen (lyPlsEtn), ethanolamine-type plasmalogen (PlsEtn) in the serum or plasma of the subject is ethanolamine-type lysozyme. Hydrolyzing to plasmalogen (lyPlsEtn):
    (2) Using a hydrolase that can hydrolyze ethanolamine-type lysoplasmalogen (lyPlsEtn) into glycero-3-phosphoethanolamine and an aldehyde and cannot hydrolyze ether-type lysophosphatidylethanolamine, (1) Hydrolyzing ethanolamine-type lysoplasmalogen (lyPlsEtn) produced in step 1 to glycero-3-phosphoethanolamine and aldehyde: and (3-1) hydrolyzing glycero-3-phosphoethanolamine A step of hydrolyzing the glycero-3-phosphoethanolamine obtained in step (2) to ethanolamine with an enzyme that is made to be ethanolamine:
    (3-2) Step of generating hydrogen peroxide from ethanolamine by an enzyme that oxidizes ethanolamine:
    (3-3) Step of quantifying hydrogen peroxide by means of hydrogen peroxide quantification: (3-4) Step of calculating the amount of PlsEtn in the serum or plasma of the subject:
    Alternatively, when the hydrolase of (2) is an enzyme capable of hydrolyzing lysophosphatidylethanolamine, the method for quantifying ethanolamine-type plasmalogen (PlsEtn) in the serum or plasma of the same subject is described in (1) A method comprising performing the steps (4) and (5) below prior to the steps (3-4) or at least before the step (2).
    (4) Step of decomposing phosphatidylethanolamine mixed in serum or plasma into lysophosphatidylethanolamine and fatty acid using an enzyme that decomposes phosphatidylethanolamine into lysophosphatidylethanolamine and fatty acid: and (5) lysophospho Step of decomposing and substantially eliminating lysophosphatidylethanolamine produced by the step (4) using lipase and / or monoglyceride lipase:
  4. 前記工程（２）の加水分解酵素が、下記（ａ）または（ｂ）の酵素のいずれかである請求項３に記載の方法。
    （ａ）配列番号１または２に記載のアミノ酸配列：
    （ｂ）配列番号１または２に記載のアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列：
    からなる酵素であって、エタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）を加水分解し、グリセロ－３－ホスフォエタノールアミンとアルデヒドとを得る反応を触媒する：     The method according to claim 3, wherein the hydrolase in the step (2) is any of the following enzymes (a) and (b).
    (A) the amino acid sequence set forth in SEQ ID NO: 1 or 2:
    (B) Amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 or 2:
    Which catalyzes the reaction of hydrolyzing ethanolamine-type lysoplasmalogen (lyPlsEtn) to give glycero-3-phosphoethanolamine and an aldehyde:
  5. 前記工程（１）のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解できる酵素が、下記（ｃ）または（ｄ）のいずれかの酵素である請求項３に記載の方法。
    （ｃ）配列番号５に記載のアミノ酸配列：
    （ｄ）配列番号５に記載のアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列であり、エタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解する作用を触媒する：      The enzyme capable of hydrolyzing the ethanolamine-type plasmalogen (PlsEtn) in the step (1) into an ethanolamine-type lysoplasmalogen (lyPlsEtn) is one of the following enzymes (c) or (d): The method described.
    (C) the amino acid sequence set forth in SEQ ID NO: 5:
    (D) An amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 5, and ethanolamine type plasmalogen (PlsEtn) is changed to ethanolamine type lysoplasmalogen (lyPlsEtn). ) Catalyze the hydrolysis action:
  6. 前記（２）の加水分解酵素が、リゾホスファチジルエタノールアミンを加水分解できる酵素である場合、同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する方法が、前記（１）～（３－４）の工程に先立つか、少なくとも前記（２）工程の前までに、下記（４）及び（５）を含む工程を行うことを特徴とする請求項３に記載の方法。
    （４）ホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する酵素を用いて、血清または血漿中に混在するホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する工程：及び
    （５）リゾホスフォリパーゼ及び／又はモノグリセリドリパーゼを用いて、前記（４）工程により生成されるリゾホスファチジルエタノールアミンを分解し、実質的に消去する工程：     When the hydrolase of (2) is an enzyme capable of hydrolyzing lysophosphatidylethanolamine, the method for quantifying ethanolamine-type plasmalogen (PlsEtn) in serum or plasma of the same subject is described in (1) to The method according to claim 3, wherein the step including the following (4) and (5) is performed prior to the step (3-4) or at least before the step (2).
    (4) Step of decomposing phosphatidylethanolamine mixed in serum or plasma into lysophosphatidylethanolamine and fatty acid using an enzyme that decomposes phosphatidylethanolamine into lysophosphatidylethanolamine and fatty acid: and (5) lysophospho Step of decomposing and substantially eliminating lysophosphatidylethanolamine produced by the step (4) using lipase and / or monoglyceride lipase:
  7. 前記工程（４）のホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する酵素としてホスフォリパーゼ（ＰＬ）を使用し、該ＰＬは、前記工程（１）のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解できる酵素としての作用を兼ねさせて新たな該ＰＬを添加することなく前記工程（１）の工程を進行せしめ、前記工程（５）を実施と同時、または前記工程（５）の実施後に、前記工程（１）が完了し、以後前記工程（２）～（３－４）を実施する請求項６に記載の方法。 Phospholipase (PL) is used as an enzyme that decomposes the phosphatidylethanolamine of the step (4) into lysophosphatidylethanolamine and a fatty acid, and the PL uses the ethanolamine-type plasmalogen (PlsEtn) of the step (1). Simultaneously with the implementation of the step (5), the step (1) is allowed to proceed without adding new PL, which also serves as an enzyme capable of hydrolyzing ethanolamine-type lysoplasmalogen (lyPlsEtn). The method according to claim 6, wherein the step (1) is completed after the step (5) is performed, and the steps (2) to (3-4) are thereafter performed.
  8. 同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する方法が、以下の（１）～（５）（枝番を付したもの、´を付したものも全て含む。以下同様）の工程を含む請求項２に記載の方法。
    （１）エタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解できる酵素を用いて、被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解する工程：
    （２）´エタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）をエタノールアミンとプラズメニルホスファチジン酸に加水分解でき、かつエーテル型リゾホスファチジルエタノールアミンを加水分解でき、かつリゾホスファチジルエタノールアミンを加水分解できる加水分解酵素を用いて、前記（１）にて生成されたエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）をエタノールアミンとプラズメニルホスファチジン酸に加水分解する工程： 
    （３－１）´エタノールアミンを酸化する酵素により、工程（２）´で得られたエタノールアミンから過酸化水素を発生せしめる工程：
    （３－２）´過酸化水素を過酸化水素定量手段により定量する工程：及び
    （３－３）´前記被験者の血清または血漿中のＰｌｓＥｔｎ量を算出する工程：     The method for quantifying ethanolamine-type plasmalogen (PlsEtn) in serum or plasma of the same subject includes all of the following (1) to (5) (numbered with branch numbers, numbered with ', etc.). The method of Claim 2 including the process of.
    (1) Using an enzyme capable of hydrolyzing ethanolamine-type plasmalogen (PlsEtn) into ethanolamine-type lysoplasmalogen (lyPlsEtn), ethanolamine-type plasmalogen (PlsEtn) in the serum or plasma of the subject is ethanolamine-type lysozyme. Hydrolyzing to plasmalogen (lyPlsEtn):
    (2) Hydrolyzing enzyme capable of hydrolyzing ethanolamine type lysoplasmalogen (lyPlsEtn) to ethanolamine and plasmenyl phosphatidic acid, hydrolyzing ether type lysophosphatidylethanolamine, and hydrolyzing lysophosphatidylethanolamine The step of hydrolyzing the ethanolamine-type lysoplasmalogen (lyPlsEtn) produced in (1) above to ethanolamine and plasmenyl phosphatidic acid using:
    (3-1) Step of generating hydrogen peroxide from the ethanolamine obtained in step (2) ′ by an enzyme that oxidizes “ethanolamine”:
    (3-2) 'Step of quantifying hydrogen peroxide by means of hydrogen peroxide quantification: and (3-3)' Step of calculating PlsEtn amount in serum or plasma of the subject:
  9. 同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する方法が、前記（１）～（３－３）´の工程に先立つか、少なくとも前記（２）´工程の前までに、下記（４）及び（５）を含む工程を行うことを特徴とする請求項８に記載の方法。
    （４）ホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する酵素を用いて、血清または血漿中に混在するホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する工程：及び
    （５）リゾホスフォリパーゼ及び／又はモノグリセリドリパーゼを用いて、前記（４）工程により生成されるリゾホスファチジルエタノールアミンを分解し、実質的に消去する工程：     A method for quantifying ethanolamine-type plasmalogen (PlsEtn) in serum or plasma of the same subject is performed prior to the steps (1) to (3-3) ′ or at least before the step (2) ′. The method according to claim 8, wherein a process including the following (4) and (5) is performed.
    (4) Step of decomposing phosphatidylethanolamine mixed in serum or plasma into lysophosphatidylethanolamine and fatty acid using an enzyme that decomposes phosphatidylethanolamine into lysophosphatidylethanolamine and fatty acid: and (5) lysophospho Step of decomposing and substantially eliminating lysophosphatidylethanolamine produced by the step (4) using lipase and / or monoglyceride lipase:
  10. 前記工程（４）のホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する酵素としてホスフォリパーゼ（ＰＬ）を使用し、該ＰＬは、前記工程（１）のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解できる酵素としての作用を兼ねさせて新たな該ＰＬを添加することなく前記工程（１）の工程を進行せしめ、前記工程（５）を実施と同時、または前記工程（５）の実施後に、前記工程（１）が完了し、以後前記工程（２）～（３－３）´を実施する請求項９に記載の方法。 Phospholipase (PL) is used as an enzyme that decomposes the phosphatidylethanolamine of the step (4) into lysophosphatidylethanolamine and a fatty acid, and the PL uses the ethanolamine-type plasmalogen (PlsEtn) of the step (1). Simultaneously with the implementation of the step (5), the step (1) is allowed to proceed without adding new PL, which also serves as an enzyme capable of hydrolyzing ethanolamine-type lysoplasmalogen (lyPlsEtn). The method according to claim 9, wherein after the step (5) is performed, the step (1) is completed, and the steps (2) to (3-3) ′ are performed thereafter.
  11. 自然排泄尿中のミオイノシトール（ＭＩ）の予め求められた閾値が、２１．６～６３．３ｍｇ／ｇＣｒの範囲から選択される値であり、血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）の予め求められた閾値が、５０～６４μＭの範囲から選択される値であって、被験者の自然排泄尿中のミオイノシトール（ＭＩ）の定量値が、前記ＭＩ閾値を越え、かつ被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）の定量値が、前記ＰｌｓＥｔｎ閾値を下回った場合に、認知症患者またはその前段階にある者と区分する請求項１に記載の方法。 A predetermined threshold value of myo-inositol (MI) in spontaneously excreted urine is a value selected from the range of 21.6 to 63.3 mg / gCr, and ethanolamine-type plasmalogen (PlsEtn) in serum or plasma Is a value selected from the range of 50 to 64 μM, and the quantitative value of myo-inositol (MI) in the spontaneously excreted urine of the subject exceeds the MI threshold, and the serum of the subject or The method according to claim 1, wherein when the quantitative value of ethanolamine-type plasmalogen (PlsEtn) in plasma falls below the PlsEtn threshold value, it is distinguished from a patient with dementia or a person in the previous stage.
  12. 前記ＭＩ閾値が３６．６ｍｇ／ｇＣｒである請求項１１に記載の方法。 The method of claim 11, wherein the MI threshold is 36.6 mg / gCr.
  13. 前記エタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）閾値が、５８μＭである請求項１１に記載の方法。 The method of claim 11, wherein the ethanolamine type plasmalogen (PlsEtn) threshold is 58 μM.
  14. 健常者と、認知症患者またはその前段階にある者とを区別することができる、自然排泄尿中のミオイノシトール（ＭＩ）の予め求められた閾値と、被験者の自然排泄尿中のミオイノシトール（ＭＩ）の定量値とを比較する工程と組み合わせることにより、被験者を認知症患者またはその前段階にある者か否かを分類する認知症検査に用いるものであって、
    同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する工程を含み、該工程中に、健常者と、認知症患者またはその前段階にある者とを区別するがことできる、血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）の、予め求められた閾値に対して、被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）の定量値を比較する工程を含む、エタノールアミン型プラスマローゲンの定量方法。     A predetermined threshold of myo-inositol (MI) in spontaneously excreted urine, which can distinguish between a healthy person and a person with dementia or in the previous stage, and myo-inositol in the subject's spontaneously excreted urine ( MI) is used for a dementia test for classifying whether a subject is a dementia patient or a person in the previous stage by combining with a step of comparing with a quantitative value of MI,
    Quantifying ethanolamine plasmalogen (PlsEtn) in the serum or plasma of the same subject, during which a healthy person can be distinguished from a patient with dementia or in its predecessor stage, Comparing a quantitative value of ethanolamine-type plasmalogen (PlsEtn) in a subject's serum or plasma against a predetermined threshold of ethanolamine-type plasmalogen (PlsEtn) in serum or plasma, A method for quantifying amine-type plasmalogens.
  15. 同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する工程が、酵素を利用する反応により、最終的に過酸化水素が生成され、該エタノールアミン型プラスマローゲンの存在量に対応して発生する過酸化水素の量を検出して、血清または血漿中のＰｌｓＥｔｎ量を算出する工程を含む請求項１４の方法。 The process of quantifying ethanolamine plasmalogen (PlsEtn) in the serum or plasma of the same subject finally generates hydrogen peroxide by the reaction using an enzyme, and corresponds to the abundance of the ethanolamine plasmalogen. 15. The method according to claim 14, further comprising the step of calculating the amount of PlsEtn in serum or plasma by detecting the amount of hydrogen peroxide generated.
  16. 同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する方法が、以下の（１）～（３－４）の工程を含む請求項１５に記載の方法。
    （１）エタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解できる酵素を用いて、被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解する工程：
    （２）エタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）をグリセロ－３－ホスフォエタノールアミンとアルデヒドに加水分解でき、かつエーテル型リゾホスファチジルエタノールアミンを加水分解できない加水分解酵素を用いて、前記（１）にて生成されたエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）をグリセロ－３－ホフォエタノールアミンとアルデヒドに加水分解する工程：及び
    （３－１）グリセロ－３－ホスフォエタノールアミンを加水分解してエタノールアミンにせしめる酵素にて、工程（２）で得られたグリセロ－３－ホスフォエタノールアミンを加水分解してエタノールアミンにする工程：
    （３－２）エタノールアミンを酸化する酵素により、エタノールアミンから過酸化水素を発生せしめる工程：
    （３－３）過酸化水素を過酸化水素定量手段により定量する工程：及び
    （３－４）前記被験者の血清または血漿中のＰｌｓＥｔｎ量を算出する工程：
    または前記（２）の加水分解酵素が、リゾホスファチジルエタノールアミンを加水分解できる酵素である場合、同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する方法が、前記（１）～（３－４）の工程に先立つか、少なくとも前記（２）工程の前までに、下記（４）及び（５）を含む工程を行うことを特徴とする方法。
    （４）ホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する酵素を用いて、血清または血漿中に混在するホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する工程：及び
    （５）リゾホスフォリパーゼ及び／又はモノグリセリドリパーゼを用いて、前記（４）工程により生成されるリゾホスファチジルエタノールアミンを分解し、実質的に消去する工程：     The method according to claim 15, wherein the method for quantifying ethanolamine-type plasmalogen (PlsEtn) in serum or plasma of the same subject comprises the following steps (1) to (3-4).
    (1) Using an enzyme capable of hydrolyzing ethanolamine-type plasmalogen (PlsEtn) into ethanolamine-type lysoplasmalogen (lyPlsEtn), ethanolamine-type plasmalogen (PlsEtn) in the serum or plasma of the subject is ethanolamine-type lysozyme. Hydrolyzing to plasmalogen (lyPlsEtn):
    (2) Using a hydrolase that can hydrolyze ethanolamine-type lysoplasmalogen (lyPlsEtn) into glycero-3-phosphoethanolamine and an aldehyde and cannot hydrolyze ether-type lysophosphatidylethanolamine, (1) Hydrolyzing ethanolamine-type lysoplasmalogen (lyPlsEtn) produced in step 1 to glycero-3-phosphoethanolamine and aldehyde: and (3-1) hydrolyzing glycero-3-phosphoethanolamine A step of hydrolyzing the glycero-3-phosphoethanolamine obtained in step (2) to ethanolamine with an enzyme that is made to be ethanolamine:
    (3-2) Step of generating hydrogen peroxide from ethanolamine by an enzyme that oxidizes ethanolamine:
    (3-3) Step of quantifying hydrogen peroxide by means of hydrogen peroxide quantification: (3-4) Step of calculating the amount of PlsEtn in the serum or plasma of the subject:
    Alternatively, when the hydrolase of (2) is an enzyme capable of hydrolyzing lysophosphatidylethanolamine, the method for quantifying ethanolamine-type plasmalogen (PlsEtn) in the serum or plasma of the same subject is described in (1) A method comprising performing the steps (4) and (5) below prior to the steps (3-4) or at least before the step (2).
    (4) Step of decomposing phosphatidylethanolamine mixed in serum or plasma into lysophosphatidylethanolamine and fatty acid using an enzyme that decomposes phosphatidylethanolamine into lysophosphatidylethanolamine and fatty acid: and (5) lysophospho Step of decomposing and substantially eliminating lysophosphatidylethanolamine produced by the step (4) using lipase and / or monoglyceride lipase:
  17. 前記工程（２）の加水分解酵素が、下記（ａ）または（ｂ）の酵素のいずれかである請求項１６に記載の方法。
    （ａ）配列番号１または２に記載のアミノ酸配列：
    （ｂ）配列番号１または２に記載のアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列：
    からなる酵素であって、エタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）を加水分解し、グリセロ－３－ホスフォエタノールアミンとアルデヒドとを得る反応を触媒する：     The method according to claim 16, wherein the hydrolase in the step (2) is one of the following enzymes (a) and (b).
    (A) the amino acid sequence set forth in SEQ ID NO: 1 or 2:
    (B) Amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 or 2:
    Which catalyzes the reaction of hydrolyzing ethanolamine-type lysoplasmalogen (lyPlsEtn) to give glycero-3-phosphoethanolamine and an aldehyde:
  18. 前記工程（１）のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解できる酵素が、下記（ｃ）または（ｄ）のいずれかの酵素である請求項１６に記載の方法。
    （ｃ）配列番号５に記載のアミノ酸配列：
    （ｄ）配列番号５に記載のアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列であり、エタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解する作用を触媒する：     The enzyme capable of hydrolyzing the ethanolamine-type plasmalogen (PlsEtn) in the step (1) into an ethanolamine-type lysoplasmalogen (lyPlsEtn) is one of the following enzymes (c) or (d): The method described.
    (C) the amino acid sequence set forth in SEQ ID NO: 5:
    (D) An amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 5, and ethanolamine type plasmalogen (PlsEtn) is changed to ethanolamine type lysoplasmalogen (lyPlsEtn). ) Catalyze the hydrolysis action:
  19. 前記（２）の加水分解酵素が、リゾホスファチジルエタノールアミンを加水分解できる酵素である場合、同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する方法が、前記（１）～（３－４）の工程に先立つか、少なくとも前記（２）工程の前までに、下記（４）及び（５）を含む工程を行うことを特徴とする請求項１６に記載の方法。
    （４）ホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する酵素を用いて、血清または血漿中に混在するホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する工程：及び
    （５）リゾホスフォリパーゼ及び／又はモノグリセリドリパーゼを用いて、前記（４）工程により生成されるリゾホスファチジルエタノールアミンを分解し、実質的に消去する工程：     When the hydrolase of (2) is an enzyme capable of hydrolyzing lysophosphatidylethanolamine, the method for quantifying ethanolamine-type plasmalogen (PlsEtn) in serum or plasma of the same subject is described in (1) to The method according to claim 16, wherein the step including the following (4) and (5) is performed prior to the step (3-4) or at least before the step (2).
    (4) Step of decomposing phosphatidylethanolamine mixed in serum or plasma into lysophosphatidylethanolamine and fatty acid using an enzyme that decomposes phosphatidylethanolamine into lysophosphatidylethanolamine and fatty acid: and (5) lysophospho Step of decomposing and substantially eliminating lysophosphatidylethanolamine produced by the step (4) using lipase and / or monoglyceride lipase:
  20. 前記工程（４）のホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する酵素としてホスフォリパーゼ（ＰＬ）を使用し、該ＰＬは、前記工程（１）のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解できる酵素としての作用を兼ねさせて新たな該ＰＬを添加することなく前記工程（１）の工程を進行せしめ、前記工程（５）を実施と同時、または前記工程（５）の実施後に、前記工程（１）が完了し、以後前記工程（２）、（３－４）を実施する請求項１９に記載の方法。 Phospholipase (PL) is used as an enzyme that decomposes the phosphatidylethanolamine of the step (4) into lysophosphatidylethanolamine and a fatty acid, and the PL uses the ethanolamine-type plasmalogen (PlsEtn) of the step (1). Simultaneously with the implementation of the step (5), the step (1) is allowed to proceed without adding new PL, which also serves as an enzyme capable of hydrolyzing ethanolamine-type lysoplasmalogen (lyPlsEtn). The method according to claim 19, wherein after the step (5) is performed, the step (1) is completed, and then the steps (2) and (3-4) are performed.
  21. 同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する方法が、以下の（１）～（３－３）´の工程を含む請求項１５に記載の方法。
    （１）エタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解できる酵素を用いて、被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解する工程：
    （２）´エタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）をエタノールアミンとプラズメニルホスファチジン酸に加水分解でき、かつエーテル型リゾホスファチジルエタノールアミンを加水分解でき、かつリゾホスファチジルエタノールアミンを加水分解できる加水分解酵素を用いて、前記（１）にて生成されたエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）をエタノールアミンとプラズメニルホスファチジン酸に加水分解する工程：及び
    （３－１）´エタノールアミンを酸化する酵素により、工程（２）´で得られたエタノールアミンから過酸化水素を発生せしめる工程：
    （３－２）´過酸化水素を過酸化水素定量手段により定量する工程：及び
    （３－３）´前記被験者の血清または血漿中のＰｌｓＥｔｎ量を算出する工程：
          The method according to claim 15, wherein the method for quantifying ethanolamine plasmalogen (PlsEtn) in serum or plasma of the same subject comprises the following steps (1) to (3-3) ′.
    (1) Using an enzyme capable of hydrolyzing ethanolamine-type plasmalogen (PlsEtn) into ethanolamine-type lysoplasmalogen (lyPlsEtn), ethanolamine-type plasmalogen (PlsEtn) in the serum or plasma of the subject is ethanolamine-type lysozyme. Hydrolyzing to plasmalogen (lyPlsEtn):
    (2) Hydrolyzing enzyme capable of hydrolyzing ethanolamine type lysoplasmalogen (lyPlsEtn) to ethanolamine and plasmenyl phosphatidic acid, hydrolyzing ether type lysophosphatidylethanolamine, and hydrolyzing lysophosphatidylethanolamine Hydrolyzing the ethanolamine-type lysoplasmalogen (lyPlsEtn) produced in the above (1) into ethanolamine and plasmenylphosphatidic acid using: and (3-1) by an enzyme that oxidizes ethanolamine The step of generating hydrogen peroxide from the ethanolamine obtained in step (2) ′:
    (3-2) 'Step of quantifying hydrogen peroxide by means of hydrogen peroxide quantification: and (3-3)' Step of calculating PlsEtn amount in serum or plasma of the subject:
  22. 同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する方法が、前記（１）～（３－３）´の工程に先立つか、少なくとも前記（２）工程の前までに、下記（４）及び（５）を含む工程を行うことを特徴とする請求項２１に記載の方法。
    （４）ホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する酵素を用いて、血清または血漿中に混在するホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する工程：及び
    （５）リゾホスフォリパーゼ及び／又はモノグリセリドリパーゼを用いて、前記（４）工程により生成されるリゾホスファチジルエタノールアミンを分解し、実質的に消去する工程：     A method for quantifying ethanolamine plasmalogen (PlsEtn) in serum or plasma of the same subject is performed prior to the steps (1) to (3-3) ′ or at least before the step (2). The method according to claim 21, wherein a process including the following (4) and (5) is performed.
    (4) Step of decomposing phosphatidylethanolamine mixed in serum or plasma into lysophosphatidylethanolamine and fatty acid using an enzyme that decomposes phosphatidylethanolamine into lysophosphatidylethanolamine and fatty acid: and (5) lysophospho Step of decomposing and substantially eliminating lysophosphatidylethanolamine produced by the step (4) using lipase and / or monoglyceride lipase:
  23. 前記工程（４）のホスファチジルエタノールアミンをリゾホスファチジルエタノールアミンと脂肪酸に分解する酵素としてホスフォリパーゼ（ＰＬ）を使用し、該ＰＬは、前記工程（１）のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解できる酵素としての作用を兼ねさせて新たな該ＰＬを添加することなく前記工程（１）の工程を進行せしめ、前記工程（５）を実施と同時、または前記工程（５）の実施後に、前記工程（１）が完了し、以後前記工程（２）～（３－３）´を実施する請求項２２に記載の方法。 Phospholipase (PL) is used as an enzyme that decomposes the phosphatidylethanolamine of the step (4) into lysophosphatidylethanolamine and a fatty acid, and the PL uses the ethanolamine-type plasmalogen (PlsEtn) of the step (1). Simultaneously with the implementation of the step (5), the step (1) is allowed to proceed without adding new PL, which also serves as an enzyme capable of hydrolyzing ethanolamine-type lysoplasmalogen (lyPlsEtn). The method according to claim 22, wherein after the step (5) is performed, the step (1) is completed, and thereafter the steps (2) to (3-3) ′ are performed.
  24. 健常者と、認知症患者またはその前段階にある者とを区別するがことできる、血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）の予め求められた閾値と、被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）の定量値とを比較する工程と組み合わせることにより、被験者を認知症患者またはその前段階にある者か否かを分類する認知症検査に用いるものであって、
    同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する工程を含み、該工程中に、健常者と、認知症患者またはその前段階にある者とを区別するがことできる、自然排泄尿中のミオイノシトール（ＭＩ）の、予め求められた閾値に対して、被験者の自然排泄尿中のミオイノシトール（ＭＩ）の定量値を比較する工程を含む、ミオイノシトール（ＭＩ）の定量方法。     A pre-determined threshold for ethanolamine plasmalogen (PlsEtn) in serum or plasma, which can distinguish between healthy and demented patients or those in its predecessor, and in the serum or plasma of the subject In combination with the step of comparing the ethanolamine-type plasmalogen (PlsEtn) with a quantitative value, the subject is used for a dementia test for classifying whether or not the subject is a dementia patient or a person in its previous stage,
    Quantifying ethanolamine plasmalogen (PlsEtn) in the serum or plasma of the same subject, during which a healthy person can be distinguished from a patient with dementia or in its predecessor stage, Quantification of myo-inositol (MI) including the step of comparing the quantitative value of myo-inositol (MI) in spontaneously excreted urine of a subject against a predetermined threshold value of myo-inositol (MI) in spontaneously excreted urine Method.
  25. 被験者を認知症患者またはその前段階にある者か否かを分類するための認知症検査方法に用いられる手段であって、
    　被験者の自然排泄尿中のミオイノシトール（ＭＩ）を定量する手段と、同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する手段とを含み、
    健常者と、認知症患者またはその前段階にある者とを区別することができる、自然排泄尿中のミオイノシトール（ＭＩ）の予め求められた閾値と、血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）の予め求められた閾値のそれぞれに対して、前記被験者のミオイノシトール（ＭＩ）の定量値とエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）の定量値を比較して用いる認知症検査手段。     A method used in a dementia test method for classifying whether a subject is a dementia patient or a person in the previous stage,
    Means for quantifying myo-inositol (MI) in spontaneously excreted urine of a subject, and means for quantifying ethanolamine-type plasmalogen (PlsEtn) in serum or plasma of the same subject,
    A pre-determined threshold of myo-inositol (MI) in spontaneously excreted urine and ethanolamine plasmalogens in serum or plasma, which can distinguish between healthy and demented patients or those in its predecessor The dementia test | inspection means which compares and uses the quantitative value of myo-inositol (MI) of said test subject, and the quantitative value of ethanolamine type plasmalogen (PlsEtn) with respect to each of the threshold value calculated | required in advance of (PlsEtn).
  26. 前記手段が検査薬キットである請求項２５に記載の認知症検査手段。 The dementia test means according to claim 25, wherein the means is a test drug kit.
  27. 健常者と、認知症患者またはその前段階にある者とを区別することができる、自然排泄尿中のミオイノシトール（ＭＩ）の予め求められた閾値と、被験者の自然排泄尿中のミオイノシトール（ＭＩ）の定量値とを比較する工程と組み合わせることにより、被験者を認知症患者またはその前段階にある者か否かを分類する認知症検査に用いるものであって、
    同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する工程を含み、該工程中に、健常者と、認知症患者またはその前段階にある者とを区別するがことできる、血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）の、予め求められた閾値に対して、被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）の定量値を比較して用いる、エタノールアミン型プラスマローゲンの定量手段。     A predetermined threshold of myo-inositol (MI) in spontaneously excreted urine, which can distinguish between a healthy person and a person with dementia or in the previous stage, and myo-inositol in the subject's spontaneously excreted urine ( MI) is used for a dementia test for classifying whether a subject is a dementia patient or a person in the previous stage by combining with a step of comparing with a quantitative value of MI,
    Quantifying ethanolamine plasmalogen (PlsEtn) in the serum or plasma of the same subject, during which a healthy person can be distinguished from a patient with dementia or in its predecessor stage, Ethanolamine used by comparing the quantitative value of ethanolamine-type plasmalogen (PlsEtn) in serum or plasma of a subject against a predetermined threshold of ethanolamine-type plasmalogen (PlsEtn) in serum or plasma Quantitative means of type plasmalogen.
  28. 該エタノールアミン型プラスマローゲンの定量手段が試薬キットである請求項２７に記載の定量手段。 28. The quantification means according to claim 27, wherein the quantification means for the ethanolamine plasmalogen is a reagent kit.
  29. 同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する試薬キットが、以下の（１ａ）～（３ａ）を含む請求項２８に記載の試薬キット。
    （１ａ）エタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解できる酵素およびその酵素反応を進めることのできる添加物。
    （２ａ）エタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）をグリセロ－３－ホスフォエタノールアミンとアルデヒドに加水分解でき、かつエーテル型リゾホスファチジルエタノールアミンを加水分解できない加水分解酵素およびその酵素反応を進めることのできる添加物。
    （３ａ）グリセロ－３－ホスフォエタノールアミンをエタノールアミンとグリセロリン酸に変換する酵素、エタノールアミンをグリコールアルデヒド、アンモニア、及び過酸化水素に変換する酵素とその酵素反応を進めるにことのできる添加物、さらに必要により過酸化水素を定量するための過酸化水素定量手段。
    　または前記（２ａ）の加水分解酵素がリゾホスファチジルエタノールアミンを加水分解できる酵素である場合、前記（１ａ）に、さらにリゾホスファチジルエタノールアミンを加水分解する酵素を含む試薬キット。     The reagent kit according to claim 28, wherein the reagent kit for quantifying ethanolamine plasmalogen (PlsEtn) in serum or plasma of the same subject comprises the following (1a) to (3a).
    (1a) An enzyme capable of hydrolyzing ethanolamine type plasmalogen (PlsEtn) to ethanolamine type lysoplasmalogen (lyPlsEtn) and an additive capable of proceeding with the enzyme reaction.
    (2a) a hydrolase capable of hydrolyzing ethanolamine-type lysoplasmalogen (lyPlsEtn) into glycero-3-phosphoethanolamine and aldehyde and not hydrolyzing ether-type lysophosphatidylethanolamine, and advancing the enzyme reaction thereof Possible additive.
    (3a) An enzyme that converts glycero-3-phosphoethanolamine into ethanolamine and glycerophosphoric acid, an enzyme that converts ethanolamine into glycolaldehyde, ammonia, and hydrogen peroxide, and an additive capable of advancing the enzymatic reaction Further, hydrogen peroxide determination means for determining hydrogen peroxide if necessary.
    Alternatively, when the hydrolase of (2a) is an enzyme capable of hydrolyzing lysophosphatidylethanolamine, the reagent kit further comprises (1a) an enzyme that hydrolyzes lysophosphatidylethanolamine.
  30. 前記（２ａ）の加水分解酵素がリゾホスファチジルエタノールアミンを加水分解できる酵素である場合、前記（１ａ）に、さらにリゾホスファチジルエタノールアミンを加水分解する酵素を含む請求項２９に記載の試薬キット。 30. The reagent kit according to claim 29, wherein, when the hydrolase of (2a) is an enzyme capable of hydrolyzing lysophosphatidylethanolamine, (1a) further contains an enzyme that hydrolyzes lysophosphatidylethanolamine.
  31. 前記（２ａ）の加水分解酵素が、下記（ａ）または（ｂ）の酵素のいずれかである請求項２９に記載の試薬キット。
    （ａ）配列番号１または２に記載のアミノ酸配列：
    （ｂ）配列番号１または２に記載のアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列：
    からなる酵素であって、エタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）を加水分解し、グリセロ－３－ホスフォエタノールアミンとアルデヒドとを得る反応を触媒する：     The reagent kit according to claim 29, wherein the hydrolase of (2a) is one of the following enzymes (a) or (b).
    (A) the amino acid sequence set forth in SEQ ID NO: 1 or 2:
    (B) Amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 or 2:
    Which catalyzes the reaction of hydrolyzing ethanolamine-type lysoplasmalogen (lyPlsEtn) to give glycero-3-phosphoethanolamine and an aldehyde:
  32. 前記（１ａ）のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解できる酵素、が下記（ｃ）または（ｄ）のいずれかの酵素である請求項２９に記載の試薬キット。
    （ｃ）配列番号５に記載のアミノ酸配列：
    （ｄ）配列番号５に記載のアミノ酸配列において、１又は複数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列であり、エタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）をエタノールアミン型リゾプラスマローゲン（ｌｙＰｌｓＥｔｎ）に加水分解する作用を触媒する：     The enzyme capable of hydrolyzing the ethanolamine-type plasmalogen (PlsEtn) of (1a) to an ethanolamine-type lysoplasmalogen (lyPlsEtn) is one of the following enzymes (c) or (d): Reagent kit.
    (C) the amino acid sequence set forth in SEQ ID NO: 5:
    (D) An amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 5, and ethanolamine type plasmalogen (PlsEtn) is changed to ethanolamine type lysoplasmalogen (lyPlsEtn). ) Catalyze the hydrolysis action:
  33. 健常者と、認知症患者またはその前段階にある者とを区別することができる、血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）の予め求められた閾値と、被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）の定量値とを比較する工程と組み合わせることにより、被験者を認知症患者またはその前段階にある者か否かを分類する認知症検査に用いるものであって、
    同一被験者の血清または血漿中のエタノールアミン型プラスマローゲン（ＰｌｓＥｔｎ）を定量する工程を含み、該工程中に、健常者と、認知症患者またはその前段階にある者とを区別することができる、自然排泄尿中のミオイノシトール（ＭＩ）の、予め求められた閾値に対して、被験者の自然排泄尿中のミオイノシトール（ＭＩ）の定量値を比較して用いる、ミオイノシトール（ＭＩ）の定量手段。     A pre-determined threshold of ethanolamine-type plasmalogen (PlsEtn) in serum or plasma, which can distinguish between healthy and demented patients or those in its predecessor, and in the serum or plasma of the subject In combination with the step of comparing the ethanolamine-type plasmalogen (PlsEtn) with a quantitative value, the subject is used for a dementia test for classifying whether or not the subject is a dementia patient or a person in its previous stage,
    Quantifying ethanolamine-type plasmalogen (PlsEtn) in the serum or plasma of the same subject, during which a healthy person can be distinguished from a patient with dementia or in its predecessor stage, Means for quantifying myo-inositol (MI), which is used by comparing the quantitative value of myo-inositol (MI) in spontaneously excreted urine of a subject with a predetermined threshold of myo-inositol (MI) in spontaneously excreted urine .
  34. 前記手段が試薬キットである請求項３３に記載の定量手段。 The quantitative means according to claim 33, wherein the means is a reagent kit.
PCT/JP2013/068965 2012-07-12 2013-07-11 Method of examining cognitive function and kit therefor WO2014010667A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2014524864A JP6185466B2 (en) 2012-07-12 2013-07-11 Cognitive function testing method and kit

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2012-156420 2012-07-12
JP2012156420 2012-07-12

Publications (1)

Publication Number Publication Date
WO2014010667A1 true WO2014010667A1 (en) 2014-01-16

Family

ID=49916108

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2013/068965 WO2014010667A1 (en) 2012-07-12 2013-07-11 Method of examining cognitive function and kit therefor

Country Status (2)

Country Link
JP (1) JP6185466B2 (en)
WO (1) WO2014010667A1 (en)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016045112A (en) * 2014-08-25 2016-04-04 株式会社 レオロジー機能食品研究所 Ether phospholipid quantifying method
JP2016111929A (en) * 2014-12-11 2016-06-23 株式会社 レオロジー機能食品研究所 Method for quantifying plasmalogen
JP2017165785A (en) * 2017-07-03 2017-09-21 丸大食品株式会社 Learning memory enhancer
WO2017221795A1 (en) * 2016-06-22 2017-12-28 旭化成ファーマ株式会社 MEASUREMENT OF Lp-PLA2 ACTIVITY
JP2018130130A (en) * 2016-05-02 2018-08-23 有限会社梅田事務所 Chicken breast meat derived plasmalogen composition and food composition containing the same for preventing forgetfulness related to language and situation of healthy subjects
WO2018212341A1 (en) * 2017-05-19 2018-11-22 国立大学法人徳島大学 Agent for preventing, ameliorating or treating acanthotic disease and/or acanthotic symptom
CN109153693A (en) * 2016-05-02 2019-01-04 有限会社梅田事务所 The judgment method of the non-diseased state of safety-stable plasmalogen and its preparation and cognitive disorder
CN113466381A (en) * 2021-06-16 2021-10-01 华南师范大学 Method for measuring antibiotics in human urine by using solid-phase extraction-high performance liquid chromatography-tandem mass spectrometry

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2669068C1 (en) * 2017-07-14 2018-10-08 Дмитрий Юрьевич Мартынов Compressor with movable flexible membrane

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007010892A1 (en) * 2005-07-19 2007-01-25 Asahi Kasei Pharma Corporation Novel phospholipid processing agent
JP2007034410A (en) * 2005-07-22 2007-02-08 Oki Electric Ind Co Ltd Dictionary information creation apparatus
JP2011093813A (en) * 2009-10-27 2011-05-12 Meiji Milk Prod Co Ltd Plasmalogen-type phospholipid
JP2011136926A (en) * 2009-12-28 2011-07-14 Hokkaido Univ Method of plasmalogen analysis
JP2011257148A (en) * 2010-06-04 2011-12-22 Teikyo Univ Detection method

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011032109A1 (en) * 2009-09-11 2011-03-17 Sma Foundation Biomarkers for spinal muscular atrophy
US20120107841A1 (en) * 2010-07-19 2012-05-03 Mcintyre John A Serum Diagnostic Method, Biomarker and Kit for Early Detection and Staging of Alzheimer's Disease

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007010892A1 (en) * 2005-07-19 2007-01-25 Asahi Kasei Pharma Corporation Novel phospholipid processing agent
JP2007034410A (en) * 2005-07-22 2007-02-08 Oki Electric Ind Co Ltd Dictionary information creation apparatus
JP2011093813A (en) * 2009-10-27 2011-05-12 Meiji Milk Prod Co Ltd Plasmalogen-type phospholipid
JP2011136926A (en) * 2009-12-28 2011-07-14 Hokkaido Univ Method of plasmalogen analysis
JP2011257148A (en) * 2010-06-04 2011-12-22 Teikyo Univ Detection method

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
RYOTA MAEBA ET AL.: "Kessei Plasmalogen no Ninchi Kino Hyoka no Biomaker to shite no Yukosei no Kento", DAI 30 KAI JAPAN SOCIETY FOR DEMENTIA RESEARCH GAKUJUTSU SHUKAI PROGRAM SHOROKUSHU, vol. 25, no. 3, 13 October 2011 (2011-10-13), pages 342 *
RYOTA MAEBA ET AL.: "Shizen Haisetsu Nyo Myoinositol no Rinshoteki Igi ni Kansuru Kento", THE JAPANESE JOURNAL OF CLINICAL PATHOLOGY, DAI 58 KAI JAPANESE SOCIETY OF LABORATORY MEDICINE GAKUJUTSU SHUKAI, vol. O-025, 2011, pages 95 *
RYOTA MAEBA: "Strange Phospholipids -Plasmalogen", OLEOSCIENCE, vol. 5, no. 9, 2005, pages 405 - 415 *
YOSHINORI FUJIWARA ET AL.: "Nyo Myoinositol o Mochiita Ninchisho Koreisha no Soki Hakken ni Muketa Seikagaku Marker no Kaihatsu", DAIWA SECURITIES HEALTH FOUNDATION KENKYU GYOSEKISHU, vol. 34, 2011, pages 62 - 65 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016045112A (en) * 2014-08-25 2016-04-04 株式会社 レオロジー機能食品研究所 Ether phospholipid quantifying method
JP2016111929A (en) * 2014-12-11 2016-06-23 株式会社 レオロジー機能食品研究所 Method for quantifying plasmalogen
JP2018130130A (en) * 2016-05-02 2018-08-23 有限会社梅田事務所 Chicken breast meat derived plasmalogen composition and food composition containing the same for preventing forgetfulness related to language and situation of healthy subjects
CN109153693A (en) * 2016-05-02 2019-01-04 有限会社梅田事务所 The judgment method of the non-diseased state of safety-stable plasmalogen and its preparation and cognitive disorder
CN109153693B (en) * 2016-05-02 2022-08-02 有限会社梅田事务所 Safe and stable plasmalogen, preparation thereof, and method for determining non-diseased state of cognitive disorder
WO2017221795A1 (en) * 2016-06-22 2017-12-28 旭化成ファーマ株式会社 MEASUREMENT OF Lp-PLA2 ACTIVITY
DE112017003142T5 (en) 2016-06-22 2019-03-21 Asahi Kasei Pharma Corporation Measurement of LP-PLA2 activity
US20200002744A1 (en) * 2016-06-22 2020-01-02 Asahi Kasei Pharma Corporation Measurement of lp-pla2 activity
US10900063B2 (en) 2016-06-22 2021-01-26 Asahi Kasei Pharma Corporation Measurement of Lp-PLA2 activity
WO2018212341A1 (en) * 2017-05-19 2018-11-22 国立大学法人徳島大学 Agent for preventing, ameliorating or treating acanthotic disease and/or acanthotic symptom
JP2017165785A (en) * 2017-07-03 2017-09-21 丸大食品株式会社 Learning memory enhancer
CN113466381A (en) * 2021-06-16 2021-10-01 华南师范大学 Method for measuring antibiotics in human urine by using solid-phase extraction-high performance liquid chromatography-tandem mass spectrometry

Also Published As

Publication number Publication date
JPWO2014010667A1 (en) 2016-06-23
JP6185466B2 (en) 2017-08-23

Similar Documents

Publication Publication Date Title
JP6185466B2 (en) Cognitive function testing method and kit
Chauhan et al. Oxidative stress in autism: Increased lipid peroxidation and reduced serum levels of ceruloplasmin and transferrin-the antioxidant proteins
JP5730344B2 (en) Quantitative reagent for small particle low density lipoprotein
Santi et al. Association of lipids with oxidative stress biomarkers in subclinical hypothyroidism
KR101586505B1 (en) METHOD AND KIT FOR QUANTIFICATION OF small, dense LDL CHOLESTEROL
EP1362925B1 (en) Compositions for assaying glycoprotein
US8697378B2 (en) Method and kit for quantitative determination for small, dense particle low density lipoproteins
EP1813680B1 (en) Compositions for lipase activity determination and method of determining activity
TWI507671B (en) Method of erasing triglyceride in lipoproteins other than low density lipoprotein
KR20130043183A (en) Method for quantifying the amount of cholesterol in high-density lipoprotein 3
EP2380989A1 (en) Method and reagent for determining mevalonic acid, 3-hydroxymethylglutaryl-coenzyme a and coenzyme a
CN111032880B (en) Method for measuring cholesterol in low density lipoprotein, reagent for measurement, and kit for measurement
EP2639310B1 (en) Method for quantification of remnant-like lipoprotein cholesterol and kit for same
EP1818412B1 (en) Method of measuring cholesterol in remnant-like lipoproteins
WO2017221795A1 (en) MEASUREMENT OF Lp-PLA2 ACTIVITY
JP4925384B2 (en) Method for quantifying N-terminal glycated protein
JP4861005B2 (en) Lipase activity measuring composition and activity measuring method
KR101943673B1 (en) Sphingomyelin measurement method and measurement kit
JP2012210179A (en) Method for measuring ether type phospholipid
JP5926801B2 (en) Plasmalogen hydrolysis method, plasmalogen measurement method
US20070224657A1 (en) Distribution of PON1 as a marker of lipid related disorders
JP2021136896A (en) Phospholipase d, and method for quantifying ethanolamine type plasmalogen
JP4244168B2 (en) Analysis method and composition of ethanolamine-containing phospholipid

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13817531

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2014524864

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13817531

Country of ref document: EP

Kind code of ref document: A1