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WO2009148050A1 - Procédé d’évaluation des effets d’atténuation et de prévention de l’artériosclérose, kit pour l’évaluation et procédé de criblage de substance - Google Patents

Procédé d’évaluation des effets d’atténuation et de prévention de l’artériosclérose, kit pour l’évaluation et procédé de criblage de substance Download PDF

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Publication number
WO2009148050A1
WO2009148050A1 PCT/JP2009/060056 JP2009060056W WO2009148050A1 WO 2009148050 A1 WO2009148050 A1 WO 2009148050A1 JP 2009060056 W JP2009060056 W JP 2009060056W WO 2009148050 A1 WO2009148050 A1 WO 2009148050A1
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Prior art keywords
mass
protein
arteriosclerosis
substance
charge ratio
Prior art date
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Ceased
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PCT/JP2009/060056
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English (en)
Japanese (ja)
Inventor
憲一 三原
準 向井
亜尚 山口
豊 高橋
泰士 矢野原
一樹 柳田
裕二 内藤
敏一 吉川
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Biomarker Science Co Ltd
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Biomarker Science Co Ltd
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Publication date
Priority claimed from JP2008146948A external-priority patent/JP5373322B2/ja
Application filed by Biomarker Science Co Ltd filed Critical Biomarker Science Co Ltd
Publication of WO2009148050A1 publication Critical patent/WO2009148050A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/76—Assays involving albumins other than in routine use for blocking surfaces or for anchoring haptens during immunisation
    • G01N2333/765—Serum albumin, e.g. HSA
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/775—Apolipopeptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/323—Arteriosclerosis, Stenosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease

Definitions

  • the present invention relates to an evaluation method and an evaluation kit for an effect of improving / preventing arteriosclerosis, and a method for screening a substance, and more specifically, a test substance in an animal that has developed arteriosclerosis or an animal that has a high risk of developing in the future.
  • An evaluation method of arteriosclerosis improvement / prevention effect which evaluates the improvement effect of arteriosclerosis of the test substance or the reduction effect of future onset risk, by indicating the concentration of the marker substance in the body fluid of the animal,
  • a kit for evaluating an arteriosclerosis improvement / prevention effect that can be easily performed by the evaluation method, and a substance for screening a substance having an effect of improving arteriosclerosis or reducing the risk of developing future using the evaluation method
  • the present invention relates to a screening method.
  • Arteriosclerosis is a general term for pathological conditions in which arterial walls are thickened and lose elasticity. There are several types of arteriosclerosis, but the most common condition is atherosclerosis (atherosclerosis). Unless otherwise noted, arteriosclerosis often refers to atherosclerosis. Atherosclerosis is a condition in which rod-shaped plaques (atheromas) are deposited on the inner wall of the artery and interfere with blood flow, and may cause serious damage to the brain, heart, kidney, limbs, and the like. That is, it may lead to life-threatening symptoms such as cerebral infarction, cerebral hemorrhage, myocardial infarction, angina pectoris, renal failure, and limb necrosis. Although the cause of arteriosclerosis has not been completely clarified yet, as described above, obesity, diabetes, hyperlipidemia, hypertension and the like are considered risk factors for the onset.
  • a prophylactic marker it is possible to easily examine whether a test substance has an effect of reducing the risk of developing the disease, which is useful for functional evaluation and screening of food materials.
  • Patent Document 1 discloses a preventive marker for diabetes and Patent Document 2 discloses a preventive marker for hyperlipidemia.
  • these are not biomarkers specialized for arteriosclerosis.
  • Patent Document 3 discloses C-reactive protein (CRP) which is a biomarker of inflammation
  • Patent Document 4 discloses ⁇ -fetoprotein which is a tumor marker.
  • CRP C-reactive protein
  • AFP AFP
  • CRP and AFP have established clinical significance (inflammation and carcinogenesis) completely different from arteriosclerosis, and are difficult to use as a biomarker of arteriosclerosis in terms of specificity. Therefore, it is desired to identify a new marker substance that enables diagnosis of future onset risk of arteriosclerosis, improvement of arteriosclerosis of the test substance, or reduction of future onset risk.
  • An object of the present invention is to newly identify a marker substance that can be a preventive marker for arteriosclerosis, and to evaluate the effect of improving the arteriosclerosis of the test substance or reducing the risk of future onset using the marker substance, etc. Is to provide.
  • One aspect of the present invention for solving the above-described problem is that an animal that has developed arteriosclerosis or an animal that has a high risk of developing the future takes in a test substance, and the following marker substance ( At least one concentration of M01) to (M29) is compared with a reference value, and the improvement effect or prevention effect of arteriosclerosis is characterized by evaluating the improvement effect of arteriosclerosis possessed by the test substance or the reduction effect of future risk of onset This is an evaluation method.
  • (M01) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 3350 when subjected to mass spectrometry;
  • (M02) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 4170 when subjected to mass spectrometry;
  • (M03) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 4280 when subjected to mass spectrometry;
  • (M04) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 4750 when subjected to mass spectrometry;
  • (M05) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a
  • the method for evaluating the effect of improving / preventing arteriosclerosis is to allow an animal that has developed arteriosclerosis or an animal with a high risk of developing in the future to ingest a test substance, and the above (M01) to (M The concentration of at least one of the 29 marker substances of M29) is compared with a reference value, and the improvement effect of arteriosclerosis or the future risk reduction effect of the test substance is evaluated.
  • Each of the marker substances (M01) to (M29) is a protein specifically detected in the body fluid of an animal immediately before the onset of arteriosclerosis, and is useful as a prophylactic marker / risk marker for arteriosclerosis. is there. According to the evaluation method of the arteriosclerosis improvement / prevention effect of this aspect, it is possible to easily and accurately evaluate the arteriosclerosis improvement effect or the future risk reduction effect of the test substance.
  • the “animal” includes humans as well as animals such as mice.
  • the values of “about 3350”, “about 8440”, “about 66000”, etc. of the mass / charge ratio (hereinafter sometimes abbreviated as “m / z”) in each marker substance are This is a value that takes into account the error range of the measured value, and has a width of approximately ⁇ 0.2%. That is, about 3350 represents about 3350 ⁇ 0.2%, about 8440 represents about 8440 ⁇ 0.2%, and about 66000 represents about 66000 ⁇ 0.2%.
  • the other mass / charge ratios have a width of approximately ⁇ 0.2% in a similar manner.
  • these marker substances are mainly proteins present in blood.
  • the marker substance (M02), (M03), (M08), (M09), (M10), (M16) in the body fluid of the animal ), (M22), (M23), and (M29) have lower concentrations, (M01), (M04), (M05), (M06), (M07), (M11), (M12) , (M13), (M14), (M15), (M17), (M18), (M19), (M20), (M21), (M24), (M25), (M26), (M27), and The concentration of (M28) shows a higher value.
  • the marker substance (M06) is apolipoprotein A2 or a modified form thereof.
  • the marker substance (M07) is serum albumin or a modified form thereof.
  • the marker substance (M09) is apolipoprotein C1 or a modified form thereof.
  • the marker substance (M10) is apolipoprotein C1 or a modified form thereof.
  • the marker substance (M15) is apolipoprotein A2 or a modified form thereof.
  • the marker substance (M16) is apolipoprotein C3 or a modified form thereof.
  • the marker substance (M21) is transthyretin or a modified form thereof.
  • the marker substance (M22) is transthyretin or a modified form thereof.
  • the marker substance (M23) is transthyretin or a modified form thereof.
  • the marker substance (M25) is a retinol binding protein or a modified form thereof.
  • the marker substance (M26) is a retinol binding protein or a modified form thereof.
  • the marker substance (M27) is apolipoprotein A1 or a modified form thereof.
  • Apolipoprotein A2 serum albumin, apolipoprotein C1, apolipoprotein C3, transthyretin, retinol binding protein, and apolipoprotein A1 are all well known for their physicochemical properties.
  • the marker substances (M06), (M07), (M09), (M10), (M15), (M16), (M21), (M22), (M23), (M25), Analysis of (M26) and (M27) is easy.
  • modified protein is a protein in which at least one of the amino acid residues constituting the protein is modified.
  • Modification includes not only addition of a compound or functional group (eg, phosphorylation) but also elimination (eg, dephosphorylation).
  • the “protein or a modified form thereof” includes an isoform of the protein.
  • protein or a modified form thereof includes substantially the same protein in which several amino acid residues are deleted, substituted or added in the primary structure of the protein.
  • protein or a modified product thereof includes a protein fragment derived from the protein that has been cleaved by a protease. In the case of a complex protein, “protein or a modified product thereof” includes its subunits.
  • Another aspect of the present invention for solving the same problem is that an animal that has developed arteriosclerosis or an animal that has a high risk of developing the disease ingests the test substance, and the following (N1) Comparing the concentration of at least one marker substance belonging to any one of (N7) to a reference value, and evaluating an improvement effect of arteriosclerosis or a future risk reduction effect of the test substance. This is a method for evaluating the curing improvement / prevention effect.
  • N1 apolipoprotein A2 or a modified product thereof, (N2) serum albumin or a modified product thereof, (N3) apolipoprotein C1 or a modified form thereof, (N4) apolipoprotein C3 or a modified product thereof, (N5) transthyretin or a modified form thereof, (N6) retinol binding protein or a modified form thereof, (N7) Apolipoprotein A1 or a modified form thereof.
  • the method for evaluating the effect of improving / preventing arteriosclerosis in this aspect is to allow an animal that has developed arteriosclerosis or an animal that has a high risk of developing in the future to ingest a test substance, and the above (N1) to (N).
  • the concentration of at least one marker substance belonging to any one of N7) is compared with a reference value, and the improvement effect of arteriosclerosis or the future risk reduction effect of the test substance is evaluated.
  • the marker substances belonging to the above (N1) to (N7) are all proteins specifically detected in the body fluids of animals immediately before the onset of atherosclerosis, and are useful as preventive markers / risk markers for arteriosclerosis. Is.
  • the evaluation method of the arteriosclerosis improvement / prevention effect of this aspect it is possible to easily and accurately evaluate the arteriosclerosis improvement effect or the future risk reduction effect of the test substance.
  • apolipoprotein A2, serum albumin, apolipoprotein C1, apolipoprotein C3, transthyretin, retinol binding protein, and apolipoprotein A1 are all well known in physicochemical properties, they are easy to analyze.
  • the term “animal” includes humans as well as animals such as mice. All of the marker substances belonging to (N1) to (N7) are mainly present in blood.
  • a representative example of a “modified protein” is a protein in which at least one of the amino acid residues constituting the protein is modified. Separation is also included.
  • “protein or a modified form thereof” includes an isoform of the protein, a substantially identical protein in which several amino acid residues are deleted, substituted or added in the primary structure of the protein, and Protein fragments derived from the protein that have been cleaved by a protease are included.
  • the “protein or a modified form thereof” includes its subunits.
  • the reference value is obtained when an animal that has developed arteriosclerosis or an animal that has a high future risk of ingesting a known substance that does not have an effect of improving arteriosclerosis or a risk of reducing future risk of development.
  • the concentration of the marker substance in the body fluid of the animal is obtained when an animal that has developed arteriosclerosis or an animal that has a high future risk of ingesting a known substance that does not have an effect of improving arteriosclerosis or a risk of reducing future risk of development.
  • the arteriosclerosis improving effect or the future risk reducing effect of the test substance can be more easily and accurately evaluated.
  • the animal that has developed arteriosclerosis or the animal that has a high risk of developing in the future is a naturally occurring model animal or a genetically engineered model animal.
  • a naturally occurring model animal or a genetically engineered model animal is used as an animal that has developed arteriosclerosis or has a high risk of developing in the future.
  • the breeding of the animal is facilitated, and the effect of improving the arteriosclerosis or the risk of reducing the future onset risk of the test substance can be evaluated very easily.
  • Examples of such animals include apolipoprotein E gene-deficient mice (hereinafter abbreviated as “apo E-deficient mice”). Apo E-deficient mice form atheroma in normal breeding and are adopted in various fields as a standard for arteriosclerosis model animals.
  • the body fluid is blood.
  • a body fluid as a measurement sample can be easily collected, and the effect of improving arteriosclerosis or the risk of future onset of the test substance can be evaluated more easily and quickly.
  • the test substance is a food material.
  • the bodily fluid or bodily fluid component is brought into contact with a carrier on which a substance having affinity for the marker substance is immobilized to capture the marker substance in the body fluid on the carrier, and the amount of the trapped marker substance Based on the above, the concentration of the marker substance in the body fluid is calculated.
  • a carrier on which a substance having affinity for the marker substance is immobilized is used. Then, the body fluid or body fluid component is brought into contact with the carrier, and the marker substance contained in the body fluid or body fluid component is captured on the carrier via a substance having affinity for the marker substance, and the amount of the marker substance captured is increased. Based on this, the concentration of the marker substance in the body fluid is calculated.
  • the concentration of the marker substance in the body fluid is calculated.
  • the carrier has a flat portion, and the substance having affinity for the marker substance is immobilized on a part of the flat portion.
  • a carrier having a planar portion is used, and a substance having affinity for the marker substance is immobilized on a part of the planar portion.
  • the substance having affinity for the marker substance can be spot-fixed at a plurality of locations on the carrier.
  • the concentration of the marker substance can be measured even from a very small amount of measurement sample.
  • An example of the carrier having a planar portion is a substrate such as a chip.
  • the substance having affinity for the marker substance is an ion exchanger or an antibody.
  • an ion exchanger or an antibody is used as a substance having affinity for the marker substance, and the marker substance in the measurement sample is placed on the carrier via the ion exchanger or the antibody.
  • the substance is an ion exchanger, various substances are easily available, and a carrier for capturing the marker substance can be easily prepared. Further, when the substance is an antibody, the marker substance can be captured more specifically. Examples of the method for measuring the amount of the captured marker substance include mass spectrometry and immunoassay (in the case of an antibody).
  • Still another aspect of the present invention is to evaluate a test substance by the method for evaluating the effect of improving / preventing arteriosclerosis according to the present invention, and to screen for a substance having an effect of improving arteriosclerosis or reducing the risk of developing future. This is a screening method for a characteristic substance.
  • This aspect relates to a screening method for substances, and at least one concentration of each of the marker substances (M01) to (M29) and each marker substance (N1) to (N7) in the body fluid of an animal is used as a reference value.
  • a substance having an effect of improving arteriosclerosis or reducing the risk of future onset is screened.
  • Each of the marker substances (M01) to (M29) and the marker substances belonging to (N1) to (N7) is a protein that is specifically detected in the body fluid of an animal immediately before the onset of arteriosclerosis It is useful as a preventive marker / risk marker for arteriosclerosis.
  • substances having an effect of improving arteriosclerosis or reducing the risk of future onset can be screened easily and with high accuracy.
  • the test substance is a food material
  • Still another aspect of the present invention is a kit for use in the method for evaluating the effect of improving / preventing arteriosclerosis according to the present invention, comprising a carrier on which a substance having affinity for the marker substance is immobilized. This is a kit for evaluating the effect of improving and preventing arteriosclerosis.
  • the kit for evaluating the effect of improving / preventing atherosclerosis in this aspect includes a carrier on which a substance having affinity for the marker substance is immobilized. According to this aspect, it is not necessary to prepare the carrier separately when measuring the concentration of the marker substance, and the concentration of the marker substance can be measured very simply.
  • the substance having affinity for the marker substance is an ion exchanger.
  • the marker substance can be more reliably captured on the carrier.
  • it is used for screening a substance having an effect of improving arteriosclerosis or reducing the risk of future onset.
  • the method for evaluating the arteriosclerosis improvement / prevention effect of the present invention it is possible to easily and accurately evaluate the arteriosclerosis improvement effect or the future risk reduction effect of the test substance.
  • a substance having an effect of improving arteriosclerosis or reducing the risk of future onset can be easily and accurately screened.
  • the evaluation kit for the arteriosclerosis improvement / prevention effect of the present invention it is not necessary to prepare the carrier separately when measuring the concentration of the marker substance, and the concentration of the marker substance can be measured very simply.
  • FIG. 6 is an ion peak diagram showing the results of SELDI-TOF-MS analysis performed in the purification process of the marker substance (M06) of Example 2.
  • 4 is a photograph showing the results of SDS-PAGE performed in the purification process of the marker substance (M07) of Example 3.
  • FIG. 6 is an ion peak diagram showing the results of SELDI-TOF-MS analysis performed in the purification process of the marker substance (M07) of Example 3.
  • FIG. 6 is a photograph showing the results of SDS-PAGE performed in the purification process of the marker substance (M09) of Example 4.
  • FIG. 6 is an ion peak diagram showing the results of SELDI-TOF-MS analysis performed in the purification process of the marker substance (M09) of Example 4.
  • 6 is a photograph showing the results of SDS-PAGE performed in the purification process of the marker substance (M10) of Example 5.
  • FIG. 6 is an ion peak diagram showing the results of SELDI-TOF-MS analysis performed in the purification process of the marker substance (M10) of Example 5. It is a photograph which shows the result of SDS-PAGE performed in the purification process of the marker substance (M15) of Example 6 and the marker substance (M16) of Example 7.
  • FIG. 6 is a photograph showing the results of SDS-PAGE performed in the purification process of the marker substance (M15) of Example 6 and the marker substance (M16) of Example 7.
  • FIG. 6 is an ion peak diagram showing the results of SELDI-TOF-MS analysis performed in the purification process of the marker substance (M15) of Example 6.
  • FIG. 10 is an ion peak diagram showing a result of SELDI-TOF-MS analysis performed in the purification process of the marker substance (M16) of Example 7.
  • 6 is a photograph showing the results of SDS-PAGE performed in the purification process of marker substances (M21) to (M23) of Example 8.
  • FIG. 10 is an ion peak diagram showing the results of SELDI-TOF-MS analysis performed in the purification process of marker substances (M21) to (M23) in Example 8.
  • 6 is a photograph showing the results of SDS-PAGE performed in the purification process of the marker substance (M25) of Example 9.
  • FIG. 10 is an ion peak diagram showing the results of SELDI-TOF-MS analysis performed in the purification process of the marker substance (M25) of Example 9.
  • 6 is a photograph showing the results of SDS-PAGE performed in the purification process of the marker substance (M26) of Example 10 and the marker substance (M27) of Example 11.
  • FIG. 10 is an ion peak diagram showing a result of SELDI-TOF-MS analysis performed in the purification process of the marker substance (M26) of Example 10.
  • FIG. 14 is an ion peak diagram showing a result of SELDI-TOF-MS analysis performed in the purification process of the marker substance (M27) of Example 11.
  • the method for evaluating the effect of improving and preventing arteriosclerosis includes two aspects.
  • an animal that has developed arteriosclerosis or an animal that has a high risk of developing in the future has the test substance ingested, and at least one concentration of the following marker substances (M01) to (M29) in the body fluid of the animal: Is compared with the reference value, and the improvement effect of arteriosclerosis or the future risk reduction effect of the test substance is evaluated.
  • (M01) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 3350 when subjected to mass spectrometry;
  • (M02) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 4170 when subjected to mass spectrometry;
  • (M03) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 4280 when subjected to mass spectrometry;
  • (M04) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 4750 when subjected to mass spectrometry;
  • (M05) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a
  • marker substances are mainly proteins present in blood, and are specifically detected in the animal body immediately before the onset of arteriosclerosis.
  • (M02), (M03), (M08), (M09), (M10), (M16), (M22), (M23), and (M29) marker substances (hereinafter referred to as these marker substances) Is sometimes referred to as “Group 1”.) Is higher in a state in which arteriosclerosis has developed or in a state in which the risk of future development of arteriosclerosis is high, the test substance is arterial. In the case of having an effect of improving hardening or reducing the risk of future onset, it shows a lower value in animals fed with the test substance.
  • (M01), (M04), (M05), (M06), (M07), (M11), (M12), (M13), (M14), (M15), (M17), (M18), (M19), (M20), (M21), (M24), (M25), (M26), (M27), and (M28) marker substances (hereinafter referred to as “Group 2”). ) May indicate a lower value in a state in which arteriosclerosis has developed or in a state in which the risk of future development of arteriosclerosis is high, the test substance has an effect of improving arteriosclerosis or In the case of having an effect of reducing the risk of future onset, the value is higher in the animal ingested with the test substance.
  • the marker substance (M06) can be identified as a fragment of apolipoprotein A2. Further, according to peptide mapping under certain conditions, the marker substance (M07) is serum albumin, the marker substances (M09) and (M10) are apolipoprotein C1, the marker substance (M15) is apolipoprotein A2, and the marker substance (M16) is Apolipoprotein C3, marker substances (M21) to (M23) are identified as transthyretin, marker substances (M25) and (M26) as retinol-binding protein, and (M27) as apolipoprotein A1.
  • the marker substance (M07) is serum albumin
  • the marker substances (M09) and (M10) are apolipoprotein C1
  • the marker substance (M15) is apolipoprotein A2
  • the marker substance (M16) is Apolipoprotein C3
  • marker substances (M21) to (M23) are identified as transthyretin
  • the marker substance (M06) is apolipoprotein A2 or a modified form thereof.
  • the marker substance (M07) is serum albumin or a modified form thereof.
  • the marker substance (M09) is apolipoprotein C1 or a modified form thereof.
  • the marker substance (M10) is apolipoprotein C1 or a modified form thereof.
  • the marker substance (M15) is apolipoprotein A2 or a modified form thereof.
  • the marker substance (M16) is apolipoprotein C3 or a modified form thereof.
  • the marker substance (M21) is transthyretin or a modified form thereof.
  • the marker substance (M22) is transthyretin or a modified form thereof.
  • the marker substance (M23) is transthyretin or a modified form thereof.
  • the marker substance (M25) is a retinol binding protein or a modified form thereof.
  • the marker substance (M26) is a retinol binding protein or a modified form thereof.
  • the marker substance (M27) is apolipoprotein A1 or a modified form thereof.
  • an animal that has developed arteriosclerosis or an animal that has a high risk of developing the future has a test substance ingested, and the following (The concentration of at least one marker substance belonging to any one of (N1) to (N7) is compared with a reference value, and the improvement effect of arteriosclerosis or the future risk reduction effect of the test substance is evaluated.
  • N1 apolipoprotein A2 or a modified product thereof, (N2) serum albumin or a modified product thereof, (N3) apolipoprotein C1 or a modified form thereof, (N4) apolipoprotein C3 or a modified product thereof, (N5) transthyretin or a modified form thereof, (N6) retinol binding protein or a modified form thereof, (N7) Apolipoprotein A1 or a modified form thereof.
  • protein modifications include methylation, acetylation, adenylylation, myristylation, etc. of N-terminal ⁇ -amino group and lysine ⁇ -amino group; addition of sugar or sugar chain to serine / threonine / asparagine; serine / threonine / Examples include phosphorylation of tyrosine, arginine, and histidine; cysteine cysteinylation, homocysteinylation, sulfonylation, etc .; ⁇ -carboxylation of glutamic acid; conversion of N-terminal glutamic acid to pyroglutamic acid, and the like. Further, elimination of these modifications (demethylation, elimination of sugars or sugar chains, dephosphorylation, etc.) is also included in “modification”.
  • Protein or modified form thereof includes isoforms of the protein. Isoforms include proteins generated by alternative splicing in addition to the various modifications described above. Furthermore, the “protein or a modified form thereof” includes substantially the same protein in which several amino acid residues are deleted, substituted or added in the primary structure of the protein. Furthermore, “protein or a modified product thereof” includes a protein fragment derived from the protein that has been cleaved by a protease. For example, a protein fragment having a length that can be recognized as derived from the protein, for example, a protein fragment composed of 20 or more amino acid residues, a protein fragment having a molecular weight of 2,000 or more, and the like can be mentioned. In the case of a complex protein, the “protein or a modified product thereof” includes its subunits.
  • a protein fragment (SEQ ID NO: 8) corresponding to the amino acid number 554 (Ala) to 565 (Ala) portion of serum albumin (SEQ ID NO: 6) is included in the “modified form of serum albumin”.
  • N-terminal amino acid sequence of apolipoprotein C1 (SEQ ID NO: 14) after cleavage of the signal sequence begins with “Ala-Pro”, but it has been reported that these two amino acids can be cleaved (Oleg Chertov et al., Proteomics, Vol. 4, No. 4, 2004).
  • Such a fragment of apolipoprotein C1 lacking two N-terminal amino acids (SEQ ID NO: 15) is included in the “modified body of apolipoprotein C1”.
  • transthyretin SEQ ID NO: 30
  • various modified transthyretins in which only the Cys residue is modified have been found (Amareth Lim et al., J. Biol. Chem., Vol. 258, No. 50, 2003).
  • S-sulfonated transthyretin in which a sulfone group is added to the Cys residue of transthyretin
  • glutathionated transthyretin in which glutathione is added are included in “modified forms of transthyretin”.
  • retinol-binding protein SEQ ID NO: 38
  • several types of protein fragments lacking some of the amino acid residues have been found in serum. These protein fragments are included in “modified forms of retinol-binding protein”.
  • only one of the marker substances (M01) to (M29) and each of the marker substances belonging to (N1) to (N7) may be used. May be used in combination. There is no particular limitation on the combination method in the case of using a plurality, but for example, the marker substance (s) selected from group 1 and the marker substance (s) selected from group 2 can be combined. .
  • the effect of improving arteriosclerosis or the future onset of an animal that has developed arteriosclerosis or an animal with a high risk of future onset is used. That is, when an animal that has developed arteriosclerosis or an animal with a high risk of future onset is ingested with a known substance that does not have an effect of improving arteriosclerosis or of reducing the risk of future onset, the above in the body fluid The concentration of the marker substance becomes an “abnormal value”.
  • the value (measured value) in the animal ingested with the test substance is compared with the reference value (abnormal value), and the measured value is significantly different from the reference value and is on the normal side (normal side) It is possible to evaluate that the test substance has an effect of improving arteriosclerosis or an effect of reducing the risk of future onset.
  • the marker substance belonging to group 1 is used as an index
  • the measured value is significantly lower than the reference value
  • the marker substance belonging to group 2 is used as an index
  • the measured value When is significantly higher than the reference value, it can be evaluated that the test substance has an effect of improving arteriosclerosis or reducing the risk of future onset.
  • a value in addition to the abnormal value described above, a value (normal value, negative control) in an animal that has not developed arteriosclerosis or an animal that has a low risk of developing arteriosclerosis can be added to the reference value.
  • test substance has an effect of improving arteriosclerosis or an effect of reducing the risk of future onset.
  • (4) a group in which an animal that has developed arteriosclerosis or an animal that has a high future risk of ingesting a known substance having an effect of improving arteriosclerosis or reducing the risk of developing future Values in animals (positive control) can also be added.
  • the group of (4) above is set and animals are raised.
  • there is a significant difference between (1) and (2) there is a significant difference between (3) and (2), and (3) is normal ((1) and (2) compared to (2). 4), it can be evaluated that the test substance has an effect of improving arteriosclerosis or reducing the risk of future onset.
  • test substance exhibits a behavior similar to that of the known substance employed in (4) and can be said to have a similar action.
  • a known substance having an effect of improving arteriosclerosis or an effect of reducing the risk of future onset includes an antihypertensive agent (calcium antagonist) such as azelnidipine.
  • an animal that has developed arteriosclerosis or an animal that has a high risk of developing in the future there is no particular limitation on the type of animal in “an animal that has developed arteriosclerosis or an animal that has a high risk of developing in the future”, and for example, a mouse, a rat, a rabbit, or a pig can be employed.
  • an “animal that has developed arteriosclerosis or an animal that has a high risk of developing in the future” a naturally occurring model animal of arteriosclerosis, or a genetically engineered model animal by transgenic or gene targeting is preferably used.
  • Specific examples of such animals include apoE-deficient mice. Since apo E-deficient mice usually develop arteriosclerosis when bred, breeding experiments can be easily performed, which is particularly preferable.
  • arteriosclerosis develops when apoE-deficient mice are normally bred, but the onset can be detected around the 20th week of breeding (for example, Arteriosclerosis, Thrombosis, and Vascular Biology; 2004; 24). ; 1006-1014). Therefore, it can be said that the apoE-deficient mice before the 20th week of breeding, for example, the 14th week of breeding, are in the stage immediately before the onset of arteriosclerosis.
  • the marker substance belonging to any of the marker substances (M01) to (M29) and (N1) to (N7) used in the method for evaluating the effect of improving / preventing arteriosclerosis according to the present invention is used in apoE-deficient mice at 14 weeks of breeding.
  • the concentration in the body fluids is significantly different. Therefore, when using an apoE-deficient mouse in the present invention, for example, the concentration of each marker substance may be measured using a body fluid around the 14th week of breeding as a measurement sample.
  • an arteriosclerosis spontaneous animal model that forms an atheroma when a high cholesterol diet is ingested can also be used.
  • the arteriosclerosis improving effect or the future risk reduction effect of the test substance can be evaluated.
  • humans can be employed as animals. In this case, the substance will be evaluated according to the results of clinical trials.
  • Blood is preferably used as the animal body fluid used in the method for evaluating the effect of improving / preventing arteriosclerosis according to the present invention.
  • serum or plasma body fluid component
  • Serum or plasma can be prepared from blood by a known method such as centrifugation.
  • test substance in the method for evaluating the effect of improving / preventing arteriosclerosis according to the present invention examples include food materials and drug substances.
  • food materials when food materials are to be evaluated, it can be used for the development of functional foods.
  • a method for measuring the concentration of a marker substance is a method generally used for protein quantification as long as it can specifically measure the concentration of the marker substance.
  • various immunoassays, mass spectrometry (MS), chromatography, electrophoresis and the like can be used.
  • the concentration of the marker substance can be accurately measured even with a sample having a lot of contaminants.
  • immunoassays include classical methods such as precipitation, agglutination, and hemolysis, which directly or indirectly measure antigen-antibody conjugates, and enzyme immunoassays (EIA) with increased detection sensitivity in combination with labeling methods. , Radioimmunoassay (RIA), fluorescent immunoassay (FIA) and the like.
  • the antibody specific for the marker substance used in these immunoassays may be monoclonal or polyclonal.
  • an ion peak derived from each marker substance can be specified, and the amount (concentration) of each marker substance can be measured with the ion peak intensity.
  • the ionization method for measuring the concentration of the marker substance by mass spectrometry either matrix-assisted laser ionization (MALDI) or electrospray ionization (ESI) can be applied.
  • MALDI matrix-assisted laser ionization
  • ESI electrospray ionization
  • MALDI is preferred because it produces less multivalent ions.
  • TOF time-of-flight mass spectrometer
  • the test material is subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to separate the target marker substance, and the gel is prepared with an appropriate dye or fluorescent substance. It is only necessary to stain and measure the intensity and fluorescence intensity of the band corresponding to the target marker substance.
  • SDS-PAGE SDS-polyacrylamide gel electrophoresis
  • two-dimensional electrophoresis combined with isoelectric focusing (IEF) can be used.
  • Western blotting can also be performed to measure the amount of marker substance on the membrane.
  • a method by liquid high-performance chromatography can be used. That is, the concentration of the marker substance in the sample can be measured by subjecting the sample to HPLC to separate the target marker substance and measuring the peak area of the chromatogram.
  • a marker substance is captured on a carrier, and the captured marker substance is a measurement target. That is, a substance having affinity for the marker substance is immobilized on the carrier, and the marker substance is captured on the carrier via the substance having the affinity. Then, the concentration of the marker substance in the body fluid is calculated based on the amount of the captured marker substance. According to this embodiment, it is possible to reduce the influence of contaminants contained in the sample, and to measure the concentration of the marker substance with higher sensitivity and higher accuracy.
  • a carrier having a flat portion such as a substrate can be used in addition to a general one such as beads, metal, glass, resin, and the like.
  • a substance having affinity for the marker substance it is preferable to immobilize a substance having affinity for the marker substance on a part of the planar portion.
  • a carrier in which a chip is used as a substrate and a substance having affinity for a marker substance is spot-fixed at a plurality of spots on the surface.
  • affinity include ion binding, affinity between metal chelate and histidine residue in protein, antigen and antibody, enzyme and substrate, bioaffinity such as hormone and receptor, and hydrophobicity. Chemical interactions such as interactions.
  • the ion exchanger When the marker substance is captured on the carrier by ion binding, the ion exchanger is immobilized on the carrier.
  • a cation exchanger or an anion exchanger can be used as the ion exchanger, and further, a strong cation exchanger, a weak cation exchanger, a strong anion exchanger, and a weak anion exchanger. Either of these can be used, but a strong anion exchanger and a weak cation exchanger are preferably used.
  • strong anion exchangers include quaternary ammonium (trimethylaminomethyl) (QA), quaternary aminoethyl (diethyl, mono-2-hydroxybutylaminoethyl) (QAE), quaternary ammonium (trimethylammonium) ( And those having a strong anion exchange group such as QMA).
  • weak cation exchangers include those having weak cation exchange groups such as carboxymethyl (CM).
  • strong cation exchangers include those having a strong cation exchange group such as sulfopropyl (SP).
  • the weak anion exchanger include those having a weak anion exchange group such as dimethylaminoethyl (DE) and diethylaminoethyl (DEAE).
  • a metal chelate such as Cu 2+ , Zn 2+ , Ni 2+ , Co 2+ , Al 3+ , Fe 3+ , Ga 3+ is fixed.
  • a fluorinated carrier can be used.
  • an antibody specific for the marker substance may be immobilized on the carrier.
  • hydrophobic groups include C4 to C20 alkyl groups, phenyl groups, and the like.
  • an immunoassay when used as a method for measuring a marker substance, it is preferable to use a carrier on which an antibody is immobilized.
  • an immunoassay system using the antibody immobilized on the carrier as the primary antibody can be easily constructed.
  • two types of antibodies specific to a marker substance and having different epitopes are prepared, one is immobilized on a carrier as a primary antibody, and the other is enzyme-labeled as a secondary antibody to construct a sandwich EIA system.
  • an immunoassay system based on a binding inhibition method or a competitive method can be constructed.
  • a substrate is used as a carrier, immunoassay using an antibody chip is possible. According to the antibody chip, the concentration of a plurality of marker substances can be measured simultaneously, and rapid measurement is possible.
  • an ion exchanger for example, an ion exchanger, a metal chelate, or a carrier on which a hydrophobic group is immobilized can be used in addition to an antibody. Since binding by these substances is not as specific as bioaffinity such as antigens and antibodies, when using a carrier on which these substances are immobilized, substances other than the marker substance can also be captured on the carrier. According to the analysis, there is no problem because it is quantified by the mass spectrometer spectrum reflecting the molecular weight.
  • a substrate as a carrier, surface-enhanced laser desorption / ionization-time-of-flight mass measurement (hereinafter referred to as "SELDI-TOF-MS") ), The concentration of the marker substance can be measured more accurately.
  • substrates that can be used, cation exchange substrates, anion exchange substrates, normal phase substrates, reverse phase substrates, metal ion substrates, antibody substrates, etc. can be used, but cation exchange substrates, particularly weak cation exchanges.
  • a substrate and an anion exchange substrate, particularly a strong anion exchange substrate are preferably used.
  • the substance screening method of the present invention is a method for screening a substance having an effect of improving arteriosclerosis or reducing the risk of future onset by evaluating a test substance by the method for evaluating the effect of improving or preventing arteriosclerosis of the present invention. is there.
  • the same embodiment as the above-described method for evaluating the effect of improving / preventing arteriosclerosis of the present invention can be employed.
  • an evaluation kit can be constructed by collecting necessary reagents.
  • the evaluation kit include those containing a carrier on which a substance having affinity for a marker substance is immobilized.
  • a carrier on which a weak cation exchanger such as CM or a strong anion exchanger such as QA or QAE is immobilized as a carrier, SELDI-TOF-MS or the like is easily performed. be able to.
  • the kit may contain other reagents such as standard substances and various pretreatment buffers.
  • this kit can be used also as a kit for screening the substance which has the improvement effect of arteriosclerosis, or the reduction effect of the future onset risk. Examples of the configuration of the kit of the present invention are given below.
  • the marker substances (M01) to (M29) and (N1) to (N7) are related to events related to arteriosclerosis, for example, whether or not arteriosclerosis has occurred, improved arteriosclerosis, and future of arteriosclerosis. There is also provided use as a biomarker for detecting the risk of developing, etc.
  • mice Animal experiments using atherosclerosis model animals ApoE-deficient mice (The Jackson Laboratory) as atherosclerosis model animals, C57BL / 6J strain mice (Charles River Japan, Inc. as normal animals, hereinafter simply referred to as “normal mice”) Abbreviated). Moreover, CE-2 (Nippon Claire Co., Ltd., hereinafter simply abbreviated as “normal feed”) and CE-2 (hereinafter simply referred to as 3 mg / kg body weight) containing azelnidipine as feed to be fed. Abbreviated as “Azelnidipine-containing feed”). Calblock (registered trademark, manufactured by Sankyo Co., Ltd.) was adopted as azelnidipine.
  • the following four groups with different combinations of mouse types and feeds were set and breeding was started. The number of individuals in each group was 5 or more.
  • Group 1 Breeding normal mice with normal feed
  • Group 2 Breeding apoE-deficient mice with regular feed
  • Group 3 Breeding apoE-deficient mice with feed containing azelnidipine
  • Group 4 Breeding normal mice with feed containing azelnidipine That is, the first group corresponds to a group that does not develop arteriosclerosis, the second group corresponds to a group that develops arteriosclerosis, and the third group corresponds to a group that suppresses the development of arteriosclerosis.
  • the fourth group is a group for verifying the action of azelnidipine.
  • SPA-H sinapinic acid
  • SPA-L sinapinic acid
  • CHCA ⁇ -cyano-4-hydroxycinnamic acid
  • SELDI-TOF-MS SELDI-TOF-MS was performed.
  • the measurement molecular weight range (m / z) was in the range of 3000 to 200000. Moreover, the measurement was performed in duplicate and the average value of m / z was calculated. Data analysis was performed using Protein® Chip® Software, Ciphergen Express® Data® Manager, and Biomarker® Patterns® Software (all from Bio-Rad).
  • Candidate peak search There is a significant difference in ionic strength between the first group and the second group (p ⁇ 0.05).
  • Azelnidipine effect verification There is a significant difference in ionic strength between the second group and the third group (p ⁇ 0.05), and the value of the third group is first than the value of the second group. It is close to the value of the group (the value of the third group has returned to the first group side).
  • FIG. 1 shows a box diagram when the peak intensity of each peak is plotted for each group.
  • the upper and lower edges of the bag are the maximum and minimum values, respectively, the upper and lower sides of the box are the third quartile (75th percentile) and the first quartile (25th percentile), respectively, Value (the same applies to FIG. 2 and subsequent figures).
  • the ROC area (first group vs second group) of this peak is 0.820
  • the p value (first group vs second group) is 0.0233
  • the p value (second group vs third group) is It was 0.0343.
  • the protein (marker substance (M01)) that produces a peak with a mass / charge ratio of about 3350 when subjected to SELDI-TOF-MS is a mouse that has developed arteriosclerosis, or the risk of future onset of arteriosclerosis. It was found to be a substance specific to high mice and could be a marker for arteriosclerosis. As a result, the arteriosclerosis of the test substance can be improved using the concentration of the marker substance (M01) in the body fluid of the animal that has developed arteriosclerosis ingested by the test substance or that has a high risk of developing in the future. It has been shown that it is possible to evaluate the effect or the effect of reducing the risk of future onset, and to screen for such substances.
  • test substance when a body fluid sample is prepared by performing the same animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 3350 is generated.
  • the test substance can be evaluated as having an effect of improving arteriosclerosis or an effect of reducing the risk of future onset.
  • FIG. 2 shows a box diagram when the peak intensity of this peak is plotted for each group.
  • the ROC area (first group vs second group) of this peak is 0.980
  • the p value (first group vs second group) is 0.0002
  • the p value (second group vs third group) is It was 0.0413.
  • a protein that produces a peak with a mass / charge ratio of about 4170 when subjected to SELDI-TOF-MS is a mouse that has developed arteriosclerosis, or the risk of future development of arteriosclerosis. It was found to be a substance specific to high mice and could be a marker for arteriosclerosis. As a result, the arteriosclerosis of the test substance is improved using the concentration of the marker substance (M02) in the body fluid of the animal that has developed arteriosclerosis ingested by the test substance or an animal that has a high risk of developing in the future. It has been shown that it is possible to evaluate the effect or the effect of reducing the risk of future onset, and to screen for such substances.
  • test substance when a body fluid sample is prepared by performing the same animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 4170 is generated.
  • the test substance When the protein concentration is maintained at a normal value, the test substance can be evaluated as having an effect of improving arteriosclerosis or an effect of reducing the risk of future onset.
  • the ROC area (first group vs second group) of this peak is 0.740
  • the p value (first group vs second group) is 0.0233
  • the p value (second group vs third group) is It was 0.0009.
  • a protein that produces a peak with a mass / charge ratio of about 4280 when subjected to SELDI-TOF-MS is a mouse that has developed arteriosclerosis, or the risk of future onset of arteriosclerosis. It was found to be a substance specific to high mice and could be a marker for arteriosclerosis. As a result, the arteriosclerosis of the test substance is improved using the concentration of the marker substance (M03) in the body fluid of the animal that has developed the arteriosclerosis ingested by the test substance or that has a high risk of developing in the future. It has been shown that it is possible to evaluate the effect or the effect of reducing the risk of future onset, and to screen for such substances.
  • test substance when a body fluid sample is prepared by performing a similar animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 4280 is generated.
  • the test substance can be evaluated as having an effect of improving arteriosclerosis or an effect of reducing the risk of future onset.
  • the ROC area (first group vs second group) of this peak is 0.860
  • the p value (first group vs second group) is 0.0126
  • the p value (second group vs third group) is It was 0.0494.
  • the protein (marker substance (M04)) that produces a peak with a mass / charge ratio of about 4750 when subjected to SELDI-TOF-MS is a mouse that has developed arteriosclerosis, or the risk of future development of arteriosclerosis. It was found to be a substance specific to high mice and could be a marker for arteriosclerosis. As a result, the arteriosclerosis of the test substance is improved using the concentration of the marker substance (M04) in the body fluid of the animal that has developed arteriosclerosis ingested by the test substance or that has a high risk of developing in the future. It has been shown that it is possible to evaluate the effect or the effect of reducing the risk of future onset, and to screen for such substances.
  • test substance when a body fluid sample is prepared by performing the same animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 4750 is generated.
  • the test substance When the protein concentration is maintained at a normal value, the test substance can be evaluated as having an effect of improving arteriosclerosis or an effect of reducing the risk of future onset.
  • the ROC area (first group vs second group) of this peak is 0.820
  • the p value (first group vs second group) is 0.0126
  • the p value (second group vs third group) is It was 0.0233.
  • the protein (marker substance (M05)) that produces a peak with a mass / charge ratio of about 5130 when subjected to SELDI-TOF-MS is a mouse that has developed arteriosclerosis, or the risk of future development of arteriosclerosis. It was found to be a substance specific to high mice and could be a marker for arteriosclerosis. As a result, the arteriosclerosis of the test substance is improved by using the concentration of the marker substance (M05) in the body fluid of the animal that has developed the arteriosclerosis ingested by the test substance or that has a high risk of developing in the future. It has been shown that it is possible to evaluate the effect or the effect of reducing the risk of future onset, and to screen for such substances.
  • test substance when a body fluid sample is prepared by performing the same animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 5130 is generated.
  • the test substance can be evaluated as having an effect of improving arteriosclerosis or an effect of reducing the risk of future onset.
  • the protein (marker substance (M06)) that produces a peak with a mass / charge ratio of about 5200 when subjected to SELDI-TOF-MS is a mouse that has developed arteriosclerosis, or the risk of future development of arteriosclerosis. It was found to be a substance specific to high mice and could be a marker for arteriosclerosis. As a result, the arteriosclerosis of the test substance is improved using the concentration of the marker substance (M06) in the body fluid of the animal that has developed the arteriosclerosis ingested by the test substance or an animal that has a high risk of developing in the future. It has been shown that it is possible to evaluate the effect or the effect of reducing the risk of future onset, and to screen for such substances.
  • test substance when a body fluid sample is prepared by performing the same animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 5200 is generated.
  • the test substance When the protein concentration is maintained at a normal value, the test substance can be evaluated as having an effect of improving arteriosclerosis or an effect of reducing the risk of future onset.
  • the ROC area (first group vs second group) of this peak is 0.780
  • the p value (first group vs second group) is 0.0082
  • the p value (second group vs third group) is It was 0.0343.
  • a protein that produces a peak with a mass / charge ratio of about 6020 when subjected to SELDI-TOF-MS is a mouse that has developed arteriosclerosis, or the risk of future development of arteriosclerosis. It was found to be a substance specific to high mice and could be a marker for arteriosclerosis. As a result, the arteriosclerosis of the test substance is improved by using the concentration of the marker substance (M07) in the body fluid of the animal that has developed the arteriosclerosis ingested by the test substance or that has a high risk of developing in the future. It has been shown that it is possible to evaluate the effect or the effect of reducing the risk of future onset, and to screen for such substances.
  • test substance when a body fluid sample is prepared by performing a similar animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 6020 is generated.
  • protein concentration is maintained at a normal value, the test substance can be evaluated as having an effect of improving arteriosclerosis or an effect of reducing the risk of future onset.
  • a protein that produces a peak with a mass / charge ratio of about 6100 when subjected to SELDI-TOF-MS is a mouse that has developed arteriosclerosis, or the risk of future development of arteriosclerosis. It was found to be a substance specific to high mice and could be a marker for arteriosclerosis. As a result, the arteriosclerosis of the test substance is improved by using the concentration of the marker substance (M08) in the body fluid of the animal that has developed arteriosclerosis ingested by the test substance or that has a high risk of developing in the future. It has been shown that it is possible to evaluate the effect or the effect of reducing the risk of future onset, and to screen for such substances.
  • test substance when a body fluid sample is prepared by performing the same animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 6100 is generated.
  • the test substance When the protein concentration is maintained at a normal value, the test substance can be evaluated as having an effect of improving arteriosclerosis or an effect of reducing the risk of future onset.
  • the ROC area (first group vs second group) of this peak is 0.980
  • the p value (first group vs second group) is 0.0003
  • the p value (second group vs third group) is It was 0.0494.
  • a protein that produces a peak with a mass / charge ratio of about 6860 when subjected to SELDI-TOF-MS is a mouse that has developed arteriosclerosis, or the risk of future development of arteriosclerosis. It was found to be a substance specific to high mice and could be a marker for arteriosclerosis. As a result, the arteriosclerosis of the test substance is improved by using the concentration of the marker substance (M09) in the body fluid of the animal that has developed the arteriosclerosis ingested by the test substance or an animal that has a high risk of developing in the future.
  • a protein that produces a peak with a mass / charge ratio of about 7010 when subjected to SELDI-TOF-MS is a mouse that has developed arteriosclerosis, or the risk of future development of arteriosclerosis. It was found to be a substance specific to high mice and could be a marker for arteriosclerosis. As a result, the arteriosclerosis of the test substance is improved using the concentration of the marker substance (M10) in the body fluid of the animal that has developed the arteriosclerosis ingested by the test substance or an animal that has a high risk of developing in the future. It has been shown that it is possible to evaluate the effect or the effect of reducing the risk of future onset, and to screen for such substances.
  • test substance when a body fluid sample is prepared by performing a similar animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 7010 is generated.
  • the test substance can be evaluated as having an effect of improving arteriosclerosis or an effect of reducing the risk of future onset.
  • a protein that produces a peak with a mass / charge ratio of about 7100 when subjected to SELDI-TOF-MS is a mouse that has developed arteriosclerosis, or the risk of future onset of arteriosclerosis. It was found to be a substance specific to high mice and could be a marker for arteriosclerosis. As a result, the arteriosclerosis of the test substance is improved by using the concentration of the marker substance (M11) in the body fluid of the animal that has developed the arteriosclerosis ingested by the test substance or the animal that has a high risk of developing in the future.
  • FIG. 12 shows a box diagram when the peak intensity of this peak is plotted for each group.
  • the ROC area (first group vs second group) of this peak is 0.940
  • the p value (first group vs second group) is 0.0007
  • the p value (second group vs third group) is 0.0082.
  • a protein that produces a peak with a mass / charge ratio of about 7570 when subjected to SELDI-TOF-MS is a mouse that has developed arteriosclerosis, or the risk of future development of arteriosclerosis. It was found to be a substance specific to high mice and could be a marker for arteriosclerosis. As a result, the arteriosclerosis of the test substance is improved using the concentration of the marker substance (M12) in the body fluid of the animal that has developed arteriosclerosis ingested by the test substance or that has a high risk of developing in the future. It has been shown that it is possible to evaluate the effect or the effect of reducing the risk of future onset, and to screen for such substances.
  • test substance when a body fluid sample is prepared by performing the same animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 7570 is generated.
  • the test substance can be evaluated as having an effect of improving arteriosclerosis or an effect of reducing the risk of future onset.
  • the protein (marker substance (M13)) that produces a peak with a mass / charge ratio of about 8440 when subjected to SELDI-TOF-MS is a mouse that has developed arteriosclerosis, or the risk of future development of arteriosclerosis. It was found to be a substance specific to high mice and could be a marker for arteriosclerosis. As a result, the arteriosclerosis of the test substance is improved by using the concentration of the marker substance (M13) in the body fluid of the animal that has developed arteriosclerosis ingested by the test substance or an animal that has a high risk of developing in the future. It has been shown that it is possible to evaluate the effect or the effect of reducing the risk of future onset, and to screen for such substances.
  • test substance when a body fluid sample is prepared by performing the same animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 8440 is generated.
  • the test substance can be evaluated as having an effect of improving arteriosclerosis or an effect of reducing the risk of future onset.
  • the ROC area (first group vs second group) of this peak is 0.980
  • the p value (first group vs second group) is 0.0002
  • the p value (second group vs third group) is It was 0.0343.
  • a protein that produces a peak with a mass / charge ratio of about 8530 when subjected to SELDI-TOF-MS is a mouse that has developed arteriosclerosis, or the risk of future development of arteriosclerosis. It was found to be a substance specific to high mice and could be a marker for arteriosclerosis. Thereby, the arteriosclerosis of the test substance is improved by using the concentration of the marker substance (M14) in the body fluid of the animal that has developed the arteriosclerosis ingesting the test substance or the animal having a high risk of developing in the future. It has been shown that it is possible to evaluate the effect or the effect of reducing the risk of future onset, and to screen for such substances.
  • test substance when a body fluid sample is prepared by performing the same animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 8530 is generated.
  • protein concentration is maintained at a normal value, the test substance can be evaluated as having an effect of improving arteriosclerosis or an effect of reducing the risk of future onset.
  • Fraction 1 (pH 9.0) is brought into contact with weak cation exchange chip CM10, washed with pH 3.0 protein chip binding buffer, and SELDI-TOF-MS (EAM: SPA-L) , An ion peak with a mass / charge ratio of 8723 (average value) was detected. As a result of the increase / decrease pattern analysis, this peak showed a high value in the first group, the third group, and the fourth group, and a low value in the second group.
  • FIG. 15 shows a box diagram when the peak intensity of this peak is plotted for each group.
  • the ROC area (first group vs second group) of this peak is 0.900
  • the p value (first group vs second group) is 0.0007
  • the p value (second group vs third group) is It was 0.0284.
  • the protein (marker substance (M15)) that produces a peak with a mass / charge ratio of about 8720 when subjected to SELDI-TOF-MS is a mouse that has developed arteriosclerosis, or the risk of future development of arteriosclerosis. It was found to be a substance specific to high mice and could be a marker for arteriosclerosis. As a result, the arteriosclerosis of the test substance is improved by using the concentration of the marker substance (M15) in the body fluid of the animal that has developed the arteriosclerosis ingested by the test substance or that has a high risk of developing in the future. It has been shown that it is possible to evaluate the effect or the effect of reducing the risk of future onset, and to screen for such substances.
  • test substance when a body fluid sample is prepared by performing the same animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 8720 is generated.
  • the test substance can be evaluated as having an effect of improving arteriosclerosis or an effect of reducing the risk of future onset.
  • the ROC area (first group vs second group) of this peak is 0.980
  • the p value (first group vs second group) is 0.0002
  • the p value (second group vs third group) is It was 0.0413.
  • a protein that produces a peak with a mass / charge ratio of about 8840 when subjected to SELDI-TOF-MS is a mouse that has developed arteriosclerosis, or the risk of future development of arteriosclerosis. It was found to be a substance specific to high mice and could be a marker for arteriosclerosis. As a result, the arteriosclerosis of the test substance is improved using the concentration of the marker substance (M16) in the body fluid of the animal that has developed arteriosclerosis ingested by the test substance or that has a high risk of developing in the future. It has been shown that it is possible to evaluate the effect or the effect of reducing the risk of future onset, and to screen for such substances.
  • test substance when a body fluid sample is prepared by performing a similar animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 8840 is generated.
  • the test substance can be evaluated as having an effect of improving arteriosclerosis or an effect of reducing the risk of future onset.
  • FIG. 17 shows a box diagram when the peak intensity of each peak is plotted for each group.
  • the ROC area (first group vs second group) of this peak is 0.980
  • the p value (first group vs second group) is 0.0002
  • the p value (second group vs third group) is 0.0032.
  • a protein that produces a peak with a mass / charge ratio of about 10,000 when subjected to SELDI-TOF-MS is a mouse that has developed arteriosclerosis, or the risk of future development of arteriosclerosis. It was found to be a substance specific to high mice and could be a marker for arteriosclerosis. As a result, the arteriosclerosis of the test substance is improved using the concentration of the marker substance (M17) in the body fluid of the animal that has developed arteriosclerosis ingested by the test substance or an animal that has a high risk of developing in the future. It has been shown that it is possible to evaluate the effect or the effect of reducing the risk of future onset, and to screen for such substances.
  • test substance when a body fluid sample is prepared by performing the same animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 10,000 is generated.
  • protein concentration is maintained at a normal value, the test substance can be evaluated as having an effect of improving arteriosclerosis or an effect of reducing the risk of future onset.
  • the ROC area (first group vs second group) of this peak is 0.940
  • the p value (first group vs second group) is 0.0004
  • the p value (second group vs third group) is It was 0.0284.
  • a protein that produces a peak with a mass / charge ratio of about 10500 when subjected to SELDI-TOF-MS is a mouse that has developed arteriosclerosis, or the risk of future development of arteriosclerosis. It was found to be a substance specific to high mice and could be a marker for arteriosclerosis. As a result, the arteriosclerosis of the test substance is improved using the concentration of the marker substance (M18) in the body fluid of the animal that has developed arteriosclerosis ingested by the test substance or that has a high risk of developing in the future. It has been shown that it is possible to evaluate the effect or the effect of reducing the risk of future onset, and to screen for such substances.
  • test substance when a body fluid sample is prepared by performing the same animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 10500 is generated.
  • protein concentration is maintained at a normal value, the test substance can be evaluated as having an effect of improving arteriosclerosis or an effect of reducing the risk of future onset.
  • a protein that produces a peak with a mass / charge ratio of about 10700 when subjected to SELDI-TOF-MS is a mouse that has developed arteriosclerosis, or the risk of future development of arteriosclerosis. It was found to be a substance specific to high mice and could be a marker for arteriosclerosis. As a result, the arteriosclerosis of the test substance is improved using the concentration of the marker substance (M19) in the body fluid of the animal that has developed arteriosclerosis ingested by the test substance or an animal that has a high risk of developing in the future. It has been shown that it is possible to evaluate the effect or the effect of reducing the risk of future onset, and to screen for such substances.
  • test substance when a body fluid sample is prepared by performing a similar animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 10700 is generated.
  • protein concentration is maintained at a normal value, the test substance can be evaluated as having an effect of improving arteriosclerosis or an effect of reducing the risk of future onset.
  • the ROC area (first group vs second group) of this peak is 0.820
  • the p value (first group vs second group) is 0.0284
  • the p value (second group vs third group) is It was 0.0494.
  • a protein that produces a peak with a mass / charge ratio of about 11700 when subjected to SELDI-TOF-MS is a mouse that has developed arteriosclerosis, or the risk of future development of arteriosclerosis. It was found to be a substance specific to high mice and could be a marker for arteriosclerosis. Thereby, the arteriosclerosis of the test substance is improved using the concentration of the marker substance (M20) in the body fluid of the animal that has developed the arteriosclerosis ingested by the test substance or the animal that has a high risk of developing in the future. It has been shown that it is possible to evaluate the effect or the effect of reducing the risk of future onset, and to screen for such substances.
  • test substance when a body fluid sample is prepared by performing a similar animal experiment using a desired test substance and SELDI-TOF-MS is performed in a similar procedure, a peak with a mass / charge ratio of about 11700 is generated.
  • the test substance can be evaluated as having an effect of improving arteriosclerosis or an effect of reducing the risk of future onset.
  • FIG. 21 shows a box diagram when the peak intensity of this peak is plotted for each group.
  • the ROC area (first group vs second group) of this peak is 0.940
  • the p value (first group vs second group) is 0.0015
  • the p value (second group vs third group) is 0.0082.
  • a protein that produces a peak with a mass / charge ratio of about 13700 when subjected to SELDI-TOF-MS is a mouse that has developed arteriosclerosis, or the risk of future development of arteriosclerosis. It was found to be a substance specific to high mice and could be a marker for arteriosclerosis. As a result, the arteriosclerosis of the test substance is improved by using the concentration of the marker substance (M21) in the body fluid of an animal that has developed arteriosclerosis ingested by the test substance or an animal that has a high risk of developing in the future. It has been shown that it is possible to evaluate the effect or the effect of reducing the risk of future onset, and to screen for such substances.
  • test substance when a body fluid sample is prepared by performing a similar animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 13700 is generated.
  • the test substance can be evaluated as having an effect of improving arteriosclerosis or an effect of reducing the risk of future onset.
  • FIG. 22 shows a box diagram when the peak intensity of this peak is plotted for each group.
  • the ROC area (first group vs second group) of this peak is 0.900
  • the p value (first group vs second group) is 0.0032
  • the p value (second group vs third group) is 0.0102.
  • a protein that produces a peak with a mass / charge ratio of about 14000 when subjected to SELDI-TOF-MS is a mouse that has developed arteriosclerosis, or the risk of future development of arteriosclerosis. It was found to be a substance specific to high mice and could be a marker for arteriosclerosis. As a result, the arteriosclerosis of the test substance is improved by using the concentration of the marker substance (M22) in the body fluid of an animal that has ingested the test substance or that has developed arteriosclerosis or that has a high risk of developing in the future. It has been shown that it is possible to evaluate the effect or the effect of reducing the risk of future onset, and to screen for such substances.
  • test substance when a body fluid sample is prepared by performing a similar animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 14,000 is generated.
  • protein concentration is maintained at a normal value, the test substance can be evaluated as having an effect of improving arteriosclerosis or an effect of reducing the risk of future onset.
  • FIG. 23 shows a box diagram when the peak intensity of this peak is plotted for each group.
  • the ROC area (first group vs second group) of this peak is 0.900
  • the p value (first group vs second group) is 0.0019
  • the p value (second group vs third group) is 0.0082.
  • the protein (marker substance (M23)) that produces a peak with a mass / charge ratio of about 14200 when subjected to SELDI-TOF-MS is a mouse that has developed arteriosclerosis, or the risk of future development of arteriosclerosis. It was found to be a substance specific to high mice and could be a marker for arteriosclerosis. As a result, the arteriosclerosis of the test substance is improved by using the concentration of the marker substance (M23) in the body fluid of the animal that has developed arteriosclerosis ingested by the test substance or the animal that has a high risk of developing in the future. It has been shown that it is possible to evaluate the effect or the effect of reducing the risk of future onset, and to screen for such substances.
  • test substance when a body fluid sample is prepared by performing the same animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 14200 is generated.
  • the test substance When the protein concentration is maintained at a normal value, the test substance can be evaluated as having an effect of improving arteriosclerosis or an effect of reducing the risk of future onset.
  • the ROC area (first group vs second group) of this peak is 0.980
  • the p value (first group vs second group) is 0.0002
  • the p value (second group vs third group) is It was 0.0343.
  • the protein (marker substance (M24)) that produces a peak with a mass / charge ratio of about 20400 when subjected to SELDI-TOF-MS is a mouse that has developed arteriosclerosis, or the risk of future development of arteriosclerosis. It was found to be a substance specific to high mice and could be a marker for arteriosclerosis. As a result, the arteriosclerosis of the test substance is improved using the concentration of the marker substance (M24) in the body fluid of the animal that has developed arteriosclerosis ingested by the test substance or an animal that has a high risk of developing in the future. It has been shown that it is possible to evaluate the effect or the effect of reducing the risk of future onset, and to screen for such substances.
  • test substance when a body fluid sample is prepared by performing the same animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 20400 is generated.
  • the test substance When the protein concentration is maintained at a normal value, the test substance can be evaluated as having an effect of improving arteriosclerosis or an effect of reducing the risk of future onset.
  • FIG. 25 shows a box diagram when the peak intensity of this peak is plotted for each group.
  • the ROC area (first group vs second group) of this peak is 0.980
  • the p value (first group vs second group) is 0.0002
  • the p value (second group vs third group) is 0.0065.
  • a protein that produces a peak with a mass / charge ratio of about 20900 when subjected to SELDI-TOF-MS is a mouse that has developed arteriosclerosis, or the risk of future development of arteriosclerosis. It was found to be a substance specific to high mice and could be a marker for arteriosclerosis. As a result, the arteriosclerosis of the test substance is improved using the concentration of the marker substance (M25) in the body fluid of an animal that has ingested the test substance or that has developed arteriosclerosis or an animal that has a high risk of developing in the future. It has been shown that it is possible to evaluate the effect or the effect of reducing the risk of future onset, and to screen for such substances.
  • test substance when a body fluid sample is prepared by performing the same animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 20900 is generated.
  • the test substance can be evaluated as having an effect of improving arteriosclerosis or an effect of reducing the risk of future onset.
  • the ROC area (first group vs second group) of this peak is 0.940
  • the p value (first group vs second group) is 0.0005
  • the p value (second group vs third group) is It was 0.0494.
  • the protein (marker substance (M26)) that produces a peak with a mass / charge ratio of about 21400 when subjected to SELDI-TOF-MS is a mouse that has developed arteriosclerosis, or the risk of future development of arteriosclerosis. It was found to be a substance specific to high mice and could be a marker for arteriosclerosis. As a result, the arteriosclerosis of the test substance is improved using the concentration of the marker substance (M26) in the body fluid of the animal that has developed arteriosclerosis ingested by the test substance or an animal that has a high risk of developing in the future. It has been shown that it is possible to evaluate the effect or the effect of reducing the risk of future onset, and to screen for such substances.
  • test substance when a body fluid sample is prepared by performing the same animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 21400 is generated.
  • the test substance When the protein concentration is maintained at a normal value, the test substance can be evaluated as having an effect of improving arteriosclerosis or an effect of reducing the risk of future onset.
  • the ROC area (first group vs second group) of this peak is 0.980
  • the p value (first group vs second group) is 0.0002
  • the p value (second group vs third group) is It was 0.0191.
  • the protein (marker substance (M27)) that produces a peak with a mass / charge ratio of about 28200 when subjected to SELDI-TOF-MS is a mouse that has developed arteriosclerosis, or the risk of future onset of arteriosclerosis. It was found to be a substance specific to high mice and could be a marker for arteriosclerosis. As a result, the arteriosclerosis of the test substance is improved using the concentration of the marker substance (M27) in the body fluid of the animal that has developed arteriosclerosis ingested by the test substance or an animal that has a high risk of developing in the future. It has been shown that it is possible to evaluate the effect or the effect of reducing the risk of future onset, and to screen for such substances.
  • test substance when a body fluid sample is prepared by performing the same animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 28200 is generated.
  • protein concentration is maintained at a normal value, the test substance can be evaluated as having an effect of improving arteriosclerosis or an effect of reducing the risk of future onset.
  • the ROC area (first group vs second group) of this peak is 0.900
  • the p value (first group vs second group) is 0.0015
  • the p value (second group vs third group) is It was 0.0156.
  • a protein that produces a peak with a mass / charge ratio of about 34400 when subjected to SELDI-TOF-MS is a mouse that has developed arteriosclerosis, or the risk of future onset of arteriosclerosis. It was found to be a substance specific to high mice and could be a marker for arteriosclerosis. As a result, the arteriosclerosis of the test substance is improved using the concentration of the marker substance (M28) in the body fluid of the animal that has developed arteriosclerosis ingested by the test substance or that has a high risk of developing in the future. It has been shown that it is possible to evaluate the effect or the effect of reducing the risk of future onset, and to screen for such substances.
  • test substance when a body fluid sample is prepared by performing a similar animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 34400 is generated.
  • protein concentration is maintained at a normal value, the test substance can be evaluated as having an effect of improving arteriosclerosis or an effect of reducing the risk of future onset.
  • a protein that produces a peak with a mass / charge ratio of about 66000 when subjected to SELDI-TOF-MS is a mouse that has developed arteriosclerosis, or the risk of future development of arteriosclerosis. It was found to be a substance specific to high mice and could be a marker for arteriosclerosis. As a result, the arteriosclerosis of the test substance is improved using the concentration of the marker substance (M29) in the body fluid of the animal that has developed arteriosclerosis ingested by the test substance or that has a high risk of developing in the future. It has been shown that it is possible to evaluate the effect or the effect of reducing the risk of future onset, and to screen for such substances.
  • test substance when a body fluid sample is prepared by performing the same animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a peak with a mass / charge ratio of about 66000 is generated.
  • the test substance can be evaluated as having an effect of improving arteriosclerosis or an effect of reducing the risk of future onset.
  • the denatured sample was centrifuged at 12,000 G for 10 minutes at 4 ° C., filtered through a 0.45 ⁇ m filter, and then added to the equilibrated column. After sequentially washing with 50 mM Tris-HCl (pH 9.0) and 100 mM sodium acetate buffer (pH 5.0), elution was performed with 50 mM sodium citrate buffer (pH 3.0), and peak fractions were collected.
  • SELDI-TOF-MS analysis was performed on each fraction, and the mass / charge ratio (average value: marker substance (M06)) of the third to fifth fractions was within the error due to oxidation modification in the purification process. A peak showing a mass / charge ratio equivalent to 5203) was confirmed.
  • FIG. 30 A part of the collected fraction was subjected to acetone precipitation, and then subjected to SDS-PAGE using a polypeptide separation gel (15-20%) NTH-5AOT gel (DRC) (FIG. 30).
  • lanes 1 to 5 are all collected fractions, and M is a molecular weight marker.
  • the separated band of about 5.2 kDa (arrow in FIG. 30) was cut out, protein was extracted, and SELDI-TOF-MS analysis was performed, and a peak showing a mass / charge ratio equivalent to that of the marker substance (M06) was confirmed. (Arrows in FIG. 31).
  • SEQ ID NO: 1 10 amino acids represented by SEQ ID NO: 1 were determined.
  • This amino acid sequence was completely identical to a part of mouse apolipoprotein A2 (SEQ ID NO: 2).
  • the predicted isoelectric point of the protein fragment corresponding to the amino acid number 15 (Phe) to 60 (Leu) portion of SEQ ID NO: 2 is 6.77, the molecular weight is 5195, and these values are SELDI-TOF- It was in good agreement with the results of MS analysis. From this, the marker substance (M06) was identified as a fragment of apolipoprotein A2.
  • the amino acid sequence of the amino acid number 15 (Phe) to 60 (Leu) part of SEQ ID NO: 2 corresponding to the protein fragment is shown in SEQ ID NO: 3.
  • FIG. 32 A portion of the collected fractions were subjected to acetone precipitation, and then subjected to SDS-PAGE using a polypeptide separation gel (15-20%) NTH-5AOT gel (DRC) (FIG. 32).
  • lanes 1 and 2 are both recovered fractions, and M is a molecular weight marker.
  • the separated band of about 6.0 kDa (arrow in FIG. 32) is cut out, protein is extracted, SELDI-TOF-MS analysis is performed, and it is equivalent to the mass / charge ratio (average value: 6023) of the marker substance (M07) A peak indicating the mass / charge ratio was confirmed (arrow in FIG. 33).
  • a known protein was searched by Mascot database (Matrix Science), and peptide mass fingerprinting was performed. These peptides were consistent with the peptides of amino acid sequences represented by SEQ ID NOs: 4 and 5, respectively.
  • the target protein was identified as “serum albumin” with a probability of 99% or more. Table 1 shows the correspondence between the exact mass, amino acid sequence, and SEQ ID NO of each peptide. The amino acid sequence of the previously reported mouse serum albumin is shown in SEQ ID NO: 6.
  • SDS-PAGE was performed again to transfer to a PVDF membrane, and the band portion of about 6.0 kDa was subjected to N-terminal amino acid analysis.
  • 12 amino acids represented by SEQ ID NO: 7 were determined.
  • This amino acid sequence was completely identical with 12 amino acids of amino acid numbers 554 (Ala) to 565 (Ala) of serum albumin (SEQ ID NO: 6).
  • the predicted isoelectric point of the protein fragment corresponding to the amino acid number 554 (Ala) to 608 (Ala) portion of SEQ ID NO: 6 is 5.65
  • the molecular weight is 6019, and these values are SELDI-TOF- It was in good agreement with the results of MS analysis.
  • the marker substance (M07) was identified as a fragment of serum albumin.
  • the amino acid sequence of the amino acid number 554 (Ala) to 608 (Ala) part of SEQ ID NO: 6 corresponding to the protein fragment is shown in SEQ ID NO: 8.
  • FIG. 34 A portion of the collected fraction was subjected to acetone precipitation, and then subjected to SDS-PAGE using a polypeptide separation gel (15-20%) NTH-5AOT gel (DRC) (FIG. 34).
  • lane 1 is a collected fraction
  • M is a molecular weight marker.
  • the isolated band of about 6.8 kDa (arrow in FIG. 34) is cut out, protein is extracted, SELDI-TOF-MS analysis is performed, and the marker substance (M09) is within the error due to oxidation modification in the purification process.
  • a peak showing a mass / charge ratio equivalent to the mass / charge ratio (average value: 6861) was confirmed (arrow in FIG. 35).
  • Example 3 The same SDS-PAGE was performed again to cut out a band of about 6.8 kDa, and in-gel digestion and mass spectrometry were performed in the same manner as in Example 3. As a result, at least two peaks were detected. “781.48” and “1042.40” were calculated. Based on these data, peptide mass fingerprinting was performed in the same manner as in Example 3. As a result, these peptides coincided with peptides having the amino acid sequences represented by SEQ ID NOs: 9 and 10, respectively, and the target protein was 99% or more. Was identified as “apolipoprotein C1”. Table 2 shows the correspondence between the exact mass, amino acid sequence, and SEQ ID NO of each peptide. The amino acid sequence of the previously reported mouse apolipoprotein C1 is shown in SEQ ID NO: 14.
  • apolipoprotein C1 “modified form of apolipoprotein C1” (SEQ ID NO: 15) lacking the N-terminal two amino acid residues (Ala-Pro) is present in serum.
  • SEQ ID NO: 15 the same protein (apolipoprotein C1) as in Example 5 described later was hit. It was thought that this was a modified form of (SEQ ID NO: 15).
  • FIG. 36 lanes 1 to 6 are all collected fractions, and M is a molecular weight marker.
  • the separated band of about 7.0 kDa (arrow in FIG. 36) is cut out, protein is extracted, SELDI-TOF-MS analysis is performed, and it is equivalent to the mass / charge ratio (average value: 7005) of the marker substance (M10) A peak indicating the mass / charge ratio of was confirmed (a in FIG. 37).
  • Example 3 The same SDS-PAGE was performed again to cut out a band of about 7.0 kDa, and in-gel digestion and mass spectrometry were performed in the same manner as in Example 3. As a result, at least three peaks were detected, and their accurate masses were , “1279.70”, “1299.71”, and “1042.53”. Based on these data, peptide mass fingerprinting was carried out in the same manner as in Example 3. As a result, the peptides having the exact masses “127.70”, “1299.71”, and “1042.53” were identified in SEQ ID NO: 11 to The protein of interest was identified as “apolipoprotein C1” (SEQ ID NO: 14) with a probability of 99% or more in agreement with the peptide of the amino acid sequence represented by 13. Table 3 shows the correspondence between the exact mass, amino acid sequence, and SEQ ID NO of each peptide.
  • apolipoprotein C1 the same protein (apolipoprotein C1) as in Example 4 was hit, but the marker substance (M10) was found to have the full length of apolipoprotein C1 from its charge / mass ratio (average value: 7005). it was thought.
  • a peak indicating a mass (about 6.8 kDa) smaller than that of the marker substance (M10) was also detected in SELDI-TOF-MS analysis (b in FIG. 37).
  • the protein of peak b is the N-terminal 2 of apolipoprotein C1 (SEQ ID NO: 14) described in Example 4. It was thought to be a “modified product of apolipoprotein C1” (SEQ ID NO: 15) lacking the amino acid (Ala-Pro).
  • the mass calculated from the amino acid sequence was 6993 Da for peak a and 6825 Da for peak b.
  • FIG. 38 lanes 1 and 2 are both recovered fractions, and M is a molecular weight marker.
  • the isolated band of about 8.7 kDa (arrow A in FIG. 38) is cut out, protein is extracted, SELDI-TOF-MS analysis is performed, and the marker substance (M15) is within the error due to oxidation modification in the purification process.
  • a peak showing a mass / charge ratio equivalent to the mass / charge ratio (average value: 8723) was confirmed (arrow in FIG. 39).
  • Example 3 The same SDS-PAGE was performed again to cut out a band of about 8.7 kDa, and in-gel digestion and mass spectrometry were performed in the same manner as in Example 3. As a result, at least two peaks were detected. It was calculated as “1832.01” and “119.69”. Based on these data, peptide mass fingerprinting was carried out in the same manner as in Example 3. As a result, the peptides with exact masses “1832.01” and “119.69” had the amino acid sequences represented by SEQ ID NOS: 16 and 17, respectively. Consistent with the peptide, the protein of interest was identified as “apolipoprotein A2” with a probability of 99% or higher. Table 4 shows the correspondence between the exact mass, amino acid sequence, and SEQ ID NO of each peptide. The amino acid sequence of the previously reported mouse apolipoprotein A2 is shown in SEQ ID NO: 2.
  • marker substance (M16) The approximately 8.8 kDa band (arrow B in FIG. 38) separated by SDS-PAGE performed in Example 6 was cut out, protein was extracted, and SELDI-TOF-MS analysis was performed. went. A peak showing a mass / charge ratio equivalent to the mass / charge ratio (average value: 8840) of the marker substance (M16) was confirmed within the range of error due to oxidation modification or the like in the purification process (arrow in FIG. 40).
  • Example 3 A similar SDS-PAGE was performed to cut out a band of about 8.8 kDa, and in-gel digestion and mass spectrometry were performed in the same manner as in Example 3. As a result, at least three peaks were detected. It was calculated as “1988.08”, “1062.48”, and “2629.26”. Based on these data, peptide mass fingerprinting was performed in the same manner as in Example 3. As a result, these peptides coincided with peptides having the amino acid sequences represented by SEQ ID NOs: 18 to 20, respectively, and the target protein was 99% or more. Was identified as “apolipoprotein C3”. Table 5 shows the correspondence between the exact mass, amino acid sequence, and SEQ ID NO of each peptide. The amino acid sequence of the previously reported mouse apolipoprotein C3 is shown in SEQ ID NO: 21.
  • FIG. 41 A portion of the collected fraction was subjected to acetone precipitation, and then subjected to SDS-PAGE using a polypeptide separating gel (15-20%) NTH-5AOT gel (DRC) (FIG. 41).
  • lanes 1 to 6 are all collected fractions, and M is a molecular weight marker.
  • the separated band of about 14.0 kDa (arrow in FIG. 41) was cut out, protein was extracted, SELDI-TOF-MS analysis was performed, and the mass / charge ratio (average value: marker substance (M21) to (M23)) Several peaks showing a mass / charge ratio equivalent to that of 13742, 13958, 14167) were confirmed (FIG. 42).
  • This peak fraction is subjected to SELDI-TOF-MS analysis, and the mass / charge equivalent to the mass / charge ratio (average value: 20908) of the marker substance (M25) within the range of error due to oxidation modification in the purification process. A peak indicating the ratio was confirmed.
  • FIG. 43 A part of the collected fraction was subjected to acetone precipitation, and then subjected to SDS-PAGE using a polypeptide separation gel (15-20%) NTH-5AOT gel (DRC) (FIG. 43).
  • lanes 1 and 2 are both recovered fractions, and M is a molecular weight marker.
  • the separated band of about 21.0 kDa (arrow in FIG. 43) is cut out, protein is extracted, SELDI-TOF-MS analysis is performed, and it is equivalent to the mass / charge ratio (average value: 20908) of the marker substance (M25)
  • a peak indicating the mass / charge ratio was confirmed (arrow in FIG. 44).
  • Example 3 The same SDS-PAGE was performed again to cut out a band of about 21.0 kDa, and in-gel digestion and mass spectrometry were performed in the same manner as in Example 3. As a result, at least 7 peaks were detected, and their accurate mass was “1155.66”, “2407.31”, “1501.83”, “1226.68”, “2705.27”, “2080.03”, and “1360.72” were calculated. Based on these data, peptide mass fingerprinting was performed in the same manner as in Example 3. As a result, accurate masses “1155.66”, “2407.31”, “1501.83”, “1226.68”, “2705” were obtained.
  • retinol-binding protein As in Example 9 described later was hit, but the marker substance (M25) was found to be retinol-binding protein from its charge / mass ratio (average value: 20908). It was considered to be a “modified form of retinol-binding protein” lacking some amino acid residues.
  • the denatured sample was diluted by adding 6.25 mL of equilibration buffer and added to the equilibrated column.
  • the flow-through fraction was collected, and the peak fraction eluted with 50 mM Tris-HCl (pH 9.0) was further collected.
  • FIG. 45 lanes 1 and 2 are both recovered fractions, and M is a molecular weight marker.
  • the isolated band of about 21.4 kDa (arrow A in FIG. 45) is cut out, protein is extracted, SELDI-TOF-MS analysis is performed, and the marker substance (M26) is within the error due to oxidation modification in the purification process.
  • a peak having a mass / charge ratio equivalent to the mass / charge ratio (average value: 21353) was confirmed (arrow in FIG. 46).
  • Example 3 The same SDS-PAGE was performed again to cut out a band of about 21.4 kDa, and in-gel digestion and mass spectrometry were performed in the same manner as in Example 3. As a result, at least 5 peaks were detected, and their accurate masses were It was calculated as “1155.62”, “1485.77”, “1226.64”, “2079.94”, and “1360.66”. Based on these data, peptide mass fingerprinting was performed in the same manner as in Example 3. As a result, these peptides coincided with peptides having amino acid sequences represented by SEQ ID NOs: 39 to 43, respectively, and the target protein was 99% or more. Was identified as “retinol-binding protein” (SEQ ID NO: 38). Table 8 shows the correspondence between the exact mass, amino acid sequence, and SEQ ID NO of each peptide.
  • the same protein (retinol-binding protein) as in Example 9 was hit.
  • the marker substance (M26) was obtained from the charge / mass ratio (average value: 21353) of the retinol-binding protein. It was considered full length.
  • Example 3 The same SDS-PAGE was performed again to cut out a band of about 28.0 kDa, and in-gel digestion and mass spectrometry were performed in the same manner as in Example 3. As a result, at least 12 peaks were detected, and their accurate masses were “1852.53”, “1394.06”, “123.86”, “15996.35”, “1340.01”, “1098.77”, “1317.196”, “826.47”, “1296 .90 ”,“ 1997.80 ”,“ 1330.93 ”, and“ 1039.71 ”. Based on these data, peptide mass fingerprinting was performed in the same manner as in Example 3.

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Abstract

La présente invention concerne un procédé d'évaluation de l'effet d'atténuation de l'artériosclérose ou de l'effet de diminution du risque d'apparition de celle-ci dans le futur d'une substance d'essai, etc. Une substance d'essai est administrée à un animal souffrant d'artériosclérose ou un animal présentant un risque élevé d'apparition de celle-ci dans le futur. Ensuite, la concentration d'au moins un membre de 29 types de substances marqueuses dans le fluide corporel de l'animal est comparée à une valeur standard de manière à évaluer l'effet d'atténuation de l'artériosclérose ou l'effet de diminution du risque d'apparition de celle-ci dans le futur de la substance d'essai. Une constitution, dans laquelle la substance marqueuse est capturée par un support sur laquelle une substance ayant une affinité pour la substance marqueuse est immobilisée et ainsi la concentration de la substance marqueuse dans le fluide corporel est calculée, est recommandée. La présente invention concerne en outre un procédé de criblage d'une substance à utiliser dans le procédé d'évaluation ci-dessus et un kit de telle manière que le procédé d'évaluation puisse être commodément appliqué.
PCT/JP2009/060056 2008-06-04 2009-06-02 Procédé d’évaluation des effets d’atténuation et de prévention de l’artériosclérose, kit pour l’évaluation et procédé de criblage de substance Ceased WO2009148050A1 (fr)

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JP2008146948A JP5373322B2 (ja) 2007-06-05 2008-06-04 動脈硬化改善・予防効果の評価方法、物質のスクリーニング方法、並びに、マーカーとしての使用
JP2008-146948 2008-06-04

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH08160042A (ja) * 1994-12-07 1996-06-21 Chemo Sero Therapeut Res Inst 動脈硬化診断用マーカー及び該マーカーを用いた動脈硬化の診断方法
JP2003504044A (ja) * 1999-07-07 2003-02-04 インサイト・ゲノミックス・インコーポレイテッド アテローム性動脈硬化に関連する遺伝子
JP2005502883A (ja) * 2001-09-07 2005-01-27 ジェンフィ 代謝症候群、循環器疾患およびアテローム性動脈硬化症を予防または処置するに有用な分子をスクリーニングする方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH08160042A (ja) * 1994-12-07 1996-06-21 Chemo Sero Therapeut Res Inst 動脈硬化診断用マーカー及び該マーカーを用いた動脈硬化の診断方法
JP2003504044A (ja) * 1999-07-07 2003-02-04 インサイト・ゲノミックス・インコーポレイテッド アテローム性動脈硬化に関連する遺伝子
JP2005502883A (ja) * 2001-09-07 2005-01-27 ジェンフィ 代謝症候群、循環器疾患およびアテローム性動脈硬化症を予防または処置するに有用な分子をスクリーニングする方法

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