JP5349857B2 - Evaluation method of arteriosclerosis improvement / prevention effect, screening method of substance, and use as marker - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method or the like for evaluating an improving effect or future risk reducing effect of arterial sclerosis possessed by a substance to be examined. <P>SOLUTION: An animal suffering from arterial sclerosis or animal having a high future risk is allowed to ingest the substance to be examined, and the concentration of at least one of five marker substances in the body fluids of the animal is compared with a reference value to evaluate the improving effect or future risk reducing effect of the arterial sclerosis possessed by the substance to be examined. The constitution is recommended that the marker substance is captured by a carrier on which a substance having the affinity to the marker substance is fixed to calculate the concentration of the marker substance in the body fluids. There are also provided a substance screening method using the evaluation method and a kit for simply performing the evaluation method. <P>COPYRIGHT: (C)2010,JPO&amp;INPIT

Description

The present invention relates to a method Evaluation of arteriosclerosis improvement and preventive effect, as well as screening method for a substance relates like, more particularly, ingest a test substance to an animal or future risk is higher animals have developed atherosclerosis is allowed, and an indicator the concentration of the marker substance in a body fluid of the animal, improvement or method evaluation of arteriosclerosis improvement and preventive effect of evaluating the effect of reducing the risk of future atherosclerotic with the test substance, and, the The present invention relates to a screening method for substances, etc., for screening substances having an effect of improving arteriosclerosis or reducing the risk of future onset using an evaluation method.

  In recent years, westernization of eating habits has progressed, and lifestyle-related diseases such as obesity, diabetes, hyperlipidemia, and arteriosclerosis, which are considered to be caused by this, are increasing. These increased incidences are not genetic but mainly due to environmental factors. For example, abnormal lipid metabolism due to ingestion of a high-fat diet or a high-calorie diet causes increased blood lipids, development of insulin resistance, adipocyte hypertrophy, insulin secretion failure, and the like. As a result, diabetes, obesity, arteriosclerosis, etc. occur with high probability, leading to the progress of the disease state.

  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.

  The importance of preventing diseases associated with lifestyle-related diseases such as arteriosclerosis is emphasized. That is, many lifestyle-related diseases gradually progress, and there are often few subjective symptoms in the early stage of onset. As a result, when it is discovered, it may have already progressed to a serious state, and prevention is particularly important. Under such circumstances, search for biomarkers capable of detecting the future risk of developing diseases, that is, marker substances called “prevention markers” or “risk markers”, has been actively conducted. By using a preventive marker for lifestyle-related diseases, for example, it becomes possible to determine the future risk of the disease, and appropriate measures such as lifestyle correction, dietary restrictions, and exercise are performed in advance to develop the disease. Can be prevented. In addition, by using 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. In addition, it is possible to easily prevent lifestyle-related diseases by daily intake of food containing food materials selected by such an evaluation / screening system.

  Regarding lifestyle related diseases, for example, Patent Document 1 discloses a preventive marker for diabetes and Patent Document 2 discloses a preventive marker for hyperlipidemia. However, these are not biomarkers specialized for arteriosclerosis. On the other hand, there is an attempt to search for a biomarker specialized for arteriosclerosis. For example, Patent Document 3 discloses C-reactive protein (CRP) which is a biomarker of inflammation, and Patent Document 4 discloses α-fetoprotein which is a tumor marker. There is a description that (AFP) can also be used as a biomarker of arteriosclerosis. However, 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.

International Publication No. 2006/073195 Pamphlet JP 2007-064747 A Special table 2001-52558 gazette JP 2007-010567 A

  An object of the present invention is to newly identify a marker substance that can be a preventive marker for arteriosclerosis, and to use the marker substance to evaluate a method for evaluating an effect of improving arteriosclerosis or a risk of reducing future onset risk of a test substance. It is to provide.

The invention according to claim 1 for solving the above-described problem is to cause 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 following marker substance ( At least one concentration of Mf1) to (Mf5) is compared with a reference value, and with respect to (Mf5), the higher concentration is used as an index, and the improvement effect of arteriosclerosis of the test substance or the risk of future onset It is a method for evaluating the effect of improving / preventing arteriosclerosis, characterized by evaluating the effect of reducing the amount of arthritis.
(Mf1) 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 3500 when subjected to mass spectrometry;
(Mf2) 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 3520 when subjected to mass spectrometry;
(Mf3) a protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M, and that produces an ion peak with a mass / charge ratio of about 5900 when subjected to mass spectrometry,
(Mf4) a protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M, and produces an ion peak with a mass / charge ratio of about 11000 when subjected to mass spectrometry,
(Mf5) 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 13600 when subjected to mass spectrometry.

  The method for evaluating the effect of improving / preventing arteriosclerosis according to the present invention allows 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 (Mf1) to (Mf5) in the body fluid of the animal ) At least one concentration of the five marker substances is compared with a reference value, and the effect of improving the arteriosclerosis or reducing the risk of future onset of the test substance is evaluated. Each of the marker substances (Mf1) to (Mf5) 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 the present invention, the arteriosclerosis improvement effect or the future risk reduction effect of the test substance can be evaluated easily and with high accuracy. The “animal” includes humans as well as animals such as mice.

  Here, the values of “about 3500”, “about 5900”, “about 13600”, etc. of the mass / charge ratio (hereinafter sometimes abbreviated as “m / z”) in each marker substance are It is a value that takes into account the error range of the measured value, and has a width of ± 0.2%. That is, about 3500 represents 3500 ± 0.2%, about 5900 represents 5900 ± 0.2%, and about 13600 represents 13600 ± 0.2%. The other mass / charge ratios have the same range of ± 0.2%. In addition, these marker substances are mainly proteins present in blood. When the test substance has an effect of improving arteriosclerosis or reducing the risk of future onset, the concentrations of the marker substances (Mf1), (Mf2), and (Mf4) in the body fluid of the animal show lower values (Mf3 ) And (Mf5) concentrations are higher.

  The invention according to claim 2 is the method for evaluating the effect of improving and preventing arteriosclerosis according to claim 1, wherein the marker substance (Mf5) is transthyretin or a modified form thereof.

  Since transthyretin is well known for its physicochemical properties, the marker substance (Mf5) can be easily analyzed according to the method for evaluating the effect of improving and preventing arteriosclerosis according to the present invention.

  A representative example of “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. Further, “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. In the case of a complex protein, “protein or a modified product thereof” includes its subunits.

  The invention according to claim 3 for solving the same problem 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 that transthyretin in the body fluid of the animal or Compare the concentration of the marker substance belonging to the modified substance with a reference value, and evaluate the effect of improving the arteriosclerosis of the test substance or reducing the risk of future onset using the higher concentration as an index. Is a method for evaluating the effect of improving and preventing arteriosclerosis.

  The method for evaluating the effect of improving / preventing arteriosclerosis according to the present invention allows 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 “transthyretin or a modification thereof in body fluid of the animal”. The concentration of the marker substance belonging to the “body” is compared with a reference value, and the effect of improving the arteriosclerosis or the risk of reducing the future onset of the test substance is evaluated. The marker substance belonging to "transthyretin or its modified form" is a protein that is specifically detected in the body fluid of an animal immediately before the onset of atherosclerosis, and is useful as a prophylactic marker / risk marker for atherosclerosis It is. According to the evaluation method of the arteriosclerosis improvement / prevention effect of the present invention, the arteriosclerosis improvement effect or the future risk reduction effect of the test substance can be evaluated easily and with high accuracy. In particular, transthyretin is easy to analyze because its physicochemical properties are well known. In the present invention, the term “animal” includes humans as well as animals such as mice.

  When the test substance has an effect of improving arteriosclerosis or reducing the risk of future onset, the concentration of the marker substance belonging to transthyretin or its modified substance in the body fluid of the animal shows a higher value.

  In the present invention, 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. The “modification” includes not only addition of a compound or functional group but also desorption. Separation is also included. In addition, “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. Furthermore, in the case of a complex protein, the “protein or a modified form thereof” includes its subunits.

  In the invention according to claim 4, the reference value is known not to have an effect of improving arteriosclerosis or an effect of reducing the risk of future onset in an animal that has developed arteriosclerosis or an animal that has a high risk of future onset. The method for evaluating the effect of improving or preventing arteriosclerosis according to any one of claims 1 to 3, wherein the marker substance concentration is in the body fluid of the animal when the substance is ingested. .

  With this configuration, the arteriosclerosis improving effect or the future risk reducing effect of the test substance can be more easily and accurately evaluated.

  The invention according to claim 5 is characterized in that the animal that has developed arteriosclerosis or the animal that has a high risk of developing in the future is a spontaneous model animal or a genetically engineered model animal. It is an evaluation method of an arteriosclerosis improvement and prevention effect given in any 1 paragraph.

  In the method for evaluating the effect of improving / preventing arteriosclerosis according to the present invention, a naturally occurring model animal or a genetically engineered model animal is used as an animal that has developed arteriosclerosis or an animal that has a high risk of developing in the future. With this configuration, it becomes easy to breed animals, and the effect of improving arteriosclerosis or the risk of reducing 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 invention according to claim 6 is the arteriosclerosis improving / preventing effect evaluation method according to any one of claims 1 to 5, wherein the body fluid is blood.

  With this configuration, it is possible to easily collect a body fluid as a measurement sample, and it is possible to more easily and quickly evaluate an effect of improving arteriosclerosis or a future risk of reducing the onset of the test substance.

  The invention according to claim 7 is the arteriosclerosis improvement / prevention effect evaluation method according to any one of claims 1 to 6, wherein the test substance is a food material.

  With this configuration, for the purpose of developing functional foods, it is possible to evaluate the effect of improving arteriosclerosis or the effect of reducing the risk of future onset.

  According to an eighth aspect of the present invention, the body fluid or body fluid component is brought into contact with a carrier on which a substance having affinity for the marker substance is immobilized, and the marker substance in the body fluid is captured on the carrier. The method for evaluating the effect of improving or preventing arteriosclerosis according to any one of claims 1 to 7, wherein the concentration of the marker substance in a body fluid is calculated based on the amount of the marker substance.

  In the method for evaluating the effect of improving / preventing arteriosclerosis according to the present invention, a carrier on which a substance having affinity for a 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. According to the evaluation method of the arteriosclerosis improvement / prevention effect of the present invention, since the marker substance captured on the carrier is the measurement object, the influence of the contaminant substance contained in the measurement sample can be reduced, and the higher The concentration of the marker substance can be measured with high sensitivity and high accuracy. Examples of the body fluid component include serum or plasma when the body fluid is blood.

  The invention according to claim 9 is characterized in that the carrier has a flat portion, and the substance having affinity for the marker substance is immobilized on a part of the flat portion. It is an evaluation method of the arteriosclerosis improvement / prevention effect of description.

  In the method for evaluating the effect of improving / preventing atherosclerosis according to the present invention, 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. With this configuration, a substance having affinity for the marker substance can be spot-fixed at a plurality of locations on the carrier. As a result, it is possible to simultaneously process a plurality of measurement samples with one carrier, and simultaneously measure the concentrations of a plurality of marker substances with one carrier, and work efficiency is improved. Furthermore, by reducing the area of each spot, 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 invention according to claim 10 is characterized in that the substance having affinity for the marker substance is an ion exchanger, a metal chelate or an antibody, and the arteriosclerosis improving / preventing effect according to claim 8 or 9 This is an evaluation method.

  In the method for evaluating the effect of improving / preventing arteriosclerosis according to the present invention, an ion exchanger, a metal chelate or an antibody is used as a substance having affinity for a marker substance, and a measurement sample is passed through the ion exchanger, metal chelate or antibody The marker substance inside is captured on the carrier. When the substance is an ion exchanger or a metal chelate, 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).

  Invention of Claim 11 evaluates a test substance by the evaluation method of the arteriosclerosis improvement / prevention effect of any one of Claims 1-10, and reduction of the onset risk of arteriosclerosis or future onset risk A screening method for a substance characterized by screening a substance having an effect.

  The present invention relates to a method for screening a substance, and compares at least one concentration of each of the marker substances (Mf1) to (Mf5) and a marker substance belonging to transthyretin or a modified substance thereof in a body fluid of an animal with a reference value. In this method, a substance having an effect of improving arteriosclerosis or reducing the risk of future onset is screened. Each of the marker substances (Mf1) to (Mf5) described above and the marker substance belonging to transthyretin or a modified form thereof 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. According to the screening method for a substance of the present invention, a substance having an effect of improving arteriosclerosis or reducing the risk of future onset can be screened easily and with high accuracy. In particular, when the test substance is a food material, it is possible to screen for a food material useful for the development of a functional food having an effect of improving arteriosclerosis or a functional food having an effect of reducing the risk of future onset.

A related invention is a kit for use in the method for evaluating an arteriosclerosis improvement / prevention effect according to any one of claims 1 to 10, wherein the carrier has an immobilized substance having affinity for the marker substance It is a kit for evaluating the effect of improving / preventing arteriosclerosis.

The kit for evaluating the effect of improving / preventing arteriosclerosis according to the present invention includes a carrier on which a substance having affinity for a marker substance is immobilized. With this configuration, 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.

A related invention is the above- described kit for evaluating an effect of improving / preventing atherosclerosis, wherein the substance having affinity for the marker substance is an ion exchanger or a metal chelate.

  With this configuration, the marker substance can be more reliably captured on the carrier.

A configuration used to screen for substances that have an effect of improving arteriosclerosis or reducing the risk of future onset is recommended .

The invention according to claim 12, the at least one protein described below is present in the body of an animal (Mf1) ~ (Mf 4) , the presence or absence of the onset of arteriosclerosis, or as a marker for the detection of the risk of developing future Is the use of .
(Mf1) 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 3500 when subjected to mass spectrometry;
(Mf2) 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 3520 when subjected to mass spectrometry;
(Mf3) a protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M, and that produces an ion peak with a mass / charge ratio of about 5900 when subjected to mass spectrometry,
(Mf4) A protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M and produces an ion peak with a mass / charge ratio of about 11000 when subjected to mass spectrometry .

  According to the evaluation method of the arteriosclerosis improvement / prevention effect of the present invention, the arteriosclerosis improvement effect or the future risk reduction effect of the test substance can be evaluated easily and with high accuracy.

  According to the screening method for a substance of the present invention, a substance having an effect of improving arteriosclerosis or reducing the risk of future onset can be screened easily and with high accuracy.

According to the evaluation kit of the arteriosclerotic improvement and prevention effect, it can be necessary to separately prepare the carrier upon concentration measurement of the marker substance without measuring the concentration of very easily marker substance.

  Hereinafter, the best mode for carrying out the present invention will be described in detail.

The method for evaluating the effect of improving / preventing arteriosclerosis according to the present invention includes two aspects. In one aspect, an animal that has developed arteriosclerosis or an animal with a high risk of developing in the future has a test substance ingested, and the concentration of at least one of the following marker substances (Mf1) to (Mf5) in the bodily fluid of the animal is set: Compared to the reference value, the effect of improving the arteriosclerosis of the test substance or reducing the risk of future onset is evaluated.
(Mf1) 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 3500 when subjected to mass spectrometry;
(Mf2) 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 3520 when subjected to mass spectrometry;
(Mf3) a protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M, and that produces an ion peak with a mass / charge ratio of about 5900 when subjected to mass spectrometry,
(Mf4) a protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M, and produces an ion peak with a mass / charge ratio of about 11000 when subjected to mass spectrometry,
(Mf5) 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 13600 when subjected to mass spectrometry.

  All of these marker substances are proteins mainly present in blood, and are specifically detected in the animal body immediately before the onset of arteriosclerosis. Each marker substance of (Mf1), (Mf2), and (Mf4) (hereinafter, a group consisting of these marker substances may be referred to as “group 1”) is in a state of developing arteriosclerosis. Or, because the test substance has a higher value at a high risk of future onset of arteriosclerosis, if the test substance has an effect of improving arteriosclerosis or reducing the risk of future onset, the test substance was ingested Lower values in animals. On the other hand, each marker substance of (Mf3) and (Mf5) (hereinafter, a group consisting of these marker substances may be referred to as “group 2”) is in a state in which arteriosclerosis has developed or the future of arteriosclerosis. Therefore, if the test substance has an effect of improving arteriosclerosis or reducing the risk of future onset, it is higher in the animal ingested by the test substance. Indicates.

  According to peptide mapping under certain conditions, the marker substance (Mf5) can be identified as transthyretin. That is, in one embodiment, the marker substance (Mf5) is transthyretin or a modified form thereof.

  In another aspect of the method for evaluating the effect of improving / preventing arteriosclerosis according to the present invention, an animal that has developed arteriosclerosis or an animal that has a high risk of developing in the future is allowed to ingest a test substance, and transthyretin in the body fluid of the animal is obtained. Or the density | concentration of the marker substance which belongs to the modification body is compared with a reference value, and the improvement effect of the arteriosclerosis which a test substance has or the reduction effect of the future onset risk is evaluated.

  Examples of protein modifications include methylation, acetylation, adenylylation, myristylation, etc. of N-terminal α-amino group and lysine ε-amino group; addition of sugar or sugar chain to serine / threonine / asparagine; serine / threonine / Examples include phosphorylation of tyrosine, arginine, histidine; cysteine cysteinylation, homocysteinylation, sulfonylation, etc .; γ-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”.

  For example, although transthyretin has one cysteine residue, the thiol group (-SH) of this only cysteine residue can be modified, and in vivo, a plurality of types of modification to cysteine residues are different. Modified transthyretin has been found. Examples of such modifications include sulfonation, cysteinylation, homocysteinelation, cysteinylglycination, and glutathioneation. The “modified form of transthyretin” in the present invention includes these modified transthyretins.

  In addition, the “protein or a modified form thereof” includes an isoform 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 comprising 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 form thereof” includes its subunits. For example, since transthyretin is a complex protein composed of four subunits, “transthyretin or a modified form thereof” includes a subunit of transthyretin.

  In the method for evaluating the effect of improving / preventing arteriosclerosis according to the present invention, only one of the marker substances (Mf1) to (Mf5) and the marker substance belonging to transthyretin or a modified form thereof may be used. You may use it 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. .

  In a preferred embodiment of the method for evaluating the effect of improving / preventing arteriosclerosis according to the present invention, as the reference value, 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. The concentration of the marker substance in the body fluid of the animal when a known substance that does not have a risk reducing effect is ingested 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”. Then, 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 (to the normal side) When maintained, it can be evaluated that the test substance has an effect of improving arteriosclerosis or reducing the risk of developing the future. Specifically, when the marker substance belonging to group 1 is used as an index, when the measured value is significantly lower than the reference value, on the other hand, when the marker substance belonging to group 2 is used as an index, the measured value 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.

  Furthermore, there may be a plurality of reference values. For example, 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. Specifically, (1) a group (group showing normal values) in which an animal that has not developed arteriosclerosis or an animal with a low risk of developing arteriosclerosis is ingested with a normal diet or test substance, (2) arteriosclerosis A group (group showing an abnormal value) of ingesting a known substance that has no effect of improving arteriosclerosis or reducing the risk of future onset to an onset animal or an animal at high risk of developing arteriosclerosis, and ( 3) A total of 3 groups are set up, and the animals are bred, in which animals that have developed arteriosclerosis or animals that have a high risk of developing arteriosclerosis are ingested. And the said marker substance in the bodily fluid of each animal is measured, and each measured value is compared. At this time, there is a significant difference between (1) and (2), there is a significant difference between (3) and (2), and (3) is closer to the normal side ((1) than (2)) It is possible to evaluate that the test substance has an effect of improving arteriosclerosis or reducing the risk of future onset.

  Furthermore, as a reference value, (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. Specifically, in addition to (1) to (3) above, the group (4) above is set and animals are raised. At this time, 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 an effect of reducing the risk of future onset. That is, such a test substance exhibits the same behavior as the known substance adopted in (4) and can be said to have a similar action. Examples of “a known substance having an effect of improving arteriosclerosis or reducing the risk of future onset” include antihypertensive agents (calcium antagonists) such as azelnidipine, acetic acid, fermented milk, safflower seed extract. Regarding the improvement effect of fermented milk on arteriosclerosis, for example, the Internet <URL: http://www.calpis.co.jp/corporate/press/nrl_00007.html> (July 13, 2007 Announced).

  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. In addition, as 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.

  In addition, it is known that arteriosclerosis will eventually develop when apoE-deficient mice are normally bred, but the onset can be detected around the 20th week of bred (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 substances (Mf1) to (Mf5) and the marker substance belonging to transthyretin or a modified form thereof used in the method for evaluating the arteriosclerosis improvement / prevention effect of the present invention are contained in the body fluid of apoE-deficient mice at 14 weeks of breeding. It shows a significant difference in the concentration in its body fluid compared to mice that are present and not in the immediate stages of onset of arteriosclerosis (eg, normal mice). 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.

  On the other hand, an arteriosclerosis spontaneous animal model that forms an atheroma when ingested a high cholesterol diet can also be used. In this case, for example, by simultaneously ingesting the test substance and cholesterol, the arteriosclerosis improving effect or the future risk reduction effect of the test substance can be evaluated. Furthermore, humans can be employed as animals. In this case, the substance will be evaluated according to the results of clinical trials.

  As an animal body fluid used in the method for evaluating the effect of improving or preventing arteriosclerosis of the present invention, blood is preferably used. In particular, it is preferable to use serum or plasma (body fluid component) prepared from blood as a measurement sample. Serum or plasma can be prepared from blood by a known method such as centrifugation.

  Examples of the test substance in the method for evaluating the effect of improving / preventing arteriosclerosis according to the present invention include food materials and drug substances. In particular, when food materials are to be evaluated, it can be used for the development of functional foods.

  In the method for evaluating the effect of improving / preventing arteriosclerosis according to the present invention, 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. Can be used as they are. For example, various immunoassays, mass spectrometry (MS), chromatography, electrophoresis and the like can be used.

  According to the immunoassay, the concentration of the marker substance can be accurately measured even with a sample having a lot of contaminants. Examples of 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.

  According to mass spectrometry, 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. As the ionization method for measuring the concentration of the marker substance by mass spectrometry, any of matrix-assisted laser desorption / ionization (MALDI) and electrospray ionization (ESI) can be applied. However, MALDI is preferred because it produces less multivalent ions. In particular, according to MALDI-TOF-MS combined with a time-of-flight mass spectrometer (TOF), an ion peak derived from a marker substance can be identified more accurately.

  When measuring the concentration of the marker substance by electrophoresis, for example, 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. When the separation of the marker substance is insufficient by SDS-PAGE alone, two-dimensional electrophoresis combined with isoelectric focusing (IEF) can also be used. Furthermore, instead of detecting directly from the gel, Western blotting can also be performed to measure the amount of marker substance on the membrane.

  When measuring the concentration of the marker substance by chromatography, for example, a method by liquid high performance chromatography (HPLC) 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.

  In a preferred embodiment, 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. As an example of the carrier that can be used in the present embodiment, 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. In the case of using a substrate, it is preferable to immobilize a substance having affinity for the marker substance on a part of the planar portion. As an example, there may be mentioned 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. Examples of “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.

  When capturing the marker substance on the carrier by ionic bonding, the ion exchanger is immobilized on the carrier. In this case, either 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. Examples of 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). Examples of weak cation exchangers include those having weak cation exchange groups such as carboxymethyl (CM). Examples of strong cation exchangers include those having a strong cation exchange group such as sulfopropyl (SP). Furthermore, examples of the weak anion exchanger include those having a weak anion exchange group such as dimethylaminoethyl (DE) and diethylaminoethyl (DEAE).

When capturing a marker substance via a metal chelate, for example, 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.

  When a marker substance is captured on a carrier by an antibody, an antibody specific for the marker substance may be immobilized on the carrier.

  When a marker substance is captured on a carrier by hydrophobic interaction, a substance having a hydrophobic group is immobilized on the carrier. Examples of hydrophobic groups include C4-C20 alkyl groups, phenyl groups, and the like.

  In this embodiment, when an immunoassay is used as a method for measuring a marker substance, it is preferable to use a carrier on which an antibody is immobilized. In this way, an immunoassay system using the antibody immobilized on the carrier as the primary antibody can be easily constructed. For example, 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. . In addition, immunoassay systems based on binding inhibition methods and competitive methods can be constructed. Further, when 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.

  On the other hand, when mass spectrometry is used in the present embodiment, for example, in addition to antibodies, ion exchangers, metal chelates, or carriers on which hydrophobic groups are immobilized can be used. 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. In particular, using a substrate as a carrier, surface-enhanced laser desorption / ionization-time-of-flight mass spectrometry (hereinafter referred to as "SELDI-TOF-MS") ), The concentration of the marker substance can be measured more accurately. As the types of 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 a metal ion substrate are preferably used.

  The method for screening a substance of the present invention evaluates a test substance by the method for evaluating the effect of improving / preventing arteriosclerosis of the present invention, and screens for a substance having an effect of improving arteriosclerosis or reducing the risk of future onset. . In the method for screening a substance of the present invention, the same embodiment as the above-described method for evaluating the effect of improving / preventing arteriosclerosis of the present invention can be employed.

In order to simply perform the method for evaluating the effect of improving / preventing arteriosclerosis according to the present invention, an evaluation kit can be constructed by collecting necessary reagents. Examples of the evaluation kit include those containing a carrier on which a substance having affinity for a marker substance is immobilized. In particular, according to the evaluation kit including a substrate on which a weak cation exchanger such as CM or a metal chelate such as copper ion is immobilized as a carrier, SELDI-TOF-MS or the like can be easily performed. . The kit may contain other reagents such as standard substances and various pretreatment buffers. In addition, 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. Given a configuration example of the kit as follows.

[Example of kit configuration]
(1) Weak cation exchange substrate: 1 sheet (2) Metal ion substrate: 1 sheet (3) Substrate cleaning buffer A (pH 3.0): appropriate amount (4) Substrate cleaning buffer B (pH 7.0 and 0.00) 5M NaCl included): Appropriate amount (5) Standard product of each marker substance: Appropriate amount

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

1. 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 containing 10% fermented milk (corresponding to 5 g / kg body weight) as feed to be given (hereinafter simply “ Abbreviated as “fermented milk-containing feed”).

The following four groups with different combinations of mouse types and feeds were set.
Group 1: Normal mice are bred with normal feed Group 2: Apo E deficient mice are bred with normal feed Group 3: Apo E deficient mice are bred with fermented milk-containing feed Group 4: Normal mice are fed with fermented milk-containing feed In other words, 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 fermented milk.

  Breeding was started under the predetermined conditions from 6 weeks of age, and 5 mice were sacrificed at each point of 14 weeks (20 weeks of age) and 34 weeks (40 weeks of age). Blood was collected from each mouse and plasma was prepared.

  Plasma in the 14th week (20 weeks old), the breeding period in which atherosclerosis begins to develop in apoE-deficient mice, was used as a body fluid sample immediately before the onset of atherosclerosis, and was subjected to the following proteome analysis.

2. Analysis by Protein Chip and Selection of Ion Peak To 20 μL of each plasma, 30 μL of denaturation buffer (9 M urea, 2% CHAPS, 50 mM Tris-HCl (pH 9.0)) was added for pretreatment to denature the protein. Next, each pretreated body fluid sample was applied to a strong anion exchange resin column (Q-Sepharose, GE Healthcare). Next, a pH 9.0 buffer solution (50 mM Tris-HCl (pH 9.0), 0.1% (w / v) 1-o-N-octyl-β-D-glucopyranoside (hereinafter referred to as “OGP”). )), PH 5.0 buffer (100 mM sodium acetate (pH 5.0), 0.1% (w / v) OGP), pH 3.0 buffer (50 mM sodium citrate (pH 3.0), 0. 1% (w / v) OGP) and organic solvent (mixed solution consisting of 0.1% trifluoroacetic acid, 50.0% acetonitrile) were eluted in order with 200 μL each, and fraction 1 (eluted at pH 9.0, passed) ), Fraction 2 (eluted at pH 5.0), fraction 3 (eluted at pH 3.0), and fraction 4 (eluted with an organic solvent).

  10 μL of each of the obtained fractions was diluted 10-fold with a pH 3.0 protein chip binding buffer (50 mM sodium citrate), and then added to a weak cation exchange chip CM10 (BioRad). Similarly, 10 μL of each of the obtained fractions was diluted 10 times with a pH 7.0 / 0.5 M NaCl protein chip binding buffer (100 mM sodium phosphate, 0.5 M NaCl, pH 7.0), and then copper-modified chip IMAC30. (Bio-Rad). Each chip was washed 3 times with each binding buffer, then once with deionized water and dried. Next, sinapinic acid (SPA-H, SPA-L) or α-cyano-4-hydroxycinnamic acid (CHCA), which is an energy absorbing molecule, is added and a protein chip reader Model PBS IIc (Bio-Rad) is used. Then, SELDI-TOF-MS was performed. In addition, the measurement molecular weight range (m / z) was performed in the range of 3000-200000. 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). Specifically, after performing baseline correction, molecular weight calibration, and spectrum normalization processing, single marker analysis and multiflow analysis combining several markers were performed. As a result, a large number of peaks were detected depending on the combination of the type of crude fraction, the type of protein chip, the washing conditions of the chip, and the like. For each peak, a p-value (Mann-Whitney test method), ROC area, and ion peak intensity were calculated, and five candidate peaks were selected using the following conditions (1) to (4) as indices.

(1) Candidate peak search (basic)
There is a significant difference in ionic strength between the first group and the second group (p <0.05).
(2) Fermented milk 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 higher than the value of the second group. It is close to the value of the first group (the value of the third group has returned to the first group side).
(3) Increase / decrease pattern analysis Only the second group shows a high value or a low value, and there is not much difference among the first group, the third group, and the fourth group. In addition, even if there is a difference between the third group and the fourth group, if the value of the fourth group is farther from the second group than the value of the third group, this condition is satisfied. handle.

3. Identification of marker substance (Mf1) Fraction 1 (pH 9.0) is brought into contact with a weak cation exchange chip CM10, washed with a protein chip binding buffer of pH 3.0, and SELDI-TOF-MS (EAM: SPA-L) , An ion peak with a mass / charge ratio of 3499 (average value) was detected. As a result of the increase / decrease pattern analysis, this peak showed a low value in the first group, the third group, and the fourth group, and a high value in the second group. FIG. 1 shows a box diagram when the peak intensity of each peak is plotted for each group. In the figure, 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, and the line in the box is the center Value (the same applies to FIG. 2 and subsequent figures). Each ROC area and each p value were as follows.

ROC area (first group vs second group): 0.98
p value (first group vs second group): 0.0002
ROC area (second group vs. third group): 0.86
p value (second group vs. third group): 0.010

  From the above, a protein (marker substance (Mf1)) that produces a peak with a mass / charge ratio of about 3500 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. Thereby, using the concentration of the marker substance (Mf1) in the bodily fluid of an animal that has developed arteriosclerosis ingesting the test substance or an animal that has a high risk of developing in the future as an index, It has been shown that it is possible to evaluate the effect of reducing the risk of future onset and to screen for such substances. For example, a protein that produces a peak with a mass / charge ratio of about 3500 when a bodily 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. When the concentration of 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.

4). Identification of marker substance (Mf2) Fraction 1 (pH 9.0) is brought into contact with weak cation exchange chip CM10, washed with a protein chip binding buffer of pH 3.0, and SELDI-TOF-MS (EAM: SPA-L) , An ion peak with a mass / charge ratio of 3518 (average value) was detected. As a result of the increase / decrease pattern analysis, this peak showed a low value in the first group, the third group, and the fourth group, and a high value in the second group. FIG. 2 shows a box diagram when the peak intensity of this peak is plotted for each group. Each ROC area and each p value were as follows.

ROC area (first group vs second group): 0.98
p value (first group vs second group): 0.0002
ROC area (second group vs. third group): 0.74
p value (second group vs. third group): 0.034

  From the above, when subjected to SELDI-TOF-MS, the protein (marker substance (Mf2)) that produces a peak with a mass / charge ratio of about 3520 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, using the concentration of the marker substance (Mf2) in the body fluid of an animal that has developed arteriosclerosis ingesting the test substance or an animal that has a high risk of developing in the future as an index, It has been shown that it is possible to evaluate the effect of reducing the risk of future onset and to screen for such substances. For example, 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 protein that produces a peak with a mass / charge ratio of about 3520 When the concentration of 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.

5. Identification of Marker Substance (Mf3) Fraction 1 (pH 9.0) was contacted with a copper-modified chip IMAC30, washed with a protein chip binding buffer of pH 7.0 / 0.5M NaCl, and SELDI-TOF-MS (EAM: CHCA). ), An ion peak having a mass / charge ratio of 5901 (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. 3 shows a box diagram when the peak intensity of this peak is plotted for each group. Each ROC area and each p value were as follows.

ROC area (first group vs second group): 0.86
p value (first group vs second group): 0.003
ROC area (second group vs. third group): 0.78
p value (second group vs. third group): 0.023

  From the above, a protein (marker substance (Mf3)) that produces a peak with a mass / charge ratio of about 5900 when subjected to SELDI-TOF-MS is a mouse that has developed arteriosclerosis, or the future risk of developing arteriosclerosis. It was found to be a substance specific to high mice and could be a marker for arteriosclerosis. Thereby, using the concentration of the marker substance (Mf3) in the body fluid of an animal that has developed arteriosclerosis ingesting the test substance or an animal that has a high risk of developing in the future as an index, It has been shown that it is possible to evaluate the effect of reducing the risk of future onset and to screen for such substances. For example, 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 protein that produces a peak with a mass / charge ratio of about 5900 When the concentration of 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.

6). Identification of marker substance (Mf4) Fraction 3 (pH 3.0) was contacted with a copper-modified chip IMAC30, washed with a protein chip binding buffer of pH 7.0 / 0.5 M NaCl, and SELDI-TOF-MS (EAM: CHCA). ), An ion peak having a mass / charge ratio of 11047 (average value) was detected. As a result of the increase / decrease pattern analysis, this peak showed a low value in the first group, the third group, and the fourth group, and a high value in the second group. FIG. 4 shows a box diagram when the peak intensity of this peak is plotted for each group. Each ROC area and each p value were as follows.

ROC area (first group vs second group): 0.86
p value (first group vs second group): 0.008
ROC area (second group vs. third group): 0.78
p value (second group vs. third group): 0.019

  From the above, when subjected to SELDI-TOF-MS, the protein (marker substance (Mf4)) that produces a peak with a mass / charge ratio of about 11000 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, using the concentration of the marker substance (Mf4) in the body fluid of an animal that has developed arteriosclerosis that has been ingested by the test substance or an animal that has a high risk of developing in the future as an index, It has been shown that it is possible to evaluate the effect of reducing the risk of future onset and to screen for such substances. For example, a protein that produces a peak with a mass / charge ratio of about 11000 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. When the concentration of 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.

7). Identification of marker substance (Mf5) Fraction 3 (pH 3.0) is brought into contact with a weak cation exchange chip CM10, washed with a pH 3.0 protein chip binding buffer, and then SELDI-TOF-MS (EAM: SPA-H). , An ion peak with a mass / charge ratio of 13648 (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. 5 shows a box diagram when the peak intensity of this peak is plotted for each group. Each ROC area and each p value were as follows.

ROC area (first group vs second group): 0.78
p value (first group vs second group): 0.028
ROC area (second group vs. third group): 0.82
p value (second group vs. third group): 0.028

  From the above, when subjected to SELDI-TOF-MS, the protein (marker substance (Mf5)) that produces a peak with a mass / charge ratio of about 13600 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. Thereby, using the concentration of the marker substance (Mf5) in the body fluid of an animal that has developed arteriosclerosis or that has a high future risk of ingesting the test substance as an index, It has been shown that it is possible to evaluate the effect of reducing the risk of future onset and to screen for such substances. For example, 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 protein that produces a peak with a mass / charge ratio of about 13600 When the concentration of 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.

  In the case where a protein equivalent to the marker substances (Mf1) to (Mf5) is also present in human body fluid, the presence or absence of onset of arteriosclerosis or the future using the concentration of the protein in human body fluid as an index The possibility of diagnosing the onset risk of this disease was also suggested.

Purification and identification of marker substance (Mf5)

  A strong anion exchange column HiTrap Q HP was equilibrated with an equilibration buffer (50 mM Tris-HCl pH 9.0, 1 M urea, 0.22% CHAPS). Denaturation buffer (50 mM Tris-HCl pH 9.0, 9 M urea, 2% CHAPS) was added to 1 mL of mouse standard plasma for denaturation treatment, and then added to the equilibrated column. The column was washed successively with 50 mM Tris-HCl (pH 9.0) and 50 mM Tris-HCl (pH 7.0), and then eluted and collected with 50 mM Tris-HCl (pH 7.0) containing 50 mM NaCl.

  The collected fraction was subjected to acetone precipitation, and then subjected to SDS-PAGE using a polypeptide separation gel DRC NTH-5A0T gel (FIG. 6). In FIG. 6, lanes 1 to 6 are collected fractions, and M is a molecular weight marker. The separated band (arrow in FIG. 6) was cut out, protein was extracted, SELDI-TOF-MS analysis was performed, and a peak showing a mass / charge ratio equivalent to that of the marker substance (Mf5) was confirmed (arrow in FIG. 7). ).

  The same SDS-PAGE was performed again to cut out a band of about 13.6 kDa, and after reduction alkylation treatment, 0.01 μg / μL trypsin solution (dissolved in 50 mM ammonium hydrogen carbonate (pH 8.0)) was allowed to act. Digested in the gel. 1 μL of the digested sample was dropped on a metal plate, 0.4 μL of a saturated CHCA solution was further dropped and dried, and then measured with a mass spectrometer Proteomics Analyzer 4700 (Applied Biosystems). At least 6 peaks Were detected, and their accurate masses were calculated as “1510.73”, “1382.63”, “2438.17”, “3123.48”, “1554.82”, and “2517.20”. . Based on these data, a known protein was searched by Mascot database (Matrix Science), and peptide mass fingerprinting was performed. These peptides were consistent with peptides having amino acid sequences represented by SEQ ID NOs: 1 to 6, respectively. The protein of interest was identified as “transthyretin” with a probability of 99% or higher. Table 1 shows the correspondence between the exact mass, amino acid sequence, and SEQ ID NO of each peptide.

  The reported amino acid sequence of mouse-derived transthyretin is as shown in SEQ ID NO: 7.

It is a box diagram about the ion peak whose mass / charge ratio is 3499 (average value). It is a box diagram about the ion peak whose mass / charge ratio is 3518 (average value). FIG. 6 is a box diagram of an ion peak having a mass / charge ratio of 5901 (average value). It is a box diagram about the ion peak whose mass / charge ratio is 11047 (average value). It is a box diagram about the ion peak whose mass / charge ratio is 13648 (average value). It is a photograph which shows the result of SDS-PAGE performed in the refinement | purification process of a marker substance (Mf5). It is an ion peak figure which shows the result of the SELDI-TOF-MS analysis performed in the refinement | purification process of a marker substance (Mf5).

Claims (12)

An animal that has developed arteriosclerosis or an animal that has a high future risk of ingesting the test substance, and compares at least one concentration of the following marker substances (Mf1) to (Mf5) in the body fluid of the animal with a reference value In addition, with regard to (Mf5), the above-mentioned concentration shows a higher value as an index, and the effect of improving or preventing arteriosclerosis of the test substance or the effect of reducing the risk of future onset is evaluated. Evaluation method.
(Mf1) 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 3500 when subjected to mass spectrometry;
(Mf2) 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 3520 when subjected to mass spectrometry;
(Mf3) a protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M, and that produces an ion peak with a mass / charge ratio of about 5900 when subjected to mass spectrometry,
(Mf4) a protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M, and produces an ion peak with a mass / charge ratio of about 11000 when subjected to mass spectrometry,
(Mf5) 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 13600 when subjected to mass spectrometry.   The method for evaluating an effect of improving or preventing arteriosclerosis according to claim 1, wherein the marker substance (Mf5) is transthyretin or a modified form thereof.   An animal that has developed arteriosclerosis or an animal that has a high risk of developing in the future, ingests the test substance, compares the concentration of the marker substance belonging to transthyretin or its modified substance in the body fluid of the animal with a reference value, and An evaluation method for an arteriosclerosis improvement / prevention effect characterized by evaluating an improvement effect of arteriosclerosis or a reduction effect of future onset risk possessed by a test substance using the higher concentration as an index.   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 onset. The method for evaluating an effect of improving or preventing arteriosclerosis according to any one of claims 1 to 3, wherein the marker substance concentration is in the body fluid of an animal.   The arteriosclerosis according to any one of claims 1 to 4, wherein the animal that has developed arteriosclerosis or an animal that has a high risk of developing in the future is a naturally occurring model animal or a genetically engineered model animal. Evaluation method of improvement / prevention effect.   The method for evaluating the effect of improving and preventing arteriosclerosis according to any one of claims 1 to 5, wherein the body fluid is blood.   The method for evaluating atherosclerosis improvement / prevention effect according to any one of claims 1 to 6, wherein 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, the marker substance in the bodily fluid is captured on the carrier, and based on the amount of the captured marker substance The method for evaluating atherosclerosis improvement / prevention effect according to any one of claims 1 to 7, wherein the concentration of the marker substance in a body fluid is calculated.   The arteriosclerosis improving / preventing effect according to claim 8, wherein the carrier has a planar portion, and the substance having affinity for the marker substance is immobilized on a part of the planar portion. Evaluation method.   The method for evaluating the effect of improving or preventing arteriosclerosis according to claim 8 or 9, wherein the substance having affinity for the marker substance is an ion exchanger, a metal chelate or an antibody.   A test substance is evaluated by the method for evaluating atherosclerosis improvement / prevention effect according to any one of claims 1 to 10, and screening for a substance having an effect of improving arteriosclerosis or a reduction effect of future onset risk is performed. A screening method for a characteristic substance. Of at least one protein described below is present in the body of an animal (Mf1) ~ (Mf 4) , used as a marker for the detection of the presence or absence, or future risk of developing arteriosclerosis.
(Mf1) 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 3500 when subjected to mass spectrometry;
(Mf2) 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 3520 when subjected to mass spectrometry;
(Mf3) a protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M, and that produces an ion peak with a mass / charge ratio of about 5900 when subjected to mass spectrometry,
(Mf4) A protein that binds to a copper ion-binding metal chelate at a pH of 7.0 and a NaCl concentration of 0.5 M and produces an ion peak with a mass / charge ratio of about 11000 when subjected to mass spectrometry .