JP5414087B2 - Evaluation method and evaluation kit for improvement / maintenance / recovery effect of motor function, screening method for substance, and use as marker - Google Patents

Description

The present invention relates to an evaluation method and evaluation kit for an improvement / maintenance / recovery effect of motor function, a method for screening a substance, and use as a marker . More specifically, the test substance is ingested and exercise load is applied. Compare the marker substance concentration in body fluids collected from animals with the reference value, and evaluate the improvement, maintenance, and recovery effects of the motor function of the test substance. The present invention relates to an evaluation kit, a screening method for a substance for screening a substance having an effect of improving / maintaining / recovering a desired motor function using the method, and use as a marker .

  With the recent increase in health orientation, interest in exercise has increased. It is empirically clear that continuous and habitual exercise is useful for disease prevention and health maintenance. In addition, whey protein is known as a food material related to motor function, and muscle strengthening agents and muscle fatigue improving agents containing whey protein have been developed (Patent Documents 1 and 2).

  The relationship between whey protein and motor function has been scientifically investigated using animals. For example, Non-Patent Document 1 describes that intake of whey protein significantly increased the liver glycogen content of treadmill-exercised rats compared to casein intake and soy protein intake. In addition, as a mechanism, it is suggested that glycosyltransferases involved in glycogen composition may change. Non-Patent Document 2 describes that rats fed whey protein significantly increased glycogen content in the liver and muscle of rats with swimming exercise compared to rats fed casein. . Furthermore, Non-Patent Document 3 describes that rats fed whey protein promoted fatty acid synthesis in the liver and muscles of swimming exercise-loaded rats compared to rats fed casein. These results indicate that the combination of whey protein intake and continuous exercise produces higher motor function improvement and maintenance / recovery effects. That is, in an animal that is subjected to exercise load and ingested whey protein, the exercise function (exercise ability, exercise performance, etc.) is particularly high.

  On the other hand, there are no reports on marker substances in body fluids that correlate with motor function, and fluctuations in motor function (improvement, maintenance, recovery, etc.) caused by the combination of ingestion of substances such as whey protein and continuous exercise There is no known marker substance to reflect. If such a marker substance is specified, it is useful for screening a substance having the same action as whey protein.

JP 2002-65212 A JP 2004-182630 A Morifuji et al., Experimental Biology and Medicine, 2004, Vol. 230, No. 1, p23-30 Morifuji et al., British Journal of Nutrition, 2005, Vol. 93, No. 1, p439-445. Morifuji et al., Nutrition, 2005, Vol. 21, No. 10, p1052-1058

  An object of the present invention is to identify a marker substance that reflects motor function and muscle condition, and to develop a series of techniques useful for screening substances that have an effect of improving and maintaining / recovering motor function using the marker substance as an index. It is to provide.

The invention according to claim 1 for solving the above-described problem is the following marker substances (A1), (A2), (A3), (A) in a body fluid collected from an animal ingested with a test substance and subjected to exercise load. By comparing at least one concentration of A4), (A5), and (A7) with a reference value, the concentration of marker substances (A1) and (A2) is measured by a method using the mass / charge ratio as an index. A method for evaluating an improvement, maintenance, and recovery effect of motor function, characterized by evaluating an improvement, maintenance, and recovery effect of motor function of the test substance .
(A1) 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 5280 when subjected to mass spectrometry;
(A2) 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 6830 when subjected to mass spectrometry;
(A3) 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 7010 when subjected to mass spectrometry;
(A4) 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 7920 when subjected to mass spectrometry;
(A5) 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 13700 when subjected to mass spectrometry;
(A7) 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 47100 when subjected to mass spectrometry,
further,
The marker substance (A3) comprises at least one amino acid sequence represented by SEQ ID NOs: 7 to 9,
The marker substance (A4) includes at least one amino acid sequence represented by SEQ ID NOs: 10 to 14,
The marker substance (A5) has reactivity with an antibody specific to transthyretin,
The marker substance (A7) includes at least one amino acid sequence represented by SEQ ID NOs: 15 to 21.

The present invention relates to a method for evaluating the effect of improving, maintaining, and recovering the motor function of a substance. The marker substance (A1) , (A2 ) in a body fluid collected from an animal ingested with a test substance and subjected to exercise load ), (A3), (A4), (A5), and (A7) by comparing at least one concentration with a reference value to evaluate the improvement / maintenance / recovery effect of the test substance. is there. The marker substances (A1) , (A2), (A3), (A4), (A5), and (A7) are proteins that increase or decrease significantly in the body fluid of an animal that is subjected to exercise load and ingested whey protein It reflects fluctuations (improvement, maintenance, recovery, etc.) of motor functions (exercise ability, exercise performance, etc.) brought about by combined use of continuous exercise and whey protein intake. ADVANTAGE OF THE INVENTION According to this invention, the improvement / maintenance / recovery effect of the motor function which a test substance has can be evaluated easily and with high precision. The “animal” includes humans as well as animals such as mice.

  Here, the “effect of improving motor function” refers to an effect of further improving motor function at a normal level. By evaluating the improvement effect of the motor function of the substance, for example, it is possible to screen food materials suitable for athletes aiming at further improvement of the motor function. Moreover, the “maintenance effect of motor function” refers to an effect of attempting to maintain a normal level while suppressing a decrease in motor function. By evaluating the maintenance effect of the motor function possessed by the substance, for example, it is possible to screen food materials and the like suitable for elderly people who want to prevent a decrease in motor function. The “motor function recovery effect” refers to an effect of trying to return a reduced motor function to a normal level. By evaluating the recovery effect of the motor function of the substance, for example, a food material suitable for an injured person who aims to recover the motor function by rehabilitation can be screened. The “improvement / maintenance / recovery effect of motor function” refers to at least one of the improvement effect, maintenance effect, and recovery effect of motor function.

Here, the values such as “about 5280”, “about 13700”, “about 47100”, 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 5280 represents about 5280 ± 0.2%, about 13700 represents about 13700 ± 0.2%, and about 647100 represents about 47100 ± 0.2%. The other mass / charge ratios have a width of approximately ± 0.2% in a similar manner. In addition, these marker substances are mainly proteins present in blood. When the test substance has an effect of improving / maintaining / recovering the motor function, the concentrations of the marker substances (A1), (A4), and (A7) in the body fluid of the animal show higher values (A2), (A3) , the concentration of and (A5) shows a lower value.

The invention according to claim 2 includes the following marker substances (A1), (A2), (A3), (A4), (A5) and (A) in a body fluid collected from an animal that is ingested and exercised. By comparing at least one concentration of A7) with a reference value, the concentration of marker substances (A1) and (A2) is measured by a method using the mass / charge ratio as an index, and the motor function of the test substance is measured . This is a method for evaluating the improvement, maintenance and recovery effects of motor function, characterized by evaluating the improvement, maintenance and recovery effects .
(A1) 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 5280 when subjected to mass spectrometry;
(A2) 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 6830 when subjected to mass spectrometry;
(A3) 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 7010 when subjected to mass spectrometry;
(A4) 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 7920 when subjected to mass spectrometry;
(A5) 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 13700 when subjected to mass spectrometry;
(A7) 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 47100 when subjected to mass spectrometry,
further,
The marker substance (A3) is apolipoprotein C1 or a modified form thereof,
The marker substance (A4) is apolipoprotein A2 or a modified form thereof,
The marker substance (A5) is transthyretin or a modified form thereof,
The marker substance (A7) is β2-glycoprotein 1 or a modified form thereof.

  Since all of apolipoprotein C1, apolipoprotein A2, transthyretin, and β2-glycoprotein 1 are well known in physicochemical properties, the method for evaluating the improvement / maintenance / recovery effect of motor function according to the present invention For example, the marker substances (A3), (A4), (A5), and (A7) can be easily analyzed.

  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 includes at least one of the following marker substances (B1) to (B4) in a body fluid collected from an animal ingested with a test substance and subjected to exercise load: This is a method for evaluating the improvement / maintenance / recovery effect of motor function, characterized by evaluating the improvement / maintenance / recovery effect of motor function of the test substance by comparing one concentration with a reference value.
(B1) Apolipoprotein C1 or a modified product thereof,
(B2) Apolipoprotein A2 or a modified product thereof,
(B3) transthyretin or a modified form thereof,
(B4) β2-glycoprotein 1 or a modified form thereof.

  The method for evaluating the improvement / maintenance / recovery effect of the motor function according to the present invention includes at least one of the marker substances (B1) to (B4) in a body fluid collected from an animal ingested with a test substance and subjected to exercise load. By comparing one concentration with a reference value, the effect of improving, maintaining and restoring the motor function of the test substance is evaluated. The marker substances (B1) to (B4) are proteins that increase and decrease significantly in body fluids of animals that are ingested and exercised whey protein, and are brought about by the combined use of continuous exercise and whey protein intake. It reflects fluctuations (improvement, maintenance, recovery, etc.) of motor function (motor ability, exercise performance, etc.). ADVANTAGE OF THE INVENTION According to this invention, the improvement / maintenance / recovery effect of the motor function which a test substance has can be evaluated easily and with high precision. Further, since apolipoprotein C1, apolipoprotein A2, transthyretin, and β2-glycoprotein 1 are all well known in physicochemical properties, they are easy to analyze. In the present invention, the term “animal” includes humans as well as animals such as mice.

All of the marker substances belonging to (B1) to (B4) are mainly present in blood. When the test substance has the effect of improving, maintaining and restoring the motor function similar to whey protein, the concentrations of the marker substances (B2) and (B 4 ) in the body fluid of the animal show higher values, and (B1) and (B3 ) Concentration is lower.

  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.

The invention according to claim 4 is characterized in that the reference value is the following (I) and / or (II): Improvement / maintenance of motor function according to any one of claims 1 to 3 -It is a method for evaluating the recovery effect.
(I) the concentration of the marker substance in a body fluid collected from an animal subjected to exercise without taking the test substance,
(II) The concentration of the marker substance in a body fluid collected from an animal ingested with the test substance without applying an exercise load.

  With such a configuration, it is possible to more easily and accurately evaluate the improvement / maintenance / recovery effect of the motor function of the test substance.

  The invention according to claim 5 is the method for evaluating the improvement / maintenance / recovery effect of motor function according to any one of claims 1 to 4, wherein the body fluid is blood.

  With this configuration, it is possible to easily collect a body fluid as a measurement sample, and to evaluate the improvement / maintenance / recovery effect of the motor function of the test substance more easily and quickly.

  The invention according to claim 6 is the method for evaluating the improvement / maintenance / recovery effect of motor function according to any one of claims 1 to 5, wherein the test substance is a food material. .

  With such a configuration, for the purpose of developing functional foods, the improvement / maintenance / recovery effect of the motor function of the test substance can be evaluated.

  In the invention according to claim 7, 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, and the marker substance in the bodily fluid is captured on the carrier. The method for evaluating the improvement / maintenance / recovery effect of motor function according to claim 1, wherein the concentration of the marker substance in a body fluid is calculated based on the amount of the marker substance. It is.

  In the method for evaluating the improvement / maintenance / recovery effect of the motor function of 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 method for evaluating the improvement / maintenance / recovery effect of the motor function according to the present invention, since the marker substance captured on the carrier is the measurement object, the influence of the contaminant contained in the measurement sample can be reduced. The concentration of the marker substance can be measured with higher sensitivity and higher accuracy. Examples of the body fluid component include serum or plasma when the body fluid is blood.

  The invention according to claim 8 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. This is a method for evaluating the improvement, maintenance, and recovery effects of motor function.

  In the method for evaluating the improvement / maintenance / recovery effect of the motor function of 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 9 is characterized in that the substance having affinity for the marker substance is an ion exchanger or an antibody, and the effect of improving, maintaining and restoring the motor function according to claim 7 or 8 It is an evaluation method.

  In the method for evaluating the improvement / maintenance / recovery effect of the motor function of the present invention, 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 added via the ion exchanger or the antibody. Capture on the carrier. When 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).

  The invention according to claim 10 evaluates the improvement / maintenance / recovery effect of the test substance possessed by the test substance by the method for evaluating the improvement / maintenance / recovery effect of the motor function according to any one of claims 1 to 9. And screening a substance having an effect of improving / maintaining / recovering a desired motor function based on the evaluation result.

The present invention relates to a method for screening a substance, wherein each of the marker substances (A1) , (A2), (A3), (A4), (A5), and (A7) in animal body fluids and (B1) to (B1) to By comparing at least one concentration of each marker substance belonging to (B 4 ) with a reference value, a substance having an effect of improving / maintaining / recovering a desired motor function is screened. Above (A1), (A2), (A3), (A4), (A5), and (A7) each marker substances belonging to each marker substance and the above (B1) ~ (B 4) , both whey protein Is a protein that significantly increases or decreases in the body fluid of an animal subjected to exercise load, and changes (improvement) in motor functions (motor ability, exercise performance, etc.) brought about by continuous exercise and whey protein intake , Maintenance, recovery, etc.). According to the screening method for a substance of the present invention, a substance having an effect of improving / maintaining / recovering a desired motor function 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 developing a functional food having an effect of improving / maintaining / recovering motor function.

  The invention described in claim 11 is a kit for use in the method for evaluating the improvement / maintenance / recovery effect of motor function according to any one of claims 1 to 9, wherein the kit has an affinity for the marker substance. It is a kit for evaluating the improvement / maintenance / recovery effect of motor function, comprising a carrier on which a substance having the substance is immobilized.

  The kit for evaluating the improvement / maintenance / recovery effect of the motor function of 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.

  The invention according to claim 12 is the kit for evaluating the improvement / maintenance / recovery effect of motor function according to claim 11, wherein the substance having affinity for the marker substance is an ion exchanger. is there.

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

  A configuration used for screening a substance having an effect of improving / maintaining / recovering motor function is recommended (claim 13).

The invention according to claim 14 is a marker substance of at least one protein of the following (A1), (A2), (A3), (A4), (A5), and (A7) present in an animal body The detection of (A1) and (A2) is performed by a method using the mass / charge ratio as an index, and is used as a marker reflecting the fluctuation of motor function .
(A1) 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 5280 when subjected to mass spectrometry;
(A2) 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 6830 when subjected to mass spectrometry;
(A3) 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 7010 when subjected to mass spectrometry;
(A4) 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 7920 when subjected to mass spectrometry;
(A5) 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 13700 when subjected to mass spectrometry;
(A7) 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 47100 when subjected to mass spectrometry,
further,
The marker substance (A3) comprises at least one amino acid sequence represented by SEQ ID NOs: 7 to 9,
The marker substance (A4) includes at least one amino acid sequence represented by SEQ ID NOs: 10 to 14,
The marker substance (A5) has reactivity with an antibody specific to transthyretin,
The marker substance (A7) includes at least one amino acid sequence represented by SEQ ID NOs: 15 to 21.

The invention according to claim 15 is a marker substance of at least one protein of the following (A1), (A2), (A3), (A4), (A5), and (A7) present in an animal body The detection of (A1) and (A2) is performed by a method using the mass / charge ratio as an index, and is used as a marker that reflects fluctuations in motor function .
(A1) 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 5280 when subjected to mass spectrometry;
(A2) 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 6830 when subjected to mass spectrometry;
(A3) 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 7010 when subjected to mass spectrometry;
(A4) 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 7920 when subjected to mass spectrometry;
(A5) 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 13700 when subjected to mass spectrometry;
(A7) 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 47100 when subjected to mass spectrometry,
further,
The marker substance (A3) is apolipoprotein C1 or a modified form thereof,
The marker substance (A4) is apolipoprotein A2 or a modified form thereof,
The marker substance (A5) is transthyretin or a modified form thereof,
The marker substance (A7) is β2-glycoprotein 1 or a modified form thereof.

The invention according to claim 16 is the use of at least one protein of the following (B1) to (B4) present in the body of an animal as a marker reflecting a change in motor function.
(B1) Apolipoprotein C1 or a modified product thereof,
(B2) Apolipoprotein A2 or a modified product thereof,
(B3) transthyretin or a modified form thereof,
(B4) β2-glycoprotein 1 or a modified form thereof.

  According to the evaluation method of the improvement / maintenance / recovery effect of the motor function of the present invention, the improvement / maintenance / recovery effect of the motor function 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 / maintaining / recovering a desired motor function can be easily and accurately screened.

  According to the kit for evaluating the improvement / maintenance / recovery effect of the motor function 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.

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

The evaluation method of the improvement / maintenance / recovery effect of the motor function of the present invention includes three aspects. In one aspect, the following marker substances (A1), (A2), (A3), (A4), (A5), and (A7) in body fluid collected from animals that have been ingested and exercised: By comparing at least one concentration with a reference value, the concentration of marker substances (A1) and (A2) is measured by a method using the mass / charge ratio as an index, and the motor function of the test substance is improved and maintained.・ Evaluate the recovery effect .
(A1) 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 5280 when subjected to mass spectrometry;
(A2) 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 6830 when subjected to mass spectrometry;
(A3) 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 7010 when subjected to mass spectrometry;
(A4) 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 7920 when subjected to mass spectrometry;
(A5) 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 13700 when subjected to mass spectrometry;
(A7) 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 47100 when subjected to mass spectrometry,
further,
The marker substance (A3) comprises at least one amino acid sequence represented by SEQ ID NOs: 7 to 9,
The marker substance (A4) includes at least one amino acid sequence represented by SEQ ID NOs: 10 to 14,
The marker substance (A5) has reactivity with an antibody specific to transthyretin,
The marker substance (A7) includes at least one amino acid sequence represented by SEQ ID NOs: 15 to 21.

All of these marker substances are proteins mainly present in the blood, and they increase or decrease significantly in the body fluids of animals subjected to exercise load as well as intake of whey protein, and combined use of continuous exercise and whey protein intake It reflects the fluctuations (improvement, maintenance, recovery, etc.) of motor function (motor ability, motor performance, etc.) brought about by. When the test substance has the same improvement / maintenance / restoration effect of motor function as whey protein, the marker substances (A1), (A4), and (A7) in the body fluids of animals (hereinafter, groups consisting of these marker substances) (A2), (A3), and (A5) (hereinafter, a group consisting of these marker substances is referred to as “Group 2”). Concentration is lower).

According to certain conditions of peptide mapping, the marker substances (A3), (A4), and (A7) can be identified as apolipoprotein C1, apolipoprotein A2, and β2-glycoprotein 1, respectively. The marker substance (A5) has reactivity with an antibody specific for transthyretin. That is, in other aspects , at least one of the following (1) , (2), (3), and (5) is satisfied.
(1) The marker substance (A3) is apolipoprotein C1 or a modified form thereof.
(2) The marker substance (A4) is apolipoprotein A2 or a modified form thereof.
(3) The marker substance (A5) is transthyretin or a modified form thereof.
(5) The marker substance (A7) is β2-glycoprotein 1 or a modified form thereof.

In another aspect of the present invention, at least one concentration belonging to any of the following marker substances (B1) to (B4) in a body fluid collected from an animal that has been ingested and exercised is compared with a reference value. Thus, the improvement / maintenance / recovery effect of the motor function of the test substance is evaluated.
(B1) Apolipoprotein C1 or a modified product thereof,
(B2) Apolipoprotein A2 or a modified product thereof,
(B3) transthyretin or a modified form thereof,
(B4) β2-glycoprotein 1 or a modified form thereof.

  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”.

  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 product thereof” includes its subunits.

  For example, the N-terminal amino acid sequence of apolipoprotein C1 (SEQ ID NO: 1, derived from mouse) after cleavage of the signal sequence starts 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 is included in the “modified product of apolipoprotein C1”.

  Similarly, the apolipoprotein A2 (SEQ ID NO: 2, derived from mouse) after cleavage of the signal sequence consists of 78 amino acids, but the protein fragment consisting of 70 amino acids on the N-terminal side (SEQ ID NO: 3) is “apolipoprotein A2 Is included in the "modified form".

  Transthyretin (also called prealbumin) is a kind of plasma protein and is a complex protein consisting of four subunits having a molecular weight of about 14,000. The amino acid sequences of mouse-derived and human-derived transthyretin are as shown in SEQ ID NOs: 4 and 5, respectively, and have one cysteine residue. The thiol group (-SH) of this unique cysteine residue can be modified, and a plurality of modified forms of transthyretin differing in the type of modification to the cysteine residue are known (for example, Lim) , Journal of Biological Chemistry, 2003, 278, p49707-49713; Lim et al., Protein Science, 2003, 12, p1775. -1785; Gericke et al., BMC Cancer, 2005, 5: 133). Specifically, a modified form of transthyretin in which a sulfone group, cysteine, homocysteine, cysteinyl glycine, or glutathione is added to a cysteine residue is known. For example, these modified transthyretins can be used as marker substances in the present invention. In the present specification, the subunit of transthyretin is also simply referred to as “transthyretin”, and the complex and the subunit are not particularly distinguished.

  As for β2-glycoprotein 1 (β2-glycoprotein1; also referred to as apolipoprotein H), the modifications include the respective modifications illustrated above. The amino acid sequence of β2-glycoprotein 1 derived from mouse is shown in SEQ ID NO: 6.

In the evaluation method of improving, maintaining and recovery effect of motor function of the present invention, even when using only one of each marker substances belonging to the (A1) ~ (A7) of and the marker substance (B1) ~ (B 4) It may be used in combination.

  The method for applying an exercise load to an animal is not particularly limited as long as it is a method that can apply a load more than the exercise performed in normal life. For example, a treadmill can be used. There is no particular limitation on the time and frequency of applying the exercise load, and it may be appropriately selected depending on the content of the load. For example, exercise load of several minutes to several tens of minutes per time is given once or more per day, and this is performed for 5 days or more per week.

In the method for evaluating the improvement / maintenance / recovery effect of the motor function of the present invention, the concentration of the marker substance in the animal body fluid is compared with a reference value. In a preferred embodiment, the reference value is (I) and / or (II) below.
(I) the concentration of the marker substance in a body fluid collected from an animal subjected to exercise without taking the test substance,
(II) The concentration of the marker substance in a body fluid collected from an animal ingested with the test substance without applying an exercise load.
When the test substance has an effect of improving / maintaining / recovering motor function, the concentration of the marker substance belonging to group 1 in the body fluid of the animal is higher than (I) and (II), and the concentration of the marker substance belonging to group 2 Indicates a lower value than (I) and (II). Note that either one of the reference values (I) and (II) may be used or may be used in combination.

  The reference value can also be set in advance. For example, using a known substance (for example, whey protein) that has an effect of improving / maintaining / restoring motor function, animal experiments are performed in advance, and a reference value (fixed value) for each marker substance is determined based on the accumulated data. Can be determined. That is, the same handling as the reference values (cut-off values, etc.) set in various clinical tests is possible.

  The animal used in the method for evaluating the improvement / maintenance / recovery effect of the motor function of the present invention is not particularly limited, and for example, humans, mice, rats, rabbits, pigs and the like can be employed.

  Blood is preferably used as the animal body fluid used in the method for evaluating the improvement / maintenance / recovery effect of the motor function of the present invention. 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 improvement / maintenance / recovery effect of the motor function of 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 improvement / maintenance / recovery effect of the motor function of the present invention, the method for measuring the concentration of a marker substance is generally used for protein quantification as long as it can specifically measure the concentration of the marker substance. Can be used as is. 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 an anion exchange substrate, particularly a strong anion exchange substrate are preferably used.

  The screening method for a substance of the present invention evaluates the improvement / maintenance / recovery effect of a test substance by the method for evaluating the improvement / maintenance / recovery effect of the motor function of the present invention, and improves / maintains the desired motor function. -Screening for substances that have a recovery effect. In the substance screening method of the present invention, the same embodiment as the above-described method for evaluating the improvement / maintenance / recovery effect of motor function of the present invention can be employed.

  In order to easily perform the method for evaluating the improvement / maintenance / recovery effect of the motor function of 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 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. The kit can also be used as a kit for screening for substances having an effect of improving / maintaining / recovering a desired motor function. Examples of the configuration of the kit of the present invention are given below.

[Example of kit configuration]
・ Weak cation exchange substrate: 1 sheet ・ Strong anion exchange substrate: 1 sheet ・ Substrate cleaning buffer A (pH 3.0): appropriate amount ・ Substrate cleaning buffer B (pH 9.0): appropriate amount ・ Standard of each marker substance Product: each appropriate amount

  Below, an example of the specific procedure which evaluates the improvement / maintenance / recovery effect of the motor function which a test substance has by this invention is given. In this example, four groups of mice are used, and SELDI-TOF-MS is employed for measuring blood and marker substances as body fluids.

The mice are divided into 4 groups and reared under the following conditions.
Group 1 (SC): no exercise load (S) + control food intake (C)
Group 2 (TC): exercise load (T) + control food intake (C)
Group 3 (TP): exercise load (T) + intake of test substance-containing food (P)
Group 4 (SP): No exercise load (S) + test substance-containing food intake (P)
The control diet does not contain any substances that have an effect of improving, maintaining, and restoring motor function (for example, whey protein), and it is known that it has no effect of improving, maintaining, or recovering motor function (for example, casein) Consists only of. The test substance-containing food has basically the same composition as the control food except that it contains the test substance. The second group and the third group are continuously subjected to exercise load by a treadmill.

  Blood is collected from each group of mice after breeding for a certain period, and a plasma sample is prepared. After denaturation with a surfactant or the like, it is applied to a strong anion exchange column, and sequentially eluted with each of pH 9.0, pH 5.0 and pH 3.0 buffers and an organic solvent for fractionation.

  The pH 9.0 elution fraction is spotted on a weak cation exchange substrate and then washed with a pH 3.0 buffer. At this time, the marker substances (A3), (A5), and (A7) are captured on the substrate. Moreover, the pH 3.0 elution fraction is spotted on a weak cation exchange substrate, and then washed with a pH 3.0 buffer. At this time, the marker substances (A2) and (A4) are captured on the substrate. The elution fraction of the organic solvent is spotted on a strong anion exchange substrate and then washed with a buffer of pH 9.0. At this time, the marker substance (A1) is captured on the substrate. Each substrate is subjected to SELDI-TOF-MS, and the concentration of each marker substance is calculated from the generated ion peak.

  The concentration values of the first group to the fourth group are arranged horizontally for each marker substance, and the increase / decrease pattern is analyzed. Regarding the value of “first group-second group-third group-fourth group”, when the marker substance belonging to group 1 shows a pattern of “low-low-high-low”, In the case of the marker substance to which it belongs, when the pattern of “high-high-low-high” is shown, it is evaluated that the test substance has an effect of improving / maintaining / recovering motor function. The values of the second group and the fourth group correspond to the reference values (I) and (II) described above, respectively. At this time, any one marker substance may be used as an index, or a plurality of marker substances may be used as an index.

  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 Breeding and Body Fluid Sample Preparation Eight week old male C57BL / 6J strain mice (Japan SLC) were preliminarily raised and divided into 4 groups. The number of individuals in each group was 7 or more.

[Raising conditions for each group]
Group 1 (SC): no exercise load (S) + control food intake (C)
Group 2 (TC): exercise load (T) + control food intake (C)
Group 3 (TP): Exercise load (T) + intake of whey protein-containing food (P)
Group 4 (SP): No exercise load (S) + whey protein containing food intake (P)

[Feed, etc.]
・ Control food: AIN93
-Whey protein-containing food (Meiji Seika Co., Ltd.): It has a composition in which casein contained in a control food is replaced with whey protein.
・ Free consumption of water and feed

[Breeding period]
・ 4 weeks (light / dark, room temperature)
・ Light: 8 o'clock to 20 o'clock, Dark: 20 o'clock to 8 o'clock ・ Room temperature: 22 ± 2 ℃

[Exercise load]
A treadmill exercise load was adopted. The first week of breeding was started at 15m / min for 15 minutes with no inclination, and gradually got used to speed and exercise time. From the second week of the breeding to the end of the breeding, an exercise load was applied at 22 m / min for 30 minutes without tilting. The frequency was once a day, 5 days per week, so that no more than 2 days of rest occurred.

  Blood was collected from each group of mice after the rearing. Plasma was prepared from the collected blood and used as a body fluid sample.

2. Analysis by Protein Chip and Selection of Ion Peak To 20 μL of each body fluid sample, 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 obtained fraction was diluted 10-fold with a pH 9.0 protein chip binding buffer (50 mM Tris-HCl (pH 9.0)) and then added to a strong anion exchange chip Q10 (Bio-Rad). did. 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. Based on these pieces of information, the peaks were narrowed down in the following steps S1 to S4.

S1: When exercise mice were compared with each other, a peak in which the expression level increased or decreased due to whey protein intake was selected. Specifically, peaks having a significant difference (p <0.05) in ionic strength were selected between the second group (TC) and the third group (TP). At this stage, 57 candidate peaks were selected.
S2: Peaks in which increase / decrease in (a) was suppressed by exercise were removed from 57 peaks. Specifically, a marker having a significant difference (p <0.05) in ionic strength between the first group (SC) and the second group (TC) was removed. This narrowed down to 43 candidate peaks.
S3: Artifacts (divalent ions, noise, etc.) were removed from 43 candidate peaks. This narrowed down to 22 candidate peaks.
S4: From the 22 candidate peaks, when the mice that ingested the whey protein were compared, the peak whose expression level was increased or decreased by exercise was selected. Specifically, a marker having a significant difference (p <0.05 or 0.1) in ionic strength was selected between the third group (TP) and the fourth group (SP). As a result, seven candidate peaks were finally selected.
In addition, as for the increase / decrease pattern of the peak finally selected, only the third group showed a high value or a low value, and there was not much difference among the first group, the second group, and the fourth group. .

3. Identification of marker substance (A1) Fraction 4 (organic solvent) was brought into contact with a weak cation exchange chip CM10, washed with a pH 3.0 protein chip binding buffer and subjected to SELDI-TOF-MS (EAM: CHCA). In some cases, an ion peak with a mass / charge ratio of 5280 (average value) was detected. As a result of the increase / decrease pattern analysis, this peak showed a low value in the first group, the second group, and the fourth group, and a high value in the third 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 (second group vs. third group): 0.774
p value (second group vs. third group): 0.009
ROC area (first group vs second group): 0.667
p value (first group vs second group): 0.184
ROC area (first group vs. fourth group): 0.506
p value (first group vs. fourth group): 0.918
ROC area (third group vs. fourth group): 0.735
p value (third group vs. fourth group): 0.031

  From the above, when subjected to SELDI-TOF-MS, the protein (marker substance (A1)) that produces a peak with a mass / charge ratio of about 5280 is significantly increased in the body fluid of the animal that is subjected to exercise load and ingested whey protein. It was found that it can increase and decrease, and can be a marker that reflects fluctuations (improvement, maintenance, recovery, etc.) of motor function (exercise ability, exercise performance, etc.) brought about by continuous exercise and whey protein intake. . This makes it possible to evaluate the improvement / maintenance / recovery effect of the test substance on the test substance using the concentration of the marker substance (A1) in the body fluid of the animal that is ingested as well as ingesting the test substance as an index. Indicated. For example, the concentration of a protein that produces a peak with a mass / charge ratio of about 5280 when a test sample is used to prepare a body fluid sample and SELDI-TOF-MS is performed according to the same procedure. When the test substance shows a pattern of “low-low-high-low” from the first group to the fourth group, the test substance should be evaluated to have the same improvement, maintenance, and recovery effects of motor function as whey protein Can do.

4). Identification of Marker Substance (A2) Fraction 3 (pH 3.0) is brought into contact with strong anion exchange chip Q10, washed with pH 9.0 protein chip binding buffer and subjected to SELDI-TOF-MS (EAM: CHCA). In this case, an ion peak having a mass / charge ratio of 6829 (average value) was detected. As a result of the increase / decrease pattern analysis, this peak showed a high value in the first group, the second group, and the fourth group, and a low value in the third 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 (second group vs. third group): 0.810
p value (second group vs. third group): 0.010
ROC area (first group vs second group): 0.639
p value (first group vs second group): 0.225
ROC area (first group vs. fourth group): 0.536
p value (first group vs. fourth group): 0.537
ROC area (third group vs fourth group): 0.658
p value (third group vs. fourth group): 0.089

  From the above, when subjected to SELDI-TOF-MS, the protein (marker substance (A2)) that produces a peak with a mass / charge ratio of about 6830 is significantly increased in the body fluid of animals that are subjected to exercise load and ingested whey protein. It was found that it can increase and decrease, and can be a marker that reflects fluctuations (improvement, maintenance, recovery, etc.) of motor function (exercise ability, exercise performance, etc.) brought about by continuous exercise and whey protein intake. . This makes it possible to evaluate the improvement / maintenance / recovery effect of the test substance on the test substance, using the concentration of the marker substance (A2) in the body fluid of the animal that is ingested as well as ingesting the test substance as an index. Indicated. For example, when a test sample is used to conduct a similar animal experiment to prepare a body fluid sample and SELDI-TOF-MS is performed according to the same procedure, the protein concentration that produces a peak with a mass / charge ratio of about 6830 When the test substance shows a pattern of “high-high-low-high” from the first group to the fourth group, the test substance should be evaluated as having the same improvement / maintenance / recovery effect of motor function as whey protein Can do.

5. Identification of Marker Substance (A3) Fraction 1 (pH 9.0) is contacted 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 7011 (average value) was detected. As a result of the increase / decrease pattern analysis, this peak showed a high value in the first group, the second group, and the fourth group, and a low value in the third 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 (second group vs. third group): 0.810
p value (second group vs. third group): 0.003
ROC area (first group vs second group): 0.583
p value (first group vs second group): 0.564
ROC area (first group vs. fourth group): 0.732
p value (first group vs. fourth group): 0.057
ROC area (3rd group vs 4th group): 0.684
p value (third group vs. fourth group): 0.039

  From the above, when subjected to SELDI-TOF-MS, a protein (marker substance (A3)) that produces a peak with a mass / charge ratio of about 7010 is significantly increased in the body fluid of an animal that is subjected to exercise load and ingested whey protein. It was found that it can increase and decrease, and can be a marker that reflects fluctuations (improvement, maintenance, recovery, etc.) of motor function (exercise ability, exercise performance, etc.) brought about by continuous exercise and whey protein intake. . This makes it possible to evaluate the improvement / maintenance / recovery effect of the test substance on the test substance, using the concentration of the marker substance (A3) in the body fluid of the animal subjected to exercise load as well as taking the test substance as an index. Indicated. For example, when the same animal experiment is performed using the test substance to prepare a body fluid sample, and the SELDI-TOF-MS is performed according to the same procedure, the concentration of the protein that produces a peak with a mass / charge ratio of about 7010 When the test substance shows a pattern of “high-high-low-high” from the first group to the fourth group, the test substance should be evaluated as having the same improvement / maintenance / recovery effect of motor function as whey protein Can do.

6). Identification of marker substance (A4) Fraction 3 (pH 3.0) is brought into contact with a weak cation exchange chip CM10, washed with a protein chip binding buffer of pH 3.0 and subjected to SELDI-TOF-MS (EAM: CHCA). In this case, an ion peak having a mass / charge ratio of 7924 (average value) was detected. As a result of the increase / decrease pattern analysis, this peak showed a low value in the first group, the second group, and the fourth group, and a high value in the third 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 (second group vs. third group): 0.714
p value (second group vs. third group): 0.035
ROC area (first group vs second group): 0.611
p value (first group vs second group): 0.488
ROC area (first group vs. fourth group): 0.542
p value (first group vs. fourth group): 0.643
ROC area (third group vs. fourth group): 0.760
p value (third group vs. fourth group): 0.019

  From the above, when subjected to SELDI-TOF-MS, the protein (marker substance (A4)) that produces a peak with a mass / charge ratio of about 7920 is significantly increased in the body fluid of the animal that is subjected to exercise load and ingested whey protein. It was found that it can increase and decrease, and can be a marker that reflects fluctuations (improvement, maintenance, recovery, etc.) of motor function (exercise ability, exercise performance, etc.) brought about by continuous exercise and whey protein intake. . This makes it possible to evaluate the improvement / maintenance / recovery effect of the test substance on the test substance by using the concentration of the marker substance (A4) in the body fluid of the animal subjected to exercise load as well as ingesting the test substance. Indicated. For example, when a test sample is used to conduct a similar animal experiment to prepare a body fluid sample, and a SELDI-TOF-MS is performed according to the same procedure, the protein concentration that produces a peak with a mass / charge ratio of about 7920 When the test substance shows a pattern of “low-low-high-low” from the first group to the fourth group, the test substance should be evaluated to have the same improvement, maintenance, and recovery effects of motor function as whey protein Can do.

7). Identification of Marker Substance (A5) Fraction 1 (pH 9.0) is contacted with a weak cation exchange chip CM10, washed with a pH 3.0 protein chip binding buffer, and SELDI-TOF-MS (EAM: SPA-HJ) , An ion peak with a mass / charge ratio of 13678 (average value) was detected. As a result of the increase / decrease pattern analysis, this peak showed a high value in the first group, the second group, and the fourth group, and a low value in the third 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 (second group vs. third group): 0.899
p value (second group vs. third group): 0.001
ROC area (first group vs second group): 0.667
p value (first group vs second group): 0.225
ROC area (first group vs. fourth group): 0.780
p value (first group vs. fourth group): 0.027
ROC area (third group vs. fourth group): 0.684
p value (third group vs. fourth group): 0.081

  As described above, when subjected to SELDI-TOF-MS, a protein (marker substance (A5)) that generates a peak with a mass / charge ratio of about 13700 is significantly increased in the body fluid of an animal that is subjected to exercise load and ingested whey protein. It was found that it can increase and decrease, and can be a marker that reflects fluctuations (improvement, maintenance, recovery, etc.) of motor function (exercise ability, exercise performance, etc.) brought about by continuous exercise and whey protein intake. . This makes it possible to evaluate the improvement / maintenance / recovery effect of the test substance on the test substance, using the concentration of the marker substance (A5) in the body fluid of the animal subjected to exercise load as well as taking the test substance as an index. Indicated. For example, the concentration of a protein that produces a peak with a mass / charge ratio of approximately 13700 when a test sample is used to prepare a body fluid sample and SELDI-TOF-MS is performed according to the same procedure. When the test substance shows a pattern of “high-high-low-high” from the first group to the fourth group, the test substance should be evaluated as having the same improvement / maintenance / recovery effect of motor function as whey protein Can do.

8). Identification of Marker Substance (A6) Fraction 1 (pH 9.0) is contacted with a weak cation exchange chip CM10, washed with a pH 3.0 protein chip binding buffer, and SELDI-TOF-MS (EAM: SPA-HJ) , An ion peak with a mass / charge ratio of 13881 (average value) was detected. As a result of the increase / decrease pattern analysis, this peak showed a high value in the first group, the second group, and the fourth group, and a low value in the third group. FIG. 6 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 (second group vs. third group): 0.810
p value (second group vs. third group): 0.003
ROC area (first group vs second group): 0.639
p value (first group vs second group): 0.248
ROC area (first group vs. fourth group): 0.780
p value (first group vs. fourth group): 0.021
ROC area (third group vs. fourth group): 0.760
p value (third group vs. fourth group): 0.019

  From the above, when subjected to SELDI-TOF-MS, the protein (marker substance (A6)) that produces a peak with a mass / charge ratio of about 13900 is significantly increased in the body fluid of the animal that is subjected to exercise load and ingested whey protein. It was found that it can increase and decrease, and can be a marker that reflects fluctuations (improvement, maintenance, recovery, etc.) of motor function (exercise ability, exercise performance, etc.) brought about by continuous exercise and whey protein intake. . This makes it possible to evaluate the improvement / maintenance / recovery effect of the test substance on the test substance by using the concentration of the marker substance (A6) in the body fluid of the animal subjected to exercise load as well as taking the test substance. Indicated. For example, the same animal experiment is performed using the test substance to prepare a body fluid sample, and the SELDI-TOF-MS is performed according to the same procedure. When the test substance shows a pattern of “high-high-low-high” from the first group to the fourth group, the test substance should be evaluated as having the same improvement / maintenance / recovery effect of motor function as whey protein Can do.

9. Identification of Marker Substance (A7) Fraction 1 (pH 9.0) is contacted with weak cation exchange chip CM10, washed with pH 3.0 protein chip binding buffer, and SELDI-TOF-MS (EAM: SPA-HJ) , An ion peak with a mass / charge ratio of 47095 (average value) was detected. As a result of the increase / decrease pattern analysis, this peak showed a low value in the first group, the second group, and the fourth group, and a high value in the third group. FIG. 7 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 (second group vs. third group): 0.786
p value (second group vs. third group): 0.009
ROC area (first group vs second group): 0.500
p value (first group vs second group): 0.954
ROC area (first group vs. fourth group): 0.655
p value (first group vs. fourth group): 0.136
ROC area (3rd group vs. 4th group) is 0.760
p value (3rd group vs 4th group) is 0.015.

  From the above, when subjected to SELDI-TOF-MS, the protein (marker substance (A7)) that produces a peak with a mass / charge ratio of about 47100 is significantly increased in the body fluid of animals that have been subjected to exercise load and ingested whey protein. It was found that it can increase and decrease, and can be a marker that reflects fluctuations (improvement, maintenance, recovery, etc.) of motor function (exercise ability, exercise performance, etc.) brought about by continuous exercise and whey protein intake. . This makes it possible to evaluate the improvement / maintenance / recovery effect of the test substance on the test substance, using the concentration of the marker substance (A7) in the body fluid of the animal subjected to exercise load as well as ingesting the test substance as an index. Indicated. For example, when the same animal experiment is performed using the test substance to prepare a body fluid sample and SELDI-TOF-MS is performed according to the same procedure, the concentration of the protein that produces a peak with a mass / charge ratio of about 47100 When the test substance shows a pattern of “low-low-high-low” from the first group to the fourth group, the test substance should be evaluated to have the same improvement, maintenance, and recovery effects of motor function as whey protein Can do.

1. Purification and identification of marker substance (A3) Spin column (1 mL) packed with strong anion exchange resin Q-Sepharose HP (GE Healthcare) was equilibrated with 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) 750 μL was added to 500 μL of C57BL mouse serum (Shimizu Experimental Materials Co., Ltd.) and added to the equilibrated column. The non-adsorbed fraction was collected and the degree of purification was confirmed by SELDI-TOF-MS.

  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 (DRC). The results are shown in FIG. In FIG. 8, 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. 8) 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 (A3) was confirmed. (A of FIG. 9).

  The same SDS-PAGE was performed again to cut out a band of about 7.0 kDa, and after reduction alkylation treatment, 0.01 μg / μL trypsin solution (dissolved in 50 mM ammonium bicarbonate (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 saturated CHCA solution was further dropped and dried, and then measured with a mass spectrometer Proteomics Analyzer 4700 (Applied Biosystems). At least three peaks Were detected, and their accurate masses were calculated as “127.70”, “1299.71”, and “1042.53”. Based on these data, a known protein was searched by Mascot database (Matrix Science), and peptide mass fingerprinting was performed. As a result, accurate masses “127.70”, “1299.71”, and “1042.53” Each of the peptides matched the peptide having the amino acid sequence represented by SEQ ID NOs: 7 to 9, and the target protein was identified as “apolipoprotein C1” 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 apolipoprotein C1 is as shown in SEQ ID NO: 1.

  In addition, as shown in FIG. 9, the peak which shows mass (about 6.8 kDa) smaller than a marker substance (A3) in the SELDI-TOF-MS analysis was also detected (b of FIG. 9). From the comparison of these two proteins with very similar properties and mass values, the protein of peak b lacks the N-terminal 2 amino acids (Ala-Pro) of apolipoprotein C1 (SEQ ID NO: 1). It was considered to be a “modified product of apolipoprotein C1”. The mass calculated from the amino acid sequence was 6993 Da for peak a and 6825 Da for peak b.

2. Purification and identification of marker substance (A4) Spin column (1 mL) packed with strong anion exchange resin Q-Sepharose HP (GE Healthcare) was equilibrated with buffer (50 mM Tris-HCl (pH 9.0), 1 M urea, 0.22% CHAPS). C57BL mouse serum (Shimizu Experimental Materials Co., Ltd.) 1 mL of denaturation buffer (50 mM Tris-HCl (pH 9.0), 9 M urea, 2% CHAPS) was denatured and added to the equilibrated column. did. The column was sequentially washed with 50 mM Tris-HCl (pH 9.0), 100 mM sodium acetate buffer (pH 5.0), and 50 mM sodium citrate buffer (pH 3.0), and then the elution buffer (33.3% isopropanol, 16 Elution with 7% acetonitrile, 0.1% TFA). The degree of purification of the eluted fraction was confirmed by SELDI-TOF-MS.

  A part of the collected fraction was subjected to acetone precipitation treatment, and then two-dimensional electrophoresis (first dimension: Immobiline DryStrip (GE Healthcare) pH 4-7, 7 cm; second dimension: polypeptide separating gel (15-20) %) NTH-5A2T (DRC)). The results are shown in FIG. The separated band of about 7.9 kDa (arrow in FIG. 10) was cut out, protein was extracted, and SELDI-TOF-MS analysis was performed. A peak showing a mass / charge ratio equivalent to that of the marker substance (A4) was confirmed (FIG. 11).

  When a two-dimensional electrophoresis was performed again under the same conditions to cut out a band of about 7.9 kDa (arrow in FIG. 10), and in-gel digestion and mass spectrometry were performed in the same manner as 1 above, at least 5 peaks were found. Detected and their exact masses were calculated as “3239.45”, “3454.62”, “28332.46”, “18331.99”, and “1193.67”. Based on these data, peptide mass fingerprinting was performed in the same manner as in the above 1. As a result, accurate masses “323.45”, “3454.62”, “28332.46”, “18331.99”, and “1193” were obtained. The peptide of .67 ”matches the peptide of the amino acid sequence represented by SEQ ID NOs: 10 to 14, respectively, and the target protein was identified as“ apolipoprotein A2 ”with a probability of 99% or more. Table 2 shows the correspondence between the exact mass, amino acid sequence, and SEQ ID NO of each peptide. Furthermore, the predicted isoelectric point of the protein fragment corresponding to the 70 amino acids on the N-terminal side of apolipoprotein A2 (SEQ ID NO: 2, 78 amino acids, isoelectric point 4.94, molecular weight 8736) is 5.13, and the molecular weight is 7925. These values were in good agreement with the physical properties of the marker substance (A4). Thereby, the marker substance (A4) was finally identified as a fragment of apolipoprotein A2 (SEQ ID NO: 3).

3. Identification of marker substances (A5) and (A6) C57BL mouse serum (Shimizu Experimental Materials Co., Ltd.) was subjected to denaturation treatment and column purification in the same manner as in Example 1 to obtain four fractions. Fraction 1 (pH 9.0, pass-through) was separated by SDS-PAGE, and then Western blotting using an anti-human transthyretin antibody (Santa Cruz) was performed. The results are shown in FIG. In FIG. 12, lane 1 is a human transthyretin preparation (positive control), lane 2 is equivalent to 50 μL of sample, and lane 3 is equivalent to 100 μL of sample. As a result, a band of about 13.7 kDa reacted in lanes 2 and 3.

  FIG. 13 (a) shows the results of SELDI-TOF-MS analysis of the fraction 1 of the third group (TP) of Example 1 and FIG. 13 (b) of the fraction 1 prepared in this example. The pattern of these ion peaks was very similar, and a peak of about 13.7 kDa corresponding to the marker substance (A5) and a peak of about 13.8 kDa corresponding to the marker substance (A6) were detected.

  It is known that transthyretin includes various modified products having different types of modification to cysteine residues. From each molecular weight value, it was considered that the marker substance (A5) was unmodified transthyretin, and the marker substance (A6) was modified transthyretin bound to SPA.

4). Purification and identification of marker substance (A7) Spin column (5 mL) packed with strong anion exchange resin Q-Sepharose HP (GE Healthcare) was equilibrated with buffer (50 mM Tris-HCl (pH 9.0), 1 M urea, 0.22% CHAPS). 1.5 mL of C57BL mouse serum (Shimizu Experimental Materials Co., Ltd.) was centrifuged at 20,000 G and 4 ° C. for 10 minutes, and the supernatant was collected. To 1.0 mL of the collected supernatant, 1.5 mL of denaturation buffer (50 mM Tris-HCl (pH 9.0), 9M urea, 2% CHAPS) was added for denaturation treatment. Further, 2.5 mL of equilibration buffer was added for dilution, and then added to the equilibrated column. The column was washed with 50 mM Tris-HCl (pH 9.0), and then eluted in two steps with 50 mM sodium acetate buffer (pH 5.0). The degree of purification of the eluted fraction was confirmed by SELDI-TOF-MS.

  HiTrap SP HP (GE Healthcare) (1 mL) was equilibrated with 50 mM MES-NaOH (pH 6.0). 2 mL of the eluted fraction was diluted 5-fold with 50 mM MES-NaOH (pH 6.0) and then added to the equilibrated column. The column was washed sequentially with 50 mM MES-NaOH (pH 6.0) containing 50, 70, 80, 90, or 100 mM NaCl, and then eluted with 50 mM MES-NaOH (pH 6.0) containing 1 M NaCl. did. The purity of the collected elution fraction was confirmed by SELDI-TOF-MS.

  A portion of the collected fraction was desalted and concentrated with Amicon Ultra (5 kDa cut; Millipore), and then SDS-PAGE (7.5% polyacrylamide gel was used). The results are shown in FIG. In FIG. 14, lanes 4 to 6 are collected fractions, and M is a molecular weight marker. The separated band of about 47 kDa (arrow in FIG. 14) 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 (A7) was confirmed (FIG. 15).

  When the same SDS-PAGE was performed again to cut out a band of about 42 kDa, and in-gel digestion and mass spectrometry were performed in the same manner as in 1 above, at least 7 peaks were detected, and their accurate masses were “2306. It was calculated as “08”, “1822.90”, “1545.82”, “1876.83”, “1105.60”, “1325.63”, and “1038.54”. Peptide mass fingerprinting was performed in the same manner as in 1 above based on these data. As a result, precise masses “2306.08”, “1822.90”, “1545.82”, “1876.83”, “1105. The peptides of “60”, “1325.63”, and “1038.54” match the peptides of the amino acid sequences represented by SEQ ID NOs: 15 to 21, respectively, and the target protein has a probability of “β-2- Identified as “Glycoprotein 1”. Table 3 shows the correspondence between the exact mass, amino acid sequence, and SEQ ID NO of each peptide. The amino acid sequence of previously reported β2-glycoprotein 1 is as shown in SEQ ID NO: 6.

It is a box diagram about the ion peak whose mass / charge ratio is 5280 (average value). It is a box diagram about an ion peak whose mass / charge ratio is 6829 (average value). It is a box diagram about the ion peak whose mass / charge ratio is 7011 (average value). It is a box diagram about the ion peak whose mass / charge ratio is 7924 (average value). It is a box diagram about the ion peak whose mass / charge ratio is 13678 (average value). It is a box diagram about the ion peak whose mass / charge ratio is 13881 (average value). It is a box diagram about the ion peak whose mass / charge ratio is 47095 (average value). It is a photograph which shows the result of SDS-PAGE performed in the refinement | purification process of a marker substance (A3). 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 (A3). It is a photograph which shows the result of the two-dimensional electrophoresis performed in the refinement | purification process of a marker substance (A4). 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 (A4). 2 is a photograph showing the results of Western blotting performed in Example 2. FIG. (A) is an ion peak diagram showing the result of subjecting the fraction 1 of the third group (TP) of Example 1 to SELDI-TOF-MS analysis, and (b) is the fraction 1 prepared in Example 2 by SELDI. -It is an ion peak diagram showing the result which used for the TOF-MS analysis. It is a photograph which shows the result of SDS-PAGE performed in the refinement | purification process of a marker substance (A7). 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 (A7).

Claims (16)

A concentration of at least one of the following marker substances (A1), (A2), (A3), (A4), (A5), and (A7) in a body fluid collected from an animal that is ingested and exercised with a test substance: By comparing with the reference value, the concentration of marker substances (A1) and (A2) is measured by the method using the mass / charge ratio as an index, and the improvement / maintenance / recovery effect of the test substance is evaluated. A method for evaluating the effect of improvement, maintenance and recovery of motor function .
(A1) 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 5280 when subjected to mass spectrometry;
(A2) 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 6830 when subjected to mass spectrometry;
(A3) 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 7010 when subjected to mass spectrometry;
(A4) 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 7920 when subjected to mass spectrometry;
(A5) 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 13700 when subjected to mass spectrometry;
(A7) 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 47100 when subjected to mass spectrometry,
further,
The marker substance (A3) comprises at least one amino acid sequence represented by SEQ ID NOs: 7 to 9,
The marker substance (A4) includes at least one amino acid sequence represented by SEQ ID NOs: 10 to 14,
The marker substance (A5) has reactivity with an antibody specific to transthyretin,
The marker substance (A7) includes at least one amino acid sequence represented by SEQ ID NOs: 15 to 21. Based on the concentration of at least one of the following marker substances (A1), (A2), (A3), (A4), (A5) and (A7) in a body fluid collected from an animal subjected to exercise load while ingesting the test substance The concentration of marker substances (A1) and (A2) is measured by the method using the mass / charge ratio as an index, and the improvement / maintenance / recovery effect on the motor function of the test substance is evaluated. This is a method for evaluating the improvement, maintenance, and recovery effects of motor function .
(A1) 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 5280 when subjected to mass spectrometry;
(A2) 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 6830 when subjected to mass spectrometry;
(A3) 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 7010 when subjected to mass spectrometry;
(A4) 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 7920 when subjected to mass spectrometry;
(A5) 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 13700 when subjected to mass spectrometry;
(A7) 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 47100 when subjected to mass spectrometry,
further,
The marker substance (A3) is apolipoprotein C1 or a modified form thereof,
The marker substance (A4) is apolipoprotein A2 or a modified form thereof,
The marker substance (A5) is transthyretin or a modified form thereof,
The marker substance (A7) is β2-glycoprotein 1 or a modified form thereof. By comparing at least one concentration belonging to any of the following marker substances (B1) to (B4) in a body fluid collected from an animal subjected to exercise load while ingesting the test substance, the test substance has A method for evaluating the improvement, maintenance and recovery effect of motor function, characterized by evaluating the improvement, maintenance and recovery effect of motor function.
(B1) Apolipoprotein C1 or a modified product thereof,
(B2) Apolipoprotein A2 or a modified product thereof,
(B3) transthyretin or a modified form thereof,
(B4) β2-glycoprotein 1 or a modified form thereof. The said reference value is following (I) and / or (II), The evaluation method of the improvement / maintenance / recovery effect of a motor function of any one of Claims 1-3 characterized by the above-mentioned.
(I) the concentration of the marker substance in a body fluid collected from an animal subjected to exercise without taking the test substance,
(II) The concentration of the marker substance in a body fluid collected from an animal ingested with the test substance without applying an exercise load.   The method for evaluating an improvement / maintenance / recovery effect of motor function according to claim 1, wherein the body fluid is blood.   The said test substance is a food material, The evaluation method of the improvement / maintenance / recovery effect of a motor function of any one of Claims 1-5 characterized by the above-mentioned.   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 the improvement / maintenance / recovery effect of motor function according to claim 1, wherein the concentration of the marker substance in a body fluid is calculated.   8. The improvement and maintenance of motor function according to claim 7, 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 of recovery effect.   9. The method for evaluating an improvement / maintenance / recovery effect on motor function according to claim 7 or 8, wherein the substance having affinity for the marker substance is an ion exchanger or an antibody.   The improvement / maintenance / recovery effect of the test substance is evaluated by the method for evaluating the improvement / maintenance / recovery effect of the motor function according to any one of claims 1 to 9, and desired based on the evaluation result A screening method for a substance, comprising screening a substance having an effect of improving, maintaining, and recovering the motor function of human.   A kit for use in the method for evaluating an improvement / maintenance / recovery effect of motor function according to any one of claims 1 to 9, comprising a carrier on which a substance having affinity for the marker substance is immobilized. A kit for evaluating the improvement, maintenance, and recovery effects of motor function.   12. The kit for evaluating the improvement / maintenance / recovery effect of motor function according to claim 11, wherein the substance having affinity for the marker substance is an ion exchanger.   13. The kit for evaluating an improvement / maintenance / recovery effect of motor function according to claim 11 or 12, which is used for screening a substance having an improvement / maintenance / recovery effect of motor function. Detection of at least one protein of the following (A1), (A2), (A3), (A4), (A5), and (A7) present in the animal body, but with marker substances (A1) and (A2) Is used as a marker that reflects fluctuations in motor function , using a method that uses the mass / charge ratio as an index .
(A1) 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 5280 when subjected to mass spectrometry;
(A2) 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 6830 when subjected to mass spectrometry;
(A3) 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 7010 when subjected to mass spectrometry;
(A4) 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 7920 when subjected to mass spectrometry;
(A5) 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 13700 when subjected to mass spectrometry;
(A7) 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 47100 when subjected to mass spectrometry,
further,
The marker substance (A3) comprises at least one amino acid sequence represented by SEQ ID NOs: 7 to 9,
The marker substance (A4) includes at least one amino acid sequence represented by SEQ ID NOs: 10 to 14,
The marker substance (A5) has reactivity with an antibody specific to transthyretin,
The marker substance (A7) includes at least one amino acid sequence represented by SEQ ID NOs: 15 to 21. Detection of at least one protein of the following (A1), (A2), (A3), (A4), (A5), and (A7) present in the animal body, but with marker substances (A1) and (A2) Is used as a marker that reflects fluctuations in motor function , using a method that uses the mass / charge ratio as an index .
(A1) 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 5280 when subjected to mass spectrometry;
(A2) 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 6830 when subjected to mass spectrometry;
(A3) 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 7010 when subjected to mass spectrometry;
(A4) 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 7920 when subjected to mass spectrometry;
(A5) 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 13700 when subjected to mass spectrometry;
(A7) 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 47100 when subjected to mass spectrometry,
further,
The marker substance (A3) is apolipoprotein C1 or a modified form thereof,
The marker substance (A4) is apolipoprotein A2 or a modified form thereof,
The marker substance (A5) is transthyretin or a modified form thereof,
The marker substance (A7) is β2-glycoprotein 1 or a modified form thereof. Use of at least one protein of the following (B1) to (B4) present in an animal body as a marker that reflects fluctuations in motor function.
(B1) Apolipoprotein C1 or a modified product thereof,
(B2) Apolipoprotein A2 or a modified product thereof,
(B3) transthyretin or a modified form thereof,
(B4) β2-glycoprotein 1 or a modified form thereof.