How to determine energy sufficiency

Abstract

A method for determining energy sufficiency simply and accurately is provided. [Solution] The concentrations of substances involved in the biosynthesis of glycerophospholipids, particularly phosphorylcholine, ethanolamine phosphate, or glycerol 3-phosphate, contained in the urine of a subject are measured, and the energy sufficiency rate is calculated from the measured values.

Description

[Technical field]

[0001] The present invention relates to a method for determining energy adequacy. [Background technology]

[0002] Energy sufficiency is one of the indicators of the nutritional state of a human or other subject. Energy sufficiency usually refers to the ratio of energy intake to energy consumption. Energy sufficiency is useful as an indicator to check, for example, whether athletes with high energy consumption are able to eat enough to compensate for their high energy consumption, whether elderly people who tend to eat less are able to eat a sufficient amount of food in proportion to their consumption, and whether patients who need to lose weight are taking in too much energy.

[0003] The energy sufficiency rate can usually be calculated by determining the energy consumption and energy intake during the same period. For example, in Non-Patent Document 1, meals for three consecutive days are recorded and the calorie intake per day is calculated. Next, the daily energy consumption is calculated from the energy metabolic rate obtained by measuring the energy metabolism during activity and the time-use survey during the same period. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Setsuko Inoue, Research Bulletin of Bunkyo University Women's Junior College, Vol. 25, pp. 29-31 (December 1, 1981) Summary of the Invention [Problem to be solved by the invention]

[0005] Conventionally, determining the energy sufficiency rate required recording meals for a certain period of time and measuring energy expenditure during the same period, which was a cumbersome procedure. In addition, it was not always easy to accurately measure energy intake and energy expenditure.

[0006] An object of the present invention is to provide a method for determining energy sufficiency simply and accurately. [Means for solving the problem]

[0007] As a result of intensive research, the present inventors have found that the concentrations of phosphorylcholine, ethanolamine phosphate, or glycerol 3-phosphate, which are substances involved in the biosynthetic pathway of glycerophospholipids and are contained in urine, are highly correlated with the energy sufficiency rate, and have completed the present invention. (1) A method for determining energy sufficiency, comprising measuring the concentration of at least one substance involved in the biosynthesis of glycerophospholipids in the urine of a subject. (2) The method according to (1), wherein the substance involved in the biosynthesis of glycerophospholipid is phosphorylcholine, ethanolamine phosphate, or glycerol 3-phosphate. (3) The method according to (2), wherein the substance involved in the biosynthesis of glycerophospholipids is phosphorylcholine. (4) The method according to (2), wherein the substance involved in the biosynthesis of glycerophospholipids is ethanolamine phosphate. (5) The method according to (2), wherein the substance involved in the biosynthesis of glycerophospholipids is glycerol 3-phosphate. (6) The method according to any one of (1) to (5), comprising using at least one of data on the subject's age, sex, physical activity level, height, weight, BMI, muscle mass, body fat percentage, visceral fat mass, and basal metabolic rate. (7) The method according to (6), comprising using age and BMI data of the subject. (8) A composition or kit for determining energy sufficiency, comprising a compound capable of measuring the concentration of at least one substance involved in the biosynthesis of glycerophospholipids contained in urine. (9) The composition or kit described in (8), wherein the substance involved in the biosynthesis of glycerophospholipid is phosphorylcholine, ethanolamine phosphate, or glycerol 3-phosphate. (10) A mail-in testing kit comprising a sealed container for holding a urine sample from a subject, the urine sample held in the sealed container being used to measure the concentration of at least one substance involved in the biosynthesis of glycerophospholipids in the subject's urine and to determine energy sufficiency. Effect of the Invention

[0008] According to the present invention, it is possible to provide a method for determining the energy sufficiency rate simply and accurately. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 is a schematic diagram showing a portion of the glycerophospholipid biosynthetic pathway. [Diagram 2] 2A is a plot of measured values ​​of the concentration of each component versus energy replenishing rate. FIG. 2B is a plot of measured values ​​of the concentration of ethanolamine phosphate versus energy replenishing rate. FIG. 2C is a plot of measured values ​​of the concentration of glycerol 3-phosphate versus energy replenishing rate. [Diagram 3] FIG. 1 is a plot diagram showing the correlation between estimated and measured energy sufficiency values ​​in Example 2(1). [Figure 4] FIG. 1 is a plot diagram showing the correlation between estimated and measured energy sufficiency values ​​in Example 3(1). [Diagram 5] FIG. 1 is a plot diagram showing the correlation between estimated and measured energy sufficiency values ​​in Example 4(1). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] In the present specification, "A to B" (A and B are numerical values) means "A or more and B or less" unless otherwise specified. In the present specification, "%" indicating concentration means weight % unless otherwise specified. In the present specification, the concentration of a urinary component is shown as a relative value of the measured concentration of the target component to the measured creatinine concentration in the same sample unless otherwise specified. Each compound includes its free form, electrolytes, salts (e.g., metal salts, sulfates, hydrochlorides, etc.), and solvates (e.g., hydrates) unless otherwise specified or inconsistent.

[0011] As used herein, the term "quantitative value" refers to a numerical value obtained directly from a sample as the concentration of a target substance in the sample or the signal intensity derived from the target substance by instrumental measurement or the like. As used herein, "measurement" refers to determining the magnitude of a target indicator, and the measurement result (measured value) may be obtained directly from the sample as a quantitative value (i.e., the term "measured value" includes "quantitative value"), or may be calculated based on the quantitative value obtained directly from the sample and / or the profile information of the subject. The measurement result may be expressed as a numerical value or may be expressed qualitatively.

[0012] 1. How to determine energy sufficiency A first embodiment of the present invention is a method for determining energy sufficiency, which is characterized by comprising measuring the concentration of at least one substance involved in the biosynthesis of glycerophospholipids, more specifically, phosphorylcholine, ethanolamine phosphate, or glycerol 3-phosphate, in urine of a subject.

[0013] In this specification, "energy sufficiency" refers to the energy intake relative to the amount of energy required by a subject (hereinafter also referred to as "energy requirement"). The "energy intake" here can be calculated, for example, from the subject's dietary record using standard calorie indications for foods such as the Ministry of Education, Culture, Sports, Science and Technology's "Standard Tables of Food Composition in Japan." The standard period for the energy sufficiency is not particularly limited, and may be one day, or any period from two days to one month. In order to use a standard that can reflect a more habitual energy intake state, it is preferable to use a period of multiple days, for example, three days, as the standard.

[0014] As used herein, "determining energy adequacy" refers to determining the degree of the subject's energy adequacy based on at least one of the substances involved in the biosynthesis of glycerophospholipids contained in the subject's urine and, if necessary, other indicators. The determined energy adequacy may be displayed as a numerical value (e.g., percentage (%)) or may be displayed qualitatively (e.g., but not limited to, "5 (very high), 4 (high), 3 (medium), 2 (low), 1 (very low)", etc.).

[0015] In this specification, the method of obtaining the "energy requirement" is not particularly limited as long as it is obtained as a value that reflects the energy consumption of the subject during a reference period. For example, the energy consumption that can be obtained by the subject wearing a wearable device for a predetermined period may be used as the energy requirement. As a wearable device, for example, a device that can calculate the actual energy consumption of the subject from the profile of the subject such as age, sex, height, weight, etc., and data such as heart rate and number of steps obtained through the device is commercially available, and this can be used. Alternatively, the energy requirement may be the energy consumption obtained by adding the energy consumption calculated based on the subject's exercise record to the basal metabolic rate of the subject. Alternatively, for example, the "estimated energy requirement" described in the Ministry of Health, Labor and Welfare's "Dietary Reference Intakes for Japanese (2020 Edition)" (December 2019, "Dietary Reference Intakes for Japanese" Development Review Committee) may be adopted as the energy requirement. The estimated energy requirement can be calculated using the following formula (I). Estimated energy requirement (kcal) = Basal metabolic rate (kcal) × α … (I) (α: Index calculated from physical activity level) The basal metabolic rate (kcal) can be calculated, for example, based on the subject's sex, age, height, and weight using the following formula (II) or (III) (Harris-Benedict equation). Male: 13.397 x weight (kg) + 4.799 x height (cm) - 5.677 x age + 88.362 ... (II) Female: 9.247 x weight (kg) + 3.098 x height (cm) - 4.33 x age + 447.593 ... (III) The subjects' physical activity levels are classified into levels I to III as follows. Level I (low): Most of your life is sedentary and involves static activities; Level II (normal): Work involves mainly sitting, but includes movement within the workplace, standing work, customer service, commuting, shopping, housework, light sports, etc.; Level III (high): People who work in jobs that require a lot of movement or standing, or who have active leisure activities such as sports. The above index α is set based on the subject's age and physical activity level as shown in Table 1.

[0016] [Table 1]

[0017] In the present specification, the above-mentioned "estimated energy requirement" for physical activity level II will be described as an example of "energy requirement" hereinafter, but this is not intended to limit the meaning of energy requirement in the present invention.

[0018] In this specification, the "energy sufficiency" is, for example, the ratio of the energy intake for three days calculated based on a three-day food record to the energy requirement for three days (here, the estimated energy requirement), but this is not intended to limit the meaning of the energy sufficiency in the present invention.

[0019] In the present specification, the term "subject" is not particularly limited as long as it is a human or animal that excretes urine, and includes, for example, humans, primates including chimpanzees, pet animals such as dogs and cats, livestock animals such as cows, pigs, horses, sheep and goats, rodents such as mice and rats, animals kept in zoos, etc. Preferably, the subject is a human.

[0020] In this specification, "urine" refers to a urine sample collected from a human or animal that is the subject of the determination of the energy sufficiency rate. The urine is not particularly limited, and may be any of early morning urine, random urine, and pooled urine, but early morning urine is particularly preferable. The "early morning urine" referred to here refers to urine collected on an empty stomach within 30 minutes after waking up. The urine sample may be liquid urine or filter paper urine soaked in filter paper. The urine sample may be fresh urine collected at a facility, or urine collected at home by a subject to check his or her health condition. In this case, the sample may be mailed from the subject's home. Whether the urine is fresh urine or a mailed sample, in order to prevent changes in the amount of components due to spoilage of the urine sample, the container (preferably a sealed container) or filter paper for transporting the urine may contain a preservative, antioxidant, etc. Examples of preservatives include toluene, xylene, hydrochloric acid, sodium formate, sodium borate, boric acid, ascorbic acid, sulfosalicylic acid, tartaric acid, metaphosphoric acid, acetic acid, citric acid, phenethyl alcohol, propylparaben, and butylene glycol. Examples of antioxidants include phenoxyethanol, isopropylmethylphenol, oxalic acid, and dibutylhydroxytoluene.

[0021] The method of the present embodiment includes measuring the concentration of at least one substance involved in the biosynthesis of glycerophospholipids in urine. In particular, the concentration of phosphorylcholine, ethanolamine phosphate, or glycerol 3-phosphate is measured. The present inventors found that the energy sufficiency rate and the measured values ​​of the concentrations of these substances in urine show a negative correlation (FIGS. 2A-C). In this specification, the measured values ​​of the concentrations of urinary components are shown as relative values ​​of the measured values ​​of the concentration of the target component to the measured values ​​of the creatinine concentration in the same sample, unless otherwise specified.

[0022] Phosphorylcholine has the structure shown in formula (IV) below, and exists mainly as the hydrophilic portion of phospholipids, which are the main components of cell membranes. [ka] Ethanolamine phosphate has a structure represented by formula (V): [ka] Glycerol 3-phosphate has a structure represented by the following formula (VI) and is a phosphate ester derived from phosphoric acid and glycerol, and is one of the components of glycerophospholipids. [ka]

[0023] Phosphorylcholine, ethanolamine phosphate, and glycerol 3-phosphate are all known to be substances involved in the biosynthetic pathway of glycerophospholipids. FIG. 1 shows a schematic diagram of the biosynthetic pathway of glycerophospholipids, particularly those related to phosphorylcholine, ethanolamine phosphate, and glycerol 3-phosphate. Glycerol 3-phosphate is used as a main component of glycerophospholipids, and is taken up into the endoplasmic reticulum in cells and converted to phosphatidic acid, which is then taken up into mitochondria and converted to glycerophospholipids, such as phosphatidylinositol, phosphatidylglycerol, and cardiolipin. Phosphatidic acid is converted to diacylglycerol in the endoplasmic reticulum, which is then converted to diacyl-type glycerophospholipids, such as phosphatidylethanolamine, phosphatidylcholine, and phosphalidylserine. Phosphatidylcholine in the endoplasmic reticulum is biosynthesized from choline, and phosphorylcholine exists as an intermediate substance. In addition, phosphatidylethanolamine in the endoplasmic reticulum is biosynthesized from ethanolamine, with ethanolamine phosphate existing as an intermediate substance.

[0024] The method for measuring the concentration of a target substance in urine may be any known method. For example, separation and quantification by a column, gas chromatography (GC), liquid chromatography (LC), high performance liquid chromatography (HPLC), or capillary electrophoresis (CE) may be included. In particular, LC, HPLC, and CE can perform quantification of each substance according to the peak area in addition to separation. Also, ionization and / or mass spectrometry such as APCI, CI, EI, ESI, FAB, FD, FI, LILBID, LSIMS, MALDI, PB, PD, SIMS, and TSP may be included. Also, the target substance may be detected using a molecule (e.g., an antibody) that specifically binds to the substance or a molecule (e.g., an enzyme) that specifically reacts with the substance. As a composition or kit containing such a molecule, a commercially available product may be used.

[0025] Below, the method of determining the energy sufficiency rate, which includes measuring the concentrations of phosphorylcholine, ethanolamine phosphate, and glycerol 3-phosphate, respectively, is described for each substance.

[0026] 1-1 Measurement of phosphorylcholine concentration 1-1-1 Energy sufficiency calculation formula using linear regression analysis The first aspect of this embodiment includes measuring the phosphorylcholine concentration in urine. As described in the Examples, a linear regression analysis was performed between the energy sufficiency rate (Y) and the measured value of the phosphorylcholine concentration in urine (X1) using 20 urine samples, and it was found that Y and X1 have a negative correlation (Y=-45358×X1+99.349) (FIG. 2A), and the absolute value of the correlation coefficient is 0.76, indicating a high correlation.

[0027] The relationship between the energy fulfillment rate (Y) and the measured value of the phosphorylcholine concentration (X1) can be expressed by the formula Y=aX1+f, and the energy fulfillment rate can be calculated by applying the actual measured value of the phosphorylcholine concentration to this X1. The values ​​of a and f vary depending on the measurement conditions of the phosphorylcholine concentration, so they must be determined in advance according to the measurement conditions. Specifically, for multiple samples with known energy fulfillment rates, the phosphorylcholine concentration is measured under the measurement conditions, and a correlation formula (Y=aX1+f) between the energy fulfillment rate (Y) and the measured value of the phosphorylcholine concentration (X1) is determined. This makes it possible to determine the constants a and f. The energy fulfillment rate can be easily calculated by applying the actual measured value of the phosphorylcholine concentration to X1 of the formula Y=aX1+f including the predetermined a and f.

[0028] 1-1-2 Energy sufficiency calculation formula using multiple regression analysis (1) In this embodiment, in addition to the measured value of the urinary phosphorylcholine concentration, at least one of the following data may be added as an index to calculate the energy sufficiency rate: the subject's age, sex, physical activity level, height, weight, BMI, muscle mass, body fat percentage, visceral fat mass, basal metabolic rate, etc. In particular, the subject's age and BMI may be added as an index.

[0029] As described in the Examples, a multiple regression analysis was performed using energy sufficiency (Y) as the dependent variable, the measured phosphorylcholine concentration (X1) as the explanatory variable, and age (X2) and BMI (X3) as covariates. As a result, Y could be expressed by the following formula. Y=(-40247.62597×X1)+(0.664881×X2)+(-0.947887×X3)+93.018149 The correlation coefficient between the energy adequacy rate calculated using the above formula and the actual energy adequacy rate (measured value) calculated from the meal records and energy expenditure was a high value of 0.85.

[0030] The relationship between the energy fulfillment rate (Y) and the measured value of phosphorylcholine concentration (X1), age (X2) and BMI (X3) is expressed by the formula Y=aX1+bX2+cX3+f. The values ​​of a, b, c and f vary depending on the measurement conditions of phosphorylcholine concentration, so they must be determined in advance according to the measurement conditions. Specifically, for a plurality of samples with known energy fulfillment rate, subject age and BMI, the phosphorylcholine concentration is measured under the measurement conditions, and the correlation formula (Y=aX1+bX2+cX3+f) between the energy fulfillment rate (Y) and the measured value of phosphorylcholine concentration (X1), age (X2) and BMI (X3) is determined. This allows the constants a, b, c and f to be determined. The energy fulfillment rate can be calculated by applying the actual measured value of phosphorylcholine concentration, age and BMI values ​​to the formula Y=aX1+bX2+cX3+f including the previously determined a, b, c and f. This makes it possible to calculate the energy sufficiency rate without needing to measure the concentrations of components other than phosphorylcholine; however, in this case, it is necessary to obtain patient information from the subject in addition to the urine sample.

[0031] 1-1-3 Energy sufficiency calculation formula using multiple regression analysis (2) In this embodiment, in addition to the measured value of the urinary phosphorylcholine concentration, the measured value of the concentration of one or more other components in the urine may be used as an index for calculating the energy sufficiency rate.

[0032] When performing multiple regression analysis with the energy fulfillment rate (Y) as the dependent variable, the measured value of phosphorylcholine concentration (X1) as the explanatory variable, and the measured values ​​of the concentrations of the other two components in urine, component A (X4) and component B (X5), as covariates, for example, the relational equation Y=aX1+dX4+eX5+f can be determined. The values ​​of a, d, e, and f vary depending on the measurement conditions of the concentrations of phosphorylcholine, component A, and component B, so they must be determined in advance according to the measurement conditions. Specifically, for multiple samples with known energy fulfillment rates, the concentrations of phosphorylcholine, component A, and component B are measured under the measurement conditions, and the correlation equation (Y=aX1+dX4+eX5+f) between the energy fulfillment rate (Y) and the measured value of the concentration of phosphorylcholine (X1), the measured value of the concentration of component A (X4), and the measured value of the concentration of component B (X5) is determined. This allows the constants a, d, e, and f to be determined. The energy sufficiency can be calculated by applying the actual measured values ​​of the concentrations of phosphorylcholine, component A, and component B to the equation Y=aX1+dX4+eX5+f, which includes predetermined a, d, e, and f.

[0033] Alternatively, in this embodiment, in addition to the measured value of the phosphorylcholine concentration in urine, at least one of the data of the profile of the subject such as age, sex, physical activity level, height, weight, BMI, muscle mass, body fat percentage, visceral fat mass, and basal metabolic rate, and the measured value of the concentration of one or more other components that have a correlation with the energy adequacy rate in urine may be added to the calculation of the energy adequacy rate.For example, the subject's age, BMI, and the measured value of the concentration of other components in urine can be added to the calculation.

[0034] 1-2 Measurement of ethanolamine phosphate concentration 1-2-1 Energy sufficiency calculation formula using linear regression analysis The second aspect of this embodiment includes measuring the ethanolamine phosphate concentration in urine. As described in the Examples, using 20 urine samples, it was found that the energy sufficiency rate (Y) and the measured value of the ethanolamine phosphate concentration in urine (X1) have a negative correlation (Y=-74383×X1+107.3) (FIG. 2B), and the absolute value of the correlation coefficient is 0.67, indicating a high correlation.

[0035] The relationship between the energy fulfillment rate (Y) and the measured value of the ethanolamine phosphate concentration (X1) can be expressed by the formula Y=aX1+f, and the energy fulfillment rate can be calculated by applying the actual measured value of the ethanolamine phosphate concentration to this X1. The values ​​of a and f vary depending on the measurement conditions of the ethanolamine phosphate concentration, so they must be determined in advance according to the measurement conditions. Specifically, for multiple samples with known energy fulfillment rates, the ethanolamine phosphate concentration is measured under the measurement conditions, and a correlation formula (Y=aX1+f) between the energy fulfillment rate (Y) and the measured value of the ethanolamine phosphate concentration (X1) is determined. This makes it possible to determine the constants a and f. The energy fulfillment rate can be easily calculated by applying the actual measured value of the ethanolamine phosphate concentration to X1 of the formula Y=aX1+f including the previously determined a and f.

[0036] 1-2-2 Energy sufficiency calculation formula using multiple regression analysis (1) In this embodiment, in addition to the measured value of the urinary ethanolamine phosphate concentration, at least one of the following data may be added as an index to calculate the energy sufficiency rate: the subject's age, sex, physical activity level, height, weight, BMI, muscle mass, body fat percentage, visceral fat mass, basal metabolic rate, etc. In particular, the subject's age and BMI may be added as indexes.

[0037] As described in the Examples, a multiple regression analysis was performed using the energy sufficiency rate (Y) as the dependent variable, the measured value of the ethanolamine phosphate concentration (X1) as the explanatory variable, and age (X2) and BMI (X3) as covariates. As a result, Y could be expressed by the following formula. Y=(-70096.79617×X1)+(0.833297×X2)+(-1.698907×X3)+111.47139 The correlation coefficient between the energy adequacy rate calculated using the above formula and the actual energy adequacy rate (measured value) calculated from the meal records and energy expenditure was high at 0.86.

[0038] The relationship between the energy fulfillment rate (Y) and the measured value of the ethanolamine phosphate concentration (X1), age (X2), and BMI (X3) is expressed by the formula Y=aX1+bX2+cX3+f. The values ​​of a, b, c, and f vary depending on the measurement conditions of the ethanolamine phosphate concentration, so they must be determined in advance according to the measurement conditions. Specifically, for a plurality of samples with known energy fulfillment rates, subject ages, and BMIs, the ethanolamine phosphate concentration is measured under the measurement conditions, and the correlation formula (Y=aX1+bX2+cX3+f) between the energy fulfillment rate (Y) and the measured value of the ethanolamine phosphate concentration (X1), age (X2), and BMI (X3) is determined. This makes it possible to determine the constants a, b, c, and f. The energy fulfillment rate can be calculated by applying the actual measured value of the ethanolamine phosphate concentration, age, and BMI values ​​to the formula Y=aX1+bX2+cX3+f, which includes the previously determined a, b, c, and f. This makes it possible to calculate the energy sufficiency rate without needing to measure the concentrations of components other than ethanolamine phosphate; however, in this case, it is necessary to obtain target profile information from the subject in addition to a urine sample.

[0039] 1-2-3 Energy sufficiency calculation formula using multiple regression analysis (2) In this embodiment, in addition to the measured value of the urinary ethanolamine phosphate concentration, the measured value of the concentration of one or more other components in the urine may be used as an index in calculating the energy sufficiency rate.

[0040] As described in the examples, when performing multiple regression analysis with the energy fulfillment rate (Y) as the dependent variable, the measured value of the ethanolamine phosphate concentration (X1) as the explanatory variable, and further adding the indexes of the measured values ​​of the concentrations of the other two components in urine, component A (X4) and component B (X5), as covariates, for example, the relational equation Y=aX1+dX4+eX5+f can be determined. The values ​​of a, d, e, and f vary depending on the measurement conditions of the concentrations of ethanolamine phosphate, component A, and component B, so they must be determined in advance according to the measurement conditions. Specifically, for multiple samples with known energy fulfillment rates, the concentrations of ethanolamine phosphate, component A, and component B are measured under the measurement conditions, and the correlation equation (Y=aX1+dX4+eX5+f) between the energy fulfillment rate (Y) and the measured value of the ethanolamine phosphate concentration (X1), the measured value of the concentration of component A (X4), and the measured value of the concentration of component B (X5) is determined. This allows the constants a, d, e, and f to be determined. The energy sufficiency can be calculated by applying the actual measured values ​​of the concentrations of ethanolamine phosphate, component A, and component B to the equation Y=aX1+dX4+eX5+f, which includes the previously determined a, d, e, and f.

[0041] Alternatively, in this embodiment, in addition to the measured value of the urinary ethanolamine phosphate concentration, at least one of the profile data of the subject, such as age, sex, physical activity level, height, weight, BMI, muscle mass, body fat percentage, visceral fat mass, and basal metabolic rate, and the measured value of the concentration of one or more other components in the urine may be added to the calculation of the energy sufficiency rate. For example, the subject's age, BMI, and the measured value of the concentration of other components in the urine can be added to the calculation.

[0042] 1-3 Measurement of glycerol 3-phosphate concentration 1-3-1 Energy sufficiency calculation formula using linear regression analysis The third aspect of this embodiment includes measuring the concentration of glycerol 3-phosphate in urine. As described in the Examples, a linear regression analysis was performed between the energy sufficiency rate (Y) and the measured concentration of glycerol 3-phosphate in urine (X1) using 20 urine samples, and it was found that Y and X1 have a negative correlation (Y = -34541 x X1 + 103.02) (Figure 2C), and the absolute value of the correlation coefficient was 0.64, indicating a high correlation.

[0043] The relationship between the energy fulfillment rate (Y) and the measured value of the glycerol 3-phosphate concentration (X1) can be expressed by the formula Y=aX1+f, and the energy fulfillment rate can be calculated by applying the actual measured value of the glycerol 3-phosphate concentration to this X1. The values ​​of a and f vary depending on the measurement conditions of the glycerol 3-phosphate concentration, so they must be determined in advance according to the measurement conditions. Specifically, for multiple samples with known energy fulfillment rates, the glycerol 3-phosphate concentration is measured under the measurement conditions, and a correlation equation (Y=aX1+f) between the energy fulfillment rate (Y) and the measured value of the glycerol 3-phosphate concentration (X1) is determined. This makes it possible to determine the constants a and f. The energy fulfillment rate can be easily calculated by applying the actual measured value of the glycerol 3-phosphate concentration to X1 of the formula Y=aX1+f including the previously determined a and f.

[0044] 1-3-2 Energy sufficiency calculation formula using multiple regression analysis (1) In this embodiment, in addition to the measured urinary glycerol 3-phosphate concentration, at least one of the following data may be added as an index to calculate the energy sufficiency: age, sex, physical activity level, height, weight, BMI, muscle mass, body fat percentage, visceral fat mass, basal metabolic rate, etc. of the subject. In particular, the age and BMI of the subject may be added as an index.

[0045] As described in the Examples, a multiple regression analysis was performed using energy sufficiency (Y) as the dependent variable, the measured value of glycerol 3-phosphate concentration (X1) as the explanatory variable, and age (X2) and BMI (X3) as covariates. As a result, Y could be expressed by the following formula. Y=(-32371.292292×X1)+(0.836491×X2)+(-1.474309×X3)+102.414497 The correlation coefficient between the energy adequacy rate calculated using the above formula and the actual energy adequacy rate (measured value) calculated from the meal records and energy expenditure was a high value of 0.82.

[0046] The relationship between the energy fulfillment rate (Y) and the measured value of the glycerol 3-phosphate concentration (X1), age (X2), and BMI (X3) is expressed by the formula Y=aX1+bX2+cX3+f. The values ​​of a, b, c, and f vary depending on the measurement conditions of the glycerol 3-phosphate concentration, so they must be determined in advance according to the measurement conditions. Specifically, for multiple samples with known energy fulfillment rates, subject ages, and BMIs, the glycerol 3-phosphate concentration is measured under the measurement conditions, and the correlation formula (Y=aX1+bX2+cX3+f) between the energy fulfillment rate (Y) and the measured value of the glycerol 3-phosphate concentration (X1), age (X2), and BMI (X3) is determined. This allows the constants a, b, c, and f to be determined. The energy adequacy ratio can be calculated by applying the actual measured value of the glycerol 3-phosphate concentration to the formula Y=aX1+bX2+cX3+f, which includes predetermined a, b, c, and f. This makes it possible to calculate the energy adequacy ratio without the need to measure the concentrations of components other than glycerol 3-phosphate, but in this case, it is necessary to obtain profile information of the subject in addition to a urine sample.

[0047] 1-3-3 Energy sufficiency calculation formula using multiple regression analysis (2) In this embodiment, in addition to the measured value of the urinary glycerol 3-phosphate concentration, the measured value of the concentration of one or more other components in the urine may be used as an index for calculating the energy sufficiency rate.

[0048] As described in the examples, when performing multiple regression analysis with the energy fulfillment rate (Y) as the dependent variable, the measured value of glycerol 3-phosphate concentration (X1) as the explanatory variable, and further adding the indexes of the measured values ​​of the concentrations of the other two components in urine, component A (X4) and component B (X5), as covariates, the relational equation Y=aX1+dX4+eX5+f can be determined. The values ​​of a, d, e, and f vary depending on the measurement conditions of the concentrations of glycerol 3-phosphate, component A, and component B, and therefore must be determined in advance according to the measurement conditions. Specifically, for multiple samples with known energy fulfillment rates, the concentrations of glycerol 3-phosphate, component A, and component B are measured under the measurement conditions, and the correlation equation (Y=aX1+dX4+eX5+f) between the energy fulfillment rate (Y) and the measured value of the glycerol 3-phosphate concentration (X1), the measured value of the concentration of component A (X4), and the measured value of the concentration of component B (X5) is determined. This allows the constants a, d, e, and f to be determined. The energy adequacy rate can be calculated by applying the actual measured values ​​of the concentrations of glycerol 3-phosphate, component A, and component B to the equation Y=aX1+dX4+eX5+f, which includes the previously determined a, d, e, and f. This demonstrated that the energy adequacy rate can be calculated more accurately using only information from a urine sample.

[0049] Alternatively, in this embodiment, in addition to the measured value of the urinary glycerol 3-phosphate concentration, at least one of the profile data of the subject such as age, sex, physical activity level, height, weight, BMI, muscle mass, body fat percentage, visceral fat mass, and basal metabolic rate, and the measured value of the concentration of one or more other components in the urine may be added to the calculation of the energy sufficiency rate. For example, the subject's age, BMI, and the measured values ​​of the concentrations of other components in the urine can be added to the calculation.

[0050] 2. Composition or kit for determining energy sufficiency The second embodiment of the present invention is a composition or kit for determining energy sufficiency. The composition or kit of this embodiment is characterized in that it contains at least one of the substances involved in the biosynthesis of glycerophospholipids contained in urine, in particular a compound capable of measuring the concentration of phosphorylcholine, ethanolamine phosphate, or glycerol 3-phosphate in urine. More specifically, the composition or kit of this embodiment is a composition or kit for use in the method described in Section "1. Method for determining energy sufficiency". Unless otherwise specified, and unless otherwise contradictory, the subject, sample, conditions, etc. for which the composition or kit of this embodiment is used are all as described in Section "1. Method for determining energy sufficiency".

[0051] In this specification, a "composition" refers to one consisting of one component (including a container), and a "kit" refers to one consisting of two or more components. The composition and kit of this embodiment are used without any particular distinction other than the number of components, so long as they are used to determine the energy sufficiency rate.

[0052] 2-1 Compounds for measuring phosphorylcholine concentration The first aspect of this embodiment includes a compound capable of measuring phosphorylcholine concentration. Examples of compounds capable of measuring phosphorylcholine concentration include compounds for subjecting a sample to CE or HPLC. In this case, the compound may also enable measurement of the concentration of other substances. Alternatively, an antibody or an antigen-binding fragment thereof that specifically binds to phosphorylcholine may be used.

[0053] 2-2 Compounds for measuring ethanolamine phosphate concentration The second aspect of this embodiment includes a compound capable of measuring the concentration of ethanolamine phosphate. Examples of the compound capable of measuring the concentration of ethanolamine phosphate include compounds for subjecting a sample to CE or HPLC. In this case, the compound may also enable the measurement of the concentration of other substances. Alternatively, examples include enzymes that react with ethanolamine phosphate, such as acetaldehyde dehydrogenase (see International Publication No. 2013 / 069645).

[0054] 2-3 Compounds for measuring glycerol 3-phosphate concentration The third aspect of this embodiment includes a compound capable of measuring the concentration of glycerol 3-phosphate. Examples of the compound capable of measuring the concentration of glycerol 3-phosphate include compounds for subjecting a sample to CE or HPLC. In this case, the compound may also enable the measurement of the concentration of other substances. Alternatively, examples of the compound include enzymes capable of reacting with glycerol 3-phosphate, such as glycerol 3-phosphate dehydrogenase (GPDH).

[0055] 2-4 Other The composition or kit of this embodiment may further include a compound capable of measuring the concentration of one or more other components in urine in addition to the above compounds. And / or the composition or kit of this embodiment may further include a compound capable of measuring the creatinine concentration in urine in addition to the above compounds. Compounds capable of measuring the creatinine concentration include compounds for subjecting a sample to CE or HPLC, as well as known compounds for measuring creatinine concentration, such as creatinase / creatininase.

[0056] The kit may further comprise a sealed container for storing a urine sample, a filter for purifying the urine sample (e.g., an ultrafiltration filter), a composition for pretreatment of a urine sample, and instructions explaining the measurement and analysis procedures.

[0057] 3. Mail-in test kits A third embodiment of the present invention is a mail-in test kit. The mail-in test kit of this embodiment is characterized in that it includes a sealed container for storing a urine sample of a subject, and the urine sample stored in the sealed container is used to measure the concentration of at least one substance involved in the biosynthesis of glycerophospholipids contained in the urine of the subject and to determine the energy sufficiency rate. The mail-in test kit is used by the subject (or his / her caregiver, etc.) who is the subject of the energy sufficiency rate determination to collect the subject's urine sample and mail the sample to a testing institution that can measure the concentrations of urinary components.

[0058] The mail-in test kit of this embodiment includes at least a sealed container for storing a urine sample. The urine sample referred to here may be liquid urine or filter paper urine soaked in filter paper. In the case of liquid urine, the sealed container may be, for example, a sealed tube with a screw cap. In the case of filter paper urine, the sealed container may be, for example, a sealed bag for sealing.

[0059] In addition to the above, the mail-in test kit of this embodiment may include a urine collection cup, instructions for the subject, a form for entering a profile (age, sex, height, weight, physical activity level, etc.), a return envelope, etc. When instructions for the subject are included, for example, the instructions may include the address of a web page (e.g., a QR code (registered trademark)) for entering a profile and / or for checking the results.

[0060] The urine sample mailed to the testing facility using the mail-in test kit is used to measure the concentration of at least one substance involved in the biosynthesis of glycerophospholipids contained in the urine, thereby determining the subject's energy sufficiency. The method for determining the energy sufficiency may be the method described in the section "1. Method for determining the energy sufficiency". EXAMPLES

[0061] The present invention will be described in more detail below by way of examples, but the present invention is not limited to these examples.

[0062] Example 1: Search for energy sufficiency markers (1) Subjects and samples Twenty volunteers were selected as subjects and asked to record and submit the names of dishes, food names, and amounts consumed over a three-day period, as well as photos of the dishes. At the same time, subjects were asked to self-collect and submit approximately 10 mL of early morning urine on the third day. Early morning urine was urine collected within 30 minutes of waking up while fasting. Age, height, and weight data were also obtained from the subjects.

[0063] (2) Calculation of energy intake Based on the submitted three-day food records, a registered dietitian performed nutritional calculations using the Ministry of Education, Culture, Sports, Science and Technology's "Standard Tables of Food Composition in Japan (2020 edition)" to calculate the calorie intake for the three days, which was used as the energy intake.

[0064] (3) Calculation of energy requirement and energy sufficiency rate (actual value) The basal metabolic rate was calculated from the sex, age, height, and weight of the subject. The estimated energy requirement was calculated using the following formula (I), and this was used as the energy requirement. Estimated energy requirement (kcal) = Basal metabolic rate (kcal) × α … (I) (α: Index calculated from physical activity level) The index α was calculated based on the criteria shown in Table 1, with the physical activity level of all subjects considered to be II. The energy intake calculated in (2) was divided by the energy requirement to calculate the energy sufficiency rate (actual value).

[0065] (4) Pretreatment of urine samples The submitted urine samples were mixed with 20 μL of an aqueous solution containing an internal standard at a concentration of 100 μM and 60 μL of ion-exchanged water, and then transferred to an ultrafiltration tube (Ultrafree MC PLHCC, centrifugal filter unit 5 kDa). The tube was centrifuged (9100 × g, 4 ° C, 60 minutes), subjected to ultrafiltration, and then subjected to measurement.

[0066] (5) Metabolomic analysis The metabolites contained in the pretreated samples were measured using a capillary electrophoresis-Fourier transform mass spectrometer (CE-FTMS) in cation and anion modes under the following conditions: The samples were diluted with ion-exchanged water 20 times in cation mode and 10 times in anion mode, and then subjected to CE-FTMS. (Cation mode: cationic metabolite analysis) CE: Agilent CE system (manufactured by Agilent) MS: Q Exactive™ Plus (ThermoFisher) Capillary: Fused silica capillary 50μm×80cm CE voltage: Positive 30kV MS Ionization: ESI Cationization MS capillary voltage: 4000V MS scan range: m / z 60-900 (Anion mode: anionic metabolite analysis) CE: Agilent CE system (manufactured by Agilent) MS: Q Exactive™ Plus (ThermoFisher) Capillary: Fused silica capillary 50μmx80cm CE voltage: Positive 30kV MS ionization: ESI anionization MS capillary voltage: 3500V MS scan range: m / z 70-1050

[0067] Peaks with a signal / noise (S / N) ratio of 3 or more were automatically extracted from the peaks detected in the cation mode or anion mode of the CE-FTMS using the automatic integration software MasterHands ver. 2.19.0.2 (developed by Keio University), and the mass-to-charge ratio, peak area value, and migration time were obtained. The peak area value of each extracted peak was corrected to a relative area value using the peak area value of creatinine as shown in the following formulas (VII) and (VIII). Since creatinine is only directly detected in the cation mode, the relative area value of creatinine corrected with the internal standard substance in the cation mode was used for the correction of the anion mode.

number

number

[0068] Based on the migration time and m / z value of the detected peak, the corresponding substance was matched and searched among all metabolites registered in the library in advance. The allowable error for the test was ±5 minutes for migration time and ±5 ppm for m / z.

[0069] Correlations were calculated between the urinary concentrations of each of the searched target substances and the energy metabolic rate, and a high correlation was found for the three compounds shown in Table 2. Figure 2 shows a correlation diagram plotting the measured concentrations of phosphorylcholine (Figure 2A), ethanolamine phosphate (Figure 2B), and glycerol 3-phosphate (Figure 2C) and the energy metabolic rate.

[0070] [Table 2]

[0071] <Example 2: Correlation with energy sufficiency using phosphorylcholine as an explanatory variable> (1) Multiple regression analysis of energy sufficiency using phosphorylcholine, age and BMI A multiple regression analysis was performed with the phosphorylcholine concentration measurement as the explanatory variable, BMI and age as the covariates, and the energy sufficiency rate (average for 3 days) as the dependent variable. When the energy sufficiency rate is Y, the phosphorylcholine concentration measurement is X1, age is X2, and BMI is X3, it was shown that the energy sufficiency rate Y can be calculated using the following formula. Y=(-40247.62597×X1)+(0.664881×X2)+(-0.947887×X3)+93.018149

[0072] As a result of the multiple regression analysis, the coefficient of determination after adjusting for the degrees of freedom was 0.67, the correlation coefficient was 0.85, and the coefficient of variation was 5.9%. Figure 3 shows the correlation between the estimated energy sufficiency calculated from the above formula and the actual measured energy sufficiency. It was shown that the energy sufficiency can be calculated more accurately by adding age and BMI as indicators compared to when the measured phosphorylcholine concentration is used as the sole indicator.

[0073] (2) Correlation between phosphorylcholine concentration and carbohydrate, lipid, and protein intake The subjects' carbohydrate, lipid, and protein intakes for three days were calculated from their food records. The required carbohydrate intake was calculated as energy consumption (kcal) x 60% x 1 / 4 (g), the required lipid intake was calculated as energy consumption (kcal) x 25% x 1 / 9 (g), and the required protein intake was calculated as the protein recommended amount in the Dietary Reference Intakes. The sufficiency rate of carbohydrate, lipid, and protein was calculated by dividing the actual intake by the required intake.

[0074] The correlation coefficients between the measured phosphorylcholine concentration and the sufficiency of carbohydrates, lipids, and proteins were calculated to be 0.24, -0.44, and -0.43, respectively. This indicates that the measured phosphorylcholine concentration has a low correlation with each of carbohydrates, lipids, and proteins. This suggests that the measured phosphorylcholine concentration in urine is useful as a specific marker for energy sufficiency.

[0075] <Example 3: Correlation with energy sufficiency using ethanolamine phosphate as an explanatory variable> (1) Multiple regression analysis of energy sufficiency using ethanolamine phosphate, age and BMI A multiple regression analysis was performed with the measured ethanolamine phosphate concentration as the explanatory variable, BMI and age as the covariates, and the dependent variable as the energy sufficiency rate (average for 3 days).When the energy sufficiency rate is Y, the measured ethanolamine phosphate concentration is X1, age is X2, and BMI is X3, it was shown that the energy sufficiency rate Y can be calculated using the following formula. Y=(-70096.79617×X1)+(0.833297×X2)+(-1.698907×X3)+111.47139

[0076] As a result of the multiple regression analysis, the coefficient of determination after adjusting for the degrees of freedom was 0.69, the correlation coefficient was 0.86, and the coefficient of variation was 5.9%. Figure 4 shows the correlation between the estimated value of the energy sufficiency calculated from the above formula and the actual measured value of the energy sufficiency. It was shown that the energy sufficiency can be calculated more accurately by adding age and BMI as indicators compared to when the measured value of ethanolamine phosphate concentration is used as the sole indicator.

[0077] <Example 4: Correlation with energy sufficiency using glycerol 3-phosphate concentration as an explanatory variable> (1) Multiple regression analysis of energy sufficiency using glycerol 3-phosphate concentration, age, and BMI A multiple regression analysis was performed with the measured glycerol 3-phosphate concentration as the explanatory variable, BMI and age as the covariates, and the dependent variable as the energy sufficiency rate (average over 3 days).When the energy sufficiency rate is Y, the measured glycerol 3-phosphate concentration is X1, age is X2, and BMI is X3, it was shown that the energy sufficiency rate Y can be calculated using the following formula. Y=(-32371.292292×X1)+(0.836491×X2)+(-1.474309×X3)+102.414497

[0078] As a result of the multiple regression analysis, the coefficient of determination after adjusting for the degrees of freedom was 0.62, the correlation coefficient was 0.82, and the coefficient of variation was 5.8%. Figure 5 shows the correlation between the estimated value of energy adequacy calculated from the above formula and the actual measured value of energy adequacy. It was shown that the energy adequacy can be calculated more accurately by adding age and BMI as indicators compared to when the measured value of glycerol 3-phosphate concentration is used as the sole indicator.

Claims

1. measuring the concentration of at least one substance involved in the biosynthesis of glycerophospholipids in urine of a subject; The substance involved in the biosynthesis of glycerophospholipid is phosphorylcholine, ethanolamine phosphate, or glycerol 3-phosphate. How to determine energy sufficiency.

2. The method according to claim 1 , wherein the substance involved in the biosynthesis of glycerophospholipids is phosphorylcholine.

3. The method of claim 1 , wherein the substance involved in the biosynthesis of glycerophospholipids is ethanolamine phosphate.

4. The method according to claim 1, wherein the substance involved in the biosynthesis of glycerophospholipid is glycerol 3-phosphate.

5. The method of claim 1 , comprising using at least one of the following data: age, sex, physical activity level, height, weight, BMI, muscle mass, body fat percentage, visceral fat mass, and basal metabolic rate of the subject.

6. The method of claim 5, comprising using age and BMI data of the subject.

7. The present invention includes a compound capable of measuring the concentration of at least one substance involved in the biosynthesis of glycerophospholipids contained in urine, The composition or kit for determining energy sufficiency, wherein the substance involved in the biosynthesis of glycerophospholipid is phosphorylcholine, ethanolamine phosphate or glycerol 3-phosphate.

8. a sealed container for storing a urine sample from the subject, the urine sample stored in the sealed container being used to measure a concentration of at least one substance involved in the biosynthesis of glycerophospholipids contained in the urine of the subject and to determine an energy sufficiency rate; The substance involved in the biosynthesis of glycerophospholipid is phosphorylcholine, ethanolamine phosphate, or glycerol 3-phosphate. Mail-in test kit.

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