Method for determining energy sufficiency rate

Abstract

To provide a method for simply and accurately determining an energy sufficiency rate.SOLUTION: The method comprises: measuring a concentration of a substance involved in biosynthesis of glycerophospholipids, particularly phosphorylcholine, ethanolamine phosphate, or glycerol 3-phosphate, in urine of a subject; and calculating an energy sufficiency rate from the measured value.SELECTED DRAWING: None

Description

[Technical Field]

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

[0002] Energy adequacy is one of the indicators of the nutritional status of subjects such as humans. Energy adequacy usually refers to the ratio of energy intake to energy expenditure. Energy adequacy is useful as an indicator to confirm, for example, whether athletes with high energy expenditure are consuming enough food to compensate for their high energy consumption, whether elderly people who tend to eat less are eating a sufficient amount of food commensurate with their consumption, and whether patients who need to lose weight are consuming excessive energy.

[0003] The energy sufficiency rate can usually be calculated by determining the energy expenditure and energy intake during the same period. For example, in Non-Patent Document 1, meals are recorded for three consecutive days and the calorie intake per day is calculated. Next, the daily energy expenditure 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] Conventional methods for determining energy sufficiency require recording dietary intake over a certain period and measuring energy expenditure over the same period, which is a cumbersome process. Furthermore, accurate measurement of energy intake and expenditure is not always easy.

[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 extensive research, the present inventors have found that the concentrations of phosphorylcholine, ethanolamine phosphate, or glycerol 3-phosphate, which are substances involved in the glycerophospholipid biosynthesis pathway and are contained in urine, are highly correlated with energy sufficiency, 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 glycerophospholipids 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) A method according to any one of (1) to (5), which comprises 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), which includes using data on the subject's age and BMI. (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 according to (8), wherein the substance involved in the biosynthesis of glycerophospholipids is phosphorylcholine, ethanolamine phosphate, or glycerol 3-phosphate. (10) A mail-in test 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 urine of the subject and to determine the energy sufficiency rate. [Effects of the Invention]

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

[0009] [Figure 1] FIG. 1 is a schematic diagram showing a portion of the glycerophospholipid biosynthetic pathway. [Figure 2] The graphs are plots of measured concentrations of each component versus energy replenishing ratio. Figure 2A is a plot of measured phosphorylcholine concentrations versus energy replenishing ratio. Figure 2B is a plot of measured ethanolamine phosphate concentrations versus energy replenishing ratio. Figure 2C is a plot of measured glycerol 3-phosphate concentrations versus energy replenishing ratio. [Figure 3] FIG. 10 is a plot diagram showing the correlation between estimated and measured values ​​of energy sufficiency in Example 2(1). [Figure 4] FIG. 10 is a plot diagram showing the correlation between estimated and measured values ​​of energy sufficiency in Example 3(1). [Figure 5] FIG. 10 is a plot diagram showing the correlation between estimated and measured values ​​of energy sufficiency in Example 4(1). DETAILED DESCRIPTION OF THE INVENTION

[0010] In this specification, "A to B" (A and B are numerical values) means "greater than or equal to A and less than or equal to B" unless otherwise specified. In this specification, "%" indicating concentration refers to weight % unless otherwise specified. In this 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 unless otherwise contradicted.

[0011] As used herein, "quantitative value" refers to a numerical value obtained directly from a sample by instrumental measurement or the like, as the concentration of a target substance in the sample or the signal intensity derived from the target substance. 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" encompasses "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 adequacy, which comprises measuring the concentration of at least one substance involved in glycerophospholipid biosynthesis, more specifically, phosphorylcholine, ethanolamine phosphate, or glycerol 3-phosphate, in the urine of a subject.

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

[0014] As used herein, "determining energy adequacy" refers to determining the level of a subject's energy adequacy based on at least one substance involved in glycerophospholipid biosynthesis contained in the subject's urine, and optionally other indicators. The determined energy adequacy may be expressed numerically (e.g., as a percentage (%)) or qualitatively (e.g., but not limited to, "5 (very high), 4 (high), 3 (moderate), 2 (low), 1 (very low)").

[0015] As used herein, the term "energy requirement" refers to a value that reflects the subject's energy expenditure during a reference period, and the method for obtaining this value is not particularly limited. For example, the energy requirement may be determined by the subject wearing a wearable device for a predetermined period. Commercially available wearable devices can calculate the subject's actual energy expenditure based on the subject's profile, such as age, gender, height, and weight, as well as data such as heart rate and step count obtained through the device. These devices can be used. Alternatively, the energy requirement may be determined by adding the subject's basal metabolic rate to the energy expenditure calculated based on the subject's exercise record. Alternatively, the "estimated energy requirement" described in the Ministry of Health, Labor, and Welfare's "Dietary Reference Intakes for Japanese (2020 Edition)" (December 2019, Committee for the Development of the "Dietary Reference Intakes for Japanese") may be used 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, using the following formula (II) or (III) (Harris-Benedict equation) based on the sex, age, height, and weight of the subject. Male: 13.397 × weight (kg) + 4.799 × height (cm) - 5.677 × age + 88.362 … (II) Women: 9.247 x weight (kg) + 3.098 x height (cm) - 4.33 x age + 447.593 ... (III) The subject's physical activity level is classified into the following levels I to III. Level I (low): Most of your life is sedentary and involves static activities; Level II (normal): Work involves mainly sitting, but also includes movement within the workplace, standing, working with customers, 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 index α is set as shown in Table 1 based on the subject's age and physical activity level.

[0016] [Table 1]

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

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

[0019] As used herein, 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, it is a human.

[0020] As used herein, "urine" refers to a urine sample collected from a human or animal whose energy sufficiency is to be determined. Urine is not particularly limited, and may be any of early morning urine, random urine, and pooled urine. However, early morning urine is particularly preferred. "Early morning urine" here refers to urine collected on an empty stomach within 30 minutes of waking up. The urine sample may be liquid urine or filter paper urine. The urine sample may be fresh urine collected at a facility, or urine collected by a subject at home to check their health status. In this case, the sample may be mailed from the subject's home. Whether the urine is fresh or mailed, the container (preferably a sealed container) or filter paper used to transport the urine may contain a preservative, antioxidant, or the like to prevent changes in the amount of components due to spoilage or the like of the urine sample. 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 this embodiment involves 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 inventors have found that the measured values ​​of the concentrations of these substances in urine show a negative correlation with energy sufficiency (Figures 2A-C). In this specification, unless otherwise specified, the measured values ​​of the concentrations of urinary components are expressed as relative values ​​of the measured concentration of the target component to the measured creatinine concentration in the same sample.

[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 the following formula (V): [ka] Glycerol 3-phosphate has a structure represented by the following formula (VI), 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 involved in the biosynthetic pathway of glycerophospholipids. Figure 1 shows a schematic diagram of the glycerophospholipid biosynthetic pathway, particularly the portion related to phosphorylcholine, ethanolamine phosphate, and glycerol 3-phosphate. Glycerol 3-phosphate is used as a major component of glycerophospholipids. It is then imported into the endoplasmic reticulum and converted to phosphatidic acid, which is then imported into mitochondria and converted to the glycerophospholipids phosphatidylinositol, phosphatidylglycerol, and cardiolipin. Phosphatidic acid is converted to diacylglycerol in the endoplasmic reticulum and then converted to the diacylglycerophospholipids phosphatidylethanolamine, phosphatidylcholine, and phosphalidylserine. Phosphatidylcholine in the endoplasmic reticulum is biosynthesized from choline, with phosphorylcholine present as an intermediate. Phosphatidylethanolamine in the endoplasmic reticulum is biosynthesized from ethanolamine, with ethanolamine phosphate present as an intermediate.

[0024] The concentration of a target substance in urine may be measured using any known method. For example, this may include separation and quantification using a column, gas chromatography (GC), liquid chromatography (LC), high-performance liquid chromatography (HPLC), or capillary electrophoresis (CE). In particular, LC, HPLC, and CE can perform separation and quantification of each substance based on its peak area. Ionization and / or mass spectrometry, such as APCI, CI, EI, ESI, FAB, FD, FI, LILBID, LSIMS, MALDI, PB, PD, SIMS, and TSP, may also be used. Furthermore, the target substance may be detected using a molecule that specifically binds to the substance (e.g., an antibody) or a molecule that specifically reacts with the substance (e.g., an enzyme). A commercially available composition or kit containing such molecules may also be used.

[0025] Below, methods for determining energy sufficiency, including measuring the concentrations of phosphorylcholine, ethanolamine phosphate, and glycerol 3-phosphate, are described for each substance.

[0026] 1-1 Measurement of phosphorylcholine concentration 1-1-1 Energy sufficiency calculation formula using linear regression analysis A first aspect of this embodiment involves measuring urinary phosphorylcholine concentration. As described in the Examples, linear regression analysis was performed using 20 urine samples to measure energy adequacy (Y) and urinary phosphorylcholine concentration (X1). It was found that Y and X1 have a negative correlation (Y = -45358 × X1 + 99.349) (Figure 2A), with an absolute value of the correlation coefficient of 0.76, indicating a high correlation.

[0027] The relationship between the energy repletion rate (Y) and the measured value of phosphorylcholine concentration (X1) can be expressed by the formula Y = aX1 + f, and the energy repletion rate can be calculated by applying the actual measured value of phosphorylcholine concentration to this X1. The values ​​of a and f vary depending on the measurement conditions of phosphorylcholine concentration, so they must be determined in advance according to the measurement conditions. Specifically, the phosphorylcholine concentration of multiple samples with known energy repletion rates is measured under the measurement conditions, and the correlation equation (Y = aX1 + f) between the energy repletion rate (Y) and the measured value of phosphorylcholine concentration (X1) is determined. This makes it possible to determine the constants a and f. The energy repletion rate can be easily calculated by applying the actual measured value of phosphorylcholine concentration to X1 in the formula Y = aX1 + f, which includes 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 urinary phosphorylcholine concentration, at least one data item selected from the subject's profile such as age, sex, physical activity level, height, weight, BMI, muscle mass, body fat percentage, visceral fat mass, and basal metabolic rate may be used as an index for calculating the energy sufficiency rate. In particular, the subject's age and BMI may be used as indexes.

[0029] As described in the Examples, a multiple regression analysis was performed using energy sufficiency (Y) as the dependent variable, the measured value of 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 sufficiency rate calculated using the above formula and the actual energy sufficiency rate (measured value) calculated from the dietary records and energy expenditure was a high value of 0.85.

[0030] The relationship between energy sufficiency (Y) and the measured 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 for phosphorylcholine concentration, and therefore must be determined in advance according to the measurement conditions. Specifically, phosphorylcholine concentrations are measured under the measurement conditions for multiple samples with known energy sufficiency, subject age, and BMI, and the correlation equation (Y = aX1 + bX2 + cX3 + f) between energy sufficiency (Y) and the measured phosphorylcholine concentration (X1), age (X2), and BMI (X3) is calculated. This allows the constants a, b, c, and f to be determined. Energy sufficiency can be calculated by applying the actual measured phosphorylcholine concentration, age, and BMI values ​​to the formula Y = aX1 + bX2 + cX3 + f, which includes the predetermined a, b, c, and f. This makes it possible to calculate the energy sufficiency rate without measuring the concentrations of components other than phosphorylcholine, but 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 to calculate the energy sufficiency rate.

[0032] When performing multiple regression analysis using energy repletion rate (Y) as the dependent variable, the measured phosphorylcholine concentration (X1) as the explanatory variable, and the measured concentrations of two other urinary components, component A (X4) and component B (X5), as covariates, the following relationship can be determined: Y = aX1 + dX4 + eX5 + f. The values ​​of a, d, e, and f vary depending on the measurement conditions for the concentrations of phosphorylcholine, component A, and component B, and therefore must be determined in advance according to the measurement conditions. Specifically, the concentrations of phosphorylcholine, component A, and component B are measured under the measurement conditions for multiple samples with known energy repletion rates, and the correlation equation (Y = aX1 + dX4 + eX5 + f) between the measured concentration of phosphorylcholine (X1), the measured concentration of component A (X4), and the measured concentration of component B (X5) can be 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 urinary phosphorylcholine concentration, at least one data item from the subject's profile, such as age, sex, physical activity level, height, weight, BMI, muscle mass, body fat percentage, visceral fat mass, and basal metabolic rate, and a measured value of the concentration of one or more other components correlated with urinary energy adequacy may be added to the calculation of energy adequacy. For example, the subject's age, BMI, and measured values ​​of the concentrations 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 A second aspect of this embodiment involves measuring urinary ethanolamine phosphate concentration. As described in the Examples, using 20 urine samples, it was found that there was a negative correlation between energy adequacy (Y) and the measured urinary ethanolamine phosphate concentration (X1) (Y = -74383 × X1 + 107.3) (Figure 2B), with the absolute value of the correlation coefficient being 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. By substituting the actual measured value of the ethanolamine phosphate concentration for this X1, the energy fulfillment rate can be calculated. The values ​​of a and f vary depending on the measurement conditions of the ethanolamine phosphate concentration, and therefore must be determined in advance according to the measurement conditions. Specifically, the ethanolamine phosphate concentrations of multiple samples with known energy fulfillment rates are measured under the measurement conditions, and the correlation equation (Y = aX1 + f) between the energy fulfillment rate (Y) and the measured value of the ethanolamine phosphate concentration (X1) is calculated. This makes it possible to determine the constants a and f. By substituting the actual measured value of the ethanolamine phosphate concentration for X1 in the formula Y = aX1 + f, which includes the predetermined a and f, the energy fulfillment rate can be easily calculated.

[0036] 1-2-2 Energy sufficiency calculation formula using multiple regression analysis (1) In this embodiment, in addition to the measured urinary ethanolamine phosphate concentration, at least one of the following data may be used 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. In particular, age and BMI of the subject may be used as indexes.

[0037] As described in the Examples, a multiple regression analysis was performed using energy sufficiency (Y) as the dependent variable, the measured value of 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 sufficiency rate calculated using the above formula and the actual energy sufficiency rate (measured value) calculated from the dietary records and energy expenditure was a high value of 0.86.

[0038] The relationship between energy adequacy (Y) and the measured 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 for ethanolamine phosphate concentration, and therefore must be determined in advance according to the measurement conditions. Specifically, the ethanolamine phosphate concentration is measured under the measurement conditions for multiple samples whose energy adequacy, age, and BMI are known, and the correlation equation (Y = aX1 + bX2 + cX3 + f) between energy adequacy (Y) and the measured ethanolamine phosphate concentration (X1), age (X2), and BMI (X3) can be determined. This allows the constants a, b, c, and f to be determined. The energy adequacy can be calculated by applying the actual measured ethanolamine phosphate concentration, age, and BMI values ​​to the formula Y = aX1 + bX2 + cX3 + f, which includes the predetermined a, b, c, and f. This makes it possible to calculate the energy sufficiency rate without measuring the concentrations of components other than ethanolamine phosphate, but in this case, it is necessary to obtain target profile information from the subject in addition to the 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 to calculate the energy sufficiency rate.

[0040] As described in the Examples, when performing multiple regression analysis using energy fulfillment rate (Y) as the dependent variable, the measured ethanolamine phosphate concentration (X1) as the explanatory variable, and additionally adding, as covariates, indicators of the measured concentrations of two other urinary components, component A (X4) and component B (X5), for example, the relationship Y = aX1 + dX4 + eX5 + f can be determined. The values ​​of a, d, e, and f vary depending on the measurement conditions for the concentrations of ethanolamine 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 ethanolamine phosphate, component A, and component B are measured under the relevant measurement conditions, and the correlation equation (Y = aX1 + dX4 + eX5 + f) between the energy fulfillment rate (Y) and the measured ethanolamine phosphate concentration (X1), the measured component A concentration (X4), and the measured component B concentration (X5) can be determined. This allows the constants a, d, e, and f to be determined. The energy adequacy 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 predetermined values ​​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 subject's profile data, 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 energy sufficiency. 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.

[0042] 1-3 Measurement of glycerol 3-phosphate concentration 1-3-1 Energy sufficiency calculation formula using linear regression analysis A third aspect of this embodiment involves measuring urinary glycerol 3-phosphate concentration. As described in the Examples, linear regression analysis was performed using 20 urine samples to measure the energy adequacy (Y) and the measured urinary glycerol 3-phosphate concentration (X1). It was found that Y and X1 were negatively correlated (Y = -34541 × X1 + 103.02) (Figure 2C), with an absolute value of the correlation coefficient of 0.64, indicating a high correlation.

[0043] The relationship between the energy adequacy ratio (Y) and the measured value of the glycerol 3-phosphate concentration (X1) can be expressed by the formula Y = aX1 + f. The energy adequacy ratio can be calculated by substituting the actual measured value of the glycerol 3-phosphate concentration for X1. The values ​​of a and f vary depending on the measurement conditions for the glycerol 3-phosphate concentration, and therefore must be determined in advance according to the measurement conditions. Specifically, the glycerol 3-phosphate concentrations of multiple samples with known energy adequacy ratios are measured under the measurement conditions, and the correlation equation (Y = aX1 + f) between the energy adequacy ratio (Y) and the measured value of the glycerol 3-phosphate concentration (X1) is calculated. This allows the constants a and f to be determined. The energy adequacy ratio can be easily calculated by substituting the actual measured value of the glycerol 3-phosphate concentration for X1 in the formula Y = aX1 + f, which includes the predetermined 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 used as an index to calculate the energy adequacy: age, sex, physical activity level, height, weight, BMI, muscle mass, body fat percentage, visceral fat mass, basal metabolic rate, etc. In particular, the age and BMI of the subject may be used as indexes.

[0045] As described in the Examples, multiple regression analysis was performed using energy adequacy (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 sufficiency rate calculated using the above formula and the actual energy sufficiency rate (measured value) calculated from the dietary records and energy expenditure was a high value of 0.82.

[0046] The relationship between energy adequacy (Y) and the measured 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 for glycerol 3-phosphate concentration, and therefore must be determined in advance according to the measurement conditions. Specifically, the glycerol 3-phosphate concentration is measured under the measurement conditions for multiple samples with known energy adequacy, age, and BMI, and the correlation equation (Y = aX1 + bX2 + cX3 + f) between energy adequacy (Y) and the measured glycerol 3-phosphate concentration (X1), age (X2), and BMI (X3) can be calculated. 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 values ​​a, b, c, and f. This makes it possible to calculate the energy adequacy ratio without measuring the concentrations of components other than glycerol 3-phosphate. However, 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 to calculate the energy sufficiency rate.

[0048] As described in the Examples, when performing multiple regression analysis using energy adequacy (Y) as the dependent variable, the measured glycerol 3-phosphate concentration (X1) as the explanatory variable, and additionally adding, as covariates, indicators of the measured concentrations of two other urinary components, component A (X4) and component B (X5), for example, the following relationship can be determined: Y = aX1 + dX4 + eX5 + f. The values ​​of a, d, e, and f vary depending on the measurement conditions for the concentrations of glycerol 3-phosphate, component A, and component B, and therefore must be determined in advance according to the measurement conditions. Specifically, the concentrations of glycerol 3-phosphate, component A, and component B are measured for multiple samples with known energy adequacy under the measurement conditions, and the correlation equation (Y = aX1 + dX4 + eX5 + f) between the energy adequacy (Y) and the measured glycerol 3-phosphate concentration (X1), the measured component A concentration (X4), and the measured component B concentration (X5) is determined. This allows the constants a, d, e, and f to be determined. The energy adequacy 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 can be calculated more accurately using only information from a urine sample.

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

[0050] 2. Composition or kit for determining energy sufficiency A second embodiment of the present invention is a composition or kit for determining energy adequacy. The composition or kit of this embodiment is characterized by including at least one of substances involved in the biosynthesis of glycerophospholipids contained in urine, particularly 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 adequacy." Unless otherwise specified or unless otherwise contradictory, the subjects, samples, conditions, etc., for which the composition or kit of this embodiment is used are all as described in Section "1. Method for determining energy adequacy."

[0051] As used herein, a "composition" refers to a single component (including a container), and a "kit" refers to a combination of two or more components. The compositions and kits of the present embodiment are used without distinction other than the number of components, as long as they are used to determine the energy sufficiency.

[0052] 2-1 Compounds for measuring phosphorylcholine concentration A 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, examples include antibodies or antigen-binding fragments thereof that specifically bind to phosphorylcholine.

[0053] 2-2 Compound for measuring ethanolamine phosphate concentration A second aspect of this embodiment includes a compound capable of measuring the concentration of ethanolamine phosphate. Examples of compounds capable of measuring the concentration of ethanolamine phosphate include compounds for subjecting samples to CE or HPLC. In this case, the compound may also be capable of measuring 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 A third aspect of this embodiment includes a compound capable of measuring the concentration of glycerol 3-phosphate. Examples of compounds 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 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-mentioned 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-mentioned compounds. Examples of 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] In the case of a kit, it may further comprise a sealed container for storing a urine sample, a filter (e.g., an ultrafiltration filter) for purifying the urine sample, a urine sample pretreatment composition, 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 testing kit. The mail-in testing kit of this embodiment is characterized in that it includes a sealed container for storing a urine sample from 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 subject's urine and determine energy sufficiency. The mail-in testing kit is used by the subject (or their caregiver, etc.) whose energy sufficiency is to be determined to collect the subject's urine sample and mail the sample to a testing institution capable of measuring the concentrations of urinary components.

[0058] The mail-in testing kit of this embodiment includes at least a sealed container for storing a urine sample. The urine sample 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.

[0059] In addition to the above, the mail-in test kit of this embodiment may also 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 viewing results.

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

[0061] The present invention will be described in more detail below with reference to 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 the dishes, foods, 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 collected within 30 minutes of waking up while fasting. Age, height, and weight data were also collected 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 the 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), centrifuged (9100 × g, 4°C, 60 minutes), ultrafiltered, 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 to 20 times the concentration in cation mode and 10 times the concentration in anion mode, and then subjected to CE-FTMS. (Cation mode: cationic metabolite analysis) CE: Agilent CE system (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: analysis of anionic metabolites) CE: Agilent CE system (Agilent) MS: Q Exactive™ Plus (ThermoFisher) Capillary: Fused silica capillary 50μm x 80cm CE voltage: Positive 30kV MS ionization: ESI anionization MS capillary voltage: 3500V MS scan range: m / z 70-1050

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

number

number

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

[0069] The correlation between the urinary concentration of each of the searched target substances and the energy metabolic rate was calculated, 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 (Fig. 2A), ethanolamine phosphate (Fig. 2B), and glycerol 3-phosphate (Fig. 2C) and the energy metabolic rate.

[0070] [Table 2]

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

[0072] The results of the multiple regression analysis showed that the coefficient of determination adjusted 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 adequacy calculated from the above formula and the actual measured energy adequacy. Compared to using the measured phosphorylcholine concentration as a sole indicator, it was shown that adding age and BMI as indicators allowed for a more accurate determination of energy adequacy.

[0073] (2) Correlation between measured phosphorylcholine concentration and carbohydrate, lipid, and protein sufficiency The subjects' dietary records were used to calculate the intake of carbohydrates, lipids, and proteins over a three-day period. The required carbohydrate intake was calculated as energy expenditure (kcal) x 60% x 1 / 4 (g), the required lipid intake was calculated as energy expenditure (kcal) x 25% x 1 / 9 (g), and the required protein intake was calculated as the recommended amount of protein in the Dietary Reference Intakes. The sufficiency rates of carbohydrates, lipids, and protein were calculated by dividing the actual intake by the required intake.

[0074] The correlation coefficients between the measured phosphorylcholine concentration and the sufficiency rates 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 urinary phosphorylcholine concentration 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 using the measured value of ethanolamine phosphate concentration as the explanatory variable, BMI and age as covariates, and energy sufficiency (3-day average) as the dependent variable.When energy sufficiency is Y, the measured value of ethanolamine phosphate concentration is X1, age is X2, and BMI is X3, it was shown that energy sufficiency Y can be calculated using the following formula. Y=(-70096.79617×X1)+(0.833297×X2)+(-1.698907×X3)+111.47139

[0076] The results of the multiple regression analysis showed that the coefficient of determination adjusted 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 energy adequacy calculated from the above formula and the actual measured energy adequacy. Compared to using the measured ethanolamine phosphate concentration as a sole indicator, it was shown that adding age and BMI as indicators allowed for a more accurate determination of energy adequacy.

[0077] Example 4: Correlation between glycerol 3-phosphate concentration and energy sufficiency 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 using the measured glycerol 3-phosphate concentration as the explanatory variable, BMI and age as covariates, and energy sufficiency (3-day average) as the dependent variable. It was shown that energy sufficiency Y can be calculated using the following formula, where Y is the measured glycerol 3-phosphate concentration, X1 is age, and X3 is BMI. Y=(-32371.292292×X1)+(0.836491×X2)+(-1.474309×X3)+102.414497

[0078] The results of the multiple regression analysis showed that the coefficient of determination adjusted 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 energy adequacy calculated from the above formula and the actual measured energy adequacy. Compared to using the measured glycerol 3-phosphate concentration as a sole indicator, it was shown that adding age and BMI as indicators allowed for a more accurate determination of energy adequacy.

Claims

1. A method for determining energy sufficiency, comprising measuring the concentration of at least one substance involved in glycerophospholipid biosynthesis in the urine of a subject.

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

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

4. The method according to claim 2, wherein the substance involved in the biosynthesis of glycerophospholipids is ethanolamine phosphate.

5. The method according to claim 2, wherein the substance involved in the biosynthesis of glycerophospholipids is glycerol 3-phosphate.

6. The method of claim 1, 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. 7. The method of claim 6, comprising using data on the subject's age and BMI.

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 according to claim 8, wherein the substance involved in the biosynthesis of glycerophospholipid is phosphorylcholine, ethanolamine phosphate, or glycerol 3-phosphate.

10. A mail-in test kit comprising a sealed container for storing a urine sample from a subject, the urine sample stored in the sealed container being 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.

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