KR101757164B1 - Prognostic metabolites for predicting intervention effect of anti-oxidative and anti-inflammatory foods - Google Patents

Prognostic metabolites for predicting intervention effect of anti-oxidative and anti-inflammatory foods Download PDF

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KR101757164B1
KR101757164B1 KR1020150164790A KR20150164790A KR101757164B1 KR 101757164 B1 KR101757164 B1 KR 101757164B1 KR 1020150164790 A KR1020150164790 A KR 1020150164790A KR 20150164790 A KR20150164790 A KR 20150164790A KR 101757164 B1 KR101757164 B1 KR 101757164B1
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권오란
김유진
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이화여자대학교 산학협력단
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Abstract

The present invention relates to an antioxidant metabolic agent for predicting the effect of an antioxidative food ingredient. As a result of observing the biochemical indicators and urinary metabolites of the antioxidant active ingredient, N-phenylacetylglycine), it was confirmed that the changes in the antioxidant capacity (GSH: GSSG ratio) due to the ingestion of food materials were better explained. Therefore, by using the above two metabolites, It can be used as a kit to predict the intervention effect of food materials.

Description

Prognostic metabolites for predicting intervention effects of anti-oxidative and anti-inflammatory foods.

The present invention relates to an indicator metabolite for predicting the intervention effect of an antioxidant food material.

The human body continuously produces reactive oxygen species during normal metabolic process. When this active oxygen group accumulates in the body excessively, it induces oxidative damage to the polyunsaturated fatty acids, amino acids and DNA of the human body. This causes aging, obesity, diabetes, It can cause chronic diseases related to oxidative stress such as diseases, cataracts and macular degeneration.

The antioxidant nutrients play a role in removing the reactive oxygen species, which can prevent accumulation of excess active oxygen groups in the body and prevent or delay oxidative damage (stress) and related chronic diseases. Low concentration of active oxygen weakens cell proliferation and defense mechanism, while high concentration of active oxygen causes aging or disease, and ultrahigh concentration of active oxygen causes cell death. Therefore, maintenance of optimal active oxygen in the cell is very important in terms of maintaining the homeostasis. Oxidative stress is an imbalance of these reactive oxygen species.

In general, active oxygen (free radical) is a compound that is generated when the oxygen in the body is not completely burned and partially incompletely reduced. The main cause of this is inevitably generated through the process of breathing or food metabolism, Tar component, soot gas, constipation, processed food, stress and ultraviolet light induce or activate active oxygen.

The reactivity of reactive oxygen species is very low, like oxygen molecules, from superoxide radicals (O 2 ), hydroxyl radicals (OH), hydrogen peroxide (H 2 O 2 ), singlet oxygen 1 O 2 ) (Wettasinghe M, Shahidi F. Scavenging of reactive oxygen species and DPPH free radicals by extracts of borage and evening primrose meals, Food Chem. 70: 17-26 (2000)). These cause lethal oxygen toxicity to the living body, and lead to cellular membrane degradation, proteolysis, lipid oxidation, DNA denaturation, and the like, thereby causing cell dysfunction and causing brain diseases such as cancer, stroke, Parkinson's disease, , Aging, and autoimmune diseases (Sawyer DT, Valentine JS, Howard, R., and G. Fridorich, 1983). Arch. Biophys. 247: 1-11 (1986); Ames BN. Dietary carcinogens and anticarcinogens. Oxygen radicals and degenerative diseases. Science. 221: 1256-1264 (1983)). In particular, the accumulation of lipid peroxidation, which is produced by attacking unsaturated fatty acids which are constituents of biomembranes, is known to cause the reduction of biological functions, aging and adult diseases (Chance B, Sies H, and Boveris A. Hydroperoxide metabolism in mammalian organs. Physiol. Rev. 59: 527-605 (1979)). Therefore, antioxidants that protect against active oxygen have been attracting attention due to the possibility of treating these diseases, and among them, studies on natural antioxidants extracted from natural products are active.

The present inventors have tried to predict the antioxidant ability of food materials and to find the criteria for judging the effect on antioxidant foods. As a result, antioxidant ability of such food materials can be predicted at a specific concentration of initial metabolites in the initial urine It was confirmed that the antioxidant foods could be used to select antioxidant food materials for personalized nutrition.

It is an object of the present invention to provide an indicator metabolite for predicting the effect of intervention of an antioxidant food material.

According to an aspect of the present invention,

1) collecting urine from a subject who has undergone a one-time treadmill exercise;

2) measuring the concentration of a metabolite comprising glycine and N-phenylacetylglycine in the urine collected in step 1);

3) selecting an individual having a glycine concentration of 0.086 ppm or more and a N-phenylacetylglycine concentration of 0.026 ppm or more in the step 2);

4) ingesting food to the selected individuals in step 3), and collecting blood before and after the one-time, medium-strength treadmill exercise; And

5) A method for predicting the antioxidant ability of a food material and a method for selecting a food having an antioxidative effect, comprising the step of measuring a change in an oxidative damage-related indicator from the blood collected in the steps 1) and 4) do.

In addition,

1) collecting urine from a subject who has undergone a one-time treadmill exercise;

2) selecting an individual having a high level of oxidative damage, comprising the step of selecting individuals having a concentration of glycine of 0.086 ppm or more and a concentration of N-phenylacetylglycine of 0.026 ppm or more in the urine collected in the step 1) .

In addition,

1) collecting urine from a subject who has undergone a one-time treadmill exercise;

2) selecting an individual having a glycine concentration of 0.086 ppm or more and a concentration of N-phenylacetylglycine of 0.026 ppm or more in the urine collected in the step 1);

3) taking the food into the selected individuals in the step 2), and collecting the blood before and after the one-time intensive treadmill exercise; And

4) measuring the change in the oxidative damage-related indicator from the blood collected in the steps 1) and 3).

The present invention relates to an antioxidant metabolic agent for predicting the effect of an antioxidative food ingredient. As a result of observing the biochemical indicators and urinary metabolites of the antioxidant active ingredient, N-phenylacetylglycine), it was confirmed that the changes in the antioxidant capacity (GSH: GSSG ratio) due to the ingestion of food materials were better explained. Therefore, by using the above two metabolites, It can be used as a kit to predict the intervention effect of food materials.

1 is a schematic diagram of an experimental procedure of the present invention.
2 is a schematic diagram of the experimental analysis direction of the present invention.
Figure 3 shows the change in the ratio of oxidized glutathione (GSH: GSSG) to the reduced glutathione of erythrocytes due to the consumption of brambles by glycine (≥ 0.086 ppm) and N-PAG (≥ 0.026 ppm) (ROC) curve indicating that the indicator is an indicator suitable for prediction:
Sens, Sensitivity; spec, specificity; PPV, positive predictive value; And NPV, negative predictive value.
FIG. 4 is a graph showing the percentages of individuals (glycine (≥0.086 ppm) and N-PAG (≥ 0.026 ppm) classified according to the basal level of urine glycine and N-PAG (N-phenylacetylglycine) And individuals with glycine (<0.086 ppm) and N-PAG (<0.026 ppm): white bars), the biomarker effect on biochemical markers is shown:
(A) geometric mean value of erythrocyte GSSG at baseline, GSH: GSSG ratio, and TNF-a; And
(B) Change in least squares mean (LSmean) (SSG, oxidized glutathione; GSH, reduced glutathione; N-PAG, N-phenylacetylglycine) in the ratio of erythrocyte GSH and GSH: GSSG from baseline and 4th week.

Hereinafter, the present invention will be described in detail.

According to the present invention,

1) collecting urine from a subject who has undergone a one-time treadmill exercise;

2) measuring the concentration of a metabolite comprising glycine and N-phenylacetylglycine in the urine collected in step 1);

3) selecting an individual having a glycine concentration of 0.086 ppm or more and a N-phenylacetylglycine concentration of 0.026 ppm or more in the step 2);

4) ingesting food to the selected individuals in step 3), and collecting blood before and after the one-time, medium-strength treadmill exercise; And

5) measuring the change in the oxidative damage-related indicator from the blood sampled in step 4).

The intact treadmill exercise of steps 1) and 4) is preferably performed for 30 minutes at an intensity of 60% of maximum oxygen consumption (VO 2max ), but is not limited thereto.

Preferably, the subject in step 1) is an adult male or female with an overweight (23? BMI (kg / m 2 ) <30) of 30 to 60 years of age.

The intake of the step 4) is preferably long-term intake, and it is most preferable to take 30 g of food per day for 4 weeks. In the present invention, the food is taken for 3 weeks and 3 times / day for 4 weeks.

The index of oxidative damage in the above step 5) is a ratio of oxidized glutathione (GSH: GSSG), malondialdehyde (MDA) and oxidized LDL (oxLDL) to reduced glutathione in red blood cells, , More preferably GSH: GSSG ratio.

In a specific example of the present invention, Rubus In order to evaluate the effects of ingestion of Coreanus Miquel on the physiological function of the body against oxidative stress induced by one-time mid- treadmill treadmill exercise, the subjects were overweight and overweight (23 ≤ BMI (kg / m 2 ) <30) for 2 weeks before the start of the experiment. After 4 weeks of the test food (bokbunja), the subjects consumed less antioxidant foods (fruits, vegetables, (3 cells / dose, 3 times / day) (intervention period). The control group (placebo) was fed with powder consisting of lactose, and the experimental group, brambles, were fed the brambles powder and about 30 g per day. One-time, medium-strength treadmill exercises were performed to give oxidative stress. Maximum oxygen consumption was measured at the time when the test food was assigned (baseline) and at the end of the 4-week test food intake , VO 2 max) at 60% intensity for 30 minutes. Blood samples were collected to observe changes in biochemical markers (antioxidant capacity, inflammation) and urinary metabolites (64 total).

The present inventors intend to identify serum antioxidative and inflammatory indicators that change significantly depending on bokbunal intake (short-term intake and long-term intake) by applying oxidative stress. As a result, long-term intake of bokbunja (4 weeks intake) The ratio of reduced glutathione (GSH) and oxidized glutathione (GSSG) (β = 1.113, P (0.015)) was found to be significantly decreased in the oxidized glutathione = 0.011) was significantly increased. It was found that malondialdehyde (MDA) and oxidized LDL (oxidized LDL; oxLDL), which are oxidative damage indicators, were significantly decreased by long-term intake of ruminants (β = -0.121, P = 0.046 for MDA; = -0.102, P = 0.02 for oxLDL). The level of IL-6, an inflammatory index, was also significantly decreased by long-term intake of rubella (4 weeks of intake) (β = -0.458, P = 0.0001) (see Table 1).

In addition, the present inventors sought to determine the changes of metabolites in plasma and urine which are significantly changed according to bramble ingestion (short-term intake and long-term intake) by oxidative stress. Depending on the long-term consumption of the bokbunja, it may be possible to use a combination of urine alanine, asparagine, glutamate, glycine, histidine, lysine, citrate, formate, It was confirmed that 3-indoxysulfate, betaine, N-phenylacetylglycine, N6-acetyllysine and phenylacetate were significantly increased (0.001?? 0.399, 0.0001? P? 0.025) ( P = 0.024) and carnitine (β = -0.555, P = 0.001) were significantly decreased (see Table 2), although adenine (β = -0.286, P = 0.024) and carnitine

In order to select the urinary metabolites which can predict the antioxidant activity or the anti-inflammatory activity change according to the ingestion of the functional food material, the present inventors have found that metabolism with positive / negative correlation with biochemical indicators Respectively. As a result, the biochemical markers and metabolic body that has a positive correlation is, the red blood cell GSH: GSSG ratio and, glycine (P = 0.007) and N-phenylacetylglycine (PAG; P = 0.002), oxLDL and serine (P = 0.040 ), IL-6 and formate ( P = 0.014). Biochemical markers and metabolites with a negative correlation showed changes in the GSSG of erythrocytes and trimethylamine ( P = 0.043) at baseline, ( P = 0.028), changes in IL-6 levels and baseline N6-acetyllysine ( P = 0.038).

In order to confirm that the selective metabolites (glycine and N-PAG) in urine are the most suitable indicators for the prediction of the change in the GSH: GSSG ratio of erythrocytes due to ruminant intake, the present inventors used the ROC curve Sensitivity, specificity, and reference point. As a result, in the ROC curve analysis, the ROC area under curve was 0.778, indicating a sensitivity of 58.1% and a specificity of 86.4% (see FIG. 3).

In order to determine the suitability of the initial urinary metabolite, glycine and N-PAG selected as the predictive index, the present inventors also compared the levels of glycine (≥0.086 ppm) and N-PAG (≥ 0.026 ppm) As a result, it was found that at the base point (Week 0), the concentration of the two metabolites was higher than that of the red blood cells (P <0.05) in the person with a specific concentration, ie, glycine (≥0.086 ppm) and N-PAG oxidized glutathione (GSSG) (P = 0.037) and was TNF-α (P = 0.085) were significantly high or high tendency appeared in the plasma and red blood cell GSH: GSSG ratio (P = 0.029) were significantly lower (See Fig. 4A). This suggests that oxidative damage is high in people with concentrations of 0.086 and 0.026 ppm glycine and N-PAG, respectively.

( P = 0.020) and GSH: GSSG ratio ( P = 0.008) were significantly higher in the two groups than in the control group (week 0) (See FIG. 4B). This indicates that the effect of food intake was good for people with high initial oxidative damage.

Therefore, it was confirmed that urinary glycine (≥ 0.086 ppm) and N-PAG (≥ 0.026 ppm) are the most suitable indexes for predicting changes in glutathione: oxidized glutathione (GSH: GSSG) ratio of erythrocytes caused by bramble ingestion.

In addition,

1) collecting urine from a subject who has undergone a one-time treadmill exercise;

2) measuring the concentration of a metabolite comprising glycine and N-phenylacetylglycine in the urine collected in step 1);

3) selecting an individual having a glycine concentration of 0.086 ppm or more and a N-phenylacetylglycine concentration of 0.026 ppm or more in the step 2);

4) Taking a food to determine whether the selected individual has antioxidative effect in step 3), collecting blood before and after the one-time intensive treadmill exercise; And

5) measuring the change in the oxidative damage-related indicator from the blood collected in the step 4).

The intact treadmill exercise of steps 1) and 4) is preferably performed for 30 minutes at an intensity of 60% of maximum oxygen consumption (VO 2max ), but is not limited thereto.

Preferably, the subject in step 1) is an adult male or female with an overweight (23? BMI (kg / m 2 ) <30) of 30 to 60 years of age.

The intake of the step 4) is preferably long-term intake, and it is most preferable to take 30 g of food per day for 4 weeks. In the present invention, the food is taken for 3 weeks and 3 times / day for 4 weeks.

The index of oxidative damage in the above step 5) is a ratio of oxidized glutathione (GSH: GSSG), malondialdehyde (MDA) and oxidized LDL (oxLDL) to reduced glutathione in red blood cells, , More preferably GSH: GSSG ratio.

In addition,

1) collecting urine from a subject who has undergone a one-time treadmill exercise;

2) selecting an individual having a high level of oxidative damage, comprising the step of selecting individuals having a concentration of glycine of 0.086 ppm or more and a concentration of N-phenylacetylglycine of 0.026 ppm or more in the urine collected in the step 1) .

The intact treadmill exercise in step 1) is preferably performed for 30 minutes at an intensity of 60% of maximum oxygen consumption (VO 2max ), but is not limited thereto.

Preferably, the subject in step 1) is an adult male or female with an overweight (23? BMI (kg / m 2 ) <30) of 30 to 60 years of age.

In addition,

1) collecting urine from a subject who has undergone a one-time treadmill exercise;

2) selecting an individual having a glycine concentration of 0.086 ppm or more and a concentration of N-phenylacetylglycine of 0.026 ppm or more in the urine collected in the step 1);

3) taking the food into the selected individuals in the step 2), and collecting the blood before and after the one-time intensive treadmill exercise; And

4) measuring the change in the oxidative damage-related indicator from the blood collected in the step 3).

The intact treadmill exercise of steps 1) and 3) is preferably performed for 30 minutes at an intensity of 60% of maximum oxygen consumption (VO 2max ), but is not limited thereto.

Preferably, the subject in step 1) is an adult male or female with an overweight (23? BMI (kg / m 2 ) <30) of 30 to 60 years of age.

The intake of the step 3) is preferably long-term intake, and it is most preferable to take 30 g of food per day for 4 weeks. In the present invention, the food is taken at 3 times / week and 3 times / day for 4 weeks.

The index of oxidative damage in step 4) is a ratio of oxidized glutathione (GSH: GSSG), malondialdehyde (MDA) and oxidized LDL (oxLDL) to reduced glutathione in erythrocytes , More preferably GSH: GSSG ratio.

Accordingly, the present invention has been made to observe changes in blood biochemical indicators and urinary metabolites when food materials having antioxidant activity are ingested. As a result, the higher the levels of glycine and N-phenylacetylglycine in the initial urine metabolism, (GSSG ratio). Therefore, it can be used as a kit for diagnosing a person having a high degree of oxidative damage and anticipating the effect of an antioxidant food material using the above two metabolites have.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

It should be noted, however, that the following examples and experimental examples are illustrative of the present invention, but the present invention is not limited by the following examples and experimental examples.

< Example  1> Bokbunja ( Rubus coreanus Miquel ) Body application test using ingestion

Human application tests of the present invention is randomized, double-blind, controlled food-controlled, parallel design study bokbunja (Rubus as Coreanus Miquel) on the physiological function of the body against oxidative stress induced by one-time mid- treadmill treadmill exercise.

Specifically, the subjects were adults over 30 years old and over 60 years old (23 ≤ BMI (kg / m 2 ) <30). In this study, the subjects who were selected for the selection and exclusion criteria were recruited and then the exercise load test was performed to determine the strength of the one - time mid - treadmill treadmill exercise. The maximum oxygen uptake was calculated by using exercise load test as a criterion for determining the exercise intensity of individuals. The test was performed by using a treadmill and respiratory gas analyzer (Quark CPET of COSMOD) to increase the treadmill slope and speed every 2 minutes Was used. Subjects were measured for resting heart rate and blood pressure, and heart rate recorded every minute. Exhalation gas, oxygen uptake, carbon dioxide production, and respiratory exchange rate (RER) were measured automatically during maximal exercise load test. The maximum oxygen uptake of each individual was calculated based on peak oxygen uptake (ml / kg / min) among the oxygen uptakes collected during the test Respectively. The mid-intensity treadmill exercise of the subjects was performed for 30 minutes at a 60% intensity of maximum oxygen consumption (VO2max) derived from the load test.

In the run-in period, foods with high antioxidant capacity (fruits, vegetables, tea, etc.) were kept low for about 2 weeks before the test food was allocated, and the control group ( n = 24) ( n = 21), followed by 4 weeks of food intake (3 rats / dose, 3 times / day) (intervention period). The control group (placebo) used in the present invention consumed powder composed of lactose, and the experimental group, brambles powder, was consumed about 30 g per day. The placebo and the test food (bokbunja) used in the present invention were manufactured and supplied by RDA, and the bokbunja were obtained by freezing and pulverizing mature bokbunja fruit.

One - time mid - intensity treadmill exercise to give oxidative stress was performed at the time point when the test food was assigned (baseline) and when the test food was completed for 4 weeks (end point). After taking steady-state blood and urine for 12 h fasting at baseline, subjects were asked to take water and test food (control food or bokbunja) and receive one-time maximum oxygen consumption (maximum oxygen consumption, VO 2 max) for 30 min at 60% intensity (single-dose procedure). Immediately after exercise, blood samples were taken and biochemical markers (antioxidant ability, inflammation) and urinary metabolites (total 64) were observed. After taking the test food for 4 weeks and then returning to the end of the 12-hour fasting period, blood and urine were collected again and then water and test food (control food or bokbunja) were taken A one-time mid-treadmill exercise was performed (long-term ingestion process). Immediately after exercise, blood samples were taken and biochemical markers (antioxidant ability, inflammation) and urinary metabolites (total 64) were observed. A schematic diagram of the entire experimental procedure of the present invention is shown in FIGS. 1 and 2. FIG.

< Experimental Example  1> Identification of biochemical indicators in blood

The present inventors sought to identify serum antioxidant and inflammation indexes that were significantly changed according to brambles intake (short-term intake and long-term intake) by applying oxidative stress as in Example 1 above.

Specifically, the beta estimate (β; estimated slope) was measured for each variable from the linear mixed-effect model of all data (N = 45; placebo, n = 24; The main fixed interaction effect between " intake of ruminants and control (placebo) "and" baseline and endpoints "can be interpreted as the mean change over the period of intake between the two treatment groups (beta for 4 weeks intake) On the other hand, the main fixation effect of the "treatment group" is interpreted as the mean change between treatment groups at baseline (beta for single ingestion) (the intercept is centered on the first measurement point). The P-value was derived from the linear mixed-effect regression model and the probability of P value <0.05 was considered significant.

As a result, as shown in Table 1, oxidized glutathione (GSSG.beta. = -1.197, P = 0.021) of red blood cells was significantly decreased due to long-term intake of brambles (intake for 4 weeks) The ratio of reduced glutathione (GSH) and oxidized glutathione (GSSG) (β = 1.113, P = 0.011) was found to be significantly increased. It was found that malondialdehyde (MDA) and oxidized LDL (oxidized LDL; oxLDL), which are oxidative damage indicators, were significantly decreased by long-term intake of ruminants (β = -0.121, P = 0.046 for MDA; = -0.102, P = 0.02 for oxLDL). The levels of IL-6, an inflammatory index, were also significantly decreased (β = -0.458, P = 0.0001) according to the long-term intake of rubella (4 weeks intake) (Table 1).

The Korean black raspberry ( Rubus) Coreanus Miquel) on Oxidation and Inflammation Single intake Long-term intake variable calculation SE P -value calculation SE P -value Inhibitors (antioxidants) GSH -1.101 1.839 0.553 0.407 1.074 0.706 GSSG -0.086 0.908 0.925 -1.197 0.513 0.021 GSH: GSSG ratio -0.043 0.076 0.577 0.113 0.044 0.011 damaged MDA 0.059 0.147 0.69 -0.121 0.06 0.046 Oxidized LDL 0.066 0.109 0.551 -0.102 0.044 0.02 Inflammation IL-6 -0.015 0.162 0.927 -0.458 0.111 <0.001 TNF-alpha 0.259 0.708 0.716 -0.298 0.656 0.65

GSH, reduced glutathione; GSSG, oxidized glutathione; MDA, malondialdehyde; LDL, low density lipoprotein; IL, interleukin; TNF, tumor necrosis factor; And SE, the standard error of the coefficient estimates.

< Experimental Example  2> Urine Metabolic  Confirm change

The present inventors tried to confirm the change of metabolites in plasma and urine which are significantly changed according to bramble intake (short-term intake and long-term intake) by oxidative stress as in <Example 1>.

Specifically, the standard error of the coefficient estimate (SE) represents the standard error of the coefficient estimates, and the beta estimate (β, estimated slope) was measured for each variable in a linear mixed-effect model of all data (N = 45; Placebo, n = 24; bokbunja, n = 21). The main fixed interaction effect between " intake of ruminants and control (placebo) "and" baseline and endpoints "can be interpreted as the mean change over the period of intake between the two treatment groups (beta for 4 weeks intake) On the other hand, the main fixation effect of the "treatment group" is interpreted as the mean change between treatment groups at baseline (beta for single ingestion) (the intercept is centered on the first measurement point). The P-value was derived from the linear mixed-effect regression model, and the probability of P value <0.05 was considered significant.

As a result, as shown in Table 2, no change was observed in the blood index due to the short-term intake (single intake) of the brambles, but the short-term intake of the brambles resulted in a significant increase in urinary trimethylamine β = -0.222, P = 0.013) and serine was significantly decreased (β = 0.008, P = 0.030) (Table 2).

As shown in Table 2, according to the long-term consumption of the bokbunja, the urinary alanine, asparagine, glutamate, glycine, histidine, lysine, citrate ), Formate, 3-indoxysulfate, betaine, N-phenylacetylglycine, N6-acetyllysine, and phenylacetate were significantly increased (0.001≤β≤0.399, 0.0001 ≤ P ≤ 0.025), but adenine (β = -0.286, P = 0.024) and carnitine (β = -0.555, P = 0.001) decreased significantly (Table 2) .

Korean black raspberry ( Rubus) for the change of urine and plasma metabolism in overweight adults coreanus Miquel): The response to treadmill exercise at baseline and endpoints during long-term ingestion Metabolism Single intake Long-term intake Estimate SE P -value 3 calculation SE P -value 3-Indoxylsulfate -0.092 0.153 0.549 0.399 0.119 0.001 Adenine 0.184 0.131 0.166 -0.286 0.125 0.024 Alanine -0.115 0.105 0.279 0.194 0.071 0.007 Asparagine -0.068 0.084 0.425 0.199 0.088 0.025 Aspartate -0.003 0.002 0.157 0.003 0.002 0.067 Betaine -0.089 0.137 0.521 0.295 0.114 0.01 Carnitine 0.132 0.2 0.515 -0.555 0.171 0.001 Citrate -0.016 0.026 0.545 0.029 0.012 0.02 Formate -0.166 0.134 0.224 0.314 0.13 0.017 Glutamine -0.125 0.074 0.098 0.22 0.056 <0.001 Glycine -0.133 0.148 0.373 0.2 0.073 0.007 Histidine -0.006 0.006 0.365 0.018 0.006 0.002 Isoleucine -0.0002 0.0002 0.348 0.0003 0.0001 0.058 Leucine -0.0003 0.0004 0.398 0.0005 0.0003 0.068 Lysine -0.314 0.208 0.138 0.329 0.122 0.008 N-Phenylacetylglycine -0.004 0.002 0.083 0.007 0.002 0.003 N6-Acetyllysine -0.001 0.001 0.336 0.001 0 0.013 Phenylacetate 0 0.001 0.708 0.002 0.001 0.008 Serine -0.222 0.086 0.013 0.248 0.089 0.006 Trimethylamine 0.008 0.004 0.03 0.008 0.004 0.047 Valine -0.107 0.079 0.181 0.134 0.069 0.054

< Experimental Example  3> Changes in blood biochemical indicators and urine Heavy metabolite  Confirm the relationship between

In order to select the urinary metabolites that can predict the antioxidant activity or the anti-inflammatory activity change depending on the ingestion of functional food materials, biochemical indices and positive / negative correlations Metabolites having a relationship were classified.

Specifically, changes in blood biochemical markers as dependent variables and analysis by hierarchical logistic regression analysis (n = 45) using the initial urine metabolite as the main predictor (covariate) Respectively. Repeated analyzes within an individual were entered into the model as random effects. Error ratios (OR) showed errors in blood biochemical changes associated with the initial metabolic status of the urine after chronic supplementation of ruminants. β, SE, and CI represent the fixed effects (log scale), standard error (log scale), and estimation results for the 95% confidence interval. Individuals with low initial metabolite values were classified as references.

As a result, biochemical indicators and metabolites having a positive correlation as shown in Table 3 are as follows:

The ratio of GSH: GSSG in red blood cells to glycine ( P = 0.007) and N-phenylacetylglycine (PAG; P = 0.002)

oxLDL and serine ( P = 0.040),

IL-6 and formate ( P = 0.014),

In addition, biochemical indicators and metabolites with a negative correlation are as follows:

GSSG changes in erythrocytes and trimethylamine ( P = 0.043) at baseline,

Changes in MDA levels and baseline adenine ( P = 0.028),

Changes in IL-6 levels and baseline N6-acetyllysine ( P = 0.038).

In a generalized mixed model, the metabolism predictable in the urine for biochemical changes Fixed effects beta SE P -value OR CI GSSG Trimethylamine -0.95 0.45 0.043 0.39 (0.15, 0.97) GSH : GSSG ratio Glycine 1.54 0.54 0.007 4.67 (1.56, 13.99) N-Phenylacetylglycine 1.78 0.55 0.002 5.95 (1.95, 18.16) MDA Adenine -One 0.44 0.028 0.37 (0.15, 0.89) oxLDL Serine 1.01 0.47 0.04 2.74 (1.05, 7.13) IL -6 Formate 1.46 0.57 0.014 4.29 (1.36, 13.49) N6-Acetyllysine -1.22 0.57 0.038 0.3 (0.09, 0.93)

GSSG, oxidized glutathione; GSH, reduced glutathione; MDA, malondialdehyde; oxLDL, oxidized LDL; IL, interleukin; CIs, confidence intervals.

< Experimental Example  4) Receiver Manipulation Characteristics for Identifying Likelihood as a Predictor of Intervention Effectiveness Receiver operating characteristic ; ROC ) Curve analysis

In order to confirm that the metabolites (glycine and N-PAG) selected in the <Experimental Example 3> in urine are the most suitable indicators for the change in the ratio of GSH: GSSG of erythrocytes caused by the consumption of bokbun, the ROC curve ) Were used to confirm the sensitivity, specificity, and reference point.

Specifically, the ROC curve analysis was performed using the SAS, ver 9.4 program (SAS Institute) in the urine glycine (≥ 0.086 ppm) and N-PAG (≥ 0.026 ppm) measured in Experimental Example 3 above. As the binomial variables (two numbers of valid categories), the change in the GSH: GSSG ratio of red blood cells was defined as a state variable (dependent variable), and the presence of high glycine and N-PAG in urine as a test variable After drawing the ROC curve, the ultimate reference point was selected using the Yudan Index (Y = sensitivity + 1-specificity) ( Sens, sensitivity, spec, PPV, positive predictive value, Predicted value).

In addition, we used the cross validation method, Leave One Out Cross Validation (LOOCV), to evaluate the suitability of the predictive indices proposed above. This is done by dividing the N datasets used into N subsets and then using (N-1) subsets as training data and the remaining one as test data do. The root mean square error (RMSE) and the mean absolute error (MAE) were calculated N times, and the fitness of the predictive index was quantitatively compared. RMSE and MAE represent the average magnitude of the relative error between the actual value and the estimated value, and the smaller the value, the better the model can be determined.

As a result, as shown in FIG. 3, in the ROC curve analysis, the ROC area under curve was 0.778, indicating a sensitivity of 58.1% and a specificity of 86.4% (FIG. 3) . As a result of performing the leave-one-out cross-validation (LOOCV), it was confirmed that the accuracy of the predicted model was not low due to the low RMSE (0.61158) and MAE (0.377778).

Thus, it was confirmed that urinary glycine (≥ 0.086 ppm) and N-PAG (≥ 0.026 ppm) are the most suitable indexes for predicting the changes in glutathione: oxidized glutathione (GSH: GSSG) ratio of erythrocytes caused by ruminant intake.

< Experimental Example  5> Urine as a predictor of intervention effect Metabolite  compatibility

To determine the suitability of the initial urine metabolites, glycine and N-PAG selected as predictable indicators in the above <Experimental Example 4>, a specific concentration level, namely glycine (≥0.086 ppm) and N-PAG (≥ 0.026 ppm ) Were compared with those who had higher levels of glycine (0.131 ppm, N-PAG 0.034 ppm) and those who did not (mean concentration: 0.076 ppm, N-PAG 0.024 ppm). Normal distribution data is expressed as mean ± standard deviation. P-values were derived from Student's t-test, and a probability of less than 5% was considered significant (GSSG, oxidized glutathione; GSH, reduced glutathione; N-PAG, N-phenylacetylglycine).

As a result, as shown in Fig. 4, oxidized glutathione (redox) of red blood cells was detected in a person having a higher concentration of two metabolites at a base point (Week 0) than a specific concentration, namely glycine (≥0.086 ppm) and N-PAG GSSG) ( P = 0.037) and plasma TNF-α ( P = 0.085) were significantly higher or higher than those of the control group ( P = 0.029) 4A). This suggests that oxidative damage is high in people with concentrations of 0.086 and 0.026 ppm glycine and N-PAG, respectively.

Based on these results, it was found that oxidant glutathione ( P = 0.020) of erythrocytes was increased in the subjects with high concentration of two metabolites by eating 4 weeks of bokbunja at the base point (Week 0) And GSH: GSSG ratio ( P = 0.008) were significantly improved (FIG. 4B). This indicates that the effect of food intake was good for people with high initial oxidative damage.

Therefore, we can confirm that the concentration of initial urinary metabolite is appropriate for assessing the degree of oxidative damage in humans, and can also predict a significant change in food intake.

Claims (18)

1) collecting urine from a subject who has undergone a one-time treadmill exercise;
2) measuring the concentration of a metabolite comprising glycine and N-phenylacetylglycine in the urine collected in step 1);
3) selecting an individual having a glycine concentration of 0.086 ppm or more and a N-phenylacetylglycine concentration of 0.026 ppm or more in the step 2);
4) ingesting food to the selected individuals in step 3), and collecting blood before and after the one-time, medium-strength treadmill exercise; And
5) measuring the change in the ratio of oxidized glutathione (GSH: GSSG) to reduced glutathione in erythrocytes, which is an indicator of oxidative damage, from the blood collected in step 4)
A method of screening individuals that require ingestion of antioxidant foods.
The method of claim 1, wherein the intact treadmill exercise of steps 1) and 4) is performed for 30 minutes at an intensity of 60% of maximum oxygen consumption (VO 2max ) How to select the required objects.
The method according to claim 1, wherein the step 1) is an adult male or female who is overweight (23? BMI (kg / m 2 ) <30) How to screen objects.
The method according to claim 1, wherein the ingestion of step (4) is ingested by 30 g per day for 4 weeks.
delete delete delete delete delete delete 1) collecting urine from a subject who has undergone a one-time treadmill exercise; And
2) An oxidized glutathione for reduced glutathione in erythrocytes was administered to individuals having a concentration of glycine of 0.086 ppm or more and a concentration of N-phenylacetylglycine of 0.026 ppm or more in the urine collected in the step 1) (GSH: GSSG).
A method for determining an individual having a high degree of oxidative damage.
12. The method of claim 11, wherein the medium-strength treadmill exercise of step 1) is performed for 30 minutes at an intensity of 60% of maximum oxygen consumption (VO 2max ) How to judge.
12. The method according to claim 11, wherein the individual in step 1) is an adult male or female overweight (23? BMI (kg / m 2 ) <30) How to judge an entity.
delete delete delete delete delete
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JP2010516805A (en) * 2007-02-01 2010-05-20 ザ アイムス カンパニー Method for reducing inflammation and oxidative stress in mammals

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Publication number Priority date Publication date Assignee Title
JP2010516805A (en) * 2007-02-01 2010-05-20 ザ アイムス カンパニー Method for reducing inflammation and oxidative stress in mammals

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Evidence-Based Complementary and Alternative Medicine, Vol.2015, Article ID 508302, 11pages(2015.10.07.)*
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