KR20200078180A - Method for measuring oxidative stress level - Google Patents

Abstract

The present invention relates to a method for measuring oxidant stress and, more specifically, to a method for quantitatively measuring oxidant stress. The method includes a step of quantifying a level of oxidative stress of a test object by using an influence factor in a level of the oxidative stress measured in the test object.

Description

Method for measuring oxidative stress level

The present invention relates to a method for measuring the level of oxidative stress. More specifically, a formula for estimating the oxidative stress in consideration of the characteristics of the subject is developed, and a method for evaluating the level of the oxidative stress in the subject based on the quantified score and using the method for determining the mediating effect of the antioxidant or food material It's about how.

Free radicals are reaction by-products that occur during metabolic processes in the body and are known to have a negative effect on the human body. Excess free radicals accumulate lipid peroxide in the cell membrane, and there is a risk of causing diseases such as stroke and myocardial infarction. It is very important to measure the amount of free radicals in the human body for prophylactic or diagnostic purposes, but reactive oxygen is reactive It is so excellent that it is difficult to measure directly.

Interest in health continues to increase, and in particular, interest in aging and disease due to oxidative stress is increasing day by day, but there is no method to objectively measure the degree of oxidation. In addition, the relationship between oxidative stress and infertility, hepatitis, arthritis, cataract, etc. has been revealed through recent studies, and thus, the need for a method to objectively measure oxidative stress is increasing, and prevention and improvement of oxidative stress The demand for food that can be made is also increasing.

In order to solve the above problems, the present invention is to provide a method for quantitatively measuring oxidative stress and a screening method for a candidate group of antioxidants using the same.

The present invention has developed a quantification method that can evaluate the oxidative stress of an individual by collecting and analyzing clinical information and oxidative stress data obtained from a population group, and conducted internal verification. In addition, in order to find out whether this method of quantifying oxidative stress is measurable even through changes in nutritional intervention, an external verification process was performed using separate clinical research data using antioxidant food ingredients.

The present invention develops a formula for estimating oxidative stress in consideration of a subject's characteristics, and a method for evaluating a subject's oxidative stress level based on a quantified score and a method for determining the mediating effect of an antioxidant or food material using the method It is about.

An example of the present invention includes the step of quantifying the degree of oxidative stress of the test subject using an influence factor of the oxidative stress level measured in the test subject, wherein the effect factor of the oxidative stress level is the test subject It relates to a method for measuring the oxidative stress level of a test subject, at least one selected from the group consisting of age, smoking status, plasma lipid peroxide concentration, whole blood white blood cell count, and alanine aminotransferase concentration.

A further embodiment of the present invention compares the oxidative stress level measured in the test subject before and after the candidate substance is administered, and the oxidative stress level measured after the candidate substance is administered, before the candidate substance is administered. If it is reduced than the measured oxidative stress level, comprising the step of selecting the candidate substance as an antioxidant,

Measuring the oxidative stress level of the test subject,

And quantifying the degree of oxidative stress of the test subject using an influence factor of the oxidative stress level of the test subject,

The oxidative stress level influencing factor, the age of the test subject, whether or not smoking, plasma lipid peroxide concentration, whole blood white blood cell count, and alanine aminotransferase concentration, at least one selected from the group consisting of, the screening method of antioxidants .

Another embodiment of the present invention, a method of providing information for the diagnosis of oxidative stress-related diseases, comprising comparing the oxidative stress level of a test subject with a oxidative stress level measured in a normal control group,

Measuring the oxidative stress level of the test subject,

The subject's age, smoking status, plasma lipid peroxide concentration, whole blood leukocyte count, and alanine aminotransferase concentration were measured, and one or more oxidative stress levels were selected.

It comprises a step of determining the degree of oxidative stress of the test subject using the measured value of the influence factor, relates to a method for providing information for the diagnosis of oxidative stress-related diseases.

Using the method for measuring the level of oxidative stress according to the present invention, it was confirmed that the degree of oxidative stress of an individual can be objectively evaluated, and that a normal individual and an oxidative stress individual can be accurately distinguished. Specifically, in the internal verification method using the Leave-One-Out Cross Validation (LOOCV) method, AUC-ROC was derived as 0.82 (sensitivity 76.8%, specificity 76.7%), and the method for measuring the oxidative stress level according to the present invention The accuracy of was confirmed.

In addition, external verification using human applied research data to confirm the mediating effect of the antioxidant food material is performed, and based on the method for measuring the oxidative stress level according to the present invention, the oxidative stress level at the base point and at the end of the intervention As a result of comparing the control group with the test food group by estimating, it was confirmed that the oxidative stress level was significantly decreased in the group ingesting the antioxidant food material, and it could be useful as a tool for determining the intervention effect of the antioxidant food material. It was also confirmed that the degree of change in antioxidant capacity in response to food ingredients intake was predictable.

Hereinafter, the present invention will be described in more detail.

An embodiment of the present invention relates to a method for measuring an oxidative stress level of a test subject, comprising quantifying the degree of oxidative stress of the test subject using an influence factor of the oxidative stress level measured in the test subject.

According to an embodiment of the present invention, the subject's age, smoking status, plasma lipid peroxide concentration, whole blood white blood cell count, and alanine aminotransferase concentration are measured, and an effect factor of at least one oxidative stress level selected from the group is measured, and the effect It is to provide a method for measuring the level of oxidative stress in a subject, comprising the step of quantifying the degree of oxidative stress in the test subject using the measured value of the factor.

The influencing factors are weighted in consideration of the estimators of each factor, and the estimators are instrumental variable estimation, ordinary least square estimation, and non-linear least square estimation. ), iterative weighted least square estimation, maximum likelihood estimation (MLE), restricted maximum likelihood estimation (restricted MLE), and limited maximum likelihood estimation without using differential equations restricted MLE, DF REML).

The test subject may be one or more selected from the group consisting of human, mouse, mouse, hamster, rat, guinea pig, rabbit, cow, dog, cat, chicken, insect, frog, mammal, primate, reptile, amphibian, and rodent. .

The degree of oxidative stress of the test subject may be determined using the following [Equation 1]:

Y is a calculated value of the degree of oxidative stress, the unit is a score,

Age is the age of the subject, and the unit is three years.

Smoking is the subject's smoking status, 1 if the subject smokes, 0 if the subject is non-smoking or has quit smoking for more than 1 year;

MDA is the subject's plasma lipid peroxide, with units of μM;

WBC is the number of white blood cells measured in a subject's whole blood sample, and the unit is x10 ³ /µl;

GPT is the alanine aminotransferase level of the subject and the unit is IU/L.

The method for measuring the oxidative stress level of the subject may further include determining that the test subject has oxidative stress when the calculated value Y of the calculated degree of oxidative stress of the test subject is 2.7 or more. have.

In the present invention, "oxidative stress (oxidative stress)" is an antioxidant defense group that restores the imbalance between the generation of free radicals (oxygen ions, free radicals, peroxides, etc.) in the living cells and the antioxidant ability to eliminate them, or damage caused by the substance It can be defined as a condition that causes oxidative damage of cellular proteins, lipids, and nucleic acids due to an imbalance of the antioxidant defense mechanism. This oxidative stress is presumed to be a major factor in the pathogenesis of metabolic syndrome, hypertension, cardiovascular disease, hyperlipidemia, diabetes, ischemia-reperfusion injury, cancer, chronic inflammation and nervous system diseases, as well as aging processes in the human body. (J Nutr Biochem 2013, 24:1183-1201; Inflammation, Aging, and Oxidative Stress.Springer; 2016: 95-106; Aging Dis 2015, 6:109-120.)

In the present invention, the "oxidative stress group" refers to a group of individuals who have already been determined to have a high level of oxidative stress, and through a method such as in vivo mechanism or clinically reviewed literature, the level of oxidative stress is high. Refers to the set of individuals determined to be.

In the present invention, "bio information" refers to biological information of an individual, for example, age, consumption level of antioxidant food, smoking or not, concentration of lipid peroxide in plasma/red blood cells/urine, plasma antioxidant nutrient level, white blood cell count, basophils Level, monocyte level, alanine aminotransferase level, aspartate aminotransferase level, gamma glutamyl transferase level, alkaline phosphatase level, albumin level, erythrocyte volume fraction, erythrocyte sedimentation rate, C-reactive protein (C-reactive protein) ) Level, uric acid level, etc., but is not limited thereto.

The present invention relates to a method for measuring the oxidative stress level of an individual, comparing a plurality of bio-information measured in the oxidative stress group and a plurality of bio-information measured in the normal group, and selecting an influence factor of the oxidative stress level To do; Weighting the selected factors in consideration of an estimated amount of the selected factors; And determining the degree of oxidative stress of the test object using the weighted factor.

In the present invention, comparing the plurality of bio-information measured in the oxidative stress group and the plurality of bio-information measured in the normal group, and selecting an influence factor of the oxidative stress level, respectively, were measured in the oxidative stress group and the normal group. It is a step of comparing bio information and selecting factors showing significant differences in the oxidative stress group as compared to the normal group as factors associated with oxidative stress. The screening of the related factors may be performed using a hierarchical mixing model. For example, in the present invention, bioinformation such as health checkups and blood analysis results of Korean adult men and women aged 19 years or older who visited the Boramae Hospital Health Check-up Center in Seoul, hierarchically mixed the normal group and the group with oxidative stress-related diseases By comparing through the model, bioinformation showing a significant difference was selected as a factor associated with oxidative stress (Examples 1-2). Specifically, as a result of a mixed hierarchical model, the subject's age, smoking status, plasma lipid peroxide (MDA), white blood cell counts (WBC), and alanine aminotransferase (glutamate pyruvate) It was confirmed that the level of transaminase (GPT) is a significant factor that can predict the level of oxidative stress.

The mixed hierarchical model is a statistical analysis method that can properly analyze data that does not fit the assumptions of the general regression model because it has a multi-layered structure in which individuals and cluster-level variables are mixed (Raudenbush SW). , Bryk AS.Hierarchical linear models: Applications and data analysis methods.Sage; 2002.). The hierarchical linear regression model is a mixed model that can be determined and included at the same time by discriminating random effects and fixed effects among factors affecting response variables or predictors. In the present invention, a hierarchical linear model was used under a multi-layered structure that takes into account individual level characteristics and bio-indicators to estimate the level of oxidative stress in individuals, and the effects of age and personal characteristic variables on individual levels of oxidative stress were investigated. For analysis, it was divided into fixed effect and random effect. The stochastic model equation is

1-level:

2-level:

는

번째 속하는

번째 개인의 산화적 스트레스 수준을 의미하고,

는 1-수준 변수이고, 회귀계수

는 고정된 상수이다. 2-수준에서 추정된 회귀계수는 고정된 상수가 아니라 확률 변수로 가정한다.

는 혼합 모형에 대한 변수를 의미하고, 회귀계수

는 랜덤효과를 나타내는 확률변수를 의미한다.

는 2-수준

에 따라서만 값이 달라지며,

군집 내에서는 모든 관측치

가 동일한 값을 갖는다.The 1-level model is a traditional linear regression model that considers only fixed variables, and these variables assume that the effect on oxidative stress levels is fixed regardless of age.

The

Belonging to the second

Means the level of oxidative stress in the first individual,

Is a 1-level variable, and the regression coefficient

Is a fixed constant. The regression coefficient estimated at 2-level is assumed to be a random variable rather than a fixed constant.

Means the variable for the mixed model, and the regression coefficient

Is a random variable representing a random effect.

Is 2-level

The value only changes depending on

All observations within the cluster

Has the same value.

In the present invention, the step of assigning weights to the selected factors in consideration of the estimated amount of the selected factors is weighted according to the influence of the selected factors on the oxidative stress level, and thus the influence on the oxidative stress level of each factor It is a step to adjust to reflect the. Among factors associated with oxidative stress levels, factors that are highly related to oxidative stress are given high weights, and factors that are relatively less relevant are excluded, so that more accurate quantification is possible when finally deriving oxidative stress. The estimator includes instrumental variable estimation, ordinary least square estimation, non-linear least square estimation, iterative weighted least square estimation, and maximum. It can be considered as one or more methods selected from the group consisting of likelihood likelihood estimation (MLE), limited maximum likelihood estimation (RMLE), and limited maximum likelihood estimation (DF REML) without differential equations, preferably through RMLE. It can be derived, but is not limited thereto.

The limited limited maximum likelihood estimation (RMLE) method generates a likelihood based on the residuals to obtain a maximum likelihood estimation of variances by maximizing the limited likelihood function to estimate the parameter value of the linear mixed effect model. Fit statistics based on this method can be used to compare different covariance models based on the same average model. In general, the solution of REML is equal to the ANOVA estimator for balanced data, and is less sensitive to outliers in the data than the maximum likelihood estimation (MLE) method, and has an unbiased variance.

For example, in the present invention, determining the degree of oxidative stress may be determined using Equation 1 below:

[Equation 1]

In Equation 1, each variable is defined as follows:

Y is the calculated value of the oxidation stress level, the unit is a score,

Age is the age of the subject, and the unit is three years.

Smoking is the subject's smoking status, 1 if the subject smokes, 0 if the subject is non-smoking or has quit smoking for more than 1 year;

MDA is the subject's plasma lipid peroxide, with units of μM;

WBC is the number of white blood cells measured in a subject's whole blood sample, and the unit is x10 ³ /µl;

GPT is the alanine aminotransferase level of the subject and the unit is IU/L.

In the present invention, the step of quantifying the degree of oxidative stress of the test subject corresponds to biological information of the test subject to the selected factors, and corrects it by reflecting the assigned weight, and finally oxidative test subject It is a step to determine the degree of stress. For example, the numerical value of the oxidative stress level can be obtained by substituting each information of the test subject into Equation (1).

Numerical values of the derived oxidative stress levels are 2.0 or more, 2.1 or more, 2.2 or more, 2.3 or more, 2.4 or more, 2.5 or more, 2.6 or more, 2.7 or more, 2.8 or more, 2.9 or more, 3.0 or more, 3.1 or more, 3.2 or more , 3.3 or more, 3.4 or more, or 3.5 or more, the test object may be determined to have oxidative stress, and more preferably, when the numerical value of the derived oxidative stress level is 2.7 or more, the test object Can be determined to have oxidative stress.

Another embodiment of the present invention comprises the step of quantifying the degree of oxidative stress of the test subject, using an influence factor of the oxidative stress level before and after the candidate substance is administered to the test subject, It is about.

According to another embodiment of the present invention, the oxidative stress level measured in the test subject before the candidate substance is administered; And comparing the oxidative stress level measured in the test subject after the candidate material is administered, and the oxidative stress level measured after the candidate material is administered is reduced compared to the oxidative stress level before the candidate material is administered. , The step of selecting the candidate substance as an antioxidant, and measuring the oxidative stress level of the test subject, age of the subject, whether smoking, plasma lipid peroxide concentration, whole blood leukocyte count, and alanine aminotransferase concentration And measuring the influence factor of at least one oxidative stress level selected from the group consisting of, and determining the degree of oxidative stress of the test subject using the measured value of the influence factor, screening of antioxidants. Is to provide a way.

The candidate substance may be a substance expected to have antioxidant activity, for example, a drug or a food, and preferably a food.

In the present invention, the term "food" includes all foods in the usual meaning, and may be in the form of beverages, candy, powders, etc., for example, may be a health functional food or a health food. Functional food refers to food having a high medical effect processed to efficiently exhibit bio-control functions in addition to the purpose of supplying nutrition. Here, "functional" means obtaining a useful effect for health use, such as adjusting nutrients or physiological effects on the structure and function of the human body. For example, the antioxidant food in the present invention may have a function of lowering the degree of oxidative stress of the individual. For example, when comparing the oxidative stress level measured in a test subject before the candidate substance is administered and the oxidative stress level measured in the test subject after the candidate substance is administered, it is measured after the candidate substance is administered. When the oxidative stress level decreases compared to the oxidative stress level measured before the candidate substance is administered, when the candidate substance is food, the candidate substance may be selected as an antioxidant food.

In the present invention, food may be prepared by a method commonly used in the art, and at the time of manufacture, raw materials and ingredients that are conventionally added in the art may be added. In addition, the formulation of the food can be prepared without limitation as long as it is a formulation recognized as food. The antioxidant food of the present invention can be manufactured in various types of formulations, and has the advantage of having no side effects that may occur when taking the drug for a long time using food as a raw material, unlike general drugs, and an auxiliary agent for reducing oxidative stress Can be consumed as. The health food refers to food having an active health maintenance or enhancement effect compared to general food, and health supplement food refers to food for health supplement purposes. In some cases, the terms health functional food, health food, and dietary supplement are used interchangeably. Specifically, the health functional food means that it has a health-specific effect when administered to an individual, but has the advantage that there is no side effect that can occur when taking the drug for a long time using food as a raw material, unlike general drugs. In the present invention, the health functional food can be consumed on a daily basis, and can bring about an effect of reducing oxidative stress.

The influence factor, as described above, is weighted in consideration of the estimated amount of each factor, and the estimated amount is instrumental variable estimation, ordinary least square estimation, and nonlinear least square estimation. (non-linear least square estimation), iterative weighted least square estimation, maximum likelihood estimation (MLE), limited maximum likelihood estimation (RMLE), and limited maximum likelihood estimation without differential equations (DF) REML) may be considered as one or more methods selected from the group consisting of, but is not limited thereto.

In the present invention, "object", "test object", "test subject", "subject", "subject", or "patient" is a human, mouse, mouse, hamster, rat, guinea pig, rabbit, cow, dog, cat, By chicken, insect, frog, mammal, non-human mammal, primate, non-human primate, reptile, amphibian, rodent, etc. means any single individual. Also included are any subjects who participated in clinical research trials that did not show any disease clinical manifestations, or subjects who participated in epidemiological studies or subjects used as controls.

The test subject, as described above, in the group consisting of human, mouse, mouse, hamster, rat, guinea pig, rabbit, cow, dog, cat, chicken, insect, frog, mammal, primate, reptile, amphibian, and rodent It may be one or more selected.

The oxidative stress level may be measured using [Equation 1]. In the screening method of the antioxidant, in the test subject before the candidate substance is administered, the calculated value of the degree of oxidative stress measured using [Equation 1] may be 2.7 or more.

An example of the present invention relates to a method for providing information for diagnosis of an oxidative stress-related disease, comprising comparing the oxidative stress level of a test subject with an oxidative stress level measured in a normal control group.

According to another embodiment of the present invention, a method of providing information for the diagnosis of oxidative stress-related diseases, comprising comparing the oxidative stress level of a test subject with an oxidative stress level measured in a normal control group, Measuring the oxidative stress level of the test subject, the subject's age, smoking status, plasma lipid peroxide concentration, whole blood leukocyte number, and alanine aminotransferase concentration selected from the group consisting of an influencing factor of at least one oxidative stress level It relates to a method for providing information for the diagnosis of oxidative stress-related diseases, comprising the steps of measuring and determining the degree of oxidative stress of the test subject using the measured value of the influence factor.

The influence factor, as described above, is weighted in consideration of the estimated amount of each factor, and the estimated amount is instrumental variable estimation, ordinary least square estimation, and nonlinear least square estimation. (non-linear least square estimation), iterative weighted least square estimation, maximum likelihood estimation (MLE), limited maximum likelihood estimation (RMLE), and limited maximum likelihood estimation without differential equations (DF) REML) may be considered as one or more methods selected from the group consisting of, but is not limited thereto.

The oxidative stress level may be measured using [Equation 1].

In the method for providing information for diagnosing the oxidative stress-related disease, the calculated value of the degree of oxidative stress measured using [Equation 1] in the normal control may be less than 2.7 points.

In the present invention, oxidative stress-related diseases include all symptoms that may occur due to free radicals in the body, or diseases that can be invented, for example, lipid peroxide production in tissues in the body, lipid peroxide accumulation in cell membranes, stroke, myocardial infarction , Infertility, hepatitis, arthritis, cataracts, may mean symptoms or diseases such as aging, but is not limited thereto.

Quantitative measurement method of oxidative stress level according to the present invention performs external verification using human applied research data to confirm the mediating effect of antioxidant food ingredients, and controls the oxidative stress level at baseline and end of mediation As a result of comparing the test food group with, it can be useful as a tool for determining the mediating effect of antioxidant food materials, and can predict the degree of change in antioxidant capacity in response to an individual's food material intake.

1 shows a process of classifying subjects into health groups and oxidative stress disease groups according to their health status.
2 is a graph showing the distribution of the estimation of the degree of oxidative stress of a subject calculated according to the method for measuring oxidative stress according to the present invention.
Figure 3 shows the ROC curve verifying the predictive ability of the oxidative stress measurement method according to the present invention through the LOOCV method, the diagonal line (gray) represents the baseline 0.5.
4 is a graph showing that the oxidative stress level of the antioxidant food material intake group and the control food group was significantly reduced in the antioxidant food material intake group as a result of measuring according to the oxidative stress measurement method according to the present invention.
5 is a graph obtained by comparing the oxidative stress level when the estimator is considered and the oxidative stress level when the estimator is not considered. Can.

Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example 1: Development of a method for quantifying oxidative stress

1-1: Classification of patients with oxidative stress-related diseases

We developed a prognosis prediction model that distinguishes healthy subjects from patients with oxidative stress-related diseases in Korean adult men and women over the age of 19.

Specifically, plasma lipid peroxide and carotenoids levels were analyzed in subjects (1,464 people as of June 2017) among adult men and women aged 19 and over who participated in the study.Recommended food score (RFS), Demographic and socio-economic characteristics and questionnaire response results were collected.

Subjects with missing dietary scores (RFS), those with at least one metabolic risk factor, or those with metabolic disease (high waist circumference, high triglyceride level, high blood sugar, low low density cholesterol, or high blood pressure) ), subjects with other diseases, subjects with a history of past illnesses or treatments, and subjects undergoing continuous drug therapy were excluded first. The risk of metabolism was established by the National Cholesterol Education Program Adult Treatment Panel III, which made the National Heart, Lung, and Blood Institute easier to diagnose metabolic syndrome clinically. (Circulation 2005; 112:2735-52). However, among the diagnostic criteria, the waist circumference standard of Koreans (Diabetes Res Clin Pract 2007;75:72-80) was applied.

(1) Waist circumference: 90 cm or more for men and 85 cm or more for women;

(2) blood pressure: systolic blood pressure of 130 mmHg or higher, or diastolic blood pressure of 85 mmHg or higher;

(3) Fasting blood sugar: 110 mg/dL or more;

(4) triglycerides: 150 mg/dL or more;

(5) HDL (high density)-cholesterol: 40 mg/dL or less for men and 50 mg/dL or less for women.

Except for the excluded subjects, 603 subjects who could be analyzed were selected. The 603 selected people were classified into a health group (159 people) and an oxidative stress disease group (444 people) according to their health status. Specifically, the subject was classified as a healthy group when no metabolic risk factor or disease related to oxidative stress was reported. Oxidative stress and related diseases include diabetes (J Biochem Mol Toxicol 2003, 17:24-38), hyperlipidemia (Obes Res Clin Pract 2013, 7:e330-341), obesity (J Clin Invest 2004, 114:1752-1761) , Cardiovascular disease (Nutr J 2007, 6:39), stroke (Free Radic Biol Med 2005, 39:841-852), hypertension (Nutr J 2007, 6:39), diet-related cancer (liver, colon, stomach, breast , Prostate, lung; Lancet 2002, 360:861-868, World Health Organization 2003).

1 shows a process of classifying subjects into health groups and oxidative stress disease groups according to their health status.

1-2: Evaluation factor and model of oxidative stress level

For the 603 subjects who were able to make final analysis, an index with a significant probability of less than 0.05 was selected between the healthy group and the oxidative stress disease group, and then a hierarchical linear mixed model was applied.

Among clinical indicators (ages) related to oxidative stress, age is determined by random effects, gender by fixed effect, whether it is smoking, plasma lipid peroxide levels, carotenoids, antioxidant food intake scores, clinical chemistry indicators (albumin) , Alkaline phosphatase, C-reactive protein, alanine aminotransferase, aspartate aminotransferase, uric acid), leukocyte differentiation factor (neutrophils, monocytes), hematologic indicators (erythrocyte sedimentation rate, erythrocyte volume, leukocyte count) Did. In order to select only those variables that are highly related to oxidative stress in the model, backward elimination and multicollinear variables are removed to remove the least helpful variables (significance probability is 0.05 or more) one by one. Did.

Then, the suitability test of a model containing different variables applies Akaike's information criterion (AIC) method, which models the assumption that the model with the least loss of information is selected as the best data and the most suitable model. The model with values was chosen as the optimal model. As a result of the final modeling, it was confirmed that the presence or absence of smoking, the plasma lipid peroxide, the number of white blood cells, and the level of alanine aminotransferase were significant factors for evaluating the level of oxidative stress (the probability of significance was less than 0.05 without multicollinearity). Finally, considering the parameter estimates of each factor derived through the limited maximum likelihood estimation (RMLE), the oxidative stress evaluation method was formulated as follows.

[Equation 1]

Y is the calculated value of the oxidative stress level, the unit is a score,

Age is the age of the subject, and the unit is three years.

Smoking is the subject's smoking status, 1 if the subject smokes, 0 if the subject is non-smoking or has quit smoking for more than 1 year;

MDA is the subject's plasma lipid peroxide, with units of μM;

WBC is the number of white blood cells measured in a subject's whole blood sample, and the unit is x10 ³ /µl;

GPT is the alanine aminotransferase level of the subject and the unit is IU/L.

1-3: Evaluation of evaluation model of oxidative stress level

As a result of recalculating the oxidative stress score through the final derived equation (1) for 603 subjects that could be analyzed, it was found that the healthy person and the person with oxidative stress disease were well distinguished through this score (FIG. 2).

Figure 2 is a graph showing the distribution of the estimation of the degree of oxidative stress of the subject calculated according to the method for measuring oxidative stress according to the present invention, the horizontal axis is the oxidative stress score (unit: point) calculated through the final derived equation (1), The vertical axis is the percentage of each section (unit: %).

Example 2: Internal verification of an evaluation model of oxidative stress levels

The predicted ability of the oxidative stress quantification method developed in Example 1 was verified through internal validation through a ROC (Receiver Operating Characteristic) curve and LOOCV analysis.

Internal verification was performed by applying the Leave-One-Out Cross Validation (LOOCV) method. This method is a total number of data (N) clustered and tested one by one to create a model corresponding to the total number of samples. When creating each model, test samples are excluded except for one sample. It is a method to calculate the performance of and average the N performances.

The area under the curve-ROC (AUC-ROC) of the entire training set was 0.91 (sensitivity 82.0%, specificity 84.3%). As a result of cross-validation using the LOOCV method, it was confirmed that the AUC-ROC was developed with 0.82 (sensitivity 76.8%, specificity 76.7%), and the developed oxidative stress prognosis prediction model has good performance (FIG. 3). Figure 3 shows the ROC curve internally verified through the LOOCV method of predicting the oxidative stress measurement method according to the present invention, the diagonal line (gray) represents the baseline 0.5. In FIG. 3, it was found that the area under the ROC curve of the Original and LOOCV (AUC-ROC) was 0.8 or more, showing good performance of the oxidative stress measurement method according to the present invention.

Example 3: Assay of evaluation model of oxidative stress level using antioxidant food material

In order to confirm whether the quantification model developed in Example 1 can change the oxidative stress evaluation score by reflecting the actual nutritional intervention effect, an external validation method using nutritional intervention research data using a separate antioxidant food material Proceeded.

Specifically, randomized, double-blind, control comparison, and parallel study data (J Funct Foods, 2018;46:356-364) were used to observe the mediating effect of antioxidant food material for 12 weeks in healthy adults without disease. . 80 adult males and females (20 males, 60 females) who are overweight or obese (BMI 25-30 kg/m ² , skeletal muscle mass 39.8% (male)/34.1% (female) or younger) between 20 and 50 years of age ; Mean age 37.3 years; mean body mass index 27.3 kg/m ² ; fasting blood glucose 91.3 mg/dL) was randomly assigned to the group consuming the antioxidant food material and the control food group. In the group ingesting antioxidant food ingredients, 2 capsules of citricol ethanol extract ( Ecklonia cava ), which has been reported to have antioxidant and anti-inflammatory activity (containing 960 mg/g of total polyphenol content), are watered immediately after breakfast and evening. And ingested with.

The model developed above was applied to the intervention data to evaluate the level of oxidative stress at baseline and at the end of intervention, and to analyze whether the estimated individual's oxidative stress level differed before and after intervention.

As a result of comparison between the group consuming the antioxidant food material and the control food group, it was confirmed that the oxidative stress level according to the quantification model developed in Example 1 was significantly reduced in the group consuming the antioxidant food material (FIG. 4).

4 is a graph showing that the oxidative stress level according to the quantification model developed in Example 1 was significantly reduced in the group consuming the antioxidant food material. Levels of oxidative stress at baseline and at the end of intervention in the group ingesting antioxidant foods and in the control food group taking into account parameter estimates derived from a mixed hierarchical model that includes age as a random effect. Was calculated. Statistical significance was confirmed by comparing the differences between the groups ingesting antioxidant food ingredients and before and after intervention (intervention end point-base point point) between groups. Each bar graph is expressed as the mean and standard error of mean. The significance probability P-value in the statistical hypothesis test was determined by Student's t- test.

Example 4: Evaluation of discriminative power in patients with oxidative stress disease

After calculating the oxidative stress level when the estimator was considered and the oxidative stress level when it was not considered, it was evaluated how well a person with oxidative stress disease can be identified.

Specifically, the oxidative stress level was derived as shown in Equation 2 below by simply summing the original measured values without applying the estimator applied to each factor derived in Example 1-2.

Y is the calculated value of the oxidation stress level, the unit is a score,

Age is the age of the subject, and the unit is three years.

Smoking is the subject's smoking status, 1 if the subject smokes, 0 if the subject is non-smoking or has quit smoking for more than 1 year;

MDA is the subject's plasma lipid peroxide, with units of μM;

WBC is the number of white blood cells measured in a subject's whole blood sample, and the unit is x10 ³ /µl;

GPT is the alanine aminotransferase level of the subject and the unit is IU/L.

When considering the estimator, AUC-ROC (Area under the curve-ROC) was 0.91 (sensitivity 82.0%, specificity 84.3%; Youden index 0.66). When the estimator was not considered, AUC-ROC was derived as 0.82 (sensitivity 68.0%, specificity 82.4%; Youden index 0.50). Therefore, it was confirmed that the case considering the estimated amount has better performance in evaluating the discriminative ability for the presence or absence of oxidative stress disease (FIG. 5 ).

Claims (17)

And quantifying the degree of oxidative stress of the test subject using the influence factor of the oxidative stress level measured in the test subject,
The oxidative stress level influencing factor is at least one selected from the group consisting of the age of the test subject, whether or not smoking, plasma lipid peroxide concentration, whole blood leukocyte number, and alanine aminotransferase concentration,
Method for measuring oxidative stress level in test subject. The method of claim 1, wherein the influence factor is weighted by taking into account the estimated amount of each of the influence factors, and the consideration of the estimated factor is instrumental variable estimation, ordinary least square estimation, and nonlinearity. Use non-linear least square estimation, iterative weighted least square estimation, maximum likelihood estimation, restricted maximum likelihood estimation, and differential equations The method is performed by one or more methods selected from the group consisting of deriveative-free restricted maximum likelihood estimation. The method of claim 1, wherein the degree of oxidative stress of the test subject is determined using Equation 1 below:
[Equation 1]

Y is the calculated value of the degree of oxidative stress, the unit is a score,
Age is the age of the subject, and the unit is three years.
Smoking is the subject's smoking status;
MDA is the subject's plasma lipid peroxide, with units of μM;
WBC is the number of white blood cells measured in a subject's whole blood sample, and the unit is x10 ³ /µl;
GPT is the alanine aminotransferase level of the subject and the unit is IU/L. The method of claim 3, further comprising determining that the subject has oxidative stress when Y of the subject is 2.7 or more. The method of claim 1, wherein the test subject is in the group consisting of human, mouse, mouse, hamster, rat, guinea pig, rabbit, cow, dog, cat, chicken, insect, frog, mammal, primate, reptile, amphibian, and rodent. Method of one or more selected. The oxidative stress level in the test subject is measured before and after the candidate substance is administered, and the oxidative stress level measured before and after the administration of the candidate substance is compared, and the oxidative stress measured after the candidate substance is administered. When the stress level is lower than the oxidative stress level measured before the candidate substance is administered, the method includes selecting the candidate substance as an antioxidant substance,
Measuring the oxidative stress level of the test subject,
And quantifying the degree of oxidative stress of the test subject using an influence factor of the oxidative stress level of the test subject,
The oxidative stress level influencing factor is at least one selected from the group consisting of the age of the test subject, whether or not smoking, plasma lipid peroxide concentration, whole blood leukocyte number, and alanine aminotransferase concentration,
Methods of screening for antioxidants. The method of claim 6, wherein the influence factor is weighted by taking into account the estimator of each factor, and the estimator is instrumental variable estimation, ordinary least square estimation, and nonlinear least square estimation ( Non-linear least square estimation, iterative weighted least square estimation, maximum likelihood estimation, restricted maximum likelihood estimation, and limited maximum without using differential equations A method that is considered as one or more methods selected from the group consisting of derivative-free restricted maximum likelihood estimation. The method according to claim 6, wherein the oxidative stress level is measured using Equation 1 below.
[Equation 1]

Y is the calculated value of the degree of oxidative stress, the unit is a score,
Age is the age of the subject, and the unit is three years.
Smoking is the subject's smoking status;
MDA is the subject's plasma lipid peroxide, with units of μM;
WBC is the number of white blood cells measured in a subject's whole blood sample, and the unit is x10 ³ /µl;
GPT is the alanine aminotransferase level of the subject and the unit is IU/L. The method of claim 6, wherein the test subject is in the group consisting of human, mouse, mouse, hamster, rat, guinea pig, rabbit, cow, dog, cat, chicken, insect, frog, mammal, primate, reptile, amphibian, and rodent. Method of one or more selected. The method according to claim 8, wherein in the test subject before the candidate substance is administered, the calculated value of the degree of oxidative stress measured using [Equation 1] is 2.7 or more. The method of claim 6, wherein the candidate substance is food. A method for providing information for the diagnosis of oxidative stress-related diseases, comprising comparing the oxidative stress level of a test subject with a oxidative stress level measured in a normal control group,
Measuring the oxidative stress level of the test subject,
The subject's age, smoking status, plasma lipid peroxide concentration, whole blood white blood cell count, and alanine aminotransferase concentration were measured, and one or more oxidative stress levels were selected.
Determining the degree of oxidative stress of the test object using the measured value of the influencer,
Methods of providing information for the diagnosis of oxidative stress-related diseases. The method of claim 12, wherein the influence factor is weighted by taking into account the estimator of each factor, and the estimator is instrumental variable estimation, ordinary least square estimation, and nonlinear least square estimation ( Non-linear least square estimation, iterative weighted least square estimation, maximum likelihood estimation, restricted maximum likelihood estimation, and limited maximum without using differential equations A method that is considered as one or more methods selected from the group consisting of derivative-free restricted maximum likelihood estimation. The method of claim 12, wherein the oxidative stress level is measured using Equation 1 below.
[Equation 1]

Y is the calculated value of the degree of oxidative stress, the unit is a score,
Age is the age of the subject, and the unit is three years.
Smoking is the subject's smoking status;
MDA is the subject's plasma lipid peroxide, with units of μM;
WBC is the number of white blood cells measured in a subject's whole blood sample, and the unit is x10 ³ /µl;
GPT is the alanine aminotransferase level of the subject and the unit is IU/L. The method of claim 14, wherein the calculated value of the degree of oxidative stress measured using [Equation 1] in the normal control is less than 2.7 points. The method of claim 12, wherein the test subject is in the group consisting of human, mouse, mouse, hamster, rat, guinea pig, rabbit, cow, dog, cat, chicken, insect, frog, mammal, primate, reptile, amphibian, and rodent. Method of one or more selected. The oxidative stress-related disease is at least one selected from the group consisting of stroke, myocardial infarction, infertility, hepatitis, arthritis, cataracts, aging, lipid peroxide overproduction in tissues, and accumulation of lipid peroxide in cell membranes. , Way.

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