KR20150097892A

KR20150097892A - Detection mean of oxygen free radical using a creatinine ratio in urinalysis and its method

Info

Publication number: KR20150097892A
Application number: KR1020140018598A
Authority: KR
Inventors: 윤중근; 박용애; 김태완; 강상훈; 김기동
Original assignee: 주식회사 디에프아이
Priority date: 2014-02-18
Filing date: 2014-02-18
Publication date: 2015-08-27

Abstract

The present invention provides a method and apparatus for detecting active oxygen in the human body through correction of creatine, which enables accurate detection of active oxygen in the human body regardless of the individual's body condition and water intake. First detection means for detecting aldehyde and second detection means for detecting creatine contained in the urine; It is possible to provide an active oxygen detection means in the human body through the creatine correction for measuring the amount of active oxygen in the human body by comparing and analyzing the values detected by the first detection means and the second detection means, A first step of detecting an amount; A second step of detecting the amount of creatine contained in urine; A third step of comparing and analyzing the values detected in the first and second steps; And a fourth step of determining the amount of active oxygen as a value comparatively analyzed in the third step. Therefore, the present invention is effective in correcting active oxygen by correcting the quantitative change of the analysis value of malondialdehyde according to the individual condition by performing correction through the creatinine analysis contained in the urine.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and apparatus for detecting active oxygen in human body through correction of creatine,

The present invention relates to an active oxygen detecting means which is a harmful element in a human body, and more particularly, to an active oxygen detecting means which detects urine by detecting the amount of active oxygen remaining in the body by urine, And a means for detecting active oxygen in the body through the correction of creatine.

Humans rely on carbohydrates and fat metabolism to supply enough energy (ATP) for survival and activity, and humans can not survive unless oxygen is supplied to the cells. In the process of oxygen metabolism in the body for this energy production, Free radicals called noxious oxygen or active oxygen are generated. These reactive oxygen species have various negative effects on the human body, for example, to produce lipid peroxides in human tissues. Peroxidation of lipid means the oxidative damage of unsaturated lipids due to the physiological action that takes place continuously in the cell membrane. That is, lipid peroxide is produced in the cell membrane by oxidizing the phospholipid in the cell membrane containing a large amount of the unsaturated fatty acid such as free radical, which is active oxygen. That is, the increase of active oxygen increases the production of lipid peroxides. Excessive lipid peroxides cause various human diseases such as stroke, myocardial infarction and hepatic diseases such as alcoholic hepatitis caused by cerebrovascular disorder.

Therefore, it is very important to quantitatively check the amount of active oxygen as an index of health prevention, and many devices have been developed for measuring the amount of active oxygen. In recent years, due to the development of biosensors, there has been a tendency to be smaller and more precise. However, currently used devices are expensive electronic sensor devices, so that anyone can not easily access them universally.

Therefore, in order to solve such a problem, the present applicant has previously filed a patent application for the active oxygen detection means and method using a test paper. That is, according to Korean Patent Laid-Open Publication No. 10-2013-0088623 (active oxygen detecting means in the human body), harmful active oxygen present in blood is very reactive and very difficult to directly measure, The amount of peroxide is detected and used as a marker of active oxygen. Malondialdehyde (MDA) is one of the measurement methods for determining the degree of lipid peroxidation. It is an index used for health impact assessment by easily detecting it in blood tissues. Measurement of malondialdehyde (MDA) (MDA) can be quantitatively detected only by color change of color by absorbing urine, so that it is possible to detect the degree of peroxidation of blood and urine, and it is possible to detect the amount of malondialdehyde (MDA) An apparatus and a method for providing active oxygen detection means in a human body have been proposed.

However, the applicant's prior patent, which utilizes the color development change according to the amount of malondialdehyde (MDA) in the urine, is very simple since it is measured with monovalent urine. However, in order to accurately measure each individual's water intake or body condition, There is a problem that can not be satisfied.

Korean Patent Publication No. 10-2014-0016055 Korean Patent Publication No. 10-2013-0088623

Accordingly, in order to solve such a fundamental problem of the prior art, the present invention has been made to use calcein to perform correction so as to accurately detect active oxygen in the human body, irrespective of the individual's body condition and water intake, (MDA) and color change of creatine, thereby enabling accurate detection of the amount of active oxygen in the human body, and a method thereof.

The objects of the present invention are not limited to the above-mentioned objects, and other objects not mentioned can be clearly understood from the following description.

According to another aspect of the present invention, there is provided a method for detecting urine, comprising: a first detecting means for detecting malondialdehyde contained in the urine; a second detecting means for detecting creatine contained in the urine; There is a technical feature in that the means for detecting active oxygen in the human body through the creatine correction which measures the amount of active oxygen in the human body by comparing and analyzing the values detected by the first detection means and the second detection means is provided.

The first detecting means is a first test strip embedded with a coloring indicator which reacts with malondialdehyde to cause color development, and the second detecting means is a second test strip embedded with a coloring indicator which causes a color change by reacting with creatine. The first test strip can be formed from a mixture of 1,3-Diethyl-2-Thiobarbituric Acid (TBA), Basic fuchsine, Pararosaniline Hydrochlpride ) Or glycine is used as a color development indicator and the second test paper is prepared by the Jaffe method using the principle of combining a urine creatinine with a picrate to generate a Janovski bond, Alternatively, Cu2 (metal) ion and urine creatinine are combined to decompose the caustic hydrogen peroxide to be a coloring indicator for color development with benzidine (ortho-toluidine, tetramethylbenzidine, 1,6-dihydroperoxide) 1 The test strip and the second test strip are attached to the upper surface of the support so that the support is immersed in the urine.

On the other hand, the present invention provides a method for detecting malondialdehyde, comprising: a first step of detecting the amount of malondialdehyde contained in the urine; A second step of detecting the amount of creatine contained in urine; A third step of comparing and analyzing the values detected in the first and second steps; And a fourth step of determining the amount of active oxygen as a value comparatively analyzed in the third step. The present invention also provides a method for detecting active oxygen in the human body through the correction of creatine.

Preferably, the first step and the second step use a color change value by color development, and the third step uses a color change value of the malon diaradehide / creatinine ratio (umol / L) / (mmol / L) . According to the ratio, it is determined that 0-0.23 is normal, 0.24-1 is low, 1 - 3 is high, and 3 or more is very high. Alternatively, in the first step, the color change value by color development is represented by four levels, the second step is represented by five levels of color change value by color development, a table showing the state of active cores by the step classification is made , The detected color change value is compared with a prepared color chart table to determine which stage belongs, and the state of active oxygen is determined by finding a corresponding point in the table. The table showing the state shows the creatine concentration on the horizontal axis And the vertical axis represents the concentration of malondialdehyde in four steps, and is shown in the form of Table 3.

The present invention is more effective in correcting active oxygen by correcting the quantitative change of the analysis value of malondialdehyde according to the individual state by performing correction through the creatinine analysis contained in the urine.

In addition, since the present invention does not require a specific facility for inspection, it can be inspected at any time, anywhere, and there are other effects that can be universally used.

Fig. 1 shows the color change of the test paper according to the concentration of creatine,
(a) is a graph showing a color change due to a Jaffe method using the principle that creatine is coupled with picrate to generate a Janovski bond,
(b) is a benzidine was coupled to the Cu ² ⁺ metal ions and Creatinine in the urine decomposition caustic hydrogen peroxide showing a color change due to the (ortho-tolyl Ruthin, tetramethylbenzidine, 1,6-di-hydroperoxide hydroxide) and the color-developing Degree.
Fig. 2 is a view showing a support provided with a test strip containing MDA and creatinine coloring indicator according to the present invention. Fig.
FIG. 3 is a graph showing the color change of creatine according to the concentration according to the present invention. FIG.
4 is a graph showing the color change according to the concentration of malondialdehyde (MDA) according to the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS The advantages and features of the present invention and the manner of achieving them will become apparent with reference to the embodiments described in detail below with reference to the accompanying drawings. However, the present invention is not limited to the embodiments described below, but may be embodied in various different forms, and these embodiments are not intended to be exhaustive or to limit the scope of the present invention to the precise form disclosed, The present invention is only defined by the description of the claims. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In the present specification, the singular form includes plural forms unless otherwise specified in the specification. Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings.

Example 1: Status according to MDA concentration in urine

1) Setting the reference range of MDA concentration

The normal range of MDA concentration in normal urine was 0-4 μmol / L, and it was 0-4 μmol / L when 30 healthy individuals were measured. It was found that the values measured by HPLC and TBARS and the results of the test paper measurement values shown in the applicant's prior patent are the same.

Therefore, the MDA concentration was divided into four stages, Normal, Low, High and Very high, based on the above-mentioned articles and generally known values. The measurement range is normal (0-4umol / L), low (5-9umol / L), high (10-19umol / L), very high (20-30umol / L). The reason is as follows.

Low (Low) (5-9umol / L) reference setting

When 30 days of 30 days in the general group are continuously measured MDA of the first morning, 99% of the measured results are overdosed the day before the measurement, or on Friday, when fatigued, 5-9umol / L range results after extreme exercise. Therefore, if the healthy person is a normal person, this concentration range is indicated as LOW because the 5-9 μmol / L step occurs depending on the situation.

Setting the standard of High (10-19umol / L), Very High (20-30umol / L)

The urine MDA level in 10 burn patients was measured to be very high, 20-25 umol / L, when the amount of urine MDA was measured 20 times or more. Based on this, 20-30 umol / L concentration was defined as Very High interval, The concentration of -19 umol / L was indicated as high.

2) Establishment of standards for color change of test paper

Since the actual test paper is displayed as a semi-quantitative amount, the concentration is expressed as Color as follows. The low concentration of low concentration was set to the color standard and the high concentration was set to the color of the medium concentration.

0-4umol / L -> Normal -> 1umol / L

5-9umol / L -> Low -> 5umol / L

10-19umol / L -> High (high) -> 10umol / L

20-30 umol / L -> very high -> 25 umol / L

3) Clinical test

In the present study, 35 patients (0-4 μmol / L), 51 patients (5-9 μmol / L) and 45 patients (10 patients) were included in this study. -19umol / L) and 19 patients (20-30umol / L). At the same time, however, 30 out of 35 (0-4 μmol / L) urine samples were within this range, but 5 were in the range of 5-9 μmol / L. Forty-two (5-9 μmol / L) were within this range, four (0-4 μmol / L) and five (10-19 μmol / L). (10-19 μmol / L), 40 were within this range, 3 (5-9 μmol / L) and 2 (20-30 μmol / L) (20-30 μmol / L), 16 were within this range and 3 were within the range (10-19 μmol / L). The results are summarized in the table below.

4) Summary of test results

Results in single urine were 85.7%, 82.3%, 88.8% and 84.2%, respectively, when compared with results in 24 hour urine. That is, 11.2% -17.7% are inconsistent results.

This discrepancy is interpreted as the fact that the urinary MDA excretion rate can be temporarily changed by various factors such as water intake, hyperglycemia, hypertension, exercise, and posture. Therefore, in consideration of each individual change factor, the value should be the same as the value of urine collected 24 hours in single urine measurement value.

Example 2)

1) Introduction of creatinine as a correction factor

Creatinine, like urea nitrogen and uric acid, is a waste of protein used as energy in the body. It is used as energy in the muscle and then formed into creatinine or creatinine phosphate, which is released into the blood and excreted from the kidneys into the urinary tract.

The effect of MDA concentration due to changes in urine volume can be avoided because the creatinine excretion is constant if the glomerular filtration function is constant. The concentration of creatinine in normal glomeruli ranges from 10 to 300 mg / dL (0.9 to 26.5 mmol / L). The correlation between the measured concentration of creatinine and the measured concentration of MDA leads to more accurate results by analyzing the quantitative ratio Will.

2) Analysis of MDA / creatinine (umol / mmol) value

The concentration of creatinine in normal glomeruli was 10-300 mg / dL (0.9-26.5 mmol / L), and the relationship between the measured concentration of creatinine and the measured concentration of MDA was examined.

In the prior patent of the present applicant, since the amount of MDA can be measured by visual observation through color change by a color development indicator and measurement by using a photometer, the present invention is also applicable to both analysis of color change of MDA and creatine and analysis through a photometer desirable.

For this, it was found that it is easy to divide the MDA into 4 stages and to observe the color change by dividing the creatine into 5 stages. In the case of the MDA, as shown in the applicant's prior patent, it is sufficient to compare the color change of the test paper with a color chart to observe the color change or to quantify the color change value using the photometer. In the case of creatine, it is relatively easy to observe the human eye by dividing it into five steps for observing color change. Such a color change may be compared with an existing color chart, and it may suffice to accurately analyze the color change value through the photometer. As shown above, MDA was divided into 4 levels and creatine was divided into 5 levels, and MDA / creatinine (umol / mmol) was shown in Table 3 below.

When the concentration of creatine in the above table is 0.9 mmol / L, the symbol ★ is diluted so that the sample is read accurately in proportion.

According to the experimental results, it is not judged positive according to the amount of cryatin in case of normal. Therefore, it was found that the normal ratio included in the range is 0-0.23 μmol / mmol.

During the first two months of the study, the results of the MDA and cryatin measurements were included in the range of 0-1 μmol / mmol for 20 months. Over-exercise, insufficient rest, and so on. After a while, it was read back to normal range. Based on this, it was appropriate to set the low range to 0.24-1 umol / mmol.

In addition, in clinical practice, cryatin <1.8 mmol / L is abnormal and diluted. Therefore, when the amount of MDA is measured at cryatin <1.8 mmol / L, it is judged that the amount of MDA is very high and the ratio of MDA umol / creatinine mmol is 3 or more. Based on this, the range is very high Respectively. Therefore, the concentration of 1-3 umol / mmol was defined as high (High).

These results are summarized as follows: When the ratio of MDA / creatinine (umol / mmol) is 0-0.23, it is normal, when it is 0.24-1, it is low, when it is 1-3, If it is 3 or more, it can be shown as very high.

The results of summarizing based on Table 2 are shown in Table 3 as normal and abnormal values.

As described above, when the normal state is indicated, the color change of MDA and the color change of creatine are visually observed, and it is possible to confirm whether or not it is normal if the range corresponding to the table is found. The creatinine concentration was measured by using biochemical equipment and the creatinine concentration was measured in single urine to determine the ratio of MDA / creatinine (umol / mmol) The results are shown in Table 4 below.

As shown in the above table, 100% agreement is obtained when the ratio of MDA / creatinine (umol / mmol) is adjusted.

Therefore, the MDA ratio for creatinine in one-time urine was calculated and displayed, indicating that the reliability of the result is high.

Example 3) Composition of test strips

Composition of indicators

The means for MDA detection is the same as that of the prior patent of the present applicant (Korean Patent Laid-open Publication No. 10-2013-0088623), and is used as it is.

Indicators of reagents for cryatin detection and methods for their attachment

The cryatin test papers used in the present invention may be satisfactorily used in a conventionally known method. For example, test papers using two principles will be described.

1) It is made by Jaffe method using the principle that urine creatinine is combined with picrate to generate Janovski bond. In this case, the color is reddish purple depending on the amount of creatinine. As shown in FIG. 1 (a), the test paper produced by the autistic method exhibited a color change according to the concentration of creatine, and the color change can be sufficiently visually confirmed as shown, Compared with a color chart, you can identify areas where the concentration of creatine is relevant.

2) It combines Cu2 (metal) ion and creatinine in the urine to decompose caustic hydrogen peroxide and develop color from yellow to green with benzidine (ortho-toluidine, tetramethylbenzidine, 1,6-dihydroperoxide) Method. As shown in Fig. 1 (b), this color change can be visually observed.

How to make test paper

A test paper that causes a color change according to the amount of creatine may be satisfactorily used in a conventional method well known in the art, and will be briefly described.

The cryatin detection test paper preparation solution is composed of a buffer solution which makes a condition to react with the color developing main coloring agent and a reagent which stabilizes the test paper. A solution such as Whatman paper or the like is added to the prepared solution A test paper of a material that can absorb moisture is immersed in the test solution to sufficiently impregnate the picrate (or benzidine (o-tolundine, tetramethylbenzidine, 1,6-dihydroperoxide) After immersion, the test paper is dried at a temperature of about 50 to 60 DEG C for about 20 to 30 minutes in a mechanism such as a dry oven to produce a final test paper.

The creatine color development test paper produced by the above method is formed at a certain interval from the MDA color development test paper, and its appearance is shown in FIG. As shown in FIG. 2, the creatine color development test paper 4 is placed on a surface of the plastic support table 1 at a position spaced apart from the MDA color development test paper 3 by a predetermined distance. Such test strips 3 and 4 may be satisfactorily bonded to the upper surface of the support 1 by using an adhesive agent 2 such as a double-sided tape or an adhesive.

When the detection means is formed as described above, the MDA color development test paper 3 and the creatine color development test paper 4 are dipped in the urine by holding the end of the plastic support 1 with one hand, then observing the color change with naked eyes, By comparing with the color chart, it is possible to determine the concentration of active oxygen by determining which section of Table 3 corresponds.

Example 4 Measurement using a photometer

Since the method of measurement by naked eye shows a semi-quantitative value, the result is limited, and it is preferable to measure using a photo-reflectometer photometer for accurate measurement. Such a photometer is currently on the market, and the present applicant has also developed a measurement-only instrument (Model: MDA R-505) and is currently in use.

The measurement method is such that when the light is emitted from the light emitting part (LED), the amount of light reflected from the test sheet is sensed by the sensor, the color of the reflector is analyzed, and the result is automatically calculated by comparing with the set parameter, And the result of the ratio can determine whether or not the active oxygen contained in the urine is normal.

In the present invention, since creatine is divided into 5 stages and MDA is divided into 4 stages, the divisional calculation method will be briefly described.

Quantitative calculation of color change and concentration change by creatinine

Table 5 shows the color change amount table according to the concentration of creatine.

Based on the above table, the graph is shown in Fig. 3 based on the following equation.

Table 6 shows the color change table according to the concentration of malondialdehyde (MDA).

Based on the above table, the graph of FIG. 4 was made based on the following equation.

Based on the above Tables 5 and 6, the amount of malondialdehyde (MDA) and creatine actually detected can be quantitatively displayed using the equation and the graphs shown in FIG. 3 and FIG. 4, / Creatinine is calculated, and the value of the amount of active oxygen can be confirmed.

Claims

A first detecting means for detecting malondialdehyde contained in the urine;
Second detecting means for detecting creatine contained in urine;
Comparison and analysis means for comparing and analyzing the values detected by the first detection means and the second detection means; And measuring the amount of active oxygen in the human body.

The method according to claim 1,
The first detecting means is a first test paper buried with a coloring indicator which reacts with malondialdehyde to cause color change,
The second detecting means is a second test paper embedded with a coloring indicator which causes color change in response to creatine,
Wherein the comparative analyzing means is a color change table for comparing color changes caused by color development or a photometer for detecting a color change value.

[Claim 3] The method according to claim 2, wherein the color change table indicates the color change value in four stages of malondialdehyde, and the creatine is shown in Table 3 showing the color change value in five levels, The amount of active oxygen in the human body through the correction of creatine.

3. The method according to claim 2, wherein the photometer is expressed by a ratio (mol / L) / (mmol / L) of malondialdehyde / creatine to creatinine correction.

5. The method according to claim 4, wherein 0 to 0.23 is determined to be normal, 0.24 to 1 is measured to be low, 1 to 3 is measured to be high, and 3 or more is determined to be very high, A means for detecting active oxygen in the body by means of creatine correction.

The method of any one of claims 2 to 5, wherein the first test strip comprises at least one of 1,3-Diethyl-2-Thiobarbituric Acid (TBA), Basic Pew Characterized in that any one of basic fuchsine, pararosaniline hydrochluride or glycine is used as a color development indicator.

The method according to claim 6, wherein the second test strip is formed by a Jaffe method using a principle of combining urine creatinine with picrate to generate a Janovski bond, And creatinine in the urine to decompose the hydrogen peroxide to produce benzidine (ortho-toluidine, tetramethylbenzidine, 1,6-dihydroperoxide) and a coloring indicator for color development. Means for detecting active oxygen in the human body.

8. The method of claim 7,
Wherein the first test strip and the second test strip are attached to the upper surface of the support so that the support is immersed in the urine.

A first step of detecting the amount of malondialdehyde contained in the urine;
A second step of detecting the amount of creatine contained in urine;
A third step of comparing and analyzing the values detected in the first and second steps; And
And a fourth step of determining an amount of active oxygen as a value comparatively analyzed in the third step. The method for detecting active oxygen in the human body through the correction of creatine.

10. The method of claim 9, wherein the first step and the second step comprise:
Characterized in that a color change value due to color development is used, thereby detecting active oxygen in the human body through the correction of creatine.

11. The method according to claim 10,
Wherein the ratio of malondialdehyde / creatine to creatine is represented by the ratio of malondialdehyde / creatine.

12. The method according to claim 11, wherein the ratio is expressed in units of (umol / L) / (mmol / L).

13. The method according to claim 12, wherein 0 to 0.23 is determined to be normal, 0.24 to 1 is measured to be low, 1 to 3 is measured to be high, and 3 or more is determined to be very high, A means for detecting active oxygen in the body by means of creatine correction.

11. The method according to claim 10, wherein in the first step, the color change value due to color development is expressed in four steps,
In the second step, the color change value due to color development is expressed in five steps, a table showing the state of active corals by the step classification is made,
The detected color change value is compared with a prepared color chart table to determine to which step belongs, and the state of active oxygen is determined by finding a corresponding point in the table, thereby detecting active oxygen in the human body through the correction of creatine .

15. The method of claim 14,
Wherein the concentration of creatine in the abscissa is represented by 5 levels, and the axis of ordinates is represented by the concentration of malondialdehyde in 4 levels, as shown in Table 3. The method for detecting active oxygen in the human body through creatine correction.