JPH10282001A - Method and apparatus for measuring oxidizing capacity of liquid sample - Google Patents

Abstract

PROBLEM TO BE SOLVED: To totally measure an antioxidizing capacity of an antioxidizing substance possessed by a sample by a method wherein a specified amount of peroxide is added to the liquid sample and a residual quantity thereof is measured after a specified time to determine a decreased amount thereof. SOLUTION: A measuring device of the antioxidizing capacity of a liquid sample such as blood is constituted of a mixing part 31 to add a known amount of peroxide such as phosphatidyl cholinehydroperoxide(PCOOH) to a sample, a detecting part 32 using a fast liquid chromatography or the like and a data processing part 33. At the mixing part 31, a switching is possible between the mode of adding peroxide and the mode of adding none. A sample such as plasma is treated in both modes for a specified time and after a pretreatment or the like, the concentration of the peroxide is measured by the detecting part 32. In the detection of the PCOOH, a liminescent sample is preferably used and the detecting part 32 may be provided with a luminescent sample pump and a luminescence detector. Based on the results of measurement, an addition recovery rate and the antioxidizing capacity of the simple is calculated.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method and an apparatus for measuring the antioxidant power of a substance. More particularly, the present invention relates to a method for measuring the total antioxidant power of a living body, especially blood, and an apparatus for performing the method.

[0002]

2. Description of the Related Art Since it has been shown that active oxygen indiscriminately oxidizes various cellular components and causes various oxygen disorders, there has been recognition of the importance of the antioxidant power of living organisms in maintaining health. Is growing.

[0003] As a method for examining the antioxidant power of a living body, a method for directly quantifying individual antioxidants in blood or tissue has been generally used. Examples of in vivo antioxidants include SOD (superoxide dismutase), catalase, glutathione peroxidase, vitamin E, ascorbic acid (vitamin C), bilirubin, β-carotene, and the like. Therefore, by examining the activity or abundance of these individual components in a living tissue such as blood, the antioxidant power of the living body is measured.

However, in the above-mentioned conventional method, it is possible to know the antioxidant power derived from individual antioxidants in body fluids such as blood or each tissue, but it is not possible to know the total antioxidant power as an individual. Can not. Because the antioxidant mechanism in the living body is complicated and the synergistic effect of multiple antioxidants is large, it is difficult to understand which antioxidant actually exerts the antioxidant effect and how much. Have difficulty. Therefore, even if the activity or abundance of each antioxidant is independently measured using the above-mentioned conventional method, and these are added, the total antioxidant power of the individual organism cannot be known.

[0005]

SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances, and does not independently measure the activity of individual antioxidants in a living body, but instead measures all antioxidants present in the living body. An object of the present invention is to provide a method capable of measuring the antioxidant power as a whole of oxidized substances, and an apparatus for easily performing the method.

Further, the present invention is applicable not only to body fluids such as blood, but also to any liquid sample, and aims to provide a method and an apparatus capable of measuring its antioxidant power.

[0007]

The object of the present invention is to provide a method for measuring the antioxidant power of a liquid sample, comprising: adding a predetermined amount of peroxide to the liquid sample; Measuring the amount of the peroxide remaining in the liquid sample after a lapse of time; antioxidation of the liquid sample based on the amount of the peroxide reduced during the predetermined time. Calculating a force index.

In the method of the present invention, the liquid sample is not particularly limited, but is preferably a biological body fluid, more preferably blood. These body fluids may be subjected to solvent extraction as necessary, and the obtained liquid may be used as a sample. Alternatively, a liquid sample obtained by homogenizing a biological tissue and performing solvent extraction may be used. Furthermore, the antioxidant power of a food or a polymer material can be measured by using the liquid food or the polymer material itself or a liquid obtained by performing the same extraction treatment as a sample.

In the present invention, the peroxide added from the outside to the sample is not particularly limited, and various peroxides such as hydrogen peroxide and various organic hydroperoxides can be used. However, when a body fluid such as blood is used as a sample, it is preferably a phospholipid peroxide, more preferably phosphatidylcholine hydroperoxide (PCOOH).
It is.

As a method for detecting the peroxide remaining in the sample, for example, the sample is separated into individual components by liquid chromatography, and the separated peroxide is reacted with a luminescent reagent. Can be performed by optically detecting the light generated by the above.

In the method of the present invention, since various peroxides added to the sample from the outside are decomposed by the overall antioxidant power of the sample, the amount or rate of the decrease depends on the sample. It directly reflects the total antioxidant power. Therefore, by measuring the amount or rate of this decrease, the total antioxidant power of the sample can be known based on the result.

In the method of the present invention, assuming that the antioxidant power of blood is measured using, for example, PCOOH as a peroxide added from the outside, blood originally contains a certain amount of PCOOH. Therefore, in order to measure the antioxidant power of blood by the method of the present invention, the amount of PCOOH added from the outside is known in advance, the amount of PCOOH originally contained in the blood, and the amount of PCOO
PCOOH remaining in the sample a predetermined time after the addition of H
Must be measured.

Therefore, the apparatus of the present invention is configured to perform such two measurements using one apparatus. That is, the antioxidant power measuring apparatus according to the present invention comprises: a mixing section for adding and mixing a predetermined amount of peroxide to a sample whose antioxidant power is to be measured; A detection unit for detecting the concentration of peroxide contained in the sample; and a data processing unit for processing the detection result obtained by the detection unit to calculate the remaining amount of the peroxide added in the mixing unit. A data processing unit.

The mixing section can be switched between a mode in which only the sample is sent to the detection section and a mode in which the peroxide is added to the sample and the mixture is sent to the detection section. Further, the detection unit includes a liquid chromatography for separating a sample supplied from the mixing unit into individual components; and reacts with a peroxide to react with the components separated by the liquid chromatography to emit light. And a light detecting means for detecting light generated by the reaction of the luminescent reagent. Furthermore, the data processing unit, the detection result in the detection unit when peroxide is not added in the mixing unit, from the result in the detection unit when peroxide is added in the mixing unit,
The recovery rate of the peroxide added in the mixing section after a predetermined time has elapsed is calculated.

[0015]

Embodiments of the present invention will be described below with reference to the drawings. First, a detection unit for detecting a peroxide concentration in a sample, which is the heart of the measurement device according to the present invention, will be described. FIG. 1 shows an embodiment of the detection unit in the device according to the present invention. In the figure, the high performance liquid chromatography HPLC includes a liquid feed pump 2 for sending an elution solvent from a solvent container 1, an injector 4 for injecting a sample 3 into the elution solvent sent by the pump, and a sample mixed therein. And a column 5 into which the elution solvent is fed. The elution section of the column 5 is provided with an ultraviolet absorption detector 6 for detecting ultraviolet absorption of various components eluted according to the adsorbing ability of the adsorbent packed in the column 5. Each component passing through the ultraviolet absorption detector 6 includes:
The luminescent reagent and the sensitizer 8 are injected by the liquid feed pump 7. The respective components into which the luminescent reagent and the sensitizer 8 have been injected are led to the flow cell 9. Opposite to the flow cell 9, for example, a photomultiplier tube of a weak photodetector 10 using a single photoelectron counting method is provided. The light generated from each component passing through the flow cell 9 is detected by the weak light detector 10. The detection results of the weak light detector 10 and the ultraviolet absorption detector 6 are recorded by a recorder 11 composed of, for example, a pen recorder.

The flow cell 9 has a capacity of about 100 μl.
For example, it is constituted by a quartz glass tube or a transparent Teflon tube. 2 and 3 show the configuration of the flow cell 9, respectively. FIG. 2 shows a configuration in which the tube 20 is straight, and FIG.
Is a spiral. The flow cell of FIG. 2 having a straight tube has low detection sensitivity, but has good peak resolution. Conversely, a flow cell with a spiral tube has good detection sensitivity but low peak resolution.

The high performance liquid chromatography HPLC
As the column 5, a column containing a chemical bond type silica gel, a hydrophilic polymer gel, silica gel, a polysaccharide gel, a polystyrene gel, a polystyrene gel derivative, a polysaccharide gel derivative, or the like as a filler is used. Preferably, octadecylsilane is used. A treated ODS reverse phase column or normal phase silica gel column is used.

The above detection unit is described in the specification of Japanese Patent Publication No. 63-233374 by the present applicant. By using this device, it is possible to detect phospholipid peroxide with high sensitivity. Can be.

In a preferred embodiment of the present invention, a recovery rate and an antioxidant power of a peroxide, which will be described later, are determined from the results obtained by the above-mentioned detecting section. In order to be able to measure this automatically, the configuration of FIG. 4 was adopted. In the figure, reference numeral 31 denotes a mixing section which adds and mixes a known amount of peroxide to plasma as a sample. The mixing section can be switched between a mode in which peroxide is added and a mode in which peroxide is not added. Therefore, it is possible to measure both the peroxide amount (A) of the plasma itself and the peroxide amount (B) after a certain period of time after the peroxide is added from the outside. Downstream of the mixing section 31, the detection section 32 of FIG. 1 is provided. The detection unit 32 includes a measurement device described with reference to FIG. 1 and a device that pre-processes the sample sent from the mixing unit 31 as necessary, as described below, and supplies the sample to the injector 4 of the measurement device in FIG. Consists of The detection output obtained by the detection unit 32 is guided to the data processing unit 33. The data processing unit 33 calculates the addition recovery rate and the antioxidant power according to, for example, a predetermined formula described later.

Next, an embodiment of a method for measuring antioxidant power according to the present invention using the above apparatus will be described. As a measurement sample,
Among the biological samples, plasma that is likely to reflect the state of the whole organism was used. Phosphatidylcholine hydroperoxide (hereinafter abbreviated as PCOOH), which is one of phospholipid peroxides, is added to the plasma, and after a predetermined time, the decomposition rate of the PCOOH is measured. By this method, how much antioxidant power of the plasma used for the sample,
That is, it was determined whether or not it had the ability to eliminate peroxide. As the PCOOH added from the outside, egg yolk-derived PCOOH (NC10S) manufactured by NOF Corporation was used.

The above-mentioned decomposition rate was determined by measuring how much PCOOH added in a certain amount was recovered after a certain time (addition recovery rate). That is, the addition recovery rate and the antioxidant power were calculated by the following equations.

Addition recovery rate (%) = B / (A + C) × 100 Antioxidant power = 1 / addition recovery rate where A: amount of PCOOH contained in plasma when PCOOH is not added from the outside B: PCOOH Is the amount of PCOOH contained in the plasma after a certain period of time after adding CCO from the outside; C; the amount of PCOOH added from the outside.

The following were used as the column elution solvent, the liquid sending pump 2, the column 5, the ultraviolet absorption detector, the luminescent reagent, the pump 7, the luminescence detector, and the recorder 11 described in the apparatus of FIG. .

Column elution solvent: 2-propanol: methanol: distilled water (390: 135: 75 (v / v); flow rate 1.0 ml / min) Pump for column elution solvent 2: PU manufactured by JASCO Corporation
-980 column 5: SIL-NH2 column manufactured by JASCO Corporation UV absorption detector: UV-970 luminescence reagent manufactured by JASCO Corporation: borate buffer solution containing cytochrome C (1 μg / ml) and luminol (10 μg / ml) 50mM, pH10) Liquid sending pump for luminescence reagent 7: PU-980 luminescence detector manufactured by JASCO Corporation: Chemiluminescence detector manufactured by Tohoku Denshi Sangyo Co., Ltd. Plasma used: 0.4 ml Externally added PCOOH: PCOOH (NC10S) derived from egg yolk manufactured by NOF Corporation The specific measurement procedure is as follows.

First, the following preprocessing is performed. That is,
Chloroform: methanol (1: 2) is added to the plasma and stirred. After centrifugation, the upper layer is collected and chloroform and distilled water are added. After stirring and centrifugation, the resulting lower layer (chloroform layer) is used as a lipid extraction sample. The lipid extraction sample thus obtained is injected by the injector 4 as the sample 3 described in FIG. It is preferable that an apparatus for automatically performing the pre-processing be connected to the injector 4 of the apparatus shown in FIG. In this case, this pretreatment device is connected to the mixing section 31 of FIG.

In order to determine the addition recovery rate, the mixing section 31
In both the cases where a known concentration of PCOOH was added to the plasma and the case where PCO was not added, the amount of PCOOH was analyzed after lipid extraction in the same manner as described above. Thereby, the amounts (A) and (B) of the PCOOH described above are measured. Accordingly, by inputting these values (A) and (B) together with the previously known amount of externally added PCOOH (C) into the data processing unit 33 in FIG. The antioxidant power is calculated.

The analysis method of the above embodiment specifically detects only PCOOH. By this method, plasma obtained from six patients known to have different concentrations of PCOOH in the normal state, PCO was obtained by the method described above.
The recovery rate of OH addition and the antioxidant power of each plasma sample were measured. In general, plasma having a lower PCOOH content in a normal state has a higher antioxidant activity, and conversely, P
Plasma with high COOH content has been found to have low antioxidant power.

FIG. 5 shows the results of the above analysis. In the figure, the horizontal axis (y) indicates the PCOOH concentration of each plasma sample when PCOOH was not added in the mixing section 31. The vertical axis (x) shows the values of the addition recovery rate (%) and the antioxidant power defined above. As is apparent from the figure, there is a relationship between the two as represented by y = 0.3462x + 9.7971. This result indicates that plasma P
Consistent with the medical community's view that COOH concentration is inversely proportional to the antioxidant power of the plasma. The correlation coefficient is R ²
= 0.81, indicating a good correlation between the two. Therefore, the above results indicate that the method according to the present invention is extremely useful for measuring the total antioxidant power of a sample.

As is clear from the above embodiment, according to the present invention, it is possible to determine the total antioxidant power of a living individual for reducing PCOOH. This has very important implications. That is, lipids in plasma are oxidized by various factors, which are known to cause aging and various diseases.In particular, phospholipids have important functions in vivo as functional lipids. It has been pointed out that the phospholipid peroxide produced by the oxidation has a deep relationship with arteriosclerosis, myocardial infarction and aging. Thus, the ability to measure the antioxidant power of plasma that destroys phospholipid peroxides allows for a pre-diagnosis of predisposition to such diseases.

[0030]

As described in detail above, according to the present invention,
It is possible to provide a method capable of measuring the total antioxidant power of a sample and an apparatus capable of easily performing the method.

[Brief description of the drawings]

FIG. 1 is a block diagram showing a configuration of a detection unit for measuring lipid peroxide in an antioxidant power measuring device according to an embodiment of the present invention.

FIG. 2 is an explanatory diagram illustrating an example of a flow cell 9 used in the detection unit in FIG. 1;

FIG. 3 is an explanatory diagram illustrating another example of the flow cell 9 used in the detection unit in FIG. 1;

FIG. 4 is a block diagram showing a configuration of an antioxidant power measuring apparatus according to an embodiment of the present invention, incorporating the detection unit of FIG. 1;

FIG. 5 is a graph showing the results of measuring the antioxidant power of a plasma sample using the apparatus of FIG.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Elution solvent, 2 ... Liquid sending pump, 3 ... Sample, 4 ... Injector, 5 ... Column, 6 ... Ultraviolet absorption detector, 7 ...
Liquid feeding pump, 8: luminescent reagent and sensitizer, 9: flow cell, 10: weak light detector, 11: recorder, 20: tube, 31: mixing unit, 32: detecting unit, 33: data processing unit.

Claims (4)

[Claims] 1. A method for measuring the antioxidant power of a liquid sample, comprising: adding and mixing a predetermined amount of peroxide to the liquid sample; Measuring the amount of said peroxide remaining in;
Calculating an antioxidant index of the liquid sample based on the amount of the peroxide decreased during the predetermined time. 2. The method of claim 1, wherein said liquid sample is a biological fluid of an organism and said peroxide is a phospholipid peroxide. 3. The method of claim 2, wherein said liquid sample is human blood and said phospholipid peroxide is phosphatidylcholine hydroperoxide. 4. A device for measuring the antioxidant power of a liquid sample, comprising: a mixing section for adding and mixing a predetermined amount of peroxide to a sample whose antioxidant power is to be measured; A detector for detecting the concentration of peroxide contained in the sample to which the peroxide has been added in the mixing unit; and data processing the detection result obtained in the detection unit to obtain the peroxide added in the mixing unit. A data processing unit for calculating the remaining amount of the substance, wherein the mixing unit has a mode of sending only the sample to the detection unit and a mode of sending the mixture to the detection unit after adding peroxide to the sample. The detection unit can be switched between two modes, wherein the detection unit includes a liquid chromatography for separating a sample supplied from the mixing unit into individual components; and a component separated by the liquid chromatography. For
A reagent mixing means for mixing a luminescent reagent which emits light by reacting with the peroxide; and a light detecting means for detecting light generated by the reaction of the luminescent reagent; The detection result of the detection unit when the peroxide is not added in the mixing unit, and the detection result of the detection unit when the peroxide is added in the mixing unit, the peroxide added in the mixing unit. An apparatus for calculating the recovery rate of the oxide after a predetermined time has elapsed.