JP2008185364A - Measuring method of blood oxidative stress marker - Google Patents

Abstract

The present invention provides a method for accurately and simultaneously fractionating a reduced low molecular thiol and an oxidized low molecular thiol in a biological sample simultaneously.
A biological sample is deproteinized, a first portion and a second portion are collected in equal amounts from the deproteinized sample, the reduced low-molecular thiol in the first portion is fluorescently labeled, After reducing the low molecular thiol of the part with a reducing agent, the reduced low molecular thiol is fluorescently labeled, and the low molecular thiol of the first part and the low molecular thiol of the second part are labeled with HPLC. The reduced low-molecular thiol and oxidized low-molecular thiol concentrations are calculated from the analytical value of the first part and the analytical value of the second part. Is quantified separately.
[Selection figure] None

Description

  The present invention relates to a method for measuring oxidative stress in a living body. More specifically, the present invention relates to a method for measuring oxidative stress using a ratio between an oxidized form and a reduced form of a low molecular thiol in a living body as a marker.

  Various arteriosclerotic changes progress with the development of lifestyle-related diseases including metabolic syndrome. It is thought that oxidative stress in the body plays an important role in the progress. Oxidative stress is defined as the difference between the oxidative damage potential of reactive oxygen groups (ROS) generated in vivo and the antioxidant potential of in vivo antioxidant systems. ROS is originally useful for energy production, invading foreign body attacks, unnecessary cell processing, cell information transmission, etc., but when excessive ROS that cannot be captured by the in vivo antioxidant system occurs It oxidizes and damages the lipids, proteins and enzymes responsible for the structure and functions of the living body, and the genetic DNA that carries the genetic information, disrupting the structure and functions of the living body. As a result, diseases such as arteriosclerosis, myocardial infarction, diabetes, and cancer are caused and aging is accelerated.

  The basis for this is epidemiological or based on findings from animal experiments. So far, glutathione peroxidase activity, superoxide dismutase (SOD) activity, and chemiluminescent substances in serum have been measured as oxidative stress indicators. However, these indicators do not reflect oxidative stress in vivo and do not represent direct oxidative stress.

  Glutathione, a kind of low-molecular thiol, is a substrate for glutathione peroxidase. When glutathione peroxidase reduces and eliminates peroxide, which is one of the chemical entities of oxidative stress, glutathione is oxidized and becomes oxidized. It has been known. Oxidized glutathione is returned to the reduced form by glutathione reductase. As described above, the oxidized-reduced form of low-molecular thiols circulates and is in a constant equilibrium state, but it is presumed that the equilibrium is lost with the increase of peroxide and the oxidized ratio increases.

  The level of glutathione peroxidase activity is not related to the oxidation-reduction type ratio in the equilibrium state of the low molecular thiol redox cycle, and even if the enzyme activity is measured, the equilibrium bias cannot be analyzed strictly. Exists.

  On the other hand, attempts to measure low molecular thiols in biological samples such as blood have been reported (see Non-Patent Documents 1 to 3, etc.). This method is intended to measure the oxidized and reduced forms of low molecular thiols.

  However, in this method, a calibration curve of only reduced thiols is prepared and reduced thiols and oxidized thiols are measured, and reduced thiols are converted to oxidized thiols during the sample processing process. However, it was not always possible to accurately measure the oxidized and reduced forms of low-molecular thiols in biological samples.

  Moreover, the relationship between the oxidation and reduction of thiol and oxidative stress has not been known.

TOYOOKA et al., Journal of Chromatography, 282 (1983) 495-500 TOYOOKA et al., Analytica Chimica Acta, 205 (1988) 29-41 TOYOOKA et al., BIOMEDICAL CHROMATOGRPHY, VOL.3, No.4, 1989, pp.166-172

  The present invention uses the reduced-oxidized ratio of low-molecular-weight thiols in biological samples as an index of oxidative stress, and accurately and simultaneously separates reduced low-molecular thiols from oxidized low-molecular-weight thiols in biological samples. The purpose is to provide a method for quantification.

  In order to know the increase in peroxide, which is one of the chemical entities of oxidative stress, the present inventors are effective in analyzing the oxidized-reduced ratio of low-molecular thiols that reflect it. I thought. That is, it was considered that the oxidation-reduction type ratio of low-molecular thiol is more appropriate as an index of oxidative stress than the conventionally measured glutathione peroxidase activity. In addition, oxidative stress may increase locally, but by analyzing the oxidation-reduction type ratio of low-molecular thiols in the blood circulating throughout the body, we thought that local imbalances could be detected. .

  Therefore, the present inventors focused on low-molecular thiols as markers representing oxidative stress, and attempted to establish a measurement method that shows a simpler and more accurate oxidation state.

  Conventionally, the oxidized-reduced fractional quantification method for low molecular weight thiols has been performed in standard samples and animal experiments where control experiments are possible. However, low molecular thiols in biological samples are very unstable, and the results of analysis using clinical materials for which control samples cannot be obtained can be expected only with low reliability, and there have been few reports. Therefore, the analytical significance of low molecular thiols was not inferred.

  In the present invention, since the instability of low molecular thiols in biological samples is due to the enzymes in the samples, it has become possible to obtain a certain analytical accuracy by performing a low temperature protein removal operation in a short time after blood collection. . Furthermore, a deproteinizing agent used for deproteinization operation and a reducing agent used for reducing oxidized low molecular thiol in a biological sample were selected that can keep the low molecular thiol stable. Furthermore, when the oxidized form is reduced to the reduced form, and both the reduced and oxidized forms are analyzed as reduced forms, a calibration curve for each of the oxidized and reduced forms is created and used, so that the reduction reaction of the oxidized form can be performed. Yield was corrected.

  As a result of analyzing several reduced and oxidized standard mixed samples using the method thus established, good accuracy was proved and the present invention was completed.

That is, the present invention is as follows.
[1] A method for measuring oxidative stress in a living body, wherein the reduced form and oxidized form of a low molecular thiol in a biological sample are separately quantified, and the abundance ratio of the oxidized form and reduced form of the low molecular thiol is used as a marker.
[2] The method for measuring oxidative stress in a living body according to [1], wherein the low molecular thiol in the biological sample is at least one selected from the group consisting of cysteine, cystenylglycine, glutathione and homocysteine.
[3] The method for measuring oxidative stress in a living body according to [1] or [2], wherein the biological sample is selected from the group consisting of blood, serum and plasma.
[4] The method for measuring oxidative stress in a living body according to any one of [1] to [3], wherein fractional quantification of reduced and oxidized forms of low-molecular thiols in a biological sample is performed using HPLC.
[5] A method for differentially quantifying reduced and oxidized low molecular thiols in a biological sample, wherein the biological sample is deproteinized, and the same amount of the first and second parts is obtained from the deproteinized sample. Each sample is collected, the reduced low molecular thiol of the first part is fluorescently labeled, the low molecular weight thiol of the second part is reduced with a reducing agent, and then the reduced low molecular thiol is fluorescently labeled. The low molecular thiol of the first part and the low molecular thiol of the second part were analyzed using HPLC, and the reduced low molecular thiol and the oxidation were determined based on the analytical value of the first part and the analytical value of the second part. A method for differentially quantifying a reduced form and an oxidized form of a low molecular thiol in a biological sample, wherein the low molecular thiol concentration is calculated.

[6] The reduced form and oxidized form of the low molecular thiol in the biological sample according to [5], wherein the low molecular thiol in the biological sample is at least one selected from the group consisting of cysteine, cystenylglycine, glutathione, and homocysteine A method for fractional quantification.
[7] The method for differentially quantifying a reduced form and an oxidized form of a low molecular thiol in a biological sample according to [5] or [6], wherein deproteinization treatment of the biological sample is performed using sulfosalicylic acid.
[8] After collecting the biological sample from the living body, cool to 0 to 4 ° C. within 1 minute, and fractionate the reduced form and oxidized form of the low molecular thiol in the biological sample of any of [5] to [7] How to quantify.
[9] The reduction treatment of the second portion of the deproteinized biological sample is performed using a water-soluble reducing agent that acts at a neutral pH. A method for differential quantification of reduced and oxidized forms of low-molecular thiols.
[10] The method for differential quantification of reduced and oxidized forms of low molecular weight thiols in a biological sample according to [9], wherein the water-soluble reducing agent acting at neutral pH is TCEP, Neutral pH.

[11] Quantification of reduced low-molecular thiols and oxidized low-molecular thiols in biological samples can be achieved by reducing calibration curves prepared using reduced low-molecular thiol standards and oxidized low-molecular thiol standards with reducing agents. The low molecular thiol in the biological sample of any one of [5] to [10], which is performed using two calibration curves of a calibration curve prepared using a reduced low molecular thiol standard product obtained by processing Method for separately quantifying a reduced form and an oxidized form.
[12] Quantification of reduced low-molecular thiol and oxidized low-molecular thiol in a biological sample is performed by a method including the following steps: [11] fractionation of reduced and oxidized low-molecular thiol in a biological sample How to quantify:
(i) Analyzing a reduced standard product of reduced low molecular thiol and oxidized low molecular thiol standard with a reducing agent by HPLC, and analyzing the HPLC chromatogram peak area of the standard product as an internal standard HPLC chromatogram Normalizing based on peak area and creating two calibration curves;
(ii) The step of determining the reducing agent efficacy by the formula 0.5 / (SHx / SSx) from the result of HPLC analysis of (i), where SHx is normalized at a specific concentration of the reduced low molecular thiol standard Represents the HPLC chromatogram peak area, and SSx represents the normalized HPLC chromatogram peak area at a specific concentration of a standard product obtained by reducing an oxidized low molecular weight thiol standard with a reducing agent;
(iii) (a) A sample obtained by fluorescently labeling the reduced low-molecular thiol of the first part of the biological sample was obtained by HPLC analysis, and normalized based on the HPLC chromatogram peak area of the internal standard From the HPLC chromatogram peak area, based on the calibration curve created using the reduced low molecular thiol standard (i), determine the reduced low molecular thiol concentration of the first part,
(b) Reductant efficacy determined in (ii), (iii) Obtained by HPLC analysis of a sample obtained by fluorescently labeling the reduced low molecular thiol of the first part of the biological sample in (a) , HPLC chromatogram peak area normalized based on HPLC chromatogram peak area of internal standard, and reduced low molecular thiol after reducing low molecular thiol in the second part of biological sample with reducing agent Sample obtained by HPLC analysis of the sample obtained by labeling and processed by the formula (reduced type + oxidized type) analysis method from the HPLC chromatogram peak area normalized based on the HPLC chromatogram peak area of the internal standard Normalized HPLC chromatogram peak area of oxidized low molecular weight thiols in biological samples by normalizing HPLC chromatogram peak area of-(reducing agent efficacy x normalized HPLC chromatogram peak area of sample processed by reducing analysis method) A correction value corresponding to the product is obtained, and from the correction value, the second part is calculated based on a calibration curve prepared using a standard product obtained by reducing the oxidized low molecular thiol standard product of (i) with a reducing agent. The process which calculates | requires the oxidation type low molecular thiol density | concentration of.

[13] Presence of oxidized form and reduced form of low molecular thiol from the quantitative value obtained by the method of differentially quantifying the reduced form and oxidized form of low molecular thiol in the biological sample of any one of [5] to [12] The method for measuring oxidative stress in a living body according to any one of [1] to [4], wherein a ratio is calculated and oxidative stress in the living body is measured from the abundance ratio.
[14] A kit for differentially quantifying a reduced form and an oxidized form of a low molecular thiol in a biological sample containing at least a deproteinizing agent that is sulfosalicylic acid and a reducing agent that is TCEP, Neutral pH.
[15] A kit for differential quantification of reduced and oxidized forms of low molecular thiols according to [14], further comprising reduced and oxidized low molecular thiol standards, and a fluorescent substance for fluorescently labeling low molecular thiols .
[16] The reduced form and oxidized form of the low molecular thiol of [14] or [15], wherein the low molecular thiol is at least one selected from the group consisting of cysteine, cystenylglycine, glutathione and homocysteine Kit for.

  According to the method of the present invention, it is possible to separate and quantify the reduced form and the oxidized form of each low molecular thiol with a certain accuracy using a human clinical material as a sample. Analysis of several cases so far reveals that the ratio of oxidized forms of glutathione has changed as originally expected in angina pectoris, for which oxidative stress is said to play an important role It became. This suggests that a change in the ratio of oxidized thiol, for example, an increase can be an indicator of an increase in oxidative stress, and a unique effect on clinical diagnosis of the present invention is expected.

Hereinafter, the present invention will be described in detail.
The low molecular thiol is a compound having an SH group and a molecular weight of about several hundreds, and as the low molecular thiol used for the index of oxidative stress of the present invention, cysteine, cystenylglycine, Glutathione and homocysteine are preferred. FIG. 1 shows the structural formula of each compound. A plurality of low molecular thiols may be measured in one measurement. For example, one, two, three or four kinds of cysteine, cystenylglycine, glutathione and homocysteine may be measured at a time. The oxidized forms of cysteine and homocysteine are called cystine and homocystin, respectively.

  In the method of the present invention, the oxidized form and reduced form of low molecular thiol are separately quantified, and the ratio of oxidized form to reduced form is calculated as an index of oxidative stress. The reduced form of the low molecular thiol exists as a reduced state of the thiol group, that is, as —SH, and the oxidized form exists as a dimer in which the thiol group is oxidized and two low molecular thiol molecules are bonded by SS bonds (FIG. 2). .

  The low molecular thiol is quantified by binding a fluorescent substance to the SH group of the reduced low molecular thiol and quantifying the complex of the low molecular thiol compound and the fluorescent substance by fluorescence (FIG. 3).

  Since the fluorescent substance cannot be bound to the oxidized low molecular thiol as it is, the oxidized low molecular thiol can be reduced, converted into a reduced low molecular thiol, and bound with the above fluorescent substance for quantification. That's fine. At this time, when the reduction rate of oxidized thiol is 100%, two molecules of reduced low-molecular thiol are generated from the dimer of oxidized low-molecular thiol, so the concentration is measured twice (FIG. 4).

  In the method of the present invention, it is possible to perform quantitative determination of oxidized low molecular thiol and reduced low molecular thiol in a biological sample. The biological sample is not limited, but whole blood, serum, and plasma are preferable.

  When collecting blood, in order to prevent oxidation of reduced low molecular thiols present in the blood, the blood collection tube is cooled in advance and used. At this time, it is preferable to add EDTA (ethylenediaminetetraacetic acid) to prevent blood coagulation. In order to prevent blood coagulation, heparin and the like are generally used. However, when heparin is used, it is highly possible that reduced low-molecular thiols are converted to oxidized forms, so it is necessary to use EDTA. is there. Further, by adding EDTA, the coenzyme of an enzyme capable of oxidizing reduced low molecular thiol can be inhibited, so that conversion of reduced low molecular thiol to oxidized form can also be prevented.

  When serum or plasma is used, the serum or plasma is separated by centrifugation. In this case as well, it is preferably performed at 0 ° C. under cooling, and in order to prevent oxidation of reduced low-molecular thiols, the one with a shorter centrifugation time is used. Is good. When the temperature at the time of centrifugation is high and the time for centrifugation is long, the reduced low molecular thiol in the sample is converted to an oxidized form. For example, in the case of plasma separation, centrifugation is performed at 1900G for 5 minutes or less, preferably 3 minutes or less. Moreover, it is necessary to collect and prepare a biological sample at the time of use. This is because, for example, when stored at −80 ° C. in the state of plasma, reduced low molecular thiol in the sample is converted to oxidized low molecular thiol.

  At this time, a standard sample containing a reduced concentration low molecular thiol or an oxidized low molecular thiol at a predetermined concentration may be prepared. In this case, the reduced low-molecular thiol standard sample can be prepared in a concentrated solution and stored frozen. For example, it can be stored at −80 ° C. and diluted before use. On the other hand, oxidized low molecular thiols need to be prepared each time they are used. Oxidized low molecular thiols are partially deposited by low temperature storage or cryopreservation at 4 ° C. or lower, and accurate measurement becomes difficult. Therefore, preparation at the time of use is necessary.

  Prior to measurement of the low molecular thiol, the biological sample is deproteinized to remove contaminating proteins and enzymes that can oxidize low molecular thiols in the biological sample. Deproteinization can be achieved by adding acid to the sample, denaturing the protein, and removing the denatured protein by filtration or centrifugation. A protein whose pH is not extremely low is preferable for deproteinization, and examples thereof include sulfosalicylic acid (SSA). Sulfosalicylic acid is desirable because it has a higher retention rate of SH groups than protein denaturing and removing agents such as trichloroacetic acid (TCA). At this time, in order to reduce the activity of the enzyme capable of oxidizing the low molecular thiol in the biological sample, the biological sample and the protein removal agent are cooled to 0 to 4 ° C. and used. The concentration of the deproteinizing agent to be used is 1 to 5%, preferably 2 to 5%, more preferably 2 to 3%, and particularly preferably 2.5%.

  The time from the collection of the biological sample to the protein removal treatment is within 5 minutes, preferably within 3 minutes, more preferably within 1 minute.

  After adding a deproteinizing agent to the biological sample, an internal standard substance is further added. As the internal standard substance, a low molecular thiol that does not exist in the living body or exists in a very small amount is used. For example, N-Acetyl-L-cysteine can be used, and it may be added to a solution used for HPLC analysis and measurement so as to have a concentration approximate to a low molecular thiol contained in a biological sample. For example, several μM to several tens μM may be added.

  After adding the internal standard, the protein is denatured by mixing and centrifugation, and the supernatant is used for measurement.

  In the method of the present invention, when a deproteinized biological sample is stored, the oxidized low molecular thiol is reduced and cannot be measured accurately. Therefore, low molecular thiol molecules are measured without storage.

  In the measurement, the same amount of the first part and the second part are taken from the deproteinized sample, and the reduced low molecular thiol is measured using the first part. At this time, as described above, the reduced low-molecular thiol contained in the first portion is labeled with a fluorescent substance. The fixed amount to be collected is not limited, but may be 5 to 100 μl, preferably 10 to 50 μl.

  Also, both the reduced and oxidized low molecular thiols are measured using the second portion. The sample contains both reduced low-molecular thiols and oxidized low-molecular thiols, but the oxidized low-molecular thiols are converted into reduced low-molecular thiols, labeled with fluorescent substances, Just measure.

  The measurement using the first part can quantitate the reduced low-molecular thiol present in the sample, and the measurement using the second part can reduce the reduced type thiol present in the sample. Low molecular thiols and oxidized low molecular thiols can be quantified. By subtracting the quantitative value of the first part from the quantitative value of the second part, the amount of oxidized low molecular thiol can be calculated.

  In the present invention, the measurement using the first part is referred to as “reduction type treatment method”, and the measurement using the second part is referred to as “(reduction type + oxidation type) treatment method”. Further, a sample processed by the “reduction type treatment method” is called a reduction type treatment sample, and a sample processed by the “(reduction type + oxidation type) treatment method” is called a (reduction type + oxidation type) treatment sample.

  Conventionally, tributylphosphine (TBP), which acts in an alkaline manner, has been used to reduce disulfide bonds, and NaOH has been added to make the reaction solution alkaline during the reduction treatment, but NaOH has a thiol structure. There was a problem of decomposing. In addition, tributylphosphine has a disadvantage that it is insoluble in water and is susceptible to air oxidation. In the present invention, a water-soluble reducing agent that does not have such disadvantages and can act neutrally is used. An example of such a reducing agent is TCEP (Tris [2-carboxyethyl] phosphine). TCEP includes TCEP, Hydrochloride, TCEP, and Neutral pH, with TCEP and Neutral pH being preferred. TCEP or Immobilized in which TCEP is immobilized on polyacrylamide gel can also be used. The concentration of the reducing agent is 1 to 5%, preferably 2 to 5%, more preferably 2 to 3%, and particularly preferably 2.5%.

  After deproteinization, the reduced low-molecular thiol in the first part of the sample and the reduced second part is then labeled with a fluorescent substance.

  Low molecular thiol is quantified by binding a fluorescent substance having a maleimide group or an aryl halide group to the SH group of a reduced low molecular thiol and quantifying the complex of the low molecular thiol compound and the fluorescent substance by fluorescence (FIG. 3). . Examples of the fluorescent substance having an aryl halide group include SBD-F (4-Fluoro-7-sulfobenzofurazan, ammonium salt), ABD-F (4-Fluoro-7-sulfamoylbenzofurazan), etc. As the fluorescent substance having a maleimide group, NAM (N- (9-Acridinyl) maleimide), DBPM, etc. are mentioned. However, any substance can be used as long as it is a labeling substance that can fluorescently label a low molecular thiol by a coupling reaction with a thiol group, and is not limited to the above substances. The concentration of the fluorescent substance during the reaction is preferably 8 to 15 mM, more preferably 9 to 11 mM, and particularly preferably 10 mM, which is a concentration adapted to the predicted low molecular thiol concentration in the biological sample.

After labeling the low molecular thiol with a fluorescent substance, the low molecular thiol is analyzed by HPLC.
As the HPLC column, for example, an SOD column can be used, and low molecular thiols can be separated by gradient elution of acetonitrile and phosphoric acid solution. Elution conditions are, for example, when moving bed A is 0.15M phosphoric acid and moving bed B is 100% acetonitrile, 0-20 minutes 2% B, 20.1-25 minutes 4% B, 25.1-27 minutes 15% B, 27.1 The distribution of peaks at -30 minutes 15% B, 30.1-45 minutes 2% B, and finer gradients can improve peak separation.
The detector may be measured using a fluorescence detector at an excitation wavelength of about 380 nm and a fluorescence wavelength of about 510 nm.

  FIG. 5 shows an example of the scheme of the method of the present invention. The conditions described in FIG. 5 are an example, and the present invention is not limited to this. As shown in FIG. 5, the HPLC analysis method in which a fluorescent substance is bound to the first part of the sample is called “reduction analysis method”, and the second part of the sample is reduced with a reducing agent. The method of HPLC analysis by binding a fluorescent substance is called “(reduced type + oxidized type) analytical method”. In the reduced analysis method, the reduced low molecular thiol in the biological sample is quantified as it is, and in the (reduced + oxidized) analytical method, the oxidized low molecular thiol in the biological sample is reduced and converted to the reduced form. In the biological sample, both reduced and oxidized low molecular thiols are quantified.

  When analyzing a low molecular thiol by HPLC, a calibration curve of reduced low molecular thiol and oxidized low molecular thiol is prepared for each measurement, and a quantitative value is calibrated based on the calibration curve. This is the possibility that oxidized low molecular thiol is mixed in the reduced low molecular thiol reagent used as the standard sample, or reduced low molecular thiol is mixed in the oxidized low molecular thiol reagent. Because there is. In addition to the conversion from oxidized low molecular thiol to reduced low molecular thiol reagent by reducing agent treatment, unwanted conversion of oxidized low molecular thiol and reduced low molecular thiol occurred during the series of steps. Because there is a possibility that. Furthermore, the conversion efficiency from the oxidized low molecular thiol to the reduced low molecular thiol by the reducing agent, that is, the efficiency of the reducing agent is likely to vary from experiment to experiment. In order to calibrate the fluctuation of the measurement value due to the mixing of impurities due to the interconversion of oxidized low molecular thiol and reduced low molecular thiol, and to calibrate the fluctuation of the measurement value due to the fluctuation of the reducing agent efficacy, Create a calibration curve for each.

For example, the calibration curve is created and the quantitative value is calibrated as follows.
(1) Prepare an oxidized standard product and a reduced standard product of low molecular thiol to be measured. At this time, when there are a plurality of low molecular thiols to be measured, a standard product in which a plurality of low molecular thiols are mixed may be prepared. In the present invention, the reduction standard product is called an SH standard product, and the oxidation standard product is called an SS standard product. Furthermore, a standard product in which a plurality of low molecular thiols are mixed is called a Mix standard product. Therefore, a standard product in which a plurality of reduced low molecular thiols are mixed is called an SH Mix standard product, and a standard product in which a plurality of oxidized low molecular thiols are mixed is called an SS Mix standard product. What is necessary is just to use a commercially available thing. The low molecular thiol oxidized form and the reduced form are serially diluted to prepare a standard sample having a plurality of concentrations.

(2) An equal amount is taken as the first part and the second part from the reduced standard sample and the oxidized standard sample, and the first part is labeled with the fluorescent substance (reduced type). Analytical method). The second part is reduced with the above reducing agent and then labeled with a fluorescent substance ((reduced type + oxidized type) analysis method). At this time, the internal standard substance is also added to the standard sample. At this time, the sample in which the first part of the reduced standard sample is labeled with a fluorescent substance is converted into an SH reduced analytical standard sample (or SH Mix reduced analytical standard sample if multiple low molecular thiols are included). The second part of the oxidized standard sample is treated with a reducing agent, and the sample labeled with a fluorescent substance is used for the SS (reduced + oxidized) analytical method standard sample (if it contains multiple low-molecular thiols) , SS Mix (reduced type + oxidized type) analytical method standard sample).
A series of processing is performed in parallel by the same method as the biological sample.

(3) The SH reduced analytical method standard sample sample and the SS (reduced + oxidized) analytical method standard sample sample obtained in (2) are analyzed by HPLC. The peak area of each peak in the chromatogram (HPLC chromatogram peak area) is obtained by HPLC analysis. Based on the peak area of the internal standard, the HPLC chromatogram peak areas of the SH reduction method standard sample and the SS (reduction type + oxidation type) standard method sample are normalized. Normalization may be performed by dividing the HPLC chromatogram peak area of the sample by the HPLC chromatogram peak area of the internal standard. A value normalized based on the peak area is referred to as a normalized HPLC chromatogram peak area.

(4) In this way, based on the normalized HPLC chromatogram peak area, a calibration curve is obtained in which the concentration of the standard sample is plotted on the horizontal axis and the normalized HPLC chromatogram peak area is plotted on the vertical axis.

(5) Next, the reduction efficiency of the reducing agent is derived based on the calibration curves of the SH reduction type analysis method standard sample and the SS (reduction type + oxidation type) analysis standard sample. The reduction efficiency can be derived based on the normalized HPLC chromatogram peak area at one point of a specific concentration of the calibration curve, for example, a point of 10 μM. At this time, the HPLC chromatogram peak area may be obtained and averaged based on a plurality of points.

For example, assume that the following normalized HPLC chromatogram peak areas SHx and SSx are obtained for a specific concentration XM of the standard sample.
Standard sample for SH reduction analysis method (10μM): SHx
SS (reduced type + oxidized type) analytical method standard sample (10μM): SSx

  In this case, 0.5 / (SHx / SSx) = SHx / 2SSx is the reducing agent efficacy rate. Here, SSx / SHx is divided by 2 because reduced low molecular thiol 2M is generated by completely reducing oxidized low molecular thiol 1M. In the above formula, SHx / SSx represents the efficacy factor of the reducing agent. When the efficacy factor is 0.5, it indicates that all oxidized low molecular thiols have been reduced by the reducing agent treatment, and the reducing agent efficacy rate and efficacy factor are When 0.625, 80% oxidized low molecular thiol was reduced by the reducing agent treatment.

(6) Next, based on the normalized HPLC chromatogram peak area of the reduced biological sample to be quantified and the normalized HPLC chromatogram peak area of the (reduced + oxidized) treated and reducing agent efficacy rate, A correction value corresponding to the normalized HPLC chromatogram peak area of the oxidized form of the biological sample is obtained. The correction formula is the normalized HPLC chromatogram peak area of the sample treated with the (reduced type + oxidized type) analysis method− (normalized HPLC chromatogram peak area of the sample treated with the reducing type analysis method) − (reducing agent efficacy rate) Represented. The correction value in this case is called an oxidation type conversion value.

The concentration of reduced low molecular thiol in the biological sample is calculated based on the calibration curve of the standard SH reduced analytical method standard sample against the normalized HPLC chromatogram peak area of the reduced biological sample. The In addition, based on the calibration curve of the standard sample sample for SS (reduced type + oxidized type) for the correction value corresponding to the normalized HPLC chromatogram peak area of the oxidized type in the biological sample, the biological sample The concentration of oxidized low molecular thiol is calculated. In this case, represented by y = mx ^h (m and h are coefficients) in advance to create a regression equation of the calibration curve may be obtained from the formula.

  Furthermore, the calculation method of the reduced type and oxidized low molecular thiol in a biological sample is demonstrated by a specific example.

  As standard products, cysteine (reduced type) standard product (Sigma Aldrich) and cystine (oxidized type) standard product (SIGMA) were used.

  As samples, two biological samples (human plasma) were used.

  To prepare a reduced calibration curve, cysteine standards (10 μM and 50 μM) were labeled with SBD-F (SH reduced analysis method standard sample) and analyzed by HPLC.

  To prepare an oxidized calibration curve, cystine standards (10 μM and 50 μM) were reduced with TCEP and neutral pH (SS (reduced + oxidized) analytical method standard sample), labeled with SBD-F, and HPLC Analyzed with

  In addition, the biological sample was labeled with SBD-F (SH reduction analysis method biological sample sample) and analyzed by HPLC. Further, the sample was subjected to reduction treatment with TCEP and neutral pH (SS (reduced + oxidized) analysis biological sample sample), labeled with SBD-F, and analyzed by HPLC. At this time, N-Acetyl-L-cysteine was added as an internal standard to both the standard product and the biological sample. At the same time, a control sample may be used (SH reduction analysis method control sample and SS (reduction type + oxidation type) analysis method control sample).

  The HPLC chromatogram peak area of each standard sample was divided by the internal standard HPLC chromatogram peak area to calculate a normalized HPLC chromatogram peak area. The normalized HPLC chromatogram area of each was as follows:

Sample Normalized HPLC chromatogram area standard sample
Standard sample for SH reduction analysis method (10μM) 0.1265
(50μM) 0.6598
SS (reduced type + oxidized type) analytical method standard sample (10μM) 0.2778
(50μM) 1.662

Biological sample 1
SH reduction analysis method biological sample 0.2175
SS (reduced + oxidized) analysis method biological sample 2.461

Biological sample 2
SH reduction analysis method biological sample 0.5874
SS (reduced type + oxidized type) analysis method biological sample 3.581

  The HPLC raw data (HPLC chromatogram area) in the biological sample is as follows, and the above normalized HPLC chromatogram area was determined by dividing each HPLC chromatogram area by the chromatogram area of the internal standard. .

Biological sample 1 HPLC chromatogram area
SH reduction analysis method biological sample 4.189 × 10 ³
Internal standard 1.930 × 10 ⁴

SS (reduced + oxidized) analysis method biological sample 4.373 × 10 ³
Internal standard 1.777 × 10 ⁴

Biological sample 2
SH reduction type biological sample 7.199 × 10 ³
Internal standard 1.226 × 10 ⁴

SS (reduction type + oxidation type) analysis method biological sample 5.350 × 10 ⁴
Internal standard 1.494 × 10 ⁴

A calibration curve was prepared based on the normalized HPLC area value of the standard sample. Two calibration curves were prepared for the standard sample for the SH reduction type analysis method and for the standard sample for the SS (reduction type + oxidation type) analysis method. FIG. 6 shows the calibration curve of the standard sample for SH reduction analysis method, and FIG. 7 shows the calibration curve for the standard sample sample of SS (reduction type + oxidation type). Each regression equation y = 0.0119x ^1.0265, was y = 0.0215x ^1.1115.

The reducing agent efficacy rate was determined using the normalized HPLC chromatogram area at 10 μM.
Calculated from the formula 0.5 / (Normalized HPLC chromatogram area of SH reduction method standard product sample / Normalized HPLC chromatogram area of SS (reduced type + oxidized type) analytical method sample) (%), 0.5 / A reducing agent efficacy rate of (0.1264 / 0.2778) = 1.099 was obtained.

Subsequently, the low molecular thiol in a biological sample was calculated.
(A) Calculation of reduced low molecular thiol in biological sample The concentration was determined based on the calibration curve of FIG. That is, to determine the concentration using the regression equation represented by y = 0.0119x ^1.0265, corresponding to a normalized HPLC chromatogram area SH reduction spectrometry biological sample samples.

Biological sample 1 y = 0.2175
From the regression equation ^{y = 0.0119x 1.0265, x = 16.95} (μM) were calculated.

Biological sample 2 y = 0.5874
From the regression equation y = 0.0119x ^1.0265 , x = 44.63 (μM) was obtained.

(B) Calculation of oxidized low molecular weight thiols in biological samples Normalized HPLC chromatogram peak area of sample processed by formula (reduced type + oxidized type)-(reducing agent efficacy rate x sample processed by reduced type analysis method) (Normalized HPLC chromatogram peak area) of the biological sample was obtained a corrected value (oxidized form conversion value) corresponding to the normalized HPLC chromatogram peak area of the oxidized form of the biological sample. Furthermore, the concentration was determined based on the calibration curve of FIG. In other words, the concentration corresponding to the oxidized conversion value was determined using a regression equation represented by y = 0.0215 × ^1.1115 .

The oxidation type conversion value of the biological sample 1 was 2.461− (1.099 × 0.2175) = 2.222 = y. The oxidized ^value was applied to the regression equation y = 0.0119x1.0265, and the concentration x = 64.90 (μM) was obtained.

The oxidation type conversion value of the biological sample 2 was 3.581− (1.099 × 0.5874) = 2.936 = y. The oxidized ^value was applied to the regression equation y = 0.0119x1.0265 to obtain a concentration x = 83.38 (μM).

  The reduced / oxidized ratio was 0.261 for biological sample 1 and 0.535 for biological sample 2.

  According to the method of the present invention, it is possible to carry out the differential quantification of the reduced form and the oxidized form of the low molecular thiol, obtain the ratio of the oxidized form and the reduced form from the obtained quantitative value, and use this ratio as an index of the increase in oxidative stress. be able to. When oxidative stress is increasing, the trend of low-part thiols in the living body changes, and the ratio of oxidized low-molecular thiols to reduced low-molecular thiols changes from when no oxidative stress is applied. That is, a change in the ratio of oxidized low molecular thiol to reduced low molecular thiol in the subject indicates a change in oxidative stress. In addition, when the ratio of oxidized low molecular thiol to reduced low molecular thiol in the subject significantly increased or decreased from a normal person who did not receive oxidative stress, this indicates that the subject had increased oxidative stress. obtain.

  Since an increase in oxidative stress can cause diseases such as arteriosclerosis, myocardial infarction, diabetes, and cancer, it is possible to determine the risk of these diseases by evaluating oxidative stress.

  The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.

Example 1 Low molecular thiol measuring method The following reagents etc. were used for the measurement of the low molecular thiol in a biological sample.
L-cystine (oxidized cysteine), DL-homocysteine, DL-homocystine, cystenylglycine (Cys-Gly), oxidized cys-Gly, GSH (reduced glutathione), GSSG (oxidized) Type glutathione), N-acetyl-L-cysteine, sodium borate and sulfosalicylic acid were purchased from SIGMA. L-cysteine used as a standard was purchased from Sigma-Aldrich. Phosphoric acid, acetonitrile and hydrochloric acid were purchased from Wako Pure Chemical Industries. 2Na-EDTA and SBD-F were purchased from Dojin Chemical. TCEP and neutral pH were purchased from PIERCE.
HPLC manufactured by Shimadzu Corporation was used. Thermo HK30103-154630 was used as the column.

The measurement was performed by the following method.
Each standard reagent was prepared with 5 mM EDTA · 2Na, and the oxidized standard was prepared in principle. Biological sample samples were prepared by collecting blood in a cooled EDTA blood collection tube and immediately centrifuging at 1900 g for 3 minutes at 0 ° C. to obtain plasma. Immediately thereafter, the next process was started.

Measurement of low molecular thiols in biological samples
500 μl of sample or standard, 600 μl of cooled 2.5% sulfosalicylic acid (SSA) in 5 mM 2Na-EDTA (final), 250 μM N-acetyl-L-cysteine in 0.1 M sodium borate (pH 9.3 2Na- (EDTA) was added, vortexed, and centrifuged at 13200 rpm, 4 ° C. for 15 minutes to obtain a supernatant. The supernatant was partially treated by the reduction treatment method and partly by the (reduction type + oxidation type) treatment method. In the reduction treatment method, 10 μl of 0.1 M sodium borate (pH9.3 2Na-EDTA) and 50 μl of 10 mM SBD-F in 0.1 M sodium borate (pH9.3 2Na-EDTA) are added to 25 μl of the supernatant. Started. After incubating in a 60 ° C. water bath for 30 minutes, it was cooled in ice. 25 μl of 2 μM hydrochloric acid and 1 ml of 0.1 M sodium borate (pH9.3 2Na-EDTA) were added to prepare a sample for HPLC measurement. The (reduced + oxidized) treatment method was started by adding 10 μl of 2.5% TCEP, neutral pH, 50 μl of 10 mM SBD-F in 0.1 M sodium borate (pH9.3 2Na-EDTA) to 25 μl of supernatant. . After incubating in a 60 ° C. water bath for 30 minutes, it was cooled in ice. 25 μl of 2 μM hydrochloric acid and 1 ml of 0.1 M sodium borate (pH9.3 2Na-EDTA) were added to prepare a sample for HPLC measurement.
FIG. 5 shows a scheme for collecting a biological sample and preparing a sample for HPLC measurement.

HPLC measurement conditions were as follows.
20 μl of the HPLC sample was passed through the SOD column under the conditions of a flow rate of 1 ml / min and a column temperature of 40 ° C., and measured at an excitation of 380 nm and an emission of 510 nm. The mobile layer A was 0.15M phosphoric acid, the mobile layer B was 100% acetonitrile, and the distribution was as follows. 0-20 minutes 2% B, 20.1-25 minutes 4% B, 25.1-27 minutes 15% B, 27.1-30 minutes 15% B, 30.1-45 minutes 2% B.

  The low molecular thiol concentration in the biological sample was calculated as follows based on the calibration curve of the reduced low molecular thiol standard product and the oxidized low molecular thiol standard product, and a quantitative value was obtained.

  Each time a biological sample was measured, the same treatment was applied to both reduced and oxidized low molecular thiol standards, and each calibration curve was drawn by HPLC analysis. At this time, using the internal standard, normalize the HPLC chromatogram peak area of the standard product by dividing the chromatogram peak area of the reduced and oxidized low molecular thiol standard HPLC by the chromatogram peak area of the internal standard. (Normalized chromatogram peak area). From any one point (XμM) on the calibration curve, the efficiency of the reducing agent is expressed by the formula 0.5 / (value obtained by SH XμM reduction type analysis method / SS XμM (reduction type + oxidation type) analysis method) The value obtained by the formula (reduced type + oxidized type) analysis method from the value obtained by the (reduced type + oxidized type) analytical method of the biological sample obtained by HPLC- Calculated by (reduced potency x value obtained by the reduced analysis method) The value obtained by the reduced analysis method of the sample was converted to the calibration curve created using the reduced standard and the converted oxidized form. The values were obtained for each low molecular thiol value from a calibration curve prepared using a standard product obtained by reducing an oxidized standard product.

Example 2 Measurement of oxidized and reduced low-molecular thiols using oxidative stress model mice Six-week-old Slc: ddY mice (obtained from Sankyo Lab Co., Ltd.) were preliminarily raised for 4 days and then subjected to experiments. The water and food were freely consumed, the temperature of the breeding room was set at 23 ± 1 ° C, and the light period was set between 6:30 a.m. And 7:00 p.m.

  After the pre-feeding period, streptozotocin (STZ) in 0.1 M citrate buffer, 200 mg / kg was injected intraperitoneally into the oxidative stress model. After confirming that the blood glucose level of the oxidative stress model was increased 11 days later (using a simple blood glucose meter advantage), serum was collected by collecting blood from the fundus and after cervical dislocation. During the experimental period, body weight and water consumption were measured at any time.

Oxidized and reduced cysteine, oxidized and reduced glutathione in the serum of oxidative stress model mice and control mice not administered with streptozotocin were measured by the method described in Example 1. Five mice were used for each of the oxidative stress model group and the control group.
Table 1 shows blood glucose levels on the 11th day.

  An increase in blood glucose level indicated that oxidative stress was increased, and that oxidative stress was increased in the STZ administration group.

  FIG. 8 and FIG. 9 show the body weight fluctuation and the amount of drinking water during the experiment. Here, the body weight is an index of the health condition of the mouse, and the amount of drinking water is an index indicating the damage caused by STZ of the mouse. As a result of various adverse effects of oxidative stress on the living body, changes in body weight and water consumption occurred, so body weight and water consumption were measured as secondary indicators of oxidative stress.

  FIG. 10 shows the reduced cysteine / oxidized cysteine ratio in oxidative stress model mice and control mice administered with STZ. FIG. 11 shows the reduced glutathione / oxidized glutathione ratio.

  As shown in the figure, there is a difference in the reduced low molecular thiol / oxidized low molecular thiol ratio between the oxidative stress model mouse administered with STZ and the control mouse, and the ratio is high in the oxidative stress model mouse and the reduced low molecular thiol Had increased.

Example 3 Measurement of low molecular thiols in angina patients and normal persons Both cysteine, homocysteine and glutathione oxidized and reduced forms of 20 angina patients and 20 healthy persons were measured by HPLC. By comparing the average ratio and distribution of each group, we investigated the relationship between the progression of arteriosclerosis and the ratio of oxidized and reduced forms of low-molecular thiols. Angina appears as a terminal symptom of arteriosclerosis, which indicates that the patient has been exposed to oxidative stress in the past or present.

  The subjects were 20 patients and 20 healthy individuals who were hospitalized at Keio University Hospital Cardiovascular Medicine for angina with coronary artery stenosis.

  Explain the purpose of the research sufficiently to the target person, obtain written consent, collect 2 cc of blood (Terumo; use Benogject vacuum blood collection tube), and separate the plasma. The low molecular thiol was measured by the method described.

  FIG. 12 shows the results of cysteine, FIG. 13 shows the results of cystenylglycine, and FIG. 14 shows the results of glutathione.

  As shown in the figure, for any low molecular thiol, the reduced / oxidized ratio was small compared to healthy individuals in patients with angina.

  According to the present invention, human oxidative stress can be measured and evaluated, and the risk of suffering from a disorder or disease caused by an increase in oxidative stress can be determined.

It is a figure which shows the structural formula of cysteine, cystenylglycine, glutathione, and a homocysteine. It is a figure which shows the structure of the reduction type of low molecular thiol, and an oxidation type. It is a figure which shows the measurement principle of a low molecular thiol. It is a figure which shows the measurement principle of an oxidation type low molecular thiol. It is a figure which shows the measuring method of a low molecular thiol. It is a figure which shows an example of the calibration curve of SH reduction | restoration type analysis method standard goods sample. It is a figure which shows an example of the calibration curve of SS (reduction type + oxidation type) analytical method standard goods sample. It is a figure which shows the body weight fluctuation | variation of the oxidative stress model mouse | mouth and control mouse | mouth in the reduction | restoration type / oxidation type ratio examination experiment of the low molecular thiol in a mouse | mouth biological body. It is a figure which shows the amount of water intake of the oxidative stress model mouse and the control mouse | mouth in the reduction | restoration type / oxidation type ratio examination experiment of the low molecular thiol in a mouse | mouth biological body. It is a figure which shows the reduced cysteine / oxidized cysteine ratio in the oxidative stress model mouse | mouth in a mouse | mouth living body low molecular thiol reduction type / oxidation type ratio examination experiment, and a control mouse. It is a figure which shows the reduced glutathione / oxidized glutathione ratio in the oxidative stress model mouse | mouth in a mouse | mouth living body low molecular thiol reduction type / oxidation type ratio examination experiment, and a control mouse. It is a figure which shows the reduced cysteine / oxidized cysteine ratio in an angina patient and a normal person. It is a figure which shows the reduced cystenyl glycine / oxidation type cystenyl glycine ratio in an angina patient and a normal person. It is a figure which shows the reduced glutathione / oxidized glutathione ratio in an angina patient and a normal person.

Claims (16)

  A method for measuring oxidative stress in a living body, wherein the reduced form and oxidized form of a low molecular thiol in a biological sample are separately quantified and the abundance ratio of the oxidized form and reduced form of the low molecular thiol is used as a marker.   The method for measuring oxidative stress in a living body according to claim 1, wherein the low molecular thiol in the biological sample is at least one selected from the group consisting of cysteine, cystenylglycine, glutathione and homocysteine.   The method for measuring oxidative stress in a living body according to claim 1 or 2, wherein the biological sample is selected from the group consisting of blood, serum and plasma.   The method for measuring oxidative stress in a living body according to any one of claims 1 to 3, wherein fractional quantification of a reduced form and an oxidized form of a low molecular thiol in a biological sample is performed using HPLC.   A method for differentially quantifying a reduced form and an oxidized form of a low molecular thiol in a biological sample, wherein the biological sample is deproteinized, and the same amount of the first part and the second part are collected from the deproteinized sample. First, the reduced low molecular thiol of the first part is fluorescently labeled, the low molecular thiol of the second part is reduced with a reducing agent, the reduced low molecular thiol is fluorescently labeled, and the fluorescently labeled first The low molecular thiol of the second part and the low molecular thiol of the second part are analyzed by HPLC, and the reduced low molecular thiol and the oxidized low molecular weight are analyzed based on the analytical value of the first part and the analytical value of the second part. A method for differentially quantifying a reduced form and an oxidized form of a low molecular thiol in a biological sample to calculate a thiol concentration.   The low molecular thiol in the biological sample is at least one selected from the group consisting of cysteine, cystenylglycine, glutathione, and homocysteine. How to quantify.   The method for differentially quantifying a reduced form and an oxidized form of a low molecular thiol in a biological sample according to claim 5 or 6, wherein the protein removal treatment of the biological sample is performed using sulfosalicylic acid.   After collecting a biological sample from a living body, the reduced form and the oxidized form of the low molecular thiol in the biological sample according to any one of claims 5 to 7, which are cooled to 0 to 4 ° C within 1 minute, are determined. how to.   The reduction in the second portion of the deproteinized biological sample is performed using a water-soluble reducing agent that acts at a neutral pH. A method for differential quantification of reduced and oxidized forms of molecular thiols.   The method according to claim 9, wherein the water-soluble reducing agent acting at a neutral pH is TCEP or Neutral pH.   Quantification of reduced low-molecular thiols and oxidized low-molecular thiols in biological samples is performed by reducing the calibration curve created using reduced low-molecular thiol standards and oxidized low-molecular thiol standards with a reducing agent. The low molecular thiol in the biological sample according to any one of claims 5 to 10, which is performed using two calibration curves of a calibration curve prepared using the obtained reduced low molecular thiol standard. A method for separately quantifying reduced and oxidized forms. The reduced low-molecular thiol and the oxidized low-molecular thiol in the biological sample are quantified by a method including the following steps. Method:
(i) Analyzing a reduced standard product of reduced low molecular thiol and oxidized low molecular thiol standard with a reducing agent by HPLC, and analyzing the HPLC chromatogram peak area of the standard product as an internal standard HPLC chromatogram Normalizing based on peak area and creating two calibration curves;
(ii) The step of determining the reducing agent efficacy by the formula 0.5 / (SHx / SSx) from the result of HPLC analysis of (i), where SHx is normalized at a specific concentration of the reduced low molecular thiol standard Represents the HPLC chromatogram peak area, and SSx represents the normalized HPLC chromatogram peak area at the specific concentration of the standard product obtained by reducing the oxidized low molecular weight thiol standard product with a reducing agent;
(iii) (a) A sample obtained by fluorescently labeling the reduced low-molecular thiol of the first part of the biological sample was obtained by HPLC analysis, and normalized based on the HPLC chromatogram peak area of the internal standard From the HPLC chromatogram peak area, based on the calibration curve created using the reduced low molecular thiol standard (i), determine the reduced low molecular thiol concentration of the first part,
(b) Reductant efficacy determined in (ii), (iii) Obtained by HPLC analysis of a sample obtained by fluorescently labeling the reduced low molecular thiol of the first part of the biological sample in (a) , HPLC chromatogram peak area normalized based on HPLC chromatogram peak area of internal standard, and reduced low molecular thiol after reducing low molecular thiol in the second part of biological sample with reducing agent Sample obtained by HPLC analysis of the sample obtained by labeling and processed by the formula (reduced type + oxidized type) analysis method from the HPLC chromatogram peak area normalized based on the HPLC chromatogram peak area of the internal standard Normalized HPLC chromatogram peak area of oxidized low molecular weight thiols in biological samples by normalizing HPLC chromatogram peak area of-(reducing agent efficacy x normalized HPLC chromatogram peak area of sample processed by reducing analysis method) A correction value corresponding to the product is obtained, and from the correction value, the second part is calculated based on a calibration curve prepared using a standard product obtained by reducing the oxidized low molecular thiol standard product of (i) with a reducing agent. The process which calculates | requires the oxidation type low molecular thiol density | concentration of.   The abundance ratio of oxidized form and reduced form of low molecular thiol from the quantitative value obtained by the method of differentially quantifying the reduced form and oxidized form of low molecular thiol in the biological sample according to any one of claims 5 to 12. The biological oxidative stress measurement method according to claim 1, wherein the biological oxidative stress is measured from the abundance ratio.   A kit for differential quantification of reduced and oxidized forms of low molecular weight thiols in a biological sample containing at least a deproteinizing agent that is sulfosalicylic acid and a reducing agent that is TCEP and Neutral pH.   The kit for differential quantification of reduced and oxidized forms of low-molecular thiols according to claim 14, further comprising reduced and oxidized low-molecular thiol standards and fluorescent substances for fluorescently labeling low-molecular thiols.   The kit for differential quantification of a reduced form and an oxidized form of a low molecular thiol according to claim 14 or 15, wherein the low molecular thiol is at least one selected from the group consisting of cysteine, cystenylglycine, glutathione and homocysteine. .

Images