Method for measuring mandelic acid and measurement kit used therefor

Abstract

The present invention provides, for example, a novel method for measuring mandelic acid and a measurement kit used therefor.　Provided is a method for measuring mandelic acid in a specimen, said method comprising a step for causing dehydrogenase to act on mandelic acid in the presence of NAD+ and / or NADP+ and measuring the amount of change of NADH and / or NADP.

Description

Method for measuring mandelic acid and measurement kit used therefor

[0001] The present invention relates to a novel method for measuring mandelic acid, and a measurement kit used therefor. ＋ and NADP ＋ The present invention relates to a method for measuring mandelic acid by reacting dehydrogenase with mandelic acid in the presence of, for example, and measuring the amount of change in NADH, NADPH, or both, and a measurement kit used therefor.

[0002] Since mandelic acid and phenylglyoxylic acid are known as metabolic products of styrene and are used as indicators of styrene exposure, measuring mandelic acid and phenylglyoxylic acid is useful in clinical testing of workers who handle these organic solvents.

[0003] Until now, mandelic acid has been measured by measuring mandelic acid in a sample by high performance liquid chromatography, as in the measurement of catecholamine metabolites (see, for example, Patent Documents 1 and 2). However, in recent years, with increasing demand for occupational health measures for workers, there has been a demand for the development of a technique for measuring mandelic acid more simply, quickly, and at low cost.

[0004] JP-A-4-122855 JP-A-5-113438

[0005] In light of the above circumstances, an object of the present invention is to provide a novel method for measuring mandelic acid, and a measurement kit and the like used therefor.

[0006] As a result of intensive research to solve the above problems, the present inventors have succeeded in creating a novel method for measuring mandelic acid, as shown below, and have found that the above object can be achieved by the above method, thereby completing the present invention.

[0007] That is, the present invention provides the following method for measuring mandelic acid.

[0008] [1] A method for measuring mandelic acid in a sample, comprising: ＋ , NADP ＋10. A method for measuring mandelic acid, comprising the steps of: allowing a dehydrogenase to act on mandelic acid in the presence of NADH, NADPH, or both; and measuring the amount of change in NADH, NADPH, or both.

[0009] [2] The measurement method according to [1], further comprising a racemase in the step.

[0010] [3] A method for measuring mandelic acid in a sample, comprising: ＋ , NADP ＋ a method for measuring the amount of formazan dye generated, the method comprising the steps of: reacting a dehydrogenase and a reducing color-developing reagent with mandelic acid in the presence of a formazan dye-producing agent or a formazan dye-producing agent;

[0011] [4] The measurement method according to [3], further comprising a racemase in the step.

[0012] [5] The measurement method according to any one of [1] to [4], further comprising diaphorase or an electron mediator in the step.

[0013] [6] The method of measuring according to [2], [4], or [5], wherein the racemase includes mandelate racemase.

[0014] [7] The method according to any one of [1] to [6], wherein the dehydrogenase includes mandelate dehydrogenase.

[0015] [8] Reduction-based color-developing reagents include 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1), 2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-3), 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8), 3,3'-[3,3'-dimethoxy-(1,1'-biphenyl)-4,4'-diyl]-bis[2-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride] (nitro-TB), and The measurement method according to any one of [3] to [7], further comprising one or more members selected from the group consisting of 3,3'-[3,3'-dimethoxy-(1,1'-biphenyl)-4,4'-diyl]bis(2,5-diphenyl-2H-tetrazolium chloride) (TB).

[0016] [9] The measurement method according to any one of [1] to [8], wherein the measurement is carried out by absorbance measurement.

[0017]

[10] The measurement method according to any one of [1] to [9], wherein the sample includes urine or other body fluids.

[0018]

[11] The measurement method according to any one of [1] to

[10] , for testing the degree of exposure to styrene, ethylbenzene, or both.

[0019] The present invention also relates to the following measurement kits:

[0020]

[12] NAD ＋ , NADP ＋ , or both, dehydrogenase, or a combination thereof, for use in the measurement method according to any one of [1], [2], [5], [6], and [8] to

[11] .

[0021]

[13] NAD ＋ , NADP ＋or both, dehydrogenase, a reducing color-developing reagent, or a combination thereof.

[0022]

[14] The assay kit according to

[12] or

[13] , further comprising a racemase, a diaphorase, an electron mediator, or any combination thereof.

[0023]

[15] The measurement kit according to any one of

[12] to

[14] , for measuring the amount of mandelic acid in urine.

[0024]

[16] The measurement kit according to any one of

[12] to

[14] for testing the degree of exposure to styrene, ethylbenzene, or both.

[0025] By using the method for measuring mandelic acid of the present invention, measurement can be performed using, for example, an automatic analyzer, etc., and mandelic acid can be measured more simply, quickly, and at lower cost than conventional measurement using high performance liquid chromatography, etc.

[0026] Furthermore, by using the measurement kit of the present invention, it becomes possible to easily carry out the above-mentioned method for measuring mandelic acid.

[0027] FIG. 1 is a schematic diagram illustrating an overview of a method for measuring mandelic acid, an example of the present disclosure. The schematic diagram illustrates a system for calculating mandelic acid (the total amount of L-mandelic acid and D-mandelic acid) by measuring the change in NADH. FIG. 2 is a schematic diagram illustrating an overview of a method for measuring mandelic acid, another example of the present disclosure. The schematic diagram illustrates a system for calculating mandelic acid (the total amount of L-mandelic acid and D-mandelic acid) by measuring the change in formazan. FIG. 3 is a graph illustrating a comparison between the measured values ​​of D-mandelic acid in a urine sample and the known concentration of D-mandelic acid in physiological saline in Example 1 of the present disclosure. FIG. 4 is a graph illustrating a comparison between the measured values ​​of L-mandelic acid in a urine sample and the known concentration of L-mandelic acid in physiological saline in Example 1 of the present disclosure. Figure 5 is a graph showing a comparison of the measured values ​​of D-mandelic acid in urine samples with known concentrations of D-mandelic acid in physiological saline in Example 2 of the present disclosure. Figure 6 is a graph showing a comparison of the measured values ​​of L-mandelic acid in urine samples with known concentrations of L-mandelic acid in physiological saline in Example 2 of the present disclosure.

[0028] Hereinafter, embodiments of the present invention will be described in detail.

[0029] [Method for measuring mandelic acid] The method of the present invention is a method for measuring mandelic acid in a sample, comprising: ＋ , NADP ＋ The method includes a step of reacting dehydrogenase with mandelic acid in the presence of NADH, NADPH, or both, and measuring the amount of change in NADH, NADPH, or both.

[0030] The measurement method of the present invention is ＋ and NADP ＋ This method measures mandelic acid by detecting the amount of change in NADH, NADPH, etc., caused by a chemical reaction in which dehydrogenase acts (catalyzes) on mandelic acid in the presence of, for example, an automatic analyzer, and enables measurement more simply, quickly, and at lower cost than conventional methods.

[0031] The above measurement method is presumed to be effective through the following mechanism of action, but the scope of the invention is not limited to this mechanism. ＋ and NADP ＋ In the presence of ＋ and NADP ＋ is reduced to NADH and NADPH. It is presumed that mandelic acid can be measured in a concentration-dependent manner by observing the changes in NADH and NADPH.

[0032] Furthermore, L-mandelic acid and D-mandelic acid are optical isomers that can be isomerized with each other by the action of racemase. In the above measurement method, the amount of mandelic acid (the total amount of L-mandelic acid and D-mandelic acid) in a sample (analyte) can be calculated by observing the changes in NADH and NADPH.

[0033] In the present invention, the above-mentioned "measurement" also includes simple "detection" aimed at simply confirming or detecting the presence of mandelic acid.

[0034] In the above measurement method, the "amount of change" typically refers to the amount of increase in NADH, NADPH, or both.

[0035] In the above measurement method, the mixing order and preparation method of each component are not particularly limited. ＋ , NADP ＋ Alternatively, the dehydrogenase and / or the dehydrogenase may be mixed or prepared in whole or in part simultaneously or sequentially as appropriate.

[0036] The mandelic acid to be measured may be L-mandelic acid, D-mandelic acid, or a mixture thereof.

[0037] The above NAD(P) ＋ is NAD(P) ＋ Any known or novel substance can be used as a source without any particular limitation.

[0038] The dehydrogenase may be a known or novel dehydrogenase, and may be a dehydrogenase that can be used to decompose mandelic acid and NAD ＋ and NADP ＋ These enzymes are not particularly limited as long as they act to convert D-mandelic acid to phenylglyoxylic acid, NADH, and NADPH, respectively, and examples thereof include mandelate dehydrogenase. In the measurement method of the present invention, it is preferable to include the mandelate dehydrogenase, which is an enzyme that reversibly acts only on D-mandelic acid. These enzymes may be used alone or in combination of two or more.

[0039] In the above measurement method, the above step is preferably carried out by measuring absorbance at, for example, 310 nm to 370 nm. Since the ultraviolet absorption maximum of NADH and NADPH is at 340 nm, it is preferable to set the range to include this range. The absorbance measurement may be, for example, 320 nm to 360 nm or 330 nm to 350 nm.

[0040] Another measurement method of the present invention is a method for measuring mandelic acid in a sample, comprising: ＋ , NADP ＋ The method includes a step of reacting mandelic acid with a dehydrogenase and a reducing color-developing reagent in the presence of the enzyme or both, and measuring the amount of formazan dye generated.

[0041] The measurement method of the present invention first ＋ and NADP ＋ In this method, mandelic acid is measured by reacting dehydrogenase (catalysis) with mandelic acid in the presence of NADH or NADPH, and then reacting it with a tetrazolium salt, which is a reducing coloring agent (and diaphorase or an electron mediator, which is a charge carrier), and detecting the amount of change in the formazan produced. This method allows for easier, faster, and lower-cost measurement than conventional methods, for example, using an automatic analyzer. ＋ and NADP ＋ can be reused in the mandelate dehydrogenase reaction.

[0042] The above measurement method is presumed to be effective through the following mechanism of action, but the scope of the invention is not limited to this mechanism. ＋ and NADP ＋ In the presence of ＋ and NADP ＋ Next, in order to eliminate this NADH and NADPH, a tetrazolium salt, which is a reducing coloring agent (and diaphorase or an electron mediator, which are charge carriers), is reacted to convert NADH and NADPH to NAD ＋ and NADP ＋ It is believed that mandelic acid can be measured in a concentration-dependent manner by observing the amount of conversion (color development) to formazan resulting from the reduction of the tetrazolium salt that is produced as the mandelic acid is oxidized to formazan.

[0043] In the above measurement method, unless otherwise specified, the method for each component is the same as that for measuring the change in the amount of NADH, etc., as described above.

[0044] As the reducing color-developing reagent, known or novel reducing color-developing reagents can be used as appropriate, and examples thereof include tetrazolium salts. Examples of the reducing color reagent include 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-1), 2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-3), 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8), 3,3'-[3,3'-dimethoxy-(1,1'-biphenyl)-4,4'-diyl]-bis[2-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride] (nitro-TB), 3,3'-[3,3'-dimethoxy-(1,1'-biphenyl)-4,4'-diyl]bis(2,5-diphenyl-2H-tetrazolium chloride) (TB) (for example, a commercially available product manufactured by Dojindo Chemical Industries, Ltd.) can be mentioned. These may be used alone or in combination of two or more.

[0045] In the above step, diaphorase or an electron mediator may be further included. By including the diaphorase or the electron mediator, when the tetrazolium salt, which is a reductive color former, undergoes an oxidation-reduction reaction with NAD(P)H, the diaphorase or the electron mediator can act as a charge carrier to more suitably promote the reaction.

[0046] The diaphorase may be a known or novel diaphorase, as appropriate. Examples of diaphorase include DAD-301 DIAPHORASE (manufactured by Toyobo Co., Ltd.). These may be used alone or in combination of two or more.

[0047] The electron mediator may be a known or novel electron mediator, as appropriate. Examples of the electron mediator include phenazine methosulfate (PMS), phenazine ethosulfate (PES), 1-methoxy-5-methylphenazinium methyl sulfate (1-mPMS), 1-methoxy-5-ethylphenazinium ethyl sulfate (1-mPES), and Meldola Blue (for example, commercially available products manufactured by Dojindo Chemical Industries, Ltd., Sigma-Aldrich, etc.). These may be used alone or in combination of two or more.

[0048] Furthermore, in the above measurement method, the above step is preferably carried out by measuring absorbance at, for example, 400 nm to 560 nm. The wavelength for the absorbance measurement can be within a range defined by any two points selected from the following: 410 nm, 420 nm, 430 nm, 440 nm, 450 nm, 460 nm, 470 nm, 480 nm, 490 nm, 500 nm, 510 nm, 520 nm, 530 nm, 540 nm, 550 nm, and 560 nm. The wavelength for the absorbance measurement is preferably set appropriately within a range that includes the absorption maximum wavelength of the formazan compound generated in the reducing color former used (e.g., −30 nm to +30 nm from the absorption maximum wavelength, −20 nm to +20 nm from the absorption maximum wavelength, or −10 nm to +10 nm from the absorption maximum wavelength).

[0049] Furthermore, in each of the above-mentioned measurement methods, the above steps may also further include a racemase.

[0050] The racemase may be any known or novel racemase, as long as it is capable of racemizing mandelic acid, and may be, for example, a mandelic acid racemase. These may be used alone or in combination of two or more.

[0051] In each of the above measurement methods, the above steps can be carried out, for example, at a pH range of 5 to 9, 6 to 8, or 6.5 to 7.5, which can be appropriately set depending on the chemical reaction and the enzyme activity.

[0052] In the above measurement method, racemase, dehydrogenase, NAD ＋ , NADP ＋ The concentrations of the components, or both of the reducing color-developing reagent, diaphorase, and electron mediator, can be adjusted appropriately depending on the purpose and application.

[0053] In the above measurement method, the sample may include urine or other body fluids.

[0054] Furthermore, the above-mentioned measurement method can be suitably used, for example, particularly for measuring the degree of exposure to styrene, ethylbenzene, or both. Since phenylglyoxylic acid and mandelic acid, which are known as metabolic products of styrene, are used as indicators of styrene exposure, the above-mentioned measurement method, which enables the measurement of mandelic acid, is useful in clinical tests of workers who handle organic solvents such as styrene and ethylbenzene.

[0055] In the above-mentioned measurement method, a known preparatory step, preparation step, post-treatment step, or the like may be provided prior to, after, or both of the above steps depending on the purpose and application.

[0056] [Measurement Kit] The measurement kit of the present invention is ＋ , NADP ＋ or both, dehydrogenase, or a combination thereof, and is used in the above-mentioned measurement method.

[0057] Another measuring kit of the present invention is ＋ , NADP ＋ or both, dehydrogenase, a reducing color-developing reagent, or a combination thereof, and is used in the above-mentioned measurement method.

[0058] Furthermore, another measuring kit of the present invention is any of the above measuring kits, further comprising racemase, diaphorase, an electron mediator, or any combination thereof.

[0059] In the above-mentioned measuring kit, each component is the same as in the above-mentioned measuring method.

[0060] In the above-mentioned measurement kit, when a dehydrogenase, a racemase, a diaphorase, an electron mediator, or a combination thereof is contained, the enzyme solution may be prepared by a known method. The specific composition of the enzyme solution is not particularly limited, and it may contain a dehydrogenase, a racemase, a diaphorase, an electron mediator, or a combination thereof in a known solvent at a concentration that is suitable for use.

[0061] In the above-mentioned measurement method, racemase, diaphorase, a reducing color-developing reagent, dehydrogenase, NAD ＋ , NADP ＋ The concentration of the electron mediator or each of these components can be adjusted appropriately depending on the purpose and application.

[0062] The above-mentioned measurement kit contains the above-mentioned racemase, diaphorase, a reducing color-developing reagent, and NAD ＋ , NADP ＋ In addition to the dehydrogenase, the electron mediator, or any combination thereof, other reagents or test tools may be included.

[0063] The measurement kit may also contain other reagents, such as a diluent for the sample, a diluent for the enzyme reaction, buffers, etc. The reagents may be provided, for example, by being dispensed into suitable sealed containers.

[0064] Furthermore, examples of test tools that can be included in the measurement kit include, but are not limited to, known disposable tools used for sampling from specimens and microtubes for mixing the various reagents.

[0065] The kit may also include instructions describing protocols and the like required for using the kit. The instructions may be printed or may be recorded as data on a known recording medium.

[0066] The above-mentioned measurement kit can be suitably used, for example, particularly for measuring the amount of mandelic acid in urine.

[0067] Furthermore, the above-mentioned measurement kit can be suitably used, for example, to test the degree of exposure to styrene, ethylbenzene, or both. Since phenylglyoxylic acid and mandelic acid, which are known as metabolic products of styrene, are used as indicators of styrene exposure, the above-mentioned measurement kit, which can measure mandelic acid, is useful in clinical tests of workers who handle organic solvents such as styrene and ethylbenzene.

[0068] Next, the present invention will be specifically explained by way of examples, but the present invention is not limited to the following examples.

[0069] Example 1

[0070] A mandelic acid measurement system utilizing the above reaction principle was investigated. NADH exhibits absorption around 340 nm, so the increase in NADH was determined by measuring absorbance at 340 nm, and the amount of mandelic acid (the total amount of L-mandelic acid and D-mandelic acid) in the sample (specimen) was calculated. MR stands for mandelate racemase, and D-MDH stands for mandelate dehydrogenase.

[0071] More specifically, the following measurement samples were prepared based on the reagent compositions shown in Table 1 below.

[0072]

[0073] For the measurement samples, L-mandelic acid (Tokyo Chemical Industry Co., Ltd.) or D-mandelic acid (Tokyo Chemical Industry Co., Ltd.) was added to physiological saline to prepare stock solutions of 100 mM L-mandelic acid and 100 mM D-mandelic acid, respectively.

[0074] Urine samples: 1 mL of 100 mM mandelic acid stock solution was added to 9 mL of Liquicheck Urine Chemistry Control Level 1 (BIO-RAD) to prepare 10 mM D-mandelic acid urine samples and 10 mM L-mandelic acid urine samples (samples 5 / 5). The 5 / 5 urine samples were further diluted five times with the control urine.

[0075] Water-soluble samples: 1 mL of 100 mM mandelic acid stock solution was added to 9 mL of physiological saline (Otsuka saline injection) to prepare 10 mM D-mandelic acid water-soluble sample and 10 mM L-mandelic acid water-soluble sample (sample 5 / 5). The 5 / 5 water-soluble sample was further diluted five times with physiological saline.

[0076] Furthermore, mandelate racemase (MR) and mandelate dehydrogenase (D-MDH) were cultured in Escherichia coli by the applicant himself and purified based on Chang Wei Fan et al., Journal of Biotechnology, 195 (2015), 67-71.

[0077]

[0078] Measurements were performed on the above samples using a Hitachi 7180 automatic analyzer (manufactured by Hitachi High-Technologies Corporation) under the conditions listed in Table 2. More specifically, 3 μL of sample was mixed with 120 μL of the first reagent, and approximately 5 minutes later, 40 μL of the second reagent was mixed. The change in absorbance at the secondary wavelength / dominant wavelength = 700 nm / 340 nm was measured immediately before the addition of the second reagent (measurement point 16) and 5 minutes after the addition (measurement point 34).

[0079] The measurement results are shown in Tables 3 and 4 below and Figures 3 and 4. The concentration was calculated from the change in absorbance between 0 mM water-soluble sample (physiological saline) and 2 mM D-mandelic acid.

[0080]

[0081]

[0082] From the results in Tables 3 and 4 and Figures 3 and 4, it was found that in Example 1, a concentration-dependent increase in absorbance was observed for both D-mandelic acid and L-mandelic acid, making quantitative measurement possible. Furthermore, urine samples showed reactivity equivalent to that of water-soluble samples. This demonstrates that the measurement method of the present invention enables effective measurement even when urine samples are used.

[0083] Example 2

[0084] As an example of a method for eliminating the reaction product of D-mandelate dehydrogenase and breaking the equilibrium state, a mandelic acid measurement system utilizing the above reaction principle was investigated. More specifically, in order to eliminate the reaction product NADH, diaphorase, a charge carrier, was reacted with tetrazolium salt (WST-8), a reducing coloring agent, to convert NADH to NAD. ＋ The method of oxidizing NADH to NAD was investigated. ＋ is recycled to the reaction of D-mandelate dehydrogenase.

[0085] When tetrazolium salt (WST-8) is reduced, a water-soluble formazan is produced that exhibits absorption around 450 nm. The amount of formazan was determined by measuring the absorbance around 450 nm, and the amount of mandelic acid (the total amount of L-mandelic acid and D-mandelic acid) in the sample was calculated. MR, D-MDH, and DI stand for mandelate racemase, mandelate dehydrogenase, and diaphorase, respectively.

[0086] More specifically, the following measurement samples were prepared based on the reagent compositions shown in Table 5 below.

[0087]

[0088] Stock solutions of L-mandelic acid and D-mandelic acid, measurement samples (urine samples, water-soluble samples), mandelate racemase (MR), and mandelate dehydrogenase (D-MDH) were all prepared and prepared in the same manner as in Example 1. Diaphorase (DI) used was a commercially available product (DIAPHORASE 3 (manufactured by Nipro Corporation)).

[0089]

[0090] Measurements were performed on the above samples using a Hitachi 7180 automatic analyzer (manufactured by Hitachi High-Technologies Corporation) under the conditions listed in Table 6. More specifically, 3 μL of diluted sample, which was prepared by mixing 20 μL of sample with 80 μL of water to dilute it 5-fold, was mixed with 120 μL of the first reagent, and approximately 5 minutes later, 40 μL of the second reagent was added. The change in absorbance at a secondary wavelength / dominant wavelength of 600 nm / 450 nm was measured immediately before the addition of the second reagent (measurement point 16) and 5 minutes after the addition (measurement point 34).

[0091] The measurement results are shown in Tables 7 and 8 below and Figures 5 and 6. The concentration was calculated from the change in absorbance between 0 mM water-soluble sample (physiological saline) and 2 mM D-mandelic acid.

[0092]

[0093]

[0094] From the results in Tables 7 and 8 and Figures 5 and 6, in Example 2, a concentration-dependent increase was observed for both D-mandelic acid and L-mandelic acid, and the ratio of the known concentration (charge value) was within 100±10% for all samples, confirming that both D-mandelic acid and L-mandelic acid had linearity up to 10 mM. Furthermore, urine samples showed reactivity equivalent to that of water-soluble samples. This demonstrates that the measurement method of the present invention can be used effectively even when urine samples are used. Thus, it was demonstrated that the measurement method of the present invention is also capable of quantitative measurement.

Claims

1. A method for measuring mandelic acid in a sample, comprising: ＋ , NADP ＋ 10. A method for measuring mandelic acid, comprising the steps of: allowing a dehydrogenase to act on mandelic acid in the presence of NADH, NADPH, or both; and measuring the amount of change in NADH, NADPH, or both.

2. The measurement method according to claim 1, further comprising a racemase in the step.

3. A method for measuring mandelic acid in a sample, comprising: ＋ , NADP ＋ a method for measuring the amount of formazan dye generated, the method comprising the steps of: reacting a dehydrogenase and a reducing color-developing reagent with mandelic acid in the presence of a formazan dye-producing agent or a formazan dye-producing agent; 4. The measurement method according to claim 3, further comprising a racemase in the step.

5. The measurement method according to claim 1 or 3, further comprising diaphorase or an electron mediator in the step.

6. The measurement method according to claim 2 or 4, wherein the racemase includes mandelic acid racemase.

7. The measurement method according to claim 1 or 3, wherein the dehydrogenase includes mandelate dehydrogenase.

8. The reducing color reagents are 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1), 2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-3), 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8), 3,3'-[3,3'-dimethoxy-(1,1'-biphenyl)-4,4'-diyl]-bis[2-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride] (nitro-TB), and The measurement method according to claim 3, wherein the compound contains one or more selected from the group consisting of 3,3'-[3,3'-dimethoxy-(1,1'-biphenyl)-4,4'-diyl]bis(2,5-diphenyl-2H-tetrazolium chloride) (TB).

9. The method of claim 1 or 3, wherein the measurement is performed by absorbance measurement.

10. The measurement method according to claim 1 or 3, wherein the sample includes urine or other body fluids.

11. The measurement method according to claim 1 or 3 for testing the degree of exposure to styrene, ethylbenzene, or both.

12. NAD ＋ , NADP ＋ 10. A measuring kit for use in the measuring method according to claim 1, comprising: a dehydrogenase; or both of them; 13. NAD ＋ , NADP ＋ 4. A measuring kit for use in the measuring method according to claim 3, comprising: a dehydrogenase; a reducing color-developing reagent; or both of them; 14. The assay kit according to claim 12 or 13, further comprising a racemase, a diaphorase, an electron mediator, or any combination thereof.

15. A measuring kit according to claim 12 or 13 for measuring the amount of mandelic acid in urine.

16. The assay kit according to claim 12 or 13, for testing the degree of exposure to styrene, ethylbenzene, or both.

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