Phenylglyoxylic acid measurement method and measurement kit to be used therein

Abstract

Provided are a novel phenylglyoxylic acid measurement method and a measurement kit to be used therein.　This method for measuring the phenylglyoxylic acid in a sample includes a step in which the sample is subjected to the action of a dehydrogenase in the presence of a decarboxylase and one or more of NAD+ and NADP+ and the amount of change in NADH and / or NADPH is measured.

Description

Method for measuring phenylglyoxylic acid and measurement kit used therefor

[0001] The present invention relates to a novel method for measuring phenylglyoxylic acid, and a measurement kit used therefor. ＋ and NADP ＋ The present invention relates to a method for measuring phenylglyoxylic acid by measuring the amount of change in NADH, NADPH, etc., caused by a chemical reaction in which a starting material, phenylglyoxylic acid, is reacted with a decarboxylase and a dehydrogenase in the presence of, for example, and a measurement kit used therefor.

[0002] Phenylglyoxylic acid and mandelic acid are known to be metabolites of styrene, and therefore are used as indicators of styrene exposure. Therefore, measuring phenylglyoxylic acid and mandelic acid is useful in clinical testing of workers who handle these organic solvents.

[0003] Up until now, phenylglyoxylic acid has been measured by measuring phenylglyoxylic 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 technology for measuring phenylglyoxylic 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 phenylglyoxylic acid, and a measurement kit and the like used therefor.

[0006] As a result of intensive investigations to solve the above problems, the present inventors have succeeded in creating a novel method for measuring phenylglyoxylic 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 phenylglyoxylic acid.

[0008] [1] A method for measuring phenylglyoxylic acid in a sample, comprising: ＋ , NADP ＋ and measuring the amount of change in NADH, NADPH, or both.

[0009] [2] The method according to [1], wherein the decarboxylase includes benzoylformate decarboxylase.

[0010] [3] The method according to [1] or [2], wherein the dehydrogenase includes benzaldehyde dehydrogenase.

[0011] [4] The method according to any one of [1] to [3], wherein the step is carried out at a pH in the range of 6 to 9.

[0012] [5] The measurement method according to any one of [1] to [4], wherein the step is carried out by measuring absorbance at 310 to 370 nm.

[0013] [6] The measurement method according to any one of [1] to [5], wherein the sample includes urine or other body fluids.

[0014] [7] The measurement method according to any one of [1] to [6] for inspecting the degree of exposure to styrene.

[0015] The present invention also relates to the following measurement kits:

[0016] [8] Decarboxylase, NAD ＋ , NADP ＋ , dehydrogenase, or any combination thereof, for use in the measurement method of any one of [1] to [7].

[0017] [9] The measurement kit according to [8], for measuring the amount of phenylglyoxylic acid in urine.

[0018]

[10] The measurement kit according to [8] or [9], for testing the degree of exposure to styrene.

[0019] By using the method for measuring phenylglyoxylic acid of the present invention, measurement can be performed using, for example, an automatic analyzer, etc., and phenylglyoxylic acid can be measured more simply, quickly, and at lower cost than conventional measurement using high performance liquid chromatography, etc.

[0020] Furthermore, by using the measurement kit of the present invention, it becomes possible to easily carry out the above-mentioned method for measuring phenylglyoxylic acid.

[0021] FIG. 1 is a schematic diagram outlining a method for measuring phenylglyoxylic acid, a representative example of the present disclosure. FIG. 2 is a graph showing a comparison of measured values ​​of phenylglyoxylic acid in a urine sample at pH 5.0 with a known concentration of phenylglyoxylic acid in physiological saline, Reference Example 1 of the present disclosure. FIG. 3 is a graph showing a comparison of measured values ​​of phenylglyoxylic acid in a urine sample at pH 6.0 with a known concentration of phenylglyoxylic acid in physiological saline, Example 1 of the present disclosure. FIG. 4 is a graph showing a comparison of measured values ​​of phenylglyoxylic acid in a urine sample at pH 8.0 with a known concentration of phenylglyoxylic acid in physiological saline, Example 2 of the present disclosure. FIG. 5 is a graph showing a comparison of measured values ​​of phenylglyoxylic acid in a urine sample at pH 9.0 with a known concentration of phenylglyoxylic acid in physiological saline, Example 3 of the present disclosure. FIG. 6 is a graph showing the reaction time course of a urine sample, Reference Example 1 of the present disclosure. Figure 7 is a graph showing the reaction time course of a urine sample in Example 1 of the present disclosure. Figure 8 is a graph showing the reaction time course of a urine sample in Example 2 of the present disclosure. Figure 9 is a graph showing the reaction time course of a urine sample in Example 3 of the present disclosure.

[0022] Hereinafter, embodiments of the present invention will be described in detail.

[0023] [Method for measuring phenylglyoxylic acid] The method of the present invention is a method for measuring phenylglyoxylic acid in a sample, comprising: ＋ , NADP ＋and measuring the amount of change in NADH, NADPH, or both.

[0024] The measurement method of the present invention is ＋ and NADP ＋ This method measures phenylglyoxylic acid by measuring the amount of change in NADH, NADPH, etc., resulting from a chemical reaction in which a starting material, phenylglyoxylic acid, is reacted (catalyzed) by decarboxylase and dehydrogenase in the presence of, for example, an enzyme such as acetylcholinesterase or acetylcholinesterase. This method enables measurement to be performed more simply, quickly, and at lower cost than conventional methods, for example, by using an automatic analyzer or the like.

[0025] The above-mentioned measurement method is believed to be effective through the following mechanism of action, but the scope of the invention is not limited to this mechanism. As shown in the outline of the present invention in Figure 1, phenylglyoxylic acid is first decarboxylated by the action of decarboxylase and converted to benzaldehyde. Then, NAD ＋ and NADP ＋ In the presence of benzoic acid (and water), the benzaldehyde is oxidized to benzoic acid by the action of dehydrogenase, and NAD ＋ or ＮＡＤＰ＋ is reduced to NADH and NADPH. It is presumed that phenylglyoxylic acid can be measured in a concentration-dependent manner by observing the changes in NADH and NADPH.

[0026] In the present invention, the above-mentioned "measurement" also includes simple "detection" aimed at simply confirming and detecting the presence of phenylglyoxylic acid.

[0027] In addition, in the present invention, the above-mentioned "amount of change" typically means the amount of increase in NADH, NADPH, or both.

[0028] In the above-mentioned measurement method, the order of mixing the components and the preparation method 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.

[0029] The above NAD(P) ＋ is NAD(P) ＋ Any known or novel substance can be used as a source without any particular limitation.

[0030] The decarboxylase can be any known or novel decarboxylase, and is not particularly limited as long as it decarboxylates phenylglyoxylic acid. Examples include benzoylformate decarboxylase, pyruvate decarboxylase, phenylpyruvate decarboxylase, indolepyruvate decarboxylase, and 2-oxoglutarate decarboxylase. In the measurement method of the present invention, it is preferable to use benzoylformate decarboxylase. These may be used alone or in combination of two or more.

[0031] The dehydrogenase may be a known or novel dehydrogenase, and may be used to convert benzaldehyde and NAD ＋ and NADP ＋ The enzyme is not particularly limited as long as it acts to convert α- and β-phosphate into benzoic acid, NADH, and NADPH, respectively, and examples thereof include benzaldehyde dehydrogenase, formate dehydrogenase, aldehyde dehydrogenase, betaine aldehyde dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, malonic semialdehyde dehydrogenase, succinic semialdehyde dehydrogenase, aminobutyraldehyde dehydrogenase, glutaric semialdehyde dehydrogenase, glycolaldehyde dehydrogenase, lactaldehyde dehydrogenase, and 2-oxoaldehyde dehydrogenase. In the measurement method of the present invention, it is preferable to include benzaldehyde dehydrogenase. These may be used alone or in combination of two or more.

[0032] In the above measurement method, the above step is preferably carried out at a pH range of 6 to 9. By carrying out the step within this range, phenylglyoxylic acid can be measured more reliably. The pH range can be, for example, a range with any two points selected from the group consisting of 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, and 9.0 as its lower and upper limits. The pH range can be, for example, 6.3 to 8.5, 6.5 to 8.0, 6.8 to 7.5, 6.9 to 7.1, or 7.0.

[0033] In the above measurement method, decarboxylase, NAD ＋ , NADP ＋ The concentrations of the components of the dehydrogenase, or both of them, can be adjusted appropriately depending on the purpose and application.

[0034] In the above measurement method, the above step is preferably carried out by measuring absorbance at, for example, 310 to 370 nm. Since the maximum ultraviolet absorption 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 to 360 nm or 330 to 350 nm.

[0035] In the above measurement method, the sample may include urine or other body fluids.

[0036] Furthermore, the above-mentioned measurement method can be suitably used, for example, particularly for measurements to examine the degree of exposure to styrene. 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 phenylglyoxylic acid, is useful in clinical tests of workers who handle organic solvents such as styrene.

[0037] 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.

[0038] [Measurement Kit] The measurement kit of the present invention is a kit for measuring decarboxylase, NAD ＋ , NADP ＋ , dehydrogenase, or any combination thereof, and is used in the above-mentioned measurement method.

[0039] In the above-mentioned measuring kit, each component is the same as in the above-mentioned measuring method.

[0040] When the above-mentioned assay kit contains decarboxylase, it may be prepared as an enzyme solution by a known method. The specific composition of the enzyme solution is not particularly limited, as long as it contains decarboxylase in a known solvent at a concentration that is suitable for the intended use.

[0041] In the above measurement method, decarboxylase, NAD ＋ , NADP ＋ The concentrations of the components containing the dehydrogenase, or both, and the dehydrogenase can be adjusted appropriately depending on the purpose and application.

[0042] The above-mentioned measurement kit is ＋ , NADP ＋ , dehydrogenase, or any combination thereof, other reagents or test tools may be included.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] The above-mentioned measurement kit can be suitably used, for example, particularly for measuring the amount of phenylglyoxylic acid in urine.

[0047] Furthermore, the above-mentioned measurement kit can be suitably used, for example, particularly for testing the degree of exposure to styrene. 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 phenylglyoxylic acid, is useful in clinical tests of workers who handle organic solvents such as styrene.

[0048] Next, the present invention will be specifically explained by way of examples, but the present invention is not limited to the following examples.

[0049] [Examples 1 to 3, Reference Example 1]

[0050] A measurement system for phenylglyoxylic acid utilizing the above reaction principle was investigated. Since NADH exhibits absorption around 340 nm, the increase in NADH was determined by measuring the absorbance at 340 nm, and the amount of phenylglyoxylic acid in the sample (analyte) was calculated. Note that BFDC and BADH stand for benzoylformate decarboxylase and benzaldehyde dehydrogenase, respectively.

[0051] More specifically, the following measurement samples were prepared based on the reagent compositions shown in Table 1 below.

[0052]

[0053] Urine sample: Control urine was prepared by adding phenylglyoxylic acid (hereinafter also referred to as PGA) (Tokyo Chemical Industry Co., Ltd.) to Liquicheck Urine Chemistry Control Level 1 (BIO-RAD) to a concentration of 10 mM (5 / 5 samples). The 10 mM PGA urine sample was further diluted five times with the control urine.

[0054] A water-soluble sample was prepared by adding PGA to physiological saline (Otsuka saline injection) to a concentration of 10 mM (5 / 5 samples). The 10 mM PGA water-soluble sample was further diluted in 5 steps with physiological saline.

[0055] Benzoylformate decarboxylase (BFDC) and benzaldehyde dehydrogenase (BADH) were cultured in Escherichia coli by the applicant himself and purified based on Choedchai Saehuan et al., Biochemica et Biophysica Acta (2007) 1770: 1585-1592 and Qingzhuo Wang et al., Journal of Applied Microbiology, (2022) 133, 273-286, respectively.

[0056]

[0057] 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, 4 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 a secondary wavelength / dominant wavelength of 700 / 340 was measured immediately before the addition of the second reagent (measurement point 16) and 5 minutes after the addition (measurement point 34).

[0058] The measurement results are shown in Tables 3 to 6 and Figures 2 to 5. A calibration curve was prepared from the measured absorbance of the sample at 0 mM (physiological saline) and 2 mM phenylglyoxylic acid, and the concentration of phenylglyoxylic acid was calculated.

[0059]

[0060]

[0061]

[0062]

[0063] From the results in Tables 3 to 6 and Figures 2 to 5, in all of Examples 1 to 3, urine samples exhibited 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. On the other hand, in Reference Example 1, no PGA concentration-dependent absorbance change was observed under the above formulation. On the other hand, in Examples 1 and 2, a PGA concentration-dependent response was observed, with the ratio of the known concentration being within 100±10% for all samples, and linearity was observed up to 10 mM. Furthermore, in Example 3, linearity was observed up to 8 mM PGA.

[0064] Next, the reaction time courses for the urine samples of Reference Example 1 and Examples 1 to 3 are shown in FIGS.

[0065] Comparing the reaction time courses in Examples 1 to 3 based on the results in Figures 6 to 9, reactivity was reduced in Example 3, and the poor linearity was likely due to the fact that the reaction did not complete within the measurement time with this formulation. However, in Example 3, it is presumed that linearity can be improved by changing the measurement conditions from the above formulation, for example, by increasing the amount of enzyme in the reagent to increase reactivity to PGA, reducing the reaction sample volume in the analysis conditions, or extending the reaction time in the analysis conditions. From the above, it is considered that in the measurement method of the present invention, the pH of the reagent (measurement sample) is more preferably in the range of 6.0 to 9.0, and particularly preferably 6.0 to 8.0.

Claims

1. A method for measuring phenylglyoxylic acid in a sample, comprising the steps of: ＋ , NADP ＋ and measuring the amount of change in NADH, NADPH, or both.

2. The method of claim 1, wherein the decarboxylase comprises benzoylformate decarboxylase.

3. The method of claim 1, wherein the dehydrogenase comprises benzaldehyde dehydrogenase.

4. The measurement method according to claim 1, wherein the step is carried out at a pH in the range of 6 to 9.

5. The measurement method according to claim 1, wherein the step is carried out by measuring absorbance at 310 to 370 nm.

6. The measurement method according to any one of claims 1 to 5, wherein the sample includes urine or other body fluids.

7. The measurement method according to any one of claims 1 to 5 for testing the degree of exposure to styrene.

8. Decarboxylase, NAD ＋ , NADP ＋ 6. A measuring kit for use in the measuring method of any one of claims 1 to 5, comprising: a dehydrogenase; a dehydrogenase; or any combination thereof.

9. The assay kit according to claim 8 for measuring the amount of phenylglyoxylic acid in urine.

10. The assay kit according to claim 8 for testing the degree of exposure to styrene.

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