Method for detecting phenylglyoxylic acid

Abstract

In a method for detecting phenylglyoxylic acid (PGA) according to the present disclosure, at least the presence of phenylglyoxylic acid in a sample is detected by measuring the decrease in a coenzyme, which occurs in association with the conversion of phenylglyoxylic acid in the sample into L-2-phenylglycine in the presence of the coenzyme and ammonium ions (NH4 +) using leucine dehydrogenase (LeuDH) that catalyzes a reverse reaction of a dehydrogenation reaction. The measurement of the decrease in the coenzyme may be performed by measuring an absorbance caused by the coenzyme. It is also possible to measure the amount of decrease in the coenzyme by measuring the change in the absorbance and to quantify the phenylglyoxylic acid in the sample on the basis of the measured amount of decrease.

Description

Method for detecting phenylglyoxylic acid

[0001] The present invention relates to a method for detecting phenylglyoxylic acid, and more particularly to a method for detecting phenylglyoxylic acid using an enzyme reaction.

[0002] Styrene is widely used as a raw material for polystyrene polymers and copolymers containing styrene in the monomer structure. It is known that long-term exposure to styrene has various effects on the human body. Therefore, workers who handle styrene are required to undergo special health examinations, including laboratory tests, to avoid or prevent the health effects of styrene exposure.

[0003] Mandelic acid and phenylglyoxylic acid (PGA) are known metabolites of styrene. Of these metabolites, the concentration of mandelic acid in urine has traditionally been measured in clinical tests for styrene exposure.

[0004] In Japan, the items in special health examinations were revised in 2020, and the clinical tests for styrene exposure were changed from measuring the concentration of mandelic acid in urine to measuring the total amount of mandelic acid and phenylglyoxylic acid in urine. Therefore, measuring phenylglyoxylic acid has become important in clinical tests in recent years.

[0005] In general, high performance liquid chromatography (HPLC) or liquid chromatography mass spectrometry (LC / MS) is used to measure mandelic acid and phenylglyoxylic acid contained in biological samples such as urine. For example, Non-Patent Document 1 discloses a case in which mandelic acid, phenylglyoxylic acid, hippuric acid, and methylhippuric acid, which are metabolic products of styrene, toluene, and xylene, are measured by HPLC.

[0006] Chompunut Onchoi et at., "High performance liquid chromatography for determination of urinary metabolites of toluene, xylene and styrene and its application", Southeast Asian J Trop Med Public Health, 2008, Vol.39, No.6, pp.1164-71

[0007] While the use of HPLC to measure metabolites in biological samples offers advantages such as high accuracy, high speed, and continuous measurement, it requires specialized equipment. In contrast, enzymatic methods for measuring metabolites in biological samples essentially require a reaction using a reagent, making it possible to use a general-purpose automated analyzer for the measurement. Furthermore, the use of a general-purpose automated analyzer also makes it possible to simultaneously measure multiple samples.

[0008] Therefore, it is considered to use an enzymatic method rather than HPLC or LC / MS to measure phenylglyoxylic acid in biological samples, but until now, no effective enzymatic method for detecting (or measuring) phenylglyoxylic acid has been known.

[0009] The present disclosure has been made to solve such problems, and aims to provide a method for detecting phenylglyoxylic acid in various samples such as biological samples using an enzymatic method.

[0010] In order to solve the above-mentioned problems, the method for detecting phenylglyoxylic acid according to the present disclosure uses leucine dehydrogenase (LeuDH) that catalyzes the reverse reaction of the dehydrogenation reaction to detect a coenzyme and ammonium ion (NH + The present invention is configured to detect the presence of at least phenylglyoxylic acid (PGA) in a sample by measuring the decrease in coenzyme that accompanies the conversion of PGA in the sample to L-2-phenylglycine in the presence of phenylglyoxylic acid (PGA).

[0011] According to the above configuration, phenylglyoxylic acid reacts with a coenzyme and ammonium ions through the catalytic action of leucine dehydrogenase to convert it to L-2-phenylglycine. Therefore, when one molecule of phenylglyoxylic acid is converted to L-2-phenylglycine, one molecule of coenzyme is reduced. Therefore, by measuring the reduction in coenzyme, it is possible to detect whether or not phenylglyoxylic acid is present in a sample. As a result, it is possible to easily detect phenylglyoxylic acid in a sample using an enzymatic method. Moreover, if the amount of coenzyme reduction can be quantified, it is also possible to quantify phenylglyoxylic acid in a sample.

[0012] In the method for detecting phenylglyoxylic acid having the above-described configuration, the decrease in the coenzyme may be measured by measuring the absorbance caused by the coenzyme.

[0013] Furthermore, the method for detecting phenylglyoxylic acid having the above configuration may be configured to measure the amount of decrease in the coenzyme by measuring the change in absorbance, and to quantify the phenylglyoxylic acid in the sample based on the amount of decrease.

[0014] In the method for detecting phenylglyoxylic acid having the above-described configuration, the coenzyme may be a pyridine nucleotide coenzyme.

[0015] In the method for detecting phenylglyoxylic acid having the above-described configuration, the sample may be a biological sample.

[0016] The present disclosure also includes a phenylglyoxylic acid detection kit used in the above-described method for detecting phenylglyoxylic acid, the kit comprising: a reagent containing leucine dehydrogenase (LeuDH) that catalyzes the reaction of converting phenylglyoxylic acid to L-2-phenylglycine, which is the reverse reaction of the dehydrogenation reaction; and a reagent containing a coenzyme.

[0017] In the phenylglyoxylic acid detection kit having the above-described configuration, ammonium ions may be added in advance to the reagent containing the coenzyme.

[0018] The above and other objects, features, and advantages of the present invention will become apparent from the following detailed description of preferred embodiments, which proceeds with reference to the accompanying drawings.

[0019] With the above-described configuration, the present invention has the effect of providing a method for detecting phenylglyoxylic acid in various samples such as biological samples using an enzymatic method.

[0020] 1 is a graph showing the relationship between the concentration of phenylglyoxylic acid in a measurement sample and absorbance, which is a representative example of the present disclosure.

[0021] [Method for detecting phenylglyoxylic acid] The method for detecting phenylglyoxylic acid according to the present disclosure uses leucine dehydrogenase (LeuDH), which catalyzes the reverse reaction of the dehydrogenation reaction, to detect a coenzyme and ammonium ion (NH + The present invention relates to a method for detecting the presence of at least phenylglyoxylic acid (PGA) in a sample by measuring the reduction of a coenzyme accompanying the conversion of PGA in the sample to L-2-phenylglycine in the presence of phenylglyoxylic acid (PGA).

[0022] Phenylglyoxylic acid, along with mandelic acid, is a metabolite of styrene. Styrene is absorbed into the body via the lungs or skin. Workers who handle styrene often absorb styrene primarily through the lungs.

[0023] When styrene is taken into the body, the vinyl group is oxidized to styrene-7,8-oxide. Styrene-7,8-oxide is hydrolyzed to styrene glycol, which is then oxidized stepwise to mandelic acid, which is further oxidized to phenylglyoxylic acid. Mandelic acid and phenylglyoxylic acid are excreted in the urine. Therefore, mandelic acid and phenylglyoxylic acid are used as indicators of styrene exposure.

[0024] Mandelic acid is excreted in greater amounts than phenylglyoxylic acid. Therefore, in the past, measurement of mandelic acid has been emphasized in clinical tests for styrene exposure. However, from the perspective of more appropriately evaluating the status of styrene exposure, it is important to consider not only mandelic acid but also the total amount of mandelic acid and phenylglyoxylic acid. Therefore, in the present disclosure, a representative sample to be detected for phenylglyoxylic acid is a biological sample such as urine that (may) contain phenylglyoxylic acid.

[0025] In the present disclosure, the biological sample to be detected is not limited to urine, but may be blood, saliva, bone marrow fluid, interstitial fluid, etc. In the case of blood, it may be whole blood or a blood sample containing only some components of blood. This also applies to biological samples derived from bodily fluids such as bone marrow fluid and interstitial fluid. In addition, the detection target in the present disclosure is not limited to biological samples, but also includes various samples that are not derived from a living organism but that (may) contain phenylglyoxylic acid.

[0026] In the present disclosure, the biological sample may be derived from a worker who handles organic solvents, i.e., a human. Therefore, the method for detecting phenylglyoxylic acid according to the present disclosure can be suitably used in health checkups to evaluate exposure to styrene in humans who handle styrene. Note that the sample may not be derived from a human but may be derived from other animals (e.g., mammals such as dogs, cats, pigs, cows, rats, and mice, or vertebrates other than mammals).

[0027] Leucine dehydrogenase (EC 1.4.1.9) catalyzes the reaction of converting L-leucine to 4-methyl-2-oxopentanoic acid in the presence of a coenzyme, as shown in the following formula (1). Note that LeuDH in formula (1) is an abbreviation for leucine dehydrogenase. In the following formula (1), the coenzyme is nicotinamide adenine dinucleotide (NADH).

[0028]

[0029] The forward reaction in formula (1) (the reaction proceeding from left to right in formula (1)) is a deamination reaction or dehydrogenation reaction that produces 4-methyl-2-oxopentanoic acid, an α-keto acid, and ammonia from the amino acid L-leucine. On the other hand, the reverse reaction (the reaction proceeding from right to left in formula (1)) is an amination reaction or hydrogenation reaction that produces L-leucine from 4-methyl-2-oxopentanoic acid, an α-keto acid. The leucine dehydrogenase used in the method for detecting phenylglyoxylic acid according to the present disclosure catalyzes the reverse reaction of formula (1).

[0030] Specifically, as shown in the following formula (2), this is a reaction that converts phenylglyoxylic acid, an α-keto acid, into L-2-phenylglycine, an amino acid, in the presence of a coenzyme (NADH) and ammonium ions.

[0031]

[0032] Thus, in the hydrogenation reaction catalyzed by leucine dehydrogenase, phenylglyoxylic acid reacts with a coenzyme and ammonium ions to be converted to L-2-phenylglycine. Therefore, when one molecule of phenylglyoxylic acid is converted to L-2-phenylglycine, one molecule of coenzyme is reduced. Thus, by measuring the reduction in coenzyme, it is possible to at least detect whether or not phenylglyoxylic acid is present in a sample. Furthermore, if the amount of reduction in coenzyme can be quantified, it is also possible to quantify the amount of phenylglyoxylic acid in a sample.

[0033] The specific leucine dehydrogenase used in the present disclosure is not particularly limited, and representative examples include those derived from Bacillus sp., Bacillus stearothermophilus, Bacillus cereus, and Bacillus sphaericus, as well as those obtained by functionally modifying these enzymes using genetic engineering techniques or the like.

[0034] The coenzyme used for the catalytic action of leucine dehydrogenase is not particularly limited. As described above, typical examples include pyridine nucleotide coenzymes such as nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH). Furthermore, coenzymes other than NADH or NADPH can be used as long as they can promote the reaction of formula (2).

[0035] As described above, the method for detecting phenylglyoxylic acid according to the present disclosure may be a method for detecting whether or not phenylglyoxylic acid is contained in a sample based on the decrease in the coenzyme. Furthermore, the method for detecting phenylglyoxylic acid according to the present disclosure may be a method for quantifying the amount of phenylglyoxylic acid in a sample by quantifying the decrease in the coenzyme, as described above, i.e., a method for detecting phenylglyoxylic acid.

[0036] In the present disclosure, the measurement of the decrease in coenzyme is not particularly limited, but typically, the decrease in coenzyme can be measured from the absorbance attributable to the coenzyme. For example, as described above, if the coenzyme is NADH, the absorbance at 340 nm attributable to the NADH can be measured. Furthermore, the measurement of the amount of decrease in coenzyme is also not particularly limited, but when absorbance is used, the amount of decrease in coenzyme can be measured by measuring the change in absorbance, and the amount of phenylglyoxylic acid in the sample can be quantified from the amount of decrease.

[0037] The method for detecting phenylglyoxylic acid according to the present disclosure converts phenylglyoxylic acid in a sample to L-2-phenylglycine in the presence of a coenzyme, as shown in formula (2) above. Therefore, an endpoint method can be applied to detect (measure) the amount of coenzyme reduction. Therefore, the method for detecting phenylglyoxylic acid according to the present disclosure can be automated using an automatic analyzer.

[0038] The endpoint method is a method for quantitatively detecting a substance by chemically reacting a substrate with the catalytic action of an enzyme and measuring the total change in the amount of reaction before and after the substrate has fully reacted (after reaching the endpoint). In this method, the reaction is allowed to proceed until the decrease in the starting material (substrate) or the increase in the product from the starting material substantially stops, and the amount of product produced is measured by absorbance, turbidity, or the like. In the endpoint method, the production of product by the chemical reaction asymptotically approaches saturation, making it easily adaptable to automated mechanical measurement.

[0039] The specific type of endpoint method used in the present disclosure is not particularly limited, and may be a one-point method or a two-point method. The two-point method is used in the examples described below. Furthermore, the endpoint method used in the present disclosure may also use a two-wavelength method in which the absorbance of a secondary wavelength is subtracted from the absorbance of a primary wavelength, as in the examples described below.

[0040] [Phenylglyoxylic Acid Detection Kit] The present disclosure includes not only the above-described method for detecting phenylglyoxylic acid, but also various fields of application of the method for detecting phenylglyoxylic acid. For example, the present disclosure includes a kit (phenylglyoxylic acid detection kit) used in the above-described method for detecting phenylglyoxylic acid.

[0041] The phenylglyoxylic acid detection kit according to the present disclosure may contain at least leucine dehydrogenase that catalyzes the reaction of converting phenylglyoxylic acid to L-2-phenylglycine (the reaction represented by formula (2)). This leucine dehydrogenase may be prepared as an enzyme solution by a known method when the kit is prepared. The specific composition of the enzyme solution is not particularly limited, and it may contain leucine dehydrogenase in a known solvent at a concentration that is appropriate for the intended use.

[0042] Furthermore, the phenylglyoxylic acid detection kit according to the present disclosure may contain other reagents or test tools in addition to leucine dehydrogenase. In the phenylglyoxylic acid detection method according to the present disclosure, phenylglyoxylic acid is reacted in the presence of a coenzyme and ammonium ions, as shown in the above formula (2). Therefore, the phenylglyoxylic acid detection kit according to the present disclosure may contain a coenzyme or a compound that serves as an ammonium ion donor, such as various ammonium salts.

[0043] In the present disclosure, as described above, a pyridine nucleotide coenzyme such as NADH or NADPH is used as the coenzyme. Therefore, the phenylglyoxylic acid detection kit according to the present disclosure may contain a pyridine nucleotide coenzyme such as NADH or NADPH. This pyridine nucleotide coenzyme may be prepared as a reagent solution using a known method. Furthermore, the ammonium salt used in the phenylglyoxylic acid detection kit according to the present disclosure is not particularly limited, but a representative example is ammonium chloride. This ammonium salt may also be prepared as a reagent solution using a known method.

[0044] For example, in the examples described below, leucine dehydrogenase is prepared as the second reagent, which is an enzyme solution, while the coenzyme NADH and the ammonium ion-donating compound ammonium chloride are both blended in a buffer solution and prepared as the first reagent. In this way, in the phenylglyoxylic acid detection kit according to the present disclosure, leucine dehydrogenase and the coenzyme may each be prepared as independent reagents.

[0045] Furthermore, the coenzyme and the ammonium ion-donating compound may be combined into a first reagent, or a third reagent containing only the ammonium ion-donating compound may be prepared separately from a second reagent containing only the coenzyme. Thus, in the phenylglyoxylic acid detection kit according to the present disclosure, some of the components contained in the detection kit may be combined into a single reagent, or may be prepared as separate, independent reagents.

[0046] In the examples described below, the pH of the first reagent containing NADH and ammonium chloride is adjusted to 8.5, and the pH of the second reagent containing leucine dehydrogenase is also adjusted to 8.5, but the pH of each reagent in the phenylglyoxylic acid detection kit according to the present disclosure is not particularly limited. For example, a suitable pH can be appropriately set depending on various conditions such as the specific type of leucine dehydrogenase used, the pH of the sample to be added, and the type of coenzyme.

[0047] As described above, the detection method for phenylglyoxylic acid according to the present disclosure is applicable to an automated measurement system. Therefore, the phenylglyoxylic acid detection kit according to the present disclosure may include various test devices that can be used in the automated measurement system. Furthermore, in the phenylglyoxylic acid detection kit according to the present disclosure, leucine dehydrogenase or other reagents may be prepared in a known form that can be used in the automated measurement system.

[0048] The phenylglyoxylic acid detection kit according to the present disclosure may also include other reagents such as a sample diluent, an enzyme reaction diluent, buffers, etc. These reagents are typically provided in suitable sealed containers. Alternatively, test tools that may be included in the phenylglyoxylic acid detection kit according to the present disclosure include, but are not limited to, known disposable devices used for sampling from samples or microtubes for mixing the various reagents. Furthermore, the phenylglyoxylic acid detection kit according to the present disclosure may also include instructions describing protocols and the like necessary for using the detection kit. Such instructions may be printed or may be recorded as data on a known recording medium.

[0049] The phenylglyoxylic acid detection kit according to the present disclosure may be a kit for clinical testing that obtains a sample from a worker who (may) handle styrene and detects or quantifies phenylglyoxylic acid, a metabolic product of styrene, contained in the sample, but the present disclosure is not limited thereto. For example, the phenylglyoxylic acid detection kit according to the present disclosure may be a research kit or an investigation kit for detecting or quantitating phenylglyoxylic acid that is not a metabolic product.

[0050] In this way, the method for detecting phenylglyoxylic acid according to the present disclosure uses leucine dehydrogenase (LeuDH), which catalyzes the reverse reaction of the dehydrogenation reaction, to detect the coenzyme and ammonium ion (NH + The present invention is configured to detect at least the presence of phenylglyoxylic acid (PGA) in a sample by measuring the decrease in coenzyme that accompanies the conversion of PGA in the sample to L-2-phenylglycine in the presence of phenylglyoxylic acid (PGA).

[0051] It has been known that L-amino acid dehydrogenases (L-amino acid dehydrogenases), including leucine dehydrogenase, catalyze both a forward reaction in which hydrogen atoms are eliminated from L-amino acids in the presence of a coenzyme to produce α-keto acids, and a reverse reaction in which hydrogen atoms are added to α-keto acids in the presence of a coenzyme to produce L-amino acids. However, it was not previously known that the reverse reaction of leucine dehydrogenase can be used to detect and measure α-keto acids. In other words, the present inventors independently discovered that the reverse reaction of leucine dehydrogenase can be used to detect the α-keto acid phenylglyoxylic acid, and have thus completed the technology of the present disclosure.

[0052] According to the present disclosure, phenylglyoxylic acid reacts with a coenzyme and ammonium ions through the catalytic action of leucine dehydrogenase to convert it to L-2-phenylglycine. Therefore, when one molecule of phenylglyoxylic acid is converted to L-2-phenylglycine, one molecule of coenzyme is reduced. Therefore, the presence or absence of phenylglyoxylic acid in a sample can be detected by measuring the reduction in coenzyme. As a result, phenylglyoxylic acid in a sample can be easily detected using an enzymatic method.

[0053] Furthermore, as shown in the Examples below, it can be seen that the measured absorbance of a test sample containing phenylglyoxylic acid and the known concentration of phenylglyoxylic acid in the test sample show good linearity. Moreover, the measured value of each test sample is within ±10% of the known concentration ratio. Therefore, according to the present disclosure, it is possible to quantify the amount of coenzyme reduction in a sample based on absorbance, and therefore it is also possible to quantify phenylglyoxylic acid in a sample based on the amount of coenzyme reduction.

[0054] Furthermore, as demonstrated in the Examples described below, in the method for detecting phenylglyoxylic acid according to the present disclosure, the detection results for an aqueous sample containing substantially only phenylglyoxylic acid (Example 1) and the detection results for a urine sample containing phenylglyoxylic acid in urine (Example 2) are almost identical. Therefore, the method for detecting phenylglyoxylic acid according to the present disclosure can satisfactorily detect (measure) the amount of coenzyme reduction, i.e., the content of phenylglyoxylic acid, not only under ideal conditions (Example 1 using an aqueous sample) but also under conditions using a biological sample such as urine (Example 2 using a urine sample).

[0055] In other words, it is believed that the catalytic action of the reverse reaction (hydrogenation reaction) of the dehydrogenation reaction by leucine dehydrogenase is hardly inhibited by components contained in biological samples such as urine. Moreover, since the endpoint method can be applied to the catalytic action of the reverse reaction by leucine dehydrogenase, it can be automated using an automatic analyzer. Therefore, the detection method of phenylglyoxylic acid according to the present disclosure can be suitably used for detecting or quantifying phenylglyoxylic acid in biological samples and is very useful in fields such as clinical testing.

[0056] The present invention will be described in more detail with reference to examples, but the present invention is not limited thereto. Those skilled in the art can make various changes, modifications, and alterations without departing from the scope of the present invention.

[0057] Example 1 A first reagent containing NADH and ammonium chloride and a second reagent containing leucine dehydrogenase (product name: LEUCINE DEHYDROGENASE (LeuDH), manufactured by Nipro Corporation) were prepared to have the compositions and pHs shown in Table 1.

[0058]

[0059] To prepare the measurement sample, first, phenylglyoxylic acid (manufactured by Tokyo Chemical Industry Co., Ltd.) was added to physiological saline to prepare a 100 mM phenylglyoxylic acid stock solution.

[0060] One mL of this phenylglyoxylic acid stock solution was added to 9 mL of physiological saline (Otsuka Saline Injection (product name), manufactured by Otsuka Pharmaceutical Factory, Inc.) to prepare a 10 mM phenylglyoxylic acid water-soluble sample (5 / 5 samples). Furthermore, the 10 mM phenylglyoxylic acid water-soluble sample was diluted with physiological saline to 2.0 mM, 4.0 mM, 6.0 mM, and 8.0 mM to prepare water-soluble samples of a total of five concentrations. Physiological saline corresponding to 0 mM phenylglyoxylic acid was also prepared as a water-soluble sample.

[0061] The absorbance of these water-soluble samples was measured using a Hitachi 7180 automatic analyzer (product name, manufactured by Hitachi High-Technologies Corporation) under the measurement conditions shown in Table 2.

[0062]

[0063] Specifically, 3 μL of the water-soluble sample was mixed with 120 μL of the first reagent, and then approximately 5 minutes later, 30 μL of the second reagent was added. The absorbance change of the water-soluble sample at the minor wavelength / major wavelength = 700 / 340 was measured immediately before the addition of the second reagent (measurement point 16) and 5 minutes after the addition (measurement point 34). The results are shown in the graph of Figure 2 by the diamond symbols and dotted line.

[0064] Example 2: 1 mL of the above-mentioned phenylglyoxylic acid stock solution was added to 9 mL of control urine (product name: Liquichek Urine Chemistry Control Level 1, manufactured by Bio-Rad Laboratories, Inc.) to prepare 10 mM phenylglyoxylic acid urine samples (5 / 5 samples). Furthermore, the 10 mM phenylglyoxylic acid urine sample was diluted with the control urine to 2.0 mM, 4.0 mM, 6.0 mM, and 8.0 mM, to prepare measurement samples (urine samples) with a total of five concentrations. A control urine corresponding to 0 mM phenylglyoxylic acid was also prepared as a urine sample.

[0065] Except for using these urine samples as measurement samples, the change in absorbance was measured in the same manner as in Example 1. The results are shown by the circular symbols and solid line in the graph of Figure 2.

[0066] (Results of Examples 1 and 2) In the graph of FIG. 2, the vertical axis represents the measured absorbance of the measurement sample, and the horizontal axis represents the known concentration of phenylglyoxylic acid in the measurement sample.

[0067] 2, no difference in reactivity was observed between the water-soluble specimen (Example 1) and the urine specimen (Example 2), and it was found that the reaction was dependent on the concentration of phenylglyoxylic acid in both Examples 1 and 2. Furthermore, the concentration conversion results show that the measured values ​​of each specimen were within ±10% of the known concentration ratio, and that both the water-soluble specimen (Example 1) and the urine specimen (Example 2) had linearity up to 10 mM phenylglyoxylic acid.

[0068] It should be noted that the present disclosure is not limited to the description of the above-described embodiment, and various modifications are possible within the scope of the claims. Therefore, embodiments obtained by appropriately combining the technical means disclosed in different embodiments or multiple modifications are also included in the technical scope of the present invention.

[0069] Furthermore, many modifications and other embodiments of the present disclosure will be apparent to those skilled in the art from the above description. Therefore, the above description should be construed as illustrative only and is provided for the purpose of teaching those skilled in the art the best mode for carrying out the present disclosure. Details of the structure and / or function thereof may be substantially changed without departing from the spirit of the present invention.

[0070] The present invention can be suitably used in the field of detecting whether or not a sample contains phenylglyoxylic acid, and particularly in the field of measuring phenylglyoxylic acid in a biological sample.

Claims

1. Leucine dehydrogenase (LeuDH), which catalyzes the reverse dehydrogenation reaction, is used to convert coenzymes and ammonium ions (NH4 + a method for detecting phenylglyoxylic acid (PGA) in a sample, the method comprising measuring a decrease in a coenzyme accompanying conversion of PGA in the sample to L-2-phenylglycine in the presence of a coenzyme selected from the group consisting of phenylglyoxylic acid (PGA) and phenylglyoxylic acid (L-2-phenylglycine), thereby detecting the presence of at least phenylglyoxylic acid in the sample.

2. The method for detecting phenylglyoxylic acid according to claim 1, wherein the decrease in the coenzyme is measured by measuring the absorbance caused by the coenzyme.

3. The method for detecting phenylglyoxylic acid according to claim 2, wherein the amount of decrease in the coenzyme is measured by measuring the change in absorbance, and the amount of phenylglyoxylic acid in the sample is quantified from the amount of decrease.

4. The method for detecting phenylglyoxylic acid according to claim 1, wherein the coenzyme is a pyridine nucleotide coenzyme.

5. The method for detecting phenylglyoxylic acid according to claim 1, wherein the sample is a biological sample.

6. A phenylglyoxylic acid detection kit used in the method for detecting phenylglyoxylic acid according to any one of claims 1 to 5, comprising: a reagent containing leucine dehydrogenase (LeuDH) that catalyzes the reaction of converting phenylglyoxylic acid to L-2-phenylglycine, which is the reverse reaction of the dehydrogenation reaction; and a reagent containing a coenzyme.

7. The phenylglyoxylic acid detection kit according to claim 6, wherein ammonium ions have been added to the coenzyme-containing reagent in advance.

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