Method for quantifying ergothioneine

Abstract

According to the present disclosure, a simple and accurate method for quantifying ergothioneine in a sample containing globin protein can be provided. A method for quantifying ergothioneine in a collected sample, comprising: (A) heating the sample; (B) mixing the heated sample with trichloroacetic acid; and (C) subjecting the sample obtained in step (B) to a quantitative operation; A method comprising:

Description

[Technical Field]

[0001] The present disclosure relates to a method for quantifying ergothioneine. [Background technology]

[0002] A method for quantifying ergothioneine in a sample is known, in which the sample is enzymatically treated to convert it into the reaction product thiourocanic acid and then measured (Patent Document 1: hereinafter sometimes referred to as the ETL method). This ETL method utilizes the fact that ergothioneine is treated with ergotinase, which is highly specific to ergothioneine, to convert it into thiourocanic acid, thereby increasing absorption at a specific wavelength. Since the amount of ergothioneine is proportional to the increase in absorption upon conversion to thiourocanic acid, the ergothioneine concentration can be quantified. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2012-065591 A (Patent Document 1) [Non-patent literature]

[0004] [Non-Patent Document 1] Cheah IK, Tang RM, Yew TS, Lim KH, Halliwell B. Administration of Pure Ergothioneine to Healthy Human Subjects: Uptake, Metabolism, and Effects on Biomarkers of Oxidative Damage and Inflammation. Antioxid Redox Signal. 2017 Feb 10;26(5):193-206. doi: 10.1089 / ars.2016.6778. Epub 2016 Sep 7. PMID: 27488221. (Non-patent document 1) Summary of the Invention [Problem to be solved by the invention]

[0005] Compared with the method of directly quantifying ergothioneine by HPLC (Non-Patent Document 1), the ETL method has the advantages of simpler sample preparation and superior processing of multiple samples, since it only requires observing an increase in absorption at a specific wavelength, eliminating the need for column separation. However, the present inventors have discovered that when attempting to quantify ergothioneine in a sample containing globin protein, such as blood, using the method of Patent Document 1 (ETL method), there are problems in that the background becomes too high, making the measurement itself difficult or making it impossible to accurately quantify ergothioneine. [Means for solving the problem]

[0006] In the course of attempting to quantify ergothioneine in a sample containing globin protein, the present inventors discovered that ergothioneine binds to globin protein, and also found that the inaccuracy of quantification is due to the binding of ergothioneine to globin protein, and that ergothioneine can be accurately quantified by dissociating the binding in advance.

[0007] For example, the present disclosure provides: [Section 1] 1. A method for quantifying ergothioneine in a collected sample, comprising: (A) heating the sample; (B) mixing the heated sample with trichloroacetic acid; and (C) subjecting the sample obtained in step (B) to a quantitative operation; Including, method. [Section 2] The step (C) of subjecting the sample to a quantitative operation is a step of subjecting the sample to an enzymatic assay, (C-1) contacting the mixture obtained in step (B) with an enzyme that acts on ergothioneine; and (C-2) a step of measuring the amount of enzyme reaction in the enzyme-treated solution obtained in step (C-1); The method according to item 1, comprising: [Section 3] Item 3. The method of item 1 or 2, wherein the sample comprises a biological sample. [Section 4] Item 4. The method according to Item 3, wherein the biological sample contains globin protein. [Section 5] Item 5. The method of item 4, wherein the globin protein is hemoglobin, myoglobin, neuroglobin, cytoglobin, or androglobin. [Section 6] Item 6. The method according to any one of Items 3 to 5, wherein the biological sample comprises blood, plasma, serum, blood cells, red blood cells, muscle, nerve, retina, liver, kidney, or semen. [Section 7] Item 7. The method according to any one of Items 1 to 6, wherein the heating temperature in step (A) is 80°C or higher. [Section 8] The method according to any one of Items 1 to 7, further comprising separating the supernatant after step (B). [Section 9] The method according to any one of Items 1 to 8, wherein the heating temperature in step (A) is 80°C or higher, preferably about 80°C to about 100°C, more preferably about 85°C to about 95°C. [Section 10] The method according to any one of Items 1 to 9, wherein the heating time in step (A) is about 1 to about 30 minutes, preferably about 5 to about 25 minutes, more preferably about 8 to about 20 minutes, and even more preferably about 10 to about 15 minutes. [Section 11] The method according to any one of items 1 to 10, wherein the final concentration of trichloroacetic acid in the mixture after step (B) is about 10 to about 500 mM, preferably about 50 to about 400 mM, more preferably about 75 to about 300 mM, even more preferably about 100 to about 200 mM, and most preferably about 150 mM. [Section 12] Item 12. The method according to any one of items 1 to 11, which does not include treating the sample with an organic solvent or acid before step (A). [Section 13] Item 13. The method according to Item 12, wherein the organic solvent or acid is trichloroacetic acid. [Section 14] Item 14. The method according to any one of Items 2 to 13, wherein the enzyme acting on ergothioneine is ergothionase. [Section 15] Item 15. The method according to any one of Items 2 to 14, wherein the step of measuring the amount of enzyme reaction comprises a step of measuring absorbance at about 311 nm or a step of adjusting the pH to 2 or less after the enzyme reaction and measuring absorbance at about 330 nm. [Effects of the Invention]

[0008] According to the present disclosure, it is possible to eliminate the influence of globin proteins on the quantitative system, and to provide a simple and accurate method for quantifying ergothioneine. [Brief explanation of the drawings]

[0009] [Figure 1] Figure 1 shows the difference in background absorbance due to sample treatment. [Figure 2] Figure 2 is a graph showing the effect of the order of heat treatment and trichloroacetic acid treatment on the quantification of ergothioneine. The left column shows the results of measurement by the ETL method, and the right column shows the results of measurement by HPLC. The Heat sample on the far left was measured only by the HPLC method. [Figure 3] FIG. 3 is a graph showing the effect of heat denaturation temperature. [Figure 4] FIG. 4 is a graph showing the ergothioneine concentration in a 10-fold diluted hemolyzed sample to which ergothioneine was added. [Figure 5] FIG. 5 is a graph showing the quantification of ergothioneine using plasma samples. [Figure 6] FIG. 6 is a graph showing the quantification of ergothioneine using samples containing purified bovine serum albumin (BSA) or hemoglobin (Hb). [Figure 7]FIG. 7 is a graph showing the quantification of ergothioneine using samples containing hemoglobin (Hb), myoglobin (Mb), or cytochrome c (Cyt c). [Figure 8] FIG. 8 is a graph showing the quantification of hercynine (Her) using a sample containing Hb. [Figure 9] FIG. 9 is a graph showing the variation of thiourocanic acid maximum absorption with pH. DETAILED DESCRIPTION OF THE INVENTION

[0010] Ergothioneine (also referred to as EGT in this disclosure) is a sulfur-containing amino acid produced by some microorganisms and is known to have diverse physiological activities, including antioxidant properties. Humans and many other animals and plants cannot synthesize ergothioneine, but they absorb it from food and accumulate it in their bodies. Its antioxidant properties have been suggested to be higher than those of vitamin C, vitamin E, cysteine, and glutathione. Ergothioneine has also been shown to have UV absorption effects, melanin production inhibitory effects, reactive oxygen species scavenging abilities, elastase activity inhibitory effects that prevent the formation of wrinkles and sagging skin, and tyrosinase activity inhibitory effects that prevent the formation of dark spots. Ergothioneine has been suggested to be effective against various diseases, but it has particularly attracted attention as a potential inhibitory effect against neurodegenerative diseases and cardiovascular diseases that cause cognitive decline. For example, a strong correlation has been suggested between cognitive decline and blood ergothioneine levels, and it is believed that a decrease in ergothioneine predisposes to diseases such as dementia. Therefore, measuring ergothioneine levels in the body is considered important.

[0011] Globin proteins are a superfamily of globular proteins. These proteins are known to those skilled in the art to have a globin fold. Globin proteins of the present disclosure include, but are not limited to, hemoglobin, myoglobin, neuroglobin, cytoglobin, or androglobin.

[0012] In a first aspect, the present disclosure provides a method for quantifying ergothioneine in a sample, comprising: (A) heating the sample; (B) mixing the heated sample with trichloroacetic acid; and (C) subjecting the sample obtained in step (B) to a quantitative operation; This relates to a method, including:

[0013] In one embodiment, the heating time and temperature of the sample in step (A) can be appropriately determined by those skilled in the art. The sample is heated, for example, to about 70, 75, 80, 85, 90, or 95°C or higher, preferably about 75°C or higher, more preferably about 80°C or higher, and even more preferably about 85°C or higher. The sample is heated, for example, to about 100°C, 95°C, 90°C, or 85°C or lower, preferably about 100°C or lower, more preferably about 95°C or lower. The sample is heated, for example, to about 75°C to about 100°C, preferably about 80°C to about 100°C, and more preferably about 85°C to about 95°C. The heating time at the above temperatures is, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 minutes or longer. The heating time at the above temperature is, for example, about 60, 55, 50, 45, 40, 35, 30 minutes or less, for example, about 1 to about 30 minutes, preferably about 5 to about 25 minutes, more preferably about 8 to about 20 minutes, and even more preferably about 10 to about 15 minutes.

[0014] In one embodiment, in step (B), the heated sample and trichloroacetic acid (sometimes referred to as TCA in the present disclosure) are only required to come into contact with each other, and there are no particular limitations on the order of addition to the container or other conditions regarding mixing. Therefore, the order, form, or means by which the heated sample and trichloroacetic acid are mixed can be appropriately determined by those skilled in the art depending on the purpose. For example, trichloroacetic acid may be in either a liquid or solid state, and trichloroacetic acid may be added first, followed by the heated sample, or the heated sample may be added first, followed by trichloroacetic acid. In the present disclosure, step (B) is performed after step (A).

[0015] In one embodiment, the concentration of trichloroacetic acid used in step (B) and the final concentration in the mixture can be appropriately determined by those skilled in the art. The concentration of trichloroacetic acid used is, for example, about 0.25, 0.5, 0.75, 1, 1.5, 2.0, 3, 4, 5 M or more, and the final concentration in the mixture is about 10, 50, 75, 100, 125, 150, 175 mM or more. The concentration of trichloroacetic acid used is, for example, about 10, 9, 8, 7, 6 M or less, and the final concentration in the mixture is about 500, 400, 300, 275, 250, 225, 200 mM or less. The concentration of trichloroacetic acid used is, for example, about 0.25 to about 10, preferably about 1 to about 9, more preferably about 2 to about 8 M, and the final concentration in the mixture is, for example, about 10 to about 500 mM, preferably about 50 to about 400 mM, more preferably about 75 to about 300 mM, even more preferably about 100 to about 200 mM, and most preferably about 150 mM.

[0016] Step (C) involves subjecting the sample obtained in step (B) to a quantitative procedure. Quantitative procedures include, for example, chemical analysis or enzymatic assay. "Chemical analysis" refers to a method for analyzing a target substance using a chemical technique such as HPLC (high-performance liquid chromatography). Examples include HPLC, which quantifies components in a sample by separation using appropriate mobile and stationary phases. Alternatively, enzymatic assays involve analysis using an enzyme reaction. "Enzymatic assays" refers to a method for analyzing a target substance using an enzyme reaction, including methods for quantifying changes in color, fluorescence, or absorbance due to a specific enzyme-substrate reaction. Enzymatic assays are preferred because they require simpler sample preparation and are more suitable for processing multiple samples than chemical assays.

[0017] The sample obtained in step (B) may be treated appropriately depending on the quantification method. For example, when an enzyme is used for quantification, the pH may be adjusted to a level suitable for the enzymatic reaction depending on the properties of the enzyme. In one embodiment, step (C) includes (C-1) contacting the mixture obtained in step (B) with an enzyme that acts on ergothioneine, and (C-2) measuring the amount of enzymatic reaction in the enzyme-treated solution obtained in step (C-1). As long as the enzymatic reaction proceeds under conditions in which the enzymatic reaction proceeds, the mixture obtained in step (B) and the enzyme that acts on ergothioneine may be contacted in step (C-1), and those skilled in the art can appropriately set the conditions. As long as the progress of the enzymatic reaction can be confirmed and the amount of ergothioneine used in the reaction can be determined, one or more enzymes may be used. The enzyme that acts on ergothioneine is not particularly limited as long as it is a protein that undergoes an enzymatic reaction using ergothioneine as a substrate, and examples thereof include hydrolases, oxidoreductases, transferases, isomerases, ligases, and lyases. In the present disclosure, ergothionase (Ergothioneine Trimethylammonia Lyase) may be used as an enzyme that acts on ergothioneine. A method for quantifying ergothioneine using ergothionase has already been reported (Patent Document 1), and by referring to this document, those skilled in the art can appropriately use ergothionase in the present disclosure.

[0018] In one embodiment, in step (C-2), the step of measuring the amount of enzyme reaction in the enzyme treatment solution quantitatively evaluates the process in which an enzyme acts on its substrate to produce a reaction product. The enzyme treatment solution is a liquid containing an enzyme, and is a liquid containing its substrate and / or enzyme reaction product. Measuring the amount of enzyme reaction means quantitatively evaluating the amount of change in the enzyme reaction product, the substrate of the enzyme reaction, or the coenzyme of the enzyme reaction before and after the enzyme reaction. If a change in the coenzyme is confirmed, NAD +Coenzymes can be measured, for example, by spectroscopic analysis. Furthermore, when changes in enzymatic reaction products are confirmed, the enzymatic reaction products may be measured by techniques such as spectroscopic analysis, chromatography, and mass spectrometry. Spectroscopic analysis is preferred for its simplicity. Ergothionase has the enzyme activity of producing thiourocanic acid and trimethylamine using L-ergothioneine as a substrate. Therefore, ergothioneine can be quantified, for example, by quantifying thiourocanic acid or trimethylamine. Measurement of thiourocanic acid can be appropriately determined by those skilled in the art; for example, absorbance at approximately 311 nm can be measured. In actual measurements, a calibration curve can be prepared, the linearity of the calibration curve confirmed, and the ergothioneine contained in the test sample can be quantified based on the absorbance at 311 nm resulting from the final production of thiourocanic acid. Furthermore, since the absorption maximum of thiourocanic acid changes when made acidic, the absorption maximum wavelength can be shifted to about 330 nm by, for example, lowering the pH to 2 or less, allowing measurement at a longer wavelength. In one embodiment, the absorbance of thiourocanic acid is measured at a neutral pH, for example, at about 300-320, 305-315, or 310-312 nm, most preferably at 311 nm, and when the pH is 2 or less, the absorbance is measured at about 320-350, 325-345, or 329-331 nm, most preferably at 330 nm.

[0019] In one embodiment, the method of the present disclosure may further include an optional step, as necessary, between step (A) and step (C) (or step (C-2)), or before step (A) or after step (C) (or step (C-2)). Optional steps include adjusting the properties of the sample, stabilizing the sample, promoting the enzymatic reaction, removing or concentrating specific components, correcting the measurement results, and the like. Specific examples include filtration, centrifugation, cooling, pH adjustment, addition of specific reagents or cofactors, changing the temperature and stirring conditions, selectively treating specific components, separating the supernatant, and correcting the measurement results. For example, an optional pretreatment step for adjusting the properties of the sample may be added before step (A). This pretreatment step includes hemolysis, filtration of the sample, centrifugation, cooling, pH adjustment, or addition of a specific reagent. For example, optional steps that may be performed between step (A) and step (C) (or step (C-2)) include stabilizing the sample, adding a cofactor to promote the enzyme reaction, changing the temperature, pH, or stirring conditions, selectively removing or concentrating specific components, or changing other conditions. For example, a step of separating the supernatant may be included after step (B). Separation of the supernatant can be carried out, for example, by centrifugation or decantation. Optional steps that may be performed after step (C) (or step (C-2)) include, for example, a step of correcting the measurement results obtained.

[0020] In the present disclosure, "hemolysis" refers to a process of destroying the cell membrane of red blood cells and releasing the components contained therein to the outside. Examples of hemolysis methods include hypotonic hemolysis, surfactant treatment, freeze-thawing, ultrasonic disruption, and chemical treatment. The method selected from these is determined appropriately depending on the stability of the target component and the measurement method.

[0021] In one embodiment, the external conditions for carrying out the step are not limited unless otherwise specified. For example, the temperature, pressure, stirring speed, etc. of the environment in which mixing is carried out can be appropriately set by a person skilled in the art depending on the purpose.

[0022] In one embodiment, the method of the present disclosure includes, but is not limited to, collecting a sample from a living organism or the environment. In the present disclosure, the sample includes, but is not limited to, a biological sample or an environmental sample, preferably a biological sample. In the present disclosure, the sample preferably contains globin protein. Biological samples include samples collected from humans or animals such as cows, pigs, chickens, dogs, cats, mice, and rats. In one embodiment, the sample is a human biological sample. Specific examples of biological samples include blood, plasma, serum, blood cells, red blood cells, muscle, nerves, retina, liver, kidneys, semen, urine, saliva, tissue extracts, cerebrospinal fluid, bile, breast milk, and cell culture medium. Preferably, blood, plasma, serum, blood cells, red blood cells, muscle, nerves, retina, liver, kidneys, or semen is used, and more preferably, blood is used. Environmental samples include liquid samples such as river water, lake water, seawater, groundwater, and industrial wastewater, as well as extracts from soil, sediments, atmospheric dust, or aerosols.

[0023] In the present disclosure, the sample may or may not contain globin protein. According to the present disclosure, by performing the process of the present disclosure on a sample containing globin protein, it is possible to more accurately quantify ergothioneine in the sample containing globin protein. However, even if it is unclear whether the sample contains globin protein, the process of the present disclosure can be used to quantify ergothioneine regardless of the presence or absence of globin protein, and therefore the present disclosure is applicable regardless of the presence or absence of globin protein in the sample. In other words, it is possible to ensure the accuracy and reliability of the measurement while eliminating the need to confirm in advance the presence or absence of globin protein in the sample.

[0024] In a second aspect, the present disclosure relates to a method for reducing the affinity between globin protein and ergothioneine in a sample. In the present disclosure, "affinity" refers to the ability of two or more chemical species to form a complex under specific conditions. Specifically, "affinity" between compounds is considered to exist when a complex is formed through physical interactions (electrostatic interactions, hydrogen bonds, hydrophobic interactions, van der Waals forces, etc.) and / or chemical bonds (covalent bonds, coordinate bonds, etc.) between molecules or atoms. The method of the present disclosure is expected to reduce the affinity between globin protein and ergothioneine, which is advantageous for measuring ergothioneine in a sample.

[0025] In a third aspect, the present disclosure relates to a method for removing globin proteins and / or pigments from a sample. According to the present disclosure, the removal of globin proteins and / or pigment components from the sample is advantageous for measuring the enzymatic reaction product of ergothioneine. In the present disclosure, the pigment components are derived from, but are not limited to, a biological sample, preferably blood.

[0026] In a fourth aspect, the present disclosure relates to a pretreatment method for treating a sample under specific conditions. Specifically, the present disclosure relates to: (A) heating the sample; and (B) mixing the heated sample with trichloroacetic acid; The present invention relates to a method for pre-treating a sample, comprising:

[0027] In a fifth aspect, the present disclosure provides a kit for quantifying ergothioneine, comprising: the kit comprises an enzyme acting on ergothioneine and / or trichloroacetic acid, and instructions; The instructions state: (A) heating the sample; (B) mixing the heated sample with trichloroacetic acid; and (C) subjecting the sample obtained in step (B) to a quantitative operation; The kit may further comprise containers for performing one or more of the steps.

[0028] When quantifying ergothioneine by measuring absorbance, the accuracy of the measurement may be reduced if the sample contains pigment components that interfere with the absorbance. By using the method of the present disclosure, the pigment components can be removed from the sample, which may be advantageous for measuring ergothioneine.

[0029] In one embodiment of the present disclosure, the sample (e.g., a sample containing or suspected of containing ergothioneine) is not treated with an organic solvent or acid (e.g., methanol, ethanol, isopropanol, ethyl acetate, formic acid, acetic acid, propionic acid, trifluoroacetic acid (TFA), trichloroacetic acid, or hydrochloric acid) before being heated in step (A). In one embodiment, the sample is not treated with trichloroacetic acid before being heated in step (A).

[0030] As used herein, the term "about" means within a range of ±10%, preferably ±5%, more preferably ±2.5%, and even more preferably ±1%.

[0031] The present invention will be specifically and in detail explained below by showing examples. However, the examples are used to illustrate the present invention and are not intended to limit the present invention. [Example]

[0032] [Example 1] Differences in background absorbance due to sample processing Hemolyzed samples were prepared by adding 175 μL of water to 20 μL of whole blood (WB). The samples were heated (or not heated) at 85°C for 8 minutes. 5 μL of 6.1 M trichloroacetic acid or water was added, and the mixture was centrifuged at 15,000 rpm (4°C). 150 μL of the supernatant was collected. 50 μL of 1 M Tris-HCl buffer, pH 9.0, was added to the sample, and the resulting mixture (200 μL) was analyzed in a 96-well plate. The hemolyzed sample and the heat-denatured hemolyzed sample supernatant (without trichloroacetic acid) were too concentrated, so they were diluted 10-fold with water before analysis (i.e., 100-fold dilution). The graphs were obtained by subtracting a blank. A blank consisting of a mixture of water and Tris-HCl buffer was used instead of the sample. The results are shown in Figure 1. As is clear from these results, the supernatants removed by centrifugation from the heat-denatured samples still retained strong coloration, and the absorbance was so high that measurement was difficult without significant dilution. On the other hand, treatment with trichloroacetic acid was found to remove almost all of the colored components and almost completely eliminated the ultraviolet light absorption, including that around 311 nm and 330 nm. Therefore, pretreatment with trichloroacetic acid was found to be important for the colorimetric determination of ergothioneine, which measures absorption around 311 nm and 330 nm, such as the ETL method.

[0033] [Example 2] Examination of whole blood sample preparation methods In this example, we investigated the effect of the order of heat treatment and trichloroacetic acid treatment on the quantification of ergothioneine. In particular, we evaluated how the order of heat treatment and trichloroacetic acid treatment affected the measurement results, and compared them with conventional chemical analysis and enzymatic measurement methods. For this investigation, we performed an existing chemical analysis based on Non-Patent Document 1, which is incorporated herein by reference, and also performed an enzymatic analysis according to the present disclosure.

[0034] Chemical analysis procedures The ergothioneine measurement method using HPLC (high performance liquid chromatography) was performed based on Non-Patent Document 1. Specifically, 20 μL of sample was mixed with 200 μL of ultrapure water and heated at 80°C for 15 minutes. After heating, the sample was centrifuged at 14,000 g for 15 minutes, and 200 μL of supernatant was recovered. 1 mL of chilled acetone was added to the recovered supernatant, and the mixture was incubated at −20°C for 2 hours. The sample was centrifuged again, and the supernatant was recovered. The acetone in the recovered supernatant was evaporated using a vacuum evaporator. The concentrate was diluted with water as needed, and the ergothioneine concentration was measured by HPLC.

[0035] Processing in enzymatic assay (ETL method) The enzymatic ergothioneine measurement method was performed as follows unless otherwise specified. Specifically, 20 μL of whole blood was hemolyzed by adding 175 μL of water and heated in a heating block at 85°C for 10 minutes. The sample was then ice-cooled, and 5 μL of 100 w / v% trichloroacetic acid (6.1 M) was added and mixed. The sample was centrifuged at 15,000 rpm at 4°C for 10 minutes. The supernatant was collected and 150 μL of the supernatant was added to a 96-well plate. Similarly, 150 μL of authentic ergothioneine (Tetraedron) / water was added to the 96-well plate. 1 M Tris HCl pH 9 was added at 60 μL / well. The absorbance at 311 nm (background) was measured, and 50 μL of 2 (v / v)% BsETL (prepared by the method described in Example 8 of Patent Document 1) / water was added and mixed. The reaction was completed by incubating at room temperature for 30 minutes, and the absorbance at 311 nm was measured again, and the difference from the absorbance before the reaction was calculated.

[0036] Treatment group In this example, whole blood samples were treated under the following different conditions, and ergothioneine was measured. Groups named Heat and #1-4 were created based on the treatment. The results are shown in Figure 2. Figure 2: Heat: Heat treatment only (comparison example) 20 μL of whole blood (hereinafter sometimes abbreviated as WB) was hemolyzed by adding 180 μL of water, followed by heating at 85°C for 8 minutes and centrifugation at 15,000 rpm at 4°C for 15 minutes. The supernatant was then subjected to HPLC (instrument: LC20A (Shimadzu Corporation), column: μBondasphere (Waters), eluent: 0.1% formic acid isocratic, flow rate: 0.5 mL / min, detection: 257 nm) to measure ergothioneine. Under these conditions, ergothioneine could be measured by chemical analysis, but was not measurable by enzymatic assay (ETL method) due to high background levels. Figure 2: #1) Trichloroacetic acid treatment only (Comparative Example) 20 μL of whole blood was hemolyzed by adding 175 μL of water, and denatured by adding 5 μL of 6.1 M trichloroacetic acid. The supernatant was then centrifuged and collected. The supernatant was then measured by ETL and HPLC. Under these conditions, the results were nearly consistent for both chemical (HPLC) and enzymatic (ETL) measurements. However, ergothioneine levels were significantly reduced compared to the results shown in Figure 2 (Heat), making proper measurement difficult. Figure 2: #2) Trichloroacetic acid treatment of the supernatant of a heat-treated sample (Example) A sample that had been hemolyzed as in Figure 2: #1 above was heated at 85°C for 10 minutes. The heat-denatured sample was centrifuged. 6.1 M trichloroacetic acid was added to the supernatant to a final concentration of approximately 152.5 mM, and the resulting supernatant was centrifuged. The resulting supernatant was measured by the ETL method and HPLC method. Under these conditions, the results of both the chemical analysis method (HPLC method) and the enzymatic measurement method (ETL method) were nearly consistent with the results of the heat treatment, and good measurement results were obtained. Figure 2: #3) After heat treatment, trichloroacetic acid treatment was performed without centrifugation to remove heat-denatured products (Example) The hemolyzed sample, as in Figure 2 #1 above, was heated at 85°C for 10 minutes. 5 μL of 6.1 M trichloroacetic acid was then added. The supernatant obtained after centrifugation was analyzed by ETL and HPLC. Under these conditions, the results of both the chemical analysis method (HPLC) and the enzymatic measurement method (ETL) were nearly identical to the results in Figure 2: Heat, demonstrating favorable results. Figure 2: #4) Trichloroacetic acid treatment followed by heat treatment 5 μL of 6.1 M trichloroacetic acid was added to a sample that had been hemolyzed as in Figure 2: #1 above. This was then heated at 85°C for 10 minutes (comparative example). The supernatant obtained after centrifugation was measured by ETL and HPLC. Under these conditions, the results of both chemical (HPLC) and enzymatic (ETL) measurements were nearly consistent, and were also nearly consistent with the results of Figure 2: #1. However, ergothioneine levels were significantly reduced compared to the results of Figure 2: Heat, making proper measurement difficult.

[0037] result These results confirmed that the order of heat treatment and trichloroacetic acid treatment significantly affects the accuracy of ergothioneine quantification. In particular, performing heat treatment first and then trichloroacetic acid treatment enabled ergothioneine measurement by both chemical and enzymatic methods. On the other hand, when trichloroacetic acid treatment was performed first (Figure 2: #4) or without heat treatment (Figure 2: #1), the sensitivity of ergothioneine measurement decreased in both chemical and enzymatic methods. Furthermore, when heat treatment alone was performed (Figure 2: Heat), ergothioneine could be analyzed by chemical analysis, but the background was too high for enzymatic analysis.

[0038] [Example 3] Examination of heat denaturation temperature The effect of heating temperature on the quantification of ergothioneine in whole blood was investigated. Unless otherwise specified, the method described in the enzymatic assay in Example 2 was followed. After treating hemolyzed samples at 65–90°C for 10 minutes, the ergothioneine concentration was measured using the supernatant after trichloroacetic acid treatment by the ergothionase method (ETL 0.4%, 30 minutes). Results showed no significant difference at 80–90°C, indicating sufficient ergothioneine release (Figure 3). Ergothioneine levels decreased dramatically below 70°C. Therefore, a heating temperature of approximately 85–90°C for 10 minutes was preferable. The color of the hemolyzed samples after heating showed no significant change and turned brown up to 90–80°C. However, at 75°C, the sample turned brown with a reddish tinge, and remained red after 10 minutes of heating at 70°C. Because the red color originates from the heme in hemoglobin (Hb), this suggests a relationship between Hb denaturation and ergothioneine release (heat treatment).

[0039] [Example 4] Hemolyzed sample: ergothioneine spiked recovery test The ergothioneine concentrations were measured using the ETL method for whole blood samples (Figure 4: #1, #2), whole blood samples with ergothioneine added (Figure 4: #3, #4), and aqueous ergothioneine solutions (Figure 4: #5, #6) under conditions of heat treatment + TCA treatment or TCA treatment without heat treatment. The measurement method followed the procedure described for the enzymatic measurement method in Figure 2: #3 in Example 2. Whole blood with an ergothioneine level of 175 μM was used. For #3 and #4, whole blood samples with ergothioneine added were prepared by adding ergothioneine so that the ergothioneine concentration at 10-fold dilution was 20 μM, resulting in a measurement sample concentration of 37.5 μM. For #5 and #6, aqueous ergothioneine solutions were prepared so that the ergothioneine concentration in the measurement sample was 40 μM. The data presented represent actual values ​​measured using 10-fold diluted samples. The results are shown in Table 1 and Figure 4 below. These results indicated that the added ergothioneine also disappeared from the supernatant when treated with TCA without heating. This suggests that both ergothioneine originally present in the blood and ergothioneine added to the blood sample later form complexes with substances that are removed by TCA treatment, resulting in a dramatic decrease in ergothioneine in the supernatant. Furthermore, it was found that ergothioneine itself can be measured without being affected by trichloroacetic acid. [Table 1] *WB: whole blood

[0040] [Example 5] We investigated the effects of plasma (Figure 5) and purified hemoglobin and purified albumin with ergothioneine added (Figure 6) on the plasma samples treated with heating and TCA, as well as TCA treatment alone. Plasma samples were diluted 2-fold with water and then treated with heating and TCA (Figure 5: Heating → ETL), and plasma samples were diluted 2-fold with water and then treated with TCA alone (Figure 5: TCA → ETL). Plasma samples for HPLC were prepared as follows, with some modifications from Non-Patent Document 1. 120 μL of methanol was added to 30 μL of plasma and incubated at -20°C for 2 hours. The supernatant obtained after centrifugation was evaporated and redissolved in water. Plasma ergothioneine concentrations were measured by HPLC (Figure 5: MeOH → HPLC). The results showed that approximately 8 μM ergothioneine was detected with both sample preparation methods, with no significant differences. This suggests that there are almost no components in plasma that bind to ergothioneine. While albumin is the major protein in plasma, hemoglobin is the main component of red blood cells, which are absent in plasma but present in large quantities in whole blood. Therefore, a comparison was performed using purified hemoglobin (Hb) and purified albumin (BSA). The ETL assay was performed using a solution containing purified Hb (Sigma-Aldrich) + ergothioneine (EGT 50 μM + Hb 30 mg / mL PBS(-) pH 7.4) and a solution containing purified BSA (Sigma-Aldrich) + ergothioneine (EGT 50 μM + BSA 30 mg / mL PBS(-)), with heat treatment and TCA treatment, and with TCA treatment only (Figure 6). The results showed that no difference was observed with BSA, whereas with Hb, when Hb was insolubilized and removed by TCA treatment without heating, the amount of ergothioneine in the supernatant decreased dramatically. This indicated that Hb has an affinity for ergothioneine. It is thought that heating completely destroys the three-dimensional structure of Hb. Therefore, this result suggests that the three-dimensional structure of Hb must be maintained in order for Hb and ergothioneine to bind.

[0041] [Example 6] Affinity with hemoglobin (Hb), myoglobin (Mb), and cytochrome c (Cyt c) A 15 mg / mL solution of purified protein in PBS(-) was added with or without ergothioneine to a concentration of 20 μM and heated at 90°C for 10 minutes. After ice-cooling, trichloroacetic acid was added to 150 mM trichloroacetic acid. After centrifugation, the supernatant was measured by the ETL assay. Hb: human, Mb: horse heart, and Cyt c: horse heart (all from Sigma-Aldrich). The results are shown in Figure 7. Treatment with trichloroacetic acid (TCA) significantly reduced ergothioneine in the supernatants of unheated Hb and unheated Mb, but unheated Cyt c showed no effect of trichloroacetic acid. These results confirm that ergothioneine has affinity not only for hemoglobin but also for myoglobin, another globin protein. On the other hand, EGT showed no (or very low) affinity for Cyt c, which, like globin proteins, is a heme protein but not a member of the globin family. Human globin proteins are currently known to include hemoglobin, myoglobin, cytoglobin, neuroglobin, and androglobin, all of which possess highly conserved globin family-specific conformations. Heat treatment of both Hb and Mb dramatically reduced their binding to ergothioneine, suggesting that ergothioneine may share a common affinity with the globin family. While globin proteins are hemoproteins, ergothioneine showed no affinity for Cyt c, a non-globin hemoprotein. This suggests that direct binding of ergothioneine to heme is unlikely. Furthermore, the addition of a large excess of ergothioneine to Hb did not alter the heme-specific optical absorption spectrum (including 414, 541, and 576 nm) (data not shown). These results suggest that ergothioneine does not directly bind to heme.

[0042] [Example 7] Instead of ergothioneine, hercynine (Her: N,N,N-trimethyl-L-histidine, Tetraedron) was added, and the treatment was carried out in the presence of Hb under the same conditions as in Example 6. Her was measured by LCMS (LC: Agilent 1260 Infinity II, MS: Agilent 6530 LC / Q-TOF, ionization: ESI method, column μBondasphere (Waters), eluent: 0.1% formic acid isocratic, flow rate: 0.15 mL / min, sample: dissolved in 0.1% formic acid). The results are shown in Figure 8. As a result, there was no effect of trichloroacetic acid, regardless of whether the sample was heated or not, confirming that the binding of ergothioneine to globin protein is ergothioneine-specific.

[0043] [Example 8] Variation of thiourocanic acid absorption maximum with pH. 0.4% (v / v) ergothionase was added to 0.2 mM ergothioneine / PBS(-) and incubated at 37°C for 30 minutes. The resulting solution was analyzed by HPLC (apparatus: LC20A (Shimadzu Corporation), eluent (A) 0.1% formic acid or PBS(-), (B) acetonitrile, gradient elution from 0 minutes (0% B) to 20 minutes (100% B), flow rate: 1 mL / min, column: μBondasphere (Waters), detector: photodiode array (Shimadzu Corporation)). The results are shown in Figure 9. (A): HPLC chart of authentic ergothioneine (eluent (A): 0.1% formic acid). (B): HPLC chart of a sample converted to thiourocanic acid by treatment with ergothionase (eluent (A): 0.1% formic acid). (C): UV absorption spectrum of authentic ergothioneine (UV absorption spectrum when 0.1% formic acid is used as eluent (A)). (D): UV absorption spectrum of thiourocanic acid (UV absorption spectrum of thiourocanic acid when 0.1% formic acid or PBS(-) is used as eluent (A)). The absorption maximum of thiourocanic acid is 311 nm in the neutral range (PBS(-) pH 7.4) to weakly basic range (pH 9), but it was confirmed that the absorption maximum extends to approximately 330 nm under acidic conditions (0.1% formic acid (pH 2 or less)). [Industrial Applicability]

[0044] According to the present disclosure, a simple and accurate method for quantifying ergothioneine can be provided by eliminating the influence of globin proteins.

Claims

1. 1. A method for quantifying ergothioneine in a collected sample, comprising: (A) heating the sample; (B) mixing the heated sample with trichloroacetic acid; and (C) subjecting the sample obtained in step (B) to a quantitative operation; Including, method.

2. The step (C) of subjecting the sample to a quantitative operation is a step of subjecting the sample to an enzymatic assay, (C-1) contacting the mixture obtained in step (B) with an enzyme that acts on ergothioneine; and (C-2) a step of measuring the amount of enzyme reaction in the enzyme-treated solution obtained in step (C-1); The method of claim 1 , comprising:

3. The method of claim 1 or 2, wherein the sample comprises a biological sample.

4. The method of claim 3 , wherein the biological sample comprises globin proteins.

5. 5. The method of claim 4, wherein the globin protein is hemoglobin, myoglobin, neuroglobin, cytoglobin, or androglobin.

6. 4. The method of claim 3, wherein the biological sample comprises blood, plasma, serum, blood cells, red blood cells, muscle, nerve, retina, liver, kidney, or semen.

7. The method according to claim 1 or 2, wherein the heating temperature in step (A) is 80°C or higher.

8. The method according to claim 1 or 2, further comprising a step of separating the supernatant after step (B).

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