How to measure Urolithin A

Abstract

To provide a simple method of measuring urolithin A.SOLUTION: A method of measuring urolithin A according to the present invention comprises irradiating a solution containing or expected to contain urolithin A with light of a first wavelength selected from a range of 220 to 360 nm, and measuring intensity of light coming out of the solution and having a second wavelength that is 100-260 nm longer than the first wavelength.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for measuring urolithin A in a sample obtained from a human. [Background technology]

[0002] Urolithins are produced by the conversion of ellagic acid and ellagitannin, which are polyphenols found in strawberries, raspberries, cranberries, blackberries, walnuts, pomegranates, wolfberries, grapes, etc., by human intestinal bacteria.

[0003] Urolithins have been reported to have a variety of physiological activities, such as antioxidant activity (Non-Patent Document 1), anti-inflammatory activity (Non-Patent Document 2), anti-glycation activity (Non-Patent Document 3), and mitophagy promotion activity (Non-Patent Document 4). It is known that the amount of urolithins, particularly urolithin A, present in the human body varies greatly depending on the state of the intestinal flora (Patent Document 1). Urolithin A in human urine can be measured, for example, by using high performance liquid chromatography (HPLC) after deconjugating a urine sample with β-glucuronidase (Non-Patent Document 5). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2013-505283 [Non-patent literature]

[0005] [Non-Patent Document 1] Biosci. Biotechnol. Biochem., 76, 395-399 (2012) [Non-Patent Document 2] J. Agric. Food Chem., 60, 8866-8876 (2012) [Non-Patent Document 3] Mol. Nutr. Food Res., 55, S35-S43 (2011) [Non-Patent Document 4] Nature Medicine, 22, 879-888 (2016) [Non-Patent Document 5] Drug Metab. Dispos. 45, 657-665 (2017) Summary of the Invention [Problem to be solved by the invention]

[0006] However, the method for measuring urolithin A described in Non-Patent Document 5 requires that each sample be subjected to HPLC, and measuring a large number of samples requires a great deal of time and cost. An object of the present invention is to provide a method capable of simply and easily measuring urolithin A. [Means for solving the problem]

[0007] As a result of extensive investigations, the inventors have discovered that urolithin A in a solution emits characteristic fluorescence when irradiated with light of a specific wavelength, and that the intensity of the fluorescence shows a high correlation with the amount of urolithin A measured by HPLC, thereby completing the present invention. (1) A method for measuring urolithin A, comprising the steps of irradiating a solution containing urolithin A or a solution suspected to contain urolithin A with light of a first wavelength selected from the range of 220 to 360 nm, and measuring the intensity of light of a second wavelength which is 100 to 260 nm larger than the first wavelength output from the solution. (2) A method for measuring urolithin A contained in a sample collected from a human, the method comprising the following steps (a) and (b): (a) preparing a sample solution from the sample; and (b) irradiating the sample solution with light of a first wavelength selected from the range of 220 to 360 nm, and measuring the intensity of light of a second wavelength output from the sample that is 100 to 260 nm larger than the first wavelength. (3) The method according to (2), wherein the sample is urine. (4) The method according to (2) or (3), wherein the step (a) comprises treating the sample with β-glucuronidase. (5) The method according to any one of (1) to (4), wherein the first wavelength is selected in the range of 260 to 320 nm. (6) The method according to any one of (1) to (5), wherein the difference between the first wavelength and the second wavelength is 120 to 200 nm. (7) A kit for measuring urolithin A, used in the method according to any one of (1) to (6). Effect of the Invention

[0008] According to the method of the present invention, urolithin A can be measured simply and easily. [Brief description of the drawings]

[0009] [Figure 1] A plot of HPLC measurement values ​​of urolithin A concentration in pretreated urine samples and fluorescence measurement values ​​under condition 1 (illumination 280 nm, detection 440 nm) is shown. [Diagram 2] A plot of the HPLC measurement values ​​of the urolithin A concentration of the pretreated urine samples and the fluorescence measurement values ​​under condition 2 (illumination 238 nm, detection 440 nm) is shown (Figure 2 [Diagram 3] A plot of the HPLC measurement values ​​of urolithin A concentrations in pretreated urine samples and the corrected values ​​obtained by subtracting the constant "3.9964" from the fluorescence measurement values ​​under condition 3 (illumination 307 nm, detection 440 nm) is shown. [Figure 4] A plot of the HPLC measurement values ​​of urolithin A concentrations in pretreated urine samples and the corrected values ​​obtained by subtracting the constant "4.7274" from the fluorescence measurement values ​​under condition 4 (irradiation 300 nm, detection 550 nm) is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] 1.Definition Urolithin A generally refers to a compound represented by formula (I) below; however, as used herein, unless otherwise specified, the term "urolithin A" encompasses the compound represented by formula (I) below, as well as derivatives, metal salts, and conjugates (e.g., glucuronide conjugates) thereof.

[0011] [ka]

[0012] Urolithin A is produced by the conversion of ellagic acid and ellagitannins contained in plants by human intestinal bacteria. Urolithin A contained in human blood, tissues and urine is known to exist mostly as urolithin A 8-O-glucuronide (formula (II) below) or urolithin A 3-O-glucuronide (formula (III) below), i.e., glucuronic acid conjugates.

[0013] [ka]

[0014] [ka]

[0015] In this specification, the "wavelength" of light means the peak wavelength unless otherwise specified. For example, light with a wavelength of 280 nm means light with a peak wavelength of 280 nm. The peak wavelength here includes an error of up to ±10 nm unless otherwise specified.

[0016] In this specification, the "first wavelength" refers to the peak wavelength of light irradiated to a sample. The light of the first wavelength is also referred to as "excitation light." In this specification, the "second wavelength" refers to the wavelength of light to be measured among the light (hereinafter also referred to as "fluorescence") output from a sample irradiated with excitation light. It is preferable that the second wavelength is the same as or very close to (for example, within ±5 nm) the peak wavelength of the fluorescence.

[0017] 2. Method for measuring Urolithin A One embodiment of the method for measuring urolithin A of the present invention is characterized by comprising the steps of irradiating a solution containing urolithin A or a solution suspected to contain urolithin A with light of a first wavelength selected from the range of 220 to 360 nm, and measuring the intensity of light of a second wavelength 100 to 260 nm longer than the first wavelength output from the solution.

[0018] In this embodiment, the solvent constituting the "solution" is not particularly limited as long as it is a liquid medium capable of dissolving urolithin A, and examples thereof include water, methanol, ethanol, isopropanol, ethyl acetate, dimethyl sulfoxide, N,N-dimethylformamide, and mixtures thereof. The use of methanol and ethyl acetate is particularly preferred. Furthermore, the "solution" of the present invention may contain, in addition to these solvents, an organic acid such as formic acid or acetic acid in any ratio.

[0019] In this embodiment, a solution containing urolithin A or a solution predicted to contain urolithin A (hereinafter also simply referred to as "urolithin A solution") is placed in a container appropriate for the fluorometer to be used, such as a quartz cell, cuvette, microwell plate, or Petri dish.

[0020] In this embodiment, the urolithin A solution is subjected to fluorescence measurement using a fluorometer. Any known fluorometer, such as a spectrofluorometer, a microplate reader, or a microfluidic device, can be used as the fluorometer, so long as it is capable of irradiating excitation light of 220 to 360 nm and detecting fluorescence of 320 to 620 nm.

[0021] In the fluorometer, the first wavelength of the excitation light with which the urolithin A solution is irradiated is selected from the range of 220 to 360 nm, preferably 260 to 320 nm, more preferably 270 to 310 nm, and even more preferably 270 to 290 nm.

[0022] The second wavelength is 100 to 260 nm, preferably 120 to 200 nm, more preferably 140 to 180 nm, longer than the first wavelength. More specifically, the second wavelength is selected from the range of 320 to 620 nm, preferably 380 to 520 nm, more preferably 410 to 470 nm.

[0023] The amount of urolithin A in a urolithin A solution may be determined using a standard solution containing a known concentration of urolithin A. Fluorescence measurement of the standard solution is carried out using a fluorometer under the same conditions as for the urolithin A solution, and a calibration curve is created from the obtained fluorescence intensity values ​​and the concentrations of urolithin A. The concentration of urolithin A in the urolithin solution can be calculated from the fluorescence intensity value of the urolithin A solution based on the calibration curve.

[0024] 3. Method for measuring urolithin A in samples obtained from humans One embodiment of the method for measuring urolithin A of the present invention is a method for measuring urolithin A contained in a sample collected from a human, and is characterized in that it comprises the following steps (a) and (b): (a) preparing a sample solution from the sample; and (b) irradiating the sample solution with light of a first wavelength selected from the range of 220 to 360 nm, and measuring the intensity of light of a second wavelength output from the sample that is 100 to 260 nm larger than the first wavelength.

[0025] 3-1. Step of preparing a sample solution from a sample (step (a)) This embodiment includes a step of preparing a sample solution for performing fluorescence measurement from a sample (hereinafter also referred to as "step (a)"). In this embodiment, the "sample" is not particularly limited as long as it is a sample taken from a human body, and for example, whole blood, plasma, serum, urine, saliva, feces, cerebrospinal fluid, tissue, etc. can be used. Liquids such as urine, serum, and plasma are preferable, and urine is particularly preferable. In this embodiment, the "sample solution" refers to a solution to be directly or diluted and subjected to fluorescence measurement. For example, when a sample containing solids such as whole blood and tissue is used, step (a) is required to include at least a step of cell disruption using a homogenizer or the like, filtration, and / or solvent extraction to obtain a sample solution having optical transparency. Hereinafter, an embodiment using urine will be described, but it is not intended to limit the sample used in the present invention to urine.

[0026] In the case of a liquid sample such as urine, the sample solution may be prepared as is or after dilution. Alternatively, the liquid sample may be used as is or after dilution and filtration.

[0027] It is known that much of the urolithin A contained in samples obtained from humans is a glucuronide conjugate. Therefore, step (a) preferably includes a step of deconjugating urolithin A contained in the sample in order to more accurately measure urolithin A. Known deconjugation techniques include, for example, a technique involving reaction with β-glucuronidase derived from Escherichia coli (e.g., Non-Patent Document 5).

[0028] Step (a) may further include a step of subjecting the sample to solvent extraction. Solvents such as ethyl acetate, hexane, and diethyl ether may be used for the solvent extraction. These solvents may be used with the addition of an organic acid such as formic acid or acetic acid. In particular, it is preferable to use ethyl acetate containing formic acid. By subjecting the sample to solvent extraction, it is possible to measure urolithin A with less interference from other substances.

[0029] Step (a) may further include a step of performing a solvent exchange for the solvent-extracted sample. For example, the solvent exchange method may include concentrating the extracted sample to dryness and then redissolving it in another solvent. The solvent used for redissolution is not particularly limited, but examples thereof include water, methanol, ethanol, isopropanol, acetonitrile, dimethyl sulfoxide, or a mixture thereof. In particular, the use of methanol, acetonitrile, or dimethyl sulfoxide is preferred.

[0030] Step (a) may include a step of filtering the sample. The number of times and timing of filtering are not particularly limited, and may be performed after dilution, deconjugation, solvent extraction, or solvent exchange. Preferably, filtering is performed after solvent exchange.

[0031] Through the above step (a), a sample solution for performing fluorescence measurement is prepared. In this specification, the "sample solution" is also called a "pretreated specimen."

[0032] 3-2 Fluorescence measurement process (step (b)) This embodiment includes a step (hereinafter also referred to as "step (b)") of irradiating the sample solution prepared in step (a) with light of a first wavelength selected from the range of 220 to 360 nm, and measuring the intensity of light of a second wavelength output from the sample solution that is 100 to 260 nm longer than the first wavelength.

[0033] In this embodiment, the sample solution is contained in a container such as a quartz cell, a cuvette, a microwell plate, or a Petri dish, depending on the fluorometer to be used.

[0034] In this embodiment, the urolithin A solution is subjected to fluorescence measurement using a fluorometer. Any known fluorometer, such as a spectrofluorometer, a microplate reader, or a microfluidic device, can be used as the fluorometer, so long as it is capable of irradiating excitation light of 220 to 360 nm and detecting fluorescence of 320 to 620 nm.

[0035] In the fluorometer, the first wavelength of the excitation light with which the urolithin A solution is irradiated is selected from the range of 220 to 360 nm, preferably 260 to 320 nm, more preferably 270 to 310 nm, and even more preferably 270 to 290 nm.

[0036] The second wavelength is 100 to 260 nm, preferably 120 to 200 nm, more preferably 140 to 180 nm, longer than the first wavelength. More specifically, the second wavelength is selected from the range of 320 to 620 nm, preferably 380 to 520 nm, more preferably 410 to 470 nm.

[0037] The amount of urolithin A contained in a sample solution may be determined using a standard solution containing a known concentration of urolithin A. Fluorescence measurement of the standard solution is performed using a fluorometer under the same conditions as for the sample solution, and a calibration curve is created from the obtained fluorescence intensity values ​​and the concentrations of urolithin A. The concentration of urolithin A in the sample solution can be calculated from the fluorescence intensity value of the sample solution based on the calibration curve.

[0038] Human urine may contain fluorescent substances other than urolithin A. Therefore, it is preferable to reduce the influence of other fluorescent substances in measuring the concentration of urolithin A. Examples of methods for reducing the influence of other fluorescent substances include the following two.

[0039] (1) Select the first wavelength / second wavelength at which the fluorescence intensity of urolithin A is high and the fluorescence intensities of other substances are low. When pretreated urine was subjected to fluorescence measurement by HPLC, it was found that the relative ratio of the peak area of ​​urolithin A to the areas of other peaks differed depending on the settings of the first and second wavelengths. By selecting the first and second wavelengths for fluorescence measurement that increase the relative ratio of the peak area of ​​urolithin A, it is possible to measure the urolithin A concentration more accurately.

[0040] (2) Subtract a constant from the urolithin A concentration calculated from the calibration curve. When multiple pretreated urine samples with known urolithin A concentrations were subjected to HPLC fluorescence measurement under conditions of an irradiation wavelength of 280 nm / detection wavelength of 440 nm, many small peaks other than urolithin A were observed along with the urolithin A peak. However, it was confirmed that the peak area of ​​urolithin A differed greatly between samples depending on the urolithin A concentration of the sample solution, whereas the areas of the other peaks showed almost no difference between samples. This suggests that the influence of substances other than urolithin A in urine on the measured value is almost constant under the same irradiation / detection conditions. A more accurate urolithin A concentration can be calculated by determining this constant value (constant) and correcting by subtracting the constant from the urolithin A concentration calculated from the calibration curve. The constant can be determined, for example, by the following method. Fluorescence measurement is performed under specified conditions for multiple urine samples with known urolithin A concentrations, and the actual measured value of urolithin A concentration is calculated from the calibration curve. A correlation curve between the known concentration and the actual measured value is created, and linear approximation is performed. The absolute value of the intercept b of the approximation equation y=ax+b is taken as the constant. Thereafter, the corrected urolithin A concentration can be obtained by subtracting the constant from the measurement value (actual value) of a urine sample of unknown concentration measured under the same conditions.

[0041] The above step (b) makes it possible to measure the urolithin A concentration in the sample solution and calculate the amount of urolithin contained in the sample from the measured value.

[0042] 4. Urolithin A Measurement Kit The present invention provides a kit for measuring urolithin A. The kit of the present invention is a kit used for any of the methods for measuring urolithin A described above.

[0043] The kit of the present invention comprises, for example, a container for containing a solution containing urolithin A or a solution predicted to contain urolithin A. The container must have a structure that does not prevent the contained solution from being irradiated with light of a first wavelength from a predetermined direction, and the solution from outputting light of a second wavelength in a predetermined direction. Examples of such containers include cuvettes, microwell plates, Petri dishes, etc.

[0044] The kit of the present invention may comprise a standard solution for quantifying urolithin A in the solution. The standard solution is preferably a plurality of sample solutions of different concentrations of urolithin A. Examples of solvents constituting the standard solution include methanol, acetonitrile, dimethyl sulfoxide, ethyl acetate, etc.

[0045] The kit of the present invention can be used to measure urolithin A contained in a sample collected from a human. When measuring such a sample, the kit may include, in addition to the above-mentioned container and standard solution, reagents and equipment for pretreatment of the sample. Examples of reagents and equipment for pretreatment include a β-glucuronidase solution, an extraction solvent (e.g., ethyl acetate containing formic acid), a centrifuge tube, a filtration filter, a dissolution solvent (e.g., methanol, acetonitrile, dimethyl sulfoxide, etc.), etc.

[0046] The kit of the invention may further comprise instructions describing the procedure for measuring urolithin A contained in a solution or sample. EXAMPLES

[0047] The present invention will be described in more detail below by way of examples, but it is not intended that the present invention be limited to the scope of the examples.

[0048] Example 1: Sample pretreatment The following pretreatment was performed on 43 human urine samples. 1 mL of urine sample and 50 μL of 0.58 M acetic acid were placed in a 15 mL tube and mixed by vortexing. 25 μL (equivalent to 360 U) of β-glucuronidase (Sigma-Aldrich Glucronidase Type H-1 No. G0751) were added and left to stand overnight at 37°C. 40 μL of 1 M hydrochloric acid was added to stop the reaction and mixed by vortexing. 2.8 mL of 1.5% formic acid / ethyl acetate solution was added and ultrasonicated for 15 minutes. The organic phase was collected by centrifugation at 1500×g for 5 minutes. The collected organic phase was dried by treating with a centrifugal evaporator for 3 hours, after which 1 mL of methanol was added and mixed by vortexing to dissolve. The solution was filtered through a cartridge filter with 0.45 μm pores to obtain a sample solution.

[0049] <Reference Example 1: HPLC measurement> HPLC measurements were performed on 43 samples of the pretreated sample solution. The HPLC measurement conditions were as follows. The time required to measure the 43 samples, including the time required to measure the standard solution, was 13 hours. HPLC: Nexera-i (Shimadzu Corporation) Column: Deverosil ODS-HG, 5 μM, 4.6 × 150 mm (maximum pressure 20 MPa) Mobile phase: A: 1% formic acid (pH 3.0), B: 1% formic acid / acetonitrile (B ratio: 20% (0 min), 30% (7.5 min), 90% (9 min), 90% (11.5 min), 20% (12 min), 20% (15 min)) Injection volume: 10μL Flow rate: 1.0mL / min Column temperature: 40℃ Detection wavelength: 360nm Data acquisition time: 11 minutes

[0050] Using a urolithin A standard sample (Sigma-Aldrich No. SML1791), several urolithin A / methanol solutions of known concentrations were prepared as standard solutions, and HPLC measurements were performed under the same conditions as for the pretreated samples. A calibration curve was created from the measurements of the standard solutions, and the concentration of urolithin A in urine was calculated from the measurements of each pretreated sample.

[0051] <Example 2: Fluorescence Measurement> Fluorescence measurements were performed on 43 samples of the pretreated sample solution. Each of the pretreated samples was diluted 10-fold with methanol and 200 μL of each was added to a 96-well microplate (Thermo Scientific, flat bottom, non-sterile, black polystyrene, No. 611F96BK). The microplate was set in a microplate reader (TECAN, Infinite M Plex), and fluorescence detection was performed under the following 4 conditions. (Condition 1) Irradiation (first wavelength) 280 nm, detection (second wavelength) 440 nm (Condition 2) Irradiation (first wavelength) 238 nm, detection (second wavelength) 440 nm (Condition 3) Irradiation (first wavelength) 307 nm, detection (second wavelength) 440 nm (Condition 4) Irradiation (first wavelength) 300 nm, detection (second wavelength) 550 nm

[0052] Using urolithin A standard (No. SML1791 manufactured by Sigma-Aldrich), multiple urolithin A / methanol solutions with known concentrations were prepared as standard solutions, and fluorescence measurements were performed on each of them under the above 4 conditions. A calibration curve was created from the measured values of the standard solutions, and the concentration of urolithin A in urine was calculated from the measured values of each pretreated sample. The time required for the measurement of all pretreated samples was within 10 minutes.

[0053] <Comparison between HPLC Measurement and Fluorescence Measurement> The measured values of the urolithin A concentration of each sample obtained in Reference Example 1 and Example 2 were compared. A correlation curve was created by plotting the HPLC measurement values on the vertical axis (y) and the fluorescence measurement values on the horizontal axis (x), and linear approximation (y = ax + b) was performed. Also, the correlation coefficient (r) between the two measurement values was calculated. When HPLC was compared with the fluorescence measurements under Conditions 1 to 4, the values of a, b, and r were as follows. (Condition 1) a: 1.1811; b: -1.8582; r: 0.9829 (Condition 2) a: 1.3795; b: -1.6174; r: 0.9075 (Condition 3) a: 0.9343; b: -3.9964; r: 0.9552 (Condition 4) a: 0.8837; b: -4.7274; r: 0.9474 A high correlation with the HPLC measurement values ​​was observed under all conditions 1 to 4. On the other hand, b was a negative value in all cases, and the overall fluorescence measurement values ​​tended to be higher than the HPLC measurements. This was presumed to be due to the influence of trace amounts of other fluorescent substances contained in the urine samples. Therefore, for conditions 3 and 4 where the absolute value of b exceeded 3, the absolute value of b was subtracted from the urolithin A concentration calculated in Example 2 to obtain a correction value. Note that the correction value was set to "0" when the value was negative. When the correction values ​​calculated in this way were compared with the HPLC measurement values, the values ​​of a, b, and r under conditions 3 and 4 were as follows. (Condition 3) a: 0.9505; b: -0.4622; r: 0.9574 (Condition 4) a: 0.9008; b: -0.7705; r: 0.9507 It was confirmed that all of the corrected values ​​showed good correlation with HPLC.

[0054] Figures 1 to 4 show plots of HPLC measurement values ​​and fluorescence measurement values ​​under conditions 1 to 4. Figure 1 is a plot of HPLC measurement values ​​and fluorescence measurement values ​​under condition 1. Figure 2 is a plot of HPLC measurement values ​​and fluorescence measurement values ​​under condition 2. Figure 3 is a plot of HPLC measurement values ​​and corrected values ​​obtained by subtracting a constant "3.9964" from the fluorescence measurement values ​​under condition 3. Figure 4 is a plot of HPLC measurement values ​​and corrected values ​​obtained by subtracting a constant "4.7274" from the fluorescence measurement values ​​under condition 4. Despite the simple method of fluorescence measurement, it was possible to measure urolithin A concentrations equivalent to those measured by HPLC measurement under all conditions.

Claims

1. A method for measuring urolithin A contained in a sample collected from a human, comprising the following steps (a) and (b): (a) preparing a sample solution from the sample; and (b) irradiating the sample solution with light having a first wavelength selected from the range of 220 to 360 nm, and measuring the intensity of light output from the sample at a second wavelength that is 100 to 260 nm larger than the first wavelength; Including, the sample is urine, The step (a) includes a deconjugation treatment of glucuronide conjugates contained in the sample, and does not include a separation treatment using a column. method.

2. The method of claim 1, wherein the first wavelength is selected from the range of 260 to 320 nm.

3. The method according to claim 1 or 2, wherein the difference between the first wavelength and the second wavelength is from 120 to 200 nm.

4. A method according to any one of claims 1 to 3, wherein the deconjugation treatment is a β-glucuronidase treatment.

5. A kit for measuring urolithin A, used in the method according to any one of claims 1 to 4.

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