Method for evaluating oxidation stress level

Abstract

This method for evaluating the level of oxidative stress according to one embodiment includes: a step for quantifying 2-methylthioadenosine (ms(1)2(2)A) in mitochondria; and a step for associating a quantitative value of ms(3)2(4)A with the level of oxidative stress.

Description

Methods for evaluating oxidative stress levels

[0001] This invention relates to a method for evaluating oxidative stress levels.

[0002] Oxidative stress is a cause of cellular damage resulting from the excessive production of reactive oxygen species (ROS). Mitochondria, in particular, are susceptible to oxidative stress because they produce large amounts of ROS during energy production.

[0003] Oxidative stress can cause abnormalities in post-transcriptional modifications of mitochondrial tRNA, for example, leading to problems in mitochondrial protein translation and potentially resulting in the development of various diseases, including mitochondrial diseases. Conversely, this means that the level of oxidative stress experienced by mitochondria may be useful in diagnosing or predicting the onset of such diseases. Therefore, there is a need for methods to assess the level of oxidative stress in mitochondria.

[0004] One of the objectives of this invention is to provide a method for evaluating oxidative stress levels using mitochondrial oxidative stress markers.

[0005] A method for evaluating oxidative stress levels according to an embodiment is a method for evaluating the level of oxidative stress experienced by mitochondria, wherein 2-methylthioadenosine (ms) in the mitochondria ２ A) A process to quantify and ms ２ The process includes a step in which the quantitative value of A is associated with the level of oxidative stress.

[0006] A method for evaluating the level of oxidative stress according to another embodiment is a method for evaluating the level of oxidative stress experienced by a subject, wherein 2-methylthioadenosine (ms) is present in a sample derived from the subject. ２ A) A process to quantify and ms ２ The process includes a step in which the quantitative value of A is associated with the level of oxidative stress.

[0007] FIG. 1 is a flowchart of the method according to the first embodiment. FIG. 2 is a flowchart of the method according to the first embodiment. FIG. 3 is a flowchart of the method according to the second embodiment. FIG. 4 is a flowchart of the method according to the second embodiment. FIG. 5 is a flowchart of the method according to the third embodiment. FIG. 6 is a flowchart of the method according to the third embodiment. FIG. 7 is a diagram showing the results of Example 1. FIG. 8 is a diagram showing the results of Example 2. FIG. 9 is a diagram showing the results of Example 3. FIG. 10 is a diagram showing the results of Example 4. FIG. 11 is a diagram showing the results of Example 5. FIG. 12 is a diagram showing the results of Example 5. FIG. 13 is a diagram showing the results of Example 6.

[0008] Hereinafter, a method for evaluating the oxidative stress level of an embodiment will be described with reference to the drawings. Each figure is a schematic diagram for promoting the understanding of the embodiment, and although there are parts where the shape, dimensions, ratio, etc. are different from the actual ones, these can be appropriately changed in design in consideration of the following description and known techniques.

[0009] [First Embodiment] The method according to the first embodiment is a method for evaluating the level of oxidative stress received by mitochondria.

[0010] As shown in FIG. 1, this method includes a step (S11) of quantifying ms ２ A in mitochondria, and a step (S12) of associating the quantified value of ms ２ A obtained in step (S11) with the level of oxidative stress.

[0011] Hereinafter, each step will be described in detail.

[0012] - Step (S11) of quantifying ms ２ A in mitochondria "Ms ２ A in mitochondria" as used in the present invention means an adenine residue modified with ms contained in mitochondria. The quantified value obtained in step (S11) is a value indicating the abundance of ms ２ A contained in the target mitochondria, and is, for example, a value of total mass, total amount of substance, mass percentage concentration, molar concentration, and molality, or a relative value calculated using these values. ２

[0013] ms ２ The quantitative determination of A can be performed by any known technique. For example, ms ２ The quantitative determination of A can be performed by mass spectrometry, particularly by mass spectrometry combined with chromatography.

[0014] ms ２ If the quantification of A is performed by mass spectrometry, then step (S11) is, for example, ms ２ The process of preparing a sample containing A, and the mass spectrometry that can detect A ２ The process involves providing the sample to an ionization source under conditions suitable for generating A ions, and then performing mass spectrometry to determine the ionization rate (ms). ２ A step to determine the amount of A ions, and the determined ms ２ The amount of A in mitochondria is ms ２ The process includes steps related to the amount of A. Furthermore, after the step of preparing the sample and before the step of providing it to the ionization source, the process may include separating the solute in the sample by chromatography.

[0015] ms ２ The process of preparing a sample containing A includes, for example, the process of extracting RNA from mitochondria. ２ The step of preparing a sample containing A may include the step of preparing mitochondria, which are the subject of the method of the first embodiment. The mitochondria preparation step can be carried out, for example, by destroying cells or their tissues and separating them from other organelles. Furthermore, ms ２ The step of preparing a sample containing A may include the step of isolating a specific RNA from the RNA extracted from the sample. RNA extraction and isolation can be carried out by known methods, such as using a dedicated kit.

[0016] The RNA extracted and isolated from mitochondria is, for example, total RNA or mitochondrial tRNA (mt-tRNA). Further details will be provided later, but ms ２Since A modification occurs in mt-tRNAs corresponding to tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe), or serine (Ser), it is preferable to isolate each of these mt-tRNAs.

[0017] For mass spectrometry, sample ionization methods include, for example, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI), electron ionization (EI), fast electron bombardment (FAB) / liquid secondary ionization (LSIMS), matrix-assisted laser desorption ionization (MALDI), field ionization, field desorption, thermal spray / plasma spray ionization, and particle beam ionization. The selection of these ionization methods can be made appropriately based on the analyte to be measured, the type of sample, the type of detector, and the selection of positive or negative ion mode. Hereinafter, the term "positive ion mode" refers to a mass spectrometry method in which positive ions are generated and detected, and the term "negative ion mode" refers to a mass spectrometry method in which negative ions are generated and detected.

[0018] Ionization and MS can be performed using a mass spectrometer. When MS is used in the present invention, the analyzers used for MS include, for example, a quadrupole (Q) analyzer, a triple quadrupole (QqQ) analyzer, a Fourier transform ion cyclotron resonance (FTICR) analyzer, an ion trap (IT) analyzer, a time-of-flight (TOF) analyzer, and a hybrid tandem analyzer (Q-TOF, IT-TOF, Q-trap, Q-FTICR).

[0019] In this invention, mass spectrometry is performed using ms ２ Since A is the target of detection, it is preferable to perform the procedure in positive ion mode. However, if the mass spectrometry is in ms ２ If it is also possible to detect modified nucleosides other than A, the procedure may be performed in negative ion mode.

[0020] In one aspect, ms ２The quantification of A can be performed using tandem mass spectrometry (MS / MS). Tandem mass spectrometry can be performed by any method known in the art, including, for example, selective reaction monitoring, precursor ion scanning, or product ion scanning. Using tandem MS, ms ２ When determining the amount of A, it is preferable to perform selective reaction monitoring (SRM). By performing SRM, if the target analyte (ms) in chromatography is determined, ２ Even if there are impurities that have a retention time similar to that of A) and the same m / z value as the precursor ion, their influence can be eliminated as long as the impurities do not produce product ions with the same m / z value as the target analyte.

[0021] Of the steps (S11), the determined ms ２ Amount of A ions in ms ２ The process associated with the quantity of A is, in one example, performed by comparison with an internal standard substance. Specifically, the target analyte (ms) ２ A) Ionize all or part of both the internal standard material and ms ２ By comparing the presence or amount of ions generated from A with the amount of ions generated from the internal standard, the ms in the sample can be determined. ２ It can be associated with the quantity of A.

[0022] In another example, ms in the sample ２ The amount of A is determined by comparison with one or more external reference standards. An example of an external reference standard is ms ２ Examples include samples spiked in area A.

[0023] Furthermore, when using SRM in tandem mass spectrometry, the ms of step (S11) ２ Amount of A ions in ms ２ The process of relating to the amount of A is, for example, the ms in the sample. ２ This can be done by comparing the SRM feature peak area of ​​A with the SRM feature peak area of ​​an internally added standard.

[0024] To avoid unwanted signals from proteins in the sample interfering with the measurement results, it is preferable that the sample be deproteinized. Methods of "deproteinization" include, for example, insolubilization by denaturation of proteins (addition of acids such as perchloric acid, trichloroacetic acid, or metaphosphoric acid; addition of organic solvents miscible with water such as acetone, acetonitrile, methanol, or ethanol; heating and cooling), or physical removal (ultrafiltration using a membrane filter (such as a centrifugal filtration device); dialysis using a dialysis tube; or ultracentrifugation). Note that the methods of deproteinization are not limited to these examples.

[0025] ms ２ Step (S12) in which the quantitative value of A is associated with the level of oxidative stress. In step (S12), the ms obtained in step (S11) ２ The quantitative value of A is correlated with oxidative stress to determine the mitochondrial oxidative stress level. In one example, step (S12) is performed by comparing it with a predetermined reference value.

[0026] A predetermined reference value is, for example, a threshold that distinguishes between a group of mitochondria known to have high oxidative stress levels (A) and a group of mitochondria known to have low oxidative stress levels (B). The reference value is, for example, the ms obtained from mitochondria group (A) and mitochondria group (B). ２ This is the mean or median of the quantitative values ​​of A. Note that the setting of the reference value is not limited to this example; the person implementing the method according to the present invention may determine it as appropriate, for example, by referring to publicly available literature or values ​​determined from past knowledge.

[0027] In this case, for example, ms ２ If the quantitative value of A is greater than the reference value, it can be determined that the level of oxidative stress is high. Also, ms ２ If the quantitative value of A is smaller than the reference value, the level of oxidative stress may be judged as low. Furthermore, multiple reference values ​​may be set, and the level of oxidative stress may be evaluated in multiple stages.

[0028] In recent years, it has become clear that mitochondrial tRNA (mt-tRNA), transcribed from mitochondrial DNA, undergoes a variety of post-transcriptional modifications. The inventors of this invention have found that oxidative stress on mitochondria imparts ms to mt-tRNA. ２ i ６ A changes, ms ２ We hypothesized that A would occur and verified this in the experimental example described below. As a result, in response to the increase in oxidative stress, ms in mitochondria increased. ２ An increase in the abundance of A was actually confirmed. Therefore, the inventors of the present invention have confirmed that ms in mitochondria ２ We found that A can serve as a biomarker indicating the level of oxidative stress experienced by mitochondria.

[0029] The method of the first embodiment, which includes steps (S11) and (S12) described above, can be used to evaluate the level of oxidative stress experienced by mitochondria.

[0030] In a further embodiment, the quantitative value associated with the level of oxidative stress may be a relative quantitative value. An example of such a relative quantitative value is the ms for all or more types of modified nucleosides in mitochondria. ２ The ratio of A can be cited.

[0031] In such cases, a further embodiment of the method involves, for example, the ms in mitochondria, as shown in Figure 2. ２ A step to quantify A (S21), a step to quantify all or more types of modified nucleosides in mitochondria (S22), and ms ２ Using the quantitative value of A, ms for all or more types of modified nucleosides ２ The process includes a step (S23) in which the ratio of A is calculated, and a step (S24) in which the ratio is associated with the level of oxidative stress.

[0032] Steps (S21) and (S22) can be carried out in the same manner as step (S11). Also, step (S24) can be carried out in the same manner as step (S12).

[0033] "All or more types of modified nucleosides" means any modified nucleoside, for example, ms ２ MS related to A ２ i ６ A, and i ６ It is preferable that A is included (ms ２ i ６ A is ms ２ It is a precursor of A, i ６ A is ms ２ i ６ (It is a precursor of A). Furthermore, the target of measurement for modified nucleosides is, for example, the modification of mt-tRNA. In this case, it is preferable that the mt-tRNA used for quantification is mt-tRNA corresponding to Trp, Tyr, Phe, or Ser.

[0034] "ms ２ Using the quantitative value of A, ms for all or more types of modified nucleosides ２ The "process in which the ratio of A is calculated" refers to the ms ratio of the total amount of modified nucleosides measured by quantitative value. ２ The objective is to determine the ratio of the quantitative values ​​of A. In particular, i ６ A, ms ２ i ６ A and ms ２ ms relative to the sum of the quantitative values ​​of A ２ It is preferable to determine the ratio of the quantitative values ​​of A.

[0035] As mentioned above, ms ２ A is ms ２ i ６ It is caused by a change from A, ms ２ i ６ A is i ６ This occurs as a result of a change from A. Even if the detection accuracy of the various detection methods applied in the quantitative process is not good, or if there is variation in the total amount of modified nucleosides contained in each mitochondria, the ms of the total amount of modified nucleosides will still be affected. ２ By determining the ratio of the quantitative values ​​of A, the level of oxidative stress experienced by mitochondria can be evaluated under more homogeneous conditions.

[0036] [Second Embodiment] The method according to the second embodiment is a method for evaluating the level of oxidative stress experienced by a subject under test. This will be explained below with reference to Figure 3.

[0037] The method for evaluating the level of oxidative stress experienced by the subject is to measure the ms in the sample derived from the subject. ２ A is quantified in step (S31), and the ms obtained in step (S31) ２ The process includes a step (S32) in which the quantitative value of A is associated with the level of oxidative stress.

[0038] In this embodiment, "subject of test" refers to, for example, a test animal or any animal cells or tissue thereof.

[0039] "Test animals" include mammals such as humans, monkeys, cattle, pigs, horses, dogs, cats, sheep, goats, rabbits, hamsters, guinea pigs, mice, and rats, as well as birds such as chickens, and are preferably mammals.

[0040] "Animal cells or tissues thereof" refers, for example, to cells or tissues derived from the animal being tested, and preferably to cells or tissues derived from mammals. Cells derived from the animal being tested include, for example, cells isolated from the animal being tested, pluripotent stem cells prepared using cells isolated from the animal being tested, and cells differentiated from said stem cells.

[0041] "Sample" refers to ms ２ The sample can be any form that may contain A, and examples include samples derived from the test animal, such as cell culture supernatant or body fluid samples. In one example, the sample used in the method of this embodiment is a human-derived sample.

[0042] "Cell culture supernatant" refers to the supernatant of a culture obtained by culturing cells of any state derived from a test subject in a culture medium for a certain period of time. Examples of cells that can be used to prepare the culture supernatant include cells isolated from the body of a test animal, cultured cells thereof, or pluripotent stem cells derived from cells isolated from the body of a test animal, or cells differentiated from said pluripotent stem cells. The cell culture conditions are, for example, 100 ml of cell culture medium in a culture dish. ５ ~10７ It is the culture supernatant of a culture in which individual cells are seeded and cultured for one day or more.

[0043] "Body fluid" is, for example, a biological sample of a liquid that can be collected from the body of a test animal. Examples of body fluids include blood, plasma, serum, bile, saliva, urine, tears, sweat, cerebrospinal fluid (CSF), etc. Preferably, the body fluid is urine or serum.

[0044] "ms ２ A" in the sample means a modified adenine residue. Also, the quantitative value obtained by step (S31) is a value indicating the abundance of ms ２ A in the sample, and is, for example, a value of total mass, total amount of substance, mass percentage concentration, molar concentration, and molality, or a relative value calculated using these values. ２ A value indicating the abundance of ms

[0045] Conventionally, oxidative stress on cells has been evaluated by nucleic acid type oxidative stress markers such as 8-oxoguanine (8OHG), which is a modification that occurs in RNA due to oxidative stress, and 8-oxodeoxyguanosine (8OHdG), which is a modification that occurs in DNA.

[0046] However, since such conventional oxidative stress markers occur in both the cytoplasm and mitochondria, they do not reflect the positional information of oxidative stress. Therefore, it has been difficult to determine, for example, whether the oxidative stress received by an arbitrary cell is the oxidative stress received by mitochondria using conventional oxidative stress markers.

[0047] As a result of focusing on modified nucleosides among such post-transcriptional modifications and conducting research, the inventors of the present invention have now found that among modified nucleosides, 2-methylthio-n6-isopentenyladenosine (ms ２ i ６ A), 5-taurinomethyluridine (tm ５ U), 5-taurinomethyl-2-thiouridine (tm ５ s ２ U), 5-formylcytidine (f ５It was revealed that (C) exists only in mt-tRNA.

[0048] As described in the first embodiment, ms in mitochondria ２ A was a biomarker indicating the level of oxidative stress received by mitochondria. Therefore, the inventors found that ms ２ A is a mitochondrial-specific oxidative stress marker, and even if various nucleic acids derived from sites other than mitochondria (for example, modified nucleosides other than ms ２ A) are contained in the sample derived from the subject, the mitochondrial oxidative stress level can be evaluated by quantifying ms ２ A in the sample. Consequently, it was shown that the level of oxidative stress received by mitochondria quantified in the second embodiment can be used as an index of the level of oxidative stress received by the subject.

[0049] As a further embodiment, the quantitative value associated with the level of oxidative stress can be a relative quantitative value, similar to the first embodiment. When the ratio of ms ２ A is associated with the level of oxidative stress, it is performed as shown in FIG. 4.

[0050] The method according to the embodiment includes a step (S31) of quantifying ms ２ A in the sample derived from the subject, a step (S32) of quantifying all or a plurality of types of modified nucleosides in the sample derived from the subject, and using the quantitative value of ms ２ A to calculate the ratio of ms ２ A to all or a plurality of types of modified nucleosides (step S33), and a step (S34) of associating the ratio of the ms ２ A with the level of oxidative stress.

[0051] Steps (S31) to (S34) can be performed in the same manner as steps (S21) to (S24) in the first embodiment. As described above, by obtaining the ratio of the quantitative value of ms ２ A to the total amount of a plurality of types of modified nucleosides, the level of oxidative stress received by mitochondria can be evaluated under more homogeneous conditions.

[0052] [Third Embodiment] The third embodiment relates to a method for evaluating the level of oxidative stress experienced by a subject, similar to the method according to the second embodiment, but further includes a step of comparing the oxidative stress level of the subject with the oxidative stress level of a control. Specifically, as shown in Figure 5, the method according to the third embodiment includes steps (S51) and (S52) similar to steps (S21) and (S22) of the first embodiment, in addition to ms in a sample derived from the control. ２ A is quantified in step (S53), and the ms obtained in step (S53) ２ The process includes a step (S54) in which the quantitative value of A is associated with the level of oxidative stress, and a step (S55) in which the oxidative stress level of the subject and the oxidative stress level of the control are compared.

[0053] In this embodiment, "control" refers to a test animal that is the same individual as the subject or a different individual, or any animal cells or tissue derived from the said test animal.

[0054] When the control is a different individual from the subject, the "control" may be an individual of the same species or an individual of a different species. Here, an individual of the same species refers to an individual belonging to the same species phylogenetically. On the other hand, an individual of a different species refers to an individual belonging to a different species from the subject. However, it is preferable that the phylogenetic classification of the individual of a different species does not differ significantly from that of the subject; for example, it is good if it belongs to the same genus as the subject.

[0055] When the control is the same individual as the subject, the "control-derived sample" is, for example, a sample collected under different conditions than the sample derived from the subject. Different conditions include, for example, a different organ from which the sample is collected and a different timing of collection. In other words, the control may be the subject at a past point in time. For example, if a different condition is to give an individual some stimulus that may cause a change in oxidative stress, the individual at the time after the stimulus was given may be used as the subject, and the individual at the time before the stimulus was given may be used as the control.

[0056] Furthermore, if the controls are different individuals, the controls may be individuals whose presence or absence of various diseases (e.g., mitochondrial diseases) is known. Alternatively, the controls may be the same individual as the subject, but at the time the subject was previously diagnosed with mitochondrial disease. Or, the controls may be the same individual as the subject diagnosed with mitochondrial disease, but before the subject was administered medication for mitochondrial disease.

[0057] In step (S53), the control sample used for quantification may be a single sample or may consist of multiple sample groups. If the control consists of multiple sample groups, the ms obtained in step (S53) ２ The quantitative value of A is, for example, the average value of quantitative values ​​measured from each of several sample groups.

[0058] Steps (S51) and (S53) can be carried out using mass spectrometry in the same manner as step (S11) in the first embodiment. However, depending on the nature of the sample to be quantified, it is preferable to subject it to various types of chromatography before performing mass spectrometry. For example, if the sample is urine collected from a test subject or control, a sample suitable for mass spectrometry can be obtained by deproteinizing it with methanol and then subjecting it to liquid chromatography.

[0059] Furthermore, steps (S52) and (S54) can be performed using the same operation as step (S12) in the first embodiment.

[0060] The third embodiment, through steps (S51) to (S55) described above, makes it possible to relatively compare the oxidative stress levels of the test subject and the control. By appropriately setting the conditions of the test subject and the control, the oxidative stress level index can be used to diagnose the progression of various diseases (e.g., mitochondrial diseases), the effectiveness of treatment, and predict the prognosis. It is also known that the oxidative stress level of cells tends to increase with the progression of aging. By using the test subject at a past point in time as the control, or by using individuals of different ages as the control, it is possible to estimate the aging level of the test subject.

[0061] In a further embodiment, the process may include a step of comparing the oxidative stress level experienced by the subject obtained in step (S34) with the oxidative stress level experienced by the control.

[0062] In this case, as shown in Figure 6, in addition to steps (S51) to (S54), the ms in the sample derived from the control is also measured. ２ A step (S55) to quantify A, a step (S56) to quantify all or more types of modified nucleosides in the control sample, and the ms obtained in step (S55) ２ Using the quantitative value of A, ms for all or more types of modified nucleosides ２ The process in which the ratio of A is calculated (S57), and ms ２ The process includes a step (S58) in which the ratio of A is associated with the level of oxidative stress, and a step (S59) in which the oxidative stress level of the subject and the oxidative stress level of the control are compared.

[0063] Steps (S55) to (S58) can also be carried out in the same way as steps (S21) to (S24) in the first embodiment, as can be steps (S51) to (S54).

[0064] According to this method, as described above, the ms of the total amount of multiple types of modified nucleosides ２ By determining the ratio of the quantitative values ​​of A, it is possible to evaluate the level of oxidative stress experienced by mitochondria, and consequently the level of oxidative stress experienced by the subjects, under more homogeneous conditions.

[0065] [Example] Experimental examples are described below. However, embodiments of the present invention are not limited to the following examples.

[0066] Example 1. Mass spectrometry (MS) ２ Measurement of A: Total RNA was purified from HeLa cells, and the RNA was digested to the nucleoside level with RNase P1. Then, the precise mass of each RNA modification in cation mode, as well as the precise mass of the fragments produced by ms / ms, was measured using an Orbitrap-type mass spectrometer. The separation column used was an ODS-3 reverse-phase column.

[0067] Analysis revealed that the ms has the structure shown in Figure 7(A).２ A had a precise mass m / z of 314.0918 and was detected at a retention time of 22.06 minutes (Figure 7(B)). Furthermore, at ms / ms, a fragment with m / z 182.0495 was produced (Figure 7(C)), and the m / z of this fragment was ms ２ We confirmed that it matches the m / z value predicted from the structure of A.

[0068] Example 2. ms ２ A. Determination of the nucleic acids present ms ２ To determine the nucleic acid in which A exists, ms in various cells and RNA groups ２ Detection of A was performed. The detection results are shown in Figures 8(A) to (C).

[0069] First, tRNA fraction, mRNA, 18S rRNA, and 28S rRNA were extracted or purified from total RNA derived from HeLa cells.

[0070] The tRNA fraction was obtained by electrophoresis of total RNA on a 15% polyacrylamide gel containing 8M urea, and then cleaving the gel containing tRNA located around 80 base pairs. mRNA was purified using the polyA-oligodT method. 18S rRNA and 28S rRNA were obtained by electrophoresis of total RNA on a 1% agarose gel, and the bands corresponding to 18S rRNA and 28S rRNA were cleaved and purified.

[0071] These RNAs were digested using RNase P1 and then measured using an Orbitrap mass spectrometer (ms). ２ A was measured. As shown in Figure 8(A), ms ２ A was detected in the total RNA and tRNA fractions.

[0072] Next, total RNA was purified from HEK293 cells and Rho0 cells from which mitochondrial DNA had been removed with ethidium bromide reagent, and ms ２ A modification was measured using a mass spectrometer. In Rho0 cells from which mitochondrial DNA had been removed, ms ２ A modification decreased significantly, so ms ２ It was suggested that A is present in mitochondrial tRNA (Figure 8(B)).

[0073] Next, mt-tRNA was added to the total RNA fraction of HeLa cells. Ｐｈｅ , mt-tRNA Ｔｙｒ mt-tRNA Ｓｅｒ（ＵＣＮ） mt-tRNA Ｔｒｐ The tRNAs were hybridized with oligoprobes having complementary biotin at their 3' ends. Each tRNA was purified with streprouvidin beads and then digested with RNase P1. As a control experiment, mt-tRNA was used. Ｖａｌ Cytoplasmic tRNA, or Cy-tRNA Ｌｙｓ The tRNAs were purified using the same method. ２ Modification A was measured using a mass spectrometer. The detection results are shown in Figure 8(C).

[0074] ms ２ A is mt-tRNA Ｖａｌ and Cy-tRNA Ｌｙｓ It was not detected, mt-tRNA Ｐｈｅ , mt-tRNA Ｔｙｒ , mt-tRNA Ｓｅｒ（ＵＣＮ） , mt-tRNA Ｔｒｐ It was detected by ms. ２ A is mt-tRNA, in particular mt-tRNA Ｐｈｅ , mt-tRNA Ｔｙｒ mt-tRNA Ｓｅｒ（ＵＣＮ） , mt-tRNA Ｔｒｐ It was confirmed to exist in [location].

[0075] Example 3. MS due to oxidative stress ２ Verification of A production: Total RNA purified from HeLa cells was mixed with 5 mM AAPH (2,2'-Azobis(2-amidinopropane) dihydrochloride) and allowed to stand at room temperature for 3 hours. The total RNA was then purified by ethanol precipitation, digested with RNase P1, and analyzed by mass spectrometry. ２ A, ms ２ i ６ A. 8OHG was detected.

[0076] Figures 9(A) to (C) are bar graphs showing the results of Example 3. Graphs (A) to (C) show the ms values ​​for the control sample (total RNA without added AAPH). ２ A, ms ２ i ６ A. These amounts in the subjects are shown relative to the amount of 8OHG, which is set to 1. In Figure 9, "*" indicates a statistically significant difference examined in the Student's t-test. In this specification, statistical significance is represented by "*", where * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001. Similarly, in the following examples, statistical significance is represented by "*".

[0077] As shown in Figures 9(A) and (B), the AAPH treatment is performed in ms ２ A, as well as 8OHG, a conventional oxidative stress marker, significantly increased. On the other hand, as shown in Figure 9(C), ms ２ i ６ A was on a downward trend. Based on these results, ms ２ A was confirmed to function as an oxidative stress marker, similar to 8OHG. Also, ms ２ A is ms ２ i ６ It was also suggested that it is an oxidative stress product derived from A.

[0078] Example 4. ms ２ Comparison of A and 8OHG: D-alanine was added to cells expressing D-amino acid oxidase (mito-DAAO) in the mitochondria (hereinafter referred to as mito-DAAO cells) at concentrations of 5 mM, 10 mM, and 20 mM. After addition, CO2 was added at 37°C. ２ The cells were left in an incubator for 7 hours to induce oxidative stress (radical stress) in the mitochondria. Meanwhile, to induce oxidative stress (radical stress) in the cytoplasm, D-alanine was added to cells expressing D-amino acid oxidase (Cyto-DAAO) in the cytoplasm (hereinafter referred to as Cyto-DAAO cells) at concentrations of 5 mM, 10 mM, and 20 mM. After addition, the cells were incubated at 37°C CO2. ２The cells were left undisturbed in an incubator for 7 hours. Afterward, total RNA was purified from each cell, digested with RNAse P1, and then saturated. ２ A and 8OHG were measured using a mass spectrometer.

[0079] Figures 10(A) to (D) show the results of Example 4. Figure 10(A) shows the ms in Mito-DAAO cells. ２ A is the amount, (B) is the amount of 8OHG in Mito-DAAO cells, and (C) is the ms in Cyto-DAAO cells. ２ Figure 10 shows the amount of (A) and (D) the amount of 8OHG in Cyto-DAAO cells. Figures 10(A) to (D) have ms on the vertical axis. ２ The horizontal axis represents the amount of A or 8OHG, and the horizontal axis represents the amount of D-Ala added. The ms of cells treated with D-Ala were measured. ２ The amounts of A and 8OHG are relative values, with the amounts in the control sample (cells without D-alanine added) set to 1.

[0080] As shown in Figure 10(A), in Mito-DAAO cells, the concentration of D-Ala added, i.e., the intensity of oxidative stress experienced by mitochondria, is dependent on the ms ２ A levels increased. On the other hand, 8OHG levels hardly increased when mitochondrial oxidative stress was weak, and only increased during strong oxidative stress caused by the addition of 20 mM D-Ala (Figure 10(B)). From Figures 10(C) and 10(D), in Cyto-DAAO cells, 8OHG levels increased strongly in response to oxidative stress. On the other hand, ms ２ The amount of A did not show a significant increase compared to the response in Mito-DAAO cells.

[0081] Based on the above results, ms ２ A was demonstrated to be a highly specific mitochondrial oxidative stress marker. Also, ms ２ A is mitochondrial-specific ms ２ i ６ Because it is produced from A, it has been shown to be an extremely specific mitochondrial oxidative stress marker compared to conventional oxidative stress markers such as 8OHG and 8OHdG.

[0082] Example 5. MS in aged mice and young mice ２ Total RNA was purified from the liver, brain, heart, and kidney of each of the aged mouse group (24 months old, 19 samples) and the juvenile mouse group (5 months old, 15 samples) to compare the amount of A. Subsequently, mt-tRNA was extracted. Ｔｒｐ , mt-tRNA Ｓｅｒ（ＵＣＮ） , mt-tRNA Ｔｙｒ , mt-tRNA Ｐｈｅ An oligoprobe with complementary biotin at its 3' end was added and hybridized. After purifying each tRNA with streprouvidin beads, it was digested with RNase P1 and analyzed by mass spectrometry. ２ A modification, ms ２ i ６ A, i ６ A was detected.

[0083] The verification results in Example 5 will be explained using Figures 11 and 12. Figures 11 (A-1 to 4) are bar graphs showing the verification results in the kidney, Figure 11 (B-1 to 4) in the heart, Figure 12 (C-1 to 4) in the liver, and Figure 12 (D-1 to 4) in the brain. The numbers after "-" indicate that 1 represents mt-tRNA. Ｔｒｐ ,2 is mt-tRNA Ｓｅｒ（ＵＣＮ） ,3 is mt-tRNA Ｔｙｒ , mt-tRNA Ｐｈｅ This shows that, for example, graph (A-1) shows mt-tRNA isolated from the kidney. Ｔｒｐ MS in ２ A modification, ms ２ i ６ A, i ６ This shows the amount of A. These amounts are relative values, with the results for young mice set to 100%. Each bar in the bar graph is the average value across multiple samples.

[0084] As shown in the figure, in all organs and all mt-tRNAs, ms in aged mice compared to young mice ２ It can be seen that the content of A has increased. Based on these results, ms as a mitochondrial oxidative stress marker in individuals ２ The usefulness of A has been proven.

[0085] Example 6. MS in each organization２ Total RNA was purified from the liver, skeletal muscle, brain, kidney, and heart of mice to investigate the tendency of A levels. ２ A was measured using a mass spectrometer. The measurement results are shown in Figure 13. The graph in Figure 13 shows the ms in the liver on the vertical axis. ２ A relative value was set, with the amount of A corrected by the amount of adenosine being set to 1.

[0086] The measurement results showed that in mice, the liver, skeletal muscle, brain, kidney, and heart were in that order of ms ２ The amount of A increased. In other words, in living organisms, ms is found in the heart muscle and kidneys, which are rich in mitochondria. ２ It was confirmed that A was present in large quantities.

[0087] This invention has great potential applications in detecting mitochondrial oxidative stress in cancer and aging, as well as in drug discovery and development, making it extremely valuable. For example, ms ２ Using the amount of A as an indicator, it is possible to screen for compounds that suppress mitochondrial oxidative stress, and its application to mitochondrial-related diseases is expected. ２ Using the amount of A as an indicator, it is also possible to screen for drugs that specifically induce mitochondrial oxidative stress, suggesting potential applications in anticancer drug development. Furthermore, ms ２ A can also be detected in urine, therefore, ms in urine ２ By analyzing A, it becomes possible to test for mitochondrial oxidative stress.

Claims

1. A method for evaluating the level of oxidative stress experienced by mitochondria, wherein (S1) 2-methylthioadenosine (ms) in the mitochondria ２ A) A step to quantify the amount, and (S2) the ms obtained in (S1) ２ A method comprising the step of relating the quantitative value of A to the level of oxidative stress.

2. The method according to claim 1, wherein in (S2), a predetermined reference value is compared with the quantitative value.

3. The method according to claim 2, wherein in (S2), the level of oxidative stress is determined to be high when the quantitative value is greater than the reference value.

4. (S3) Further comprising the step of quantifying all or more types of modified nucleosides in the mitochondria, wherein in (S2), the ms obtained in (S1) ２ Using the quantitative value of A, the ms for all or more of the modified nucleosides ２ The method according to claim 1, wherein the ratio of A is calculated and associated with the level of oxidative stress.

5. The method according to claim 4, wherein the modified nucleosides are all modifications of tRNA.

6. The method according to claim 5, wherein the tRNA is selected from at least one of the following: a tRNA corresponding to tryptophan (Trp), a tRNA corresponding to tyrosine (Tyr), a tRNA corresponding to phenylalanine (Phe), and a tRNA corresponding to serine (Ser).

7. A method for evaluating the level of oxidative stress experienced by a subject, comprising: (S1) 2-methylthioadenosine (ms) in a sample derived from the subject ２ A) A step to quantify the amount, and (S2) the ms obtained in (S1) ２ A method comprising the step of relating the quantitative value of A to the level of oxidative stress.

8. The method according to claim 7, wherein the subject of the test is animal cells or a test animal, and the sample derived from the subject of the test is the cell culture supernatant of the animal cells or a body fluid sample collected from the test animal.

9. The method according to claim 8, wherein the test animal is a human.

10. The method according to claim 8, wherein the bodily fluid sample is blood or urine.

11. (S3) Further comprising the step of quantifying all or more types of modified nucleosides in the sample, wherein in (S2), the ms obtained in (S1) ２ Using the quantitative value of A, the ms for all or more of the modified nucleosides ２ The method according to claim 7, wherein the ratio of A is calculated and associated with the level of oxidative stress.

12. The method according to claim 11, wherein the modified nucleoside is a modification of mitochondrial tRNA.

13. The method according to claim 12, wherein the mitochondrial tRNA is selected from at least one of the following: mitochondrial tRNA corresponding to tryptophan (Trp), mitochondrial tRNA corresponding to tyrosine (Tyr), mitochondrial tRNA corresponding to phenylalanine (Phe), and mitochondrial tRNA corresponding to serine (Ser).

14. (Comparison of reference value and quantitative value) The method according to claim 7 or 11, wherein in (S2), a predetermined reference value is compared with the quantitative value.

15. The method according to claim 14, wherein, in (S2), the level of oxidative stress is determined to be high when the quantitative value is greater than the reference value.

16. Step of quantifying ms ２ A in the sample derived from the control; (S4) ms ２ The step of associating the quantification value of A with the level of oxidative stress received by the control; and (S5) further including the step of comparing the level of oxidative stress of the subject obtained in (S2) with the level of oxidative stress of the control obtained in (S4), the method according to claim 7.

17. The method according to claim 16, wherein in (S5), if the level of oxidative stress obtained in (S2) is greater than the level of oxidative stress obtained in (S4), it is determined that the level of oxidative stress experienced by the subject is high.

18. The method according to claim 16, wherein the subject and the control are animal cells or test animals, and the sample derived from the subject and the sample derived from the control are the cell culture supernatant of the animal cells or a body fluid sample taken from the test animal.

19. The method according to claim 18, wherein the control is a different individual from the subject under test.

20. The method according to claim 18, wherein the control is a group of samples different from the subject being tested, and the quantitative value obtained in (S3) is the average value of each of the quantitative values ​​of the group of samples.

21. The method according to claim 18, wherein the control is the same individual as the subject under test, and the sampling conditions for the sample derived from the control are different from the sampling conditions for the sample derived from the subject under test.

22. (S4) ms in the control sample ２ Steps to quantify A; (S5) Steps to quantify all or more types of modified nucleosides in the sample; (S6) Using the quantitative values ​​obtained in (S4) and the quantitative values ​​obtained in (S5), the ms of all or more types of modified nucleosides in the control ２ The method according to claim 11, further comprising: (S7) calculating the ratio of A and relating it to the level of oxidative stress experienced by the control; and (S3) comparing the level of oxidative stress of the control obtained in (S6).

23. The method according to claim 22, wherein in (S7), if the level of oxidative stress obtained in (S3) is greater than the level of oxidative stress obtained in (S6), it is determined that the level of oxidative stress experienced by the subject is high.

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