Method for quantifying nicotinamide adenine dinucleotide (NAD +) or nicotinamide mononucleotide (NMN), and kit and filter paper for performing the method

Abstract

To provide a method for accurately quantifying NAD + or an NAD + precursor such as NMN using a DBS method.SOLUTION: A method for quantifying nicotinamide adenine dinucleotide (NAD +) or nicotinamide mononucleotide (NMN) in a test subject, the method comprising step 1 of bringing a filter paper impregnated with a radical inhibitor, to which blood derived from the test subject has been added, into contact with a solvent, and step 2 of quantifying the nicotinamide adenine dinucleotide (NAD +) or nicotinamide mononucleotide (NMN) in the solvent obtained in step 1.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for quantifying nicotinamide adenine dinucleotide (NAD+) or nicotinamide mononucleotide (NMN), as well as a kit and filter paper for carrying out the method. [Background technology]

[0002] Nicotinamide adenine dinucleotide (NAD+) is a coenzyme essential for energy production reactions. It has been reported that NAD+ levels decrease with age in rodents and humans (Non-Patent Document 1).

[0003] Nicotinamide mononucleotide (NMN) is also present in various biological species and, together with nicotinamide riboside (NR), is a precursor of NAD+, which is collectively known as the vitamin B3 group. It has also been reported that these NAD+ precursors can improve the pathology of aging-related diseases in rodents and other animals by increasing NAD+ in vivo (Non-Patent Document 2). In recent years, NAD+ precursors such as NMN have been sold as anti-aging supplements, and increasing NAD+ levels in vivo has attracted attention.

[0004] Therefore, it is expected that quantifying the levels of NAD+ and NAD+ precursors such as NMN in vivo will lead to understanding of pathological conditions and the degree of aging. Various methods for quantifying the levels of NAD+ and NMN, etc., have been investigated. For example, Non-Patent Document 3 reports that NAD+ can be detected by the dried blood spot method (DBS method). The DBS method involves dropping (also called spotting) blood from a test subject onto specialized filter paper, drying it, and then quantifying the concentration of a substance to be measured in the blood through an extraction procedure. The DBS method offers the following advantages: (1) the blood is used as is, eliminating the need for procedures such as separating plasma components from the blood; (2) a smaller volume of blood can be collected than conventional blood tests, allowing for self-collection; and (3) the use of filter paper facilitates transportation to testing facilities. For these reasons, the method is attracting attention and is currently being used in newborn mass screening, therapeutic drug monitoring (TDM) in remote locations, and other such applications. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Experimental gerontology,2020,134,110888 [Non-patent document 2] Biochemistry, Vol. 87, No. 2, pp. 239-244, 2015 [Non-patent document 3] Metabolites,2017,7,35 Summary of the Invention [Problem to be solved by the invention]

[0006] However, when attempting to quantify the above-mentioned NAD+ precursors such as NAD+ and NMN using the DBS method described in Non-Patent Document 3, etc., there is a problem that accurate quantification cannot be achieved. This is because, firstly, the degree of decomposition of NAD+ precursors such as NAD+ and NMN tends to vary significantly depending on their storage conditions, and special treatment such as rapid freezing is required before attempting to quantify them. Furthermore, the DBS method described in Non-Patent Document 3, etc., has poor storage stability after dripping onto filter paper, depending on the substance to be quantified, which can lead to inaccurate quantification.

[0007] An object of the present invention is to provide a method for accurately quantifying NAD+ or NAD+ precursors such as NMN using the DBS method. [Means for solving the problem]

[0008] The present invention has been made to solve the above problems and comprises the following configurations. [1] A method for quantifying nicotinamide adenine dinucleotide (NAD+) or nicotinamide mononucleotide (NMN) in a subject, the method comprising the following steps 1 and 2: (Step 1) contacting a filter paper impregnated with a radical inhibitor or a chaotropic agent to which blood derived from the subject has been added with a solvent; and (Step 2) A step 2 of quantifying the nicotinamide adenine dinucleotide (NAD+) or nicotinamide mononucleotide (NMN) in the solvent obtained by the step 1. [2] The method described in [1], which is a method for quantifying nicotinamide adenine dinucleotide (NAD+). [3] The method according to [1] or [2], wherein the filter paper is impregnated with a chaotropic agent. [4] The method according to [1] or [2], wherein the radical inhibitor comprises a surfactant. [5] The method according to [4], wherein the surfactant is an anionic surfactant. [6] [5] The method according to [5], wherein the anionic surfactant is sodium dodecyl sulfate. [7] The method according to [3], wherein the chaotropic agent comprises a guanidine salt. [8] [7] The method according to [7], wherein the guanidine salt is guanidine thiocyanate. [9] The method according to any one of [1] to [8], wherein the filter paper is made of cellulose.

[10] The method according to any one of [1] to [9], wherein the solvent is water and / or acetonitrile.

[11]

[10] The method according to

[10] , wherein the solvent is water.

[12] The method according to any one of [1] to

[11] , wherein the quantification in step 2 is carried out by a method using a mass spectrometer or a colorimetric method.

[13] A kit for carrying out the method according to any one of [1] to

[12] , comprising a blood collection device and filter paper impregnated with a radical inhibitor or a chaotropic agent.

[14] A filter paper impregnated with a radical inhibitor or a chaotropic agent for carrying out the method according to any one of [1] to

[12] . [Effects of the Invention]

[0009] According to the present invention, since NAD+ or NAD+ precursors such as NMN have good storage stability, it is possible to provide a method for accurately quantifying NAD+ or NAD+ precursors such as NMN using the DBS method. DETAILED DESCRIPTION OF THE INVENTION

[0010] <Method for quantifying nicotinamide adenine dinucleotide (NAD+) or nicotinamide mononucleotide (NMN)> The method of the present invention for quantifying nicotinamide adenine dinucleotide (NAD+) or nicotinamide mononucleotide (NMN) (hereinafter sometimes abbreviated as the quantification method of the present invention) comprises step 1 (hereinafter sometimes abbreviated as step 1 of the present invention) of contacting a solvent with filter paper impregnated with a radical inhibitor or a chaotropic agent to which blood from a test subject has been added, and step 2 (hereinafter sometimes abbreviated as step 2 of the present invention) of quantifying the NAD+ or NMN in the solvent obtained by step 1. The quantitative determination method of the present invention will be described in detail below.

[0011] [Nicotinamide adenine dinucleotide (NAD+)] As described above, nicotinamide adenine dinucleotide (NAD+) in the quantification method of the present invention is a coenzyme essential for energy production reactions, and is also known as an electron transport system in all eukaryotes, archaea, eubacteria, etc. Nicotinamide adenine dinucleotide can exist in two forms in vivo: oxidized and reduced. In the present invention, however, oxidized nicotinamide adenine dinucleotide (i.e., NAD+) is referred to. In addition, the old names for NAD+, such as diphosphopyridine nucleotide, coenzyme I, coenzyme I, and codehydrogenase I, are also included in the nicotinamide adenine dinucleotide of the present invention.

[0012] [Nicotinamide mononucleotide (NMN)] As described above, nicotinamide mononucleotide (NMN) in the quantification method of the present invention is a NAD+ precursor that is present in various biological species and is collectively known as the vitamin B3 group. NMN is also known to have various functions, such as activating mitochondria and the sirtuin gene, a so-called longevity gene. The structure of NMN is as follows:

[0013] [ka]

[0014] As described above, the substance to be quantified in the quantification method of the present invention is NAD+ and / or NMN, and these can be accurately quantified by the quantification method of the present invention. Among them, from the viewpoint of the accuracy of quantification, NAD+ is more preferable as the substance to be quantified in the quantification method of the present invention. It goes without saying that the above-mentioned "quantification" in the present invention refers to calculating the concentration, mass, molar amount, etc. of NAD+ or NMN in the blood derived from a subject, as described below.

[0015] [Subjects] The subject in the quantification method of the present invention may be a mammal such as a human, monkey, mouse, rat, dog, cat, pig, or rabbit, with a human, monkey, mouse, or rat being preferred, and a human being being more preferred. Examples of such a human include healthy individuals, patients suffering from a disease associated with an increase or decrease in NAD+ and / or NMN, and individuals suspected of suffering from a disease associated with an increase or decrease in NAD+ and / or NMN. Specific examples of such diseases include age-related diseases.

[0016] [Process 1] Step 1 according to the present invention is a step of contacting a solvent with a filter paper impregnated with a radical inhibitor or a chaotropic agent to which blood derived from a test subject has been added. Step 1 will be described in detail below.

[0017] The blood in step 1 of the present invention refers to blood derived from the above-mentioned subject, and specific examples include whole blood, blood cells, plasma, serum, etc., with whole blood being preferred among these. Furthermore, the method for collecting blood from a subject may be any method commonly used in this field, such as a method using an autologous blood collection device. Examples of such an autologous blood collection device include the MBS Micro Blood Collection Kit (Micro Blood Science, Inc.). When the subject is a human, blood can be collected from the tips of the human's fingers or toes using the autologous blood collection device.

[0018] The filter paper in step 1 of the present invention is a target to which the blood derived from the subject is added, and is impregnated with a radical inhibitor or a chaotropic agent. Among them, from the viewpoint of the accuracy of quantification, filter paper impregnated with a chaotropic agent is more preferable.

[0019] The radical inhibitor is a substance that destroys protein-protein interactions and denatures proteins, and specifically includes, for example, surfactants, buffer solutions, and the like.

[0020] Examples of such surfactants include anionic surfactants, cationic surfactants, amphoteric surfactants, and nonionic surfactants, among which anionic surfactants are preferred. Examples of such anionic surfactants include sodium dodecyl sulfate (sometimes called sodium lauryl sulfate), lithium dodecyl sulfate, sodium cholate, and sodium deoxycholate, among which sodium dodecyl sulfate is preferred.

[0021] Examples of such buffer solutions include Tris buffer solutions such as tris(hydroxymethyl)aminomethane buffer solution, phosphate buffer solution, citrate buffer solution, glycine buffer solution, 3-morpholinopropanesulfonic acid buffer solution, acetate buffer solution, veronal buffer solution, borate buffer solution, and Good's buffer solution such as carbonate buffer solution, and among these, Tris buffer solution is preferred.

[0022] The chaotropic agent is a substance that reduces the interaction between water molecules, thereby destabilizing the structure of proteins, and contains at least a guanidine salt.

[0023] Examples of such guanidine salts include guanidine thiocyanate, guanidine hydrochloride, guanidine nitrate, and guanidine sulfate, with guanidine thiocyanate being preferred among these.

[0024] The method for impregnating the filter paper with the radical inhibitor and chaotropic agent may be any method commonly used in this field, such as vacuum impregnation or vacuum pressure impregnation, in which the filter paper is immersed in a solution containing the radical inhibitor or chaotropic agent for a certain period of time, or a method in which the radical inhibitor or chaotropic agent is directly applied or coated on the filter paper.

[0025] Examples of the main component (base) of the filter paper in step 1 of the present invention include cellulose, cotton, pulp, etc., with cellulose being preferred among these. A method for obtaining such cellulose-based filter paper may be any method commonly used in this field. For example, such a method involves mixing water and cellulose, removing water from the mixture using a paper machine, drying the mixture, and then producing filter paper using an automatic filter paper punching machine. Reference may also be made to the method described in JP-B 08-032995.

[0026] The thickness of the filter paper in step 1 according to the present invention may be in the range of 500 μm to 800 μm, and preferably in the range of 550 μm to 750 μm.

[0027] The weight of the filter paper in step 1 according to the present invention is, for example, the weight per unit area (mg / cm 2 ) is 5mg / cm 2 ~50mg / cm 2 Preferably, 15 mg / cm 2 ~35mg / cm 2 is. In particular, when the filter paper in step 1 according to the present invention is impregnated with a radical inhibitor, the weight of the filter paper is, for example, the weight per unit area (mg / cm 2 ) is 5mg / cm 2 ~50mg / cm 2 Preferably, 15 mg / cm 2 ~20mg / cm 2 In addition, when the filter paper in step 1 according to the present invention is impregnated with a chaotropic agent, the weight of the filter paper is, for example, the weight per unit area (mg / cm 2 ) is 5mg / cm 2 ~50mg / cm 2 Preferably, 30 mg / cm 2 ~35mg / cm 2 is. When measuring the weight per unit area, for example, a Sartorius electronic balance (MSA225S-100-DI) can be used.

[0028] Furthermore, other parameters of the filter paper in step 1 according to the present invention, such as the length and width of the filter paper, the shape, and the impregnation concentration of the radical inhibitor and the chaotropic agent, can be set arbitrarily in consideration of the actual conditions of use, etc. In particular, such a shape is not particularly limited, but examples thereof include a circle, a square, etc.

[0029] Any filter paper having the above-described properties may be used in step 1 of the present invention. Examples of filter paper that satisfy these properties include Whatman FTA DMPK-A Card (GE Healthcare), QIAcard FTA DMPK-A Card (QIAGEN), Whatman FTA DMPK-B Card (GE Healthcare), and QIAcard FTA DMPK-B Card (QIAGEN), and these may also be used. As described in the Examples below, the Whatman FTA DMPK-A Card (GE Healthcare) and QIAcard FTA DMPK-A Card (QIAGEN) are impregnated with a radical inhibitor (sodium dodecyl sulfate), and the main component of the filter paper is cellulose. Furthermore, as described in the Examples below, the Whatman FTA DMPK-B Card (GE Healthcare) and QIAcard FTA DMPK-B Card (QIAGEN) are impregnated with a chaotropic agent (guanidine thiocyanate), and the main component of the filter paper is cellulose.

[0030] The solvent used in step 1 of the present invention is used to extract components containing NAD+ and NMN from the filter paper containing the blood of the subject. Specific examples include water, acetonitrile, a mixture of water and acetonitrile, and alcohols such as ethanol. Of these, water or a mixture of water and acetonitrile is preferred, with water being more preferred. Examples of such water include ion-exchanged water, deionized water, pure water, ultrapure water, and distilled water. Of these, ultrapure water is preferred. When using a mixture of water and acetonitrile, the ratio of the two components may range from 80% water:20% acetonitrile to 20% water:80%, preferably 80% water:20% acetonitrile to 40% water:60% acetonitrile. When preparing the solvent, an internal standard substance corresponding to the substance to be quantified may be added to the solvent.

[0031] In step 1 of the present invention, the method for contacting the filter paper with the solvent may be any method commonly used in this field, and specifically, for example, a method in which an amount of solvent sufficient to fully immerse the filter paper is added to the filter paper and mixed in. The amount is, for example, 100 μL to 1000 μL, and can be appropriately adjusted depending on the conditions of the filter paper and the performance of the equipment used for quantification, which will be described later. More specifically, the above method may be carried out, for example, as follows. First, a certain amount of blood from the subject is collected in advance, and 1 μL to 10 μL of the blood is spotted at a predetermined location on filter paper. The spot on the filter paper where the blood was spotted is then cut out and administered into a tube such as a 2 mL tube. Next, 100 μL to 1000 μL of the solvent is added to the tube. After adding the solvent, the mixture may be mixed at room temperature for a certain period of time (approximately 10 to 60 minutes) using a mixer such as a micromixer, if necessary. Furthermore, when carrying out the above-mentioned method, if necessary, reference may be made to techniques described in Non-Patent Document 1, Non-Patent Document 3, JP-T-2015-508493A, JP-T-2018-530766A, JP-T-2014-503197A, JP-T-2011-506922A, JP-T-2004-505277A, and "Uniformity of Dry Matrix Spots Application Note" (Agilent Technologies, Inc.).

[0032] Thus, in step 1 of the present invention, by contacting a filter paper containing blood from a test subject with a solvent, components including NAD+ and NMN can be extracted from the filter paper containing blood from the test subject.

[0033] Furthermore, after carrying out step 1 of the present invention and before carrying out step 2 of the present invention described below, a step of adding a certain amount of organic solvent may be carried out to stabilize the substance to be quantified in the solvent obtained by step 1 of the present invention. Examples of such organic solvents include acetonitrile, methanol, and ethanol, with acetonitrile being preferred. The amount of organic solvent added may be, for example, twice the amount of solvent 1 used in step 1 of the present invention.

[0034] [Process 2] Step 2 according to the present invention is a step of quantifying NAD+ or NMN in the solvent obtained by Step 1 according to the present invention. Step 2 will be described in detail below.

[0035] The method for quantifying NAD+ or NMN in step 2 of the present invention refers to a method for quantifying NAD+ or NMN present in the solvent obtained by the operation of step 1 of the present invention, and any method commonly used in this field may be used, such as a method using a mass spectrometer, a colorimetric method, or a fluorimetric method.

[0036] Examples of methods using such mass spectrometers include methods using a liquid chromatograph mass spectrometer (LC-MS / MS), a gas chromatograph mass spectrometer (GC / MS), a capillary electrophoresis mass spectrometer (CE / MS), etc. These methods may involve, for example, separating components in a biological sample (such as blood) in a separation section having a chromatograph such as a liquid chromatograph, ionizing the separated components in a mass analysis section, and then separating them by mass-to-charge ratio (m / z). More specific techniques may be performed with reference to the accompanying documentation for the device or to the techniques described in, for example, WO2014 / 038524, WO2017 / 19588, WO2018 / 034346, etc.

[0037] The colorimetric method is a method in which a target substance is quantified using a microplate reader such as a microplate spectrophotometer based on the degree of color development when a biological sample (blood, etc.) is reacted with a coloring agent. Such a color former may be any that is commonly used in this field, and specific examples thereof include orthocresolphthalein complexone (OCPC), arsenazo-III, chlorophosphonazo-III, eriochrome black T, glyoxal-bis(2-hydroxyanil), NN (2-hydroxy-1-(2-hydroxy-4'-sulfo-1'-naphthylazo)-3-naphthoic acid), hydroxynaphthol blue, etc. Furthermore, the specific technique for the colorimetric method may be performed according to a method commonly used in this field, and may be performed with reference to, for example, JP-A 2003-262629. When quantification is performed by the colorimetric method, commercially available kits may be used. Specific examples of such kits include the NAD / NADH Assay Kit-WST (Dojindo Laboratories, Inc.) and the NAD / NADH Assay Kit (Colorimetric) (CELL BIOLABS).

[0038] Such a fluorescence method is a method in which a biological sample (such as blood) containing a target substance is irradiated with light, and the light generated when it is excited and returns to the ground state is detected. Specifically, it is a method for quantifying the target substance using a plate reader such as a fluorescence plate reader based on the degree of light generated when the excited electrons return to the ground state. Also, regarding the specific method of the above fluorescence method, it may be carried out according to the methods usually used in this field. For example, it may be carried out by referring to JP-A-2012-202742, JP-A-2009-276162, etc. In addition, when quantification is performed by the above fluorescence method, a commercially available kit or the like may be used. Specific examples of such kits include, for example, NAD+ / NADH Assay Kit (Fluorometric) (CELL BIOLABS).

[0039] <Kit for implementing a method for quantifying NAD+ or NMN> The kit for implementing the method for quantifying NAD+ or NMN of the present invention (hereinafter, may be abbreviated as the kit of the present invention) includes a blood collection device and a filter paper impregnated with a radical inhibitor or a chaotropic agent. Hereinafter, the details of the kit of the present invention will be described.

[0040] Such a blood collection device is for collecting blood from a test subject and is as described in <the quantification method of the present invention>. The same applies to specific examples, preferred examples, etc.

[0041] Such a filter paper is impregnated with a radical inhibitor or a chaotropic agent and is as described in <the quantification method of the present invention>. The same applies to specific examples, preferred examples, etc.

[0042] The kit of the present invention includes at least the above blood collection device and a filter paper impregnated with a radical inhibitor or a chaotropic agent, but may include an instruction manual, a container for storing the filter paper, etc. as necessary.

[0043] Such instructions essentially describe the principles and operating procedures of the quantification method of the present invention through text, drawings, etc., and may take the form of an instruction manual, package insert, pamphlet, leaflet, etc.

[0044] Such a container may be any container capable of storing filter paper, preferably one having the ability to absorb moisture. Specific examples of such containers include standard moisture-absorbing bags. Specific examples of such standard moisture-absorbing bags include Absorbent Kun (Maruto Sangyo Co., Ltd.) (registered trademark).

[0045] The kit of the present invention allows the quantification method of the present invention to be carried out more simply, quickly, and accurately. Furthermore, the kit of the present invention allows a subject (e.g., a human) to prepare filter paper containing blood from the subject at a specific location such as their home, mail the filter paper to an institution (e.g., a testing institution or medical institution equipped with a mass spectrometer such as LC-MS / MS), where NAD+ and NMN are quantified, and, if necessary, the quantification results are fed back to the subject, making it possible to provide an integrated service. As described above and in the examples below, the filter paper included in the kit of the present invention provides excellent storage stability for NAD+ and NMN, making the kit of the present invention fully applicable to the above-mentioned services that require mailing of filter paper.

[0046] <Application of the quantification method of the present invention> The above-mentioned quantification method of the present invention can be applied to various fields. For example, as mentioned above, age-related diseases are known to be diseases related to the increase or decrease of NAD+ and NMN. Therefore, it is considered possible to carry out analyses (e.g., determination, diagnosis, etc.) utilizing the correlation between the quantification results obtained by the quantification method of the present invention and the above-mentioned diseases. [Example]

[0047] The present invention will be explained in more detail below with reference to examples, but the scope of the present invention is not limited to these examples.

[0048] <Example 1: Examination of optimization of extract> We searched for the optimal solvent (i.e., this corresponds to the solvent in step 1 of the present invention; hereinafter, this may be abbreviated as the extraction solution) to be used to extract components containing NAD+ and NMN from a specific filter paper to which blood from the test subject had been added. To further explain the purpose of this Example 1, filter paper was not used in this Example 1 because the focus was on optimizing the extract. The specific method is as follows: various candidate extracts were added to blood, and the resulting solution was used as is to quantify NAD+ and NMN using a mass spectrometer.

[0049] [Experimental Method] Three 6-week-old male SD rats underwent laparotomy under isoflurane inhalation anesthesia, and whole blood was collected from the abdominal aorta. 200 μL of the collected blood (i.e., whole blood) was dispensed into 1.5 mL microtubes and centrifuged (13,200 × g, 5 minutes, 4°C). The supernatant was removed, and the centrifugal residue was used as the blood cell fraction. Then, a total of six types of extracts were prepared by mixing ultrapure water (Milli-Q integral 5 system / Merck Ltd.) and acetonitrile (Thermo Fisher Scientific) in ratios of 100:0, 80:20, 60:40, 40:60, 20:80, and 0:100, respectively. 5 μL of individual blood or blood cells, 10 μL of a mixed aqueous solution of internal standards (NAD-d4 and NMN-d5 / Toronto Research Chemicals, Inc.), and 295 μL of extract were added to a 1.5 mL microtube and vortexed. The mixture was then mixed at room temperature for 30 minutes using a micromixer (TOMY MICRO TUBE MIXER MT-400, stirring speed 5). 100 μL of the mixed blood or blood cell extract was dispensed into microtubes, 200 μL of acetonitrile was added, vortexed, and centrifuged (24,250 × g, 10 minutes, 4°C). After centrifugation, 100 μL of the supernatant was dispensed into a vial, and 0 μL to 50 μL of the mixed aqueous solution of internal standards, diluted according to the extract used, was added and mixed to a final organic solvent content of 67%. NAD+ and NMN were then measured by LC-MS / MS under the following analytical conditions.

[0050] [LC-MS / MS measurement conditions]

[0051] [Table 1]

[0052] [Table 2]

[0053] [Table 3]

[0054] [Experimental Results] The results of the above experiments are shown in Table 4.

[0055] [Table 4]

[0056] As can be seen from Table 4, when six different extracts with different acetonitrile content ratios were used, the highest concentrations of NAD+ and NMN in blood were found when only water was used as the extract (i.e., 100% water and 0% acetonitrile). Furthermore, when the acetonitrile content of the extract was adjusted to about 60%, the concentrations of NAD+ and NMN were lower than when the acetonitrile content was 0% (i.e., water only), but a certain level of NAD+ and NMN was still observed. However, when the acetonitrile content was adjusted to 80% or more, the concentrations of NAD+ and NMN were found to be significantly reduced. This trend in results was also observed when blood cells were used. The results of Example 1 showed that in quantifying NAD+ and NMN in blood, the solvent that can most efficiently extract them from blood is water. Considering these results, it is assumed that the NAD+ and NMN to be quantified are present in blood cells, and that the osmotic pressure of water destroys the blood cell components, allowing for their efficient detection.

[0057] Example 2: Optimization of filter paper based on refrigerated storage stability Regarding the filter paper to which the blood from the test subject is added (i.e., the filter paper in step 1 of the present invention), we searched for the optimal filter paper for quantifying NAD+ and NMN when the filter paper to which the blood from the test subject is added is stored in a refrigerator for a certain period of time. The specific experimental method is as follows.

[0058] [Experimental Method] Approximately 120 μL of blood (the equivalent of two collection containers) was collected from five subjects (adult men and women) per test using the MBS Micro Blood Sampling Test Kit (Micro Blood Science, Inc.) via self-collection, and the sample was left on ice until processing. The subjects fasted on the morning of the collection day, and the sample was collected around 9:00 AM. The test was conducted four times in total, and evaluation was carried out on a total of 19 subjects, excluding one subject who was absent due to insufficient blood volume. The collected blood was collected in 1.5 mL microtubes for each subject and mixed by gentle pipetting. Five μL of blood was spotted (dropped / added) at multiple locations on four types of filter paper (Whatman FTA DMPK-A Card / GE Healthcare, QIAcard FTA DMPK-B Card / QIAGEN, QIAcard FTA DMPK-C Card / QIAGEN, Whatman 903 protein saver card / Cytiva), sealed in moisture-wicking bags (Kyushukun® / Maruto Sangyo Co., Ltd.), and refrigerated. After storage for 2 days, 2 weeks (14 or 15 days), or 1 month (30 days), the spotted area was excised and collected in a 2 mL microtube. 300 μL of a mixed aqueous solution of internal standards (NAD-d4 and NMN-d4 / Toronto Research Chemicals, Inc.) was added and vortexed, followed by mixing at room temperature for 30 minutes using a micromixer (TOMY MICRO TUBE MIXER MT-400, stirring speed 5). 150 μL of the blood extract solution was dispensed into microtubes, 300 μL of acetonitrile was added, vortexed, and centrifuged (24,250 × g, 10 minutes, 4°C). 40 μL of the supernatant after centrifugation was dispensed into vials and analyzed by LC-MS / MS. Separately, 5 μL of blood was collected into a 2 mL microtube (without a filter paper spot) as a sample for initial values. 300 μL of a mixed aqueous solution of internal standards (NAD-d4 and NMN-d4 / Toronto Research Chemicals, Inc.) was added and vortexed, followed by mixing at room temperature for 30 minutes using a micromixer (TOMY MICRO TUBE MIXER MT-400, stirring speed 5). 150 μL of the blood extract solution was dispensed into microtubes, 300 μL of acetonitrile was added, vortexed, and then centrifuged (24,250 × g, 10 minutes, 4°C). 40 μL of the supernatant after centrifugation was dispensed into vials and analyzed by LC-MS / MS. The Whatman FTA DMPK-A Card may be abbreviated as DMPK-A card hereinafter. The QIAcard FTA DMPK-B Card may be abbreviated as DMPK-B card hereinafter. The QIAcard FTA DMPK-C Card may be abbreviated as DMPK-C card hereinafter. Furthermore, the Whatman 903 protein saver card may be abbreviated as 903 card hereinafter.

[0059] [LC-MS / MS measurement conditions]

[0060] [Table 5]

[0061] [Table 6]

[0062] [Table 7]

[0063] [Experimental Results] The results of the above experiments are shown in Tables 8 to 11.

[0064] [Table 8]

[0065] [Table 9]

[0066] [Table 10]

[0067] [Table 11]

[0068] As is clear from Tables 8 and 9, the NAD+ concentrations after 2 days, 2 weeks, and 1 month of storage using the DMPK-A card were 81.5%, 79.1%, and 71.8% of the initial value (day 0), respectively, demonstrating good refrigerated storage stability.Furthermore, the NAD+ concentrations after 2 days, 2 weeks, and 1 month of storage using the DMPK-B card were 80.7%, 87.8%, and 79.8% of the initial value (day 0), respectively, demonstrating even better refrigerated storage stability than when using the DMPK-A card. On the other hand, as is clear from Table 10 above, when the DMPK-C card was used, the NAD+ concentrations after 2 days, 2 weeks, and 1 month of storage were 70.1%, 66.6%, and 54.3%, respectively, of the initial value (day 0). As is clear from Table 11 above, when the 903 card was used, the NAD+ concentrations after 2 days, 2 weeks, and 1 month of storage were 74.1%, 77.2%, and 82.6%, respectively, of the initial value (day 0). These results demonstrate that, with regard to NAD+, the DMPK-A card and B card (especially the DMPK-B card) were superior on average to the DMPK-C card and the 903 card in terms of refrigerated storage stability throughout each period.

[0069] Furthermore, as is clear from Table 8 above, when using the DMPK-A card, the NMN concentration after 2 days, 2 weeks, and 1 month of storage was 73.1%, 80.6%, and 99.9%, respectively, of the initial value (day 0), demonstrating good refrigerated storage stability. Furthermore, as is clear from Table 9 above, when using the DMPK-B card, the NMN concentration after 2 days, 2 weeks, and 1 month of storage was 95.4%, 93.6%, and 78.6%, respectively, of the initial value (day 0), demonstrating good storage stability similar to that of the DMPK-A card. On the other hand, when the DMPK-C card was used, the NMN concentrations after 2 days, 2 weeks, and 1 month of storage were 68.4%, 52.9%, and NC, respectively, of the initial value (day 0), as is clear from Table 10 above, and when the 903 card was used, the NMN concentrations after 2 days, 2 weeks, and 1 month of storage were 60.1%, 64.5%, and 56.3%, respectively, of the initial value (day 0), as is clear from Table 11 above. These results demonstrate that the DMPK-A card and B card also have superior refrigerated storage stability for NMN compared to the DMPK-C card and 903 card.

[0070] Considering the above results, the DMPK-A card and B card were treated with chemical agents, whereas the DMPK-C card and 903 card were not treated with chemical agents (Am. J. Trop. Med. Hyg., 99(2), 2018, pp. 256-265, or Bioanalysis, 2013, 5, 2613-30, etc.). Therefore, it is presumed that the presence or absence of treatment with the chemical agent and the type of treatment contribute to the stability of the product when stored in a refrigerator. The chemical agent mentioned above is a radical inhibitor (sodium dodecyl sulfate) on the DMPK-A card, and a chaotropic agent (guanidine thiocyanate) on the DMPK-B card (Am. J. Trop. Med. Hyg., 99(2), 2018, pp. 256-265, or Bioanalysis, 2013, 5, 2613-30, etc.).

[0071] <Example 3: Optimization of filter paper based on room temperature storage stability> Regarding the filter paper to which the blood from the test subject is added (i.e., the filter paper in step 1 of the present invention), we searched for the optimal filter paper for quantifying NAD+ and NMN when the filter paper to which the blood from the test subject is added is stored at room temperature. The specific experimental method is as follows.

[0072] [Experimental Method] A portion of the blood spot filter paper prepared in Example 2 was sealed in a standard moisture-absorbing bag (Kunshukun (registered trademark) / Maruto Sangyo Co., Ltd.) and stored at room temperature. After storage at room temperature for 2 days, 1 week (7 days), or 2 weeks (15 days), the blood spotted area was excised and collected in a 2 mL microtube. After the same treatment as in Example 2, the sample was measured by LC-MS / MS under the same conditions as in Example 2.

[0073] [Experimental Results] The results of the above experiments are shown in Tables 12 to 15.

[0074] [Table 12]

[0075] [Table 13]

[0076] [Table 14]

[0077] [Table 15]

[0078] As is clear from Table 12 above, when the DMPK-A card was used, the NAD+ concentrations after 2 days and 2 weeks of storage were 71.6% and 51.6% of the initial value (day 0), respectively, demonstrating good storage stability. Furthermore, as is clear from Table 13 above, when the DMPK-B card was used, the NAD+ concentrations after 2 days, 1 week, and 2 weeks of storage were 82.0%, 87.2%, and 67.6% of the initial value (day 0), respectively, demonstrating even better room temperature storage stability than when the DMPK-A card was used. On the other hand, as is clear from Table 14 above, when the DMPK-C card was used, the NAD+ concentrations after 2 days and 2 weeks of storage were 55.9% and 50.2%, respectively, of the initial value (day 0), and as is clear from Table 15 above, when the 903 card was used, the NAD+ concentrations after 2 days and 2 weeks of storage were 66.2% and 50.3%, respectively, of the initial value (day 0). These results demonstrate that, with regard to NAD+, the DMPK-A card and B card are superior to the DMPK-C card and 903 card in terms of room temperature storage stability.

[0079] Furthermore, as is clear from Tables 12 and 13 above, although there was some fluctuation in the values ​​for NMN, it was shown that when DMPK-A and B cards were used, NMN could be quantified without significantly impairing storage stability. On the other hand, as is clear from Table 14 above, when the DMPK-C card was used, the NMN concentration after 2 days and 2 weeks of storage could not be quantified. Furthermore, as is clear from Table 15 above, when the 903 card was used, the NMN concentration after 2 days of storage was 79.8% of the initial value (0 days), but the NMN concentration after 2 weeks of storage could not be quantified. These results demonstrate that the DMPK-A card and B card are superior to the DMPK-C card and 903 card in terms of room temperature storage stability for NMN as well.

[0080] Considering the above results, it is presumed that, as in Example 2, the presence or absence of treatment with chemical agents and the type of treatment are factors.

[0081] Example 4: Optimization of filter paper based on extraction efficiency Regarding the filter paper to which the blood from the test subject is added (i.e., the filter paper in step 1 of the present invention), we searched for the optimal filter paper for quantifying NAD+ and NMN from the viewpoint of the extraction efficiency of NAD+ and NMN from the filter paper to which the blood from the test subject is added. The specific experimental method is as follows.

[0082] [Experimental Method] The extraction efficiency of NAD+ and NMN was evaluated with and without filter paper. The preparation method for samples without filter paper was the same as in Example 2. Meanwhile, the preparation method for samples with filter paper was the same as that in Example 2. Specifically, 5 μL of blood was spotted onto each of four types of filter paper (Whatman FTA DMPK-A Card / GE Healthcare, QIAcard FTA DMPK-B Card / QIAGEN, QIAcard FTA DMPK-C Card / QIAGEN, Whatman 903 protein saver card / Cytiva), and then subjected to various treatments similar to those in Example 2. After that, the samples were measured by LC-MS / MS under the same conditions as in Example 2, without storing them in a refrigerator or at room temperature for a certain period as in Examples 2 and 3.

[0083] [Experimental Results] The results of the above experiments are shown in Tables 16 and 17.

[0084] [Table 16]

[0085] [Table 17]

[0086] As is clear from Table 16 above, when DMPK-A and B cards were used, the extraction efficiency of NAD+ from the filter paper (calculated based on the concentration value without filter paper) was 95.1% and 90.9%, respectively, indicating good extraction efficiency for NAD+. On the other hand, as is clear from Table 17 above, when the DMPK-C card and 903 card were used, the extraction efficiency of NAD+ from the filter paper was 72.9% and 77.2%, respectively. These results demonstrate that the DMPK-A and B cards are superior in terms of extraction efficiency for NAD+ compared to the DMPK-C card and 903 card.

[0087] Furthermore, as is clear from Table 16 above, when DMPK-A and B cards were used, the extraction efficiency of NMN from the filter paper (calculated based on the concentration value without filter paper) was 125.6% and 87.7%, respectively, demonstrating good extraction efficiency. On the other hand, as is clear from Table 17 above, when the DMPK-C card and 903 card were used, the extraction efficiency of the filter paper was 87.9% and 73.2%, respectively. These results show that the DMPK-A and B cards are superior in terms of extraction efficiency for NMN as well, compared to the DMPK-C card and 903 card.

[0088] Further commenting on the results of Examples 2 to 4 above, DBS methods such as those of the present invention are generally performed on the premise that autologous blood collection is involved. Therefore, for example, the step of adding blood from a test subject to filter paper and the step of quantifying the target substance contained in the filter paper are often performed in physically or temporally separate stages. More specifically, the step of adding blood to the filter paper is performed, for example, by the test subject (e.g., a human) at home. The filter paper is then transported to an institution (e.g., a testing institution, medical institution, etc.) equipped with a mass spectrometer such as LC-MS / MS, where the step of quantifying the target substance is performed. Therefore, when performing the DBS method, the filter paper must be stored at room temperature or in a refrigerator for a certain period of time (e.g., several days to a month). Furthermore, it is also necessary to prevent the degradation of the target substance during storage. Therefore, the filter paper used in the DBS method is required to have excellent storage stability for the target substance. From the above Examples 2 to 4, it can be said that the DMPK-A card and B card (i.e., corresponding to the filter paper in step 1 of the present invention) were shown to be able to satisfy the above properties. In other words, it was found that they are optimal for the quantification method of NAD+ and NMN using the DBS method.

[0089] <Example 5: Examination of storage stability of post-extraction solution> The storage stability of the solvent (extraction solution) after contacting the filter paper containing blood from the test subject with the solvent (hereinafter sometimes abbreviated as post-extraction solution) was examined. The specific method is as follows:

[0090] [Experimental Method] A portion of the blood extract solution (100% water) obtained in Example 2 after filter paper storage (refrigerated for 15 days) and a portion of the solution for LC-MS / MS measurement (water containing 67% acetonitrile) were each frozen and stored. After 71 days of frozen storage, the former was mixed with twice the amount of acetonitrile, and the supernatant was centrifuged (24,250 × g, 10 minutes, 4°C). The latter was centrifuged (24,250 × g, 10 minutes, 4°C) and the supernatant was measured by LC-MS / MS under the same conditions as in Example 2.

[0091] [Experimental Results] The results of the above experiments are shown in Tables 18 to 21.

[0092] [Table 18]

[0093] [Table 19]

[0094] [Table 20]

[0095] [Table 21]

[0096] As is clear from Tables 18 and 19, when the extracts from each filter paper were stored in the extract form (100% water), the storage stability of NAD+ for the DMPK-B card was 93.1%, demonstrating good storage stability. The extracts from the DMPK-A, C, and 903 cards were 145.8%, 200.6%, and 266.3%, respectively, all of which were significantly higher than the initial values. On the other hand, as is clear from Tables 20 and 21, the storage stability of NAD+ when the extracts from each filter paper were stored in the form of a solution to which acetonitrile was added was 89.6%, 73.6%, and 92.1% for DMPK-A, C, and 903 cards, respectively, which was an improvement from the value when the extract was in the extract state (100% water).

[0097] Considering the above results, it is inferred that the DMPK-B card is stable even in 100% water because NAD+ is sufficiently inactivated at the time of extraction. On the other hand, for the DMPK-A, C, and 903 cards, NAD+ is not sufficiently inactivated during extraction, and when stored in 100% water, there is a tendency for the values ​​to fluctuate significantly compared to the initial value due to the progression of the enzyme reaction, etc. Therefore, it is inferred that inactivating these cards by adding acetonitrile will make them stable when stored.

[0098] These results suggest that the DMPK-B card eliminates the need for adding organic solvents such as acetonitrile to the extracted solution, which is typically used in this field to stabilize the solution during storage. This simplifies NAD+ quantification. Furthermore, because the addition of organic solvents such as acetonitrile is no longer necessary, it suggests the possibility of using more convenient methods, such as colorimetric and fluorimetric methods, to quantify NAD+ in the extracted solution. This is because the addition of organic solvents such as acetonitrile reduces the NAD+ concentration in the sample, reducing sensitivity. Furthermore, the compatibility of acetonitrile with surfactants and other reagents included in colorimetric and fluorimetric assay kits makes these methods unfeasible. [Industrial Applicability]

[0099] The present invention can be used in industry to quantify NAD+ or NAD+ precursors such as NMN using the DBS method.

Claims

1. A method for quantifying nicotinamide adenine dinucleotide (NAD+) or nicotinamide mononucleotide (NMN) in a subject, the method comprising the following steps 1 and 2: (Step 1) contacting a filter paper impregnated with a radical inhibitor to which blood derived from the subject has been added with a solvent; and (Step 2) A step 2 of quantifying the nicotinamide adenine dinucleotide (NAD+) or nicotinamide mononucleotide (NMN) in the solvent obtained by the step 1.

2. The method of claim 1, which is a method for quantifying nicotinamide adenine dinucleotide (NAD+).

3. The method according to claim 1 or 2, wherein the radical inhibitor comprises a surfactant.

4. The method of claim 3 , wherein the surfactant is an anionic surfactant.

5. The method of claim 4, wherein the anionic surfactant is sodium dodecyl sulfate.

6. The method according to any one of claims 1 to 5, wherein the filter paper is made of cellulose.

7. The method according to any one of claims 1 to 6, wherein the solvent is water and / or acetonitrile.

8. The method of claim 7 wherein the solvent is water.

9. The method according to any one of claims 1 to 8, wherein the quantification in step 2 is carried out by a method using a mass spectrometer or a colorimetric method.

10. A kit for carrying out the method according to any one of claims 1 to 9, comprising a blood collection device and a filter paper impregnated with a radical inhibitor.

11. A filter paper impregnated with a radical inhibitor for carrying out the method according to any one of claims 1 to 10.

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