Method for stabilizing nicotinamide adenine dinucleotide and composition for stabilization

Abstract

Provided are a method and composition for stabilising nicotinamide adenine dinucleotide (NAD) in a biosample.　A sample derived from a subject is brought into contact with nicotinamide and / or nicotinamide derivative to stabilise NAD in the sample.

Description

[Technical Field]

[0001] The present invention relates to a method and composition for stabilizing nicotinamide adenine dinucleotide, and a method and kit for measuring nicotinamide adenine dinucleotide. [Background technology]

[0002] Nicotinamide adenine dinucleotide (NAD) is a pyridine nucleotide that functions as an electron carrier in the cellular metabolism of aerobic respiration in many of the main redox reactions in the body, and plays an important role in maintaining the balance of redox homeostasis. As shown in Non-Patent Documents 1 and 2, it is known that NAD in human tissues and plasma decreases with age. Patent Document 1 also describes the use of blood NAD as an index for determining the degree of aging in humans. + It is disclosed that amounts of metabolites such as the above can be used.

[0003] Non-Patent Document 3 discloses a simple quantification method using a microplate reader as a method for measuring NAD concentrations in biological samples. It is described that in this method, blood samples for NAD measurement are stable for 5 hours when collected from a subject, suspended in an extraction buffer solution, and cooled on ice. In other words, it is indicated that, to ensure sample stability, it is preferable to store biological samples at room temperature, and that it is not preferable to store them on ice for long periods of time. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2019-509489 [Non-patent literature]

[0005] [Non-Patent Document 1] Hassina Massudi et al., PLoS One. 2012;7(7):e42357 [Non-patent document 2] James Clement et al., Rejuvenation Res 2019 Apr; 22(2): 121-130 [Non-patent document 3] Katsumi Shibata et al., Vitamin Vol. 75, No. 9 (September), pp. 455-462 (2001) Summary of the Invention [Problem to be solved by the invention]

[0006] NAD in biological samples is very unstable, making it difficult to store samples for long periods of time, even under refrigerated conditions. In particular, whole blood samples require immediate deproteinization after collection. However, in practice, it is often difficult to immediately measure NAD in biological samples or deproteinize them in clinical settings. Therefore, a simple method for stabilizing NAD in biological samples is desired.

[0007] An object of the present invention is to provide a method and composition for stabilizing NAD in a biological sample, and also to provide an NAD measurement method and kit for measuring the amount of NAD in a biological sample while maintaining a stable NAD content. [Means for solving the problem]

[0008] As a result of extensive research, the present inventors discovered that the NAD concentration in a biological sample can be stabilized by contacting NAD in the biological sample with nicotinamide and / or a nicotinamide derivative, and thus completed the present invention.

[0009] That is, the present invention provides the following. (1) A method for stabilizing nicotinamide adenine dinucleotide (NAD) in a sample removed from a subject, comprising: The method comprises a contacting step of contacting the sample with nicotinamide and / or a nicotinamide derivative. (2) The method according to (1), wherein the nicotinamide and / or nicotinamide derivative is nicotinamide, nicotinamide mononucleotide (NMN) and / or nicotinamide riboside (NR). (3) The method according to (1) or (2), wherein the nicotinamide and / or nicotinamide derivative comprises two or more compounds selected from the group consisting of nicotinamide, NMN, and NR. (4) In the contact step i) the final concentration of nicotinamide is 10 to 500 mM; ii) the final concentration of NMN is 1 to 100 mM; and / or iii) the final concentration of NR is 1–100 mM; The method according to (2) or (3). (5) The method according to any one of (1) to (4), wherein the sample is whole blood or tissue fluid of the subject. (6) The method according to any one of (1) to (5), wherein the subject is a human. (7) A method for measuring NAD, comprising the step of measuring NAD in a sample stabilized by the method according to any one of (1) to (6). (8) The method according to (7), further comprising a step of pretreating the sample before the step of measuring NAD. (9) The step of measuring NAD comprises measuring the amount of NAD in the pretreated sample. + (8) The method according to (8), comprising reacting in a system comprising a dehydrogenase having the above coenzyme as a coenzyme, its substrate, diaphorase, and a tetrazolium salt. (10) A composition used for stabilizing NAD in a sample, comprising nicotinamide and / or a nicotinamide derivative. (11) The composition according to (10), wherein the nicotinamide and / or nicotinamide derivative is nicotinamide, NMN, and / or NR. (12) The composition according to (10) or (11), wherein the nicotinamide and / or nicotinamide derivative comprises two or more compounds selected from the group consisting of nicotinamide, NMN, and NR. (13) The composition according to any one of (10) to (12), which is an aqueous solution. (14) The composition according to any one of (10) to (13), which is used for stabilizing NAD in a sample taken from a subject. (15) The composition according to any one of (10) to (14), wherein the sample is whole blood or tissue fluid of a subject. (16) The composition according to any one of (10) to (15), wherein the subject is a human. (17) The composition according to any one of (10) to (16), for use in the method according to any one of (1) to (6). (18) A kit for measuring NAD, comprising the composition according to any one of (10) to (17) and a reagent for measuring NAD in a sample. (19) The kit for measuring NAD according to (18), further comprising a pretreatment reagent for pretreatment of a sample. (20) The reagent for measuring NAD is NAD + (19) The kit for measuring NAD according to (19), comprising a first reagent solution containing a dehydrogenase having as a coenzyme NAD as a coenzyme and a tetrazolium salt, and a second reagent solution containing a substrate for the dehydrogenase and diaphorase. This specification includes the disclosure of Japanese Patent Application No. 2022-011518, from which the present application claims priority. [Effects of the Invention]

[0010] The present invention provides a method and composition for stabilizing NAD in a biological sample, as well as an NAD measurement method and kit for measuring the amount of NAD in a biological sample while maintaining the NAD content stable. [Brief explanation of the drawings]

[0011] [Figure 1] This is a line graph showing the change in the content of NAD added to pooled human serum during refrigeration and incubation at 25°C and 37°C. The relative values ​​(%) of NAD concentration in serum under each incubation condition are shown, with the concentration at the time of NAD addition (0 hr) set to 100%. [Figure 2]1 is a line graph showing the change in NAD content in rat whole blood samples containing an NAD stabilizer or PBS added thereto, stored at 25°C under refrigeration. (A) shows the change in NAD concentration over time in samples stored under refrigeration. (B) shows the change in NAD concentration over time in samples stored at 25°C. DETAILED DESCRIPTION OF THE INVENTION

[0012] 1. Nicotinamide adenine dinucleotide (NAD) stabilization method 1-1.Configuration A first embodiment of the present invention provides a method for stabilizing nicotinamide adenine dinucleotide (NAD) in a biological sample taken from a subject (hereinafter also referred to as an "NAD stabilization method"). The NAD stabilization method of this embodiment is characterized by including a contacting step of contacting nicotinamide and / or a nicotinamide derivative with the biological sample taken from the subject. The NAD stabilization method of this embodiment is capable of suppressing a decrease in the amount of NAD in the biological sample by allowing nicotinamide and / or a nicotinamide derivative to coexist with NAD.

[0013] 1-2.Definition As used herein, the term "subject" refers to a mammal. Mammals refer to animals belonging to the phylum Chordata, subphylum Vertebrates, class Mammalia, including both humans and non-humans, and include, for example, primates including humans and chimpanzees, pet animals such as dogs and cats, livestock animals such as cows, pigs, horses, sheep, and goats, rodents such as mice and rats, and mammals kept in zoos. The subject herein is preferably a human.

[0014] As used herein, the term "sample" or "biological sample" is not particularly limited, and may refer to, for example, a sample taken from a subject, such as blood (e.g., whole blood, serum, plasma), urine, feces, saliva, milk, tissue fluid or cell extract, hair, sweat, or a mixture thereof. A preferred biological sample herein is whole blood or tissue fluid. Unless otherwise specified, "whole blood" refers to whole blood that has been mixed with an anticoagulant immediately after collection to prevent blood clotting. Typical anticoagulants used include dipotassium EDTA, disodium EDTA, heparin, and trisodium citrate, but any anticoagulant may be used.

[0015] In this specification, nicotinamide adenine dinucleotide (NAD) refers to an electron carrier having a structure in which nicotinamide mononucleotide and adenosine are bound via a phosphate bond, and functions as a coenzyme for oxidoreductase. NAD includes an oxidized form (NAD) represented by the following formula (I): + ) and reduced form (NADH), but NAD as used herein encompasses both the oxidized and reduced forms. [ka] The oxidized form is converted to the reduced form, and vice versa, by a reaction occurring at the nicotinamide moiety, as shown in formula (II) below. This conversion reaction is reversible. [ka]

[0016] As used herein, the term "nicotinamide" refers to a compound represented by the following formula (III): [ka]

[0017] As used herein, the term "nicotinamide derivative" refers to a compound having a nicotinamide skeleton represented by formula (III). There are no particular limitations on the compound as long as it has a nicotinamide skeleton, and examples thereof include nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR), and both oxidized and reduced forms thereof are encompassed.

[0018] The oxidized form of nicotinamide mononucleotide (NMN) has the structure shown in formula (IV): [ka]

[0019] The oxidized form of nicotinamide riboside (NR) has the structure shown in formula (V): [ka]

[0020] 1-3. Contact process The NAD stabilization method of this embodiment includes a contacting step in which nicotinamide and / or a nicotinamide derivative (hereinafter also referred to as "nicotinamides") is contacted with a sample to be measured for NAD. The method for contacting the sample with nicotinamides is not particularly limited, but for example, in the case of a liquid sample, a method of dissolving a solid composition, such as a solid or powdered composition, containing nicotinamides in the sample or a method of mixing an aqueous composition containing nicotinamides with the sample may be used. In particular, a method of mixing an aqueous composition with the sample is preferred.

[0021] The nicotinamides used are not particularly limited as long as they are compounds having a nicotinamide skeleton, but are particularly preferably nicotinamide, nicotinamide dimononucleotide (NMN) and / or nicotinamide riboside (NR).More preferably, two or more compounds selected from the group consisting of nicotinamide, NMN, and NR.For example, a mixture of nicotinamide and NMN, a mixture of nicotinamide and NR, or a mixture of NMN and NR can be used.

[0022] When nicotinamide is used as the nicotinamide, the final concentration of nicotinamide when mixed with the sample is preferably 10 to 500 mM. The lower limit of the final concentration of nicotinamide can be 10 mM or more, 20 mM or more, 30 mM or more, 40 mM or more, 50 mM or more, 60 mM or more, 70 mM or more, 80 mM or more, 90 mM or more, 100 mM or more, 150 mM or more, 200 mM or more, 250 mM or more, or 300 mM or more. The upper limit of the final concentration of nicotinamide can be 500 mM or less, 450 mM or less, 400 mM or less, 350 mM or less, 300 mM or less, 250 mM or less, 200 mM or less, 150 mM or less, 100 mM or less, 90 mM or less, 80 mM or less, 70 mM or less, 60 mM or less, or 50 mM or less. When NMN is used as the nicotinamide, the final concentration of NMN when mixed with the sample is preferably 1 to 100 mM. The lower limit of the final concentration of NMN can be 1 mM or more, 2 mM or more, 3 mM or more, 4 mM or more, 5 mM or more, 6 mM or more, 7 mM or more, 8 mM or more, 9 mM or more, 10 mM or more, 15 mM or more, 20 mM or more, 25 mM or more, 30 mM or more, 40 mM or more, 50 mM or more, 60 mM or more, 70 mM or more, 80 mM or more, or 90 mM or more. The upper limit of the final concentration of NMN can be 100 mM or less, 90 mM or less, 80 mM or less, 70 mM or less, 60 mM or less, 50 mM or less, 45 mM or less, 40 mM or less, 35 mM or less, 30 mM or less, 25 mM or less, 20 mM or less, 15 mM or less, 10 mM or less, 9 mM or less, 8 mM or less, 7 mM or less, 6 mM or less, or 5 mM or less. When NR is used as a nicotinamide, the final concentration of NR when mixed with the sample is preferably 1 to 100 mM. The lower limit of the final concentration of NR can be 1 mM or more, 2 mM or more, 3 mM or more, 4 mM or more, 5 mM or more, 6 mM or more, 7 mM or more, 8 mM or more, 9 mM or more, 10 mM or more, 15 mM or more, 20 mM or more, 25 mM or more, 30 mM or more, 40 mM or more, 50 mM or more, 60 mM or more, 70 mM or more, 80 mM or more, or 90 mM or more.In addition, the upper limit of the final concentration of NR can be 100 mM or less, 90 mM or less, 80 mM or less, 70 mM or less, 60 mM or less, 50 mM or less, 45 mM or less, 40 mM or less, 35 mM or less, 30 mM or less, 25 mM or less, 20 mM or less, 15 mM or less, 10 mM or less, 9 mM or less, 8 mM or less, 7 mM or less, 6 mM or less, or 5 mM or less.

[0023] By using the NAD stabilization method of this embodiment, it was confirmed that the amount of NAD contained in a biological sample, such as a whole blood sample, is stable for at least 24 hours under refrigerated or room temperature storage conditions. The method of this embodiment can be achieved by a simple technique, such as mixing a collected sample with a composition containing nicotinamides, and therefore can be performed at a clinical site immediately after sample collection (blood collection). Therefore, by using this method, a biological sample, such as whole blood, for NAD measurement can be transported from a clinical site to a testing institution under refrigerated or room temperature conditions, and NAD measurement can be performed at the testing institution.

[0024] 2. NAD measurement method A second embodiment of the present invention provides a method for measuring NAD. The method for measuring NAD of this embodiment is characterized by including a step of measuring NAD in a sample stabilized using the method described in Section "1. Method for stabilizing nicotinamide adenine dinucleotide (NAD)." The method for measuring NAD of this embodiment enables more accurate quantification of the amount of NAD in a living body. Since the amount of NAD in a living body can be an indicator of the degree of aging, it becomes possible to more accurately determine the degree of aging in a subject. In this embodiment, the definitions of terms are the same as those described in Section "1. Method for stabilizing nicotinamide adenine dinucleotide (NAD)," unless otherwise specified.

[0025] This embodiment includes a step of measuring NAD in a sample. Preferably, this embodiment includes a step of pretreating a sample stabilized using the method described in Section "1. Nicotinamide adenine dinucleotide (NAD) stabilization method" prior to the step of measuring NAD in the sample. Sample pretreatment can include, for example, deproteinization. Any known deproteinization method can be used, including, for example, protein denaturation and precipitation. As used herein, the term "protein denaturation and precipitation" refers to a method in which a protein precipitant containing an organic solvent such as ethanol, acetone, or acetonitrile, or an acid such as trichloroacetic acid or perchloric acid, is added to a sample, mixed, cooled, and then centrifuged to recover the supernatant. Because NAD is particularly susceptible to degradation due to the influence of proteins derived from the sample, deproteinization is preferred.

[0026] In the step of measuring NAD in a sample, preferably a pretreated sample, the method for measuring NAD is not particularly limited, and known methods such as gas chromatography-mass spectrometry (GC / MS), liquid chromatography-mass spectrometry (LC / MS), competitive immunoassay, and enzyme cycling can be used.

[0027] When using the enzymatic cycling method, for example, the pretreated sample is subjected to a + The reaction can be carried out in a system containing a dehydrogenase (e.g., glucose dehydrogenase) that uses 1-(2-methyl-2-propanol) as a coenzyme, its substrate (e.g., glucose), diaphorase, and a tetrazolium salt (e.g., WST-8). In this system, the enzymatic cycling reaction shown in formula (VI) below occurs. [ka]

[0028] In the reaction shown in formula (VI), NAD + The reaction of glucose dehydrogenase with glucose in the presence of NAD +is reduced to produce NADH. On the other hand, when diaphorase reacts with WST-8 in the presence of NADH, NADH is oxidized to NAD + Both enzyme reactions result in NAD + A cycling reaction of NAD and NADH occurs. During this reaction, the absorbance (at a wavelength close to the maximum absorption wavelength of 450 nm) of the water-soluble formazan produced by the reduction of WST-8 is measured over time, and the rate of increase in absorbance is calculated. By performing a similar reaction in advance using a standard solution containing a known concentration of NAD and creating a calibration curve of the NAD concentration and the rate of increase in absorbance, the NAD concentration of the sample can be calculated based on the absorbance rate obtained from the sample.

[0029] NAD + Dehydrogenases that use NAD as a coenzyme include + There are no particular limitations on the enzyme as long as it reacts using the coenzyme, but examples that can be used include glucose dehydrogenase, lactate dehydrogenase, alcohol dehydrogenase, etc. When glucose dehydrogenase is used, the reaction concentration is preferably 50 to 5000 U / L, particularly 100 to 3000 U / L or 250 to 2000 U / L.

[0030] Diaphorase (also called NADH dehydrogenase) uses NADH to reduce the tetrazolium salt to form formazan and simultaneously oxidizes NADH to NAD + Diaphorase is known as an enzyme that induces the reaction. There are no particular limitations on the species of origin or structure of the enzyme as long as it induces such a reaction, but for example, diaphorase derived from Clostridium kluyveri can be used. The reaction concentration of diaphorase is preferably 100 to 5000 U / L, particularly 200 to 3000 U / L or 500 to 2000 U / L.

[0031] The tetrazolium salt that serves as a substrate for diaphorase is not particularly limited as long as it generates a water-soluble formazan upon reaction with diaphorase. For example, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium (also known as WST-8), 2-(4-indophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium, and 2-(4-indophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium can be used.

[0032] The above-mentioned enzymatic cycling method can be easily performed, for example, using an existing automated analyzer for clinical testing (e.g., Hitachi 7180 automated analyzer (manufactured by Hitachi High-Technologies Corporation)). Alternatively, instead of the above-mentioned enzymatic cycling method, a method using a microplate reader as described in Non-Patent Document 3 can also be used.

[0033] 3. NAD stabilizing composition A third embodiment of the present invention is a composition used for stabilizing NAD in a biological sample (hereinafter also referred to as an "NAD stabilizing composition"). The NAD stabilizing composition of this embodiment is characterized by containing nicotinamide and / or a nicotinamide derivative. The NAD stabilizing composition of this embodiment can stabilize the NAD concentration in a sample by contacting (mixing or dissolving) the composition with the sample.

[0034] The NAD stabilizing composition of this embodiment can be used in the NAD stabilizing method described in the section "1. Nicotinamide adenine dinucleotide (NAD) stabilizing method." In this embodiment, the definitions of terms are the same as those described in the section "1. Nicotinamide adenine dinucleotide (NAD) stabilizing method," unless otherwise specified.

[0035] The NAD stabilizing composition of the present embodiment contains nicotinamide and / or a nicotinamide derivative (nicotinamides) as an active ingredient, and as long as it is possible to contact the sample with the active ingredient, its form is not particularly limited, and it may be a solid composition such as a solid or powder, or may be an aqueous solution composition containing nicotinamides. In particular, it is preferable that the composition be an aqueous solution that can be easily mixed with the sample.

[0036] The nicotinamides contained in the NAD stabilizing composition are not particularly limited as long as they are compounds having a nicotinamide skeleton, but are particularly preferably nicotinamide, NMN, and / or NR. Furthermore, it is more preferable to use two or more compounds selected from the group consisting of nicotinamide, NMN, and NR. For example, a mixture of nicotinamide and NMN, a mixture of nicotinamide and NR, or a mixture of NMN and NR can be used.

[0037] The amount of nicotinamides contained in the NAD-stabilizing composition can be adjusted so that the desired final concentration is achieved when the composition is brought into contact with a sample (by mixing, dissolving, etc.). For example, when the composition is prepared as an aqueous solution, the nicotinamide concentration in the composition may be 10 times the desired final concentration, and the sample and composition may be mixed at a volume ratio of 9:1. In this case, the concentration of nicotinamides in the composition may be, for example, 100 to 5000 mM for nicotinamide, 10 to 1000 mM for NMN, or 10 to 1000 mM for NR.

[0038] When the NAD stabilizing composition is in the form of an aqueous solution, the composition may contain, in addition to the nicotinamides as active ingredients, phosphoric acid, citric acid, pH buffers such as Tris, MES, HEPES, and PIPES, chelating agents such as EDTA, surfactants, antioxidants, preservatives, etc. The pH of the composition as an aqueous solution is preferably 3.0 to 8.0, more preferably 4.0 to 7.0, in consideration of the stability of the nicotinamides.

[0039] When the NAD stabilizing composition is a solid (solid, powder, etc.), the composition may contain, as necessary, a solubilizing agent, a pH buffer, a chelating agent, an antioxidant, a preservative, a bulking agent, an excipient, etc., in addition to the nicotinamides that serve as the active ingredient. When the solid composition is mixed with a biological sample, the pH of the mixture is preferably adjusted to 3.0 to 8.0, particularly 4.0 to 7.0.

[0040] 4. NAD measurement kit A fourth embodiment of the present invention is a kit for measuring NAD. The kit for measuring NAD of this embodiment is characterized by including the composition described in Section "3. NAD stabilizing composition" and a reagent for measuring NAD. The reagent for measuring NAD referred to here is preferably a reagent for use in the NAD measurement method described in Section "2. NAD measurement method." In this embodiment, the definitions of terms are the same as those described in Section "1. Nicotinamide adenine dinucleotide (NAD) stabilization method," unless otherwise specified.

[0041] In this embodiment, the NAD measurement kit preferably further includes a pretreatment reagent for pretreating the sample. For example, a reagent for deproteinization of the sample can be used as the pretreatment reagent. Protein denaturation and precipitation can be used as the deproteinization treatment. In this case, the pretreatment reagent includes a protein precipitant containing an organic solvent such as ethanol, acetone, or acetonitrile, or an acid such as trichloroacetic acid or perchloric acid.

[0042] In this embodiment, the "reagent for measuring NAD (hereinafter also referred to as "NAD measurement reagent")" is not particularly limited in terms of the measurement principle, etc., as long as it is a reagent that can quantify NAD. NAD measurement reagents suitable for known methods such as gas chromatography-mass spectrometry (GC / MS), liquid chromatography-mass spectrometry (LC / MS), competitive immunoassay, and enzymatic cycling can be used. Preferably, an NAD measurement reagent suitable for enzymatic cycling can be used.

[0043] The enzymatic cycling method can use, for example, the principle described in formula (VI) described in the section "2. NAD measurement method." When using the principle described in formula (VI), the NAD measurement reagent preferably contains the following reagent solution. First reagent: NAD + A dehydrogenase (glucose dehydrogenase in formula (VI)) with a coenzyme of 1,2-dihydroxybenzoate (WST-8 in formula (VI)) and a tetrazolium salt (WST-8 in formula (VI)). Second reagent solution: substrate of the dehydrogenase (D-glucose in formula (VI)) and diaphorase. The first reagent solution is mixed with a sample containing NAD, and the second reagent solution is added to the mixture to cause a reaction, resulting in an enzyme cycling reaction as shown in formula (VI). This reaction reduces the tetrazolium salt to produce formazan. The absorbance at wavelengths near the maximum absorption wavelength of this formazan is measured over time, and the rate of increase in absorbance is calculated, allowing the amount of NAD in the sample to be quantified.

[0044] In this embodiment, the NAD measurement reagent included in the kit preferably further includes a standard solution containing NAD of known concentrations (NAD standard solution). A calibration curve can be created from the rate of absorbance increase obtained by performing the same reaction with the NAD standard solution as with the sample. When using an NAD standard solution, the NAD standard solution preferably contains nicotinamide and / or a nicotinamide derivative to stabilize NAD, similar to the composition described in "3. NAD stabilizing composition."

[0045] The first reagent solution, the second reagent solution, and the NAD standard solution may contain, as necessary, pH buffers such as phosphoric acid, citric acid, Tris, MES, HEPES, and PIPES, chelating agents such as EDTA, surfactants, antioxidants, preservatives, and the like.

[0046] The pH of the second reagent solution is preferably 7.5 to 9.0, particularly 7.5 to 8.5, in order to stabilize diaphorase, while the pH of the first reagent solution is preferably 5.0 to 7.4, particularly 6.0 to 7.4.

[0047] In this embodiment, the NAD measurement kit may further include, in addition to the NAD stabilizing composition and the NAD measurement reagent, a sample storage container suitable for a measurement device (e.g., a Hitachi 7180 automatic analyzer (manufactured by Hitachi High-Technologies Corporation)), an instrument setting manual, etc. [Example]

[0048] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to the following examples.

[0049] [Reference Example] NAD stability test (spiking and recovery test in human pooled serum) NAD was added to 2250 μL of human pooled serum to a concentration of 50 μmol / L, and the mixture was refrigerated and incubated at 25° C. and 37° C. The NAD concentration in the serum was measured 1, 2, and 19 hours after the start of incubation.

[0050] The NAD concentration of a sample (here, serum) was measured according to the following procedure. (1) Deproteinization Three volumes of 0.4 M trichloroacetic acid solution were added to the sample and mixed, and the mixture was left to stand at room temperature for 10 minutes. After that, the mixture was centrifuged and the supernatant was collected to obtain the NAD extract.

[0051] (2) NAD measurement The NAD in the extract was measured using a Hitachi Model 7180 automatic analyzer (Hitachi High-Tech Corporation). Measurements were performed using the following measurement parameters: 1st reagent solution (phosphate buffer (pH 7.0), containing 0.6 mM WST-8 and 1.7 U / mL glucose dehydrogenase (GLUCDH "Amano" 2, manufactured by Amano Enzyme Co., Ltd.)) and 2nd reagent solution (phosphate buffer (pH 7.8), containing 320 mM D-glucose and 15 U / mL diaphorase (No. 46445003, manufactured by Oriental Yeast Co., Ltd.)). ·Analysis Method ··· Rate A ·Measurement wavelength (secondary / main) 600nm / 450nm Reaction time: 10 minutes Metering points 27-34 Sample solution (NAD extract) 2 μL R1 Reagent 120 μL R2 Reagent 40 μL Specifically, 2 μL of NAD extract and 120 μL of the first reagent were mixed and incubated at 37°C for 4.5 minutes (photometric points 1-16), after which 40 μL of the second reagent was added to initiate the reaction (photometric point 17). The absorbance of the water (blank) at the same photometric points was subtracted from the absorbance of the sample 8-10 minutes after the start of the reaction (photometric points 27-34) to calculate the change in absorbance per minute (ΔAbs / min).

[0052] The NAD concentration of the sample was calculated from a calibration curve of NAD concentration-rate of increase in absorbance (ΔAbs / min) previously obtained by measuring multiple NAD solutions with known concentrations.

[0053] Figure 1 shows the time course of the relative NAD concentration (%) in serum under each incubation condition, with the concentration at the time of NAD addition (0 hours) taken as 100%. When stored at 25°C and 37°C, the NAD concentration fell to almost zero% within 2 hours. When stored in a refrigerator, the concentration also fell to about half within 2 hours and to almost zero% within 20 hours. This indicates that NAD is in a very unstable state in serum.

[0054] [Example 1] Blood NAD stabilization test using NAD stabilizers (observation of changes over time) Whole blood was collected from retired SD rats using EDTA-containing blood collection tubes. 450 μL of whole blood was placed in a 1.5 mL centrifuge tube, and 50 μL of 10x stabilizer (PBS (pH 6.1) containing 2 M nicotinamide, 100 mM NMN, and 40 mg / mL EDTA) was immediately added and mixed to prepare the measurement sample. A control sample was prepared by adding PBS to the whole blood sample instead of the 10x stabilizer. The measurement and control samples were immediately frozen and stored at -80°C. Each frozen sample was thawed at room temperature for approximately 30 minutes, mixed thoroughly, and then placed in a refrigerator or at 25°C. Tests under each condition were performed in triplicate.

[0055] After leaving the samples to stand for a predetermined time, NAD extraction was carried out using the same procedure as in Reference Example (1). NAD measurement was carried out using the same procedure as in Reference Example (2).

[0056] The measurement results for each sample are shown in Figure 2. Figure 2A shows the change in NAD concentration over time in a sample stored in a refrigerator. Figure 2B shows the change in NAD concentration over time in a sample stored at 25°C. In the control sample, almost all NAD was undetectable upon freezing and thawing, and the NAD concentration decreased further by storing in a refrigerator and at 25°C. On the other hand, in the measurement sample containing a stabilizer, the NAD concentration hardly decreased even after storing for 24 hours, both in a refrigerator and at 25°C, demonstrating that NAD was present stably in the sample.

[0057] [Example 2] Blood NAD stabilization test using nicotinamide and NMN Whole blood was collected from retired SD rats using EDTA-containing blood collection tubes. PBS (pH 6.0) containing nicotinamide and NMN at 10x concentrations shown in Table 1 was prepared as various 10x stabilizers. 450 μL of whole blood was placed in a 1.5 mL centrifuge tube, and 50 μL of each of the various 10x stabilizers was added and mixed. NAD was added to each tube to a concentration of 100 μM to prepare the measurement samples.

[0058] After standing overnight at 25°C, each measurement sample was subjected to NAD extraction and NAD measurement in the same manner as in Example 1. The concentration at the time of NAD addition (0 hours) was set as 100%, and the relative value (%) of the NAD concentration of each measurement sample was calculated. The results are shown in Table 1. It was confirmed that the amount of remaining NAD increased with increasing amounts of nicotinamide and NMN added.

[0059] [Table 1]

[0060] [Example 3] Blood NAD stabilization test using nicotinamide and NR Whole blood was collected from retired SD rats using EDTA-containing blood collection tubes. PBS (pH 6.0) containing nicotinamide and NR at 10x concentrations shown in Table 2 was prepared as various 10x stabilizers. 450 μL of whole blood was placed in a 1.5 mL centrifuge tube, and 50 μL of each of the various 10x stabilizers was added and mixed. NAD was added to each tube to a concentration of 100 μM to prepare the measurement samples.

[0061] After standing overnight at 25°C, each measurement sample was subjected to NAD extraction and NAD measurement in the same manner as in Example 1. The concentration at the time of NAD addition (0 hours) was set as 100%, and the relative value (%) of the NAD concentration of each measurement sample was calculated. The results are shown in Table 2. It was confirmed that the amount of remaining NAD increased with increasing amounts of nicotinamide and NR added.

[0062] [Table 2]

[0063] [Example 4] Blood NAD stabilization test using NMN and NR Whole blood was collected from retired SD rats using EDTA-containing blood collection tubes. PBS (pH 6.0) containing NMN and NR at 10x concentrations shown in Table 3 was prepared as various 10x stabilizers. 450 μL of whole blood was placed in a 1.5 mL centrifuge tube, and 50 μL of each of the various 10x stabilizers was added and mixed. NAD was added to each tube to a concentration of 100 μM to prepare the measurement samples.

[0064] After standing overnight at 25°C, each measurement sample was subjected to NAD extraction and NAD measurement in the same manner as in Example 1. The concentration at the time of NAD addition (0 hr) was set as 100%, and the relative value (%) of the NAD concentration of each measurement sample was calculated. The results are shown in Table 3. It was confirmed that the amount of remaining NAD increased with increasing amounts of NMN and NR added.

[0065] [Table 3]

[0066] [Example 5] Effect of pH on blood NAD stabilization Whole blood was collected from retired SD rats using EDTA-containing blood collection tubes. PBS or citric acid solutions containing 2 M nicotinamide (Stabilizer 1) and 100 mM NMN (Stabilizer 2) at the pH values ​​shown in Table 4 were prepared as 10x stabilizers. 450 μL of whole blood was placed in a 1.5 mL centrifuge tube, and 50 μL of each of the 10x stabilizers was added and mixed. NAD was added to each tube to a concentration of 100 μM, and these were used as measurement samples.

[0067] After standing overnight at 25°C, each measurement sample was subjected to NAD extraction and NAD measurement in the same manner as in Example 1. The concentration at the time of NAD addition (0 hours) was set as 100%, and the relative value (%) of the NAD concentration of each measurement sample was calculated. The results are shown in Table 4. It was confirmed that there was no significant difference in the stabilizing effect of blood NAD in the pH range of 10x stabilizer pH 4.29 to pH 7.11.

[0068] [Table 4] [Industrial Applicability]

[0069] The present invention can be used mainly in the industrial fields of clinical testing and in vitro diagnostic pharmaceuticals. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. 1. A method for stabilizing nicotinamide adenine dinucleotide (NAD) in a biological sample removed from a mammalian subject, the method comprising: a contacting step of contacting the biological sample with nicotinamide and / or a nicotinamide derivative, A method that does not include a step of culturing cells.

2. 2. The method of claim 1, wherein the nicotinamide and / or nicotinamide derivative is nicotinamide, nicotinamide mononucleotide (NMN) and / or nicotinamide riboside (NR).

3. In the contacting step i) the final concentration of nicotinamide is 10 to 500 mM; ii) the final concentration of NMN is 1 to 100 mM; and / or iii) the final concentration of NR is 1 to 100 mM; The method of claim 2.

4. The method of claim 1 , wherein the sample is whole blood or tissue fluid of a subject.

5. The method of claim 1 , wherein the subject is a human.

6. A method for measuring NAD, comprising the step of measuring NAD in a sample stabilized by the method according to any one of claims 1 to 5.

7. The method of claim 6, further comprising a step of pre-treating the sample prior to the step of measuring NAD.

8. The step of measuring NAD comprises: The pretreated sample was ＋ reacting the compound in a system containing a dehydrogenase having the compound as a coenzyme, a substrate thereof, diaphorase, and a tetrazolium salt; The method of claim 7.

9. A composition comprising nicotinamide and / or a nicotinamide derivative for use in stabilizing NAD in a biological sample (not including cultured cells) removed ex vivo from a mammalian subject.

10. The composition of claim 9, wherein the nicotinamide and / or nicotinamide derivative is nicotinamide, NMN and / or NR.

11. A composition according to claim 9 or 10 for use in a method according to any one of claims 1 to 5.

12. A kit for measuring NAD, comprising the composition according to claim 9 or 10 and a reagent for measuring NAD in a sample.

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