Method for measuring concentrations of nicotinamide mononucleotide, nicotinamide adenine dinucleotide, and nicotinamide

Abstract

To provide a method that can highly sensitively measure concentrations of three components of nicotinamide mononucleotide, nicotinamide adenine dinucleotide, and nicotinamide in a sample.SOLUTION: Provided is a method for measuring nicotinamide mononucleotide (NMN), nicotinamide adenine dinucleotide (NAD+), and isolating nicotinamide (NAM), including: a step A of simultaneously separating NMN and NAD+ in a sample by reversed-phase liquid chromatography; and a step B of separating NAM in the sample, wherein the separation step A uses a mixture of a highly volatile ammonium salt solution containing an amine-based reagent and an organic solvent as a mobile phase.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for highly sensitive measurement of the concentrations of three components, nicotinamide mononucleotide, nicotinamide adenine dinucleotide, and nicotinamide, contained in a sample.

Background Art

[0002] Nicotinamide mononucleotide (NMN) is a major precursor related to the biosynthesis of nicotinamide adenine dinucleotide (NAD + ) in mammals, and it is known that administration of NMN promotes the biosynthesis of NAD + and can be used for the treatment of various age-related diseases (for example, Patent Document 1, Non-Patent Document 1).

[0003] NAD + is a conventionally known coenzyme and is considered to play an important role in reduction reactions in the cells of various organisms, and it has been reported in many biological pathways such as metabolism, cancer, stress response, inflammation, and aging. Also, NAD + decreases in many tissues such as the pancreas, adipose tissue, skeletal muscle, liver, skin, and brain with aging, and it has also been reported in many cases that a decrease in the amount of NAD + leads to the onset of age-related diseases such as cancer, heart disease, type II diabetes, obesity, hypertension, and age-related macular degeneration (for example, Patent Document 1, Non-Patent Document 1).

[0004] NAD + 's biosynthesis pathways are mainly known as two pathways: the de novo pathway starting from tryptophan and the salvage pathway using NAM. Especially in mammals, NAD +The salvage pathway in which NAM generated with the consumption of + is reused is very important for maintaining NAD levels. In the salvage pathway, nicotinamide (NAM), an amide form of vitamin B3, and 5'-phosphoribosyl pyrophosphate (PRPP) are converted to NMN by nicotinamide phosphoribosyltransferase (NAMPT), and this NMN is + adenylated by nicotinamide / nicotinic acid mononucleotide adenylyltransferase (NMNAT) to generate + NAD (for example, Patent Document 1). In order to promote the biosynthesis of

[0005] NAD, many attempts have been made to administer NMN, a major precursor of + NAD, and the application of NMN in the treatment of various pathological conditions including age-related diseases has attracted attention (for example, Patent Document 1).

[0006] However, since NMN is rapidly metabolized in vivo and converted to other metabolites such as + NAD, it has been reported that it is difficult to directly detect NMN in biological samples such as blood (for example, Patent Document 1).

[0006] In actual measurement target samples, it is considered that the above-mentioned NMN, + NAD, and NAM are mixed. If the concentrations of these three components in the sample can be measured with high sensitivity, it is considered that it can contribute to elucidating the more detailed mechanism of the + salvage pathway of + NAD, its utilization as a biomarker in various diseases, and more accurate analysis of the time-course changes in the concentrations of NMN, + NAD, and NAM in NMN administration studies aimed at promoting the biosynthesis of

[0007] NAD. As an attempt to measure the concentrations of the three components of NMN, + NAD, and NAM in a sample, Non-Patent Document 2 is known. In Non-Patent Document 2, the concentration measurement is performed according to the following procedure.

[0008] As a pretreatment for the sample, samples containing 2 - 4.5×10 7 (cultured yeast cells) or 4 - 20×10 6 (cultured mammalian cells) are collected, washed once with ice-cold potassium buffered saline, resuspended in 300 μL of 75% ethanol / 25% 10 mM HEPES (v / v), pH 7.1 solution at 80°C, and then shaken (1000 rpm, 80°C, 3 minutes) to extract ethanol-soluble metabolites. Centrifuge at 16,000×g for 10 minutes, and dry the obtained supernatant at 40°C. Resuspend in ammonium acetate buffer and subject to LC-MS / MS analysis (Liquid chromatography / tandem mass spectrometry). (For NMN and NAD + measurement, 13 C-labeled substances are used, and for NAM measurement, 18 O-labeled substances are used as internal standards)

[0009] The following conditions are adopted as LC-MS / MS conditions. 1. For NMN and NAD + measurement: 1) LC (Liquid chromatography) conditions (alkaline separation): Analytical column: Hypercarb, 1 mm x 100 mm Column temperature: 60°C A mobile phase mixture of mobile phase A and mobile phase B is used. Mobile phase A: 7.5 mM ammonium acetate containing 0.05% (v / v) ammonium hydroxide Mobile phase B: Acetonitrile containing 0.05% (v / v) ammonium hydroxide Time program: Time (min) Proportion of mobile phase B (%) 0 5 1.8 5 14 54 14.1 90 17.1 90 17.2 5 32.25 Flow rate: 0.08 mL / min Injection volume: 2.5 μL Measurement time: 32.2 min 2) MS / MS conditions: MS / MS: Acquity TQD (manufactured by Waters) Scan type: MRM (Multiple reaction monitoring) Ion polarity: Positive Monitored ions: Compound name Precursor ion > Product ion (m / z) CE (Collision energy) (eV) NMN 335 > 123 12 NAD + 664 > 428 26

[0010] 2. In the NAM measurement: 1) LC conditions (acidic conditions) Analysis column: Hypercarb, 2.1 mm x 100 mm Column temperature: 60 °C Mobile phase A: 10 mM ammonium acetate containing 0.1% (v / v) formic acid Mobile phase B: 10 mM acetonitrile containing 0.1% (v / v) formic acid Time program: Time (min) Ratio of mobile phase B (%) 0 5 1.8 5 11.2 35.9 11.3 90 13.3 90 13.4 5 23.4 5 Flow rate: 0.2 mL / min Injection volume: 2.5 μL Measurement time: 23.4 min 2) MS / MS conditions: MS / MS: Acquity TQD (manufactured by Waters) Scan type: MRM Ion polarity: Positive Monitor ion: Compound name Precursor ion > Product ion (m / z) CE (eV) NAM 123 > 96 16

[0011] As a result of the concentration measurement in Non-Patent Document 2, the detection limit of each component is as follows. NMN: 400 nmol / L NAD + : 76 nmol / L NAM: 240 nmol / L

[0012] Thus, in Non-Patent Document 2, although attempts were made to measure the concentrations of the three components of NMN, NAD + , and NAM in the sample, the measurement sensitivity could not be said to be sufficiently high.

[0013] On the other hand, in Patent Document 1, attempts were made to measure the concentration of NMN alone, and it has been reported that high-sensitivity concentration measurement was possible. In Patent Document 1, the concentration measurement was performed according to the following procedure.

[0014] As a pretreatment for the sample, 960 μL of ice-cold methanol was added to 20 μL of the plasma sample, and 20 μL of an internal standard solution (125 μM 10-camphorsulfonic acid) was added as an IS (Internal standard). After stirring with a vortex mixer and sonication (for 5 minutes), centrifugation was performed at 16000×g, 4°C for 5 minutes, and the obtained supernatant was analyzed by LC-MS / MS.

[0015] As the LC-MS / MS conditions for NMN measurement, the following conditions are adopted. As the LC conditions: Analysis column: Discovery HS F5 (2.1x150 mm, 3 mm) Column temperature: 40°C Mobile phase A: Water containing 0.1% (v / v) formic acid Mobile phase B: Acetonitrile Time program: Time (min) Ratio of mobile phase B (%) 0 0 5 0 15 40 15.1 100 18 100 18.1 0 25 0 Flow rate: 0.25 mL / min Injection volume: 2 μL Measurement time: 25 min As MS / MS: Triple quadrupole mass spectrometer Scan type: MRM Ion polarity: Positive and Negative Collision gas: 0.19 MPa (argon) Nebulizer gas: 2.0 L / min -1 (N2) Heater gas: 10.0 L / min -1 (N2) Ionization voltage: -3.0 kV Turbo probe temperature: 400 °C Monitor ion: Compound name Precursor ion Product ion (m / z) (m / z) NMN 335.05 123.2 97.2 80.2 78.2 41.2 10-Camphorsulfonic acid (IS) 232.85 215.1 151.15 81.1 107.2

[0016] As a result of the concentration measurement in Patent Document 1, the detection limit of NMN is as follows. NMN: 5 nmol / L

[0017] Thus, it has been reported in Patent Document 1 that highly sensitive detection of NMN was possible, but the concentrations of NAD + , NAM in the sample were not measured, and for a more detailed elucidation of the salvage pathway mechanism and for NAD + salvage pathway, no further study has been conducted on the more detailed mechanism of the NAD+ In the NMN administration study aimed at promoting the biosynthesis of + , it cannot contribute to a more accurate analysis of the temporal changes in the concentrations of NMN, NAD

Prior Art Documents

Patent Documents

[0018]

Patent Document 1

Non-Patent Documents

[0019]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0020] Therefore, an object of the present invention is to provide a method for highly sensitive measurement of the concentrations of three components, NMN, NAD + , and NAM, in a sample.

Means for Solving the Problems

[0021] To solve the above problems, the present invention has the following configuration. (1) Step A of simultaneously separating nicotinamide mononucleotide and nicotinamide adenine dinucleotide in a sample by reverse-phase liquid chromatography, and step B of separating nicotinamide in the sample, wherein in the separation step A, a mixture of a highly volatile ammonium salt solution containing an amine-based reagent as a mobile phase and an organic solvent is used. A method for measuring the concentrations of nicotinamide mononucleotide, nicotinamide adenine dinucleotide, and nicotinamide. (2) The measurement method according to (1), wherein a strong acid is added to the sample as a pretreatment for the reverse-phase liquid chromatography. (3) The measurement method according to (1) or (2), wherein a porous graphite carbon column is used in the separation steps A and B. (4) The measurement method according to any one of (1) to (3), wherein in the separation step B, a mixture of a highly volatile ammonium salt solution containing an acid as a mobile phase and an organic solvent is used. (5) The measurement method according to (4), wherein acetonitrile is used as the organic solvent of the mobile phase in the separation steps A and B. (6) In the separation steps A and B, the ratio of acetonitrile in the mobile phase is changed over time, and in the separation step A, the ratio of acetonitrile is in the range of 0 to 100% (v / v), and in the separation step B, a gradient in which the ratio of acetonitrile gradually increases in the range of 0 to 100% (v / v) is utilized. The measurement method according to (5). (7) The measurement method according to any one of (1) to (6), wherein the sample is selected from biological samples. (8) The measurement method according to (7), wherein the sample is selected from blood, urine, cerebrospinal fluid and aqueous humor, or other biological tissues. (9) The measurement method according to any one of (1) to (8), further comprising a step of quantifying nicotinamide mononucleotide, nicotinamide adenine dinucleotide, and nicotinamide separated in the separation steps A and B by mass spectrometry, respectively.

Advantages of the Invention

[0022] According to the present invention, by reverse-phase liquid chromatography, a separation step A for simultaneously separating nicotinamide mononucleotide and nicotinamide adenine dinucleotide, which are relatively close components in a sample, and a separation step B for separating nicotinamide in the sample are included. As a result, nicotinamide mononucleotide and nicotinamide adenine dinucleotide as measurement targets and nicotinamide as a measurement target can be efficiently extracted. Further, in separation step A, by using a mixture of a highly volatile ammonium salt solution containing an amine-based reagent as a mobile phase and an organic solvent, nicotinamide mononucleotide and nicotinamide adenine dinucleotide can be specifically and selectively separated. By subjecting each separated component to mass spectrometry, it becomes possible to measure the concentrations of the three components of nicotinamide mononucleotide, nicotinamide adenine dinucleotide, and nicotinamide as measurement targets with high sensitivity.

[0023] In addition, by adding a strong acid (for example, a 10 vol% perchloric acid solution in the examples described later) to the sample as a pretreatment for the reverse-phase liquid chromatography, the conversion from nicotinamide to nicotinamide adenine dinucleotide via nicotinamide mononucleotide in a biological sample can be sufficiently blocked. As a result, it becomes possible to measure the concentrations of the three components of nicotinamide mononucleotide, nicotinamide adenine dinucleotide, and nicotinamide existing in the sample with high sensitivity and high precision.

Brief Description of the Drawings

[0024]

Figure 1

Figure 2

Figure 3

BEST MODE FOR CARRYING OUT THE INVENTION

[0025] The present invention will be described in detail below together with embodiments. First, before the description of the examples regarding the specific samples described later, the method of the present invention common to these examples will be described below based on specific processing examples including pretreatment. First, the meanings of the abbreviations used in the following description are summarized in Table 1.

[0026]

Table 1

[0027] 1. Pretreatment As pretreatment, 20 μL of distilled water (substitute matrix), or blood, urine, cerebrospinal fluid, aqueous humor sample is taken into a micro test tube, and 10 vol% perchloric acid (100 μL) as a strong acid, 3 mol / L K2CO3 (35 μL), and an internal standard substance (IS; 20 μL) are added and stirred with a vortex mixer. Centrifuge at 15,000×g at 4°C for 5 minutes, and use the obtained supernatant as a sample solution for LC-MS / MS analysis.

[0028] In the above, when a biological sample (blood, urine, cerebrospinal fluid, aqueous humor) is collected and pretreatment is not immediately performed, 5 volumes of 10 vol% perchloric acid can be added immediately after the collection of blood, urine, cerebrospinal fluid, and aqueous humor, stirred with a vortex mixer, and then stored at -65°C or lower. When pretreating the stored sample, add the stirred sample (120 μL) after thawing with a vortex mixer, 3 mol / L K2CO3 (35 μL), and an internal standard substance (IS; 20 μL), and stir with a vortex mixer. Centrifuge at 15,000×g at 4°C for 5 minutes, and use the obtained supernatant as a sample solution for LC-MS / MS analysis.

[0029] In the above pretreatment, the strong acid added to the sample is not limited to the above perchloric acid, and other acids can be used, but it should deactivate the activities of conversion enzymes such as NAMPT and NMNAT. Note that the above alkaline solution is not limited to 3 mol / L K2CO3, and types, concentrations, and amounts capable of neutralizing strong acids are used. Also, in the following LC-MS / MS analysis, as the column used in separation steps A and B of the present invention, a porous graphite carbon column is preferably used. Further, in separation steps A and B of the present invention, in separation step A, a mixture of a highly volatile ammonium salt solution containing an amine-based reagent and an organic solvent is used as the mobile phase, while in separation step B, a mixture of a highly volatile ammonium salt solution containing an acid and an organic solvent is preferably used as the mobile phase. As the organic solvent of the mobile phase in separation steps A and B, for example, acetonitrile is used. In separation steps A and B, the ratio of acetonitrile in the mobile phase is changed over time. In separation step A, the ratio of acetonitrile is in the range of 0 to 100% (v / v) (preferably in the range of 5 to 40% (v / v)), and in separation step B, a gradient in which the ratio of acetonitrile gradually increases in the range of 0 to 100% (v / v) (preferably in the range of 10 to 40% (v / v)) is utilized.

[0030] 2. LC-MS / MS Conditions 2.1 NMN and NAD + Measurement The LC conditions are shown in Table 2, and the MS / MS conditions are shown in Table 3.

[0031]

Table 2

[0032]

Table 3

[0033] 2.2 NAM Measurement The LC conditions are shown in Table 4, and the MS / MS conditions are shown in Table 5.

[0034]

Table 4

[0035]

Table 5

[0036] Considering the results of the examples for the specific samples described below, from the results of the above LC-MS / MS analysis, the lower limits of detection of the concentrations of the three components of NMN, NAD + , and NAM are as shown in the following table, and high-sensitivity measurement of the concentrations of the three components was possible.

[0037]

Table 6

[0038] Next, examples for more specific samples (specific samples) will be described. First, the meanings of the abbreviations used in the following description of the examples are summarized in Table 7.

[0039]

Table 7

[0040] Example 1 (Analytical method validation) (Outline of the analytical method) A quantitative method (internal standard method) using LC-MS / MS for NMN, NAD + and NAM in biological samples was developed, and validation was carried out for the following items. After neutralizing the sample added with 10 vol% perchloric acid with K2CO3, an internal standard solution (a mixed solution of NMN-d4, NAD-d4, and NAM-d4) was added and measured by LC-MS / MS. NMN, NAD +Since NMN and NAM are endogenous compounds, calibration curves and QC samples were prepared using an alternative matrix (distilled water).

[0041] (Summary of Validation Results) NMN and NAD in biological samples (20 μL each of human plasma, human urine, human cerebrospinal fluid, human aqueous humor, monkey plasma, dog plasma, rat plasma, and mouse plasma) + had a quantification range of 5.00 - 2000 nmol / L, and NAM had a quantification range of 10.0 - 2000 nmol / L. Also, storage stability (-65°C or lower, NMN and NAD + : 160 days, NAM [human plasma, human urine, human cerebrospinal fluid, human aqueous humor, monkey plasma, rat plasma, and mouse plasma: 153 days], [dog plasma: 67 days]) was confirmed. Exemplary chromatograms are shown in Figures 1 - 3.

[0042] (Calibration Curves) Calibration curve samples were prepared using an alternative matrix (distilled water). The pretreated calibration curve samples were measured by LC - MS / MS. NMN and NAD + in the concentration range of 5.00 - 2000 nmol / L and NAM in the concentration range of 10.0 - 2000 nmol / L met the criteria for evaluation, i.e., accuracy: 85% - 115% (LLOQ: 80% - 120%), and more than 3 / 4 of the points including LLOQ and ULOQ met the above accuracy. The results are shown in Tables 8 - 10.

[0043] [Table 8]

[0044] [Table 9]

[0045] [Table 10]

[0046] (Reproducibility) QC samples (NMN and NAD + : 5.00 nmol / L to 1600 nmol / L, NAM: 10.0 nmol / L to 1600 nmol / L) were prepared using a substitute matrix (distilled water). The pretreated QC samples were measured by LC-MS / MS. The accuracy and precision of the average values at each concentration with n = 5 were calculated. NMN, NAD + and NAM met the criteria of accuracy: 85% - 115% (80% - 120% for LLQC) and precision: 15% or less (20% or less for LLQC). The results are shown in Tables 11 - 13.

[0047]

Table 11

[0048]

Table 12

[0049]

Table 13

[0050] (Lower limit of quantification) It is defined as the lowest concentration at which the calibration curve and reproducibility results meet the criteria. As shown in Table 6 above, the lower limit of quantification for NMN and NAD + was 5 nmol / L, and the lower limit of quantification for NAM was 10 nmol / L.

[0051] (Quantification in various matrices) QC samples were prepared for human plasma, human urine, human cerebrospinal fluid, human aqueous humor, monkey plasma, dog plasma, rat plasma, and mouse plasma. The pretreated QC samples were measured by LC-MS / MS. The accuracy and precision of the average values at each concentration with n = 3 were calculated. NMN, NAD +And NAM met the evaluation criteria of truth degree: 80% - 120% and accuracy: 20% or less. The results are shown in Tables 14 - 16.

[0052]

Table 14

[0053]

Table 15

[0054]

Table 16

[0055] (Dilution validity) NMN and NAD + For human aqueous humor, NAM for human cerebrospinal fluid, human aqueous humor, monkey plasma, rat plasma and mouse plasma, QC samples were prepared. These were diluted 1 / 10 or 1 / 100 with a solution prepared by mixing distilled water and 10 vol% perchloric acid at a ratio of 20:100 (v / v) to prepare diluted QC samples. The pretreated diluted QC samples were measured by LC - MS / MS. The truth degree and accuracy of the average value of each concentration with n = 3 were calculated. NMN, NAD + and NAM met the evaluation criteria of truth degree: 80% - 120% and accuracy: 20% or less. The results are shown in Tables 17 - 19.

[0056]

Table 17

[0057]

Table 18

[0058]

Table 19

[0059] (Storage stability (below -65°C)) For human plasma, human urine, human cerebrospinal fluid, human aqueous humor, monkey plasma, dog plasma, rat plasma and mouse plasma, QC samples were prepared and stored frozen at -65°C or below. After the storage period expired, the QC samples were pretreated and measured by LC-MS / MS. The residual rate relative to the initial value (before storage) was calculated from the average value of n = 3 for each concentration. When the residual rate was between 80% and 120%, which was the evaluation criterion, it was judged to be stable. The results are shown in Tables 20 to 22.

[0060]

Table 20

[0061]

Table 21

[0062]

Table 22

[0063] Example 2 (Measurement of NMN, NAD + and NAM concentrations in rat plasma samples) Using the analytical method constructed in Example 1, the concentrations of NMN, NAD + and NAM in rat plasma samples were measured, and an attempt was made to detect minute fluctuations in the concentrations of NMN, NAD + and NAM by this measurement method.

[0064] (Collection of rat plasma samples) For 7-week-old rats (5 rats), approximately 0.25 mL of blood was collected over time (0, 5, 10, 15, 30 minutes, 1, 2, and 4 hours) under fasting conditions. The blood was centrifuged (15,000×g, 5 minutes, 4°C) to prepare plasma.

[0065] (Preparation of samples for measuring NMN, NAD + and NAM) In a micro test tube, 5 volumes of 10 vol% perchloric acid was added to the above plasma and mixed. 120 μL of this mixed solution was taken into another micro test tube, 3 mol / L K2CO3 (35 μL) and an internal standard solution (IS; 20 μL) were added, and it was stirred with a vortex mixer. It was centrifuged at 15,000×g for 5 minutes at 4°C, and the obtained supernatant was used as a sample solution for LC-MS / MS analysis.

[0066] (Analysis of NMN and NAD by LC-MS / MS) + Analysis) 10 μL of the measurement sample prepared above was injected into LC-MS / MS. The analysis conditions are shown in Tables 23 and 24 below.

[0067]

Table 23

[0068]

Table 24

[0069] (Analysis of NAM by LC-MS / MS) 10 μL of the same measurement sample as above was injected into LC-MS / MS. The analysis conditions are shown in Tables 25 and 26 below.

[0070]

Table 25

[0071]

Table 26

[0072] (Concentration measurement results) The concentrations of NMN, NAD + and NAM in the rat plasma obtained under the above analysis conditions were 5.23 - 37.4 nmol / L (NMN), 0 - 26.7 mmol / L (NAD +) was 743 to 3440 nmol / L (NAM). As a result, it was verified that the fine fluctuations in the concentrations of NMN, NAD + and NAM can be detected by this measurement method. The results of quantifying the concentrations of NMN, NAD + and NAM in plasma are shown in Table 27.

[0073]

Table 27

Claims

1. Step A of simultaneously separating nicotinamide mononucleotide and nicotinamide adenine dinucleotide in a sample by reverse-phase liquid chromatography, and step B of separating nicotinamide in the sample, wherein in the separation step A, a mixture of a highly volatile ammonium salt solution containing an amine-based reagent as a mobile phase and an organic solvent is used. A method for measuring the concentrations of nicotinamide mononucleotide, nicotinamide adenine dinucleotide, and nicotinamide.

2. The measuring method according to claim 1, wherein a strong acid is added to the sample as a pretreatment for the reverse-phase liquid chromatography.

3. The measuring method according to claim 1, wherein a porous graphite carbon column is used in the separation steps A and B.

4. The measuring method according to claim 1, wherein in the separation step B, a mixture of a highly volatile ammonium salt solution containing an acid as a mobile phase and an organic solvent is used.

5. The measuring method according to claim 4, wherein acetonitrile is used as the organic solvent of the mobile phase in the separation steps A and B.

6. In the separation steps A and B, the ratio of acetonitrile in the mobile phase is changed over time. In the separation step A, the ratio of acetonitrile is in the range of 0 to 100% (v / v), and in the separation step B, a gradient in which the ratio of acetonitrile gradually increases in the range of 0 to 100% (v / v) is utilized. The measuring method according to claim 5.

7. The measuring method according to claim 1, wherein the sample is selected from biological samples.

8. The measuring method according to claim 7, wherein the sample is selected from blood, urine, cerebrospinal fluid, and aqueous humor, or other biological tissues.

9. The measuring method according to any one of claims 1 to 8, further comprising the step of quantifying nicotinamide mononucleotide, nicotinamide adenine dinucleotide, and nicotinamide separated in the separation steps A and B by mass spectrometry, respectively.

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