JPH0943153A - Nitrogen monoxide measuring method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To simply measure nitrogen monoxide with high sensitivity by reacting 2,3-diaminonaphthalene or its derivative with nitrogen monoxide under substantially neutral condition in the presence of an oxygen source, and measuring the fluorescence of the generated naphthalene triazole or its derivative. SOLUTION: 2,3-diaminonaphthalene or its derivative is reacted with nitrogen monoxide under physiologically allowed neutral condition of, for example, pH of 6.0 to 8.0 or particularly preferably pH of 6.9 to 7.5 in the presence of an oxygen source selected from a group consisting of oxygen, ozone and oxide compound. When the fluorescent of naphthalene triazole or its derivative generated by this reaction is measured, trace amount of the nitrogen monoxide can be accurately detected and determined under the physiological condition with high detecting sensitivity.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for measuring nitric oxide and a reagent for measuring nitric oxide used in the method.

[0002]

2. Description of the Related Art It has been revealed that nitric oxide (NO) is a short-lived unstable radical species and has an important function as a biologically active substance in vivo (Hyundai Kagaku, 1994
April issue special issue). Method for measuring nitric oxide, NO ₂ is oxidative degradation product of nitric oxide ^- or NO ₃ ^- and indirect method of measuring, it is roughly classified into methods of measuring the nitrogen monoxide directly. Although the direct method has attracted attention from the viewpoint of detecting and quantifying nitric oxide under physiological conditions, at present,
No specific and sensitive detection method applicable to in vitro systems has been developed.

[0003] For example, a chemiluminescence method (Palmer, RM,
et al., Nature, 327, pp.524-526, 1987), a method for measuring the absorption spectrum of metHb produced by the oxidation of oxyhemoglobin (O ₂ Hb) (Kelm, M., et al., Circ. Res. .
66, pp.1561-1575, 1990), a method of quantifying the current flowing when the electrode is inserted into tissue and oxidized (Shibuki,
K., Neurosci. Res. 9, pp. 69-76, 1990; Malinski, T.,
Nature, 356, pp. 676-678, 1992) and the grease reaction method (Green, LC, et al., Anal.Biochem., 126, pp. 13).
1-1138, 1992) are known as representative methods (for a review, Noboru Toda, "Approach from the Latest Medicine 12, NO", pp.42-52, 3.NO measurement method, Nagano Tetsuo Author,
Published by Medical View; Archer, S., FASEB J., 7, pp.
349-360, 1993).

[0004] Grease reaction method, nitrogen radicals monoxide has been generated NO ₂ oxidation ^- is a method of detecting by using a diazonium salt compound according to the azo coupling of naphthylethylenediamine. This method does not directly measure nitric oxide radicals, but has the advantage that no special equipment or technique is required. Also cadmium
(Stainton, MP, Anal.Chem., 46, p.1616, 1974; Gr
een, LC, et al., Anal.Biochem., 126, pp.131-13
8, 1982) and hydrazine (Sawicki, CR and Scaringell
i, FP, Microchem. J., 16, pp. 657-672, 1971)
₃ ^- NO ₂ ^- since it can be measured by reduction, there is also characterized in that enable the determination of nitric oxide-related metabolites.

[0005] Similar to the Griess reaction method NO ₂ ^- as a reagent for measuring nitric oxide by detecting the 2,3
Diaminonaphthalene (2,3-diaminonaphthalene) is known. This reagent, NO ₂ under acidic conditions ^- naphthalenetriazole reaction to a fluorescent adducts with (chemical name: 1-
(H) -naphtho [2,3-d] triazole) (Wiersma, J. et al.
H., Anal. Lett., 3, pp.123-132, 1970). 2,3-diaminonaphthalene and NO ₂ ^- are examined in detail the reaction conditions, the reaction proceeds fastest pH = 2 or less, completed in about 5 minutes at room temperature (Wiersma, JH, Anal . Le
tt., 3, pp.123-132, 1970; Sawicki, CR, Anal.
t., 4, pp. 761-775, 1971). The generated adduct is p
It fluoresces most efficiently at H = 10 or more (Damiani, P. and
Burini, G., Talanta, 8, pp. 649-652, 1986).

The method for measuring nitric oxide using 2,3-diaminonaphthalene has a characteristic that the detection limit is about several tens of nM and the sensitivity is 50 to 100 times higher than that of the grease reaction method ( Misko, TP, Anal. Biochem. 214, pp.11-
16, 1993). This method is also excellent in that it does not require any special equipment or technology and can be easily performed. (The above is summarized in DOJIN News, No. 74,
Information ^. NO measurement reagent: 2,3-Diaminonaphthalen
e, published by Dojin Chemical Research Institute, 1995). However, instead of nitric oxide itself is its oxide NO ₂ ^- because the are the reactive species, is an indirect method as compared to the method of measuring nitric oxide directly, also with 2,3-diaminonaphthalene nO ₂ ^- reaction from where carried out in strongly acidic under (pH = 2 or below) of, as a method of detecting and quantifying nitric oxide under physiological conditions there is a problem that can not be adopted.

[0007]

An object of the present invention is to provide a method for directly measuring nitric oxide, which is highly sensitive and simple under physiological conditions. Another object of the present invention is to provide a reagent for measuring nitric oxide used in the above method.

[0008]

Means for Solving the Problems As a result of intensive efforts to solve the above-mentioned problems, the present invention has shown that dissolved oxygen and oxide compounds (for example, PTIO and its derivatives) can be used for 2,3-diaminonaphthalene or its derivatives. It was found that by reacting nitric oxide with an oxygen source such as carboxy PTIO) under neutral conditions, a fluorescent naphthalenetriazole or its derivative is efficiently produced. It was also found that the method for measuring nitric oxide applying this reaction has extremely high detection sensitivity and can accurately quantify a trace amount of nitric oxide. The present invention has been completed based on these findings.

That is, the present invention provides a method for measuring nitric oxide, which comprises the steps of: (a) using 2,3-diaminonaphthalene or its derivative and nitric oxide as an oxygen source under substantially neutral conditions. And a step (b) of measuring the fluorescence of the naphthalenetriazole or its derivative produced in the above step. According to a preferred embodiment thereof, the method wherein the oxygen source is selected from the group consisting of oxygen, ozone, and oxide compounds; the method wherein oxygen is dissolved oxygen; and the oxide compound is
There is provided the above method, which is PTIO or a derivative thereof.

According to another aspect of the present invention, from 2,3-diaminonaphthalene or a derivative thereof which reacts with nitric oxide in the presence of an oxygen source under substantially neutral conditions to form a naphthalenetriazole or a derivative thereof. A reagent for measuring nitric oxide, and, in a preferred embodiment thereof, the reagent for measuring selected from the group consisting of oxygen, ozone, and oxide compounds; the reagent for measuring oxygen, wherein oxygen is dissolved oxygen; and oxide There is provided the above measuring reagent, wherein the compound is PTIO or a derivative thereof.

Further, a reaction reagent comprising PTIO or a derivative thereof, wherein 2,3-diaminonaphthalene or a derivative thereof is reacted with nitric oxide under substantially neutral conditions to form a naphthalenetriazole or a derivative thereof. A reagent for measuring nitric oxide for use in the method for measuring nitric oxide, comprising: (c) 2,3-diaminonaphthalene or a derivative thereof, which is a reagent for measuring nitric oxide. , (D) a kit containing PTIO which is a reaction reagent of nitric oxide and the above 2,3-diaminonaphthalene or a derivative thereof.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The method for measuring nitric oxide of the present invention comprises reacting 2,3-diaminonaphthalene or its derivative with nitric oxide under substantially neutral conditions to produce naphthalenetriazole or its derivative. And
It is characterized in that the reaction is carried out in the presence of an oxygen source.
In this specification, measurement is a concept including measurement for various purposes such as detection, quantification, and qualification. Further, the substantially neutral conditions, for example, in the range of pH 6.0 ~ 8.0, preferably pH
6.5 to 7.8, more preferably pH 6.8 to 7.6, particularly preferably pH 6.9 to 7.5, and the like, and preferably a physiologically acceptable pH range. .

2,3-Diaminonaphthalene used as a measuring reagent is commercially available and can be easily obtained. This compound is unstable to light and should be recrystallized or extracted from a solvent before use, or a high-purity reagent that is commercially available as a reagent for measuring nitric oxide under strongly acidic conditions should be used. Is. Further, the 2,3-diaminonaphthalene derivative has an unsubstituted amino group at the 2-position and 3-position of the naphthalene ring, and is one or more selected from the 1-position and 4-position to 8-position. A naphthalene derivative having an arbitrary substituent at the position can be used. Although the kind of the substituent is not particularly limited, for example, a halogen atom, C
_1-6 alkyl group, C _1-6 halogenated alkyl group, hydroxyl group, C
_{A 1-6} alkoxy group, a substituted or unsubstituted amino group, a carboxyl group, a C _1-6 alkoxycarbonyl group, a sulfonic acid group, a sulfonylamide group or the like can be preferably used. The halogen atom may be any of fluorine, chlorine, bromine or iodine, and the alkyl group or alkoxy group may be linear or branched.

More specifically, preferred compounds as the measuring reagent used in the method of the present invention are, for example, 2,3-diaminonaphthalene, 6,7-diamino-naphthalenesulfonamide and the like. However, the reagent for measurement is not limited to these, and can be appropriately selected depending on the purpose of measurement, the type of target sample, and the like. The amount of 2,3-diaminonaphthalene or a derivative thereof used for measurement is not particularly limited, and the amount of 2,3-diaminonaphthalene used for the conventionally known nitric oxide measurement method under strong acidity is the same as the amount used. (Eg Wiersma, JH, Anal. Lett., 3, p
p.123-132, 1970; Sawicki, CR, Anal. Lett., 4, pp.
761-775, 1971; Damiani, P. and Burini, G., Talant
a, 8, pp.649-652, 1986 and other publications). For measurement of a biological sample, it is suitable to use, for example, a concentration of about 10 μmol.

As the oxygen source, it is possible to use oxygen, ozone, an oxide compound, or the like. Generally, dissolved oxygen can be used as oxygen, but if necessary, oxygen gas may be introduced into the reaction system, or an oxygen generating reagent (for example, hydrogen peroxide) may be added. Good.
The oxide compound is not particularly limited as long as it is a compound having an oxide bond in which an oxygen atom is easily cleaved, such as NO, SO, PO, and for example, PTIO (2-phenyl-4,4,
5,5-Tetramethylimidazoline-1-oxyl-3-oxide: Maeda, H., et al., J. Leuk. Biol., 56, pp.58
8-592, 1994; Akaike, T., et al., Biochemistry, 32,
pp.827-832, 1993) or its derivative (carboxyl PTIO having a carboxyl group introduced at the p-position of the phenyl group of PTIO).
Etc.), triphenylphosphine oxide, triethylamine oxide and the like can be used.

Whether or not the oxide compound can be suitably used in the measuring method of the present invention can be appropriately assayed by those skilled in the art by the method described in detail in Examples. Of the above oxide compounds, PTIO and its derivatives (eg carboxy PT
IO and the like) are particularly preferable compounds and compounds easily available to those skilled in the art (Tokyo Kasei Co., Ltd., Organic).
Chemicals Catalog, 32, 1994 etc.). The oxide compound itself may be used as a reaction reagent, but a compound encapsulated in a liposome or the like can also be used.

In the above reaction, the amount of the oxygen source coexisted in reacting 2,3-diaminonaphthalene and nitric oxide is not particularly limited, but at least 1 μmol or more, preferably at least 1 mol based on nitric oxide to be measured. The amount is preferably 10 to 30 μmol, more preferably about 10 to 20 μmol. For measurement of biological samples, etc.
Although it is preferable to add a molar amount, the required amount of oxygen source is generally supplied by dissolved oxygen. If the amount of the oxygen source is extremely small, the measurement sensitivity may decrease, and if the amount of the oxygen source is extremely large, there may be a problem in the emission of light by fluorescence. Therefore, it is preferable to predict the amount of nitric oxide to be measured by a preliminary test or a known method and add an oxygen source in an appropriate concentration range. Reaction is 10 ~ 25 ℃
It is possible to perform in the temperature range of.

The naphthalenetriazole or its derivative formed by the reaction of 2,3-diaminonaphthalene or its derivative with nitric oxide may be measured according to a conventionally known fluorescence measuring method (eg, Wiersma, JH, Ana
l. Lett., 3, pp.123-132, 1970; Sawicki, CR, Ana
l. Lett., 4, pp.761-775, 1971; Damiani, P. and Bur
ini, G., Talanta, 8, pp. 649-652, 1986; Damiani, P.
and Burini, G., Talanta, 8, pp.649-652, 1986; Mis
ko, TP, Anal. Biochem. 214, pp. 11-16, 1993, etc.). Hereinafter, the present invention will be described more specifically with reference to Examples, but the scope of the present invention is not limited to the following Examples.

[0019]

【Example】

Example 1: Production of reagent for measuring nitric oxide (6,7-diaminonaphthalenesulfonamide) of the present invention 2,3-diaminonaphthalene was dissolved in anhydrous tetrahydrofuran, trifluoroacetic anhydride was added to this solution, and the solution was fed under an argon stream. The mixture was stirred for 2-3 hours to form 2,3-di- (trifluoroacetylamido) naphthalene. Post-treatment was carried out by a conventional method, and the obtained crude crystals were recrystallized from benzene. The 2,3-di- (trifluoroacetylamido) naphthalene was treated with chlorosulfone to chlorsulfonate the 5-position of the naphthalene ring, and the obtained crystals were recrystallized from chloroform. This compound was converted to a sulfonamide form by treatment with ammonia, and recrystallized from water-ethanol.
6,7-Diaminonaphthalenesulfonamide (mp 183-184
℃, decomposition) was obtained. C ₁₀ H ₁₁ N ₃ SO ₂ : Molecular weight 237.29, M ⁺ 237 ¹ H-NMR (d ⁶ -DMSO, 400 MHz) 5.22 (s, 2H, 6-NH ₂ ), 5.25
(s, 2H, 7-NH ₂ ), 6.90 (s, 1H, 8-H), 7.04 (t, 1H, J = 7.7Hz, 3-
H), 7.19 (s, 2H, SO ₂ NH ₂ ), 7.55 (s, 1H, 5-H), 7.57 (d, 1H, J
= 7.7Hz, 2-H), 7.61 (d, 1H, J = 7.7Hz, 4-H)

When the above 6,7-diaminonaphthalenesulfonamide was reacted with nitric oxide in the presence of oxygen under the following conditions, a triazole ring was formed at the 6,7-position. A compound having
The hydrogen atom numbers in the data correspond to the case where the three nitrogen atoms of the triazole are at the 1-, 2-, and 3-positions, respectively, and the sulfonylamide group is at the 5-position). C ₁₀ H ₈ N ₄ SO ₂ : molecular weight 248.26, M ⁺ 248 ¹ H-NMR (d ⁶ -DMSO / D ₂ O, 400 MHz) 7.59 (dd, 1H, J = 7.3Hz,
8.8Hz, 7-H), 8.16 (d, 1H, J = 7.3Hz, 6-H), 8.39 (d, 1H, J = 8.
8Hz, 8-H), 8.73 (s, 1H, 4-H), 9.16 (s, 1H, 9-H)

Example 2 NOC which is a spontaneous NO generator as a source of nitric oxide
(Hrabie, JA, J. Org. Chem., 58, pp. 1472-1476,
1993), NOC-10 (half-life of 40 minutes) was used, and nitric oxide produced in the reaction solution was reacted with 2,3-diaminonaphthalene using PTIO which is an oxide compound as an oxygen source.
Phosphate buffer was used as the reaction solvent, 2,3-diaminonaphthalene (manufactured by Dojindo Laboratories Ltd.) 0.1 mM, pH of reaction solution
The reaction was performed at 25 ° C. as 7.4. In order to remove the influence of oxygen, the reaction was carried out using a solvent (completely degassed state) in which argon gas was blown and degassed for 1 hour. An increase in fluorescence intensity was observed by the method of adding the degassed NOC-10 solution and PTIO solution into the degassed fluorescence cell. As a control, the same reaction was performed in the absence of PTIO using a solvent containing dissolved oxygen before degassing. The results of changing the concentration of nitric oxide in the reaction solution by changing the concentration of NOC-10 are shown in Figures 1 to 3 (final concentration of NOC-10 is 100 μM and 150 μM, respectively).
And 250 μM results are shown).

Example 3 Mouse-derived macrophages (RAW264) were cultured in a 24-well plate at a concentration of 1 × 10 ⁶ cells / well for about half a day, and the culture solution was mixed with LPS (10 μg / ml) and INF-γ (100 U). (/ ml) was exchanged for a culture solution mixed with the mixture and stimulation was applied. Then, wait a few hours, L-Arg (1mM), 2,3-diaminonaphthalene, PTIO (
200 μl in total) was dissolved in Krebs-Ringer-phosphate buffer (KRP) and further cultured. After a certain period of time, collect the culture solution (supernatant KRP) and measure it with a fluorometer (ex. 375 n
m, em. 425 nm). 2,3-diaminonaphthalene 10
μM, L-Arg 1 mM, BSA 0.2% was added to all reaction solutions, and PTIO10 μM, Cu, Zn-SOD (copper, zinc superoxide dismutase) 1 mg / ml was added to 1: PTIO (-), SOD ( -);
2: PTIO (+), SOD (-); 3: PTIO (-), SOD (+); 4: PTIO
(+) And SOD (+) are added. FIG. 4 shows the results. P
It was shown that the addition of TIO increased the measurement sensitivity 2-3 times.

As a control experiment, PTIO 10 μM and L-Arg 1 mM were added without addition of 2,3-diaminonaphthalene, and the measurement was performed 9 hours after stimulation. It was clear that the increased fluorescence obtained was from 2,3-diaminonaphthalene. Also,
For the culture solution without stimulation and without the addition of L-Arg, 2,
When the measurement was performed in the presence of 10 μM of 3-diaminonaphthalene and 10 μM of PTIO, almost no increase in fluorescence intensity was observed. In addition, 2,3-diaminonaphthalene 60 μM, PT
L-, which is a nitric oxide synthase (NOS) inhibitor, was added to the culture medium for 9 hours after stimulation with the addition of IO 20 μM and L-Arg 1 mM.
When NMMA (N-ω-methyl-L-arginine) was added and the fluorescence intensity was measured 2 hours later, a decrease in fluorescence intensity was observed as the concentration of L-NMMA increased, and 20 mM L-NMMA
The fluorescence intensity decreased to about 1/20 in the presence of. From these results, it is clear that the increase in fluorescence intensity in the results shown in FIG. 4 is derived from nitric oxide.

[0024]

INDUSTRIAL APPLICABILITY The measuring method of the present invention is useful because it can measure nitric oxide highly sensitively and easily under physiological conditions.

[Brief description of drawings]

FIG. 1 is a diagram showing the results of measuring nitric oxide by the method of the present invention using NOC-10 at a final concentration of 100 μM.

FIG. 2 is a diagram showing the results of measuring nitric oxide by the method of the present invention using NOC-10 at a final concentration of 150 μM.

FIG. 3 is a diagram showing the results of measuring nitric oxide by the method of the present invention using NOC-10 at a final concentration of 250 μM.

FIG. 4 is a diagram showing the results of measuring nitric oxide in macrophages by the measuring method of the present invention. In the figure, □:
PTIO (-), SOD (-); ◇: PTIO (+), SOD (-); ○: PTIO
(-), SOD (+); △: shows the results of PTIO (+) and SOD (+).

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Hirokazu Kojima 4-19, Mejiro 2-chome, Toshima-ku, Tokyo Adachikata

Claims (7)

[Claims] 1. A method for measuring nitric oxide, which comprises the steps of: (a) 2,3-diaminonaphthalene or a derivative thereof and nitric oxide in the presence of an oxygen source under substantially neutral conditions. A step of reacting; and (b) a step of measuring the fluorescence of the naphthalenetriazole or its derivative produced in the above step. 2. The method of claim 1, wherein the oxygen source is selected from the group consisting of oxygen, ozone, and oxide compounds. 3. The method according to claim 2, wherein the oxide compound is PTIO or a derivative thereof. 4. A reagent for measuring nitric oxide comprising 2,3-diaminonaphthalene or its derivative, which reacts with nitric oxide in the presence of an oxygen source under substantially neutral conditions to produce naphthalenetriazole or its derivative. . 5. The measuring reagent according to claim 4, wherein the oxygen source is selected from the group consisting of oxygen, ozone, and an oxide compound. 6. A reaction reagent comprising PTIO or a derivative thereof, wherein naphthalenetriazole or a derivative thereof is formed by reacting 2,3-diaminonaphthalene or a derivative thereof with nitric oxide under substantially neutral conditions. For reaction reagents. 7. A kit for measuring nitric oxide for use in the method for measuring nitric oxide according to claim 3, wherein (c) a reagent for measuring nitric oxide, 2,3-diaminonaphthalene or a derivative thereof. And (d) PTIO which is a reaction reagent of nitric oxide with the above 2,3-diaminonaphthalene or its derivative
Or a kit containing the derivative thereof.