JP2002223794A - Method for measuring activity of platelet activating factor acetyl hydrolase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for safely, rapidly, simply, accurately and directly measuring activity of a platelet activating factor(PAF) acetyl hydrolase in high precision, using PAF analogue compound containing a coloring substituent group in the structure as a substrate. SOLUTION: The activity of platelet activating factor acetyl hydrolase can safely, rapidly, simply, correctly, highly accurately and directly be measured by using PAF analogue compound as a substrate in the presence of an inhibitor which is an esterolytic activity related substance other than PAF acetyl hydrolase.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

FIELD OF THE INVENTION The present invention relates to a method for measuring platelet activating factor acetylhydrolase activity which is clinically important, a reagent for measuring platelet activating factor acetylhydrolase activity, and a platelet activating factor acetylhydrolase utilizing the reagent. The present invention relates to an activity measuring kit.

[0002]

2. Description of the Related Art Platelet activatin
g Factor: 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (hereinafter abbreviated as PAF)
It is a mediator for a wide range of onset of various disease states such as inflammation and allergic reactions. On the other hand, platelet activating factor acetylhydrolase (hereinafter referred to as PAF-AH)
It has a function to eliminate the strong biological activity of F,
It regulates PAF levels in vivo and is involved in maintaining homeostasis in living organisms. And Kawasaki disease, systemic lupus erythematosus,
In diseases such as hemolytic uremic syndrome, with the progression of these conditions, serum PAF-
Variations in AH activity have been observed.

[0003] Thus, PAF- in serum or plasma
Since AH activity reflects the onset and pathological changes of various inflammatory patients, measurement of PAF-AH activity is important as a novel clinical test method that provides useful information for diagnosis of these diseases. Furthermore, there are patients with serum PAF-AH deficiency, and this enzyme deficiency is regarded as a risk factor for severe pediatric asthma, suggesting that measurement of PAF-AH activity is important from the viewpoint of preventive medicine. ing.

Hitherto, PAF-AH activity has been measured by a bioassay method or a method using a radioisotope.

[0005] In the bioassay method, PAF-AH is reacted with PAF as a substrate for a certain period of time, and the ability of the remaining PAF to aggregate platelets is monitored.
However, there are drawbacks in that platelets must be prepared at the time of use and expensive reagents have to be used, and there is a problem that is difficult to use as a general test method.

On the other hand, a method using a radioisotope (RI
Method A) is to react the PAF-AH by labeling the acetyl group of PAF with ³ H or ¹⁴ C and measure the released radioactivity, or to label the alkyl group and react it with PAF-AH. , The reaction product lyso-PAF or unreacted P
This is a method of collecting AF and measuring radioactivity. However, this method using radioisotopes limits the users and equipment of radioactive materials, and also raises safety issues,
There are problems such as disposal of radioactive reagents. Furthermore, when collecting labeled lyso-PAF or unreacted labeled PAF and measuring the remaining radioactivity, lyso-PAF
Alternatively, there is a problem that it is difficult to use PAF as a general inspection method, such as a complicated operation such as extraction of PAF with an organic solvent and separation by chromatography.

[0007] Therefore, a method for measuring PAF-AH activity without requiring such complicated operations has been studied. One of the methods is a method using a synthetic substrate. In recent years, various PAF-AH substrates and PAF-AH
Have been developed.

For example, Japanese Patent Application Laid-Open No. 7-59597 describes a method for measuring PAF-AH activity using a PAF-like compound which releases a dicarboxylic acid by the action of PAF-AH as a substrate. In this method, PAF-AH in a sample is allowed to act on the substrate to generate a dicarboxylic acid, and then an enzyme that specifically reacts with the generated dicarboxylic acid is caused to act. ⁺ , NADP ⁺ , NADH, NADPH, hydrogen peroxide, or other commonly used marker substances, and the production rate or amount of dicarboxylic acid is determined by measuring these marker substances by a known method. Is a method of measuring PAF-AH activity by Since this method is an enzymatic measurement method in which a large number of enzymes are combined with a detection system, there are problems such as stability and specificity of the enzyme to be used, and it is necessary to pay attention to maintaining the performance of the reagent. And so on.

[0009] JP-A-4-346797 describes a method for measuring PAF-AH activity using thioPAF and a thioPAF analog compound as substrates. This method uses a color reagent that reacts with an SH group and quantitatively develops a color. This publication states that the lysothio PAF produced from a substrate such as thio PAF by PAF-AH
As an example, a method of following the yellow color of 2-nitro-5-mercaptobenzoic acid released by a substitution reaction between an H group and 5,5′-dithiobis (2-nitrobenzoic acid) is described. Susceptible to interference from other thiol compounds or redox substances coexisting in the reaction solution or sample, such as ascorbic acid, bilirubin, cysteine or glutathione, and has a problem in maintaining the performance of reagents as in the enzymatic assay. There is.

Further, a PAF-like compound, 1-dodecanoyl-2- (4-nitrophenylglutaryl) -sn
-A method for measuring PAF-AH activity using glycero-phosphocholine as a substrate is known (Arteriosclerosis, Thrombos
is, and Vascular Biology Vol. 16, No 4 591-599 (199
6)). However, in this assay, since the substrate is also hydrolyzed by various esterases or esterase-like substances present in the sample such as plasma or serum, the measured value is greatly positively affected, and accurate PAF-
Measurement of AH activity was difficult.

[0011]

Accordingly, a method for measuring PAF-AH activity which is not affected by various esterases or esterase-like substances, which is safe and easy to use without using a radioactive substrate. A direct measurement method was desired. That is, there has been a demand for the development of a measurement system that is less affected by a substrate having high specificity for PAF-AH or various esterases or esterase-like substances.

[0012]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, have found that the effects of substrates having relatively high specificity on PAF-AH and various esterases and esterase-like substances are considered. PAF-AH without receiving
The present inventors have found a measurement system, developed a safe and easy-to-use method for directly measuring PAF-AH activity without using a radioactive substrate, and completed the present invention.

The present invention relates to a method for measuring PAF-AH activity, which comprises the general formula (I):

[0014]

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(Wherein R ₁ represents an acyl group, an alkyl group or an alkenyl group, and A represents a saturated or unsaturated, substituted or unsubstituted hydrocarbon having 2 to 7 carbon atoms which may be interposed by an oxygen atom. X represents a group that becomes a coloring compound or a fluorescent coloring compound when released, and R ₂ represents a monomethylaminoethyl group, a dimethylaminoethyl group, or a trimethylaminoethyl group.) And a platelet-activating factor acetylhydrolase-containing sample, in the presence of an inhibitor that does not inhibit platelet-activating factor acetylhydrolase, but inhibits other esterolytic activity-related substances, and reacts to thereby release a chromogenic compound. Or a method comprising measuring the amount of a fluorescent compound.

In a preferred embodiment, the color-forming compound is an aromatic hydroxy compound or an aromatic thiol compound.

In a preferred embodiment, the inhibitor used in the measurement method of the present invention is selected from the group consisting of an anionic surfactant, a nonionic surfactant, a bile salt, a bile salt derivative and a chelating agent. At least one inhibitor.

In a preferred embodiment, the anionic surfactant used in the measurement method of the present invention is an alkali metal salt of an alkyl sulfate or an alkali metal salt of an alkyl sulfonic acid.

In a preferred embodiment, the nonionic surfactant used in the measurement method of the present invention is a polyoxyethylene alkyl allyl ether or a polyoxyethylene sorbitan fatty acid ester.

In a preferred embodiment, the bile salts and bile salt derivatives used in the measurement method of the present invention are sodium cholate, sodium deoxycholate, sodium chenodeoxycholate, sodium dehydrocholate, taurocholate. Sodium, sodium taurolithocholic acid, sodium taurodeoxycholate, sodium taurochenodeoxycholate, sodium tauroursodeoxycholate, sodium taurodehydrocholate, CHAPS, CHAPSO, BIGC
HAP, or deoxy-BIGCHAP.

The present invention also relates to a reagent for measuring PAF-AH activity, which is represented by the general formula (I):

[0022]

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(Wherein, R ₁ represents an acyl group, an alkyl group or an alkenyl group, and A represents a saturated or unsaturated, substituted or unsubstituted hydrocarbon having 2 to 7 carbon atoms which may be interposed by an oxygen atom. X represents a group that becomes a coloring compound or a fluorescent coloring compound when released, and R ₂ represents a monomethylaminoethyl group, a dimethylaminoethyl group, or a trimethylaminoethyl group.) And PAF-
And an inhibitor that does not inhibit AH but inhibits other esterolytic activity-related substances.

In a preferred embodiment, PAF-AH
The aromatic hydroxy compound or aromatic thiol compound is released from the substrate by the reaction with the compound.

In a preferred embodiment, the inhibitor contained in the reagent of the present invention is selected from the group consisting of an anionic surfactant, a nonionic surfactant, a bile salt, a bile salt derivative and a chelating agent. At least one inhibitor.

In a preferred embodiment, the anionic surfactant contained in the reagent of the present invention is an alkali metal salt of an alkyl sulfate or an alkali metal salt of an alkyl sulfonic acid.

[0027] In a preferred embodiment, the nonionic surfactant contained in the reagent of the present invention is a polyoxyethylene alkyl allyl ether or a polyoxyethylene sorbitan fatty acid ester.

In a preferred embodiment, the bile salts and bile salt derivatives contained in the reagent of the present invention are sodium cholate, sodium deoxycholate, sodium chenodeoxycholate, sodium dehydrocholate, sodium taurocholate. , Sodium taurolithocholic acid, sodium taurodeoxycholate, sodium taurochenodeoxycholate, sodium tauroursodeoxycholate, sodium taurodehydrocholate, CHAPS, CHAPSO, BIGCHAP, or deoxy-BIGCHAP.

The present invention further relates to a kit for measuring platelet activating factor acetylhydrolase activity, comprising the above-mentioned reagent.

The present invention also provides a kit for measuring PAF-AH activity, which comprises the above-mentioned general formula (I):

[0031]

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(Wherein R ₁ represents an acyl group, an alkyl group or an alkenyl group, and A is a saturated or unsaturated, substituted or unsubstituted hydrocarbon having 2 to 7 carbon atoms which may be interposed by an oxygen atom. X represents a group that becomes a coloring compound or a fluorescent coloring compound when released, and R ₂ represents a monomethylaminoethyl group, a dimethylaminoethyl group, or a trimethylaminoethyl group.) And a container containing a solution containing an inhibitor that does not inhibit PAF-AH but inhibits other esterolytic activity-related substances.

In a preferred embodiment, PAF-AH
The aromatic hydroxy compound or aromatic thiol compound is released from the substrate by the reaction with the compound.

[0034] In a preferred embodiment, the substrate is
Dissolved in solution or solvent.

[0035] In another preferred embodiment, the kit of the present invention further comprises a container containing a solution or a solvent for dissolving the substrate.

Further, the present invention provides a compound represented by the general formula (Ia):

[0037]

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Where R₁Is an acyl having 6 to 20 carbon atoms
X represents an alkyl group or an alkenyl group;
Sometimes a group that becomes a coloring compound or a fluorescent coloring compound
Then R ₂Is a monomethylaminoethyl group, dimethylamino
Represents an ethyl group or a trimethylaminoethyl group. )
I do.

[0039] In a preferred embodiment, the color-forming compound is an aromatic hydroxy compound or an aromatic thiol compound.

The present invention also relates to a method for measuring PAF-AH activity, and relates to a compound represented by the general formula (Ia):

[0041]

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(Wherein, R ₁ represents an acyl group, an alkyl group or an alkenyl group having 6 to 20 carbon atoms, X represents a group which becomes a color-forming compound or a fluorescent color-forming compound when released, and R ₂ represents A substrate represented by a monomethylaminoethyl group, a dimethylaminoethyl group or a trimethylaminoethyl group) is reacted with a PAF-AH-containing sample, and the amount of the coloring compound or fluorescent coloring compound released thereby is determined. The present invention relates to a measuring method including measuring.

In a preferred embodiment, the color-forming compound is an aromatic hydroxy compound or an aromatic thiol compound.

According to the above-mentioned invention, a substrate having relatively high specificity for PAF-AH and a system for directly measuring PAF-AH which is not affected by various esterases or esterase-like substances are provided. Furthermore, since the present invention does not use a radioactive substrate, it provides a method which can be used as a safe, rapid and simple measurement method for daily inspection. As described above, the method for measuring PAF-AH of the present invention comprises (1) a shorter measurement time than conventional methods, (2) not affected by various esterases and esterase-like substances, and (3) a chromogenic compound. Alternatively, it has the advantage that the PAF-AH activity can be measured directly by monitoring the fluorescent chromogenic compound, so that it can be measured more accurately than the conventional method using enzymes or reagents that lead to a chromogenic system. is there. Therefore, the present invention is a highly useful invention because it allows the diagnosis of disease and the progress of prognosis to be diagnosed in a short time.

Further, the present invention provides a reagent for measuring PAF-AH and a kit containing the same. Since PAF-AH activity can be measured directly and easily with these reagents and kits, it is possible to diagnose a disease based on a change in PAF-AH activity and grasp the progress of the disease state. Therefore, the contribution of the present invention in medicine is great.

[0046]

BEST MODE FOR CARRYING OUT THE INVENTION (Features of Measurement Method of the Present Invention) Platelet activating factor acetylhydrolase (PAF) of the present invention
The characteristic of the method for measuring -AH) activity is that, by adding an inhibitor that inhibits a substance related to ester degradation activity other than PAF-AH, together with the substrate represented by the general formula (I),
The object is to prevent the release of a coloring compound or a fluorescent coloring compound due to non-specific hydrolysis by these ester-decomposing activity-related substances, and to measure the PAF-AH activity more accurately. That is, the color-forming or fluorescent-color-forming compound released from the substrate is converted to PAF-A as much as possible.
The purpose of the present invention is to design the measurement system so as to be derived only from H. Further, when the substrate represented by the general formula (Ia) is used among the substrates represented by the general formula (I), the measurement can be performed with higher sensitivity.

When the substrate used in the present invention reacts with PAF-AH, it releases a dicarboxylic acid derivative bound to a chromogenic or fluorescent compound, and when the dicarboxylic acid derivative is released, it develops a chromogenic or fluorescent compound. It has the characteristic of releasing (releasing) a coloring compound. That is, when the substrate used in the present invention reacts with PAF-AH, a color-forming or fluorescent-colored substituted aromatic compound is released in an amount proportional to the PAF-AH activity. By monitoring sex compounds, PAF-AH activity can be measured directly.

The conventional measuring method is to introduce a dicarboxylic acid generated by contacting a substrate with PAF-AH enzymatically into a color-forming system, or to replace the dicarboxylic acid with 5,5′-dithiobis (2-nitrobenzoic acid). Through the color system of free 2-nitro-5-mercaptobenzoic acid, and not a direct color, so that not only the decomposition of the substrate itself, but also the deactivation of the enzyme and the composition leading to other color systems There were many factors related to measurement inaccuracies, such as deterioration, and the PAF-AH activity value could not be obtained accurately.

The measuring method of the present invention has a special effect of solving these problems. That is, in the measurement method according to the present invention, a chromogenic or fluorescent chromogenic compound that can be directly detected by PAF-AH is generated, so that the measurement error due to the ester-decomposing activity-related substances contained in serum samples, plasma samples, and the like. Is made as small as possible, and the PAF-AH activity can be accurately measured.

As used herein, the term "substances related to ester degradation activity" includes enzymes or substances that cleave ester bonds, such as esterases (esterases) and esterase (esterase) -like substances. Examples of esterase (esterase) -like substances include, for example,
Proteins having an esterase activity, such as apolipoprotein B contained in LDL, may be mentioned.

Hereinafter, the substrate used in the present invention, the method for producing the same, the inhibitor used in the present invention, the reagent for measuring PAF-AH, the measuring method using the reagent, and the kit for measuring PAF-AH will be sequentially described. .

(Substrate Used in the Present Invention) The PAF-
The substrate used in the AH measurement method is represented by the general formula (I). General formula (I):

[0053]

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In the formula, R ₁ is preferably an acyl group, an alkyl group or an alkenyl group. The number of carbon atoms is preferably about 6 to about 20. More preferably, from about 8 to about 18.

A is preferably a saturated or unsaturated, substituted or unsubstituted hydrocarbon group having 2 to 7 carbon atoms. Among them, compounds having 2 carbon atoms are preferred. Further, the hydrocarbon group may be interposed with an oxygen atom. That is, -COC
-And the like.

X represents a reaction between PAF-AH and a substrate,
It is a compound that is released (released) when the dicarboxylic acid derivative is released, and preferably has a coloring property or a fluorescent coloring property. X is preferably a group that becomes an aromatic hydroxy compound or an aromatic thiol compound when hydrolyzed, for example, a 4-nitrophenyl group, a 2-chloro-4-nitrophenyl group, a 2-fluoro- 4-nitrophenyl group, 3-fluoro-4-nitrophenyl group, 4-
Nitrothiophenyl group, 5-indolyloxy group, 2,
And a 6-diphenyl-4-nitrophenyl group.

Examples of the fluorescent coloring compound include a 4-methylcoumaryl group, a coumaryl group, and a flavoneoxyl group.

Examples of R ₂ include a monomethylaminoethyl group, a dimethylaminoethyl group and a trimethylaminoethyl group. Trimethylamino groups are preferred.

Among the substrates, those having 2 carbon atoms of A are represented by the general formula (Ia).

[0060]

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In the general formula (Ia), R₁, X, and R
₂Is, respectively, the R₁, X, and R ₂Same as
You. As the compound represented by the general formula (Ia), for example, 1
-Myristoyl-2- (4-nitrophenylsuccini
G) -sn-glycero-phosphocholine is preferred. this
The compound is 1-myristo when A has 3 carbon atoms.
Yl-2- (4-nitrophenylglutaryl) -sn-
4 times more sensitive than glycerophosphocholine
It has the effect of doing.

Examples of the substrate used in the present invention include the following substrates in addition to the above-mentioned substrates, but the substrate is not limited thereto. 1-octanoyl-2- (4-nitrophenylglutaryl) -sn-glycero-phosphocholine,
1-decanoyl-2- (4-nitrophenylglutaryl) -sn-glycero-phosphocholine, 1-lauroyl-2- (4-nitrophenylglutaryl) -sn-glycero-phosphocholine, 1-myristoyl-2- (4 -Nitrophenylglutaryl) -sn-glycero-phosphocholine, 1-oleoyl-2- (4-nitrophenylglutaryl) -sn-glycero-phosphocholine, 1-alkyl-2- (4-nitrophenylglutaryl)- sn-glycero-phosphocholine, 1-alkenyl-2- (4-nitrophenylglutaryl) -sn-glycero-phosphocholine 1-myristoyl-2- (4-nitrophenyladipoyl) -sn-glycero-phosphocholine, 1- Myristoyl-2- (4-nitrophenylpimeloyl) -sn
-Glycero-phosphocholine, 1-myristoyl-2-
(4-nitrophenylsuberoyl) -sn-glycero-
Phosphocholine, 1-myristoyl-2- (4-nitrophenylazoloyl) -sn-glycero-phosphocholine,
1-myristoyl-2- (2-chloro-4-nitrophenylglutaryl) -sn-glycero-phosphocholine,
-Myristoyl-2- (4-methylcoumarylglutaryl) -sn-glycero-phosphocholine, 1-myristoyl-2- (2-fluoro-4-nitrophenylglutaryl) -sn-glycero-phosphocholine, 1-myristoyl-2 -(3-fluoro-4-nitrophenylglutaryl) -sn-glycero-phosphocholine, 1-myristoyl-2- (4-nitrothiophenylglutaryl) -sn
-Glycero-phosphocholine, 1-myristoyl-2-
(5-Indolyloxyglutaryl) -sn-glycero-phosphocholine, 1-myristoyl-2- (2,6-diphenyl-4-nitrophenylglutaryl) -sn-glycero-phosphocholine, 1-myristoyl-2- ( 4-
(Nitrophenylglycolyl) -sn-glycero-phosphocholine, 1-myristoyl-2- [5-dimethylamino-2- (2-thiazolylazo) phenylsuccinyl]
-Sn-glycero-phosphocholine, 1-myristoyl-
2- [1- (2-triazolylazo) -2-naphthylsuccinyl] -sn-glycero-phosphocholine, 1-myristoyl-2-[(4-nitrophenyl) dimethylsuccinyl] -sn-glycero-phosphocholine, 1-myristoyl- 2-[(6-flavonoxyl) succinyl]
-Sn-glycero-phosphocholine, and 1-palmitoyl-2- (4-nitrophenylsuccinyl) -sn-glycero-phosphocholine.

(Method for Producing Substrate Used in the Present Invention) The substrate for PAF-AH used in the present invention can be produced according to a method known per se. Details will be described in a synthesis example described later.
For example, the method described in JP-A-52-89622 discloses 1-acylglycerophosphocholine, 1-O-alkylphosphocholine (commercially available lysoPAF), 1-O-alkenylphosphocholine obtained using phospholipase A2. Choline (commercially available lysophosphocholine plasmalogen) or the like is used as a starting material, and these starting materials are reacted with dicarboxylic anhydride (for example, succinic anhydride) in a mixed solution of anhydrous chloroform and anhydrous dichloromethane using triethylamine as a catalyst, Introducing the desired dicarboxylic acid at position 2,
1-Acyl-2-monodicarbonyl-phosphocholine is obtained. When succinic anhydride is used, 1-acyl-2-succinyl-phosphocholine is obtained.

The thus obtained 1-acyl-2-
Monodicarbonyl-phosphocholine was purchased from William N. Washb.
urn et al., J. Am. Chem. Soc. 112, 2040-2041 (199
According to the description of 0), the chromophore aromatic hydroxy compound such as p-nitrophenol is esterified in the presence of triethylamine to give 1-acyl-2- (4-nitrophenylsuccinyl) -phosphocholine. obtain.

When a dicarboxylic acid having a long methylene group at the 2-position is introduced, a monobenzylcarboxylic acid ester obtained by reacting a dicarboxylic anhydride with a protecting group benzyl alcohol (4th edition Experimental Chemistry Lecture 22 Organic Synthesis IV) 131) can be used. Monobenzyl carboxylate is reacted with 1-acylglycerophosphocholine by the method described in JP-A-52-89622 to obtain 1-acyl-2-monobenzylcarbonylphosphocholine. Next, the obtained monobenzyl compound is debenzylated using palladium / carbon under a hydrogen gas atmosphere to obtain 1-acyl-2-monocarbonylphosphocholine. 1 obtained in this way
-Acyl-2-monocarbonylphosphocholine is chlorinated with oxalyl chloride, and an aromatic hydroxy compound as a chromophore such as p-nitrophenol is esterified in the presence of triethylamine to give 1-acyl-2- (4
-Nitrophenylazolyl) -phosphocholine is obtained.

(Inhibitor used in the present invention) As a measuring method of the present invention, a method which does not inhibit PAF-AH, but is preferably carried out in the presence of an inhibitor which inhibits another ester degradation activity-related substance is preferable. Here, “an inhibitor that does not inhibit PAF-AH but inhibits other ester degradation activity-related substances”
Means that it does not inhibit PAF-AH but inhibits other substances related to esterification activity.
It does not inhibit F-AH, but includes both meanings as inhibitors which inhibit other related substances for esterolytic activity.

In the measurement of the PAF-AH activity, the ester bond of the substrate is non-specifically hydrolyzed by another ester-decomposing activity-related substance present in the serum or plasma sample, and the compound represented by the general formula (I) or (Ia) (E.g., a chromogenic compound) is undesirably released, resulting in noise of PAF-AH activity. Therefore, when measuring PAF-AH of a serum or plasma sample, PAF-AH
However, it is preferable that an inhibitor (hereinafter also referred to as a non-specific reaction inhibitor) that inhibits other ester degradation activity-related substances (activity) is present.

Examples of such inhibitors include, but are not limited to, anionic surfactants, nonionic surfactants, bile salts, bile salt derivatives, and the like. One inhibitor may be used alone, or two or more inhibitors may be used in combination. Preferred combinations are anionic surfactants and nonionic surfactants, or anionic surfactants and bile salts or bile salt derivatives. Two or more inhibitors of the same type may be used.

Examples of the anionic surfactant include fatty acid salts (eg, sodium stearate, potassium oleate, etc.), alkyl sulfate salts (eg, sodium lauryl sulfate, lithium lauryl sulfate, etc.), and alkylbenzene sulfonates (eg, Sodium laurylbenzenesulfonate, sodium 4-n-octylbenzenesulfonate), alkylnaphthalenesulfonates (eg, sodium 2-naphthalenesulfonate), alkylsulfosuccinates (eg, sodium dialkylsulfosuccinate), alkyl Diphenyl ether disulfonate (eg, sodium alkyl diphenyl ether disulfonate), alkyl phosphate (eg, potassium alkyl phosphate), polyoxyethylene alkyl sulfate (eg, For example, sodium polyoxyethylene lauryl ether sulfate, polyoxyethylene lauryl ether triethanolamine sulfate), polyoxyethylene alkyl allyl sulfate (eg, sodium polyoxyethylene nonyl phenyl ether sulfate), alkyl sulfonate (eg, , Sodium octanesulfonate, sodium nonanesulfonate, sodium decanesulfonate, sodium tridecanesulfonate, etc.), naphthalenesulfonic acid formalin condensate (eg, sodium salt of β-naphthalenesulfonic acid formalin condensate, etc.), polyoxyethylene Preferably, it is at least one anionic surfactant selected from the group consisting of alkyl phosphate esters.

Examples of the nonionic surfactant include polyoxyethylene alkyl ether (for example, polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene stearyl ether, polyoxyethylene oleyl ether, etc.), polyoxyethylene alkyl Allyl ethers (eg, polyoxyethylene octyl phenyl ether, polyoxyethylene nonyl phenyl ether, polyoxyethylene isooctyl phenyl ether, etc.), polyoxyethylene derivatives (polyoxyethylene-polyoxypropylene condensate, etc.), sorbitan fatty acid esters (Eg, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate , Sorbitan tristearate, sorbitan trioleate, sorbitan distearate, etc.), polyoxyethylene sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate,
Polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan tristearate, polyoxyethylene sorbitan trioleate, etc.), polyoxyethylene sorbitol fatty acid esters (eg, polyoxyethylene sorbite tetraoleate, etc.), glycerin fatty acid esters (eg, Glycerol monostearate, glycerol monooleate, etc.), polyoxyethylene fatty acid esters (eg, polyethylene glycol monolaurate, polyethylene glycol monostearate, polyethylene glycol monooleate, polyethylene glycol distearate, etc.), polyoxyethylene At least one nonionic surfactant selected from the group consisting of alkylamines and alkylalkanolamides is preferred. Arbitrariness.

Examples of bile salts and bile salt derivatives include sodium cholate, sodium deoxycholate, sodium chenodeoxycholate, sodium dehydrocholate, sodium taurocholate, sodium taurolithocholic acid, sodium taurodeoxycholate, and sodium taurochenodeoxycholate. , Sodium tauroursodeoxycholate, sodium taurodehydrocholate, CHAPS: 3-[(3-Cholamidopropyl) dimethy
lammonio] propanesulfonic acid, CHAPSO: 3-
[(3-Cholamidopropyl) dimethylammonio] -2-hydroxypr
opanesulfonic acid, BIGCHAP: N, N-Bis (3-Dg
luconamidopropyl) cholamide, deoxy-BIGCHAP:
Preferred are at least one bile salt and bile salt derivative selected from N, N-Bis (3-D-gluconamidopropyl) deoxycholamide.

(PAF-AH Activity Measuring Reagent) The PAF-AH activity measuring reagent of the present invention contains the PAF-AH substrate represented by the above general formula (I) or (Ia). The substrate may be 0.001 mM to 250 mM, more preferably 0.01 mM to 10 mM.
It is used in a concentration range of 0 mM, more preferably 0.01 mM to 10 mM.

The reagent for measuring PAF-AH activity is preferably a solution, and the substrate of PAF-AH is preferably selected from those having a stable pH of about 3 to about 9 by selecting a stable pH for each substrate. Buffers (eg, HEPES buffer, citrate buffer,
Dissolved in a tartrate buffer, a phosphate buffer, an acetate buffer, a Tris buffer, a borate buffer, a good buffer, etc.).

The reagent for measuring PAF-AH activity of the present invention preferably contains the above-mentioned non-specific reaction inhibitor. The concentration of the non-specific reaction inhibitor is not particularly limited when the inhibitor does not inhibit PAF-AH. However, when the inhibitor inhibits PAF-AH, the concentration at which the inhibitor does not inhibit PAF-AH is determined in advance. The following concentrations may be used.

The reagent for measuring PAF-AH activity of the present invention
In addition to these components, a water-miscible organic solvent may be contained as necessary to dissolve the PAF-AH substrate. As the water-miscible organic solvent, an alcohol (for example,
Methanol, ethanol, n-propyl alcohol, isopropyl alcohol), ketone (for example, acetone,
Methyl isobutyl ketone), esters (eg, methyl acetate, ethyl acetate, propyl acetate, methyl propionate) and the like. The water-miscible organic solvent is PAF-
Preferably not more than about 30% by weight, more preferably not more than about 20% by weight, based on the final solution weight, so as not to affect the activity of AH.

The reagent for measuring PAF-AH activity of the present invention
It may contain a substance that promotes dissociation of the color-forming or fluorescent-coloring compound. Examples of these substances include α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, and quaternary functional groups such as a basic functional group (for example, diethylaminoethyl, dimethylaminoethyl, and trimethylammonioethyl). Ammonium salts) into which cyclodextrins are introduced. These compounds may comprise about 0.5%.

Further, it is preferable that a chelating reagent coexist with the reagent for measuring PAF-AH activity of the present invention. In the coexistence of the chelating agent, by the phospholipase A ₂ of arylesterase and Ca ²⁺ dependency, it is possible to suppress the decomposition reaction of the substrate used in the present invention, PA
It becomes possible to specifically measure F-AH activity. As the chelating reagent, EDTA or EG
A suitable chelating agent such as TA can be used. The chelating agent may comprise about 20 mM.

Further, the reagent for measuring PAF-AH activity of the present invention is generally used for measuring the activity of enzymes such as buffers, stabilizers, activators and excipients in addition to the above. May be included.

As will be described later in detail, in the present invention, HDL associated-PAF-AH activity, LDLas
sociated-PAF-AH activity and total serotype PAF-AH
The activity can each be measured. HDLassociat
In order to measure ed-PAF-AH activity, the PAF of the present invention was used.
-AH activity measuring reagents include anionic compounds (for example, dextran sulfate,
Ionic compounds such as sulfated cyclodextrin, sulfated oligosaccharides, heparin, peparan sulfate, phosphotungstic acid, etc., cations such as Mg ²⁺ , Mn ²⁺ , polyclonal antibodies or monoclonal antibodies (eg, anti-apolipoprotein B) Antibodies)).

In order to separate and quantify LDL-associated-PAF-AH, the reagent for measuring PAF-AH activity of the present invention contains a polyclonal antibody or a monoclonal antibody (anti-apolipoprotein A antibody, Anti-apolipoprotein E antibody).

(Measurement of PAF-AH activity) In the measurement method of the present invention, a sample having (possibly) PAF-AH activity was added to the reagent for measuring PAF-AH activity prepared as described above, and the mixture was allowed to react. This is a method of qualitatively or quantitatively measuring the absorbance of a chromogenic or fluorescent compound and the excited fluorescence.

As a sample for measuring PAF-AH activity, body fluids such as human or animal blood, serum, plasma, urine or amniotic fluid, and human or animal cells, organs or extracts of cells and organs are used. Can be A test sample containing PAF-AH such as a purified product of PAF-AH and serum, plasma, etc.
It is appropriately diluted with a buffer having a pH of 6 to 9 (for example, a HEPES buffer, a Tris buffer, a phosphate buffer, a borate buffer, a good buffer, etc.), and kept at 20 to 40 ° C. use. To these buffers, NaCl, KCl, etc. may be added so as to have an appropriate salt concentration.

In the method for measuring PAF-AH activity of the present invention, the amount of chromogenic or fluorescent chromogenic released by hydrolysis of the substrate is measured. The quantification is performed by measuring the absorbance. The measurement can be performed by either the end method or the rate method. In the case of the endo method, first, it is necessary to react a measurement reagent from which a substrate has been removed with a sample to measure a sample blank. In the case of the rate method, the absorbance may be measured within a time period in which the measurement can be performed quantitatively. In addition, PAF-AH
Can be calculated from the molecular extinction coefficient of the released chromogenic or fluorescent chromogenic substance.

The method for measuring PAF-AH activity of the present invention can be carried out using a manual method or an automatic analyzer. The change in absorbance of the chromogenic or fluorescent compound released from the substrate is measured. For example, in the case of a coloring compound, the change in absorbance at a wavelength of 280 to 560 nm
Further, in the case of a fluorescent coloring compound, a laser beam is irradiated to excite fluorescence to detect spectroscopically, and the amount of change in absorbance per unit time is obtained (so-called rate assay method). AH activity can be determined.

For example, in the general formula (I) according to the present invention, A is a substrate having 2 carbon atoms (ie, a 1-type substrate represented by the general formula (Ia)).
Myristoyl-2- (4-nitrophenylsuccinyl)
Using -sn-glycero-phosphocholine produces 4-nitrophenol. At this time, as the amount of 4-nitrophenol increases, the absorbance at 405 nm, which is the absorption wavelength of 4-nitrophenol, also increases quantitatively. Therefore, the rate of increase or the amount of increase in absorbance at 405 nm is calculated.

Further, the HDL associated-PAF-AH activity, LD
Lassociated-PAF-AH activity and total serotype PAF-
AH activity can be fractionated and each can be accurately measured. L
By using a measuring reagent containing a substance that aggregates DL, LDL is aggregated and removed, and HDL associated-P
AF-AH can be quantified. In addition, by using a measurement reagent containing a substance that aggregates HDL,
HDL is aggregated and removed, and LDL associated-PAF-AH
Can be determined. If neither is added, total serotype PAF-AH activity is measured.

A specific measuring method will be described below. First,
Inhibitor reagents containing the inhibitor dissolved in a suitable buffer (eg, 50 mM HEPE containing the inhibitor and 150 mM NaCl)
S buffer (pH 7.4), substrate solution (eg, 50 mM HEPE containing 16% ethanol, substrate and 150 mM NaCl)
S buffer (pH 7.4)). After an appropriate amount of human pool serum or PAF-AH purified product solution is mixed with the inhibitor solution, a substrate solution is added thereto and reacted. Reaction p
H is preferably from about 6.5 to about 8.0, most preferably about 7.4. The reaction temperature is between 20C and 40C, preferably 37C. After the addition of the substrate solution, the absorbance is measured for an appropriate time (for example, from the second minute to the fifth minute), and the change in the absorbance per unit time is determined. , PAF-
The AH activity value (nmol / min / sample 1 ml) can be determined.

In the case of automatic measurement by the rate method, first, the above-mentioned inhibitor solution and substrate solution are prepared. For example, the measurement is performed using an automatic analyzer H-7170 (Hitachi, Ltd.) according to the measurement parameters. The purified human pool serum or PAF-AH is mixed with the inhibitor solution, and after a certain period of time, the reaction is started by mixing the substrate solution. The change in absorbance (time course) at this time is monitored, and the change in absorbance per unit time (1 minute) from the second minute to the fifth minute after the start of the reaction is determined. The amount of change in absorbance per unit time obtained from the sample (ΔEs);
Amount of change in absorbance per unit time (ΔE _B ) obtained by adding purified water instead of a sample, and the molecular extinction coefficient (ε) of a chromogenic compound or a fluorescent chromogenic compound in the reaction solution
Then, the PAF-AH activity value of each serum sample is calculated by the following equation.

[0089]

(Equation 1)

(PAF-AH Activity Measurement Kit) The PAF-AH activity measurement kit of the present invention comprises the above-described PAF-AH activity measurement kit to which a substrate, and if necessary, a non-specific reaction inhibitor and other additives are added. Includes reagents for The kit may be, for example, in the form of an ampule containing a reagent for measuring PAF-AH activity, a form in which a fixed amount of the reagent for measuring PAF-AH activity is injected into a well, or a container containing a reagent for measuring PAF-AH activity and a container for measuring. And a well. The PAF-AH activity is measured by adding the serum sample to the reagent for measuring PAF-AH activity attached to the kit of these forms.

The kit for measuring PAF-AH activity of the present invention comprises a test containing the reagent for measuring PAF-AH activity to which a substrate and, if necessary, a nonspecific reaction inhibitor and other additives are added. It can be a piece. If the reagent is a solution,
The test piece may be a dried test piece after immersion in the reagent. By immersing this in serum or the like, PAF-AH activity can be qualitatively confirmed. The approximate activity can also be known using a colorimetric table. Accordingly, the container includes such a test piece.

Further, the kit of the present invention comprises a substrate represented by the above general formula (I) and an inhibitor which does not inhibit PAF-AH but inhibits other ester degradation activity-related substances (non-specific reaction inhibitor). May be filled in independent containers, and may be mixed at the time of use. In this case, the substrate may be dissolved in a solution or a solvent, and a solution and / or a solvent for dissolving the substrate may be separately contained in a separate container. The form you choose depends on the stability of the substrate, operability,
The amount may be determined in consideration of the amount of use and the like.

Further, in addition to the above-mentioned substrates and the like, tools and devices for measuring absorbance may be included in the kit.

[0094]

EXAMPLES Hereinafter, the present invention will be described more specifically based on examples, but the present invention is not limited to these examples.

Example 1 Preparation of Substrate Compound No. 1
The following is an example of the production of 25.

(Compound No. 1: Synthesis of 1-octanoyl-2- (4-nitrophenylglutaryl) -sn-glycero-phosphocholine) 340 mg (0.68 mmol) of 1-octanoyl-2-glutarylphosphatidylcholine was treated with dichloromethane (dehydrated). ) In 3 ml, 800 μl (1.6 mmol) of 2M oxalyl chloride was added under ice cooling, and the mixture was stirred at room temperature for 1.5 hours. After concentration under reduced pressure, dichloromethane (dehydrated) 3 ml
And stirred at room temperature, 4-nitrophenol 104mg
(0.75 mmol), 138 μL of triethylamine (1.
36 mmol) and 3 ml of dichloromethane (dehydrated). After stirring at room temperature for 2.5 hours, the mixture was concentrated under reduced pressure.
To this, 60 ml of chloroform: methanol (2: 1) was added and washed with water (15 ml). The lower layer was separated, dried over anhydrous sodium sulfate, and the filtrate was concentrated. Diethyl ether was added to make the mixture cloudy, and the mixture was allowed to stand overnight. The supernatant was collected, and the residue was purified with a silica gel column using a chloroform-methanol-water system to obtain 218 mg (52%) of a yellow oil.

(Compound No. 2: Synthesis of 1-decanoyl-2- (4-nitrophenylglutaryl) -sn-glycero-phosphocholine) 272 mg (0.55 mmol) of 1-decanoyl-2-glutarylphosphatidylcholine was treated with dichloromethane (dehydrated). ) Dissolve in 3 ml and add 2M oxalyl chloride 5 under ice cooling.
After adding 50 μl (1.1 mmol), the mixture was stirred at room temperature for 1.5 hours. After concentration under reduced pressure, the residue was dissolved in 3 ml of dichloromethane (dehydrated), and stirred at room temperature with 84 mg of 4-nitrophenol (0.1 ml).
6 mmol), 101 μL of triethylamine (1.1 mm
ol) and 3 ml of dichloromethane (dehydrated).
After stirring at room temperature for 2.5 hours, the mixture was concentrated under reduced pressure. To this, 60 ml of chloroform: methanol (2: 1) was added and washed with water (15 ml). The lower layer was collected, dried over anhydrous sodium sulfate, and the filtrate was concentrated, purified with a silica gel column using methanol, and further purified with a chloroform-methanol-water system using a silica gel column.
g (32%).

(Compound No. 3: Synthesis of 1-lauroyl-2- (4-nitrophenylglutaryl) -sn-glycero-phosphocholine) 290 mg (0.52 mmol) of 1-lauroyl-2-glutarylphosphatidylcholine was treated with dichloromethane (dehydrated). ) Dissolve in 3 ml and add 2M oxalyl chloride 5 under ice cooling.
After adding 20 μl (1.04 mmol), the mixture was stirred at room temperature for 1.5 hours. After concentration under reduced pressure, dichloromethane (dehydrated) 3 ml
And stirred at room temperature with 4-nitrophenol 79mg
(0.57 mmol), 115 μL of triethylamine (1.
04 mmol) and 3 ml of dichloromethane (dehydrated). After stirring at room temperature for 2.5 hours, the mixture was concentrated under reduced pressure.
To this, 60 ml of chloroform: methanol (2: 1) was added and washed with water (15 ml). The lower layer was collected, dried over anhydrous sodium sulfate, and the filtrate was concentrated. The residue was purified with a silica gel column using a chloroform-methanol-water system.
1 mg (49%) was obtained.

(Compound No. 4: Synthesis of 1-myristoyl-2- (4-nitrophenylglutaryl) -sn-glycero-phosphocholine) 283 mg (0.49 mmol) of 1-myristoyl-2-glutarylphosphatidylcholine was added to chloroform (dehydrated). ) Dissolve in 3 ml and add 2M oxalyl chloride 4 under ice cooling.
After adding 90 μl (0.98 mmol), the mixture was stirred at room temperature for 1.5 hours. After concentration under reduced pressure, the residue was dissolved in 3 ml of chloroform (dehydrated), and 102.1 m of 4-nitrophenol was stirred at room temperature.
g (0.74 mmol), 137 μL of triethylamine
(0.98 mmol) and 3 ml of chloroform (dehydrated) were added. After stirring at room temperature for 2 hours, 0.1N hydrochloric acid 1
ml was added. 75 ml of chloroform: methanol (2: 1) was added to this solution, and the mixture was washed with water (15 ml). The lower layer is removed, dried over anhydrous sodium sulfate, and the filtrate is concentrated.
Purification was performed on a silica gel column using a chloroform-methanol-water system to obtain 182 mg (53%) of a yellow amorphous substance.

NMR (CDCl ₃ ): 0.89 (t, 3H, J = 7.0 Hz), 1.25
(s, 20H), 150-1.65 (m, 2H), 1.95-2.12 (m, 2H), 2.29 (t,
2H, J = 7.7 Hz), 2.40-2.55 (m, 2H), 2.71 (t, 2H, J = 7.3 Hz)
, 3.38 (s, 9H), 3.80 (br, 2H), 3.92-4.05 (m, 2H), 4.10
-4.20 (m, 1H), 4.25-4.45 (m, 3H), 5.15-5.30 (m, 1H), 7.3
1 (d, 2H), 8.29 (d, 2H); MS (SIMS): 703 (MH ⁺ )

(Compound No. 5: Synthesis of 1-oleoyl-2- (4-nitrophenylglutaryl) -sn-glycero-phosphocholine) 350 mg (0.55 mmol) of 1-oleoyl-2-glutarylphosphatidylcholine was treated with dichloromethane. (Dehydrated) Dissolve in 3 ml and add 2M oxalyl chloride 5 under ice cooling.
After adding 50 μl (1.1 mmol), the mixture was stirred at room temperature for 1.5 hours. After concentration under reduced pressure, the residue was dissolved in 3 ml of dichloromethane (dehydrated), and stirred at room temperature with 84 mg of 4-nitrophenol (0.1 ml).
61 mmol), 153 μL of triethylamine (1.1 m
mol) and 3 ml of dichloromethane (dehydrated). After stirring at room temperature for 2.5 hours, the mixture was concentrated under reduced pressure. 60 ml of chloroform: methanol (2: 1) was added to this,
It was washed with water (15 ml). The lower layer was separated, dried over anhydrous sodium sulfate, and the filtrate was concentrated and purified by a silica gel column (methanol) to obtain 303 mg (73%) of the desired fraction.

(Compound No. 6: Synthesis of 1-alkyl-2- (4-nitrophenylglutaryl) -sn-glycero-phosphocholine) 1-alkyl-2-glutarylphosphatidylcholine
267 mg (0.45 mmol) was dissolved in 3 ml of dichloromethane (dehydrated), and 2M oxalyl chloride 450 ml was added under ice-cooling.
After adding μl (0.9 mmol), the mixture was stirred at room temperature for 1.5 hours. After concentration under reduced pressure, the residue was dissolved in 3 ml of dichloromethane (dehydrated) and stirred at room temperature with 69 mg of 4-nitrophenol (0.5 m
mol), 125 μL of triethylamine (0.9 mmol)
l), a solution of 3 ml of dichloromethane (dehydrated) was added. After stirring at room temperature for 2.5 hours, the mixture was concentrated under reduced pressure. To this, 60 ml of chloroform: methanol (2: 1) was added and washed with water (15 ml). The lower layer was collected, dried over anhydrous sodium sulfate, and the filtrate was concentrated, purified with a silica gel column using methanol, and further purified with a chloroform-methanol-water system using a silica gel column.
g (45%).

(Compound No. 7: Synthesis of 1-alkenyl-2- (4-nitrophenylglutaryl) -sn-glycero-phosphocholine) This was carried out in the same manner as in the synthesis of compound No. 6, and 98 mg (60%) of yellow amorphous crystals I got

(Compound No. 8: Synthesis of 1-myristoyl-2- (4-nitrophenylsuccinyl) -sn-glycero-phosphocholine) 1-myristoyl-2-succinylphosphatidylcholine 200 mg (0.35 mmol) in 3 ml of dichloromethane (dehydrated) And then add 350 μl (0.71 mmol) of 2M oxalyl chloride under ice-cooling, and then add 1.5 ml at room temperature.
Stirred for hours. After concentration under reduced pressure, dichloromethane (dehydrated) 3m
1-nitrophenol 154m
g (0.39 mmol), 98 μL of triethylamine (0.30 mmol).
71 mmol) and 3 ml of chloroform (dehydrated). After stirring at room temperature for 1.5 hours, 60 ml of chloroform: methanol (2: 1) was added to the solution, followed by washing with water (15%).
ml). The lower layer was collected, dried over anhydrous sodium sulfate, concentrated, and purified on a silica gel column with a chloroform-methanol-water system to obtain 229 mg of a pale yellow oil (64 mg).
%).

NMR (CDCl ₃ ): 0.80-0.95 (m, 3H), 1.26 (br,
20H), 1.57 (br, 2H), 2.22-2.29 (m, 2H), 2.75-2.82 (m, 2
H), 2.82-2.93 (m, 2H), 3.34 (s, 9H), 3.79 (br, 2H), 4.0
1 (br, 2H), 4.10-4.22 (m, 1H), 4.22-4.48 (m, 4H), 5.27
(br, 1H), 7.30-7.36 (m, 2H), 8.27-8.32 (m, 2H); MS (SIM
S): 689 (MH ⁺ )

(Compound No. 9: Synthesis of 1-myristoyl-2- (4-nitrophenyladipoyl) -sn-glycero-phosphocholine) The same procedure as in the synthesis of compound No. 8 was carried out, and 167 mg (52%) of a yellow oil was obtained. ) Got.

(Compound No. 10: 1-Myristoyl-2- (4-
Synthesis of nitrophenylpimeloyl) -sn-glycero-phosphocholine) The same operation as in the synthesis of compound No. 8 was carried out to obtain 341 mg (82%) of a yellow oily substance.

(Compound No. 11: 1-myristoyl-2- (4-
(Synthesis of nitrophenylsuberoyl) -sn-glycero-phosphocholine) In the same manner as in the synthesis of compound No. 8, 114 mg (34%) of a yellow oily substance was obtained.

(Compound No. 12: 1-myristoyl-2- (4-
Synthesis of nitrophenylazeroyl) -sn-glycero-phosphocholine) The same operation as in the synthesis of compound No. 8 was carried out to obtain 190 mg (76%) of a yellow oil.

NMR (CDCl ₃ ): 0.89 (m, 3H), 1.20-1.50 (m,
26H), 1.60 (br, 4H), 1.70-1.85 (m, 2H), 2.30 (m, 4H),
2.61 (t, 2H, J = 7.46 Hz), 3.38 (s, 9H), 3.83 (br, 2H), 3.9
0-4.02 (m, 2H), 4.10-4.22 (m, 1H), 4.25-4.50 (m, 3H),
5.23 (br, 1H), 7.21-7.35 (m, 2H), 8.20-8.35 (m, 2H).

(Compound No. 13: 1-myristoyl-2- (2-
Chloro-4-nitrophenylglutaryl) -sn-glycero-
Synthesis of phosphocholine) 177 mg (0.3 mmol) of 1-myristoyl-2-glutarylphosphatidylcholine is dissolved in 3 ml of dichloromethane (dehydrated), and 300 μl (0.6 mmol) of 2M oxalyl chloride is added under ice-cooling, followed by 2 hours at room temperature. Stirred. After concentration under reduced pressure, dichloromethane (dehydrated)
The solution was dissolved in 3 ml, and stirred at room temperature, 104 mg (0.6 mmol) of 2-chloro-4-nitrophenol and triethylamine 8
4 μL (0.6 mmol), 3 ml of dichloromethane (dehydrated)
Was added. After stirring at room temperature for 3 days, the mixture was concentrated under reduced pressure. 60 ml of chloroform: methanol (2: 1)
Was added and washed with water (15 ml). The lower layer was separated, dried over anhydrous sodium sulfate, and the filtrate was concentrated. The residue was purified with a silica gel column using a chloroform-methanol-water system to obtain 156 mg (71%) of a yellow oil.

(Compound No. 14: 1-myristoyl-2-[(4
Synthesis of 1-myristoyl-2-glutarylphosphatidylcholine (191 mg, 0.33 mmol) in 3 ml of dichloromethane (dehydrated) was dissolved in 3 ml of dichloromethane (dehydrated). (.67 mmol), and the mixture was stirred at room temperature for 2 hours. After concentration under reduced pressure, dichloromethane (dehydrated) 3
and 4- (methyl) coumarin 1 under stirring at room temperature.
15 mg (0.67 mmol), triethylamine91.
4 μL (0.67 mmol), dichloromethane (dehydrated) 3 m
1 solution was added. After stirring at room temperature for 4 days, the mixture was concentrated under reduced pressure. Chloroform: methanol (2: 1) 60m
was added and washed with water (15 ml). The lower layer was collected, dried over anhydrous sodium sulfate, and the filtrate was concentrated.
Purification on a silica gel column was performed using a methanol-water system to obtain 183.2 mg (76.5%) of a colorless amorphous substance.

NMR (CDCl ₃ ): 0.89 (t, 3H, J = 6.9 Hz), 1.2
5 (br, 20H), 1.50-1.65 (m, 2H), 1.95-2.12 (m, 2H), 2.29
(t, 2H, J = 7.7 Hz), 2.44 (s, 3H), 2.47-2.51 (m, 2H), 2.7
0 (t, 2H, J = 7.2 Hz), 3.38 (s, 9H), 3.82 (br, 2H), 3.98-4.
03 (m, 2H), 4.11-4.22 (m, 1H), 4.30-4.48 (m, 3H), 5.19-5.
32 (m, 1H), 6.27 (s, 1H), 7.00-7.11 (m, 1H), 7.11-7.20
(m, 1H), 7.58-7.70 (m, 1H); MS (SIMS): 726 (MH ⁺ ).

(Compound No. 15: 1-Myristoyl-2- (2-
Synthesis of fluoro-4-nitrophenylglutaryl) -sn-glycero-phosphocholine) 266 mg (0.46 mmol) of 1-myristoyl-2-glutarylphosphatidylcholine
Was dissolved in 3 ml of chloroform (dehydrated), 457 μl (0.91 mmol) of 2M oxalyl chloride was added under ice cooling, and the mixture was stirred at room temperature for 1.5 hours. After concentration under reduced pressure, the residue was dissolved in 3 ml of chloroform (dehydrated), and stirred at room temperature to give 2-fluoro-4.
-Nitrophenol 107.7 mg (0.69 mmol
l), triethylamine 127.4 μL (0.91 mmol
l), 3 ml of chloroform (dehydrated) was added. After stirring at room temperature for 3 hours, 1 ml of 0.1 N hydrochloric acid was added.
70 ml of chloroform: methanol (2: 1) is added to this solution.
Was added and washed with water (15 ml). The lower layer was separated, dried over anhydrous sodium sulfate, and the filtrate was concentrated. The residue was purified by a silica gel column using a chloroform-methanol-water system to obtain 260 mg (79%) of a yellow amorphous substance.

(Compound No. 16: 1-myristoyl-2- (3-
Synthesis of fluoro-4-nitrophenylglutaryl) -sn-glycero-phosphocholine) Performed in the same manner as in the synthesis of compound No. 15, and 171 mg of yellow amorphous crystals (62%)
I got

(Compound No. 17: 1-myristoyl-2- (4
Synthesis of -nitrothiophenyl) glutarylphosphatidylcholine) 227 mg (0.39 mmol) of 1-myristoyl-2-glutarylphosphatidylcholine was dissolved in 3 ml of chloroform (dehydrated), and 2M oxalyl chloride was dissolved under ice cooling.
After adding 90 μl (0.78 mmol), the mixture was stirred at room temperature for 2 hours. After concentration under reduced pressure, the residue was dissolved in 3 ml of chloroform (dehydrated), and 4-nitrothiophenol 90.8 m was stirred at room temperature.
g (0.59 mmol), triethylamine 108.3 μ
A solution of L (0.78 mmol) and 3 ml of chloroform (dehydrated) was added. After stirring at room temperature for 2 hours, 0.1N hydrochloric acid
1 ml was added. 70 ml of chloroform: methanol (2: 1) was added to this solution, and the mixture was washed with water (15 ml). The lower layer is removed, dried over anhydrous sodium sulfate, and the filtrate is concentrated.
Purification on a silica gel column was performed using a chloroform-methanol-water system to obtain 202 mg (72%) of a brown oily substance.

NMR (CDCl ₃ ): 0.80-0.98 (m, 3H), 1.27 (br,
20H), 1.59 (br, 2H), 1.92-2.12 (m, 2H), 2.20-2.35 (m, 2
H), 2.35-2.52 (m, 2H), 2.70-2.88 (m, 2H), 3.37 (s, 9H),
3.82 (br, 2H), 3.90-4.02 (m, 2H), 4.02-4.20 (m, 1H), 4.
22-4.47 (m, 3H), 5.22 (br, 1H), 7.51-7.65 (m, 2H), 8.15-
8.31 (m, 2H); MS (SIMS): 719 (MH ^<+> ).

(Compound No. 18: 1-myristoyl-2- (5-
Synthesis of indolyloxyglutaryl) -sn-glycero-phosphocholine 205 mg (0.35 mmol) of 1-myristoyl-2-glutarylphosphatidylcholine was dissolved in 3 ml of chloroform (dehydrated), and 352 μl of 2M oxalyl chloride (0. After 7 mmol), the mixture was stirred at room temperature for 2 hours. After concentration under reduced pressure, the residue was dissolved in 3 ml of chloroform (dehydrated), and stirred at room temperature with 5-hydroxyindole 70.4.
mg (0.53 mmol), triethylamine 98.2μ
A solution of L (0.7 mmol) and 3 ml of chloroform (dehydrated) was added. After stirring at room temperature for 2 hours, 0.1N hydrochloric acid 1
ml was added. 70 ml of chloroform: methanol (2: 1) was added to this solution, and the mixture was washed with water (15 ml). The lower layer was separated, dried over anhydrous sodium sulfate, and the filtrate was concentrated. The residue was purified with a silica gel column using a chloroform-methanol-water system to give 143 mg (59%) of a yellow oil.

NMR (CDCl ₃ ): 0.80-1.00 (m, 3H), 1.20-1.4
0 (m, 20H), 1.50-1.65 (m, 2H), 1.91-2.15 (m, 2H), 2.18-
2.35 (m, 2H), 2.35-2.51 (m, 2H), 2.51-2.70 (m, 2H), 2.78
(s, 9H), 3.00-3.29 (m, 3H), 3.89-4.27 (m, 5H), 4.27-4.5
0 (m, 1H), 5.21 (br, 1H), 6.41 (s, 1H), 6.70-6.90 (m, 1H),
7.21 (s, 1H), 7.40-7.65 (m, 1H), 11.18 (s, 1H); MS (SI
MS): 697 (MH ^<+> ).

(Compound No. 19: 1-Myristoyl-2- (2,
Synthesis of 6-diphenyl-4-nitrophenylglutaryl) -sn-glycero-phosphocholine) 1-myristoyl-2-glutarylphosphatidylcholine 254 mg (0.44 mmol)
l) was dissolved in 3 ml of chloroform (dehydrated), and
After adding 437 μl (0.87 mmol) of M oxalyl chloride, the mixture was stirred at room temperature for 2 hours. After concentration under reduced pressure, the residue was dissolved in 3 ml of chloroform (dehydrated) and stirred at room temperature with 191 mg of 2,6-diphenyl-4-nitrophenol (0.65 mm).
ol), triethylamine 122 μL (0.87 mmol
l), 3 ml of chloroform (dehydrated) was added. After stirring at room temperature for 16 hours, 1 ml of 0.1 N hydrochloric acid was added. To this solution was added chloroform: methanol (2: 1) 70
Then, the mixture was washed with water (15 ml). The lower layer was collected, dried over anhydrous sodium sulfate, and the filtrate was concentrated.
The residue was purified by a silica gel column using a methanol-water system to obtain 219 mg (58%) of a yellow oily substance.

NMR (CDCl ₃ ): 0.89 (t, 3H, J = 7.2 Hz), 1.26
(br, 20H), 1.26-1.61 (m, 4H), 1.91 (t, 2H, J = 7.6 Hz), 2.
14 (t, 2H, J = 7.1 Hz), 2.26 (t, 2H, J = 7.8 Hz), 3.31 (s, 9
H), 3.74 (br, 2H), 3.92 (t, 2H, J = 6.0 Hz), 4.00-4.15 (m,
1H), 4.28 (br, 2H), 4.30-4.42 (m, 1H), 5.14 (br, 1H), 7.
32-7.50 (m, 10H), 8.28 (s, 2H); MS (SIMS): 855 (MH ^<+> ).

(Compound No. 20: 1-Myristoyl-2- (4-
Synthesis of nitrophenylglycolyl) -sn-glycero-phosphocholine) 164 mg (0.28 mmol) of 1-myristoyl-2-glycolylphosphatidylcholine was dissolved in 3 ml of chloroform (dehydrated), and 2M oxalyl chloride was cooled on ice.
After adding 281 μl (0.56 mmol), the mixture was stirred at room temperature for 2 hours. After concentration under reduced pressure, the residue was dissolved in 3 ml of chloroform (dehydrated), and 43.0 mg (0.31 mg) of 4-nitrophenol was stirred at room temperature.
mmol), 78 μL of triethylamine (0.56 mmol
l), 3 ml of chloroform (dehydrated) was added. After stirring at room temperature for 2 hours, 1 ml of 0.1 N hydrochloric acid was added.
56 ml of chloroform: methanol (2: 1) was added to this solution, and the mixture was washed with water (14 ml). The lower layer was collected, dried over anhydrous sodium sulfate, and the filtrate was concentrated.The residue was purified with a silica gel column using a chloroform-methanol-water system to give a yellow oil.
102 mg (51%) were obtained.

(Compound No. 21: 1-myristoyl-2- [5-
Synthesis of dimethylamino-2- (2-thiazolylazo) phenylsuccinyl] -sn-glycero-phosphocholine) 1-myristoyl-2-succinylphosphatidylcholine 419 mg
(0.74 mmol) was dissolved in 6 ml of chloroform (dehydrated), and 738 μl (1.48 mmol) of 2M oxalyl chloride was added under ice cooling.
After l) was added, the mixture was stirred at room temperature for 2.5 hours. After concentration under reduced pressure, the residue was dissolved in 6 ml of chloroform (dehydrated) and stirred at room temperature with 5-dimethylamino-2- (2-thiazolylazo) phenol 202
mg (0.81 mmol), triethylamine 206 μL (1.48
mmol) and 6 ml of chloroform (dehydrated). After stirring at room temperature for 2 hours, 2 ml of 0.1 N hydrochloric acid was added. Add chloroform: methanol (2: 1) 15 to this solution.
0 ml was added and washed with water (30 ml). The lower layer was collected, dried over anhydrous sodium sulfate, and the filtrate was concentrated.
The residue was purified by a silica gel column using a methanol-water system to obtain 247 mg (42%) of a red oily substance.

(Compound No. 22: 1-Myristoyl-2- [1-
(2-triazolylazo) -2-naphthylsuccinyl]-
Synthesis of sn-glycero-phosphocholine) The synthesis was carried out in the same manner as in the synthesis of compound No. 21 to obtain 64 mg (14%) of a reddish brown oil.

(Compound No. 23: 1-myristoyl-2-
[Synthesis of [(4-nitrophenyl) dimethylsuccinyl] -sn-glycero-phosphocholine) The same procedure as in the synthesis of compound No. 21 was carried out, and white amorphous crystals 150 mg (49%)
I got

(Compound No. 24: 1-Myristoyl-2-
[(6-Flavoneoxyl) succinyl] -sn-glycero
-Synthesis of phosphocholine) The same operation as in the synthesis of compound No. 21 was carried out to obtain 295 mg (68%) of white amorphous crystals.

NMR (CDCl ₃ ): 0.87 (t, 3H, J = 6.9 Hz), 1.24
(br, 20H), 1.46-1.64 (m, 2H), 2.25 (t, 2H, J = 7.7 Hz), 2.
65-3.09 (m, 6H), 3.33 (s, 9H), 3.78 (br, 2H), 4.00 (t, 2H,
J = 6.7Hz), 4.09-4.20 (m, 1H), 4.23-4.43 (m, 3H), 5.17-
5.31 (m, 1H), 5.41-5.56 (m, 1H), 7.10-7.65 (m, 8H).

(Compound No. 25: 1-palmitoyl-2-
(Synthesis of 4-nitrophenylglutanyl) -sn-glycero-phosphocholine 320 mg (0.51 mmol) of 1-palmitoyl-2-glutarylphosphatidylcholine was dissolved in 3 ml of dichloromethane (dehydrated), and 765 μl (1.53 ml) of 2M oxalyl chloride under ice cooling. (mmol) was added, followed by stirring at room temperature for 1.5 hours. After concentration under reduced pressure, the residue was dissolved in 3 ml of dichloromethane (dehydrated) and stirred at room temperature with 104 mg of 4-nitrophenol (0.
75 mmol), 139 μL of triethylamine (1.02 mmol
l), a solution of 3 ml of dichloromethane (dehydrated) was added. After stirring at room temperature for 2.5 hours, the mixture was concentrated under reduced pressure. To this is added 75 ml of chloroform: methanol (2: 1) and washed with water (15 m
l) Take the lower layer, dry with anhydrous sodium sulfate,
The filtrate was concentrated. The residue was purified by a silica gel column (methanol) to obtain 203.6 mg (55%) of a yellow amorphous substance.

Example 2 Measurement of PAF-AH Activity; Substrate Specificity (Example of Purification of Enzyme PAF-AH) About 400 ml of human plasma was adjusted to a specific gravity d = 1.0063 with KBr, and centrifuged.
Fractions containing chylomicron, VLDL, and LDL were obtained. 50 mM Tris-HCl containing 10 mM CHAPS (Tri
s-HCl) buffer (pH 7.4)
The mixture was applied to a Q Sepharose column equilibrated with 50 mM Tris-HCl buffer (pH 7.4) containing APS. After washing the column with the same buffer, it was eluted with a linear gradient of NaCl (0-0.5M). The eluted PAF-AH active portion was concentrated with Centrip 30 and 10 mM CHAPS,
50 mM Tris-HCl buffer containing 0.3 M NaCl (pH
It was applied to Sephacryl S-200 equilibrated in 7.4).
The PAF-AH active portion eluted with the same buffer was concentrated with a centrip 30 and the buffer was diluted with 10 mM CHAP.
It was applied to a bull-Sepharose column equilibrated with 25 mM Tris-HCl buffer (pH 7.4) containing S, 0.3 M NaCl. After washing with the same buffer, 10 mM CHAPS,
50 mM Tris-HCl buffer containing 1.5 M NaCl (pH
8.0). The PAF-AH active portion was concentrated with Centrip 30 to obtain purified PAF-AH.

(Measurement of activity) Compound No. 1 synthesized above
-13, 15-17, 20 and 25, PAF-
AH activity was measured. The following solutions, buffer: 50 mM HEPES buffer (pH 7.4) containing 150 mM NaCl, 10 mM EDTA; and substrate solution: 4 mM substrate, 90 mM NaCl, 6 mM
30 mM HE with EDTA and 40% ethanol
A PES buffer (pH 7.4) was prepared.

The PAF-AH purified product 10 obtained by the above method
ul and 900 ul of the buffer solution were mixed and left in a thermostat at 37 ° C. for 5 minutes, and then 100 ul of the substrate solution was added to start the reaction.
After the addition of the substrate solution, the change in absorbance (ΔE _S ) per unit time (1 minute) from the second minute to the fifth minute and the molecular extinction coefficient ε = 12000 of paranitrophenol (compounds 13, 15 and For the 16 compounds, the molecular absorption coefficient ε = 18000 was used, and the PAF-AH activity value (nm)
ol / min / 1 ml of sample) was calculated by the above equation. Table 1 shows the results. Wherein, Delta] E _B is the value measured by adding purified water instead of the sample.

[0132]

[Table 1]

From these results, it was found that 1-myristoyl-2- (4-nitrophenylsuccinyl) -sn of compound No. 8
-Glycero-phosphocholine (A = 2 in general formula (I)) was found to have the highest substrate specificity. This corresponds to the commonly used compound number 4 1-myristoyl-2-
(4-nitrophenylglutaryl) -sn-glycero-
This indicates that the substrate specificity is at least 4 times higher than that of phosphocholine (A = 3 in the general formula (I)).

The compounds of the general formula (I) where A = 4 to 7 (Compound Nos. 9 to 12) also showed PAF-AH activity,
The smaller A is, the higher the activity tends to be. The activity was also detected when the acyl group (R ₁ ) of the general formula (I) had 8 to 18 carbon atoms (compound numbers 1 to 5 and 25). The activity was also detected when R ₁ was an alkyl group or an alkenyl group (Compound Nos. 6 and 7). R ₁ is, it did not appear to have any significant effect on the very activity. Furthermore, substrates that release halogen-substituted nitrophenyl compounds (substrate numbers 13, 15, and 16) have also been shown to be useful.

Hereinafter, a highly specific substrate (compound No. 8:
An example using 1-myristoyl-2- (4-nitrophenylsuccinyl) -sn-glycero-phosphocholine (hereinafter, referred to as a substrate of the present invention) will be described.

Example 3 Measurement of PAF-AH Activity; Effect of Inhibitor The following inhibitor solution and substrate solution were prepared respectively. Inhibitor solution: The inhibitors described in Table 2 were added to 150 mM NaCl
Was dissolved in a 50 mM HEPES buffer solution (pH 7.4) containing at the concentration shown in Table 2. Substrate solution: 4 mM of the substrate of the present invention,
30m with 40% ethanol and 90mM NaCl
M HEPES buffer (pH 7.4).

Human pooled serum or PAF-AH purified product 10
ul and 900 ul of the inhibitor solution were mixed, left in a thermostat at 37 ° C. for 5 minutes, and 100 ul of the substrate solution was added to start the reaction. From the change in absorbance (ΔE) per unit time (1 minute) from the second minute to the fifth minute after the addition of the substrate solution and the molecular extinction coefficient ε = 12000 of paranitrophenol, PA
The F-AH activity value (nmol / min / sample 1 ml) was calculated. Table 2 shows the results.

[0138]

[Table 2]

In the presence of various inhibitors, a great difference was observed in the degree of inhibition between the human pooled serum and the purified PAF-AH product. That is, the purified PAF-AH product almost retains the activity of 90% or more (the decrease in the activity by the inhibitor is small), whereas the human pooled serum contains
There is no one that retains the activity of 90% or more. this is,
As a result of purification of the purified PAF-AH purified product, the relative activity is considered to be unlikely to be reduced by the inhibitor due to the low content of a substance that causes a nonspecific hydrolysis reaction. Suggests that the relative activity was reduced as a result of the suppression of this non-specific hydrolysis activity by the inhibitor.

Example 4 Measurement of PAF-AH Activity: Correlation with Conventional Method The following reagents were prepared. Reagent 1 (A): 50 mM HEPES containing 150 mM NaCl
Buffer (pH 7.4) Reagent 2 (A): Substrate solution; 50 mM HEPE containing 1.6 mM of the substrate of the present invention, 16% ethanol and 150 mM NaCl
S buffer (pH 7.4); Reagent 1 (B): inhibitor solution; 150 mM NaCl, 10 mM ED
TA, 1 mM sodium lauryl sulfate, 5 mM CHAPS
50 mM HEPES buffer (pH 7.4) containing: and a reagent 2 (B): a substrate solution containing an inhibitor; 150 mM NaC
1, 10 mM EDTA, 1 mM sodium lauryl sulfate, 5 m
50 mM HEPES buffer (pH 7.4) containing M CHAPS, 16% ethanol and 1.6 mM of the substrate of the present invention. Serum samples: 5 types of samples S1, S2, S3, S4 and S5.

The PAF-AH activity was measured using H-717.
A type 0 automatic analyzer (Hitachi, Ltd.) was used. The parameters at the time of measurement are as follows.

[0142]

[Table 3]

According to the above parameters, the serum sample 5u
l and reagent 1 (A) (buffer) or 1 (B) (inhibitor solution) 240u
After mixing at a constant temperature (37 ° C., 5 minutes), 80 ul of reagent 2 (A) (substrate solution) or reagent 2 (B) (substrate solution containing inhibitor) is added. , Mixed and the reaction started. The change in absorbance (time course) at this time is shown in FIG.
And shown in FIG. When a solution containing the substrate of the present invention is added to a mixture of a sample and reagent 1 (A) or 1 (B), PAF-A
An increase in absorbance derived from 4-nitrophenol generated from the substrate of the present invention by the reaction of H with the substrate of the present invention was confirmed.

As is clear from the comparison between FIG. 1 and FIG.
The increase in absorbance is more suppressed when the inhibitor is present (FIG. 2) than when the inhibitor is not present (FIG. 1).
This also suggests that the inhibitor suppresses non-specific hydrolysis of the substrate.

The PAF-AH activity is determined by the change in absorbance (ΔE) per unit time (1 minute) from 2 minutes to 5 minutes after the addition of the substrate (reagent 2 (A) or reagent 2 (B)). The PAF-AH activity value of each serum sample was calculated by using the molecular extinction coefficient ε = 12000 of paranitrophenol and paranitrophenol.

On the other hand, radioactively labeled P
Human serum PAF-AH activity was measured using AF as a substrate to confirm the usefulness of the method of the present invention.

1-O-octadecyl [octadecyl-9,10-
³ H (N)] 2-O-acetyl-sn-glycerophosphocholine (N
EN, NET-1009) 5pmole (18.5kBq), 1-O-octadecyl-2-O-acetyl-sn-glycerophosphocholine (BACHEM
FEINCHEMIKALIEN AG, O-1355) 34 pmole and an appropriate amount of human serum (50-100 fold dilution, 6.5-37.5 ul, 3 concentration steps for each sample) in 100 mM Tris-HCl buffer (pH 7.4), 5 mM EDT
Suspended in A, the total volume was adjusted to 0.1 ml and incubated at 37 ° C for 10 minutes. After completion of the reaction, lipids were extracted by the Bligh & Dyer method. That is, chloroform 0.125ml and methanol 0.25m
l and shake for 2 minutes, then 0.125 ml of chloroform
And shake for 30 seconds, and finally add 0.1 ml of water and 30
Shake for seconds. Then, centrifuge (400g, 3 minutes),
0.04 ml of the lower layer (organic layer) was spotted on TLC (Merck, 5554-1M), and the developing solvent (chloroform: methanol: water =
65: 35: 6). The radioactivity was measured using a bioimaging analyzer MacBAS5000 manufactured by Fujifilm. Enzyme activity is the dose of human serum sample (μl)
Using the amount of lyso-PAF formed (nmol) converted by the radioactivity measured by the reaction time (10 minutes) and the reaction time (10 minutes), the number of moles of lyso-PAF generated per unit time and per unit liquid volume was determined, and nmol / min. / Ml.

The correlation between the measurement result (Y) using the substrate of the present invention and the measurement result (X) using the radiolabeled PAF is shown below.
FIG. 3 shows the case without the inhibitor, and FIG. 4 shows the case with the inhibitor. The regression line without inhibitor (FIG. 3) was Y = 206X + 307, and the correlation coefficient was R = 0.97.
It was 0. The regression line with inhibitor (FIG. 4) is
Y = 179X + 93.6, correlation coefficient R = 0.997
Met. Thus, in each case, a strong correlation was observed, and when an inhibitor was present, a very strong correlation was observed, confirming the usefulness of the substrate of the present invention and the measurement method of the present invention. Was.

(Example 5: Measurement of PAF-AH) In the same manner as in Example 4, using the substrate of the present invention, a normal-range pooled serum (trade name: Nescol-X, manufactured by Chemistry and Serum Therapeutics Research Institute, released by: PAF-A in Aswell Corporation
H activity was measured. Table 4 shows the measurement results obtained by repeating these tests 10 times.

[0150]

[Table 4]

As shown in Table 4, it was confirmed that the method of the present invention is a method for accurately measuring PAF-AH activity in serum.

[0152]

According to the method for measuring PAF-AH activity of the present invention, PAF in a sample can be obtained without using a radiolabeled substrate.
-Since AH activity can be measured directly, it can be used as a safe, quick and simple measurement method for daily tests. In addition, the PAF-AH activity measurement method of the present invention can directly measure PAF-AH by monitoring the measurement time shorter than conventional methods, being free from various esterases and esterase-like substances, and monitoring chromogenic compounds. -AH
Since it has the advantage of being able to measure the activity, it is more accurate and quicker than the conventional method using enzymes or reagents that induce a chromogenic system. Thus, the prognosis can be diagnosed in a short time. Therefore, the present invention provides a highly useful PA
A method for measuring F-AH activity is provided.

[Brief description of the drawings]

FIG. 1 is a diagram showing a time course when measuring PAF-AH activity in a sample in the absence of an inhibitor.

FIG. 2 is a diagram showing a change in absorbance when measuring PAF-AH activity in a sample in the presence of an inhibitor.

FIG. 3 is a diagram showing a correlation between a measurement method using no inhibitor and a measurement method using a radiolabeled substrate.

FIG. 4 is a diagram showing a correlation between a measurement method using an inhibitor (the measurement method of the present invention) and a measurement method using a radiolabeled substrate.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Koji Mizuno 2-1-2-1, Oharano Nishisakaya-cho, Nishikyo-ku, Kyoto, Kyoto (72) Fumetsu Iwakura 2-13-7 Toyosato, Higashiyodogawa-ku, Osaka-shi, Osaka − 406 (72) Inventor Yasuji Soda 4-5-1, Motoyamakita-cho, Higashinada-ku, Kobe-shi, Hyogo F-term (reference) QQ03 QQ08 QQ30 QR41 QR54 QR57 QR66 QR67 QS02 QS20 QX01 QX02

Claims (21)

[Claims] 1. A method for measuring platelet activating factor acetylhydrolase activity, which comprises the general formula (I): (Wherein, R ₁ represents an acyl group, an alkyl group, or an alkenyl group, and A represents a saturated or unsaturated, substituted or unsubstituted hydrocarbon group having 2 to 7 carbon atoms which may be interposed by an oxygen atom. , X represents a group that becomes a coloring compound or a fluorescent coloring compound when released, and R ₂ represents a monomethylaminoethyl group, a dimethylaminoethyl group or a trimethylaminoethyl group.) Activating factor acetylhydrolase-containing sample is reacted in the presence of an inhibitor that does not inhibit platelet activating factor acetylhydrolase but inhibits other esterolytic activity-related substances, thereby releasing a chromogenic compound or fluorescent color. A method for measuring, comprising measuring the amount of an acidic compound. 2. The method according to claim 1, wherein the color-forming compound is an aromatic hydroxy compound or an aromatic thiol compound. 3. The method according to claim 1, wherein the inhibitor is an anionic surfactant,
The method according to claim 1 or 2, wherein the method is at least one inhibitor selected from the group consisting of a nonionic surfactant, a bile salt, a bile salt derivative, and a chelating agent. 4. The method according to claim 3, wherein the anionic surfactant is an alkali metal salt of an alkyl sulfate or an alkali metal salt of an alkyl sulfonic acid. 5. The method according to claim 3, wherein the nonionic surfactant is a polyoxyethylene alkyl allyl ether or a polyoxyethylene sorbitan fatty acid ester. 6. The bile salt and the bile salt derivative are sodium cholate, sodium deoxycholate, sodium chenodeoxycholate, sodium dehydrocholate, sodium taurocholate, sodium taurolithocholic acid, sodium taurodeoxycholate, tauro sodium. Sodium chenodeoxycholate, sodium tauroursodeoxycholate, sodium taurodehydrocholate, CHAPS, CHAPSO, BIGCHAP,
4. The method according to claim 3, which is deoxy-BIGCHAP. 7. A reagent for measuring platelet activating factor acetylhydrolase activity, which reagent has the general formula (I): (Wherein, R ₁ represents an acyl group, an alkyl group, or an alkenyl group, and A represents a saturated or unsaturated, substituted or unsubstituted hydrocarbon group having 2 to 7 carbon atoms which may be interposed by an oxygen atom. , X represents a group that becomes a coloring compound or a fluorescent coloring compound when released, and R ₂ represents a monomethylaminoethyl group, a dimethylaminoethyl group or a trimethylaminoethyl group.) An inhibitor that does not inhibit the activator acetylhydrolase but inhibits other related substances related to esterification activity. 8. The reagent according to claim 7, wherein the color-forming compound is an aromatic hydroxy compound or an aromatic thiol compound. 9. The method according to claim 8, wherein the inhibitor is an anionic surfactant,
The reagent according to claim 7 or 8, which is at least one inhibitor selected from the group consisting of a nonionic surfactant, a bile salt, a bile salt derivative, and a chelating agent. 10. The reagent according to claim 9, wherein the anionic surfactant is an alkali metal salt of an alkyl sulfate or an alkali metal salt of an alkyl sulfonic acid. 11. The reagent according to claim 9, wherein the nonionic surfactant is a polyoxyethylene alkyl allyl ether or a polyoxyethylene sorbitan fatty acid ester. 12. The bile salt and bile salt derivative,
Sodium cholate, sodium deoxycholate, sodium chenodeoxycholate, sodium dehydrocholate, sodium taurocholate, sodium taurolithocholate, sodium taurodeoxycholate, sodium taurochenodeoxycholate, sodium tauroursodeoxycholate, sodium taurodehydrocholate , CHAPS, CHAPSO, BIGCHA
The reagent according to claim 9, which is P or deoxy-BIGCHAP. 13. A kit for measuring platelet activating factor acetylhydrolase activity, comprising the reagent according to claim 7. Description: 14. A kit for measuring platelet activating factor acetylhydrolase activity, which kit has the general formula (I): (Wherein, R ₁ represents an acyl group, an alkyl group, or an alkenyl group, and A represents a saturated or unsaturated, substituted or unsubstituted hydrocarbon group having 2 to 7 carbon atoms which may be interposed by an oxygen atom. , X represents a group which becomes a coloring compound or a fluorescent coloring compound when released, and R ₂ represents a monomethylaminoethyl group, a dimethylaminoethyl group or a trimethylaminoethyl group.) And a container comprising a solution containing an inhibitor that does not inhibit platelet activating factor acetylhydrolase but inhibits other esterolytic activity-related substances. 15. The kit according to claim 14, wherein the color-forming compound is an aromatic hydroxy compound or an aromatic thiol compound. 16. The kit according to claim 14, wherein the substrate is dissolved in a solution or a solvent. 17. The method according to claim 15, further comprising a container containing a solution or a solvent for dissolving the substrate.
The kit according to 1. 18. A compound represented by the general formula (Ia):(Where R₁Is an acyl group having 6 to 20 carbon atoms, alkyl
X represents a group or an alkenyl group;
Represents a compound or a group that becomes a fluorescent coloring compound; ₂Is
Nomethylaminoethyl group, dimethylaminoethyl group or
Represents a trimethylaminoethyl group. ) 19. The compound according to claim 18, wherein the color-forming compound is an aromatic hydroxy compound or an aromatic thiol compound. 20. Platelet activating factor acetylhydrolar
A method for measuring zease activity, the method comprising the general formula (Ia)
A compound represented by the following formula:(Where R₁Is an acyl group having 6 to 20 carbon atoms, alkyl
X represents a group or an alkenyl group;
Represents a compound or a group that becomes a fluorescent coloring compound; ₂Is
Nomethylaminoethyl group, dimethylaminoethyl group or
Represents a trimethylaminoethyl group. ) Substrate
And a sample containing platelet activating factor acetylhydrolase,
And the resulting chromogenic compound or firefly
A measuring method comprising measuring the amount of a photochromic compound. 21. The method according to claim 20, wherein the color-forming compound is an aromatic hydroxy compound or an aromatic thiol compound.