WO2000032808A1 - Method for assaying platelet-activating-factor acetylhydrolase activity - Google Patents

Abstract

Platelet-activating-factor acetylhydrolase activity can be directly assayed safely, quickly, conveniently and accurately at a high sensitivity by using a PAF-analogous compound containing a chromophore substituent in its structure as a substrate and, further, using an inhibitor for an esterase activity-related substance other than platelet-activating-factor acetylhydrolase.

Description

Platelet activator method for measuring drolase activity

 The present invention relates to a method for measuring the activity of a clinically important platelet-activating factor, acetyltyl hydrolase, a reagent for measuring the activity of a platelet-activating factor, acetyltyl hydrolase,

The present invention relates to a kit for measuring platelet activating factor acetyl hydrolase activity, which utilizes the above reagent. Background art

 Platelet activating factor (platelet activating factor) is a mediator for the development of a wide variety of pathological conditions, such as inflammation and allergic reactions, and is abbreviated as PAF. You. On the other hand, platelet-activating factor acetylhydrolase (hereinafter referred to as PAF-AH) has the function of eliminating the powerful biological activity of PAF, and regulates the level of PAF in vivo to maintain homeostasis in the organism. Are involved. In addition, with diseases such as Kawasaki disease, systemic erythematosus, hemolytic uremic syndrome, etc., changes in serum PAF-AH activity were observed with the progression of these conditions, which were not observed in healthy subjects. ing.

Thus, PAF-AH activity in serum or plasma reflects the onset and pathological changes of various inflammatory patients, so that measurement of PAF-AH activity provides useful information for diagnosis of these diseases. This is an important new clinical test method. In addition, 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. Conventionally, the measurement of PAF-AH activity has been carried out using a bioassay method and a method using radioisotopes.

 The bioassay method is a method of measuring PAF-AH by reacting PAF-AH with a substrate PAF for a certain period of time and monitoring platelet aggregation by the remaining PAF. However, there are drawbacks such as the need to use expensive reagents, and the like, and there is a problem that is difficult to use as a general test method.

On the other hand, a method using a radioactive isotope (RIA method), ³ H or ¹⁴ C the Asechiru group PA F is reacted with labeled to PAF- AH, the force ,, or alkyl measuring the liberated radioactivity This is a method in which a group is labeled and reacted with PAF-AH, and the reaction product lyso-PAF or unreacted PAF is recovered and radioactivity is measured. However, this method using radioisotopes limits the users of radioactive substances and equipment used, and has safety problems and disposal of radioactive reagents. In addition, when collecting labeled lyso-PAF or unreacted labeled PAF and measuring the remaining radioactivity, extract lyso-PAF or PAF with an organic solvent and separate by chromatography. There was a problem that it was difficult to use as a general detection method, such as the necessity of complicated operations such as the necessity of having to perform it.

 Therefore, methods for measuring PAF-AH activity without such complicated operations are being studied. One of these methods is a method using a synthetic substrate. In recent years, various PAF-AH substrates and methods for measuring 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 that 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 this substrate to generate a dicarboxylic acid, and then to the generated dicarboxylic acid. Enzyme that reacts specifically reacts, and further continuous enzyme reaction system is combined to generate commonly used marker substances such as NAD +, NADP +, NADH, NADPH or hydrogen peroxide, and these marker substances Is measured by a known method to determine the production rate or amount of dicarboxylic acid, and thereby the PAF-AH activity is measured. Since this method is an enzymatic assay in which a large number of enzymes are combined with a detection system, there are problems such as the stability and specificity of the enzyme used, and care must be taken to maintain the performance of the reagent. There are problems such as.

 Japanese Patent Application Laid-Open No. Hei 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. According to this publication, 2-AF is released by the substitution reaction between 5,5'-dithiobis (2-nitrobenzoic acid) and one SH group of lysothio PAF generated from a substrate such as thio PAF by PAF-AH. A method for tracking the yellow color of Toro-5-mercaptobenzoic acid is described as an example, but this method uses other thiol compounds and redox substances coexisting in the reaction solution and the sample, such as ascorbic acid and pyrinolevin. It is susceptible to interference such as cysteine or daltathione, and has a problem in maintaining the performance of the reagent as in the enzymatic assay.

In addition, Japanese Patent Publication No. 4-66864 (corresponding patent: US Pat. No. 5,091,527) describes a PAF-like compound as a substrate for phospholipase. However, this publication does not show experimental data that the PAF analog can actually be a substrate for PAF-AH. On the other hand, PAF-AH activity measuring method using 1-decanoyl 1- (4-ditrophenyl dartaryl), which is a similar compound of PAF, and 1-sn-glycerose-phosphocholine as substrate, is well known. Arteriosclerosis, Thrombosis, and Vascular Biology, Vol. 16, No. 4591-599 (1996)). However, in this assay, plasma Since the substrate is also hydrolyzed by various esterases or esterase-like substances present in samples such as serum or serum, the measured values are greatly positively affected, and it is difficult to measure the PAF-AH activity accurately. Was.

 Therefore, a method for measuring PAF-AH activity that is not affected by various esterase-esterase-like substances and that is safe and easy to operate without using a radioactive substrate has been desired. That is, P A F—

It is desired to develop a measurement system that is less affected by a substrate having high specificity for AH or various esterases or esterase-like substances. Disclosure of the invention

 The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, have found that PAF-AH is not affected by a substrate having relatively high specificity for PAF-AH and various esterases and esterase-like substances. They found a measurement system, developed a safe and easy-to-operate method that can directly measure PAF-AH activity without using a radioactive substrate, and completed the present invention.

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

CH ₂ -0-R,

 ί ο ο

 I

CH-O-C-A-C-X (I)

 O—

CH ₂ -0-P-0-R ₂

 o

(In the formula, represents an acyl group, an alkyl group or an alkenyl group, A represents a saturated or unsaturated, substituted or unsubstituted hydrocarbon group having 2 to 7 carbon atoms optionally interposed by an oxygen atom, X Represents a group that becomes a coloring compound or a fluorescent coloring compound when released, and represents a monomethylaminoethyl group or a dimethylaminoethyl group. And a trimethylaminoethyl group. ) Is reacted with a platelet-activating factor acetylhydrolase-containing sample in the presence of an inhibitor that does not inhibit the platelet-activating factor acetylhydrolase but inhibits other ester degradation activity-related substances. And measuring the amount of the chromogenic or fluorescent compounds released thereby.

 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 anionic surfactants, nonionic surfactants, bile salts, bile salt derivatives, and chelating agents. 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 alkyl sulfate or an alkali metal salt of alkyl sulfonic acid.

 In a preferred embodiment, the nonionic surfactant used in the measurement method of the present invention is a polyoxyethylene alkylaryl 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, and dehydrocholate. Sodium, tau Sodium cholate, sodium taurolitocholate, sodium taurodeoxycholate, sodium tauronodeoxycholate, sodium tauroursodexoxycholate, sodium taurodehydrocholate, CHA

It is PS, CHAPSO, BIGCHAP, or Deoxy BIGCHAP.

The present invention also relates to a reagent for measuring PAF-AH activity, which has a general formula (I): CHz-OR,

 O O

CH-O-C-A-C-X (I)

 O "

CH ₂ -0-P-0-R ₂

 o

(In the formula, R 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. PAF—an inhibitor that does not inhibit AH but inhibits other related substances for esterolytic activity.

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

 In a preferred embodiment, the inhibitor contained in the reagent of the present invention is at least selected from the group consisting of anionic surfactants, nonionic surfactants, bile salts, bile salt derivatives and chelating agents. One type of inhibitor.

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

 In a preferred embodiment, the nonionic surfactant contained in the reagent of the present invention is polyoxyethylene alkylaryl ether or polyoxyethylene sorbitan fatty acid ester.

Further, in a preferred embodiment, the bile salt and the bile salt derivative contained in the reagent of the present invention are sodium cholate, sodium deoxycholate, sodium chenodeoxycholate, sodium dehydrocholate, tauroko. Sodium oxalate, sodium taurolitocholate, sodium taurodeoxycholate, sodium taurochenodeoxycholate, sodium tauroursodeoxycholate, sodium taurodehydrocholate, CHAPS: ό— [(3—Cholamidopropyl) dimethyla onio "propanesulfonic acid, Cri APSO: 3— [(3— Cholamidopropyl) diraethylamraonio] ― 2-hydroxypro panesulfonic acid, BI GCHAP: N, N— Bis (3— D— gluconamidopropy Dcholamide, or Deoxy BI GCHAP: N, N— Bis (3— D— gluconamid opropy 1) deoxycho 1 ami de ₀

 The present invention further relates to a kit for measuring the activity of platelet activating factor acetylhydrolase, which comprises the above reagent.

 The present invention also provides a kit for measuring P AF-AH activity, wherein the kit has the general formula (I)

 o

(In the formula, represents an acyl group, an alkyl group or an alkenyl group, 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.) ,

A container containing a solution containing an inhibitor that does not inhibit PAF-AH but inhibits other related substances related to esterification activity.

In a preferred embodiment, an aromatic hydroxy compound or an aromatic thiol compound is released from the substrate upon reaction with PAF-AH. In a preferred embodiment, the substrate is dissolved in a solution or a solvent.

 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):

CH-0-C-CH ₂ -CH ₂ -CX (I _a )

 I o "

CH ₂ -0- P-0-R ₂

 O

(In the formula, represents an acyl group, an alkyl group, or an alkenyl group having 6 to 20 carbon atoms, X represents a group that becomes a coloring compound or a fluorescent coloring compound when released, and R ₂ represents a monomethylaminoethyl group. Represents a dimethylaminoethyl group or a trimethylaminoethyl group.

 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, which relates to a compound represented by the general formula (la):

CH-0-C-CH ₂ -CH ₂ -CX (I _a )

 O "

CH ₂ -0- P-0-R ₂

 o

(In the formula, represents an acyl group, an alkyl group or an alkenyl group having 6 to 20 carbon atoms, X represents a group that becomes a coloring compound or a fluorescent coloring compound when released, and R ₂ represents a monomethylaminoethyl group. , Dimethylaminoethyl group or Represents a limethylaminoethyl group. ) And a PAF-AH-containing sample, and measuring the amount of a chromogenic compound or a fluorescent chromogenic compound released by the reaction.

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

 According to the above invention, a substrate having relatively high specificity for PAF-AH, and a direct measurement system for PAF-AH that is not affected by various estera-esterase-like substances are provided. Furthermore, since the present invention does not use a radioactive substrate, it provides a method that can be used as a safe, rapid, and simple measurement method for daily inspection. As described above, the PAF-AH measurement method of the present invention (1) has a shorter measurement time than conventional methods, (2) is not affected by various esterases and esterase-like substances, and (3) has a chromogenic property. The ability to directly measure PAF-AH activity by monitoring a compound or a fluorescent compound is more accurate than the conventional method using enzymes or reagents that lead to a chromogenic system. Is possible. 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, according to the present invention, there are provided a reagent for measuring PAF-AH and a kit containing the same. The PAF-AH activity can be measured directly and easily with these reagents and kits, so that the diagnosis of disease based on the change in PAF-AH activity and the progress of the disease state can be grasped. Therefore, the medical contribution of the present invention is great. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a diagram showing a time course when the PAF-AH activity in a sample without an inhibitor was measured.

FIG. 2 is a graph 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 the correlation between the measurement method using no inhibitor and the measurement method using a radioactively labeled 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. BEST MODE FOR CARRYING OUT THE INVENTION

 (Features of the measuring method of the present invention)

 The feature of the method for measuring platelet activating factor acetyl hydrolase (PAF-AH) activity of the present invention is that, together with the substrate represented by the above general formula (I), it inhibits an ester-decomposing activity-related substance other than PAF-AH. By adding an inhibitor that inhibits the release of color-forming compounds or fluorescent compounds due to non-specific hydrolysis by these ester-decomposing activity-related substances, the PAF-AH activity can be more precisely improved. The point is to measure. In other words, the chromogenic compound released from the substrate is to design the measurement system so that the fluorescent compound is derived only from PAF-AH as much as possible. Further, when the substrate represented by the general formula (Ia) among the substrates represented by the general formula (I) is used, the measurement can be performed with higher sensitivity. When the substrate used in the present invention reacts with PAF-AH, it releases a dicarponic acid derivative bound to a chromogenic or fluorescent compound, but when the dicarbonic acid derivative is released, it develops a chromogenic or fluorescent chromogenic compound. It has the feature of releasing (releasing) the compound. That is, when the substrate used in the present invention reacts with PAF-AH, an amount of a chromogenic or fluorescent chromogenic substituted aromatic compound that is proportional to the PAF-AH activity is released. By monitoring fluorescent compounds, PAF-AH activity can be measured directly.

The conventional assay method is based on the ability to enzymatically introduce the dicarboxylic acid generated by contacting a substrate with PAF-AH into a chromogenic system, or 5,5'-dithiobis (2-nitro) (Benzoic acid), which leads to the color development system of released 2-twotro-5-mercaptobenzoic acid, which is not a direct color development, so it not only degrades the substrate itself but also deactivates the enzyme. There were many factors related to the inaccuracy of the measurement, such as the deterioration of the composition leading to the system, and the PAF-AH activity value could not be obtained accurately. When these problems are solved by the measuring method of the present invention, a special effect is exhibited. In other words, in the measurement method according to the present invention, a chromogenic or fluorescent compound that can be directly detected by PAF-AH is generated, and therefore, it is possible to use a compound that is related to ester degradation activity contained in serum samples, plasma samples, and the like. PAF-AH activity can be measured with high accuracy while minimizing measurement errors.

 In the specification of the present application, the term “substances related to ester degradation activity” includes enzymes or substances that cleave ester bonds, such as ester esterases (esterases) and esterase (esterase) -like substances. Examples of the ester-degrading enzyme (esterase) -like substance include proteins having an esterase activity such as apolipoprotein B contained in LDL.

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

 (Substrate used in the present invention)

 The substrate used in the method for measuring P AF-AH of the present invention is represented by the general formula (I). General formula (I):

CH ₂ -0-R,

 J o o

 CH-0-C-A-C-X (I)

 I O "

CH ₂ -0-P-0-R ₂

o In the formula, R 1 is preferably an acyl group, an alkyl group or an alkenyl group. Preferably, the carbon number is from about 6 to about 20. More preferably, it is about 10 to about 16, and even more preferably, about 12 to 14.

 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. In addition, an oxygen atom may be interposed in the hydrocarbon group. That is, it may have a structure such as -C-0-C-.

 X is a compound that is released (released) when the PAF-AH reacts with the substrate to release the dicarboxylic acid derivative, and preferably has a color-forming property or a fluorescent color-forming property. Is preferably a group that becomes an aromatic hydroxy compound or an aromatic thiolic compound when hydrolyzed, for example, 412 trophenyl, 2-chloro-14-nitrophenyl, 2- Examples thereof include a fluoro-412 trophenyl group, a 3-phenyl group, a 4-nitrophenyl group, a 4-nitrophenyl group, a 5-indoloxy group, and a 2,6-diphenyl-4-nitrophenyl group.

 Examples of the fluorescent chromogenic compound include a 4-methylcoumaryl group, a coumaryl group, and a flavonoxyl 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 (la).

OO II II CH-0-C-CH ₂ -CH ₂ -CX (I a)

o In the general formula (la), Ri, X, and R _2, respectively, wherein Ri, the same X, and the R _2. Examples of the compound represented by the general formula (la) include 1-Miris Toinole 2- (4-ditrophenylsuccinyl) -sn-glycerose-phosphocholine (compound 8) and 1-palmitoyl 2- (4- Nitrophenylsuccinyl) 1 sn-glycerose-phosphocholine (compound 26), 1 _alkyl-12- (4-nitrophenylsuccinyl) 1 sn-glycerophosphocholine

(Compound 27), 1-oleoyl-1-2- (412-trophenylsuccinyl) -sn-glycose-phosphocholine (compound 28), 1-octanoyl-2- (4-nitrophenylsuccinyl) -sn-glycerophosphocholine (compound 27) compound 29), 1- Dekanoiru 2- (4-nitrophenyl succinyl) _ sn Gurise opening one phosphocholine (compound 30), 1-lauroyl one 2- (4-two Toro phenylalanine succinyl) Single _sn - glyceryl Theroux phosphocholine (Compound 31).

 When the compound with A is 2 (compound having a succinyl group) is used as the substrate, the PAF is about 2 to 4 times larger than when the compound with A is 3 (compound having a daltalyl group) is used as the substrate. —AH activity was observed. From this, it is considered that a compound in which A is 2 (a compound having a succinyl group) is most effective as a substrate.

 1 _ myristoyl- 1 2-(4-ditrophenylsuccinyl) 1 sn-dali cello-phosphocholine (compound 8) has 3 carbon atoms of A, 1-myristoinole 1-(4- (Nitrophenyl dartaryl) It has the effect of increasing the sensitivity by a factor of 4 or more compared to 1-sn-glycerose phosphocorin.

 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-octanoinole 2- (4 nitrophenyl dalaryl) sn glycerophosphocholine (compound 1), 1-decanoyl 2- (4-1-2 trophenyl dartaryl) one sn-glycerose one phosphocholine (compound 2),

 1 1 lauroy / leh 2— (4—2 trofeninole gnoretarinole) 1 sn—glycerose—phosphocholine (compound 3),

1-Miris Toinore 2- (4-Trofueninoregunoretarinore) 1 sn-glycerophosphocholine (compound 4),

 1-oleoinole 2- (4-ditrophenyl dartaryl) 1 sn-glycerose 1 phosphocholine (compound 5),

 1-anorequil-1 2-((412-trophenylenyldarthalyl) -1 sn-glycerone phosphocholine (compound 6),

 1-alkenyl 2- (4-nitrophenyldartarinole) 1 sn—glycerose—phosphocholine (compound 7)

 (1) One stolyl 2-((4-12 Trofel-adiboinole)) 1 sn-glycerose-phosphocholine (compound 9),

1—Miris Toileu 2— (4_Nitrofeninolepime Leinole) 1 sn—glycerophosphocholine (Compound 10),

 1-Miris toy / ray 2— (4-Nitrofeninoles veroninole) 1-sn-glycerophosphocholine (Compound 11),

 1—Miris Toileu 2— (4-Nitrophenylenyl) 1 sn—Glyce Mouth—Phosphocholine (Compound 12),

 1 _ iris toinore 2- (2-chloro-1-4-nitrophenyl dartaryl) 1 sn-glycerophosphocholine (compound 13),

 1-Miris Toileu 2— [(4-Methyl) tamarylglutarinole] —sn—Glyceline-phosphocholine (compound 14),

1-Miris Toileu 2- (2-Hunooleol 4-12-trophenylenyl dararyl) sn-Glycetophosphocholine (Compound 15), 1-Miristoyl-1-2- (3-fluoro-412-tohenylphenyldaltharyl) — sn—glycerophosphocholine (compound 16),

 1-Miris Toinole 2- (4-Nitrothiophenyldartalyl) 1 sn-glycerophosphocholine (compound 17),

1-Miris Toileu 2-(5-Indolyloxyglutaryl) -sn-Dali cellophosphocholine (compound 18),

 1-Miris Toinole 2- (2,6-diphenyl-1-nitrophenyl dartaryl) —sn-glycerophosphocholine (compound 19),

 1-Miris Toinole 2- (4-Nitrophenylglycolyl) -1-sn-glycerophosphocholine (compound 20),

1-Miristoyl-1- [5-dimethylamino-2- (2-thiazolylazo) phenylsuccinyl] -sn-glycerophosphocholine (compound 21), 1-millistoyl 2- [1- ( 2-triazolyl Honoré § zo) Single 2- Nafuchirusa Kushiniru] one s _n - glycerin opening one phosphocholine (compound 22),

1 _ Myris Toileu 2— [(412-Trophenyl) dimethylsuccinyl] 1 sn-glycerophosphocholine (Compound 23),

 1-Milis Toino 2- 1-[(6-flavonoxyl) succinyl] -1-sn-glycerophosphocholine (compound 24), and

 1-Palmi Tonole 2 -— (4-Nitrophenyl danoletarinole) _sn-glycerophosphocholine (Compound 25).

 (Method for producing substrate used in the present invention)

The PAF-AH substrate 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 Japanese Patent Application Laid-Open No. Sho 52-89622 discloses a method in which 1-glycol-lipophosphocholin, 110-alkylphosphocholine (commercially available lyso-PAF), I-O-norekeninolephosphocholine (commercially available lysophosphocholine plasma The starting material is used as a starting material, and these starting materials are reacted with dicarboxylic anhydride (for example, succinic anhydride) in a mixed solution of chloroform anhydride and anhydrous dichloromethane using triethylamine as a catalyst to give a desired 2-position. The dicarboxylic acid is introduced to give 1-acyl-12-monodicarboxyphosphocholine. When anhydrous succinic acid is used, 1-acyl-2-succinylphosphocholine is obtained.

 The 1-acyl 1-2-monodicarboxyphosphocholine obtained in this way was purified with oxalyl chloride according to the description of William N. Washburn et al., J. Am. Chem. So 112, 2040-2041 (1990). An aromatic hydroxy compound as a chromophore, for example, p-ditrophenol is esterified in the presence of triethylamine to give 1-acyl-1- (4-nitrophenyl-sacshell) -phosphocholine.

 When a dicarboxylic acid having a long methylene group is introduced at the 2-position, a monobenzyl carboxylate obtained by reacting dicarboxylic anhydride with a protecting group benzyl alcohol (4th edition Experimental Chemistry Course 22 Synthesis IV page 131) can be used. According to the method described in the above-mentioned Japanese Patent Application Laid-Open No. 52-89662, a monobenzyldicarboxylate is reacted with 1-acylglycerophosphocholine to give 1-acyl-2-monobenzylcarbonylphosphocholine. Get. Next, the obtained monobenzyl compound is debenzylated using palladium / carbon under a hydrogen gas atmosphere to obtain 1-acyl-2-monocarbonylphosphocholine. The thus obtained 1-acyl-12-monopropionylphosphonylcholine is chloridized with oxalyl chloride, and an aromatic hydroxy compound as a chromophore, for example, p-ditrophenol is present in triethylamine. Esterification is performed under the following conditions to obtain 1-acyl-1- (4-1-2-trophenyl-2-yl) -phosphocholine.

(Inhibitor used in the present invention) As a measurement method of the present invention, a method that does not inhibit PAF-AH, but is preferably performed in the presence of an inhibitor that 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 ester degradation activity-related substances. Concentrations do not inhibit PAF-AH, but include both meanings as inhibitors that inhibit other substances related to esterolytic activity.

 In the measurement of 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 X in formula (I) or (Ia) (Eg, a chromogenic compound) is undesirably released, resulting in noise of PAF-AH activity. Therefore, when measuring PAF-AH in a serum or plasma sample, an inhibitor that does not inhibit PAF-AH but inhibits other ester degradation activity-related substances (activity) (hereinafter referred to as a nonspecific reaction inhibitor) It is preferable to exist). 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 non-ionic surfactants, or anionic surfactants and bile salts or bile salt derivatives. Two or more inhibitors of the same type may be used.

Examples of anionic surfactants include fatty acid salts (eg, sodium stearate, potassium oleate, etc.), alkyl sulfate salts (eg, sodium lauryl sulfate (SDS), lithium lauryl sulfate, etc.), alkylbenzenes Sulfonates (for example, sodium laurylbenzenesulfonate, sodium 4-octylbenzenesulfonate, etc.), alkyl naphthalene sulfonates (for example, sodium 2-naphthalene sulfonate, etc.), alkyl sulfonates Succinates (eg, sodium dialkylsulfosuccinate), alkinoresylphenylethenoresulfonates (eg, sodium alkyldiphenyl etherdisulfonate), alkyl phosphates (eg, potassium alkylphosphate) ), Polyoxyethylene alkyl sulfates (eg, sodium polyoxyethylene lauryl ether sulfate, triethanolamine sulfate, etc.), polyoxyethylene alkylaryl sulfates (eg, polyoxyethylene nonyl sulfate) Sodium enyl ether sulfate), alkyl sulfonates (for example, sodium octane sulfonate, sodium nonane sulfonate, sodium decane sulfonate, tridecane sulfone) Sodium), at least one anionic interface selected from the group consisting of naphthalenesulfonic acid formalin condensate (for example, sodium salt of naphthalenesulfonic acid formalin condensate) and polyoxyethylene alkyl phosphate ester Preferably, it is an activator. Examples of nonionic surfactants include polyoxyethylene alkyl ethers (for example, polyoxyethylene lauryl ether, polyoxyethylene acetyl ether, polyoxyethylene stearyl ether ether, polyoxyethylene oleyl ether, etc.). , Polyoxyethylene alkyl aryl ethers (for example, polyoxyethylene octyl phenyl ether, polyoxyethylene noninolephenyl ether, polyethylene X-100 (registered trademark), etc.), polyoxyethylene derivatives (polyoxyethylene-polyoxypropylene condensate, etc.), sorbitan fatty acid esters (eg, sorbitan monolaurate, sorbitan monopanolemitate, sonolebitan monostearate) Sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleate, sorbitan distearate, etc., polyoxyethylene sorbitan fatty acid esters (for example, polyoxyethylene sorbitan monolaurate) (Trade name Tween 20 (registered trademark)), polyoxydiethylenesorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene sonorevitan monooleate, polyoxyethylene sorbitan tristearate, polyoxy Ethylene sorbitan trioleate, etc., polyoxyethylene sorbitol fatty acid ester (for example, polyoxyethylene sorbite tetraoleate, etc.), glycerin fatty acid ester (for example, glycerol monostearate, glyceronol monooleate, etc.), Polyoxyethylene fatty acid esters (eg, polyethylene glycol monolaurate, polyethylene glycol monostearate, polyethylene glycol monooleate, polyethylene glycol monooleate) Over distearate, etc.), polyoxyethylene alkyl § Min, and is the group or al selection consisting of alkyl Al force Noruami de, at least one non-ionic surfactants are preferred.

 Examples of bile salts and bile salt derivatives include sodium cholate, sodium deoxycholate, sodium chenodeoxycholate, sodium dehydrocholate, sodium taurocholate, sodium taurolithate. , Sodium taurodoxycholate, sodium tauronodeoxycholic acid, sodium tauroursodoxycholate, sodium taurodehydrocholate, CHAP S, CHAP S〇, BIGCHAP and Deoxy BIGC HAP Preferred are at least one bile salt as well as a bile acid salt derivative selected from the group.

 Any one of the anionic surfactant, the nonionic surfactant, the bile salt and the bile salt derivative may be used alone, or two or more may be used in combination. Further, two or more different types of inhibitors may be used in combination.

 (Reagent for PAF-AH activity measurement)

The reagent for measuring PAF-AH activity of the present invention contains a PAF-AH substrate represented by the above general formula (I) or (la). Substrate is 0.001 mM-250m M, more preferably 0.01 mM to 100 mM, even more preferably 0.01 mM to 10 mM.

 The reagent for measuring PAF-AH activity is preferably a solution, and the substrate for PAF-AH is preferably a buffer having a pH of about 3 to about 9 by selecting a stable pH for each substrate. (Eg, HE PES buffer, citrate buffer, tartrate buffer, phosphate buffer, acetate buffer, Tris buffer, borate buffer, good buffer, etc.).

 Further, the reagent for measuring PAF-AH activity of the present invention preferably contains the above-mentioned non-specific reaction inhibitor. The concentration of the nonspecific reaction inhibitor is not particularly limited if the inhibitor does not inhibit PAF-AH, but if it inhibits PAF-AH, the concentration at which the inhibitor does not inhibit PAF-AH is determined in advance. It may be used at a concentration lower than that. The reagent for measuring PAF-AH activity of the present invention may contain, in addition to these components, a water-miscible organic solvent as needed to dissolve the PAF-AH substrate. Examples of water-miscible organic solvents include alcohols (eg, methanol, ethanol, n-propyl alcohol, isopropyl alcohol), ketones (eg, acetone, methyl isobutyl ketone), and esters (eg, methyl acetate, ethyl acetate, acetic acid). Propyl, methyl propionate) and the like. The water-miscible organic solvent is preferably about 30% by weight, based on the final solution weight, so as not to affect the activity of PAF-AH. /. Or less, more preferably about 20% by weight or less.

The reagent for measuring PAF-AH activity of the present invention may contain a substance that promotes dissociation of a chromogenic or fluorescent compound. These substances include ct-cyclodextrin, 3-cyclodextrin, _γ -cyclodextrin, or basic functions of these cyclodextrins (for example, ethylaminoethyl, dimethylaminoethyl, or trimethylammonioethyl). Quaternary ammonium salts). This 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 Ariruesute hydrolase Ya C a ²⁺ dependent phospholipase eight _2, it is possible to suppress the decomposition reaction of the substrate used in the present invention, to specifically measure the PAF- AH activity become able to. As this chelating reagent, an appropriate chelating agent such as EDTA or EGTA can be used. Chelating agents may be included at about 20 mM.

 Furthermore, the reagent for measuring PAF-AH activity of the present invention includes, in addition to the above, those generally used for measuring the activity of enzymes such as buffers, stabilizers, activators, and excipients. You may go out.

As will be described in detail later, in the present invention, HDL-associated-PAF-AH activity, LDL-associated-PAF-AH activity, and total serotype PAF-AH activity can be measured. In order to measure HD Lassociated-PAF-AH activity, the reagent for measuring PAF-AH activity of the present invention contains an anionic compound (eg, dextran sulfate, sulfated cyclodextrin, sulfate) as a substance that aggregates LDL. Oligosaccharides, heparin, へ paran sulfate, phosphotungstic acid, etc.), cations such as Mg ²⁺ , Mn ² \, etc., polyclonal antibodies or monoclonal antibodies (eg, anti-apolipoprotein B antibodies) Etc. can be included in appropriate amounts.

 In addition, in order to fractionate and quantify LD Lassociated-PAF-AH, the reagent for measuring PAF-AH activity of the present invention includes a polyclonal antibody or a monoclonal antibody (anti-apolipoprotein A antibody, A polypoprotein E antibody).

 (P AF—AH activity measurement)

The measurement method of the present invention uses the PAF-AH activity measuring reagent Add a sample with F-AH activity (probably) and react with it. Measure the absorbance of the released chromogenic or fluorescent compound and the excited fluorescence to measure qualitatively or quantitatively Is the way.

 Samples for measuring PAF-AH activity include 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. Is used. Test samples containing PAF-AH purified products and serum, plasma, etc., PAF-AH are buffered solutions of pH 6-9 (for example, HEPES buffer, Tris buffer, phosphate buffer, borate buffer, Dilute appropriately with a good buffer, etc.), keep at 20-40 ° C, and use for the measurement reaction. To these buffers, NaCl, KCl, etc. may be added so as to obtain 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 a 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 measure the sample blank by reacting the measurement reagent without the substrate with the sample. In the case of the rate method, the absorbance may be measured within a time when the measurement can be performed quantitatively. The activity value of PAF-AH can be calculated based on the molecular extinction coefficient of the fluorescent chromogenic substance.

In addition, the method for measuring PAF-AH activity of the present invention can be carried out using a manual technique or an automatic analyzer. The change in absorbance of a chromogenic or fluorescent compound released from the substrate is measured. For example, in the case of a chromogenic compound, a change in absorbance at a wavelength of 280 to 560 nm is detected, and in the case of a fluorescent chromogenic compound, spectroscopic detection is performed by irradiating a laser beam to excite fluorescence, By determining the amount of change in absorbance per unit time (the so-called rate-assay method), PAF-AH activity can be determined with high accuracy. For example, in the general formula (I) of the present invention, the substrate having two carbon atoms of A in general formula (I) (that is, general formula (la)) is composed of 1-millis-toyl 2- (4-ditrophenylsuccinyl) 1 sn— Glycetophosphocholine (compound 8) produces 4-nitrophenol. At this time, the absorbance at 405 nm, which is the absorption wavelength of 412 trophenol, increases quantitatively with the increase of 412 trophenol, so the rate of increase or the amount of increase in absorbance at 405 nm is calculated.

 Further, using the method for measuring PAF-AH activity of the present invention, HD Lassociated—PAF—AH activity, LD Lassociated—PAF—AH activity, and total serotype PAF—AH activity are separated and accurately measured, respectively. it can. By using a measurement reagent containing a substance that aggregates LDL, LDL can be aggregated and removed, and HDLassociated-PAF-AH can be quantified. In addition, by using a measurement reagent containing a substance that aggregates HDL, HDL can be aggregated and removed, and LDLassociated-PAF-AH can be quantified. If neither is added, total serotype PAF-AH activity is measured.

A specific measuring method will be described below. First, an inhibitor reagent containing the inhibitor dissolved in an appropriate buffer (for example, 50 mM HEPES buffer (pH 7.4) containing the inhibitor and 150 mM NaC1), a substrate solution (for example, 16% ethanol) A 50 mM HEPES buffer (ρΗ7.4) containing a substrate and 150 mM NaCl is prepared. After mixing an appropriate amount of human serum or PAF-AH purified product solution with the inhibitor solution, add the substrate solution and react. The pH of the reaction is preferably from about 6.5 to about 8.0, most preferably about 7.4. The reaction temperature is 20 ° C to 40 ° C, preferably 37 ° C. 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), the amount of change in the absorbance per unit time is determined, and the molecular absorbance coefficient of the chromogenic compound or the fluorescent chromogenic compound is determined. , P AF-AH activity value (nmol / min / ral sample) can be determined. In addition, when performing the automatic measurement by the LT method, first, the inhibitor solution and the base solution are prepared. For example, use an H-7170 automatic analyzer (Hitachi, Ltd.) and measure according to the measurement parameters. Mix the purified human serum or PAF-AH with the inhibitor solution, and after a certain period of time, mix the substrate solution to start the reaction. 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 change in absorbance per unit time obtained from the sample (Δ Ε s), the change in absorbance per unit time obtained by adding purified water instead of the sample (Δ Ε _にお け_る ), and the amount of change in the reaction solution. The PAF-activity value of each serum sample is calculated from the molecular extinction coefficient (ε) of the chromogenic compound or the fluorescent chromogenic compound by the following formula. f _{Bruno,, (AE S - ΔΕ Β} ) X final reaction volume ^{X 1 0 6 PAF-AH (} nmol / min / ml) = ·

 ε X sample amount X layer length

(P A F—ΑΗ activity measurement kit)

 The kit for measuring PAF-ΑΗ activity of the present invention includes the reagent for measuring PAF-AH activity to which a substrate, and if necessary, a nonspecific reaction inhibitor and other additives are added. The kit may be, for example, in the form of an ampoule containing a reagent for measuring PAF-AH activity, in a form in which a fixed amount of the reagent for measuring PAF-AH activity is injected into a well, or in a container containing a reagent for measuring PAF-AH activity. And a form including a hole for use. The PAF-AH activity is measured by adding the serum sample to the PAF-AH activity measurement reagent attached to these kits.

The kit for measuring PAF-AH activity of the present invention is a test strip containing the reagent for measuring PAF-AH activity to which a substrate, and if necessary, a nonspecific reaction inhibitor and other additives are added. Can be When the reagent is a solution, it may be a test piece that has been immersed in the reagent and then dried. By immersing this in serum etc., it can be qualitatively PAF—AH activity can be confirmed. The approximate activity can also be determined using a colorimetric table. Therefore, the container includes such a test piece.

 Further, the kit of the present invention contains a substrate represented by the above general formula (I) and an inhibitor (a non-specific reaction inhibitor) that does not inhibit PAF-AH but inhibits another ester degradation activity-related substance. Each solution may be filled in an independent container, and may be mixed at the time of use. In this case, the substrate may be dissolved in a solution or a solvent, and the solution for dissolving the substrate and Z or the solvent may be contained in a separate container. The form to be selected may be determined in consideration of substrate stability, operability, amount used, and the like.

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

 (Example)

 Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to the examples.

 (Example 1: Synthesis of substrate)

The synthesis examples of Compounds 1 to 31 are shown below.

 (Compound 1: 1-Otanoyl 2- (4-nitrophenyl dalaryl) -sn-Synthesis of glycerol-phosphocholine)

Dissolve 340 mg (0.68 mmo 1) of 1-octanoyl-2-glutarylphosphatidylcholine in 3 ml of dichloromethane (dehydrated), add 2 M oxalyl chloride 800/1 (1.6111111101) under ice-cooling, and add room temperature The mixture was stirred for 1.5 hours below. After concentration under reduced pressure, dissolve in dichloromethane (dehydrated) 3m1 and stir at room temperature under stirring at room temperature for 4-12 trophenol 104mg (0.75mmo1), triethylamine 138mg (190ML: 1.36mmo1), dichloromethane ( (Dehydration) 3 ml of solution was added. After stirring at room temperature for 2.5 hours, the mixture was concentrated under reduced pressure. To this was added 60 ml of chlorophenol: methanol (2: 1) and washed with water (15 ml). Lower layer The filtrate was concentrated after drying over anhydrous sodium sulfate, decyl ether was added thereto, and the mixture was turned cloudy and allowed to stand overnight. The supernatant was collected, and the residue was purified by silica gel column purification using a chloroform-form aqueous methanol solution to obtain 21.8 mg (52%) of a yellow oily substance.

 (Compound 2: Synthesis of 1-decanoyl 1-2- (412 trophenyl dartaryl) 1 sn-glycerophosphocholine)

 272 mg (0.55 mmo 1) of 1-decanoinoleate 2-glutarylphosphatidinolecoline was dissolved in 3 ml of dichloromethane (dehydrated), and 550 μl of 2M oxalyl chloride (1. Immo 1) was added under ice-cooling. The mixture was stirred at room temperature for 1.5 hours. After concentration under reduced pressure, dissolve in 3m1 of dichloromethane (dehydrated), and stir at room temperature under stirring at room temperature, forty-one nitrophenol 84mg (0.6mmo1), triethylamine 1.1mg (153μL: 1.1mmo1) 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 was added 60 ml of methanol (2: 1), and washed with water (15 ml). The lower layer is removed, dried over anhydrous sodium sulfate, and the filtrate is concentrated, purified by silica gel column chromatography with methanol, and further purified by silica gel column chromatography with form-methanol-aqueous-water system. 14 mg (32%) of yellow amorphous crystals I got

 (Compound 3: Synthesis of 1-Lauroyl 2- (4-Ditrophenyldarthalyl) -sn-Glycetophosphocholine)

 1 1 Lauroi Lou 2 _ Glutaryl phosphatidylcholine 290 mg (0.

52 mmol) was dissolved in 3 ml of dichloromethane (dehydrated), and 520/1 of oxalyl chloride (1.04 mmol) was added under ice-cooling, followed by stirring at room temperature for 1.5 hours. After concentration under reduced pressure, dissolve in 3m1 of dichloromethane (dehydrated), and stir at room temperature under stirring at room temperature. Forty-nine phenol (79mg, 0.57mmo1), triethylamine 105mg (145μL: 1.04mmol), dichloromethane (dehydrated) 3 ml of solution was added. After stirring at room temperature for 2.5 hours, the mixture was concentrated under reduced pressure. To this Lum: 60 ml of methanol (2: 1) was added and washed with water (15 ml). The lower layer was removed, dried over anhydrous sodium sulfate, and the filtrate was concentrated. The residue was purified by silica gel column purification using a form-aqueous form-methanol solution to obtain 171 mg (49%) of the desired fraction.

 (Compound 4: Synthesis of 1-myristoylu-2- (4-12-trophenyldartaryl) -sn-glycerophosphocholine)

 1 Dissolve 283 mg (0.49 mmo 1) of 1-Miristoyl 2-glutarylphosphatidylcholine in 3 ml of chloroform (dehydrated) and add 490 μl (0.98 mmo 1) of 2 M oxalinole chloride under ice-cooling. After the addition, the mixture was stirred at room temperature for 1.5 hours. After concentration under reduced pressure, dissolve in 3 ml of chloroform (dehydrated) and stir at room temperature under stirring at room temperature for 42.1 tol phenol 102.1 mg (0.74 mmo 1), triethylamine 99 mg (136 μL: 0.98 mmo 1) A solution of 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. 75 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 by silica gel column chromatography using chloroform / methanol / aqueous system to obtain 182 mg (53%) of a yellow amorphous substance.

Negation _{R (CDC1 3): 0.89 (} t, 3H, J 7. ΟΗζ), 1.25 (s, 20H), 150-1.65 (ra, 2H), 1.9 5-2.12 (ra, 2H), 2.29 (t, 2H , J = 7.7 Hz), 2.40-2.55 (ra, 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 (ra, 1H), 4.

25- 4.45 (ra, 3H), 5.15-5.30 (m, 1H), 7.31 (d, 2H), 8.29 (d, 2H);

MS (SIMS): 703 (MH ⁺ )

 (Compound 5: Synthesis of 1-oleoyl-1-2- (412-trophenyldartaryl) -sn-glycemic-phosphocholine)

Dissolve 350 mg (0.55 mmo 1) of 1-oleoyl-1-2-glutarinolephosphatidinorecholine in 3 ml of dichloromethane (dehydrated) and add 2 M o-chloride under ice-cooling. After adding 550 μl (1.1 mmo 1) of quizalil, the mixture was stirred at room temperature for 1.5 hours. After concentration under reduced pressure, dissolve in 3 ml of dichloromethane (dehydrated) and stir at room temperature under stirring at room temperature for 84 mg (0.61 mm o 1) of 4 12 trofenol, 11 mg of triethylamine (153 μL: 1.1 mm o) 1), 3 ml of dichloromethane (dehydrated) solution was added. After stirring at room temperature for 2.5 hours, the mixture was concentrated under reduced pressure. To this, 60 ml of form: 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 by a silica gel column using a form-form / methanol / aqueous solution containing water to obtain 303 mg (73%) of the desired fraction.

 (Compound 6: Synthesis of 1-alkyl-1 2 _ (4-nitrophenyl dalaryl) —sn-glycerophosphocholine)

First, 1-alkyl one 2-arsenide Dorokishi one sn- glyceryl port one 3- Hosufachi phosphatidylcholine (lyso one PAF: AVANT I POLAR LIPI DS, I NC ( abbreviation, AVT) made by alkyl chains, and mainly C ₁₆ C ₁₈₎ was used to prepare 1-alkyl one 2-glutaryl phosphatidyl choline by atmospheric method. 267 mg (0.45 mmo 1) of the obtained 1-alkyl-1-2-glutarylphosphatidylcholine was dissolved in 3 ml of dichloromethane (dehydrated), and 450 μl (0.9 mmo 1) of 2 M oxalyl chloride was added under ice cooling. Then, the mixture was stirred at room temperature for 1.5 hours. After concentration under reduced pressure, dissolve in 3 ml of dichloromethane (dehydrated), and stir at room temperature under stirring, 4-nitrophenol 69 mg (0.5 mmo 1), triethylamine 90.

A solution of 9 mg (125 μL: 0.9 mmo 1) and 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 was added 60 ml of chloroform: methanol (2: 1) and washed with water (15 ml). The lower layer was removed and dried over anhydrous sodium sulfate.The filtrate was concentrated, purified by silica gel column purification with methanol, and further purified by silica gel column chromatography using a form-methanol-aqueous solution with a cuff. Yellow amorphous crystals 146 mg (45%) I got (Compound 7: Synthesis of 1-alkenyl-2- (4-nitrophenyl dalaryl) -sn-glycerophosphocholine)

First, lysophosphatidylcholine plasmalogen (manufactured by DOOS AN SER DARY RESEARCH LABORATOR IES (abbreviated as SRL): the alkenyl chain is mainly C ₁₆ and C ₁₈ ), and 1-alkenyl 1-2-glutamate is obtained in a conventional manner. Ril phosphatidylcholine was made. Then, using this 1-anololecenyl-12-gnoletaryl phosphatidylcholine (137 mg, 0.23 mmol) as a starting material, a synthesis was carried out in the same manner as in the case of compound 6, to obtain 98 mg (60%) of yellow amorphas crystals. Was.

 (Compound 8: 1—Myristoyl-1-2 -— (412-trophenylsuccinyl)

-sn—Synthesis of Glycetophosphocholine)

 Dissolve 200 mg (0.35 mmo 1) of 1-Miris Toylol 2-succinylphosphatidylcholine in 3 ml of dichloromethane (dehydrated) and add 350 μl of 2 M oxalyl chloride (0.71 mmo 1) under ice-cooling. The mixture was stirred at room temperature for 1.5 hours. After concentration under reduced pressure, dissolve in 3 ml of dichloromethane (dehydrated), and stir at room temperature under stirring at room temperature. Forty-one nitrophenol 54 mg (0.39 mmo 1), triethylamine 71.7 mg (99 1: 0.7 1 mmo 1), Cloguchi honolem (Dehydration) 3 ml of the solution was added. After stirring at room temperature for 1.5 hours, 60 ml of chloroform: methanol (2: 1) was added to this solution, followed by washing with water (15 ml). The lower layer was separated, dried over anhydrous sodium sulfate, concentrated, and purified with a silica gel column using a form-form-methanol-aqueous solution with a black mouth to obtain 154 mg (64%) of a pale yellow oil.

_{NMR (CDC1 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, 2H), 2.82-2.93 (m, 2H), 3.34 (s, 9H), 3.79 (br, 2H), 4.01 (br, 2H), 4.10-4.22 (m, 1H), 4.22-4.48 (ra, 4H), 5.27 (br, 1H ), 7.30-7. 36 (m, 2H), 8.27-8.32 (m, 2H); MS (SIMS): 689 (MH ⁺ )

(Compound 9: 1—Miris Toinole 1— (4—Nit phenyladip. Synthesis of one sn—glyceic monophosphocholine)

 (1) Starting with 265 mg (0.45 mmo 1) of 1-myristoyl-1-2-aziboylphosphatidylcholine as a starting material, the synthesis was carried out in the same manner as in the synthesis of compound 8; 16.7 mg (52%) of a yellow oil of 1-sn-glycose-l-phosphocholine was obtained.

 (Compound 10: Synthesis of 1-Miris Toyl 2- (4-Nitrophenylphenylpimelole) sn-glycerophosphocholine)

 (1) Starting with 347 mg (0.57 mmo 1) of 1-myristoyl 2-pimeloylphosphatidylcholine as a starting material, the synthesis was carried out in the same manner as in the synthesis of compound 8, and 1-Miris Toyl 2- (4-nitrophenylvimeirole) was synthesized. 341 mg (82%) of a yellow oil of 1 sn-glycerophosphocholine was obtained.

 (Compound 11: 1 _ Synthesis of 1-myris toino-one 2- (4-nitrophenylsperonole) -sn-glycerose-phosphocholine)

 Starting from 284 mg (0.46 mmo 1) of 1-Miris Toinole 2-suberoylphosphatidylcholine, synthesis was carried out in the same manner as in the synthesis of Compound 8, and 1-Miris Toinore-1 2- (4-Ni Tolufenii / resveroinole) A single sn-glycerophosphocholine yellow oil was obtained in an amount of 114 mg (34%).

 (Synthesis of compound 1 2: 1—Miris Toyl 2— (4-Nitral phenol)

 1 One Millis Toileu 2—Azeroyl Phosphatidylcholine 21 Omg (0.

33mmo 1) was used as a starting material, and the synthesis was carried out in the same manner as in the synthesis of compound 8. 1-millistoylone / ray 2- (4-nitropheninola zeroisole) 1 sn-glycerophosphocholine yellow oil 1 9 Omg (76%) was obtained.

Negation _{R (CDC1 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), 3.90-4.02 (m, 2H), 4.10-4.22 (m, 1H), 4.25-4.50 (ra, 3H) , 5.23 (br, 1H), 7. 21-7.35 (m, 2H), 8.20-8.35 (m, 2H) ₀

 (Compound 13: 1—Myristoyl-1-2 -— (2-chloro-4-2-trophenyl glutarinole) -sn—Glyceto-phosphocholine synthesis)

 Dissolve 1-77 mg (0.3 mmo 1) of 1-Miris Toinole 1 in 3 ml of dichloromethane (dehydrated) and add 2 M oxalyl chloride 300 1 (0.6 mmo 1) under ice-cooling. After the addition, the mixture was stirred at room temperature for 2 hours. After concentration under reduced pressure, dissolve in 3 ml of dichloromethane (dehydrated), and stir at room temperature, with 2-chloro-4-nitronitrophenol 104 mg (0.6 mm o 1), 84 μL of triethylamine (0.6 mmo 1) A solution of 3 ml of dichloromethane (dehydrated) was added. After stirring at room temperature for 3 days, the mixture was concentrated under reduced pressure. To this was added 60 ml of chloroform: methanol (2: 1) 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 by a silica gel column with a water-soluble form-methano-l-water system to obtain a yellow oil (156 mg, 71%).

 (Compound 14: Synthesis of 1-myristoyl-1-2-((4-methyl) coumarylglutaryl) -1-sn-glycerose-phosphocholine)

 (1) Dissolve 191 mg (0.33 mmo 1) of 1-Millis Toylol 2-glutarylphosphatidylcholine in 3 ml of dichloromethane (dehydrated), and add 2 M oxalyl chloride 328 1 (0.6711111101) under ice-cooling. Thereafter, the mixture was stirred at room temperature for 2 hours. After concentration under reduced pressure, dissolve in 3 ml of dichloromethane (dehydrated), and stir at room temperature. 4- (Methyl) coumarin 1 15 mg (0.67 mm o 1), triethylamine 9

A solution of 1.4 AL (0.67 mmo 1) and 3 ml of dichloromethane (dehydrated) was added. After stirring at room temperature for 4 days, the mixture was concentrated under reduced pressure. To this was added 60 ml of form: methanol (2: 1) 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 silica gel column purification using a form-form-methanol-aqueous system with a black mouth to obtain 183.2 mg (76.5%) of a colorless amorphous substance. NMR (CDCI3): 0.89 (t, 3H, J = 6.9 Hz), 1.25 (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 (ra, 2H), 2.70 (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, IH), 4.30-4.48 (m, 3H), 5.19-5.32 (m, lH), 6.27 (s, IH), 7.00-7.11 (m, 1H), 7.11- 7.20 (m, IH), 7.58-7.70 (m, IH); MS (SIMS): 726 (MH ⁺ )

 (Compound 15: 1 Synthesis of 1-myristoyl 2- (2-fluoro-1-412-trophenylenyldartharyl) -sn-glycerophosphocholine)

 Dissolve 266 mg (0.46 mmo 1) of 1-Miristoyl-1-2-glutarylphosphatidylcholine in 3 m of chloroform (dehydrated) and add 2 M oxalyl chloride 457 1 (0.91 mmo 1) under ice-cooling. ) And stirred at room temperature for 1.5 hours. After concentration under reduced pressure, dissolve in 3 ml of chloroform (dehydrated), and stir at room temperature. 2-Fluoro 4-nitrophenol 107.7 mg (0.69 mmo 1), triethylamine 1 27.4 μΙ ^ (0.9 1ππιιηο 1), 3 ml of a solution of black-mouthed form (dehydrated) was added. After stirring for 3 hours at room temperature, 1 ml of 0.1 N hydrochloric acid was added. To this solution was added 7 Om1 of chloroform: methanol (2: 1), and the mixture was washed with water (15 m1). The lower layer was removed, dried over anhydrous sodium sulfate, and the filtrate was concentrated. The residue was purified by silica gel column chromatography using chloroform-methanol-aqueous system to obtain 260 mg (79%) of yellow amorphous.

 (Compound 16: 1—Miris Toyl 2- (3-Fluoro 1-412 trophenyl dartalyl) 1sn—Synthesis of glycerophosphocholine)

 1 Starting with 223 mg (0.38 mmol) of 2-glutarylphosphatidylcholine as a starting material, synthesis was carried out in the same manner as in the synthesis of compound 15; 1-Miris Toyl 2- (3-Fnoroleol 4 —Nitrophenyl dartaryl) was obtained as a yellow amorphous crystal of sn-glycerophosphocholin (171 mg, 62%).

(Compound 17: 1—Miris Toinole 1—2- (4-12 Trotiophenyl) Dal Synthesis of Tallylphosphatidylcholine)

 Dissolve 227 mg (0.39 mmo 1) of 1-myristoyl-1-2-glutarylphosphatidylcholine in 3 ml of chloroform (dehydrated) and add 390 μl of 2 M oxalyl chloride (0.78 mmo 1) under ice-cooling. After the addition, the mixture was stirred at room temperature for 2 hours. After concentrating under reduced pressure, dissolve in 3 ml of black mouth form (dehydrated) and stir at room temperature. 41.2 Trotiofenol 90.8 mg (0.59 mmo 1), triethylamine 108.3 L (0.78 mmo 1), black mouth form (dehydrated) ) 3 ml of solution were added. After stirring for 2 hours at room temperature, 1 ml of 0.1 N hydrochloric acid was added. To this solution was added 70 ml of form: methanol (2: 1) and washed with water (15 ml). The lower layer was removed, dried over anhydrous sodium sulfate, and the filtrate was concentrated. The residue was purified by silica gel column chromatography using a form-form / methanol / aqueous solution system to obtain 202 mg (72%) of a brown oil.

_{NMR (CDC1 3): 0.80-0.98 (} ra, 3H), 1.27 (br, 20H), 1.59 (br, 2H), 1.92-2.12 (m, 2H), 2.20-2.35 (m, 2Η), 2.35-2.52 (m, 2Η), 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 ⁺ ) ₀ (compound 18: 1— Miris Toileu 2- (5-Indolyloxyglutarinyl) -sn-Synthesis of glycerol phosphocholine)

(1) Dissolve 205 mg (0.35 mmo 1) of 1-myristol 2-yl gnoletaryl phosphatidylcholine in 3 ml of chloroform (dehydrated) and add 2 M oxalyl chloride 352 ^ 1 (0.7 mmo 1 ) Was added, followed by stirring at room temperature for 2 hours. After concentration under reduced pressure, dissolve in 3 ml of black mouth form (dehydrated) and stir at room temperature, 70.4 mg (0.53 mm o 1) of 5-hydroxyindole, 98.2 μL of triethylamine (0.7 mmo 1), black mouth form (Dehydration) 3 ml of solution was added. After stirring at room temperature for 2 hours, 1 ml of 0.1 N hydrochloric acid was added. To this solution was added 70 ml of methanol: 2: 1 methanol and washed with water (15 ml). Take the lower layer After drying over anhydrous sodium sulfate, the filtrate was concentrated and purified with a silica gel column using a chloroform-form-methanol-aqueous system to give 143 mg (59%) of a yellow oil.

_{NMR (CDC1 3): 0.80-1.00 (} m, 3H), 1.20-1.40 (ra, 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 (ra, 3H), 3.89-4.27 (m, 5H), 4.27- 4.50 (m, 1H), 5.21 (br, 1H), 6.41 (s, lH), 6.70-6.90 (ra, 1H), 7.21 (s, 1H), 7.40-7.65 (m, 1H), 11.18 (s, 1H); MS (SIMS): 697 (MH ⁺ ) o

 (Compound 19: 1-Miris Toyl 2- (2,6-Diphenyl 2- 4-Trojerdanoreletari / re) 1-sn-Glyceto-phosphocholine synthesis)

 Dissolve 254 mg (0.44 mm o 1) of 1-Miris Toileu 2-Gnoletarinole phosphatidylcholine in 3 ml of black-mouthed form (dehydrated) and cool under ice-cooling 2 M oxalyl chloride 43 7 μ1 (0.87 mm After adding o1), the mixture was stirred at room temperature for 2 hours. After concentrating under reduced pressure, dissolve in 3 ml of black-mouthed form (dehydrated), and stir at room temperature under stirring at room temperature for 2,6-dipheninole 4-12 trofenanol 19 1 mg (0.65 mmo 1), triethylamine 122 / i L (0.87 mmo) l), 3 ml of a solution of black-mouthed form (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 70 ml of form: methanol (2: 1), 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 by a silica gel column using a form-form-methanol-aqueous system with a black mouth to obtain 219 mg (58%) of a yellow oil.

_{NMR (CDC1 3): 0.89 (} t, 3H, J = 7.2 Hz), 1.26 (br, 20Η), 1.26-1.61 (ra, 4Η), 1. 91 (t, 2Η, J = 7.6 Hz), 2.14 ( t, 2H, J = 7.1 Hz), 2.26 (t, 2H, J = 7.8 Hz), 3.31 (s, 9H), 3.74 (br, 2H), 3.92 (t, 2H, J = 6.0 Hz), 4.00- 4.15 (ra, 1H), 4.28 (br, 2H), 4.30-4.42 (ra, 1H), 5.14 (br, 1H), 7.32-7.50 (m, 10H), 8.28 (s, 2H); MS (SI

MS): 855 (MH ⁺ ) o (Compound 20: 1—Miris Toyl 2- (4-12-Trophenylenylaicillyl) 1-synthesis of glycerophosphocholine)

 Dissolve 164 mg (0.28 mmo 1) of 1-Miris Toileu 2-glycolylphosphatidylcholine in 3 ml of chloroform (dehydrated), and add 2 M oxalyl chloride 281 μ1 (O.56 mmo I) under ice cooling. After the addition, the mixture was stirred at room temperature for 2 hours. After concentration under reduced pressure, dissolve in 3 ml of chloroform (dehydrated), and stir at room temperature with 4-nitrophenol 43. Omg (0.31 mm o 1), 78 L of triethylamine (0.56 mm o 1), chloroform (dehydrated) 3 ml of solution was added. After stirring for 2 hours at room temperature, 1 ml of 0.1 N hydrochloric acid was added. To this solution was added 56 ml of chloroform: methanol (2: 1) and washed with water (14 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 form-form-methanol-aqueous system using a filter to give 102 mg (51%) of a yellow oil.

 (Compound 21: Synthesis of 1-myristoyl-1- [5-dimethylamino-2- (2-thiazolylazo) phenylsuccinyl] -1-sn-glycerophosphocholine)

419 mg (0.74 mmo 1) of 1-Miris Toilu 2-succinylphosphatidylcholine was dissolved in 6 ml of chloroform (dehydrated), and 738 μ1 of 2 M oxalyl chloride (1.48 mmo I) was added under ice-cooling. Thereafter, the mixture was stirred at room temperature for 2.5 hours. After concentration under reduced pressure, dissolve in 6 ml of chloroform (dehydrated) and then stir at room temperature under stirring at room temperature, 202 mg (0.81 mmo 1) of 5-dimethylamino-2- (2-thiazolylazo) phenol, 206 μL of triethylamine (1.48 mmo 1), Mouth form (dehydrated) 6 ml of solution was added. After stirring at room temperature for 2 hours, 2 ml of 0.1 N hydrochloric acid was added. 150 ml of chloroform: methanol (2: 1) was added to this solution, and the mixture was 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 silica gel column purification using a form-aqueous-methanol-aqueous system to give 247 mg (42%) of a red oil. (Compound 22: Synthesis of 1-Miristoyl-1-2- (1- (2-triazolylazo) -12-naphthylsuccinyl) -1-sn-glycerophosphocholine)

 Starting from 330 mg (0.58 mmo 1) of 1-Miris Toyl 2-succinylphosphatidylcholine, synthesis was carried out in the same manner as in the synthesis of compound 21. 1-Miris Toyl 2- [1- (2-triazolylazoso) ) — 2-naphthylsuccinyl] -l-sn-glycerol-phosphocholine was obtained as a red-brown oil (64 mg, 14%).

 (Compound 23: Synthesis of 1-myristoyl-1-2-((4-ditrophenyl) dimethinolesuccino-nore) 1-sn-glycose-phosphocholine)

 Dimethinole succininophosphatidylcholine 256 mg (0.43 mm o

Using 1) as the starting material, the synthesis was carried out in the same manner as in the synthesis of compound 21. 1-Miris toyl 2-[(4-Ditrophenyl) dimethylsuccinyl] -1-sn-griseose-phosphocholine white amorphous crystal 1 5 Omg (49%) was obtained.

 (Compound 24: 1-Myristoyl 2-[(6-flavonoxyl) sax shell] —sn—Synthesis of glycerophosphocholine)

 1—Myristoyl 2-Succinylphosphatidylcholine 310 mg (0.55 mmo 1) as the starting material, 1—Myristoyl 1—2 — [(6-Flavone oxyl) succinyl] —sn—Glycose monophosphocholine white amorpha This gave 295 mg (68%) of sucrose.

_{NMR (CDC1 3): 0.87 (} t, 3H, J = 6.9 Hz), 1.24 (br, 20Η), 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 (ra, 1H), 7.10—7.65 (m, 8H).

 (Synthesis of compound 25: 1—palmi tonole 1— (4—2 1-port phenyl daltarinole) 1 sn—glyce 1-phosphocholine)

1Palmi Toinole 2-Glutarinolephosphatidylcholine 320 mg (0.51 mmo 1) was dissolved in 3 ml of dichloromethane (dehydrated), 765 μl of 2M oxalyl chloride (1.53 mmo 1) was added under ice cooling, and the mixture was stirred at room temperature for 1.5 hours. After concentration under reduced pressure, dissolve in 3 ml of dichloromethane (dehydrated) and stir at room temperature under stirring at room temperature for 104 mg (0.75 mmol) of 412 trophenol, 139 μL of triethylamine (1.02 mmol), and 3 ml of dichloromethane (dehydrated). The solution was added. After stirring at room temperature for 2.5 hours, the mixture was concentrated under reduced pressure. To this was added 75 ml of methanol (2: 1) and washed with water (15 ml). The lower layer was removed, dried over anhydrous sodium sulfate, and the filtrate was concentrated. The residue was purified on a silica gel column using a form-form / methanol / aqueous solution with a black mouth to obtain 203.6 mg (55%) of a yellow amorphous substance.

 (Compound 26: 1 Synthesis of 1-palmi tonone 2- (4-122 phenyl succinyl) 1 sn-glycose 1-phosphocholine)

1Palmi Tolue 2-Succinylphosphatidylcholine 217 mg (0.36 mm o 1) is dissolved in 5 ml of chloroform (dehydrated), and cooled with ice. 2 M oxalyl chloride 3 6.5 _μ 1 ( After adding 0.73 mmo 1), the mixture was stirred at room temperature for 1.5 hours. After concentrating under reduced pressure, dissolve in 5 ml of chloroform (dehydrated), and stir at room temperature under stirring at room temperature for 4-2-trophenol 55.8 mg (0.4 OmmoI), triethylamine 74 mg (102 μL) : 0.73 mm o 1), 3 ml of a solution of black mouth form (dehydrated) was added. After stirring at room temperature for 17 hours, the mixture was concentrated under reduced pressure. 75 ml of black form: methanol (2: 1) was added thereto, followed by washing with water (14 ml). The lower layer was separated, dried over anhydrous sodium sulfate, and the filtrate was concentrated. The residue was purified by silica gel column purification using chloroformmethanol / aqueous system to obtain 160 mg (62%) of yellow amorphous crystals.

_{NMR (CDC1 3): 0.88 (} t, 3H, J = 6.9Hz), 1.20-1.39 (ra, 24Η), 1.48-1.63 (ra, 2H), 2.20-2.35 (m, 2H), 2.70-2.82 (m , 2H), 2.85-2.98 (m, 2H), 3.32 (s, 9H), 3.72- 3.85 (m, 2H), 4.00 (t, 2H, J = 7.0Hz), 4.08-4.21 (m, 1H), 4.25- 4.45 (m, 3H), 5.15-5.35 (m, 1H), 7.28-7.31 (m, 2H), 8.25-8.30 (ra, 2H) (Compound 27: 1-Alkyl-2- (4-Nitrophenylsuccinyl) -synthesis of sn-glycerophosphocholine)

First, 1 primary alkyl one 2-arsenide Dorokishi one sn- glyceryl port one 3- Hosufachi phosphatidylcholine (lyso one PAF: alkyl chains, mainly C _{1 6} and C ₁₈₎ using a conventional method by 1-alkyl one 2 -Succinylphosphatidylcholine was prepared. Using the obtained 1-alkyl-12-succinylphosphatidinorecholine (189 mg, 0.33 mmo 1) as a starting material, it was synthesized in the same manner as in the synthesis of compound 26 to obtain 1-alkyl-12- ( 4-159 mg (69%) of brown amorphous crystals of 4-nitropheninolesuccinyl) -sn-glycero-phosphocholine were obtained.

_{MR (CDC1 3): 0.82- 0.95} (m, 3H), 1.18- 1.39 (m, 28H), 1.40 - 1 · 69 (m, 2Η), 2.2

5 (t, 2H, J = 7.8Hz), 2.70—2.82 (m, 2H), 2.82-2.92 (m, 2H), 3.32 (s, 9H), 3.77 (br, 2H), 4.00 (t, 2H, J = 7.0Hz), 4.08- 4.22 (m, 1H), 4.22-4.45 (m, 3H), 5.15- 5.33 (m, 1H), 7.29-7.34 (m, 2H), 8.24-8.30 (m, 2H)

 (Compound 28: Synthesis of 1-oleoyl-1- (4-nitrophenylsuccinyl) -sn-glycose monophosphocholine)

 1-oleoyl-1-2-succinylphosphatidylcholine 229 mg (0.37 mmo 1) was used as a starting material, and was synthesized in the same manner as in the synthesis of compound 26, and 1-oleoinole 2- (4-12-trofeninole succinyl) -1 There were obtained 2 2 mg (85%) of yellow amorphous crystals of sn—glyce mouth—phosphocholine.

_{NMR (CDC1 3): 0.88 (} t, 3H, J = 6.9Hz), 1.15-1.40 (m, 20Η), 1.55 (t, 2Η, J = 6.8H z), 1.59-2.08 (m, 4H), 2.25 (t, 2H, J = 7.7Hz), 2.70-2.81 (m, 2H), 2.83-2.94 (m, 2H), 3.32 (s, 9H), 3.77 (br, 2H), 4.00 (t, 2H, J = 5.7Hz), 4.08-4.21 (m, 1H), 4.29 (br, 2H), 4.33-4.48 (m, 1H), 5.16-5.40 (m, 3H), 7.28-7.34 (ra, 2H) '8.24- 8.30 (m, 2H)

(Compound 29: 1—Octanoyl-1 2— (4-Nitrofenilzacino 2) 1—Synthesis of sn-glycero-phosphocholine) CT / JP99 / 06745

 39

1-Octanoyl 2-Succinylphosphatidylcholine 15 lmg (0.33 mmo 1) was used as a starting material and synthesized in the same manner as in the synthesis of compound 26. 1-Otanoinoyl 2- (4-Nitrofufenii / resuccinyl) As a result, 159 mg (80%) of a yellow oil of sn—glyce monophosphocholine was obtained.

_{MR (CDC1 3): 0.87 (} t, 3H, 7. ΟΗζ), 1 · 10- 1.38 (m, 8H), 1.43-1.65 (ra, 2H), 2.

25 (t, 2H, J = 7.5Hz), 2.65-2.81 (ra, 2H), 2.81-2.95 (m, 2H), 3.33 (s, 9H), 3.77 (br, 2H), 3.89—4.05 (m, 2H), 4.08-4.20 (m, 2H), 4.20—4.32 (m, 1H), 4.32-4.45 (m, 3H), 5.10—5.30 (m, 1H), 7.31 (d, 2H), 8.27 ( d, 2H)

 (Compound 30: Synthesis of 1-decanoyl 2- (4-nitrophenylsuccinyl) -sn-glycerophosphocholine)

 1-decanoyl 2-succinylphosphatidinolecoline 247 mg (0.5 2 mmo 1) was used as a starting material and synthesized in the same manner as in the synthesis of compound 26. 1-decanoyl-1- (4-nitropheninolesuccinyl) 1) sn-glycerose 208 mg (64%) of yellow amorphous crystals of monophosphocholine were obtained.

_{NMR (CDC1 3): 0.87 (} t, 3H, J = 6.9Hz), 1.25 (s, 12H), 1.45- 1.65 (m, 2Η), 2.25

(t, 2Η, J = 7.7Hz), 2.65-2.82 (m, 2H), 2.85-2.95 (m, 2H), 3.33 (s, 9H), 3.77 (br, 2H), 3.99 (t, 2H, J = 5.5Hz), 4.08-4.22 (m, 1H), 4.25-4.48 (m, 3H), 5.20-5.30 (ra, 1H), 7.27-7.35 (m, 2H), 8.22-8.30 (m, 2H )

 (Compound 31: 1—Lauroyl 2- (4-12 trophenylsuccinyl) —sn—Synthesis of glycerophosphocholine)

 1-Lauroinore 2- 2-succinylphosphatidinorecholine Starting from 56 mg (0.5 mmo 1) of starting material, it was synthesized in the same manner as in the synthesis of compound 26, and 1-lauroinore-1 2- (4-ni 177 mg (53%) of a yellow oily substance of one sn-glycerose and one phosphocholine was obtained.

Negation _{R (CDC1 3): 0.87 (} t, 3H, J = 7.0Hz), 1.15-1.36 (m, 16H), 1.49-1.62 (m, 2H), 2.

25 (t, 2H, J = 7.7Hz), 2.70-2.81 (m, 2H), 2.85-3.00 (m, 2H), 3.33 (s, 9H), 3.77 T / JP99 / 06745

 40

(br, 2H), 4.00 (t, 2H, J = 5.6Hz), 4.11-4.22 (m, 1H), 4.25-4.42 (m, 3H), 5.20-5.30 (m, 1H), 7.28-7.35 (m , 2H), 8.23-8.31 (m, 2H)

 (Example 2: PAF-AH activity measurement; substrate specificity)

 (Example of purification of the enzyme p AF—AH)

 About 40 Om1 of human plasma was prepared with KBr to a specific gravity of d = 1.063, and centrifuged to obtain a fraction containing chylomicron, VLDL, and LDL. l Q Sepharose dialyzed against 5 OmM Tris-HCl buffer (pH 7.4) containing OmM CHAP S, and previously equilibrated with 5 OmM Tris-HCl buffer (pH 7.4) containing 1 OmM CHAP S Attached to the column. After washing the column with the same buffer, the column was eluted with a linear gradient of NaCl (0-0.5M). The eluted PAF-AH active part was concentrated with Centrip 30 and applied to Cefacryl S-200 equilibrated with 50 mM Tris-HCl buffer (pH 7.4) containing 10 mM CHAP S, 0.3 M Na C1. did. The active part of PAF-AH eluted with the same buffer was concentrated with Centrip 30 and the buffer was equilibrated with 25 mM Tris-HCl buffer (pH 7.4) containing 10 mM CHAP S, 0.3 M Na C1. Attached to a Blue Sepharose column. After washing with the same buffer, elution was carried out with 50 mM Tris-HCl buffer (pH 8.0) containing lOmM CHAP S, 1.5 M NaCl. The active part of PAF-AH was concentrated with Centrip 30 to obtain purified PAF-AH.

 (Measurement of activity)

 PAF-AH activity was measured for Compounds 1 to 13, 15 to 17, 20, and 25 synthesized above.

 The following solution,

 Buffer: 50 mM HEPS buffer (pH 7.4) containing 150 mM NaCl, 10 mM EDTA; and

Substrate solution: 4 mM substrate, 90 mM NaCl, 6 mM EDTA, and 4 mM 41

A 30 mM HEPES buffer solution (ρΗ7.4) containing 0% ethanol was prepared.

Mix 10 u1 of the purified PAF-AH product obtained by the above method with 900 u1 of the buffer solution, leave the mixture in a thermostat at 37 ° C for 5 minutes, add 100 μl of the substrate solution, and start the reaction. Was. After the addition of the substrate solution, the change in absorbance per unit time (1 minute) from the second minute to the fifth minute (ΔΕ ₅ ) and the molecular extinction coefficient f of paranitrophenol f = 12000 (compound 13, 13 The PAF-ΑΗ activity value (nraol / rain / sample lral) was calculated from the above formula based on the molecular extinction coefficient ε = 18000 for the compounds 15 and 16. The results are shown in Table 1. Wherein the [Delta] [epsilon] _beta is a value measured by adding purified water instead of the sample. table 1

vn mol / min / mlgSilJ From the results in Table 1, it can be seen that the compounds of A == 2 to 7 in the general formula (I) are the bases of PAF-AH. The smaller the A, the higher the PAF-AH activity tends to be, indicating that the case of A = 2 in the general formula is most excellent as a PAF-AH substrate. It was also shown that when the acyl group (R!) In the general formula (I) has 8 to 18 carbon atoms, it can be a substrate. When the number of carbon atoms is 10 to 16, the activity tends to be relatively high, and when the number of carbon atoms is 12 to 14, the activity tends to be higher. Furthermore, it was shown that when is an alkenyl group (compound 7), it can also be a substrate.

 Compounds that release halogen-substituted ditrophenyl compounds as color formers (compounds 13, 15, and 16) have also been shown to be useful as substrates.

 Considering the results in Table 1, it is considered that compounds of the general formula (I) in which A = 2 and where 12 to 14 are suitable as a substrate for PAF-AH. In particular, the compound 8 (1—myristolyl 2) in the general formula (I) wherein A is 2 and is 14

-It was found that (4-12 trophininole succino)-1 sn-glycemic-1 phosphocholine) was the best substrate. This compound 8 is a compound of the general formula 4 used as a substrate for PAF-AH 4 (1-1-millistoyl 2- (4-nitrophenyl dartaryl)-1-sn-glycerophosphocholin (general formula ( This indicates that it is a substrate of PAF-AH that is more than 4 times more sensitive than A = 3 and R _x = 14) of I).

 (Example 3: PAF-AH activity measurement; effect of inhibitor)

 The following inhibitor solution and substrate solution were prepared respectively. As the substrate, compound 8 (1-myristoyl-1- (4-nitrophenylsuccinyl) -sn-glycose-phosphocholine) was used.

Inhibitor solution:

 The inhibitors shown in Table 2 were dissolved in 50 mM HEPES buffer (pH 7.4) containing 150 mM NaCl at the concentrations shown in Table 2.

Substrate solution:

30 mM HEPE S buffer (pH 7.4) containing 4 mM compound 8, 40% ethanol and 90 mM NaC1. 43 Mix 10 u1 of purified human serum or PAF-AH with 900 u1 of the inhibitor solution, leave in a thermostat at 37 ° C for 5 minutes, add 100 u1 of substrate solution, and react. Started. Change in absorbance per unit time (1 minute) from the 2nd to 5th minute after addition of the substrate solution ^: The amount (ΔΕ) and the molecular extinction coefficient of paranitrophenol £ = 12000 PAF-AH activity value (Nmo1 / min Zml sample) was calculated. The results are shown in Table 2. Table 2

As is evident from Table 2, in the presence of various inhibitors,

A large difference in the behavior of the activity was observed between the purified PAF and the purified AH. In other words, the purified PAF-AH product retains almost 90% or more of the activity (the decrease in activity by the inhibitor is small), whereas the activity of human pool serum is 90% or more. Nothing is retained. This is because the relative content of the PAF-AH purified product, which results in a non-specific hydrolysis reaction as a result of purification, is low, and the relative activity is unlikely to be reduced by the inhibitor. Topur serum It contains a lot of substances that cause non-specific hydrolysis reactions, suggesting that the relative activity was reduced as a result of the suppression of this non-specific hydrolysis activity by inhibitors. That is, compounds (inhibitors) listed in Table 2 rarely inhibit PAF-AH, but inhibit substances related to esterolytic activity other than PAF-AH, ie, esters other than PAF-AH. It was shown to be an inhibitor of degradation-related substances.

 (Example 4: Measurement of PAF-AH activity: correlation with conventional method)

 The following reagents were prepared.

Reagent 1 (A) (buffer):

 50 InM HEPS buffer containing 15 OmM NaCl (pH 7.4) Reagent 2 (A) (substrate solution):

 1. Contains 6 mM Compound 8, 16% ethanol and 15 OmM NaC1

 5 OmM HE PES buffer (pH 7.4);

Reagent 1 (B) (inhibitor solution):

 5 OmM HEPS buffer (pH 7.4) containing 15 OmM NaCl, 1 OmM EDTA, 1 mM sodium lauryl sulfate, and 5 mM CHAPS;

 as well as

Reagent 2 (B) (solution containing substrate and inhibitor):

 5 OmM HEPES buffer (pH 7.4) containing 15 OmM NaCl, 1 OmM EDTA, 1 mM sodium lauryl sulfate (SDS), 5 mM CHAPS, 16% ethanol and 1.6 mM compound 8.

Serum samples: 5 samples S1, S2, S3, S4 and S5.

For the measurement of PAF-AH activity, an automatic analyzer H-7170 (Hitachi, Ltd.) was used. The parameters at the time of measurement are as follows. 45 Table 3

 According to parameter 1 above, 5 µl of serum sample (S1 to S5) and 240 2401 of reagent 1 (Α) (buffer) or 1 (Β) (inhibitor solution) are dispensed and mixed, and then mixed. ° C, left for 5 minutes. To this, a substrate or a substrate and an inhibitor solution were added as follows to form a group without an inhibitor and a group with an inhibitor:

 Group without inhibitor: 80 µl of reagent 2 (A) (substrate solution) added to the test solution obtained by mixing serum sample and reagent 1 (A) (buffer);

 Inhibitor group: A test solution obtained by mixing a serum sample with reagent 1 (Β) (inhibitor solution) and adding 80 µl of reagent 2 (Β) (solution containing substrate and inhibitor).

 In each group, compound 8 was hydrolyzed by the reaction of PAF-II in the serum sample with the substrate (compound 8), and an increase in the absorbance derived from the generated 4-12 trophenol was observed. This change in absorbance (time course) is shown in FIGS. 1 and 2. FIG. 1 shows the case without an inhibitor, and FIG. 2 shows the case with an inhibitor.

 As is evident from the comparison between Figs. 1 and 2, when no inhibitor is present (Fig.

From 1), it can be seen that the inhibitor (SDS + CHAP S + EDTA) is present (Fig.

In 2), the increase in absorbance is suppressed. This suggests that the inhibitor (SDS + CH APS + EDTA) inhibits the activity of related substances other than PAF-II, and suppresses nonspecific hydrolysis of the substrate (compound 8). I have. Next, the method for measuring human serum PAF-AH activity of the present invention was compared with the conventional method for measuring human serum PAF-AH activity using radiolabeled PAF as a substrate. Was found to be useful.

 The PAF-AH activity according to the method of the present invention is determined by the change in absorbance 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-AΕ activity value of each serum sample was calculated using the amount (Δ) and the molecular extinction coefficient ε = 12000 of paranitrophenol.

 The measurement of human serum PAF-AH activity using a radiolabeled PAF as a substrate, which was a conventional method, was performed by the method described below.

1 one O- Okutadeshiru [Okutadeshiru ^{9, 10- 3 H (N)} ] 2- O- Asechiru one sn- glyceryl port phosphocholine (NEN Corp., NET- 1009) 5 pmo 1 e (1 8. 5 k B q), 1-O-octadecyl-2-O-acetinolate sn-glycerophosphocholine (BACHEM FEINCHEMIKALIEN AG, 0-1355) 34 pmo 1 e, and appropriate amount of human serum (diluted 50-100 times, 6.5-37.5 u Each sample (3 concentration steps) was suspended in 10 OmM Tris-HCl buffer (pH 7.4) and 5 mM EDTA, and the total volume was adjusted to 0.1 lm 1 and incubated at 37 ° C for 10 minutes. After completion of the reaction, lipids were extracted by the Bligh & Dyer method. That is, add 0.125 ml of chloroform and 0.25 ml of methanol to the reaction solution and shake for 2 minutes, then add 0.125 ml of chloroform and shake for 30 seconds, and finally shake Then, 0.1 ml of water was added and shaken for 30 seconds. After that, centrifugation (400 g, 3 minutes) was performed, and the lower layer (organic layer) 0.041111 was spotted on the same plate (Merck, 5554-1M). Metano: No. 65: 35: 6). The radioactivity was measured using a bioimaging analyzer Mac BAS 5000 manufactured by Fujifilm. Enzyme activity is expressed in units of the amount of lyso-PAF produced (nmo 1), which is calculated based on the radioactivity measured by the dose (μ 1) of the human serum sample and the reaction time (10 minutes). The number of moles of lyso-PAF generated per unit time and liquid volume was determined and expressed as nmol / min / m1.

 Figure 3 shows the correlation between the measurement results (Y) using compound 8 and the measurement results (X) using radiolabeled PAF, with and without the inhibitor shown in Fig. 3 and Fig. 4, respectively. . The regression line without inhibitor (FIG. 3) was Y == 206X + 307, and the correlation coefficient was R = 0.970. The regression line with the inhibitor (Fig. 4) was Y = 179X + 93.6, and the correlation coefficient was R = 0.997. Thus, in each case, a strong correlation was observed, and when an inhibitor was present, an extremely strong correlation was observed, confirming the usefulness of the measurement method of Compound 8 and the present invention. Was.

 (Example 5: PAF-AH measurement)

 Using compound 8, normal range pooled serum (trade name: Nescor I-X, manufacturer: Chemistry and Serological Therapy Research Institute, release: Azdell Co., Ltd.) PAF

—AH activity was measured. As in Example 4, Table 4 shows the results of 10 repeated experiments using a system containing the inhibitor and a system not containing the inhibitor. As an inhibitor,

SDS, CHAP S and EDTA were used.

 Table 4 Activity (nmole minZ ml sample)

 Experiment No Without inhibitor With inhibitor

 1 640 348

 2 671 329

 3 598 348

 4 603 346

 5 656 344

 6 603 336

 7 593 336

 8 585 332

 9 613 344

 10 681 329

 Average 624 339

 Standard deviation 34.8 7.7

CV (%) 5.6 2.3 As shown in Table 4, when Compound 8 was used without an inhibitor, the measured values were large and the standard deviation was large because the substances related to esterolytic activity other than PAF-AH were also measured. The standard deviation was small, indicating that PAF-AH activity could be measured accurately. From these results, it was confirmed that Compound 8 was excellent as a substrate, and that PAF-AH activity in serum could be accurately measured by using Compound 8 as a substrate and using an inhibitor.

 (Example 6)

 Using compound 5 (1-oleoyl-1- (4-nitrophenyldartalyl) -sn-glycerophosphocholine) and the inhibitors listed in Table 5, PAF was performed in the same manner as in Example 2. — AH activity was measured. Table 5 shows the results. Table 5

 * 1) nmol / min / ml sample

Sodium decanesulfonate, CHAPS, sodium cholate, and SDS + CHAPS act to activate purified PAF-AH; ^, apparent PAF-AH activity when using human serum Greatly reduced / 32808

 PCT / JP99 / 06745

49. This indicates that these inhibitors do not inhibit PAF-AH itself, but do inhibit substances that cause nonspecific hydrolysis reactions. For other inhibitors, the decrease in PAF-AH activity in human serum is greater than the decrease in PAF-AH purified product activity, inhibiting substances that cause nonspecific hydrolysis. It is shown that.

 Therefore, it is shown that good results can be obtained by measuring PAF-AH activity using compound 5 as a substrate in the presence of an inhibitor.

 (Example 7)

 Using compound 12 (1-myristoyl-2- (4-1-2trophenylenyl) -sn-glycerophosphocholine) and the inhibitors listed in Table 6, the same procedure as in Example 2 was carried out. PAF-AH activity was measured. Table 6 shows the results. Table 6

 * 1) nmol / min / ml! I fee

SDS, Trito nX-100, CHAPS, sodium cholate, SDS + Triton x-100, and SDS + CHAP S activate or do not inhibit purified PAF-AH blood It was shown that the use of Qing significantly reduced the apparent PAF-AH activity. This indicates that these inhibitors do not inhibit PAF-AH itself, but do inhibit substances that cause nonspecific hydrolysis reactions. As for other inhibitors, the effect of reducing the activity of PAF-AH in human serum serum is greater than the decrease in the activity of purified PAF-AH products. Is shown.

 Therefore, good results can be obtained by measuring PAF-AH using compound 12 as a substrate in the presence of an inhibitor.

 (Example 8)

 Using compound 23 (1-myristoyl-2-[(4-1-2trophenyl) dimethylsuccinyl] -sn-glycerophosphocholine) and the inhibitors listed in Table 7, the procedure was as in Example 2. PAF-AH activity was measured. Table 7 shows the results.

* 1 nmol / min / mlg¾¾- All inhibitors used in Table 7 act in a direction that activates or does not inhibit purified PAF-AH. PAF—showed to significantly reduce AH activity. This means this These inhibitors have been shown not to inhibit PAF-AH itself, but to inhibit non-specific hydrolysis reactions.

 Therefore, good results can be obtained by measuring PAF-AH using compound 23 as a substrate in the presence of an inhibitor.

 (Example 9)

 Using compound 28 (1-oleoyl-1- (4-ditrofuer-luzacinyl) -sn-glycerophosphocholine) and the inhibitors listed in Table 8, in the same manner as in Example 2, PAF- AH activity was measured. Table 8 shows the results. Table 8 shows the results.

 Table 8

* 1 nmo min / ml sample Trito nX—100, Tween—20, CHAPS, sodium cholate, SDS + Trito nX—100, and 303 + 11? S acts in a direction that activates or does not inhibit purified PAF-AH, but apparently reduces the apparent PAF-AH activity when using human serum. . This indicates that these inhibitors do not inhibit PAF-AH itself, but do inhibit substances that cause non-specific hydrolysis reactions. Sodium decanesulfonate also contains PAF in human serum. (1) The decrease in AH activity is greater than the decrease in PAF-AH purified product activity, indicating that substances that cause non-specific hydrolysis reactions are inhibited.

 Therefore, good results can be obtained by measuring PAF-AH using Compound 28 as a substrate in the presence of an inhibitor.

 As described above, it has been clarified that PAF-AH activity can be accurately performed in the presence of an inhibitor using a compound represented by the general formula (I) and a compound of a different kind of A as a substrate. Industrial applicability

 According to the method for measuring PAF-AH activity of the present invention, it is possible to directly measure PAF-AH activity in a sample without using a radioactively labeled substrate, so that it is safe, quick, and simple for daily inspection. It can be sufficiently used as a measuring method.

In addition, the PAF-AH activity measurement method of the present invention can be directly measured by reducing the measurement time compared to the conventional method, not being affected by various esterases and esterase-like substances, and monitoring the chromogenic compound. PAF—Has advantages such as the ability to measure AH activity, enabling more accurate and faster measurement than conventional methods that use enzymes or reagents that induce color development. Therefore, it will be possible to diagnose the disease and diagnose the prognosis in a short time. Therefore, the present invention provides an extremely useful method for measuring PAF-AH activity.

Claims

53 Claims

1. A method for measuring platelet activating factor acetylhydrolase activity, which comprises the general formula (I):

CH ₂ -0-R,

 O 0

 CH-0-C-A-C-X (I)

 O "

CH ₂ -0-P-0-R ₂

O (In the formula, represents an acyl group, an alkyl group or an alkenyl group, and A represents a saturated or unsaturated, substituted or unsubstituted, substituted or unsubstituted hydrocarbon group having 2 to 7 carbon atoms even if an oxygen atom is interposed. X represents a group which becomes a color-forming compound or a fluorescent color-forming compound when released, and R ₂ represents a monomethylaminoethyl group, a dimethylaminoethyl group or a trimethylaminoethyl group.) The platelet-activating factor acetylhydrolase-containing sample is reacted in the presence of an inhibitor that does not inhibit the platelet-activating factor acetylhydrolase, but inhibits other ester-degrading activity-related substances, thereby releasing the color. A measuring method, comprising measuring the amount of a luminescent compound or a fluorescent 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 inhibitor according to claim 1, wherein the inhibitor is at least one inhibitor selected from the group consisting of an anionic surfactant, a nonionic surfactant, a bile salt, a bile salt derivative, and a chelating agent. 2. The measurement method according to 2.

 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 nonionic surfactant is a polyoxyethylene alkyl arylate. 4. The measuring method according to claim 3, wherein the measuring method is a polyoxyethylene sorbitan fatty acid ester.

 6. The bile salts and bile salt derivatives are sodium cholate, sodium deoxycholate, sodium chenodeoxycholate, sodium dehydrocholate, sodium taurocholate, sodium taurocholate, tadolo. The sodium chloride according to claim 3, which is sodium deoxycholate, sodium tauronodeoxycholic acid, sodium tauroursodoxycholate, sodium taurodehydrocholate, CHAPS, CHAP SO, BI GCHAP, or deoxy BIG CHAP. Measuring method.

 7. A reagent for measuring platelet activating factor acetylhydrolase activity, wherein the reagent has a general formula (I):

CH ₂ -0-P-0-R ₂

 o

(In the formula, represents an acyl group, an alkyl group or an alkenyl group, 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 chromogenic compound or a fluorescent chromogenic compound when released, and R ₂ represents a monomethylaminoethyl group, a dimethylaminoethyl group or a trimethylaminoethyl group.) And a platelet activity A reagent which does not inhibit the activation factor acetylhydrolase, but which inhibits other 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 inhibitor comprises an anionic surfactant, a nonionic surfactant, a bile salt, 9. The reagent according to claim 7, which is at least one inhibitor selected from the group consisting of 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.

 1 1. The reagent according to claim 9, wherein the nonionic surfactant is a polyoxyethylene alkylaryl ether or a polyoxyethylene sorbitan fatty acid ester.

 12. The bile salts and bile salt derivatives are sodium cholate, sodium deoxycholate, sodium chenodeoxycholate, sodium dehydrocholate, sodium taurocholate, sodium taurocholate, tauro sodium. 10.The method according to claim 9, which is sodium deoxycholate, sodium taurokenodeoxycholate, sodium tauroursodoxycholate, sodium taurodehydrocholate, CHAPS, CHAP SO, BI GCHAP, or deoxy-BIG CHAP. Reagents.

 13. A kit for measuring platelet activation factor acetylhydrolase activity, comprising the reagent according to any one of claims 7 to 12.

 14. A kit for measuring platelet activating factor acetylhydrolase activity, which kit has the general formula (I):

CH ₂ -0-P-0-R ₂

 O

(Wherein, represents an acyl group, an alkyl group or an alkenyl group, 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 is a coloring compound or a fluorescent coloring compound when released R ₂ represents a monomethylaminoethyl group, a dimethylaminoethyl group or a trimethylaminoethyl group. A kit comprising: a container containing the substrate represented by the formula: and a solution containing an inhibitor that does not inhibit the platelet-activating factor acetylhydrolase but inhibits other ester-decomposing 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 kit according to claim 15 or 16, further comprising a container containing a solution or a solvent for dissolving the substrate.

 18. Compound represented by general formula (la):

CH ₂ -0-R,

 0 O

 II II

CH-0-C-CH shi-λ (la)

 0—

CH ₂ -0-P-0-R ₂

 o

(Ashiru group wherein from 6 to 20 carbon atoms, an alkyl group or an alkenyl group, X represents a group a chromogenic compound or a fluorescent chromogenic compound when liberated, R ₂ is monomethyl aminoethyl group, Represents a dimethylaminoethyl group or 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. A method for measuring platelet activating factor acetylhydrolase activity, which comprises the step of: 57

CHz-O-R,

OO II II CH-0-C-CH ₂ -CH ₂ -CX (la)

 O "

CH ₂ -0-P-0-R ₂

 O

(Wherein, R, represents an acyl group, an alkyl group or an alkenyl group having 6 to 20 carbon atoms, X represents a group that becomes a coloring compound or a fluorescent coloring compound when released, and R ₂ represents monomethylamino. Ethyl group, dimethylaminoethyl group or trimethylaminoethyl group) is reacted with a sample containing platelet-activating factor acetyl hydrolase, and the resulting chromogenic compound or fluorescence is released. A measuring method comprising measuring the amount of a chromogenic compound.

21. The method according to claim 20, wherein the color-forming compound is an aromatic hydroxy compound or an aromatic thiol compound.