JP2002017398A - Method of measuring lysophospholipid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of specifically, simply and exactly measuring the lysophospholipid in specimens. SOLUTION: The lysophospholipid is determined by allowing a lysophospholipase-active enzyme that hydrolyzes lysophospholipid but not acyl type phospholipid to act on lysophospholipid and determining the product. In the case of lysophosphatidylcholine, glycerol-3-phosphorylcholine(GPC) or a free fatty acid is liberated and the liberated GPC or free fatty acid is specifically determined. In the case of lysophosphatidic acid(LPA), glycerol-3-phosphoric acid(G-3-P) or a free fatty acid is liberated and specifically determined. Thus, a simple and exact process for specifically determining LPC or LPA and a kit therefor can be provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a lysophospholipid, particularly lysophosphatidylcholine (hereinafter referred to as LPC), which is mainly used in the field of clinical examination and basic biological research.
A specific measurement method for lysophosphatidic acid (hereinafter sometimes referred to as LPA) and a kit therefor.

[0002]

2. Description of the Related Art LPC is a lysophospholipid containing choline and accumulates in ischemic myocardial tissue (Yuko Hashizume et al., Nihon Pharmaceutical Journal 114: 287-293, (1999)). LPC increases with the oxidative denaturation of low-density lipoprotein, which is important for the occurrence of hardening (Noriaki Kume, Molecular Me
dicine 37 (1): 22-28, (2000)). LPC also shows high levels in blood in patients with multiple myeloma and ovarian cancer (Sasagawa T. et al., Lipids 34: 1
7-21, (1998), Okita M. et al., Int.J. Cancer 71: 31-34,
(1997)), etc., and is expected to be a new diagnostic marker.

On the other hand, LPA is the simplest glycerophospholipid having one fatty acid. Since LPA exists only in a trace amount in a living body, it has been conventionally thought that LPA only plays a role as an intermediate metabolite of phospholipid biosynthesis or as a component of cell membrane. However, in recent years, it has been shown that LPA has various physiological activities, and since LPA-specific receptors have been discovered, its role as a physiologically active lipid has attracted attention.

[0004] Representative physiological activities of LPA that have been reported include platelet aggregation, cell growth promotion, and the like.
Formation of stress fibers, promotion of cancer cell invasion, and the like.

Furthermore, similarly to LPC, blood LPA is significantly higher in ovarian cancer patients than in healthy individuals (Misako Okita, Bulletin of Okayama Prefectural University School of Health and Welfare 1 (1): 29-3)
5, (1994), Xu Y. et al., JAMA.280 (8): 719-723, (199
8), and US Pat. No. 5,994,141), and that even in patients with multiple myeloma, LPA in serum is higher than in healthy subjects (Takayo Sasakawa et al., Nutrition-Evaluation and Treatment 12 (4): 303-
306, (1995)), and is expected to be a new tumor diagnostic marker.

[0006] The method of measuring LPC or LPA, which is also expected to serve as a tumor diagnostic marker, is rather complicated and not versatile. As a general method for measuring lysophospholipid, for example, Tokumura A. et al., Am. J. Ph.
Ysiol. 267: C204-210, (1994) is known. This method extracts a lipid component from a sample, separates and separates each lysophospholipid from other lipid components by thin-layer chromatography, and then extracts the lipid component. This is a method for analyzing fatty acid methyl esters obtained through a methyl group transfer reaction by gas chromatography, and lacks practicality.

[0007] By the way, International Patent Publication WO 00/23612
Include lysophospholipase (enzyme name lysophospholipase (abbreviation LYP) as an enzyme having specificity for lysophospholipids.
L): manufactured by Asahi Kasei Corporation). However, also in this method, the lipid is once extracted from the sample, and the lysophospholipid is further concentrated to hydrolyze the obtained lysophospholipid. This is this L
This suggests that YPL is not strictly specific for lysophospholipids. In fact, LPC measurement performed by the present inventors showed that LYPL partially decomposes and measures phosphatidylcholine, which is a diacyl-type phospholipid (see Example 1 described later). Furthermore, a method of measuring LPC by combining the enzymatic cycling method is also known (Hideo Misaki et al., 126th Annual Meeting of the Kinki Branch of the Japan Society for Clinical Chemistry 3
2-50, (1986)) used lysophospholipase (L
YP: Toyo Brewing Co., Ltd. (now Asahi Kasei Corporation)
It is the same enzyme as LYPL and has the same problems as above.

As described above, there is still no enzyme specific to lysophospholipid, and the measurement of lysophospholipid still has to be performed by a complicated method.

[0009]

Therefore, the lysophospholipid (particularly, LP) in the sample does not require sample pretreatment.
A method for specifically and simply and accurately measuring C and LPA) and a kit therefor are desired.

[0010]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above problems, and as a result, have found an enzyme that degrades lysophospholipid but hardly degrades diacyl-type phospholipid. , LPA, LPC) and a kit for specifically and simply measuring the same, and a kit therefor have been developed, thereby completing the present invention.

The present invention relates to a method for measuring lysophospholipids in a sample, comprising: (a) applying a reagent containing lysophospholipase which degrades lysophospholipid but does not degrade diacyl phospholipid to the sample; and (B) (a)
Measuring the reaction product of the above.

In a preferred embodiment, the method does not include a step of extracting lipids from the sample in advance.

[0013] In a preferred embodiment, the lysophospholipase is a lipase derived from a microorganism of the genus Bacillus.

In a preferred embodiment, the object to be measured is lysophosphatidylcholine, which is measured by measuring glycerol-3-phosphorylcholine or a free fatty acid which is a reaction product generated by the decomposition of lysophosphatidylcholine.

In a preferred embodiment, the glycerol-3-phosphorylcholine acts on (i) glycerophosphorylcholine phosphodiesterase,
(Ii) a step of causing choline oxidase to act; and (ii)
i) a step of measuring the generated hydrogen peroxide.

In another preferred embodiment, the glycerol-3-phosphorylcholine is reacted with (i) glycerophosphorylcholine phosphodiesterase, (ii) glycerol-3-phosphate oxidase, and (iii) And measuring hydrogen peroxide.

In a preferred embodiment, the lysophospholipid is lysophosphatidic acid and is measured (a).
Is glycerol 3-phosphate or free fatty acids.

In a preferred embodiment, the glycerol-3-phosphate comprises: (i) reacting glycerol-3-phosphate with glycerol-3-phosphate oxidase; and (ii) producing hydrogen peroxide. Is measured.

In another preferred embodiment, the glycerol-3-phosphate is (i) glycerol-3-phosphate.
Glycerol-3-phosphate dehydrogenase is added to phosphoric acid, and oxidized nicotinamide adenine dinucleotide (NAD ⁺ )
Or oxidized nicotinamide adenine dinucleotide phosphate (NADP ⁺ ), and (ii) the amount of reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) produced Is measured.

The present invention also provides a kit for specifically measuring lysophospholipid in a sample, comprising: (a) a reagent containing lysophospholipase that degrades lysophospholipid but does not degrade diacyl phospholipid; And (b) (a)
And a reagent for measuring the reaction product of the above.

[0021] In a preferred embodiment, the lysophospholipase is a lipase derived from a microorganism of the genus Bacillus.

In a preferred embodiment, the kit of the present invention is a kit for measuring lysophosphatidylcholine, wherein the reagent (b) comprises (i) glycerophosphorylcholine phosphodiesterase, (ii) choline oxidase, and (iii) Reagent for measuring hydrogen peroxide,
including.

In a preferred embodiment, the kit of the present invention is a kit for measuring lysophosphatidylcholine, wherein the reagent (b) is (i) glycerophosphorylcholine phosphodiesterase, (ii) glycerol-3.
-A phosphate oxidase, and (iii) a reagent for measuring the generated hydrogen peroxide.

In a preferred embodiment, the kit of the present invention is a kit for measuring lysophosphatidic acid, wherein said reagent (b) comprises (i) glycerol-3-phosphate oxidase, and (ii) A reagent for measuring hydrogen peroxide.

In a further preferred embodiment, the kit of the present invention is a kit for measuring lysophosphatidic acid, wherein said reagent (b) further comprises glycerol-
3-phosphate dehydrogenase, and reduced nicotinamide adenine dinucleotide (NADH).

In another embodiment, the kit of the present invention is a kit for measuring lysophosphatidic acid, wherein the reagent (b) comprises (i) glycerol-3-
Phosphate dehydrogenase, and (ii) oxidized nicotinamide adenine dinucleotide (NAD ⁺ ) or oxidized nicotinamide adenine dinucleotide phosphate (NAD
P ⁺ ).

[0027]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The basic principle of the method for measuring lysophospholipid in the present invention is that an enzyme having lysophospholipase activity is allowed to act on lysophospholipid as a substance to be measured.
The measurement of the resulting reaction product.

When the lysophospholipid is lysophosphatidylcholine (LPC), the basic principle of the measurement method is that a lysophospholipase (hereinafter referred to as “the present invention”) is decomposed into LPC, which is a substance to be measured, which degrades lysophospholipid but does not degrade diacyl phospholipid. The glycerol-3-phosphorylcholine (hereinafter sometimes referred to as GPC) produced by the action of the lysophospholipase of the present invention) is further reacted with glycerophosphorylcholine phosphodiesterase (hereinafter referred to as GPC).
P) and measure the resulting choline or glycerol-3-phosphate (hereinafter sometimes referred to as G-3-P) using their oxidase or dehydrogenase. It is.

When the lysophospholipid is lysophosphatidic acid (LPA), the measurement principle is that the lysophospholipase of the present invention is allowed to act on LPA, which is a substance to be measured, to produce G-3-P or free fatty acid. Is measured using enzymes specific to them.

As used herein, “lysophospholipase” refers to an enzyme that degrades lysophospholipid but does not degrade diacyl phospholipid, and “reagent containing lysophospholipase” refers to a composition containing lysophospholipase. . For example, it refers to a composition containing an enzyme having lysophospholipase activity and one or more combinations of a buffer, a coenzyme, a surfactant, or an enzyme stabilizer or activator. Also, "a reagent containing lysophospholipase"
May contain only lysophospholipase.

When measuring LPC by measuring GPC generated by lysophospholipase, lysophospholipase need not necessarily be specific only to LPC. That is, as long as LPC can be decomposed to generate GPC, it may have activity against other lysophospholipids. This is because other lysophospholipids that do not contain choline are not measured if the generated GPC is further subjected to GPCP and measured using an oxidase specific for the generated choline.

On the other hand, if lysophospholipase is an enzyme specific only to LPC, or if GPCP is an enzyme specific only to GPC, G-3-P is measured using its oxidase or dehydrogenase. Thereby, LPC can be measured.

When lysophospholipase is used in combination with GPCP and choline oxidase, LPC and LP
The reaction of A is as shown in FIG.

Also, G produced by lysophospholipase
Even when measuring the amount of LPA by measuring -3-P, lysophospholipase does not necessarily need to be specific only to LPA. That is, it may have activity against other lysophospholipids as long as LPA can be decomposed to generate G-3-P. because,
This is because lysophospholipid degradation products other than G-3-P are not measured by measurement using the generated oxidase or dehydrogenase specific to G-3-P.

More specifically, the lysophospholipase used in the present invention is not only LPA but also LPA.
It also acts on C, but does not pose a problem when measuring LPA. Because the LPC in the sample is decomposed and GPC
Glycerol-3-, which produces PC but then acts
Phosphate oxidase (hereinafter sometimes referred to as GPO) or glycerol-3-phosphate dehydrogenase (hereinafter G3P
DH) is G-3-P specific,
This is because LPC is not measured because it does not affect GPC. When lysophospholipase and GPO are used in combination, the reaction of LPA and LPC is as shown in FIG.

The lysophospholipase used in the present invention is not particularly limited as long as it is an enzyme that degrades lysophospholipid but does not degrade diacyl phospholipid. As a preferred enzyme, an enzyme name monoglyceride lipase (abbreviated as MGLPL (also referred to as MGLP)), which is derived from a microorganism of the genus Bacillus and is commercially available as monoglyceride lipase from Asahi Kasei Corporation, is used.

However, when measuring the generated free fatty acids, those having extremely high specificity for LPA or LPC must be used. This is because, if the specificity is low, when a sample containing various kinds of lysophospholipids such as blood is measured, other lysophospholipids other than the measurement target are also detected. As an enzyme having lysophospholipase activity highly specific for LPA used in such a measurement method, for example, an enzyme derived from rat brain (Fiona J. Thompson and Mike A. Clark,
Biochem. J. 300: 457-461, (1994)).

As a method for measuring G-3-P, G-3-P
Method for measuring hydrogen peroxide generated using GPO specific to GAD, or a method for measuring the amount of NADH or NADPH generated from NAD ⁺ or NADP ⁺ using G3PDH specific for G-3-P ( Edited by Masamitsu Kanai, Clinical Examination Method Proposal Revised 30th Edition (1993, p. 560), etc., but any method can be used as long as it can accurately measure G-3-P.

GPO includes, for example, enzymes derived from Streptococcus, Aerococcus, and Pediococcus, and any GPO specific to G-3-P can be used in the present invention.

The G3PDH includes, for example, those derived from rabbit muscle. Any G3PDH specific to G-3-P can be used in the present invention.

As a method for measuring the generated free fatty acids,
Is an enzymatic assay for serum free fatty acids (Shimizu S.
Anal. Biochem. 98 341-345, 1979).
However, if it is a method that can accurately measure free fatty acids,
Any method may be used. For example, acyl CoA synthetic yeast
And acyl-CoA oxidase or acyl-CoA
Using dehydrogenase, the amount of hydrogen peroxide produced or
NAD⁺Or NADP ⁺NADH generated from
Measure the amount of free fatty acids by measuring the amount of NADPH.
Can be.

Further, hydrogen peroxide, NADH or NADPH generated by these reactions can be measured in combination with a known high-sensitivity measurement technique. Known high-sensitivity measurement techniques include, for example, a method of measuring hydrogen peroxide generated by an oxidase using a fluorescence reaction or a luminescence reaction, and a method of amplifying and measuring hydrogen peroxide by an enzymatic cycling reaction (Hideo Misaki , Inspection and technology 27
(8): 973-980, (1999), and Registration No. 1594750), a method using a high-sensitivity color former having a molar extinction coefficient superior to that of the existing Trinder reagent (Japanese Patent Laid-Open No. 5-22993 and Norihito Aoyama) , Laboratory Test, 14 (9): 1014-1019, (199
7)) are known.

The sample to be measured is not limited at all, and may be a sample in basic research such as a culture medium of LPA or LPC-producing cells, or a sample in a clinical test such as a body fluid component. As body fluid components,
For example, whole blood, serum, plasma, urine, saliva, tears, ascites,
Cerebrospinal fluid, amniotic fluid, vaginal fluid and the like.

The kit for measuring lysophospholipid of the present invention comprises lysophospholipase that degrades lysophospholipid but does not degrade diacyl phospholipid, and a reagent for measuring a reaction product.

As used herein, the term "reagent for measuring a reaction product" means G-3-P, GPC, free fatty acid or the like produced by the action of lysophospholipase, as described above, directly or indirectly. Refers to a single component or a composition containing the same used in the method for measuring the temperature.

As used herein, the term "reagent for measuring the generated hydrogen peroxide" refers to the method for directly or indirectly measuring the hydrogen peroxide generated by the action of an oxidase as described above. And a composition containing the same. Examples of this component include a color former. For example, a combination of N-ethyl- (2-hydroxy-3-sulfopropyl) -4-methylaniline as a Trinder reagent and 4-aminoantipyrine as a coupler (Norihito Aoyama) Et al., Supra).

Usually, the kit of the present invention is provided as a reagent kit formulated so that each substance including the main reaction enzyme has an appropriate concentration. Examples of containers containing reagents include vials, ampules, plates, and microplates formed of glass, plastic, and the like.
Materials and materials that do not affect the contained reagents
The form is not particularly limited. At this time, the form of the reagent is not particularly limited, such as a dry powder or a liquid, and may include an activator of the enzyme, a stabilizer, a preservative, and the like.

The kit of the present invention can be used not only for quantitative measurement but also for qualitative measurement. Suitable forms for the qualitative measurement include, for example, a combination of a form such as a microplate, a test strip, and an ampoule or a vial.

The reagent kit may be a combination of two or more reagents having different addition times into the reaction solution, such as a combination of the first reaction reagent and the second reaction reagent. It can be a system. It is also preferable that the kit is contained in one container and mixed during the reaction.

Further, when a substance that affects the measured value is contained in the sample, it is necessary to remove the influence.
It is preferable to more accurately measure lysophospholipase activity. As such a method, for example, LP in a sample
When A is measured by the amount of generated G-3-P, G-3-P originally present in the sample affects the measured value, so that G-3-P is reacted with GPO or the like in advance. A method of removing G-3 without adding an enzyme having lysophospholipase activity as a control
There is a method in which the amount of P is measured, the measured value is subtracted from a system in which an enzyme having lysophospholipase activity is added and measured, and the true activity is determined.

[0051]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, which should not be construed as limiting the present invention.

(Example 1: Comparison of specificity of lysophospholipase) As lysophospholipase, LYPL derived from a microorganism of the genus Vibrio (manufactured by Asahi Kasei Corporation) and MGLPL derived from a microorganism of the genus Bacillus (manufactured by Asahi Kasei Corporation)
Was used.

First, the following reagents for LPC measurement were prepared. Reagent A (pH 8.0) Tris 100 mmol / L Triton X-100 0.01% calcium chloride 1 mmol / L N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline 5 mmol / L peroxidase (horseradish Origin) 20U / mL Glycerophosphorylcholine phosphodiesterase (Derived from Gliocladium) 1U / mL Choline oxidase (Derived from Arthrobacter) 10U / mL Reagent B (pH 8.0) Tris 100mmol / L Triton X-100 0.01% 4-aminoantipyrine 10 mmol / L lysophospholipase (MGLPL) 50 U / mL Reagent C (pH 8.0) Tris 100 mmol / L Triton X-100 0.01% 4-aminoantipyrine 10 mmol / L lysophospholipase (LYPL) 10 U / mL

1-oleoyl LPC, 1-oleoyl L
PA, 1-palmitoyl-2-linoleoyl PC, and sphingomyelin were each dissolved in physiological saline containing 1% of a surfactant Triton X-100 (trade name) so as to have a concentration of 1.0 mmol / L. 300 μL of reagent A was added to 10 μL of each sample, and reacted at 37 ° C. for 5 minutes. To this, 100 μL of reagent B was added and reacted at 37 ° C. for 5 minutes. This 37
The change in absorbance of the reaction solution at a main wavelength of 570 nm and a sub-wavelength of 700 nm at 5 ° C. for 5 minutes was measured. The results are shown in FIG.

In FIG. 3, 1-oleoyl LPC is LPC
1-oleoyl LPA is abbreviated as LPA, 1-palmitoyl-2-linoleoyl PC is abbreviated as PC, and sphingomyelin is abbreviated as SM. LYPL and MG
LPLs are both lysophospholipids, LPC and LPA
To generate GPC and G-3-P, respectively. Subsequently, GPCP degrades GPC generated from LPC, and choline oxidase acts on the generated choline to generate H ₂ O ₂ . On the other hand, G-3-P generated from LPA is GPCP.
And does not serve as a substrate for choline oxidase (see FIG. 1).

As a result, LPC is measured but LPA is not measured. LYPL and MGLPL are SM
Is not measured, so SM is not measured. Therefore, LY
PL and MGLPL have the same action on LPC, LPA and SM. However, LYPL acts on PC by about 10% to generate GPC, and GPCP generates GPC.
The difference is that by acting on the PC, the PC is measured, whereas MGLPL has no effect on the PC, and therefore no PC is detected. That is, MGLPL
Does not degrade diacyl phospholipids. PC is the most abundant phospholipid in serum and is said to exist several tens of times as much as LPC, so if 10% of it is measured, the measured value will be more than twice as much as true LPC. Therefore, in order to accurately measure the amount of LPC without pre-treating the sample such as extracting lipids in advance, a lysophospholipase having extremely high specificity for lysophospholipids such as the MGLPL type is required. .

The same applies to the LPA measurement reagent as well as the LPC measurement reagent. When the lysophospholipid is accurately measured without performing a pretreatment such as lipid extraction, the LPA measurement reagent is required. As for the reagent, lysophospholipase having extremely high specificity for lysophospholipid like MGLPL is required.

(Example 2: Preparation of LPC calibration curve)
The following reagents were prepared. Reagent A (pH 8.0) Tris 100 mmol / L Triton X-100 0.01% calcium chloride 1 mmol / L N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline 3 mmol / L peroxidase (horseradish 10U / mL glycerophosphorylcholine phosphodiesterase (derived from genus Gliocladium) 0.1U / mL choline oxidase (derived from Arthrobacter) 10U / mL Reagent B (pH 8.0) Tris 100mmol / L Triton X-100 0.01% 4-amino Antipyrine 5mmol / L lysophospholipase (MGLPL) 30U / mL

1% of palmitoyl LPC (synthetic product)
Was dissolved in a physiological saline solution containing the surfactant Triton X-100 (trade name) to prepare a 1.0 mmol / L LPC solution.
300 μL of reagent A was added to 10 μL of the sample, and reacted at 37 ° C. for 5 minutes. To this, 100 μL of reagent B was added and reacted at 37 ° C. for 5 minutes. The change in the absorbance of the reaction solution at a main wavelength of 570 nm and a sub-wavelength of 700 nm at 37 ° C. for 5 minutes was measured. FIG. 4 shows the results.

A good linear relationship passing through the origin was obtained between the LPC concentration and the amount of change in absorbance. LPC can be measured using the measurement reagent of the present invention.

(Example 3: Correlation with conventional method) 15 samples of human serum, 5 samples of control serum (Consera: Nissui Pharmaceutical Co., Ltd., Lipid Salem I: Eiken Chemical Co., Ltd., Lipid Salem II: Eiken Chemical) Co., Ltd., Ceraclear-L
P: Aswell Co., Ltd., High Level Check Lipid: Kokusai Reagent Co., Ltd.) was used as a sample, and the amount of LPC was measured in the same manner as in Example 2.

On the other hand, lipids were extracted from the 20 samples by the following conventional method, the amount of fatty acids in LPC was analyzed, and the total amount of LPC was converted from the amount of fatty acids. 0.1 mL sample
0.1 mL of 2% KCl solution, 0.1 mL of 2N-HCl and 7.2 μg of 1-heptadecanoyl LPC as an internal standard
And chloroform and methanol (2:
1) After adding and mixing 1.5 mL of the solution, the mixture was centrifuged at 1000 × g for 10 minutes, and 1 mL of the lower layer was collected in another container. After the fractionated lipid component was dried to nitrogen, it was redissolved in 50 μL of a chloroform: methanol (2: 1) solution, loaded on a single lane of a silica gel TLC plate, and chloroform: methanol: ammonia (60: 35: 8). And developed at room temperature. 8-anilino-1-naphthalenesulfonic acid was sprayed and visually observed under ultraviolet light, and LP was removed from the plate.
The portion corresponding to C was scraped off and collected in another container. 2 mL of boron trifluoride was added to the collected container, and the mixture was heated at 105 ° C. for 30 minutes to perform a methyl group rearrangement reaction. Thereafter, 1 mL of distilled water and 2 mL of hexane were added, and the mixture was vigorously stirred for 10 seconds.
The mixture was centrifuged at 1000 × g for 10 minutes, and 1.2 mL of the upper layer was collected in another container. Subsequently, after drying to dryness with nitrogen, the residue was dissolved in 40 μL of toluene to obtain an analysis sample for gas chromatography.

In order to measure the amount of fatty acids contained in each sample, a GC-14B (manufactured by Shimadzu Corporation) gas chromatograph equipped with a SUPELCO WAX 10 column (30 m × 0.25 mm, manufactured by SUPELCO) was used. Oven starting temperature is 100 ℃
After holding for 1 minute, raise the temperature to 200 ° C at 40 ° C / minute,
Hold for 2 minutes. Thereafter, the temperature was further increased to 240 ° C. at a rate of 10 ° C./min, and held for 15 minutes. Carrier gas is helium gas, sample vaporization chamber temperature is 250 ℃, detector temperature is 250 ℃
Met.

The retention time of each fatty acid methyl was confirmed in advance, and the peak area corresponding to each fatty acid methyl was converted to the peak area corresponding to 1-heptadecanoyl LPC, which is an internal standard, to obtain methyl palmitate, Methyl stearate, methyl oleate, methyl linoleate,
The sum of the respective LPCs corresponding to methyl arachidonic acid and methyl docosahexaenoate was calculated using the LPCs contained in each sample.
Amount.

FIG. 5 shows the correlation between the measurement result by the method of the present invention and the measurement result by the conventional method. As shown in FIG. 5, a good correlation with a correlation coefficient of 0.935 was recognized. In the method of the present invention, in the measurement of LPC,
It is possible to easily and quickly obtain a measurement result equivalent to that of a conventional fatty acid analysis method requiring complicated operations. In FIG. 5, ● represents human serum, ○ represents Concera, ▲ represents Lipid Serum I, and Δ represents Lipid Serum I.
I and ■ represent CERACLEAR-LP, and □ represents high-level check lipid.

Example 4 Preparation of LPA Calibration Curve The following reagents were prepared. Reagent A (pH6.8) Bis-Tris 100 mmol / L Triton X-100 0.01% N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline 3 mmol / L Peroxidase (from horseradish) 10 U / mL GPO (from Streptococcus sp.) 15 U / mL Flavin adenine dinucleotide 0.05 mmol / L Reagent B (pH 6.5) Bis-Tris 20 mmol / L Triton X-100 0.01% 4-aminoantipyrine 5 mmol / L Lysophospholipase (MGLPL) 20U / mL

1-oleoyl LPA (synthetic product) was dissolved in a physiological saline solution containing 1.0% of surfactant Triton X-100 (trade name) to prepare a 0.5 mmol / L LPA solution. The sample was diluted five times to prepare a sample for a calibration curve. Sample 10
300 μL of reagent A was added to the μL, and reacted at 37 ° C. for 5 minutes.
To this, 100 μL of reagent B was added and further reacted at 37 ° C. for 5 minutes. After adding reagent B, the main wavelength 570 nm (sub wavelength 700 n
The change in absorbance of the reaction solution in m) was measured. FIG. 6 shows the results. A good linear relationship was obtained between the LPA concentration and the amount of change in absorbance, indicating that LPA can be quantified using this measurement reagent. Also, 0.5 mmol /
FIG. 7 shows a reaction time course when the LPA of L was measured. The reaction converged instantly and showed a constant absorbance.

Example 5 Detection of Various LPAs with a Highly Sensitive Color Developing Agent A reagent using a triphenylmethane derivative as a high sensitive color developing agent was prepared. Reagent A (pH 6.5) Bis-Tris 100 mmol / L Triton X-100 0.05% Lysophospholipase (MGLPL) 20 U / mL N, N, N ′, N ′, N ″, N ″ -hexa (3-sulfo Propyl) -4,4 ',
4 ''-triaminotriphenylmethane salt (manufactured by Dojindo Laboratories Inc.) 3 mmol / L Reagent B (pH 6.5) Bis-Tris 100 mmol / L Triton X-100 0.05% peroxidase (from horseradish) 30 U / mL GPO (from Streptococcus sp.) 15U / mL Flavin adenine dinucleotide 0.05mmol / L

1-palmitoyl LPA, 1-stearoyl L
PA, 1-oleoyl LPA, 2-oleoyl LPA, 1-
Palmitoyl LPC and 1-palmitoyl LPE (lysophosphatidylethanolamine) were dissolved in a physiological saline solution containing 1.0% of a surfactant Triton X-100 to prepare samples each corresponding to 2 mmol / L. Reagent A in 4 μL of sample
After adding 300 μL and heating at 37 ° C. for 5 minutes, reagent B 100
μL was added and reacted at 37 ° C. for 5 minutes. The change in absorbance of the reaction solution at a wavelength of 600 nm for 5 minutes after the addition of the reagent B was measured. FIG. 8 shows the result. Various LPAs could be measured by this assay regardless of the fatty acid species of LPA and the binding position of the fatty acid. Also, LP
Other lysophospholipids other than A were hardly detected.

(Example 6: Measurement of the amount of LPA in human serum using enzymatic cycling method) An enzymatic cycling method as shown below (Hideo Misaki, Test and Technology 27 (8): 9)
73-980, (1999), and Registration No. 1594750). Reagent A (pH 7.6) Bis-Tris 100 mmol / L Triton X-100 0.01% N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline 3 mmol / L β-NADH 0.05 mmol / L 12α-hydroxysteroid dehydrogenase (from Bacillus) 2 U / mL lysophospholipase (MGLPL) 10 U / mL Reagent B (pH 7.6) Bis-Tris 20 mmol / L Triton X-100 0.01% peroxidase (from horseradish) 20 U / mL 4-Aminoantipyrine 5mmol / L GPO (from Streptococcus sp.) 50U / mL G3PDH (from rabbit muscle) 5U / mL Sodium cholate 1mmol / L

Human serum was measured using 1-oleoyl LPA 0.1 mmol / L as a standard. The reaction was carried out in the same manner as in Example 1, and the absorbance of the reaction solution after the addition of the reagent B was measured with time at a main wavelength of 570 nm (sub wavelength 700 nm). This measurement includes the absorbance resulting from the reaction of G-3-P, which is naturally present in human serum. Therefore, in place of the reagent A, a G-3-P measurement reagent C containing no enzyme having the lysophospholipase activity shown below was used, and Example 1 was used.
The reaction was performed in the same manner as described above, and the absorbance depending on G-3-P present in the human serum was measured, and the true measured value was obtained by subtracting from the measured value.

Reagent C (pH 7.6) Bis-Tris 100 mmol / L Triton X-100 0.01% N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline 3 mmol / L β-NADH 0.05 mmol / L 12α-hydroxysteroid dehydrogenase (from Bacillus) 2U / mL

FIG. 9 shows a reaction time course corrected by subtracting the measured value obtained by using the reagent C from the measured value obtained by using the reagent A. From this result, the change in absorbance per unit time was calculated, and the value of LPA converted from the change in absorbance per unit time of 1-oleoyl LPA 0.1 mmol / L standard solution measured at the same time is shown in Table 1.

[0074]

[Table 1]

As shown in Table 1, it was found that LPA can be easily measured even in a sample containing contaminants such as human serum.

[0076]

According to the present invention, complicated methods such as lipid extraction and lysophospholipid separation are not required as compared with the conventional method, and even if the sample is a body fluid, pretreatment such as lipid extraction can be performed. The lysophospholipids such as LPC and LPA can be easily and accurately measured without the necessity. In addition, the method of the present invention requires only a small amount of sample to be used for measurement,
Since pretreatment of the sample is not required, it can be applied to a general-purpose biochemical automatic analyzer and can measure a large number of samples in a short time. For these reasons, the present invention is extremely useful both in the field of basic biochemical research and in the field of medical clinical examination.

[Brief description of the drawings]

FIG. 1 is a diagram showing the reaction of LPC and LPA when lysophospholipase is used in combination with GPCP and choline oxidase.

FIG. 2 is a view showing the reaction of LPA and LPC when lysophospholipase and GPO are used in combination.

FIG. 3 is a diagram showing the difference in reactivity between LYPL and MGLPL for diacyl phospholipids.

FIG. 4 is a graph showing the relationship between the concentration of 1-palmitoyl LPC and the amount of change in absorbance.

FIG. 5 is a diagram showing a correlation between the LPC measurement result by the method of the present invention and the LPC measurement result by the conventional fatty acid analysis method.

FIG. 6 is a graph showing the relationship between the concentration of 1-oleoyl LPA and the amount of change in absorbance.

FIG. 7 is a graph showing a reaction time course when measuring 0.5 mmol / L of LPA.

FIG. 8 is a graph showing the amount of change in absorbance when various LPAs are measured.

FIG. 9 is a graph showing a reaction time course in the measurement of LPA in human serum.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Koji Mizuno, Inventor 2-1-4-201 Oharano Nishisakaya-cho, Nishikyo-ku, Kyoto-shi, Kyoto (72) Inventor Shigeyuki Imamura 632-1 Mifuku, Oni-cho, Tata-gun, Shizuoka Prefecture Asahi Kasei Kogyo F term in the company (reference) 2G045 AA25 BB01 BB10 CA25 CA26 DA61 FA11 FA26 FA29 FB01 FB04 FB06 GC10 4B063 QA01 QA18 QA19 QQ03 QQ70 QR03 QR04 QR12 QR13 QR23 QR42 QR66 QS10 QS26 QS36 QX01

Claims (16)

[Claims] 1. A method for measuring lysophospholipid in a sample, comprising: (a) applying a reagent containing lysophospholipase which degrades lysophospholipid but does not degrade diacyl-type phospholipid to the sample. , And (b)
Measuring the reaction product of (a). 2. The method according to claim 1, wherein the method does not include a step of extracting lipid from a sample in advance. 3. The lysophospholipase according to claim 1, wherein the lysophospholipase is a lipase derived from a microorganism of the genus Bacillus.
The method described in. 4. The lysophospholipid is lysophosphatidylcholine, and the reaction product to be measured is glycerol-3.
2. A phosphorylcholine or free fatty acid.
The method according to any one of Items 1 to 3, above. 5. The method according to claim 5, wherein the glycerol-3-phosphorylcholine comprises (i) a step of causing glycerophosphorylcholine phosphodiesterase to act, (ii) a step of causing choline oxidase to act, and (iii) a step of measuring the generated hydrogen peroxide. 5. The method of claim 4, wherein the method is measured in a step. 6. The glycerol-3-phosphorylcholine is subjected to (i) a step of causing glycerophosphorylcholine phosphodiesterase to act, (ii) a step of causing glycerol-3-phosphate oxidase to act, and (iii) measuring the generated hydrogen peroxide. The method of claim 4, wherein the method comprises: 7. The method according to claim 1, wherein the lysophospholipid is lysophosphatidic acid, and the reaction product to be measured (a) is glycerol 3-phosphate or free fatty acid. . 8. The method according to claim 1, wherein the glycerol-3-phosphate is
(I) Glycerol-3-phosphate is replaced with glycerol-3-
A step of allowing a phosphate oxidase to act, and (ii) a step of measuring the generated hydrogen peroxide.
The method according to claim 7. 9. The method according to claim 9, wherein the glycerol-3-phosphate is
(I) Glycerol-3-phosphate is replaced by glycerol-3-
Allowing phosphate dehydrogenase to act with oxidized nicotinamide adenine dinucleotide (NAD ⁺ ) or oxidized nicotinamide adenine dinucleotide phosphate (NADP ⁺ ); and (ii) reducing nicotinamide adenine dinucleotide to be produced (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NA
9. The method of claim 7, wherein the method comprises measuring the amount of DPH). 10. A kit for specifically measuring lysophospholipid in a sample, comprising: (a) a reagent containing lysophospholipase that degrades lysophospholipid but does not degrade diacyl-type phospholipid; and (b) A kit for measuring the reaction product of (a). 11. The kit according to claim 10, wherein the lysophospholipase is a lipase derived from a microorganism of the genus Bacillus. 12. The lysophospholipid is lysophosphatidylcholine, and the reagent (b) is (i) glycerophosphorylcholine phosphodiesterase, (ii) choline oxidase, and (iii) a reagent for measuring generated hydrogen peroxide. The kit according to claim 10, comprising: 13. The lysophospholipid is lysophosphatidylcholine, and the reagent (b) is selected from the group consisting of (i) glycerophosphorylcholine phosphodiesterase, (ii) glycerol-3-phosphate oxidase, and (iii) generated hydrogen peroxide. The kit according to claim 10, comprising a reagent for measurement. 14. The lysophospholipid is lysophosphatidic acid, and the reagent (b) is (i) glycerol-3-
The kit according to claim 10, comprising a phosphate oxidase, and (ii) a reagent for measuring the generated hydrogen peroxide. 15. The kit according to claim 14, wherein said reagent (b) further comprises glycerol-3-phosphate dehydrogenase and reduced nicotinamide adenine dinucleotide (NADH). 16. The lysophospholipid is lysophosphatidic acid, and the reagent (b) is (i) glycerol-
3-phosphate dehydrogenase, and (ii) oxidized nicotinamide adenine dinucleotide (NAD ⁺ ) or oxidized nicotinamide adenine dinucleotide phosphate (NAD
The kit according to claim 10, which comprises P ⁺ ).