JP5800830B2 - Phosphatidylserine quantitative method and quantitative kit - Google Patents

Description

  The present invention relates to a method for quantifying phosphatidylserine (hereinafter sometimes referred to as PS), and a kit for quantifying phosphatidylserine.

  Phosphatidylserine is a kind of glycerophospholipid. PS refers to a structure in which serine phosphate is bonded to the 3-position of the glycerol skeleton and acyl chains of various chain lengths and double bonds are bonded to the 1- and 2-positions (Fig. 1). PS is one of phospholipid components constituting the cell membrane of prokaryotic cells and eukaryotic cells. The proportion of PS to total phospholipids in the cell membrane is generally small but plays an important role in various cell functions. Phosphatidylserine is known to act as a cofactor of blood coagulation reaction, and exposure of phosphatidylserine to the cell surface is observed in cells in which apoptosis is induced. PS is present as a component of lipoproteins in human and animal blood.

  Therefore, there is a demand for an excellent quantitative method for PS for studying the function of PS. Methods for analyzing PS that have been used to date include TLC-phosphorus quantification (for example, Non-Patent Document 1), HPLC, mass spectrometry (for example, Non-Patent Document 2), and fluorescence-labeled annexin V staining. .

  The TLC-phosphorus quantification method has been widely used heretofore, but has low detection sensitivity, takes time and labor to operate, and is unsuitable for handling a large number of samples.

  In PS analysis using a combination of HPLC and UV absorption detector, the double bond of the PS acyl chain is detected, so accurate measurement of the total amount of PS independent of the type of acyl chain cannot be performed. In addition, in PS analysis using a combination of HPLC and an evaporative light scattering detector, PS can be quantified without depending on the acyl chain of PS, but detection sensitivity is not sufficient for quantifying small amounts of PS contained in cultured cells. It is enough.

  In the analysis of PS using mass spectrometry, the detection sensitivity is high, but since the difference in the acyl chain of PS molecules is also detected and detected, it is very difficult to measure the total amount of PS.

  Fluorescence-labeled annexin V staining is used to determine apoptosis by staining PS that appears on the cell surface using the specific binding between annexin V and PS. However, it is not quantitative for PS.

Stone, S. J., Cui, Z. and Vance, J. E. (1998) Cloning and expression of mouse liver phosphatidylserine synthase-1 cDNA.Overexpression in rat hepatoma cells inhibits the CDP-ethanolamine pathway for phosphatidylethanolamine biosynthesis.J Biol Chem 273, 7293-7302 Taguchi, R., Houjou, T., Nakanishi, H., Yamazaki, T., Ishida, M., Imagawa, M. and Shimizu, T. (2005) Focused lipidomics by tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 823, 26-36

  Conventional PS analysis methods have the above-mentioned problems, and there is no method for quantifying a small amount of PS with high accuracy.

  Therefore, an object of the present invention is to provide a phosphatidylserine quantification method and a phosphatidylserine quantification kit capable of easily and easily quantifying PS with high sensitivity and high accuracy.

The present inventor has found that the above object can be achieved by using a series of enzyme reactions shown in FIG. The method for quantifying phosphatidylserine shown in FIG. 2 will be described below.
(i) By hydrolyzing PS with phospholipase D, serine and phosphatidic acid are generated.
(ii) Oxidize serine with L-amino acid oxidase to produce H ₂ O ₂ , NH ₃ and 2-oxo-3-hydroxypropionic acid.
(iii) 10-acetyl-3,7-dihydroxyphenoxazine and H ₂ O ₂ are reacted with peroxidase to produce resorufin. By measuring the fluorescence intensity generated from resorufin, the amount of PS can be determined.

  The present invention has been completed based on these findings and has been completed. The present invention provides the following phosphatidylserine quantification method and phosphatidylserine quantification kit.

(I) Quantification method of phosphatidylserine
(I-1) A method for quantifying phosphatidylserine in a sample, comprising:
(1) a step of allowing phospholipase D and L-amino acid oxidase to act on a sample, and
(2) A method comprising measuring the amount of H ₂ O ₂ produced in the step (1) and quantifying phosphatidylserine from a previously determined calibration curve.
(I-2) The measurement of the amount of H ₂ O _{2 in the} step (2) is performed by measuring the fluorescence intensity, absorbance, or luminescence amount of a compound produced by allowing peroxidase to act on H ₂ O _2. The method according to (I-1), characterized in that
(I-3) In place of the step (2), (2 ′) (a) a decrease in oxygen in the step (1), (b) an increase in ammonia, or (c) 2-oxo-3- The method according to (I-1), further comprising a step of quantifying phosphatidylserine by measuring a production amount of hydroxypropionic acid.
(I-4) A method for quantifying phosphatidylserine in a sample, comprising:
(1 ′) allowing phospholipase D to act on the sample; and
(2 ″) A method comprising the step of quantifying phosphatidylserine by measuring the amount of serine produced in the step (1 ′).
(I-5) In the step (1), in place of phospholipase D, phospholipase C and phosphomonoesterase are used, according to any one of (I-1) to (I-3). the method of.
(I-6) The method according to (I-4), wherein phospholipase C and phosphomonoesterase are used in place of phospholipase D in the step (1 ′).
The (I-7) wherein (2) Measurement of H ₂ O ₂ amount in the step, the fluorescence intensity of resorufin to produce by the action of 10-acetyl-3,7-dihydroxyphenoxazine and peroxidase H ₂ O ₂ The method according to (I-1) or (I-2), which is performed by measurement.

(II) Kit for quantification of phosphatidylserine
(II-1) A phosphatidylserine quantification kit containing phospholipase D or phospholipase C and phosphomonoesterase.
(II-2) The kit according to (II-1), further comprising L-amino acid oxidase.
(II-3) The kit according to (II-2), further comprising peroxidase.
(II-4) The kit according to (II-3), further comprising 10-acetyl-3,7-dihydroxyphenoxazine.

  The phosphatidylserine quantification method and quantification kit of the present invention are capable of quantifying phosphatidylserine with high sensitivity, high accuracy and simpleness, and enabling high-throughput quantification of phosphatidylserine. Furthermore, the quantitative method of the present invention enables high-accuracy quantitative determination of a small amount of PS contained in cultured cells, which has been difficult until now. It can also be applied to the quantification of PS contained in blood.

It is a figure which shows the structure of a phosphatidylserine. It is a figure which shows reaction of the determination method of the phosphatidylserine of this invention. It is a graph which shows the calibration curve (low concentration) of phosphatidylserine. It is a graph which shows the calibration curve (high concentration) of phosphatidylserine. It is a figure which shows the influence of an acyl chain and an amine head in fixed_quantity | quantitative_assay of phosphatidylserine. The vertical axis is a relative value with the fluorescence intensity of POPS as 100%. 3 is a graph showing the correlation between the enzyme quantification method of the present invention (vertical axis) and the TLC-phosphorus quantification method (horizontal axis) in cell-extracted PS quantification.

  Hereinafter, the phosphatidylserine quantification method and the phosphatidylserine quantification kit of the present invention will be described in detail.

Method for quantifying phosphatidylserine The method for quantifying phosphatidylserine in the sample of the present invention includes (1) a step of allowing phospholipase D and L-amino acid oxidase to act on the sample, and (2) H ₂ O produced in the step (1). It is characterized by having a step of measuring _two quantities and quantifying phosphatidylserine from a previously determined calibration curve.

  Hereinafter, each step will be described.

<(1) Process>
In step (1), phospholipase D and L-amino acid oxidase are allowed to act on the sample.

By allowing phospholipase D to act on the sample, serine and phosphatidic acid are generated from phosphatidylserine and H ₂ O. Further, by reacting the product with L-amino acid oxidase, H ₂ O ₂ , NH ₃ and 2-oxo-3-hydroxypropionic acid are produced from serine, O ₂ and H ₂ O.

  Phospholipase D is a phospholipid hydrolase that hydrolyzes the base side of the phosphodiester bond of glycerophospholipid. The phospholipase D used in the present invention can be used from any microorganism, animal, or plant, as long as it can hydrolyze phosphatidylserine to produce serine and phosphatidic acid. And those derived from Streptomyces chromofuscus (GenBank Accession Number: AF523823.1) are particularly preferred. Also preferred is phospholipase D consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence of Streptomyces chromofuscus-derived phospholipase D.

  Of the major phospholipids, only PS and lysophosphatidylserine (LPS) produce substrates for L-amino acid oxidase by reaction with phospholipase D. However, in most biological samples, the LPS content is much lower than the PS content. This allows specific quantification of PS.

L-amino acid oxidase is an enzyme that catalyzes a reaction in which L-amino acid is oxidatively deaminated to 2-oxo acid using molecular oxygen. As long as L-amino acid oxidase used in the present invention is capable of oxidatively deaminating serine to produce H ₂ O ₂ , NH ₃ and 2-oxo-3-hydroxypropionic acid, a microorganism, Although those derived from both animals and plants can be used, those derived from animals are preferred, and those derived from Crotalus adamanteus venom (GenBank Accession Number: AF071564.1) are particularly preferred. Also preferred is an L-amino acid oxidase comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence of the L-amino acid oxidase derived from the crotalus adamanteus venom.

  In the method for quantifying PS of the present invention, when the above two types of enzymes are allowed to act on a sample, the two types of enzymes may be added together and reacted at the same time or sequentially added and reacted. May be.

  The conditions for allowing phospholipase D to act on the sample can be appropriately set according to the characteristics of the enzyme used, but the pH is usually 7 to 9, and the temperature is usually 15 to 40 ° C. The time for which phospholipase D is allowed to act on the sample can be appropriately set according to the characteristics of the sample to be analyzed, but is usually 1 minute or longer.

  Conditions for allowing the L-amino acid oxidase to act on the sample can be appropriately set according to the characteristics of the enzyme used, but the pH is usually 7 to 8.5 and the temperature is usually 15 to 40 ° C. The time for which L-amino acid oxidase is allowed to act on the sample can be appropriately set according to the characteristics of the sample to be analyzed, but is usually 10 minutes or longer.

  When the action temperature and pH of the two types of enzymes are the same, all enzyme reactions can be performed simultaneously.If the action temperature and pH differ depending on the enzyme, the temperature and pH are set to the required step by step. The reaction can be performed.

  In the PS quantification method of the present invention, the amounts of the two types of enzymes in the reaction solution in which two types of enzymes are allowed to act on the sample should be appropriately adjusted to an appropriate amount of enzyme for analysis in consideration of the amount of PS contained. However, phospholipase D is usually 0.1 to 1000 U / ml, and L-amino acid oxidase is usually 0.1 to 100 U / ml.

  The reaction solution for allowing phospholipase D and L-amino acid oxidase to act on the sample may contain a buffer solution, a metal salt, and the like in addition to the sample and the enzyme. Examples of the buffer include Tris-hydrochloric acid buffer, potassium phosphate buffer, glycine-hydrochloric acid buffer, acetic acid buffer, citrate buffer, and the like. Examples of the metal salt include potassium salt and sodium salt.

  The sample in the present invention is not particularly limited as long as PS is required to be quantified, for example, cultured cells, culture fluid, human or animal tissue and blood-containing body fluid, plant tissue and plant body fluid, Examples include fungi, fungi, bacteria and bacterial cultures, pharmaceuticals, foods, supplements, and the like. The sample may be diluted with a diluent, and examples of such a diluent include a buffer. Examples of the buffer solution include those described above. The sample may be pretreated before the enzyme reaction, and examples of such treatment include heat treatment.

  In the present invention, phospholipase C and phosphomonoesterase may be used in place of phospholipase D in the step (1). Serine can also be generated in the same manner by allowing these two enzymes to act on PS. Phospholipase C can be used from microorganisms, animals and plants as long as it can hydrolyze phosphatidylserine to produce diacylglycerol and phosphoserine. The phosphomonoesterase can be used from any microorganism, animal or plant as long as it can hydrolyze phosphoserine to produce serine and phosphoric acid.

<(2) Process>
In step (2), the amount of H ₂ O ₂ produced in step (1) is measured, and phosphatidylserine is quantified from a calibration curve determined in advance.

As a result of a series of reactions, one molecule of H ₂ O ₂ is generated from one molecule of PS, and therefore PS can be quantified by measuring the amount of H ₂ O ₂ .

The H ₂ O ₂ amount of quantitative, fluorescence intensity can be used a variety of methods that can detect and quantify H ₂ O ₂ amount, for example, compounds produced by the action of peroxidase to H ₂ O ₂ The amount of H ₂ O ₂ can be determined by measuring the absorbance or the amount of luminescence. As such a method, specifically, a compound (N, N'-Bis (2-hydroxy-3-sulfopropyl) tolidine, etc.) that reacts with H ₂ O ₂ by peroxidase to obtain a new absorption wavelength is used. Absorbance measurement using a compound that reacts with H ₂ O ₂ with peroxidase to oxidatively condense multiple compounds to obtain a new absorption wavelength (such as oxidative condensation of phenol and 4-aminoantipyrine). A method for measuring fluorescence intensity using a compound (10-acetyl-3,7-dihydroxyphenoxazine, etc.) that newly reacts with H ₂ O ₂ by peroxidase to generate fluorescence, and H ₂ O ₂ by peroxidase And a method of measuring the amount of luminescence using a compound (luminol or the like) that newly emits light by reacting with the compound.

Among them, preferred is a method of measuring fluorescence intensity using a compound that reacts with H ₂ O ₂ by peroxidase to newly generate fluorescence, and particularly preferred is 10-acetyl-3 for H ₂ O ₂ , This is a method for measuring the fluorescence intensity of resorufin produced by the action of 7-dihydroxyphenoxazine (Amplex (registered trademark) Red) and peroxidase. Resorufin is a fluorescent compound having a maximum excitation wavelength of 571 nm and a maximum fluorescence wavelength of 585 nm. On the other hand, 10-acetyl-3,7-dihydroxyphenoxazine is a non-fluorescent compound and does not produce fluorescence even when irradiated with light having a wavelength of about 571 nm. As a result of a series of reactions, one molecule of resorufin is produced from one molecule of PS, so it is possible to quantify PS by measuring the amount of resorufin. The amount of resorufin can be measured, for example, by using a fluorescent microplate reader and selecting a filter having an excitation wavelength of 544 nm and a fluorescence wavelength of 590 nm and measuring the fluorescence intensity.

  The peroxidase used in the present invention can be derived from any microorganism, animal or plant, but is preferably derived from a plant, particularly preferably from horseradish (GenBank Accession Number: E01651.1). . In addition, a peroxidase having an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of the horseradish peroxidase is also preferable.

  Conditions for allowing the peroxidase to act on the sample can be appropriately set according to the characteristics of the enzyme used, but the pH is usually 6 to 8 and the temperature is usually 15 to 40 ° C. The time for which the peroxidase is allowed to act on the sample can be appropriately set, but is usually 5 to 120 minutes.

In the PS quantification method of the present invention, the amount of peroxidase enzyme in the reaction solution in which peroxidase is allowed to act can be appropriately adjusted to an enzyme amount suitable for analysis in consideration of the amount of H ₂ O ₂ contained therein. Usually, 0.5 to 50 U / ml.

The reaction solution for allowing peroxidase to act on H ₂ O ₂ may contain a buffer solution, a metal salt, and the like, and examples of the buffer solution and the metal salt include those described above.

The amount of 10-acetyl-3,7-dihydroxyphenoxazine can be adjusted as appropriate when 10-acetyl-3,7-dihydroxyphenoxazine is contained in the reaction solution for allowing peroxidase to act on H ₂ O _2. Is usually 10-500 μM.

  In the method of the present invention, PS can be quantified by reacting these three enzymes simultaneously under conditions suitable for all enzyme reactions of phospholipase D, L-amino acid oxidase and peroxidase.

  In the present invention, the activities of phospholipase D, L-amino acid oxidase and peroxidase are activities measured by the methods described in the Examples.

  The range of “one or two or more” is not particularly limited, but for example, 1 to 50, preferably 1 to 25, more preferably 1 to 12, further preferably 1 to 9, and particularly preferably 1 Means ~ 5. Techniques in which one or more amino acids are substituted, deleted, or added in a specific amino acid are known.

  In the present invention, a microorganism, animal or plant-derived enzyme refers to an enzyme produced by a microorganism, animal or plant, and one or more amino acids substituted, added, deleted or inserted in the amino acid sequence of the enzyme. The modification obtained by is widely included.

  Each of the above-mentioned enzymes can be obtained as a commercial product, or can be produced by obtaining a gene using information on a known gene sequence and preparing a transformant. The produced enzyme can be purified by affinity chromatography, ion exchange chromatography, hydroxyapatite column chromatography, ammonium sulfate salt folding method, or the like.

The following method is mentioned as an example of the quantification method of PS of this invention. First, H ₂ O ₂ is measured by a method of the present invention for a standard sample obtained by appropriately diluting a solution with a known PS concentration, and a calibration curve of the amount of H ₂ O ₂ (such as fluorescence intensity of resorufin) with respect to the PS concentration is prepared. Then, the amount of H ₂ O ₂ can be measured according to the present invention using a sample whose PS content is unknown, and the amount of PS can be determined from the calibration curve.

<(2 ') Process>
In the PS quantification method of the present invention, instead of the step (2), (2 ′) (a) the amount of decrease in oxygen in the step (1), (b) the amount of increase in ammonia, or (c) 2- You may have the process of quantifying phosphatidylserine by measuring the production amount of oxo-3-hydroxypropionic acid. Phosphatidylserine can be quantified from a calibration curve determined in advance.

As shown in FIG. 2, one molecule of O ₂ , NH ₃ and 2-oxo-3-hydroxypropionic acid corresponds to one molecule of PS. Therefore, by measuring the increase / decrease of the molecule, PS Can be quantified. The method for measuring (a) oxygen decrease, (b) ammonia increase, or (c) 2-oxo-3-hydroxypropionic acid production is particularly limited as long as the amount of the molecule can be measured. However, for example, (a) is a measurement using an oxygen electrode, (b) is a measurement using an ammonia electrode, and (c) is an enzyme quantification method using 2-oxo-3-hydroxypropionic reductase. .

<(1 ') and (2'')steps>
The PS quantification method of the present invention includes (1 ′) a step of allowing phospholipase D to act on a sample, and (2 ″) a step of quantifying phosphatidylserine by measuring the amount of serine produced in the step (1 ′). It may be a method having Phosphatidylserine can be quantified from a calibration curve determined in advance.

  As a result of the series of reactions described above, one molecule of serine is generated from one molecule of PS, and therefore PS can be quantified by measuring the amount of serine. The method for measuring the amount of serine is not particularly limited as long as it is a method capable of measuring the amount of serine, and examples thereof include an enzyme quantification method using serine dehydrogenase.

  The phospholipase D used in step (1 ′), the conditions for causing phospholipase D to act, and the like are the same as those in step (1). In addition, phospholipase C and phosphomonoesterase can be used in place of phospholipase D in the step (1 ′).

  The method for quantifying phosphatidylserine according to the present invention enables quantification of phosphatidylserine with high sensitivity, high accuracy, and simplicity.

  In the examples shown below, the steps of the PS quantification method of the present invention are (1) incubation with reaction reagent S1, (2) incubation with reaction reagent S2, and (3) fluorescence intensity measurement. The time required for the work is about 260 minutes, and even if the number of samples to be measured is increased, only the dispensing operation of the reaction reagent is increased, and the work time does not change much.

  On the other hand, the steps of PS quantification by TLC-phosphorus quantification method are as follows: (1) Sample landing on silica gel plate, (2) Solvent (chloroform / methanol / acetic acid / formic acid / water = 70/30/12/4 / 2) development, (3) drying, (4) spot detection with iodine, (5) spot scraping recovery with spatula, (6) decomposition to inorganic phosphoric acid with 70% perchloric acid, (7) Reaction with ammonium molybdate, (8) Absorbance measurement. The time required for the work is about 400 minutes, and the time spent for the steps (1) and (5) greatly increases as the number of samples to be measured increases.

  Thus, when the PS quantification method of the present invention is compared with the conventional method (PS quantification by TLC-phosphorus quantification method), the method of the present invention can be said to be a simple method due to the small number of steps.

Phosphatidylserine quantification kit The phosphatidylserine quantification kit of the present invention comprises phospholipase D, preferably further comprising L-amino acid oxidase, more preferably further comprising peroxidase, and particularly preferably further 10- Contains acetyl-3,7-dihydroxyphenoxazine.

  By performing the PS quantification method using the PS quantification kit of the present invention, it is possible to quantify PS with high sensitivity, high accuracy, and simpleness.

  The PS quantification method described above can be applied to the method of using the PS quantification kit of the present invention. Phospholipase D, L-amino acid oxidase and peroxidase are the same as described above.

  The PS quantification kit of the present invention may contain phospholipase D, L-amino acid oxidase and peroxidase as an enzyme solution, or may contain a dry powder form. The PS quantification kit of the present invention may further contain a buffer, a metal salt and the like. Examples of the buffering agent and the metal salt include those described above. The buffer and the metal salt are preferably included in the kit in the form of an aqueous solution or powder.

  The phosphatidylserine quantification kit of the present invention may also contain phospholipase C and phosphomonoesterase instead of phospholipase D. Phospholipase C and phosphomonoesterase are the same as described above.

  Examples are given below to illustrate the present invention in more detail. However, the present invention is not limited to these examples.

[Method]
600 U / ml phospholipase D (derived from Streptomyces chromofuscus) (Enzo Life Sciences BML-SE301), 20 U / ml L-amino acid oxidase (derived from Crotalus adamanteus venom) (Worthington Biochemical LAO), 50 mM NaCl , And a solution containing 50 mM Tris-HCl (pH 7.4) was used as a reaction reagent solution S1. The amount of phospholipase D enzyme that liberates 1 μmol of choline per hour from 5 mM phosphatidylcholine at 37 ° C. and pH 8.0 is defined as 1 U. The amount of L-amino acid oxidase enzyme that oxidizes 1 μmol of L-leucine per minute at 25 ° C. and pH 7.6 is defined as 1 U.

  6.25 U / ml peroxidase (from horseradish root) (Oriental Yeast 46261003), 187.5 μM 10-acetyl-3,7-dihydroxyphenoxazine (Invitrogen A12222), 0.125% Triton X-100, 50 mM A solution containing NaCl and 50 mM Tris-HCl (pH 7.4) was used as a reaction sample solution S2. At 25 ° C. and pH 7.0, 4-aminoantipyrine and phenol are oxidized and condensed, and the amount of peroxidase enzyme that produces 1 μmol of quinoneimine dye per minute is 1 U.

  10 μl of a sample or standard solution containing 1% Triton X-100 was injected into a microplate, 10 μl of reaction reagent S1 was added, and incubated at 25 ° C. for 240 minutes. Thereafter, 80 μl of reaction reagent S2 was added and incubated at room temperature for 15 minutes, and 20 μl of Amplex Red / UltraRed Stop Reagent (A33855 manufactured by Invitrogen) was added to stop the reaction. After stopping the reaction, a fluorescence microplate reader (Fluoroskan Ascent FL manufactured by Thermo Scientific) was used to select a filter having an excitation wavelength of 544 nm and a fluorescence wavelength of 590 nm, and the fluorescence intensity was measured.

  The amount of PS in the sample was determined from the calibration curve obtained by measuring the standard solution and the fluorescence intensity of the sample. Fluorescence is stable and measurable for at least 3 hours after adding Amplex Red / UltraRed Stop Reagent.

  Addition of Amplex Red / UltraRed Stop Reagent can be omitted if it is possible to rapidly measure fluorescence after adding reaction reagent S2 and incubating for 15 minutes.

The TLC-phosphorus determination method was performed as follows.
(1) 100μl of sample on the silica gel plate
(2) Development using solvent (chloroform / methanol / acetic acid / formic acid / water = 70/30/12/4/2)
(3) Dry
(4) Spot detection with iodine
(5) Spot scraping collection using a spatula
(6) Decomposition into inorganic phosphoric acid by adding 70% perchloric acid (500μl) and heating at 160 ° C for 2 hours
(7) Ammonium molybdate: Add 5 ml of malachite green reagent and incubate at room temperature for 20 minutes
(8) Absorbance measurement (wavelength 660 nm)
Ammonium molybdate: how to prepare malachite green reagent solution (100 ml)
(1) Stir 0.3% malachite green aqueous solution (50 ml) for 30 minutes
(2) 2.1% ammonium molybdate: 2.5N HCl aqueous solution (50 ml) was added and stirred for 30 minutes
(3) Filtration with No.2 filter paper
(4) Add Tween20 (60μl) and stir for 30 minutes

[result]
Test example 1
A calibration curve was prepared by the method of the present invention using a purified sample of L-α-palmitoyl-oleoyl-phosphatidylserine (POPS), and a straight line was shown in the range of 0 to 0.5 nmol (r = 0.9987) and the detection limit was 0.05 nmol (FIG. 3). In the range of 0.5 to 10 nmol, a hyperbola was shown (r = 0.9998) (FIG. 4).

Test example 2
Soy-derived PS and porcine brain-derived PS are a mixture of PS with various types of acyl chains attached. There was no difference in fluorescence intensity after the enzyme quantitative reaction of the present invention between 1 nmol POPS and 1 noml of soybean PS or 1 nmol pig brain PS. It was found that there was no effect (Fig. 5).

  Lysophosphatidylserine (LPS) is a single-chain phospholipid in which a fatty acid at the 2-position of the glycerol skeleton is dissociated from PS. There was no difference in fluorescence intensity after the enzyme quantitative reaction of the present invention between 1 nmol POPS and 1 nmol LPS (FIG. 5).

  Phosphatidylethanolamine (PE) and phosphatidylcholine (PC) have an amine at the head, like phosphatidylserine, and liberate ethanolamine and choline when phospholipase D acts. However, in the enzyme quantitative reaction of the present invention, fluorescence was not generated and detected with PE or PC (FIG. 5).

Test example 3
Lipids were extracted from HEK293 cells cultured on a plastic petri dish by Bligh-Dyer method, and a PS addition recovery test was performed on the cell-extracted lipid solution (Table 1). As shown in Table 1, almost 100% could be recovered at the added PS amount of 0.25, 0.50, 1.25 and 2.50 nmol. From this result, it was found that the quantification of the added PS was not inhibited by other cell extract substances, and the quantification method of the present invention could accurately quantitate intracellular PS.

Test example 4
The TLC-phosphorus quantification method has been the most widely used method for quantifying PS in cultured cells. When the amount of PS obtained by the TLC-phosphorus quantification method and the amount of PS obtained by the quantification method of the present invention were compared for the same sample obtained from HEK293 cells, they almost coincided (y = −0.8007 + 0.9999x R = 0.9803) (FIG. 6). From these facts, the amount of PS in cultured cells obtained using the quantification method of the present invention developed this time is reliable.

  Lipids extracted from HEK293 cells by Bligh-Dyer method were once dried. When performing the enzyme quantification method of the present invention, the dried lipid was dissolved in 1% TritonX-100 aqueous solution (200 μl), and 10 μl thereof was injected into the microplate. On the other hand, when performing the TLC-phosphorus determination method, the dried lipid was dissolved in a methanol: chloroform (1: 1) mixture (100 μl), and the total amount (100 μl) was spotted on a silica gel plate. In this case, the amount of sample used is 20 times, but in the case of the enzyme quantification method of the present invention, even smaller amounts can be quantified.

  Since the PS quantification method of the present invention has a sensitivity 100 times that of the TLC-phosphorus quantification method, the amount of sample required is 1/100.

Test Example 5
Using the PS quantification method of the present invention, high-density lipoprotein (HDL) isolated from human plasma (EMD Chemicals, Inc. (Cat # 437641), human plasma 150 mM NaCl, 0.01% EDTA, pH 7.4 The amount of PS contained in 10 μl was determined. As a result, the amount of PS in HDL was 19.8 nmol / mg protein.

  From the above results, it can be seen that the PS quantification method of the present invention is simple, highly sensitive, and highly accurate, and can achieve high throughput. In addition, with the quantification method of the present invention, high-accuracy quantification of a small amount of PS contained in cultured cells, which has been difficult until now, was also possible. Furthermore, it can be applied to quantification of PS contained in blood.

  The phosphatidylserine quantification method and quantification kit of the present invention include PS quantification in experiments using cultured cells, PS quantification in experiments using dispersion systems (liposomes and micelles), quantification of purified PS, and cultured cells. Quantification of PS contained in PS and contained in culture solution, quantification of PS contained in body fluid containing human tissue and blood, quantification of PS contained in body fluid containing animal tissue and blood, plant tissue and plant body fluid Can be used for quantification of PS contained in foods, PS contained in fungi and fungi, PS contained in bacteria and PS contained in bacterial cultures, and quantification of PS contained in foods and pharmaceuticals. .

Claims (3)

A method for quantifying phosphatidylserine in a sample, comprising:
(1) A step of causing phospholipase D and L-amino acid oxidase to act on a sample, and (2) a step of measuring the amount of H ₂ O ₂ produced in step (1) and quantifying phosphatidylserine from a calibration curve obtained in advance. And measuring the amount of H ₂ O _{2 in the} step (2) by measuring the fluorescence intensity, absorbance, or luminescence amount of a compound produced by allowing peroxidase to act on H ₂ O _2. And the method. The method according to claim 1, wherein phospholipase C and phosphomonoesterase are used in place of phospholipase D in the step (1). A kit for quantification of phosphatidylserine comprising (I) phospholipase D or phospholipase C and phosphomonoesterase, (II) L-amino acid oxidase, and (III) peroxidase.

Images