JP2013255436A - Method and kit for quantification of sphingomyelin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method and kit for quantification of sphingomyelin which enable highly specific, highly sensitive, and easy quantification of sphingomyelin.SOLUTION: A method for quantification of sphingomyelin in a sample includes following steps: (1) acting a sphingomyelinase, an alkaline phosphatase, a choline oxidase, and a peroxidase on the sample in the presence of a nonionic surfactant (the sphingomyelinase is derived from a microorganism belonging to the genus Bacillus); and (2) measuring the fluorescence intensity, absorbance or light emission amount of compounds generated in the step (1) to determine the amount of sphingomyelin from a predetermined calibration curve. A kit for quantification of sphingomyelin comprises a sphingomyelinase, an alkaline phosphatase, a choline oxidase, a peroxidase, and a nonionic surfactant (the sphingomyelinase is derived from a microorganism belonging to the genus Bacillus).

Description

  The present invention relates to a method for quantifying sphingomyelin using an enzyme and a kit for quantifying sphingomyelin.

  Sphingomyelin (SM) is the major sphingolipid and an essential component of the cell membrane in all animals and plants, and certain prokaryotes. In animals, SM is present in cell membranes, myelin sheaths, and plasma lipoproteins.

  SM is a component of lipid rafts, which are microdomains on cell membranes, and is involved in the regulation of various intracellular signal transduction and membrane protein activities such as receptors, channels, and transporters.

  SM metabolites, including ceramide, sphingosine, and sphingosine 1-phosphate, act as important regulators of cell proliferation, differentiation, and apoptosis. Neiman-Pick disease, a type of lysosomal disease, lacks the activity of the acid sphingomyelinase (SMase) enzyme and is caused by the accumulation of SM in cells, tissues, and body fluids. Human plasma SM levels have been reported to be a risk factor for coronary artery disease. SM is a structural and functional component of the lipoprotein surface and plays an important role in lipoprotein metabolism.

  Therefore, SM quantification is important for the understanding of various biological processes. Mass spectrometry has been used for identification and relative quantification of SM molecular species. However, it is difficult to determine the total concentration of SM by mass spectrometry. HPLC with an evaporative light scattering detector is used to quantify SM, but the sensitivity limit is 200 ng (about 280 pmol).

  The enzyme quantification method of SM is simple, rapid, high sensitivity, and high throughput, and does not require a special device (for example, Patent Document 1, Non-Patent Documents 1 to 5). Enzyme quantification is useful for studying various cellular processes, protein functions, and diseases related to SM metabolism. The enzyme fluorescence method for quantifying SM is more sensitive than a similar method using a colorimetric detection system (Non-patent Document 1). However, the specificity and accuracy of enzyme quantification methods have not been evaluated much.

  In Non-Patent Document 1, sphingomyelin, alkaline phosphatase, choline oxidase, and peroxidase four enzymes are used, and sphingomyelin is quantified by this quantification method. Is possible in the range of 0.02 to 20 nmol.

  In Non-Patent Document 2 and Patent Document 1, the range of linearity in the determination of sphingomyelin using the same four-enzyme system as described above and SM measurement is 0.5-5 μg (about 0.7-7 nmol). Have been described.

  Non-Patent Document 3 describes a method for quantifying sphingomyelin using the same four-enzyme system as described above, and that the linearity range in this assay is 10 to 120 μg / dL.

  Non-patent documents 4 and 5 also describe a method for quantifying sphingomyelin using the same four-enzyme system as described above.

International Publication No. 2007/078806

He, X., et al. Anal Biochem 2002, 306, 115-123. Hojjati, M.R., et al. J Lipid Res 2006, 47, 673-676. Jiang, X.C., et al. Arterioscler Thromb Vasc Biol 2000, 20, 2614-2618. Shigeyuki Imamura, Hideo Misaki, History of Medicine 1983, 127, 114-120. Hidaka, H., et al. Clin Biochem 2008, 41, 1211-1217.

  As described above, conventionally, SM is quantified by thin layer chromatography / phosphorus determination, high performance liquid chromatography, or mass spectrometry. However, these methods have drawbacks such as low detection sensitivity and quantitative accuracy, and time and labor.

  In recent years, an enzyme quantification method for SM has been developed, but its SM specificity and quantification accuracy have not been studied at all.

  Accordingly, an object of the present invention is to provide a sphingomyelin quantification method and a sphingomyelin quantification kit capable of easily quantifying SM with high specificity and high sensitivity.

  The present inventor uses (1) a sphingomyelinase derived from Bacillus cereus in the known enzyme reactions disclosed in Patent Document 1 and Non-Patent Documents 1 to 5 (FIG. 1). And (2) the use of a nonionic surfactant in the enzyme reaction, the knowledge that the above object can be achieved.

A method for quantifying phosphatidylserine shown in FIG. 1 will be described below.
(i) SM is hydrolyzed with SMase to produce ceramide and phosphorylcholine.
(ii) Phosphorylcholine is hydrolyzed by alkaline phosphatase to produce choline.
(iii) Oxidize choline with choline oxidase to generate H ₂ O ₂ .
(iv) Resorufin is produced by reacting 10-acetyl-3,7-dihydroxyphenoxazine (Amplex® Red) with H ₂ O ₂ with peroxidase. The amount of SM can be measured by measuring the fluorescence intensity generated from resorufin.

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

(I) Determination of sphingomyelin
(I-1) Method for quantifying sphingomyelin in a sample having the following steps:
(1) a step of allowing a sphingomyelinase, alkaline phosphatase, choline oxidase, and peroxidase to act on a sample in the presence of a nonionic surfactant (the sphingomyelinase is derived from a microorganism belonging to the genus Bacillus), and
(2) A step of measuring the fluorescence intensity, absorbance or luminescence amount of the compound produced in step (1), and quantifying sphingomyelin from a calibration curve determined in advance.
(I-2) The method according to (I-1), wherein the concentration of the nonionic surfactant when the enzyme is allowed to act in the step (1) is 0.01 to 1% by volume.
(I-3) In step (1), the enzyme is allowed to act in two steps: (a) a step of causing sphingomyelinase and alkaline phosphatase to act; and (b) a step of acting of choline oxidase and peroxidase. The method according to (I-1) or (I-2).
(I-4) The method according to any one of (I-1) to (I-3), wherein the nonionic surfactant is polyoxyethylene octylphenyl ether.
(I-5) The method according to any one of (I-1) to (I-4), wherein the sphingomyelinase is derived from a microorganism belonging to Bacillus cereus.

(II) Sphingomyelin quantification kit
(II-1) Sphingomyelinase, alkaline phosphatase, choline oxidase, and peroxidase, and a sphingomyelin quantification kit containing a nonionic surfactant (the sphingomyelinase is derived from a microorganism belonging to the genus Bacillus) .
(II-2) The kit according to (II-1), wherein the nonionic surfactant is used at a concentration of 0.01 to 1% by volume in the enzyme reaction.
(II-3) The kit according to (II-1) or (II-2), wherein the nonionic surfactant is polyoxyethylene octylphenyl ether.
(II-4) The kit according to any one of (II-1) to (II-3), wherein the sphingomyelinase is derived from a microorganism belonging to Bacillus cereus.

  The sphingomyelin quantification method and quantification kit of the present invention can quantitate sphingomyelin with high specificity, high sensitivity, and high accuracy.

  According to the present invention, SM-specific compounds sphingosylphosphocholine (SPC), phosphatidylcholine (PC), and lysophosphatidylcholine (LPC) are not detected, and are highly SM specific. Further, the detection limit of the present invention is 5 pmol, which is very sensitive compared to the conventional SM quantification method, and it is possible to perform quantification with high accuracy.

  Furthermore, the necessary operation in the present invention is mainly the dispensing of the sample and the reaction solution to the microplate with a pipette, which is very simple and enables high-throughput quantification.

It is a figure which shows the reaction in the fixed_quantity | assay method of SM of this invention. SMase hydrolyzes SM into ceramide and phosphorylcholine. Alkaline phosphatase catalyzes the production of choline from phosphorylcholine. Choline oxidation is catalyzed by choline oxidase to produce _two H ₂ O ₂ molecules. In the presence of peroxidase, Amplex Red reacts with H ₂ O ₂ to produce the fluorescent compound resorufin, and the fluorescence intensity is measured. 4 is a graph showing a standard curve in SM measurement of Test Example 1. Palmitoyl SM standard solution was added to reaction reagent M1 and incubated at 37 ° C. for 30 minutes. Then, reaction reagent M2 was added. After incubation at room temperature for 30 minutes, a reaction stop solution was added. The fluorescence intensity was measured using a fluorescence microplate reader. The background fluorescence was 11.0 ± 0.3 and was subtracted from each value. Each point represents the mean ± SD of 3 measurements. Lines were obtained by linear regression analysis (A) and quadratic regression analysis (B). The correlation coefficients were r = 0.9985 (A) and r = 0.9993 (B). 6 is a graph showing changes in fluorescence in response to palmitoyl SM, chicken egg SM, pig brain SM, milk SM, SPC, PC, and LPC (all 100 μM) in the SM measurement of Test Example 1. Each bar represents the mean ± SD of 3 measurements. Multiple comparisons were performed using Bonferroni test according to ANOVA. There were no statistically significant differences between palmitoyl SM, chicken egg SM, pig brain SM, and milk SM. 6 is a graph showing the linearity of SM measurement in Test Example 2. Lipid extracts from HEK293 cells were serially diluted with 1% by volume Triton® X-100. The correlation coefficient was r = 0.9991.

  The sphingomyelin quantification method and sphingomyelin quantification kit of the present invention will be described in detail below.

Method for quantifying sphingomyelin The method for quantifying sphingomyelin in the sample of the present invention is characterized by the following steps.
(1) a step of allowing a sphingomyelinase, alkaline phosphatase, choline oxidase, and peroxidase to act on a sample in the presence of a nonionic surfactant (the sphingomyelinase is derived from a microorganism belonging to the genus Bacillus), and
(2) A step of measuring the fluorescence intensity, absorbance or luminescence amount of the compound produced in step (1), and quantifying sphingomyelin from a calibration curve determined in advance.

  Hereinafter, each step will be described.

<Process (1)>
In step (1), sphingomyelinase, alkaline phosphatase, choline oxidase, and peroxidase are allowed to act on the sample in the presence of a nonionic surfactant.

By allowing sphingomyelinase to act on the sample, ceramide and phosphorylcholine are produced from SM and H ₂ O. Next, by causing alkaline phosphatase to act on the product, choline and phosphate are produced from phosphorylcholine and H ₂ O. Next, choline oxidase is allowed to act on the product to produce H ₂ O ₂ and betaine from choline, O ₂ and H ₂ O.

  Sphingomyelinase is an enzyme that hydrolyzes the ester bond of sphingophospholipids. The sphingomyelinase used in the present invention is one that hydrolyzes sphingomyelin to produce ceramide and phosphorylcholine and is not particularly limited as long as it is derived from a microorganism belonging to the genus Bacillus, but a microorganism belonging to Bacillus cereus Those derived from are preferred. Also preferred is a sphingomyelinase 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 a sphingomyelinase derived from a microorganism belonging to the Bacillus cereus.

  Alkaline phosphatase is a zinc enzyme that has an optimum pH alkaline and hydrolyzes phosphate monoester bonds to produce inorganic phosphate. The alkaline phosphatase used in the present invention may be derived from any microorganism, animal, or plant, as long as it can hydrolyze phosphorylcholine to produce choline. Those derived from the intestines are particularly preferred. Moreover, alkaline phosphatase consisting of an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of bovine intestinal alkaline phosphatase is also preferred.

  The choline oxidase used in the invention can be any microorganism, animal, or plant, as long as it can oxidize choline and generate hydrogen peroxide. -Those derived from SP (Alcaligenes sp.) Are particularly preferred. In addition, choline oxidase having an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of the alkali genus sp. Choline oxidase is also preferable.

  The peroxidase used in the present invention can be derived from any microorganism, animal or plant, but is preferably derived from a plant, and particularly preferably derived from horseradish. 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 preferred.

  In the SM quantification method of the present invention, when the above four types of enzymes are allowed to act on a sample, the four types of enzymes may be added together and reacted at the same time or sequentially added and reacted. Also good. However, the enzyme is preferably allowed to act in two steps: (a) sphingomyelinase and alkaline phosphatase, and (b) choline oxidase and peroxidase. Thus, accuracy can be improved by reacting four types of enzymes stepwise.

  Conditions for allowing sphingomyelinase to act on the sample can be appropriately set according to the characteristics of the enzyme used, but the pH is usually 6 to 9, and the temperature is usually 15 to 40 ° C. The time for which sphingomyelinase is allowed to act on the sample can be appropriately set according to the characteristics of the sample to be analyzed, but is usually 5 minutes or longer.

  Conditions for allowing alkaline phosphatase to act on the sample can be appropriately set according to the characteristics of the enzyme used, but the pH is usually 6 to 9, and the temperature is usually 15 to 40 ° C. The time for which alkaline phosphatase is allowed to act on the sample can be appropriately set according to the characteristics of the sample to be analyzed, but is usually 5 minutes or longer.

  Conditions for allowing choline oxidase to act on the sample can be appropriately set according to the characteristics of the enzyme used, but the pH is usually 6 to 9, and the temperature is usually 15 to 40 ° C. The time for which choline 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 5 minutes or longer.

  The conditions for allowing 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 9, 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 according to the characteristics of the sample to be analyzed, but is usually 5 minutes or longer.

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

  In the SM quantification method of the present invention, the amount of the four types of enzymes in the reaction solution in which the four 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 SM contained. Sphingomyelinase is usually 0.01 to 10 U / ml, preferably 0.1 to 5 U / ml, alkaline phosphatase is usually 1 to 100 U / ml, preferably 5 to 40 U / ml, and choline oxidase is usually 0.1 to 20 U / ml, preferably 1-10 U / ml, peroxidase is usually 0.5-50 U / ml, preferably 1-10 U / ml. Since these four types of enzymes can obtain high accuracy by completing the reaction almost completely within the reaction time, it is desirable to use a sufficient amount of enzymes.

  In the present invention, the reaction solution for allowing sphingomyelinase, alkaline phosphatase, choline oxidase, and peroxidase to act on the sample contains a nonionic surfactant in addition to the sample and the enzyme. In addition, when reacting four types of enzymes sequentially, the nonionic surfactant should just be contained in the reaction liquid at the time of making sphingomyelinase react. The concentration of the nonionic surfactant in the reaction solution is preferably 0.01 to 1% by volume, more preferably 0.05 to 0.5% by volume, and particularly preferably 0.1 to 0.4% by volume.

  Known nonionic surfactants can be used such as sorbitan monooleate, sorbitan monoisostearate, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan sesquioleate, sorbitan trioleate. Ate, penta-2-ethylhexyl diglycerol sorbitan, tetra-2-ethylhexyl diglycerol sorbitan, mono cottonseed oil fatty acid glycerin, glyceryl monoerucate, glyceryl sesquioleate, glyceryl monostearate, diglycerin diisostearate, α, α ' -Glycerol oleate, glyceryl monostearate, malic monostearate, propylene glycol monostearate, hardened castor oil attractant, glycerin alkyl ether, polyoxye Tylene (POE) sorbitan monooleate, POE sorbitan monostearate, POE sorbitan monooleate, POE sorbitan tetraoleate, POE sorbite monolaurate, POE sorbite monooate, POE sorbite monooleate, POE sorbite monostearate, POE monooleate , POE monooleate, ethylene glycol distearate, POE lauryl ether, POE oleyl ether, POE stearyl ether, POE behenyl ether, POE-2-octyldodecyl ether, POE cholestanol ether, POE octyl phenyl ether, POE nonyl phenyl ether, POE diethyl ether Nonylphenyl ether, polyoxyethylene / polyoxypropylene (POE / POP) cetyl ether, POE / POP monobutyl ether, POE / POP-2-decyltetradecyl ether, POE / POP monobutyl ether POE / POP hydrogenated lanolin, Tetronic, POE castor oil, POE hydrogenated castor oil, POE hydrogenated castor oil monoisostearate, POE hydrogenated castor oil triisostearate, POE hydrogenated castor oil monopyroglutamic acid diester, POE cured Castor oil maleic acid, POE sorbite beeswax, palm oil fatty acid diethanolamide, lauric acid monoethanolamide, fatty acid isopropanolamide, POE fatty acid amide, sucrose fatty acid ester, POE nonylphenylformaldehyde condensate, alkylethoxydimethylamine oxide, trioleyl phosphorus An acid etc. are mentioned. These may be used alone or in combination of two or more. The nonionic surfactant is preferably polyoxyethylene octyl phenyl ether (eg, Triton X-100).

In the present invention, a reaction solution for allowing sphingomyelinase, alkaline phosphatase, choline oxidase, and peroxidase to act on a sample has fluorescence intensity, absorbance, or luminescence amount by reacting with H ₂ O _{2 in} the presence of peroxidase. Increasing compounds are included. In addition, when reacting 4 types of enzymes sequentially, the said compound should just be contained in the reaction liquid at the time of making peroxidase react. An example of such a compound is 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red). The concentration of 10-acetyl-3,7-dihydroxyphenoxazine in the reaction solution can be adjusted as appropriate, but is usually 10 to 500 μM.

  In addition, the reaction solution for allowing sphingomyelinase, alkaline phosphatase, choline oxidase, and peroxidase to act on the sample may contain a buffer solution, a metal salt, or the like. 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 magnesium salt, potassium salt, sodium salt and the like.

  The sample in the present invention is not particularly limited as long as SM 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.

<Process (2)>
In step (2), the fluorescence intensity, absorbance or luminescence amount of the compound produced in step (1) is measured, and sphingomyelin is quantified from a previously determined calibration curve.

As a result of a series of reactions, two molecules of H ₂ O ₂ are generated from one molecule of SM, and therefore it is possible to quantify SM by measuring the amount of H ₂ O ₂ .

As the measurement method in step (2), specifically, a compound that reacts with H ₂ O ₂ by peroxidase to obtain a new absorption wavelength (for example, N, N′-bis (2-hydroxy-3-sulfopropyl) ), A method for measuring absorbance using tolidine, etc., a compound that reacts with H ₂ O ₂ by peroxidase to oxidize and condense multiple compounds to obtain a new absorption wavelength (for example, oxidative condensation of phenol and 4-aminoantipyrine) Etc.), fluorescence intensity is measured using a compound that reacts with H ₂ O ₂ with peroxidase to generate new fluorescence (for example, 10-acetyl-3,7-dihydroxyphenoxazine, etc.) And a method of measuring the amount of luminescence using a compound that reacts with H ₂ O ₂ by peroxidase to newly generate luminescence (eg, luminol).

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 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, two molecules of resorufin are produced from one molecule of SM, so it is possible to quantify SM 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.

In this specification, the amount of sphingomyelinase enzyme that hydrolyzes 1 μmol of trinitrophenylaminolauroyl sphingomyelin per minute at 37 ° C. and pH 7.4 is 1 U. The amount of alkaline phosphatase enzyme is 1U per international enzyme unit. The amount of choline oxidase enzyme that produces 1 μmol of H ₂ O ₂ per minute at 37 ° C. and pH 8.0 is defined as 1U. The amount of peroxidase enzyme is 1 U for 1 international enzyme unit.

  The range of the “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 for substituting, deleting, or adding one or more amino acids 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.

  An example of the SM quantification method of the present invention is the following method. First, the fluorescence intensity of a standard sample obtained by appropriately diluting a solution with a known SM concentration is measured by the method of the present invention, and a calibration curve of the fluorescence intensity with respect to the SM concentration is created. Then, the fluorescence intensity can be measured according to the present invention using a sample whose SM content is unknown, and the SM amount can be obtained from the calibration curve.

  The sphingomyelin quantification method of the present invention enables highly specific, highly sensitive and highly accurate sphingomyelin quantification.

Sphingomyelin quantification kit The sphingomyelin quantification kit of the present invention comprises sphingomyelinase, alkaline phosphatase, choline oxidase, peroxidase, and a nonionic surfactant.

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

  The SM quantification method described above can be applied to the method of using the SM quantification kit of the present invention. Sphingomyelinase, alkaline phosphatase, choline oxidase, peroxidase, and nonionic surfactant are the same as those described above.

The SM quantification kit of the present invention may contain sphingomyelinase, alkaline phosphatase, choline oxidase, and peroxidase as an enzyme solution, or may contain a dry powder form. The SM quantification kit of the present invention may contain a compound that generates a compound capable of measuring fluorescence intensity, absorbance, or luminescence amount by allowing peroxidase to act in the presence of H ₂ O ₂ . The SM quantitative determination 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.

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

Materials SMase derived from Bacillus cereus was purchased from Sigma-Aldrich (St. Louis, MO). Choline oxidase derived from Alkaligenes sp was purchased from Wako Pure Chemical Industries (Osaka, Japan). Bovine intestinal alkaline phosphatase and horseradish root peroxidase were purchased from Oriental yeast (Osaka, Japan). Amplex Red reagent was purchased from Molecular Probes (Eugene, OR). Palmitoyl SM, chicken egg-derived SM, pig brain-derived SM, milk-derived SM, sphingosylphosphocholine (SPC), L-α-palmitoyl-oleyl PC, and L-α-monooleyl PC are Avanti Polar Lipids (Alabaster, AL) Purchased from. Other chemicals were of special grade.

The enzyme measurement of SM was performed using a three-reaction reagent system. The reaction reagent solution M1 contains 1 U / ml SMase, 20 U / ml alkaline phosphatase, 1.5 mM MgCl ₂ , 50 mM NaCl, and 50 mM Tris-HCl (pH 7.4). The reaction reagent M2 contains 4 U / ml choline oxidase, 5 U / ml peroxidase, 300 μM Amplex Red, 0.2% by volume Triton X-100, 50 mM NaCl, and 50 mM Tris-HCl (pH 7.4). Amplex Red Stop reagent was purchased from Molecular Probes. The SM standard solution was dissolved in a 1% by volume Triton X-100 aqueous solution.

  Sample (10 μl) was added to reaction reagent M1 (40 μl) and incubated at 37 ° C. for 30 minutes. After incubation, reaction reagent M2 (50 μl) was added. After incubating at room temperature for 30 minutes, Amplex Red Stop reagent (20 μl) was added. The fluorescence intensity was measured using a fluorescence microplate reader (Fluoroskan Ascent FL, Thermo Fisher Scientific, Rockford, IL), and the excitation wavelength side and fluorescence wavelength side filters were set to 544 nm and 590 nm, respectively.

Measurement of SM content in cells
HEK293 cells were cultured at 37 ° C. in a humidified incubator (5% CO ₂ ) using DMEM containing 10% heat-inactivated FBS. Cells were seeded in 6-well plates and incubated at 37 ° C. for 48 hours. After incubation, the cells were cooled on ice, washed with chilled PBS, scraped, and the cells were disrupted by sonication. Cell lipids were extracted by Bligh and Dyer's method (Bligh, EG, Dyer, WJ, 1959. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37, 911-917.) Dissolved in 1% by volume Triton X-100. SM in lipid extracts from cells was measured by the enzyme assay method of the present invention.

<Result>
Test Example 1: An SM measurement calibration curve was prepared using an SM standard solution. The calibration curve for SM measurement was linear between 10 and 100 μM (r = 0.9985) (FIG. 2A). When SM was at low concentrations, the curve was slightly non-linear and fitted to a quadratic regression curve (r = 0.9993) (Figure 2B). As shown in the inset of FIG. 2B, the detection limit was 0.5 μM (5 pmol in the reaction mixture). This was 140 times more sensitive than the colorimetric analysis reported in Non-Patent Document 2, and four times more sensitive than the fluorescence analysis reported in Non-Patent Document 1.

  The fluorescence change of the response to palmitoyl SM was the same as that of egg SM, pig brain SM and milk SM with mixed acyl chains. This indicates that the method can measure SM regardless of the type of acyl chain. SPC (lyso form of SM) is present in a small amount in cells and is known as a lipid mediator. In this analysis, SPC showed only a negligible increase in fluorescence (˜1% of the increase in fluorescence due to SM), and other choline-containing phospholipids, PC and LPC, did not lead to an increase in fluorescence (FIG. 3). By using Triton X-100, SPC and LPC can be excluded from the measurement method. The structures of SM, SPC, PC and LPC are shown below.

Test Example 2: Measurement of SM in cultured cells
In order to confirm the accuracy of SM measurement, a known amount of palmitoyl SM was added to the cell lipid extract and a recovery test was performed (Table 1). As a result, the average recovery rate of the added SM was 100.3%. This indicates that there was no interference of other hydrophobic compounds extracted from the cells with respect to the SM measurement.

  In order to test the linearity of the quantification, lipid extracts from HEK293 cells were serially diluted with a 1% by volume Triton X-100 aqueous solution. As shown in FIG. 4, a well-fitted regression line was obtained (r = 0.9991).

  From the above results, it can be seen that the SM quantification method of the present invention has high specificity, high sensitivity, and high accuracy.

Claims (9)

Method for quantifying sphingomyelin in a sample having the following steps:
(1) a step of allowing a sphingomyelinase, alkaline phosphatase, choline oxidase, and peroxidase to act on a sample in the presence of a nonionic surfactant (the sphingomyelinase is derived from a microorganism belonging to the genus Bacillus), and
(2) A step of measuring the fluorescence intensity, absorbance or luminescence amount of the compound produced in step (1), and quantifying sphingomyelin from a calibration curve determined in advance.   The method according to claim 1, wherein the concentration of the nonionic surfactant when the enzyme is allowed to act in the step (1) is 0.01 to 1% by volume.   In the step (1), the enzyme is allowed to act in two steps: (a) a step of causing sphingomyelinase and alkaline phosphatase to act, and (b) a step of acting of choline oxidase and peroxidase. The method described in 1.   The method according to claim 1, wherein the nonionic surfactant is polyoxyethylene octylphenyl ether.   The method according to any one of claims 1 to 4, wherein the sphingomyelinase is derived from a microorganism belonging to Bacillus cereus.   A kit for quantifying sphingomyelin containing sphingomyelinase, alkaline phosphatase, choline oxidase, and peroxidase, and a nonionic surfactant (the sphingomyelinase is derived from a microorganism belonging to the genus Bacillus).   The kit according to claim 6, wherein the nonionic surfactant is used at a concentration of 0.01 to 1% by volume during an enzymatic reaction.   The kit according to claim 6 or 7, wherein the nonionic surfactant is polyoxyethylene octylphenyl ether.   The kit according to any one of claims 6 to 8, wherein the sphingomyelinase is derived from a microorganism belonging to Bacillus cereus.

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