Hemoprotein stabilization method

Abstract

To provide a method for stabilizing hemoprotein, that is suitable for use in the biochemical field and the analytical chemistry field including clinical diagnostic agent applications.SOLUTION: Provided is a method for stabilizing hemoprotein, in which an aromatic heterocyclic compound is added.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for stabilizing heme proteins. More specifically, the present invention relates to a method for stabilizing the activity of peroxidase, which is a heme protein, by adding imidazole, which is an aromatic heterocyclic compound.

Background Art

[0002] Conventionally, in clinical diagnosis, measurement of biological components by an enzymatic method has been carried out. In particular, a method using an oxidase-peroxidase-redox chromogenic reagent system, that is, a method in which a substance to be measured in a sample is subjected to an enzymatic reaction to generate hydrogen peroxide, and this is reacted with a chromogenic agent in the presence of peroxidase for colorimetric determination, is widely used. In order to make the composition containing the enzyme used in this method usable for a long period of time, it is of course important to stably maintain the enzyme activity.

[0003] Patent Document 1 describes a method for suppressing inactivation by an organic solvent by adding a protein folding factor to an organic solvent-containing solution containing a protein.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] Patent Document 1 has found a method for suppressing inactivation of proteins by organic solvents, and is limited to stabilization methods under limited conditions. On the other hand, the present inventor has found that heme proteins such as catalase and peroxidase are liable to be decomposed and denatured in an aqueous solution and are deactivated by long-term storage. An object of the present invention is to provide a method for improving the stability of heme proteins such as catalase in an aqueous solution.

Means for Solving the Problems

[0006] As a result of intensive studies to solve the above problems, the present inventors have found that the addition of an aromatic heterocyclic compound stabilizes heme proteins, and have thus completed the present invention. That is, the present invention has the following configuration. (Item 1) A method for stabilizing a heme protein, characterized by coexisting an aromatic heterocyclic compound. (Item 2) The method according to Item 1, wherein the heme protein is peroxidase. (Item 3) The method according to Item 1 or 2, wherein the aromatic heterocyclic compound has a nitrogen atom. (Item 4) The method according to any one of Items 1 to 3, wherein the aromatic heterocyclic compound containing a nitrogen atom has a molecular weight of 100 g / mol or less. (Item 5) The method according to any one of Items 1 to 4, wherein the aromatic heterocyclic compound having a nitrogen atom is imidazole. (Item 6) The method according to any one of Items 1 to 5, wherein the concentration of the imidazole is 1.8 mmol or more in a reagent containing a heme protein. (Item 7) The method according to any one of Items 1 to 6, wherein the aqueous solution containing the heme protein is at least one selected from the group consisting of Tris buffer, citrate buffer, borate buffer, phosphate buffer, MES, Bis-Tris, ADA, ACES, BES, PIPES, MOPS, TES, HEPES, Tricine, Bicine, POPSO, TAPS, CHES, and CAPS. (Item 8) The method according to any one of Items 1 to 7, wherein the concentration of the buffer solution is 1 mmol / L to 1000 mmol / L. (Item 9) A reagent kit for measuring biological components, characterized by coexisting an aromatic heterocyclic compound and containing at least the following components (a) to (d). (a) Peroxidase (b) A redox chromogenic reagent that reacts with hydrogen peroxide in the presence of peroxidase to develop color (c) Buffer (d) Aromatic heterocyclic compound (Item 10) The method according to claim 9, wherein the biological component is at least one selected from the group consisting of creatinine, triglyceride, inorganic phosphorus, creatine, cholesterol ester, sialic acid, α-amylase, GOT, GPT, guaase, and phospholipid. [Effect of the Invention]

[0007] According to the present invention, a method for stabilizing hemoproteins can be provided by adding an aromatic heterocyclic compound. [Modes for Carrying Out the Invention]

[0008] Hereinafter, the present invention will be described in more detail while showing embodiments of the present invention, but the present invention is not limited thereto. All non-patent documents and patent documents described in this specification are incorporated herein by reference. In this specification, "~" means "above and below". For example, if "x~y" is described in the specification, it means "x or more and y or less". In this specification, the singular expression is understood to include the concept of its plural form unless otherwise stated.

[0009] In addition, the enzyme to be stabilized in the present invention is not particularly limited in terms of its intended use, concentration, form of existence, etc. For example, as long as it is present in a composition that uses the enzyme as a constituent and utilizes the function of the enzyme, examples include analytical reagents for molecular biology applications, analytical reagents for biochemical applications, in vitro diagnostic agents, liquid in vitro diagnostic agents, dry-type in vitro diagnostic agents processed into chip or slit shapes, enzyme sensors, enzyme electrodes, and pharmaceutical compositions. Manufacturing techniques and utilization techniques for these compositions have already been established in their respective industrial fields. Therefore, by applying the knowledge thereof to the present invention, the enzyme present in various compositions can be stabilized, and the mode thereof is not particularly limited.

[0010] (Method for stabilizing hemoprotein) The method of the present invention is to stabilize a hemoprotein by coexisting an aromatic heterocyclic compound.

[0011] (Hemoprotein) A hemoprotein is a protein containing heme. Heme means a complex composed of a divalent iron atom and porphyrin. Porphyrin is a cyclic planar structure composed of four pyrrole molecules, and heme has a divalent iron atom at the center of this cyclic planar structure. Examples of hemoproteins include hemoglobin, which is an oxygen carrier, cytochromes involved in the electron transport system, catalase having enzyme activity, peroxidase, etc. The hemoprotein may or may not have enzyme activity, but it is preferably one having enzyme activity. Examples of hemoproteins having enzyme activity include catalase, peroxidase, etc.

[0012] (Peroxidase) As the peroxidase used in the present invention, any type of enzyme may be used as long as it is an enzyme that catalyzes the reaction between hydrogen peroxide and a redox chromogenic reagent. Examples include peroxidases derived from plants, bacteria, and basidiomycetes.

[0013] Among these, peroxidases derived from horseradish, rice, and soybeans are preferred for reasons such as purity, ease of availability, and price, and peroxidase derived from horseradish is more preferred.

[0014] As commercially available products, PEO-131 (manufactured by Toyobo), PEO-301 (manufactured by Toyobo), PEO-302 (manufactured by Toyobo), etc. are preferably used.

[0015] The peroxidase used in the present invention may be peroxidase of any size, for example, peroxidase having a molecular weight (Mw) of about 30,000 to 50,000. In one embodiment, the peroxidase used in the present invention preferably has a molecular weight of 40,000 to 45,000.

[0016] The peroxidase used in the present invention preferably has an optimum pH of pH 5.5 to 9.0, more preferably pH 6.0 to 7.0.

[0017] The peroxidase used in the method of the present invention may use only one type of peroxidase, or may use a mixture of two or more types of peroxidases. Further, when using two or more types of peroxidases, they may be peroxidases derived from the same type of microorganism or peroxidases derived from different types of microorganisms.

[0018] (Aromatic heterocyclic compound) It is a cyclic compound containing at least two different elements in the ring. Examples of aromatic heterocyclic compounds include nucleic acids, drugs, biomass, and many natural dyes and synthetic dyes. In the stabilization of the heme protein according to the present invention, it is considered that the aromatic heterocyclic compound acts as a ligand to prevent the decrease in enzyme activity by metal ions. Further, it is considered that the hydrogen atom of the aromatic heterocyclic compound forms a hydrogen bond with the non-bonding electron pair of the heme protein, preventing the denaturation of the heme protein.

[0019] (Aromatic heterocyclic compound containing a nitrogen atom) Among complex cyclic aromatic compounds, those containing a nitrogen atom are useful skeletons that are widely used in the field of pharmaceutical development. Examples of basic skeletons include imidazole, pyrrole, pyrazole, oxazole, thiazole, pyridine, pyrimidine, indole, purine, quinoline, and isoquinoline. All of these can be obtained as commercially available products, etc.

[0020] (Imidazole) It is an aromatic heterocyclic compound containing nitrogen atoms at the 1,3 positions on a five-membered ring. As uses, industrially, it is used as a raw material for medical and agricultural chemicals, a curing agent for epoxy resins, a curing catalyst for urethanes, and a copper rust inhibitor. Also, as pharmaceuticals, azole antifungal drugs are known as typical examples containing an imidazole structure. Examples of imidazoles in the broad sense having substituents include cimetidine, losartan, ozagrel, and the like.

[0021] In the present invention, the type and concentration of the aromatic heterocyclic compound containing a nitrogen atom in the reagent are not particularly limited. Preferably, the concentration in the reagent is adjusted to be 0 to 500 mmol / L, preferably 1 to 100 mmol / L, more preferably 1.5 to 50 mmol / L, and still more preferably 2 to 25 mmol / L.

[0022] In the present invention, the type and concentration of the aromatic heterocyclic compound containing a nitrogen atom in the reagent are not particularly limited. It contains a substance in which the molecular weight of the aromatic heterocyclic compound containing a nitrogen atom is 500 g / mol or less, preferably 300 g / mol or less, more preferably 200 g / mol or less, and still more preferably 100 g / mol or less.

[0023] (Reagent for measuring biological components) Reagents for measuring biological components include analytical reagents for biochemical applications, in vitro diagnostic agents, liquid in vitro diagnostic agents, etc. Also, in a broad sense, it includes dry-type in vitro diagnostic agents processed into chip or slit shapes, enzyme sensors, enzyme electrodes, and the like.

[0024] The biological component measurement reagent of the present invention is not particularly limited as long as it contains the following components (1) to (4). It can take various forms as described above, and those skilled in the art can appropriately set the constitution such as the reagent composition based on the description in this specification. The biological component measurement reagent kit may further include a specimen sampler, an instruction manual, etc. in addition to the bottle containing the first reagent and the bottle containing the second reagent. (1) Peroxidase (2) A redox chromogenic reagent that reacts with hydrogen peroxide in the presence of peroxidase to develop color (3) Buffer (4) Aromatic heterocyclic compound

[0025] As the biological component measurement reagent, a CRE measurement reagent is preferred. The CRE measurement reagent is not particularly limited as long as it can quantify creatinine, but a reagent containing creatinine amidohydrolase, creatinase, sarcosine oxidase, and peroxidase is preferred. Such a CRE measurement reagent can quantify the CRE concentration by a peroxidase-chromogenic agent system by designing a so-called conjugate reaction in which creatine generated by the reaction of creatinine amidohydrolase using creatinine as a substrate reacts further with creatinase to generate sarcosine, and further hydrogen peroxide is generated from sarcosine using sarcosine oxidase.

[0026] The UA measurement reagent is not particularly limited as long as it can quantify uric acid, but a reagent containing uricase and peroxidase is preferred. Such a UA measurement reagent can quantify the UA concentration by a peroxidase-chromogenic agent system using hydrogen peroxide generated by the reaction of uricase using uric acid as a substrate.

[0027] The TG measurement reagent is not particularly limited as long as it can quantify triglyceride, but a reagent containing lipoprotein lipase, glycerol kinase, glycerol-3-phosphate oxidase, and peroxidase is preferred. Such a TG measurement reagent can quantify the TG concentration by means of a peroxidase-chromogenic agent system by using lipoprotein lipase with triglyceride as a substrate and generating hydrogen peroxide using glycerol kinase and glycerol-3-phosphate oxidase as conjugate enzymes.

[0028] The LDL and HDL measurement reagents are not particularly limited as long as they can quantify LDL cholesterol and HDL cholesterol, but a reagent containing cholesterol esterase and cholesterol oxidase is preferred. Such LDL and HDL measurement reagents can quantify LDL and HDL by means of a peroxidase-chromogenic agent system by allowing cholesterol esterase and cholesterol oxidase to act and generating hydrogen peroxide.

[0029] As a means for implementing the above method, it is preferably a reagent (or kit) configured to be applicable to a general-purpose automatic analyzer (for example, Hitachi 7170 type automatic analyzer). As such a form, for example, a reagent (or kit) composed of a combination of a dry preparation produced by means such as freeze-drying and a dissolving solution can be mentioned. Preferably, it is a liquid reagent, for example, a liquid reagent in which the reagent is divided into two packages (hereinafter, also referred to as a two-reagent system liquid reagent). In this method, first, the first type of reagent (hereinafter, also referred to as the first reagent) is added to the sample and allowed to react for a certain period of time, and then the second type of reagent (hereinafter, also referred to as the second reagent) is further added and allowed to react, and the target component can be quantified by measuring the change in absorbance during this period.

[0030] (Redox chromogenic reagent) As the redox coloring reagent used in the present invention, any type of dye may be used as long as it reacts with hydrogen peroxide to develop color. Examples thereof include a hydrogen donor, a coupler, a leuco form, a tetrazolium salt, etc. The amount used and the form of addition are not particularly limited. Any of these can be obtained as commercially available products.

[0031] A typical example using a hydrogen donor and a coupler is the Trinder method in which a hydrogen donor and a coupler are oxidatively condensed with hydrogen peroxide in the presence of peroxidase to form a dye. As hydrogen donors used in the Trinder method and the like, phenol, phenol derivatives, aniline derivatives, naphthol, naphthol derivatives, naphthylamine, naphthylamine derivatives, etc. are known. For example, N-ethyl-N-sulfopropyl-3-methoxyaniline, N-ethyl-N-sulfopropylaniline, N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline, N-sulfopropyl-3,5-dimethoxyaniline, N-ethyl-N-sulfopropyl-3,5-dimethylaniline, N-ethyl-N-sulfopropyl-3-methylaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline, N-sulfopropylaniline, N-(2-hydroxy-3-sulfopropyl)-2,5-dimethylaniline, N-ethyl-N-(3-methylphenyl)-N'-succinylethylenediamine, N-ethyl-N-(3-methylphenyl)-N'-acetylethylenediamine, etc. can be mentioned. Further, these hydrogen donors can be used in combination with a coupler. Further, as the coupler, 4-aminoantipyrine (4AA), aminoantipyrine derivatives, vanillylaminesulfonic acid, methylbenzothiazolinone hydrazone (MBTH), sulfonated methylbenzothiazolinone hydrazone (SMBTH), etc. are known.

[0032] Examples of the leuco form include triphenylmethane derivatives, phenothiazine derivatives, diphenylamine derivatives, etc. Specifically, 4,4'-benzylidenebis(N,N-dimethylaniline), 4,4'-bis[N-ethyl-N-(3-sulfopropylamino)-2,6-dimethylphenyl]methane, 1-(ethylaminothiocarbonyl)-2-(3,5-dimethoxy-4-hydroxyphenyl)-4,5-bis(4-diethylaminophenyl)imidazole, 4,4'-bis(dimethylamino)diphenylamine, N-(carboxymethylaminocarbonyl)-4,4'-bis(dimethylamino)diphenylamine salt (DA64), 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine salt (DA67), etc. can be mentioned.

[0033] Examples of the tetrazolium salt include 2,3,5-triphenyltetrazolium salt, 2,5-diphenyl-3-(1-naphthyl)-2H-tetrazolium salt, 3,3'-[3,3'-dimethoxy-(1,1'-biphenyl)-4,4'-diyl]-bis[2-(4-nitrophenyl)-5-phenyl-2H-tetrazolium] salt, 3,3'-[3,3'-dimethoxy-(1,1'-biphenyl)-4,4'-diyl]-bis(2,5-diphenyl-2H-tetrazolium) salt, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium salt, 3,3'-(1,1'-biphenyl-4,4'-diyl)-bis(2,5-diphenyl-2H-tetrazolium) salt, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium salt, etc.

[0034] In the present invention, preservatives, salts, enzyme stabilizers, chromogen stabilizers, etc. may be added within a range that does not affect the reaction. Their usage amounts, forms of addition, etc. are not particularly limited. All of these can be obtained as commercially available products. Examples of preservatives include azides, chelating agents, antibiotics, antibacterial agents, etc. Examples of chelating agents include ethylenediaminetetraacetic acid and its salts. Examples of antibiotics include gentamicin, kanamycin, chloramphenicol, etc. Examples of antibacterial agents include methylisothiazolinone, imidazolidinyl urea, etc. Examples of salts include sodium chloride, potassium chloride, aluminum chloride, etc. Examples of enzyme stabilizers include sucrose, trehalose, cyclodextrin, gluconates, amino acids, etc. Examples of chromogen stabilizers include chelating agents such as ethylenediaminetetraacetic acid and its salts, cyclodextrin, etc.

[0035] (Buffer solution) When the reagent for measuring biological components using the present invention is a liquid reagent, it is preferable to contain a buffer solution component. Examples of buffer solutions include Tris buffer solution, phosphate buffer solution, borate buffer solution, carbonate buffer solution, Good's buffer solution, etc. Their usage amounts, set pH, forms of addition, etc. are not particularly limited. All of these can be obtained as commercially available products.

[0036] Examples of GOOD's buffers include N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), 2-morpholinoethanesulfonic acid (MES), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), N-tris(hydroxymethyl)methyl-2-aminomethanesulfonic acid (TES), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), N-cyclohexyl-2-hydroxy-3-aminopropanesulfonic acid (CAPSO), 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO), 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (EPPS), 2-hydroxy-3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (HEPPSO), 3-morpholinopropanesulfonic acid (MOPS), 2-hydroxy-3-morpholinopropanesulfonic acid (MOPSO), piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid) (POPSO), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPSO), N-(2-acetamido)iminodiacetic acid (ADA), N,N-bis(2-hydroxyethyl)glycine (Bicine), N-[tris(hydroxymethyl)methyl]glycine (Tricine), and the like.

[0037] In the present invention, the type and concentration of the buffer in the reagent are not particularly limited. Preferably, the concentration in the reagent is adjusted to be 0 to 500 mmol / L, more preferably 5 to 100 mmol / L, still more preferably 10 to 75 mmol / L, and even more preferably 20 to 50 mmol / L.

Example

[0038] Hereinafter, the present invention will be specifically described by way of examples. It should be noted that the present invention is not particularly limited by the examples.

[0039] <Example 1: Measurement of peroxidase activity> To an aqueous solution containing peroxidase, imidazole was added at a concentration of 1.8 - 22.0 mmol / L. A reagent without added imidazole was also prepared, and the activity of peroxidase was measured.

[0040] (Preparation of reagents) Bicine (pH 9.0) 50.0 mmol / L Peroxidase 8.0 U / mL

[0041] For the measurement, two types of the above reagents were used: one stored at 4°C and the other stored at 35°C for one week.

[0042] (Measurement method) Peroxidase (PEO) (Principle) The generated purpurogallin is extracted with ether and measured by the change in absorbance at 420 nm. (Definition) The amount of enzyme that generates 1.0 mg of purpurogallin in 20 seconds under the following conditions is defined as 1 purpurogallin unit (U).

[0043] (Reagents) (a) 5% (w / v) pyrogallol aqueous solution (prepared as needed) (b) 0.147 mol / L h 2 o 2 aqueous solution (1.67 mL of 30% (w / v) H 2 O 2 solution is diluted with distilled water to 100 mL (c) 0.1 M phosphate buffer pH 6.0 (for reaction mixture and enzyme dilution) (d) 2.0 N H 2 SO 4 solution (e) Enzyme solution: Pre-cool the enzyme standard in ice-cold (c), dissolve it in (c), dilute it with (c) to 3.0 - 6.0 purpurogallin U / mL, and store it in ice-cold condition.

[0044] (Procedure) 1. Prepare the following reaction mixture in a test tube and pre-warm it at 20°C for about 5 minutes. 14.0 mL distilled water 2.0 mL pyrogallol aqueous solution (A) 1.0 mL H 2 O 2 aqueous solution (B) 2.0 mL phosphate buffer solution (C) 2. Add 1.0 mL of (e) to start the reaction. 3. After reacting exactly for 20 seconds at 20°C, add 1.0 mL of (d) to stop the reaction. Extract the generated purpurogallin from the mixture after the reaction stop with 15 mL of ether. Repeat this operation 5 times, combine the extracts, and add more ether to make the total volume 100 mL. Measure the absorbance at 420 nm for this solution (OD test ). 4. For the blank test, leave the reaction mixture standing at 20°C for 20 seconds, add 1.0 mL of (d) and mix, then add 1.0 mL of (e) to prepare. Perform ether extraction on this solution in the same manner as above and measure the absorbance (OD blank ).

[0045] The calculation formula for PEO activity is described below. [Number] U / mg = U / mL × 1 / c 0.117: Absorbance at 420 nm of 1 mg% purpurogallin ether solution c: Enzyme concentration at dissolution (c mg / mL) (Note) 1 purpurogallin unit corresponds to 13.5 international units (using o-dianisidine as the substrate, under the reaction conditions of 25°C).

[0046] The calculation formula for PEO residual rate is described below. (Number 2) PEO residual rate (%) = PEO activity value (U / mL) of the reagent stored at 35°C for 1 week / PEO activity value (U / mL) of the reagent stored at 4°C

[0047] Table 1 shows the measurement results of peroxidase activity when imidazole was added at each concentration.

Table 1

[0048] When imidazole was not added, it was confirmed that the residual rate of the enzyme decreased significantly (0.66 U / mL) due to storage at 35°C for 1 week. On the other hand, when imidazole was added, a result of higher PEO residual rate was obtained. From this result, it was confirmed that the addition of imidazole in the solution stabilized the peroxidase activity.

Industrial Applicability

[0049] The measurement method of the present invention can be used in application fields such as pharmaceuticals for in vitro diagnosis, and it is of great contribution to the industrial world.

Claims

1. A method for stabilizing a heme protein, characterized by co-existing an aromatic heterocyclic compound.

2. The method according to claim 1, wherein the heme protein is peroxidase.

3. The method according to claim 1, wherein the aromatic heterocyclic compound has a nitrogen atom.

4. The method according to claim 1, wherein the aromatic heterocyclic compound containing the nitrogen atom has a molecular weight of 100 g / mol or less.

5. The method according to claim 3, wherein the aromatic heterocyclic compound having the nitrogen atom is imidazole.

6. The method according to claim 1, wherein the concentration of the imidazole is 1.8 mmol or more in a reagent containing the heme protein.

7. The aqueous solution containing the heme protein is at least one selected from the group consisting of Tris buffer, citrate buffer, borate buffer, phosphate buffer, MES, Bis-Tris, ADA, ACES, BES, PIPES, MOPS, TES, HEPES, Tricine, Bicine, POPSO, TAPS, CHES and CAPS. The method according to claim 1.

8. The method according to claim 1 or 2, wherein the concentration of the buffer solution is 1 mmol / L to 1000 mmol / L.

9. A reagent kit for measuring a biological component, characterized by co-existing an aromatic heterocyclic compound and containing at least the following (a) to (d) as constituent elements. (a) Peroxidase (b) A redox chromogenic reagent that reacts with hydrogen peroxide in the presence of peroxidase to develop color (c) Buffer (d) Aromatic heterocyclic compound

10. The method according to claim 9, wherein the biological component is at least one selected from the group consisting of creatinine, triglyceride, inorganic phosphorus, creatine, cholesterol ester, sialic acid, α-amylase, GOT, GPT, guaase, and phospholipid.

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