JPS5889199A - Quantitative analysis of phosphatidylglycerol - Google Patents

Abstract

PURPOSE:To determine phosphatidylglycerol in a specimen solution in a short time easily even at extremely low concentration, by using an enzyme for catalyzing a reaction for converting phosphatidylglycerol into glycero-3-phosphoric acid. CONSTITUTION:A specimen solution such as amniotic fluid, etc. containing various kinds of lipids is treated with phosphalipase C an enzyme for catalyzing a reaction for preparing glycero-3-phosphoric acid and diglyceride from phasphatidylglycerol and water. The liberated glycero-3-phosphoric acid is reacted with glycerophosphate oxidase, and the amount of oxygen consumed by the reaction of the amount of hydrogen peroxide produced by it is determined by an electrochemical means or using an indicator.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel method for the determination of phosphatidylglycerol.

In recent years, neonatal respiratory distress syndrome (RDS) in the perinatal period has become a major problem in neonatal management, accounting for a large proportion of deaths. RDS is thought to be caused by a deficiency in lung surfactant, which is important in preventing the shrinkage of alveoli that swell immediately after birth. This deficiency of pulmonary surfactant causes atelectasis during breathing, which triggers the onset of RDS. Therefore, the onset of RDS can be predicted early,
If we can start treating newborns early, RD8
The onset of the disease can be prevented or the symptoms can be kept mild. Therefore, it is necessary to measure this pulmonary surfactant. Broadly speaking, there are currently two methods: methods that utilize the physical properties of lung surfactants to limit them, and methods that biochemically elucidate the constituent components of lung surfactants. In this latter method, the ratio of each lipid composition, such as the ratio of lecithin to sphingomyelin, the ratio of phosphatidylglycerol to phosphatidylinositol, and the determination of dipalmitoylphosphatidylcholine, was determined by thin layer chromatography. Am, J, 0bste
t Gynecol, 440:109 (1971)
.

Am, J. ()bstet Gynecol,, 61
8:125. (1976).

Am, J. Obstet Gynecol,, 894
:188. (1979).

0bstet & Gynecol, 2'95:
57. (1981).

Am, J. Obstet Gynecol, 697:
188. (1980) etc.]. In addition, based on the results of these various tests, it has recently been recognized that the presence or absence of phosphatidylglycerol is an important factor in the development of RD8. Its quantification was extremely difficult. Conventionally, the method for quantifying cyphosphatidylglycerol involves centrifuging amniotic fluid, a body fluid, to remove cellular components, then extracting lipid components from the supernatant, and then analyzing the components in this extract using thin-layer chromatography. After separation, each phospholipid is quantified, the ratio of various phospholipids is determined, and phosphatidylglycerol is indirectly determined from the total phospholipid value determined in advance by a phosphoric acid determination method using a wet ashing method. Chromatography method (
Am, J. Obstet, Gynecol, .
618:125, (1976), Am, J., 0bste.
t Gynecol, 1079:185, (197
9), Am, J., Obstet Gynecol, .
8'99: 1BB, (1979), Am, J, 0bs
tet Gynecol, 440:109, (1
971)) and quantitative methods using high performance liquid chromatography (Journal of chromatography)
phy.

277:228. (1981)) is known. However, this thin layer chromatography method requires 2 to 8 days to extract lipid components from amniotic fluid, requires complicated operations, and has the drawbacks of requiring high temperature heating for spot detection. Furthermore, since it is an indirect measurement method based on the relative amount ratio from a spot and total phospholipids, and it is thin layer chromatography, it has various drawbacks such as the inability to simultaneously process a large number of samples.

The present inventors conducted various studies on a method for easily and accurately quantifying only phosphatidylglycerol from sample fluids such as amniotic fluid containing various lipid components, and found that, quite unexpectedly, it acts on lecithin. Phospholipase C (phosph
olipaseC: 8.1.4.8 phosphe
tidyl ch Tang wo-choline phosph
It was discovered that phosphatidylglycerol (ohydrolase) acts on phosphatidylglycerol to produce glycero-8-phosphate and diglyceride, and by quantifying this glycero-8-phosphate produced and liberated in the reaction, it was possible to determine the quality of phosphatidylglycerol alone. The quantitative method was completed. More preferably, glycerophosphate oxidase is allowed to act on glycero-8-phosphate generated and liberated by allowing phospholipase C to act on the test solution,
Next, by quantifying the amount of oxygen consumed or the amount of hydrogen peroxide produced in the reaction, we completed a method that allows phosphatidylglycerol alone to be determined extremely well. The present invention has been completed based on the above findings, and has the ability to act on an enzyme that catalyzes the reaction that produces glycero-8-phosphate and diglyceride from phosphatidylglyceronyl and water in the determination of phosphatidylglycerol in a test solution. At least, a method for quantifying phosphatidylglycerol is characterized in that the liberated glycero-8-phosphate is then quantified, preferably in the quantification of phosphatidylglycerol in a test solution, comprising: (a) allowing phospholipase C to act on phosphatidylglycerol; to release glycero-8-phosphate, (
b) Allow glycerophosphate oxidase to act on glycero-8-phosphate, (e) Then quantify the amount of oxygen consumed and the amount of hydrogen peroxide produced in the reaction, (a) (b>
(This is a method for quantifying phosphatidylglycerol, characterized by quantitative determination by a combination of the steps of (→).In addition, in the present invention, it is preferable to prepare a composition for quantitative determination in which each reagent is prepared. The operation can be performed at room temperature around 37° C., is extremely simple, and can be performed in an extremely short reaction time.

Furthermore, according to the present invention, it is possible to accurately measure phosphatidylglycerol concentrations down to extremely low concentrations, and it is extremely useful because it directly measures phosphatidylglycerol values, and it can be done simply and in a short time. This method also enables simultaneous measurement of multiple samples, and provides a useful method for quantifying phosphatidylglycerol.

First, the enzyme that catalyzes the reaction of producing glycero-8-phosphate and diglyceride from phosphatidylglycerol and water in the present invention includes, for example, 1 mole of lecithin and 1 mole of water to 1 mole of diglyceride and 1 mole of diglyceride.
Phospholipase C (E, C, 8, 1,
4, 8).

As this phospholipase C, 1. If it catalyzed the above enzymatic reaction, it was extracted and collected from cells containing phospholipase C. It may also be a commercially available enzyme reagent, such as Streptomyces
s) belonging to the genus Streptomyces hachijiyounsis A-1148 (11, hach i joens i
s A-1148; FERM-PNa 1829)
Streptomyces realibileticyu IJ IF
012787 (fit, albireticuli
IFO12787), Streptomyces cinnamoneus IFO12852 (gt, cinnamoneu
m IFO12852; $treptovertic
illium cinnamoneum subsp, c
innamoneum IFO12852), Streptomyces griseocarnensis IFO12776 (
st, griseocarinensis IFO
12776; streptoverticillium
griseocarneum IFO12776) (
In addition, among bacteria belonging to the genus Streptomyces, the bacterial reservoir that forms a whirl in the aerial system is separated from the genus Streptomyces as the genus Streptobayateicillium (Ba1dacci; 195
8 years)] and Clostridium velci (clostridium
idiumwelchii), Bacillus cereu x (
Microbial-derived enzymes, which are enzymes obtained from cultures of phospholipase C-producing bacteria such as Bacillus cereus [Special Publication No. 58-1856, Lipid Meta
bolism P214 (1960)) and commercially available phospholipase C enzyme reagents can be used. The amount of phospholipase C to be used is not particularly limited, and can be changed as appropriate depending on the measurement time and the concentration of phosphatidylglycerol. For example, 0.1 or more units of phospholipase C may be used per test, preferably about 1 to 100 units. Furthermore, this phosphorinase C preferably contains a weakly acidic to weakly alkaline Tris-HCl buffer, citrate buffer, borate buffer,
It may be used by dissolving it in a buffer such as FTPES-NaOH buffer or imidazole buffer, and may be adjusted by adding a surfactant such as sodium deoxycholate or serum albumin as necessary. Next, the phospholinase C enzyme solution prepared in this way and the test solution are mixed, water is consumed from the phosphatidylglycerol in the test solution, and glycero-8-phosphate is liberated. However, the mixing ratio of both strains is not particularly limited, and they may be mixed at a ratio that preferably contains about 1 to 100 units of phosphorinase C per test solution, and the reaction temperature is The reaction time may preferably be around 87°C, and the reaction time may be sufficient as long as glycero-8-phosphoric acid is produced, and is usually 5 minutes or more, preferably 10 minutes or more. Next, by quantifying the glycero-8-phosphate produced in the reaction, the phosphatidylglycerol in the test liquid is determined from this measured value.

Further, as a method for quantifying glycero-8-phosphate, various known measurement methods such as chemical assays and enzymatic assays can be used. Preferably, an enzymatic quantitative method is convenient, which involves using one or more enzymes that use glycero-8-phosphate as a substrate, and quantifying detectable changes in the enzyme's action during the reaction. . For example, a GPO-based quantitative method in which glycerophosphate oxidase (opo) is applied to liberated glycero-3-phosphate to consume oxygen in the 54 reaction solution and generate hydrogen peroxide,
It is especially convenient and good.

Furthermore, in this GPO-based quantitative method, the amount of oxygen consumed in the reaction solution is quantified as an electrical change at an oxygen electrode, or the amount of hydrogen peroxide produced is determined as an electrical change at a hydrogen peroxide electrode. Quantify by electrical means, or
Alternatively, GPO or GPO and phospholipase C can be immobilized using known enzymatic means, such as entrapping immobilization with acrylamide, mixing with proteins such as albumin, and then cross-linking the proteins to immobilize them, collagen or A method of immobilizing a car by enclosing it in fibroin etc. by covalent bonding, a method of immobilizing it by adsorption or covalent bonding to a porous organic polymer resin, a method of entrapping immobilization using a photocurable resin. The immobilized enzyme may be made by various entrapment, adsorption, binding, etc. methods, and the shape of the immobilized enzyme may be a shape suitable for enzyme electrodes for incorporation into oxygen electrodes or hydrogen peroxide electrodes. It is preferable to use it as a film-like fibrous material, granular or ~tubular material, and use it as an enzyme electrode equipped with this immobilized enzyme in the detection part of an oxygen electrode or hydrogen peroxide electrode for quantitative determination by electrical means. In addition, as a means of quantifying the amount of hydrogen peroxide, an indicator composition that reacts with hydrogen peroxide and converts into a detectable product may be used as a useful enzyme in a significantly reduced amount. Quantification may also be performed using In addition, this indicator composition is usually a color-forming drug composition that causes a visible change in color tone, a fluorescent drug composition that emits fluorescence when irradiated with ultraviolet rays, or a luminescent drug composition that develops color. A composition is used in which the changes can be quantified. For example, as a coloring agent composition, a composition containing a substance having a peroxidase action and a dye precursor is used. Substances that have this peroxidase action include:
Peroxidase derived from horseradish is commonly used, and a combination of an electron acceptor and a phenolic compound is commonly used as a dye precursor. Further, as the electron acceptor, for example, 4-aminoantipyrine, 2-hydrazinobenzothiazole, 3-methyl-2-benzothiazolone hydratene, 2-aminobenzothiazole, etc. are used, and as the phenolic compound, for example, phenol , 3-methyl-No-ethyl-N-(β-hydroxyethyl)aniline, 3.5-xylenol, N,
N-dimethylaniline, N,N-diethylaniline, etc. are used, such as bis(2,4,6-drichlorophenol oxalate, phenylthiohydantoin, homovanillic acid, 4-hydroxyphenylacetic acid, vanillylamine, 8-methoxytyramine). , phloretic acid, hordenine,
Examples include luminol monoanion, lucigenin, waffin, etc., and hydrogen peroxide may be quantified by using them together with a substance having an electron acceptor/peroxidase action, if necessary. Furthermore, the amount of enzyme reagents and dye precursors used is not particularly limited; for example, the GPO per test is usually 0.05 units or more, preferably 0.1 to
200 units, peroxidase usually 0.05 units or more
Preferably, 0.1 to 500 units may be used, and electron acceptors and phenolic compounds are usually prepared using distilled water or a weakly acidic or weakly alkaline buffer solution to a concentration of 0.1mM or more. Just do it. In addition, these reagents may be used separately or in combination with the phospholipase C enzyme solution described above, and may also be used in quantitative compositions, paper, films, etc. in which these reagents are freeze-dried. It may also be made into a laminated integral quantitative composition formed by applying the composition to the composition. The GPO-based quantitative method constructed in this way has good sensitivity to glycero-3-phosphate liberated in the test solution, and is still susceptible to the effects of impurities in the test solution. This is an excellent method for accurate measurement.

In addition, the GPOs used in this GPO-based quantitative method include:
Any enzyme that catalyzes the reaction of producing dihydroxyacetone phosphate and hydrogen peroxide from glycero-8-phosphate and oxygen may be used, for example, belongs to the genus Streptococcus, Lactobacillus, Leuconostoc, or Pediococcus. Glycerophosphate oxidase-producing bacteria (Japanese Unexamined Patent Publication No. 72892/1982) and glycerophosphate oxidase-producing bacteria belonging to the genus Aerococcus (Aerococcus fistula viridans F012219 strain, IP012)
An enzyme obtained from a culture of 817 strain (JP-A-55-15746) or a commercially available enzyme can be used.

Another enzymatic method for quantifying glycero-8-phosphate involves catalyzing the reaction that produces dihydroxyacetone phosphate and reduced NAD from glycero-8-phosphate and nicotine adenine dinucleotide (NAD). Glycerophosphate dehydrogenase is allowed to act on glycero-3-phosphate liberated in the test solution in the presence of NAD to produce dihydroxyacetone phosphate and reduced NAD.
An example is a quantitative method in which the amount of glycero-8-phosphate is determined by producing NAD and then quantifying this reduced NAD. When quantifying reduced NAD, 840n is usually used.
Absorbance measurements at wavelengths around m or diaphorase or water-soluble tetrazolium salts such as 3-(4,5-dimethyl)-2-thiazolyl-2H-tetrazolium bromide, 2-(P-
iodophenyl)-8-(P-nitrophenyl)-5-
Phenyl-2H-tetrazolium chloride, 8.8
'-(8,3'-dimethoxy-4,41-biphenylylene)-bis(2-(P-nitrophenyl-5-phenyl-2H-tetrazolium chloride)]) The color may be set using chlorophenol indophenol or the like, and the color tone may be measured by absorbance at its specific absorption wavelength.

Furthermore, it is convenient to use commercially available enzymes and reagents, and the amounts used can be designed as appropriate, and surfactants and stabilizers may be used as necessary. It may also be processed into various compositions.

Furthermore, the test liquid targeted in the present invention may be any sample containing phosphatidylglycerol,
Examples include collected body fluids such as amniotic fluid.

In addition, when using amniotic fluid as the test fluid, the amniotic fluid may be used as it is, or if the content of phosphatidylglycerol in the amniotic fluid is extremely low, the amniotic fluid may be concentrated; for example, 5 to 6 ml of the collected amniotic fluid may be concentrated. Extract with 15-18 m/chloroform-methanol (2:1),
The chloroform layer was collected by centrifugation at Orpm and evaporated to dryness in nitrogen gas to obtain a lipid fraction. This lipid fraction may then be dissolved in a fixed amount of 1% Triton X to 100 solution and then used as a sample of amniotic fluid components.

In this way, the enzyme solution of phospholipase C and, for example, each enzyme and other reagents based on the above-mentioned GPO-based quantitative method may be applied to the test solution sequentially or simultaneously. The ratio of the solution to the solution is not particularly limited, and usually about 0.1 to 8 ml of a solution such as an enzyme reagent is used per 0.01 ml to 1 ml of the test liquid. Further, the reaction conditions are preferably around 37°C, and the reaction time may be selected as appropriate to ensure sufficient reaction time, and the reaction is usually carried out for 5 minutes or more, preferably 10 minutes or more. As the reaction medium, water or a weakly acidic or weakly alkaline buffer solution as a solvent for each reagent is used.

By quantifying the sample solution in this way, phosphatidylglycerol can be directly determined down to an extremely low concentration in an extremely short period of time, and can be performed at room temperature without the need for complicated operations. It is a quantitative method.

Furthermore, the method for measuring the activity of each enzyme, phospholipase C and GPO, used in the later-described examples of the present invention is not particularly limited, and examples thereof are as follows.

(a) Phospholipase C activity measurement method 0.1 ml of a 4% aqueous solution of phosphatidyl cochloride isolated and purified from egg yolk;
0.1M Tris-HCl buffer (pH 8,5) O, J3a
/Oyohi 20mM MgCl2 aqueous solution (0.1ml) is mixed, 0.1ml of phospholipase C-containing enzyme solution is added thereto, and the mixture is reacted at 87°C for 15 minutes. After reaction, 8.6%
0.8 ml trichloroacetic acid and 5.5% serum 7A,
The reaction was stopped by adding 1 ml of Pumi:yO, and the mixture was left in a water bath for 20 minutes. Filter through Toyo F paper (m5B), transfer 0.5ml of the filtrate to a 1z Kjeldahl digestion flask, and
%HCl0.0.65a/10. After decomposition at 170°C for 2 hours, the liberated inorganic phosphorus was dissolved in amitor sulfite test solution 0.
5mJ, 8.8% ammonium molybutate 0.25m
/, water 8.2mA! was added to develop color, and after being left at room temperature for 20 minutes, 0. D, read the absorbance at 650 mμ,
The amount of enzyme that releases 1 μM/ml of inorganic phosphorus in 15 minutes is defined as 1 unit. (b) GPO activity measurement method 0.2M) Lis-HCl buffer (pH 8,0)
0.2 wLt peroxidase (0,51rQ/at
, 45U/m7ri 0.1ydo, 8(W/V
) 4-aminoantipyrine 0.110.1
MDL-glycero-8-phosphate 0.1
dO, 2% (V/V) N, N-di) 7=') 7
0.2 ml distilled water
0.8yal reaction solution with the above composition,
1. Qm/ was placed in a small test tube, prewarmed at 87°C for 8 minutes, and 20 μl of GPO-containing enzyme solution was added to it for 10 minutes.
The reaction was allowed to proceed for a minute, and then 0.25% (W/V) sodium laurylbenzenesulfonate 2.0- was added to stop the reaction, and the absorbance was measured at a wavelength of 565 nm.

Enzyme activity is calculated according to the following formula (where ΔA indicates the absorbance value at 565 nm for 10 minutes).Next, the present invention will be specifically described with reference to Examples, but the present invention is not limited thereby. It's not a thing.

Example 1 0.2M dimethylglutaric acid-NaOH buffer (pH
7,5) 0.2m1 10mMCaCl2
0.2 ml 110% sodium deoxycholate
0.05m11% bovine albumin
0.1 m140U/1
1Ll phospholipase C and 50U/mJGPo containing solution 0.1m/45U/m/peroxidase
0.1 ml 10.8% 4-aminoantipyrine
0.2I″1O08%8-methyl-N-
Ethyl-N-(β-hydroxyethyl)aniline
0.2 ml meter
2. Od Prepare 2.0 ml of the reaction composition for quantification of phosphatidylglycerol having the above composition, and add 50 μl of the test solution containing various phosphatidylglycerols (phosphatidylglycerol content n-moles to 100 n-moles).
moles) and reacted at 37°C for 15 minutes, then the color of the pigment produced after the reaction was measured at a wavelength of 550 nm.
Absorbance (OD5sO) was measured at m.

As a result, as shown in FIG. 1, a quantitative curve showing good linearity with respect to the phosphatidylglycerol content in the test solution was obtained.

Example 2 0.2M dimethylglutaric acid-NaOH buffer (pH 7
,5) 0.2 10% Sodium Deoxycholate
0.051L 120U/ml phospholipase C1 and 50U/mJGPO containing solution 0, lIE/45U/m/peroxidase
0.1wLlO08% 4-aminoantipyrine
0.2m10, a% phenol 0.2m
1 1% bovine albumin Q, 1ml steamed
Retained water 1
．． 05 ml total 2.0 ml Prepare 2.0 ml of the reaction composition for quantification of phosphatidylglycerol having the above composition, and add 50 μl of the test solution containing various phosphatidylglycerols (phosphatidylglycerol content: 10 n moles to 1
00 μmoles) was added and reacted at 37°C for 15 minutes, and then the color tone of the pigment produced after the reaction was measured at a wavelength of 500 μmoles.
Absorbance (ODsoo) was measured at nm. the result,
As shown in FIG. 2, a quantitative curve showing good linearity with respect to the phosphatidylglycerol content in the test solution was obtained.

[Brief explanation of the drawing]

Figures 1 and 2 show quantitative curves for phosphatidylglycerol. Patent Applicant Toyo Jozo Co., Ltd. Representative Ito Fujima No. 1 Figure 0 20 40 60 80
100 phosphatidylglycerol content (n mo
les) Figure 2 Phosphatidylglycerol content (n moles)
) Procedural amendment May-6, 1980 Haruki Shimada, Commissioner of the Patent Office 1, Indication of the case Showa S-Year Patent Application No. 1g711gA 2, Name of the invention Method for quantifying phosphatidylglycerol 3, Person making the amendment Case and Relationships Patent applicant address: 63, Mifuku, Ohito-cho, Tagata-gun, Shizuoka Prefecture, /. The detailed description of the invention in the targeted description of the amendment is as follows: 0.2M dimethylglutaric acid-hydroxide tll lamb's buffer solution 0.20d, 10m・M ultrasonicated purified egg yolk A reaction solution containing 0.20 ml of lecithin emulsion, 0.1 A Oml of distilled water, 0.20 ml of lecithin emulsion, and 0.1 A Oml of distilled water was prewarmed at 37°C for S minutes, and the enzyme solution 0. / ml was added and reacted at 37°C for 70 minutes, and then 0 ml of Lis-HCl buffer containing 0.2% Zodium deoxycholate was added to stop the reaction. /
, Q ff1j color source solution [composition: 0.3 * *
-AmimiAntivin+)nQ,/m,0.2'A7e/-
le 0. /u, choline oxidase iA OU /id)
0. / ml, peroxidase (RIOU/l) 0
．． 1m, alkaline postfutase (4 (OU/l1j)
0.1ml 1% 50mM) Lys-hydrochloric acid (pHff,
0)0.3d] was added and reacted at 37°C for 20 minutes, and its absorbance (ΔAsoon
m) E 31Jt73° Enzyme activity is calculated according to the following formula t. / l Enzyme activity (TJ/mj) = ΔA 500Hz x −x
/ Ox 775 J Specification No. 2/Page line D r O
, 3 (W/V) J is corrected as "0, 3rd Section (W/V) J" 1966 Patent Application No. 1g'lda g6ko1 Invention Name: Method for quantifying phosphatidylglycerolRelationship with the case of the person who made the 3S amendment Patent applicant address: 76S of 632 Mifuku, Ohito-cho, Tagata-gun, Shizuoka Prefecture Contents of the amendment (+) 0 threat r(,?) of the lIS attachment of the application Delete one document certifying that the storage of the documents has been entrusted.

Claims (1)

[Scope of Claims] (1) In the determination of phosphatidylglycerol in a test solution, an enzyme that catalyzes the reaction of producing glycero-8-phosphate and diglyceride from phosphatidylglycerol and water is acted upon, and then released. A method for quantifying phosphatidylglycerol, which comprises quantifying glycero-8-phosphate. (2) The assay method according to claim 1, wherein the enzyme that catalyzes the reaction of producing glycero-8-phosphate and diglyceride from phosphatidylglycerol and water is phospholipase C. (3) The quantitative method according to claim 1 or 2, wherein the test fluid is a body fluid. (4) In quantifying phosphatidylglycerol in a test solution, (a) phospholipase C is applied to phosphatidylglycerol to release glycero-3-phosphate, (b) glycerophosphate oxidase is applied to glycero-8-phosphate. (C) The amount of oxygen consumed or hydrogen peroxide produced in the reaction is then determined. The quantitative method according to claim 1, 2, or 3, which is performed by a combination of the above steps (a), (b), and (C). (5) The quantitative method according to claim 4, wherein the amount of oxygen is determined by electrochemical quantitative means using an oxygen electrode. (6) The quantitative method according to claim 4, wherein the amount of hydrogen peroxide is determined by electrochemical quantitative means using a hydrogen peroxide electrode. (7) The quantitative method according to claim 4, in which the amount of hydrogen peroxide is determined using an indicator composition that reacts with hydrogen peroxide and converts into a detectable product. (8) The quantitative method according to claim 7, wherein the indicator composition is a coloring agent composition, a fluorescent agent composition, or a luminescent agent composition. (9) Claim 8, wherein the coloring agent composition contains a substance having a peroxidase action, a dye precursor, and a dye precursor.
Quantitative method described in section. 01 The assay method according to claim 9, wherein the dye precursors are 4-aminoantipyrine and phenol. (11) The quantitative method according to claim 9, wherein the dye precursors are 4-aminoantipyrine and 8-methyl-N-ethyl-N-(β-hydroxyethyl)aniline. (12) The quantitative method according to claim 10, wherein in the measurement, absorbance is measured at a wavelength of around 5000 m. (18) The quantitative method according to claim 11, wherein in the measurement, absorbance is measured at a wavelength of around 5500 m.