CA1202869A

CA1202869A - Method for the quantitative measurement of the phosphatidyl glycerol

Info

Publication number: CA1202869A
Application number: CA000415626A
Authority: CA
Inventors: Shigeyuki Imamura; Hideo Misaki
Original assignee: Toyo Jozo KK
Current assignee: Toyo Jozo KK
Priority date: 1981-11-20
Filing date: 1982-11-16
Publication date: 1986-04-08
Also published as: GB2110822A; IT1153093B; JPS5889199A; IT8224350A0; FR2516936A1; FR2516936B1; DE3242652A1; GB2110822B

Abstract

ABSTRACT

METHOD FOR THE QUANTITATIVE MEASUREMENT OF
PHOSPHATIDYL GLYCEROL

A method for quantitative measurement of phosphatidyl glycerol in the liquid such as body fluid in which the fluid is treated with an enzyme to obtain glycero-3-phos-phate, said enzyme being able to play as a catalyst in a reaction to produce said glycero-3-phosphate and diglyceride from phosphatidyl glycerol and water, and the liberated glycero-3-phosphate is observed.

Description

~2();Z~69 METHOD FOR THE QUANTITATIV~ MEASUREMENT OF
PHOSPHATIDYL GLYCEROL

The present invention relates to a novel method for the quantitative measurement of phosphatidyl glycerol.

Recently, in the management of newborn babies, the respi~atory distress syndrome (RDS) in the perinatal period constitutes a big problem and the deaths due to it occupy a high percentage of total deaths. It is regarded that RDS occurs due to the lack of pulmonary surfactant. This material is important to prevent contraction of alveoli pulmonum which expands immediately after the birth. The lack of this pulmonary surfactant brings about atelectasis in respiration and occurrence of RDS. Consequently, if the occurrence of RDS can be estimated in an earlv ~eriod and the treatment o~ the newborn baby can be starte~ early, th~
occurrence of RDS may be prevented or it may be restrained to a mild case. Accordingly, the measurement of this pul-monary surfactant is the means of necessity. The means are roughly classified into the method for the measurement wherein the physical property of pulmonary surfactant is utili~ed and the one wherein compositive components of pulmonary surfactant are biochemically resolved. The lat-ter method has been practised by measuring the ratio of lipids such as the ratio of lecithin to sphingomyelin, that of phosphatidyl glycerol to phosphatidyl inositol or the quantitative measurement of dipalmitoyl phosphatidyl cho-line by means of the thin-layer chromatography ~Am. J.
Obstet. Gynecol., 440: 109 (1971); Am. J. Obstet. Gynecol., 613: 125 (1976); Am. J. Obstet. ~ynecol., ~94: 133 (1979);
Obstet. & Gynecol., 295: 57 (1981); Am. J. Obstet. Gynecol., 697: 138 (1980); etc.]. From the results of these various inspections, it has been recently recognized that the ex-istence of phosphatidyl glycerol is an important factor ~2V2869 for the occurrence of RDS. However, the quantitative measurement of phosphatidyl glycerol was very difficult because the quantity thereof in existence is as little as about one tenth of the amount of lecithin. Heretofore, as the method for the quantitative measurement of phosphatidyl glycerol, there has been known the method of thin-layer chromatography wherein, after the removal of cell components by the centrifugal separation from the liquor amnii, lipid components are extracted from the supernatant, the compo-n~nts of this extract are separated by the thin-layer chro-matography, each phospholipid is quantitatively measured, the ratios of various phospholipids are obtained and the quantity of phosphatidyl glycerol is obtained indirectly from the total phospholipid value preliminarily obtained by the quantitative measurement of phosphoric acid accord-ing to the wet combustion method [Am. J. O~stet. Gynecol., 613: 125 (1976); Am. J. Obstet. Gynecol., 1079: 135 (1979);
Am. J. Obstet. Gynecol., 899: 133 (1979); Am. J. Obstet.
Gynesol., 440: 109 (1971)], or the method for the quantita-tive measurement wherein the high speed liquid chromato-graphy is used [Journal of Chromatography, 277: 223 (1981)].
However, this method of thin-layer chromatography had dis-advantages that it takes two to three days for the extrac-tion of lipid components from liquor amnii, it requires complicated operations and, further, a heating at a high temperature is necessary because of the spot detection.
Also it had such other defects that it is an indirect method for the measurement based on the ratios of relative quanti-ties from spots and total phospholipid and it can not per-form simultaneous treatment of many samples because itemploys thin-layer chromatography.

The present inventors carried out various studies about the method whereby, in a liquid containing various lipid components to be inspected such as liquor amnii, only 12(~;~869 phosphatidyl glycerol can be quantitatively measured simp-ly, conveniently and accurately in a short time. As the result, they found ~uite unexpectedly that phospholipase C (phospholipase C: 3.1.4.3 phosphatidyl choline choline phosphohydrolase), which has the enzymatic action to act on lecithin and catalyze the reaction to produce 1.2-digly-ceride and phosphatidyl choline acts on phosphatidyl glycerol producing glycero-3-phosphate and diglyceride and they achieved a satisfactory method for the quantitative measurement of phosphatidyl glycerol only by quantitatively measuring this glycero-3-phosphate produced by the reaction and extricated. More preferably, they have achieved a method whereby only phosphatidyl glycerol may be quantita-tively measured highly satisfactorily by allowing phos-pholipase C to act on the liquid to be inspected to produce glycero-3-phosphate allowing glycerophosphate oxidase to act on this glycero-3-phosphate and measuring the quantity of oxygen consumed or hydrogen peroxide produced in the reaction. Thus the present invention relates to a method for the quantitative measurement of phosphatidyl glycerol in the liquid to be inspected, which comprises liberating glycero-3-phosphate by the action of an enzyme which plays a role of the catalyst in the reaction to produce glycero-3-phosphate and diglyceride from phosphatidyl ~lycerol and water and then quantitatively measuring glycero-3-yhosphate produced; preferably, it relates to a method for the quan-titative measurement of phosphatidyl glycerol in the liquid to be inspected, which comprises carrying out the quantita-tive measurement by the combination of the following steps ~0 (a), (b) and (c):
(a) liberating glycero-3-phosphate by allowing phos-pholipase C to act on phosphatidyl glycerol, (b3 allowing glycerophosphate oxidase to act on glycero-3-phosphate,and then (c) measuring the quantity of oxygen consumed or .., hydrogen peroxide produced in the reaction.

In the present invention, it is advantageous to pre-pare each reagent in an adjusted kit for the quantitative measurement and besides the operation for the quantitative measurement can be conducted at the room temperature of about 37C and it is a very simple operation and in addi-tion, the time required for the reaction is very short.
Moreover, based on the present invention, phosphatidyl glycerol can be accurately measured down to a markedly low concentration and the value of phosphatidyl glycerol can be directly measured revealing a great usefulness. Also, since the measurement can be conducted simply and con-veniently in a short time, many samples can be simultane-ously measured. Thus, the present invention provides a useful method for the quantitative measurement of phos-phatidyl glycerol.

First, as an example of the enzyme which plays a role of the catalyst in the reaction to produce glycero-3-phos-phate and diglyceride from phosphatidyl glycerol and water in the present invention, phospholipase C may be included.
This phospholipase C (E.C.3.1.4.3) has been known as an enzyme which is a catalyst in the reaction to produce each 1 mole of diglyceride and phosphatidyl choline from each 1 mole of lecithin and water. As far as the substance can be a catalyst in the enzyme reaction mentioned above, there may be used any one such as the one obtained by the ext~ac-tion of phospholipase C-containing cells or an enzyme re-agent sold on the market, for example, a microorganism-originated enzyme obtained from the culture of a phos-pholipase C-producing microorganisms which belongs to a Streptomyces genus such as Streptomyces hachijoensis A-1143 strain ¦FERM-P No. 1329), Streptomyces albireticuli IFO 12737, Streptomyces cinn~mnneum IFO 12852 (Strepto-~20286g verticillium cinnamoneum subsp. cinnamoneum IFO 12852).
Streptomyces griseocarinensis IFO 12776 (Streptoverticil-lium griseocarneum IFO 12776~ [among the bacteria belong-ing to Streptomyces genus, those which form whirl in its aerobacteria system are separated from Streptomyces genus to be named Streptoverticillium genus (Baldacci; 1958)]
or Clostridium welchii and Bacillus cereus [Japanese Ex-amined Published Patent Application No. 1356/1978, I,ipid Metabolism P214 (1960)] or enzyme reagent of phospholipase C sold on the market. The quantity of phospholipase C to be used should be appropriately modified and designed ac-cording to the time required for the measurement and the concentration of phosphatidyl glycerol and no particular limitation is to be imposed on it. For instance, per one test, there may be used phospholipase C of usually not less than 0.1 unit, preferably about 1 - 100 units. In addition, this phospholipase C is preferably used after it is dis-solved in a buffer such as weakly acidic to weakly alkaline Tris-HCl buffer, citric acid buffer, boric acid buffer, PIPES-NaOH buffer or imidazole buffer andl if necessary, it may be adjusted by adding thereto a surfactant such as sodium deoxycholate or serum albumin. Then, the enzyme solution containing phospholipase C thus adjusted and the liquid to be inspected are mixed and glycero-3-phosphate is produced from phosphatidyl glycerol in the liquid to be inspected with the consumption of water. The mixing ratio of the both is not particularly limited, and they may be mixed at such ratio that preferably about 1 - 100 units of phospholipase C is contained per one test of the liquid to be inspected. The reaction temperature may be about 37C and the reaction time may be what is sufficient for the liberation of glycero-3-phosphate,usually not less than 5 minutes, preferably not less than 10 minutes. Then, glycero-3-phosphate liberated by the reaction is quantita-tively measured. From this value of quan~itative ~easure-., , ~ i, 2136~

~ 6 -ment, the value of phosphatidyl glycerol in the liquid to be inspected is obtained.

For the quantitative measurement of glycero-3-phos-phate, various known methods for the quantitative chemicalmeasurement or those using enzymes may be employed. Pre-ferably, an enzymatic method ~or the quantitative measure-ment wherein one species or more of enzymes whose substrate is glycero-3-phosphate is employed for action and the de-tectable change of enzyme action in the reaction is quanti-tatively measured is simple and convenient. For instance, the GPO type method for quantitative measurement wherein glycerophosphate oxidase (GPO) is allowed to act on the liberated glycero-3-phosphate so that the oxygen in the re action mixture is consumed to produce h~drogen peroxide is particularly simple and convenient.

Further, in this GPO type method for the quantitative measurement, the quantity of oxygen consumed in the reac-tion liquid is measured with an oxygen electrode; thequantity of hydrogen peroxide produced is measured with a hydrogen peroxide electrode as the electrical change. The quantity of oxygen or h~drogen peroxide can be measured by an enzyme electrode wherein an oxygen electrode or a hydrogen peroxide electrode and immobilized enzyme are assembled.
Immobilized enzymes may be formed by various means such as inclusion, adsorption and bonding; for instance, GPO or GPO
and phospholipase C are immobilized by a known immobiliza-tion means such as a method wherein GPO or GPO and phos-pholipase C are included and immobilized with acrylamide;or a method wherein GPO or GPO and phospholipase C are blended with a protein such as albumin and the proteins are cross linked with each other to cause immobilization; or a method wherein GPO or GPO and phospholipase C are included with collagen or fibroin or are covalent-bonded with them; or a l~U2~;9 method wherein GPO or GPO and phospholipase C are immobiliz-ed by the adsorption onto a porous organic polymer resin or by the covalent bonding with the same, a method wherein GPO
or GPO and phospholipase C are included and immobilized us-ing a photosetting resin. The form of the immobilizedemzyme may be the one wherein the immobilized enzyme is processed into a membranelike, fibrous, granular or tubular shape which is preferable in use for an enzyme electrode to be assembled into an oxygen electrode or a hydrogen peroxide electrode and the immobilized enzyme may be used as an enzyme electrode wherein this immobilized enzyme is incorporated into the detecting part of the electrode to be used so that the amount of the effective enzyme used for the quantitative measurement by an electrical means is markedly small.

Further, the quantitative measurement of hydrogen per-oxide may be carried out by using an indicator composition which produces a detecta~le material by the reaction with hydrogen peroxide. As the indicator composition,there usually are used the compositions whose change may be quantitatively - measured by a spectroscopic means, for instance, a colora-tion reagent composition whose color change occurs in the visible range, a fluorescence reagent composition which fluoreSces by ultraviolet rays irradiation or a photogenic reagent composition which gives forth light. For example, as a coloration reagent composition, a material containing a substance having peroxidase action and chromogen may be used. As the substance having the peroxidase action, usual-ly the peroxidase originated from horseradish is often usedand, as the chromogen, usually the combination of an elec-tron acceptor and a phenolic compound is often used.
Further, as an electron acceptor, such compound is used as, for example, 4-aminoantipyrine, 2~hydrazinobenzothiazole, 3-methyl-2-benzothiazolone hydrazine, 2-aminobenzothiazole ~,' ~ ~Z~Z869 . _ ~

or the like. As a phenolic compound, such compound is used as, for example~ phenol, 3-methyl-N-ethyl~N-(B-hydroxyethylJaniline , 3,5-xylenol, N,N-dimethylaniline, N,N-diethylaniline or the like.

As luminous substrates in a fluorescence reagent com-position or a photogenic reagent composition, there may be illustrated various known ones, for example, bis(2,4,6-trichlorophenol)oxalate, phenylthiohydantoin, homovanillic acid, 4-hydroxyphenylacetic acid, vanillylamine, 3-methoxy-tyramine, phloretic acid, hordenine, luminol monoanion, lucigenine, wa~in and the like. Each of them may be, if necessary, used together with an electron acceptor and/or a substance having peroxidase action for the quantitative measurement of hydrogen peroxide.

There is no particular limitation on the quantity of the enzyme reagent or the chromogen used. For instance, there may be used per one test usually not less than 0.05 unit, preferably 0.1 - 200 units of GPO and usually not less than 0.05 unit, preferably 0.1 - 500 units of per-oxidase. ~lso there may be used a solution so adjusted by distilled water or weakly acidic or weakly alkaline buffer solution that the concentration of an electron ac-ceptor or a phenolic compound i5 usually not less than 0.1 mM. These reagents may be used separately from the enzyme solution of phospholipase C mentioned above or may be blended therewith, or further, these reagents may be formed into such composition for quantitative measurement as a freeze-dried composition or an integrate~ laminate by ap-plying them onto filter papers, films or the li~e.

The GPO type method for the quantitative measurement thus composed shows a high sensitivity to the glycero-3-phosphate in the liquid to be inspected and, since v ~ZV'~136~

g it is not affected by the impurity in the liquid to be inspected, it is an excellent method whereby an accurate measurement can be carried out.

The GP0 used for this GPO type method may be any enzymes as far as it plays the role of a catalyst in the reaction to produce dihydroxyacetone phosphoric acid and hydrogen peroxide from glycero-3-phosphate and oxy-gen, for example, Streptococcus genus, Lactobacillus genus, Lenconostoc genus, glycerophosphate oxidase producing bacteria which belong to Pediococcus genus (Japanese unexamined patent publication No. 7289211978), a enzyme obtained from the culture of glycerophosphate oxidase-producing bacteria which belongs to Aerococcus genus (Aerococcus viridans IFO 12219 strain and IFO 12317 strain, Japanese unexamined patent publication No. 15746/1980) and enzymes sold on the market.

As another enzymatic method for the quantitative measurement of glycero-3-phosphate, glycerophosphate dihydrogenase which catalyses the reac~ion to produce dihydroxyacetone phosphoric acid and reduced NAD
from glycero-3-phosphate and nicotine adenine dinucleotide (NAD) is allowed to act on glycero-3-phosphate liberated in the solution to be tested in the presence of NA~ to produce dihydroxyacetone phosphoric acid and re-duced NAD and then this reduced NAD is quan-titatively measured so that the quantity of glycero-3-phosphate may be determined. When the quantitative measurement of this reduced NAD is conducted, such method may be usually used as the measurement of absorbance by a wave length of about 340 nm or, after it is aIlowed to develop color by a water-soluble tetrazolium salt such as 3-(4,5-dimethyl)-2-thiazolyl-2H-tetrazolium bromide,

2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl-2H-tetrazolium 12~)Z8~9 chloride, 3,3'-(3,3'-dimethoxy-4,4'-biphenylene)-bis[2-(p-nitrophenyl-5-phenyl-2H-tetra~olium chloride)](Nitrotetra-zolium Blue) or 2,6-dichlorophenolindophenol in the pre-sence of diaphorase or phenazine methosulfate, the color may be measured in accordance with absorbances using their special absorption wave lengths.

In addition, as to these enzymes, reagents and the like, it is simple and convenient to use those sold on the market and the quantity to be used may be appropriately designed. Also, if necessary, a surfactant or a stabilizer may be used and they may ~e processed into various composi-tions.

In addition, as the liquid to be inspected which is the object in the present invention, any sample may be used as far as it contains phosphatidyl glycerol, for in-stance, body fluids such as amniotic fluid sampled.

When amniotic fluid is the liquid to be inspected, the sample obtained may be used as it is or when the content of phosphatidyl glycerol is remarkably small 7 the sample may be concentrated for instance, 5 - 6 ml of liquor amniotic fluid sampled is extracted with 15 - 18 ml of chloroform-methanol (2:1), the chloroform layer is collected by cen-trifuging at 2000 rpm and it is evaporated to dryness in nitrogen gas to obtain the total lipid. Then, after dis-solving this total lipid in a definite amount of 1%"Triton X-lOO"solution, the obtained solution may be used as the sample of amniotic fluid. In this way, each enzyme solu-tion of phospholipase C and other enzymes such as used in GPO type methods of quantitative measurement mentioned above and other reagents may be allowed to act on the solu-tion to be inspected successively or simultaneously. In this case, there is no particular limitation on the use * Trademark for octylphenoxy polyethoxy ethanol; it is a nonionic surfactant.

12{)Z869 ratio of the liquld to be inspected to the enzyme reagent and the like and usually, about 0.1 - 3 ml of the solution of enzyme reagent or the like is used for 0.01 ml - 1 ml of the liquid to be inspected. As the reaction condition, it is preferable to conduct the reaction at about 37C and, as the reaction time, any length may be selected as far as the reaction is completely terminated; usually the reaction is continued for not less than 5 minutes, preferably not less than 10 minutes. Also as the reaction medium, water or a weakly acidic to weakly alkaline buffer as a solvent of each rea~ent and the like is used.

Thus, by the quantitative measurement of the liquid to be inspected, phosphatidyl glycerol may be directly and quantitatively measured in a very short time down to an extremely low concentration. In addition, no complicated operation is needed and the operation may be carried out at the normal temperature. Consequently, this is a good method for the quantitative measurement.
Also there is no particular limitation to the method for the measurement of the activity of phospholipase C and GPO used in the practical examples mentioned later in the present invention and they may be illustrated by examples as follows:

(a] Method for the measurement of the activity of phospholipase C
0.1 ml of 4% phosphatidyl choline(separated from egg yolk and purified) solution, 0.3 ml of 0.1 M Tris-hydro-chloric acid buffer (pH 8.5) and 0.1 ml of 20 mM aqueous solution of MgC12 are mixed together and to this is added 0.1 ml of enzyme solution containing phospholipase C.
The mixture is allowed to react at 37C for 15 minutes and the reaction is suspended by adding 0.8 ml of 3.6%

~L2~;~8~9 trichloroacetic acid and 0.1 ml of 5.5~ serum albumin and the reaction mixture is allowed to stand in water bath for 20 min.,then filtered by Toyo filter paper (No. 5B).
0.5 ml of the filtrate is taken into a Kjeldahl flask, 0.55 ml of 60% HC104 is added to decompose for 2 hours at 170GC, and liberated inorganic phosphor is allowed to develop color by addition of 0.5 ml of amidol sulfite testing solution, 0.25 ml of 3.3% ammonium molybdate and 3.2 ml of water. After being left to stand at room temperature for 20 min.,the absorbance is measured at OD 650 m~. The activity of enzyme which extricates 1 ~mole of inor~anic phosphor per 15 minutes is defined as 1 unit (U).

(b3 Method for the measurement of the activity of GPO
0.2 M Tris-hydrochloric acid buffer (pH 8.0) 0.2 ml peroxidase (0.5 mg/ml, 45 U/ml) 0.1 ml 0.3% tW/V) 4-aminoantipyrine 0.1 ml 0.1 M DL-glycero-3-phosphate 0.1 ml 0.2% (V/V) N,N-dimethylaniline 0.2 ml Distilled water 0.3 ml One mililiter of the reaction liquid having the com-position mentioned above is charged in a small test tube and preheated at 37C for 3 minutes. To this is added 20 ~1 of enzyme solution containing GPO and the mixture is allowed to react for 10 minutes. Then, the reaction is suspended by the addition of 2.0 ml of 0.25% (W/V) sodium laurylbenzene sulfonate and the absorbance of the product is measured at a wave length of 565 nm.

The activity of the enzyme is calculated in accordance with the following equation:

Activity of enzyme (U/ml) = ( 6 o ) x ( 10 ) .~
, .

l~Z86g wherein ~A shows the absorbance for 10 minutes at a wave length of 565 nm.

The ~mbodiment of the present invention will be men-S tioned in the following examples. However, the present in-vention is not intended to be limited by them.

Fig. 1 and Fig. 2 indicate the quantitative measure-ment curve of phosphatidyl glycerol.
Example 1 0.2 M dimethylglutarate-NaOH buffer (pH 7.5) 0.2 ml 10 mM CaC12 0.2 ml 10% sodium deoxycholate 0.05 ml 1% Bovine serum albumin 0.1 ml Solution containing 40 U/ml phospholipase C
and 50 U/ml GPO 0.1 ml 45 U/ml peroxidase 0.1 ml 0.3% 4-aminoantipyrine 0.2 ml 0.3% 3-methyl-N-ethyl-N-~-hydroxyethyl~aniline 0.2 ml Distilled water 0.~5 ml Total 2.0 ml 2.0 ml of reaction composition for quantitative measure-ment of phosphatidyl glycerol having the above compositionis prepared and thereto is added 50 ~1 of solution to be in-spected which contains various phosphatidyl glycerol ~con-tent of phosphatidyl glycerol being 20 n moles ~ 100 n moles~
and the mixture is reacted at 37C for 15 minutes and then the color development of the pigment produced after reaction is measured by absorbance at the wave length of 550 nm (OD
550).

The results are as shown by Fig. 1. A satisfactory tendency of linear relation was obtained as against the , ~;~0~8~g content of phosphatidyl glycerol of the inspected solution.

Example 2 0.2 M dimethyl glutarate-NaOH buffer (pH 7-5) 0 2 ml 10% sodium deoxycholate 0.05 ml Solution containing 20 U/ml phospholipase C
and 50 U/ml GPO 0.1 ml ~5 U/ml peroxidase 0.1 ml 0.3% 4-aminoantipyrine 0.2 ml 0.3~ phenol 0.2 ml 1% Bovine serum albumin 0.1 ml Distilled water 1.05 ml Total 2.0 ml 2.0 ml of reaction composition for quantitative measurement of phosphatidyl glycerol having the aforesaid composition is prepared, to such solution is added 50 ~l of the solution to be inspected containing various phos-phatidyl glycerol (content of phosphatidyl glycerol being10 n moles ~100 n moles), the mixture is allowed to react for 15 minutes at 37C and the color shade of the pigment produced after reaction was measured by absorbance at wave length of 500 nm (OD 500).
The results are as shown in Fig. 2, where a quantita-tive measurement curve having satisfactory linearity against the content of phosphatidyl glycerol was obtained.

Claims

1. A method for the quantitative measurement of phos-phatidyl glycerol, in a liquid containing lipid components which is to be inspected, which method comprises the sequential steps of (a) liberating glycero-3-phosphate by allowing phos-pholipase C to act on phosphatidyl glycerol, (b) allowing glycerophosphate oxidase to act on glycero-3-phosphate, and then (c) measuring the quantity of oxygen consumed or hydrogen peroxide produced in the reaction.

2. The method for the quantitative measurement according to Claim 1, wherein the liquid to be inspected is a body fluid.

3. The method for the quantitative measurement accord-ing to Claim 1, wherein the quantitative measurement of oxygen is made by an electrochemical method using an oxygen electrode.

4. The method for the quantitative measurement accord-ing to Claim 1, wherein the quantitative measurement of hydrogen peroxide is carried out by an electrochemical method using an hydrogen peroxide electrode.

5. The method for the quantitative measurement accord-ing to Claim 1, wherein the quantitative measurement of hydrogen peroxide is carried out by using an indicator com-position which changes into a detectable product produced by the reaction with hydrogen peroxide.

6. The method for the quantitative measurement accord-ing to Claim 5, wherein the indicator composition is a color developing composition, fluorescent composition or luminating composition.

7. The method for quantitative measurement according to Claim 6, wherein the color developing composition con-tains a substance having peroxidase action and a dye pre-cursor.

8. The method for quantitative measurement according to Claim 7, wherein the dye precursor is 4-aminoantipyrine and phenol.

9. The method for quantitative measurement according to Claim 7, wherein the dye precursor is 4-aminoantipyrine and 3-methyl-N-ethyl-N-(.beta.-hydroxyethyl) aniline.

10. The method for quantitative measurement according to Claim 8, wherein the measurement consists of an absorbance measurement at a wave length of about 500 nm.

11. The method for quantitative measurement according to Claim 9, wherein the measurement consists of an absorbance measurement at a wave length of about 550 nm.