GB2111677A

GB2111677A - Method for quantitative measurement of phosphatidyl glycerol

Info

Publication number: GB2111677A
Application number: GB08233333A
Authority: GB
Inventors: Shigeyuki Imamura; Hideo Misaki
Original assignee: Toyo Jozo KK
Current assignee: Toyo Jozo KK
Priority date: 1981-12-16
Filing date: 1982-11-23
Publication date: 1983-07-06
Also published as: IT8224763A0; DE3246090A1; DE3246090C2; GB2111677B; CA1201047A; FR2518117B1; JPS58107199A; FR2518117A1; IT8224763A1; IT1155384B

Abstract

A method for quantitative measurement of phosphatidyl glycerol in a liquid to be inspected, especially in a body fluid such as amniotic fluid according to which only the phosphatidyl glycerol can be simply, conveniently and accurately measured in a short time. This method comprises allowing an enzyme such as phospholipase D to act on the phosphatidyl glycerol in the liquid to liberate glycerol, which is quantitatively measured preferably by GK-GPO type method.

Description

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

Recently, the number of deaths of light body weight newborn babies has been descreased owing to the improved management of newborn babies and the like. However, the respiratory distress syndrome (RDS) of a newborn baby in the perinatal period is still a big problem and the deaths due to it amount for a high percentage of deaths. It is regarded that RDS occurs owing to the lack of pulmonary surfactant. This material is important to prevent alveoli pulmonum which expanded immediately after the birth from contraction. The lack of this pulmonary surfactant brings about atelectasis in respiration and occurrence of RDS. Consequently, if the occurrence of RDS can be early estimated so that the remedy of the newborn baby can be early started, the occurrence of RDS can be prevented or it can be restrained to a mild case.

Accordingly, the measurement of this pulmonary surfactant is necessary.

The means are roughly classified into the method for the measurement wherein the physical property of pulmonary surfactant is utilized and one wherein compositive components of pulmonary surfactant are biochemically resolved. The latter method has been carried out by obtaining the ratio of each lipid such as that of phosphatidyl choline to sphingomyelin, that of phosphatidyl glycerol to phosphatidyl inositol or the quantitative measurement of dipalmitoyl phosphatidyl choline by means of the thin-layer chromatography (Am. J. Obstet. Gynecol., 440: 109 (1971); Am. J. Obstet, Gynecol., 613:125(1976); Am. J. Obstet. Gynecol., 894:133 (1979); Obstet. 8 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 existence of phosphatidyl glycerol is an important factor for the occurrence of RDS. However, the quantitative measurement of phosphatidyl glycerol was very difficult because the quantity thereof in existence is one tenth as little as that of phosphatidyl choline.

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 component by the centrifugal separation from the liquor amnii, lipid components are extracted from the supernatant, the components of this extract are separated by the thin-layer chromatography, each phospholipid is quantitatively measured, the ratios of various phospho-lipids are obtained and the quantity of phosphatidyl glycerol is obtained indirectly from the total phospholipid value preliminarily obtained by the quantitative measurement of phosphate according to the wet combustion method [Am. J. Obstet. Gynecol., 613:125 (1976); Am. J.

Obstet. Gynecol., 1079; 135 (1979); Am. J. Obstet. Gynecol., 899:133 (1979); Am. J.

Obstet, Gynecol., 440: 109 (1971)], or the method for the quantitative measurement wherein the high speed liquid chromatography is used [Journal of Chromatography, 277: 223 (1981)].

However, this method of thin-layer chromatography had disadvantages that it takes two to three days for the extraction 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 various defects that it is an indirect method for the measurement based on the ratios of relative quantities from spots and total phospholipid and it can not carry out the simultaneous treatment of many samples because of the thin-layer chromatography.

The present inventors carried out various studies about the method whereby, from the liquid containing various lipid components to be inspected such as liquor amnii, the quantitative measurement of only phosphatidyl glycerol can be simply, conveniently and accurately achieved in a short time.

As the results, they found quite unexpectedly that phospholipase D acts on phosphatidyl glycerol producing glycerol and phosphatidic acid and they achieved a satisfactory method for the quantitative measurement of only phosphatidyl glycerol by quantitatively measuring this glycerol produced by the reaction and extricated.

More preferably, there has been achieved a method whereby only phosphatidyl glycerol can be markedly satisfactorily measured quantitatively by allowing phospholipase D to act on the liquid to be inspected to produce glycerol, then allowing glycerolkinase to act on this glycerol in the presence of a phosphate donor such as adenosine triphosphate (ATP), further allowing glycerophosphate oxidase to act on said product and then measuring the quantity of oxygen consumed or hydrogen peroxide produced in the reaction.

The present invention has been achieved based on the finding mentioned above and it relates to a method for the quantitative measurement of phosphatidyl glycerol in the liquid to be inspected which comprises liberating glycerol by the action of an enzyme which plays a role of the catalyst in the reaction to produce glycerol and phosphatidic acid from phosphatidyl glycerol and water and then quantitatively measuring glycerol produced.Preferably, it relates to a method for the quantitative measurement of phosphatidyl glycerol in the liquid to be inspected which comprises the combination of the following processes (a), (b), and (c) and (d): (a) liberating glycerol by allowing phospholipase D to act on phosphatidyl glycerol, (b) producing glycero-3-phosphate by allowing glycerolkinase to act on glycerol in the presence of a phosphate donor, (c) allowing glycerophosphate oxidase to act on glycero-3-phosphate, and then (d) measuring the quantity of oxygen consumed or hydrogen peroxide produced in the reaction.

In the present invention, it is advantageous to prepare each reagent in an adjusted kit for the quantitative measurement and the operation for the quantitative measurement can be conducted at the room temperature of about 37"C and, in addition, in a very short time for the reaction.

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 conveniently in a short time, many samples can be simultaneously measured. Thus, the present invention provides a useful method for the quantitative measurement of phosphatidyl glycerol.

Fig. 1 shows the calibration curve in Example 1 of the present invention, Fig. 2 shows the results of quantitative measurement of phosphatidyl glycerol when various amniotic fluid components sampled were used and Fig. 3 shows the calibration curve in Example 3 of the present invention.

At first, there can be illutrated phospholipase D as the enzyme which plays a role of the catalyst in the reaction of phosphatidyl glycerol and water to produce glycerol and phosphatidic acid in the present invention.

This phospholipase D has been known as an enzyme which is a catalyst in the reaction to produce each 1 mole of phosphatidic acid and 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 one obtained by the extraction of phospholipase D-containing cells or an enzyme reagent sold on the market, for example, a microorganism-originated enzyme obtained from the culture of a phospholipase D-producing bacterium which belongs to a Streptomyces genus [Streptomyces hachijoensis A-l 43 strain (FERM-P No. 1 329); Japanese examined patent publication No. 39918/1977, Streptomyces chromofussusA-0848 strain (FERM-P No.

3519); Japanese unexamined patent publication No. 130984/1977], etc. and enzyme reagents of phospholipase D sold on the market.

The quantity of phospholipase D to be used should be appropriately modified and designed according 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 D of usually not less than 0.1 unit, preferably about 1-10 units. In addition, this phospholipase D is preferably used after it is dissolved in a buffer such as weak acidic to weak alkali Tris-HCI buffer, citric acid buffer, boric acid buffer, PlPES-NaOH buffer or imidazole buffer and, if necessary, it may be adjusted by adding thereto a non ironic surfactant such as Triton X-1 00 or serum albumin.Then, the enzyme solution containing phospholipase D thus adjusted and the liquid to be inspected are mixed and glycerol and phosphatidic acid are 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 a ratio so that preferably about 1-10 units of phospholipase D is contained per one test of the liquid to be inspected. The reaction temperature may be about 37"C and the reaction time may be what is sufficient for the liberation of glycerol, usually not less than 5 minutes, preferably not less than 10 minutes. Then, glycerol liberated by the reaction is quantitatively measured. From this value of quantitative measurement, there can be obtained the value of phosphatidyl glycerol in the liquid to be inspected.

As the methods for the quantitative measurement of glycerol, there can be utilized various known methods for the quantitative chemical measurement and those using enzymes. Preferably, an enzymatic method for the quantitative measurement wherein one species or more of enzymes whose substrate is glycerol are allowed to act on the glycerol and the detectable change of enzyme action in the reaction is quantitatively measured is simple and convenient.

For instance, as the method for the measurement of glycerol, the GK-GPO type method wherein glycero-3-phosphate and adenosine diphosphate (ADP) are produced by the action on glycerol in the presence of a phosphate donor such as adenosine triphosphate (ATP) and glycerolkinase (GK), then glycerophosphate oxidase (GPO) is allowed to act on glycero-3phosphate so that the oxygen in the reaction liquid is consumed to produce hydrogen peroxide is particularly simple and convenient.

In this GK-GPO type method, quantity of oxygen consumed in the reaction liquid is measured with an oxygen electrode or quantity of hydrogen peroxide produced is measured with a hydrogen peroxide electrode as the electrical change. The quantity of oxygen or hydrogen peroxide can also be measured by an enzyme electrode in which an oxygen electrode or hydrogen peroxide electrode and an immobilized GPO are assembled. The GPO immobilized may be in the form of membranes, fibers, pellets or tubes in order to facilitate having the enzyme installed at the detecting part of the oxygen or hydrogen peroxide electrode. In this embodiment, the electrode can work with a small amount of enzyme. Phospholipase D or GK employed may also be similarly or appropriately immobilized.For immobilization, any processes are available, such as, for example, polymerized inclusion method using, for example, acrylamide; immobilizing method using crosslinking agent for mixture with a protein such as albumin; inclusion method using collagen or fibroin; covalent-bonding method using collagen or fibroin; adsorption onto or covalent-bonding with a porous orgainc polymer resin; inclusion method using a photosetting resin, or covalent-bonding method using an aminated glass.

Further, as another means for quantitative measurement of hydrogen peroxide, the quantitative measurement may be carried out by a spectrophotometric methods using an indicator which is produced by the reaction with hydrogen peroxide. As the indicator, there are usually used that wherein change can be quantitatively measured by a spectrophotometric means, for instance, a coloration reagent wherein color change occurs in the visible range, a fluorescence reagent composition which fluorescents or a luminescent reagent composition which is luminescent by ultraviolet rays irradiation. For example, as a coloration reagent composition, there is used a material containing a substance having peroxidase action and a chromogen.As the substance having the peroxidase action, usually the peroxidase originated from horseradish is often used and, as the chromogen, usually the combination of an electron acceptor and a hydrogen donor is often used. Further, as an electron acceptor, there is used, for example, 4-aminoantipyrine, 2hydrazinobenzothiazole, 3-methyl-2-benzothiazolone hydrazine, 2-aminobenzothiazole or the like.

As a hydrogen donor, there is used, for example, phenol, 3-methyl-N-ethyl-N-(ss-hydroxyethyl)an- iline, 3,5-xylenol, N,N-dimethylaniline, N,N-diethylaniline or the like.

As luminous substrates in a fluorescence reagent composition or a luminescent reagent composition, mention may be made of various known ones, for example, bis(2,4,6-trichlorophenol)oxalate, phenylthiohydantoin, homovanillic acid, 4-hydroxyphenylacetic acid, vanillylamine, 3-methoxy-ethylamine, phloretic acid, hordenine, luminol monoanion, lucigenine 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 0.01 unit, preferably 0.05-100 units of GK, 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 peroxidase. Also there may be used a solution so adjusted that the concentration of an electron acceptor or a hydrogen donor is usually not less than 0.1 mM and a solution so adjusted that the concentration of a phosphate donor such as ATP or cytosine triphosphate is not less than 0.1 mM. More preferably, in order to raise up the enzyme activity of GK, a water-soluble salt to release magnesium ions, for example, magnesium chloride is used and it may be dissolved in distilled water or a weakly acidic to weakly alkali buffer.These reagents may be used separately from the enzyme solution of phospholipase D mentioned above or may be blended therewith, or further, these reagents may be formed into an integrated laminate by applying them onto filter papers, films or the like.

The GK-GPO type method for the quantitative measurement shows a high sensitivity to the glycerol produced in the liquid to be inspected and, since 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.

As GK used for this GK-GPO type method for the quantitative measurement, there may be used any enzymes as far as they produce glycerol-3-phosphate and ADP from glycerol and ATP, for example, enzymes originated from the liver of a rat or a pigeon [J. Biol. Chem., 211, 951 (1954) and Biochem. Z., 329, 320 (1 957)], those originated from microorganisms which are enzymes obtained from the culture medium of GK-producing microorganisms such as Candida mycoderma or Streptomyces canus A-2408 (FERM-P No. 4977) [Biochem. Z., 333, 471 (1961), Japanese unexamined patent publication No. 162987/1980], etc., and other ones sold on the market.

Furthermore, as GPO, there may be used any enzymes as far as they play a role of a catalyst in the reaction to produce dihydroxyacetonephosphate and hydrogen peroxide from glycero-3phosphate and oxygen, for example, Streptococcus genus, Lactobacillus genus, Leuconostoc genus, glycerophosphate oxidase producing bacteria which belong to Pediococcus genus (Japanese unexamined patent publication No. 72892/1978), an enzyme obtained from the culture of glycerophosphate oxidase-producing bacteria which belong to Aerococcus genus (Aerococcus viridans IFO 1221 9 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 glycerol, glycerolkinase is allowed to act on glycerol in the presence of a phosphate donor such as ATP to produce glycero3-phosphate and ADP, then glycerophosphate dehydrogenase is allowed to act on this glycero 3-phosphate in the presence of nicotine adenine dinucleotide (NAD) to produce dihydroxyacetone and reduced NAD and then this reduced NAD is quantitatively measured so that the quantity of glycerol may be determined.When the quantitative measurement of this reduced NAD is conducted, there may be used usually the measurement of absorbance at about 340 nm or, after it is allowed to give forth color using a water-soluble tetrazolium salt such as 3-(4,5 dimethyl)-2-thiazolyl-2 H-tetrazoliu m bromide, 2-(p-iodophenyl)-3-(p-nitro-phenyl)-5-phenyl-2 H-tetrazolium chloride, 3, 3'-(3, 3'-dimethoxy-4, 4'-biphenylene)-bis[2-(p-nitrophenyl-5-phenyl-2 H-tetra- zolium chloride] (Nitrotetrazolium Blue) or 2,6-dichlorophenol-indophenol in the presence of diaphorase or phenazine methosulfate, the color may be measured in accordance with absorbances using their special absorption wave lengths.

As a method different from the above-mentioned, there can be illustrated a means for the quantitative measurement wherein glycerol dehydrogenase is allowed to act on glycerol in the presence of NAD to produce dihydroxyacetone and reduced NAD and this reduced NAD is quantitatively measured.

Further, there can be illustrated a means which is based on a procedure wherein glycerolkinase is allowed to act on glycerol in the presence of a phosphate donor such as ATP to produce glycerophosphate and ADP, pyruvate kinase is allowed to act on this ADP in the presence of phosphoenol-pyruvate to produce pyruvic acid and ATP and this pyruvic acid is quantitatively measured.As the methods for the quantitative measurement of this pyruvic acid, there are used means such as one wherein a hydrazine compound such as 2,4-dinitrophenylhydrazine is allowed to act on pyruvic acid to make it give forth color and the color is measured in accordance with the absorbance using a wavelength of about 440 nm, one wherein lactate dehydrogenase is allowed to act on pyruvic acid in the presence of reduced NAF to produce oxidized NAD and lactic acid and the decreased quantity of reduced NAD in the reaction system is measured by such means as an absorbance measurement at a wave length of about 340 nm, one wherein pyruvate oxidase is allowed to act on pyruvic acid and the quantity of oxygen consumed or that of hydrogen peroxide in the reaction system is measured as an electrical change and one wherein pyruvic acid is measured by a spectrophotometric means using an indicator composition for hydrogen peroxide.

Other than those mentioned above, there may be used a method for the quantitative measurement of glycerol wherein glycerol oxidase is allowed to act on glycerol and the quantity of oxygen consumed or that of hydrogen peroxide or glyceraldehyde produced in the reaction system is measured.

As 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.

As the liquid to be inspected which is the object in the present invention, there can be illustrated any sample as far as it contains phosphatidyl glycerol, for instance, body fluids such as amniotic fluid sampled.

When amniotic fluid is the liquid to be inspected, it is preferable to use the sample obtained from the region containing phosphatidyl glycerol; 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 centrifuging at 2000 rpm and it is evaporated to dryness in nitrogen gas to obtain the total lipid. Then, after this total lipid is dissolved in a definite amount of 1% Triton X-100 solution, an enzyme solution of phospholipase D and, for instance, each enzyme based on the method for the quantitative measurement of GK-GPO mentioned above and other reagents may be allowed to act on the total lipid successively or simultaneously.In this case, there is no particular limitation on the use ratio of the liquid to be inspected to the enzyme reagent and the like and usually, about 0. 1-3 ml of the 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 37"C 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, there is used water or a weakly acidic to weakly alkali buffer as a solvent of each reagent and the like.

Thus, by the quantitative measurement of the liquid to be inspected, phosphatidyl glycerol can be directly and quantitatively measured in a very short time and the value of phosphatidyl glycerol as small as 2.0 n moles, that is the value of phosphatidyl glycerol of 0.03 mg/dl when the liquor amnii of 5-6 ml is used, can be measured. This means that the quantity of phosphatidyl glycerol in an extremely low concentration compared to the value of 0.2 mg/l thereof which is the critical level for the occurrence of RDS can be measured. In addition, no complicated operation is needed and the operation can be carried out at the normal temperature. Consequently, this is a good method for the quantitative measurement.

Also there is no particular limitation on the method for the measurement of each activity of phospholipase D, GK and GPO used in practical examples mentioned later in the present invention and there can be illustrated following methods: (a) Method for the measurement of the activity of phospholipase D To 0.1 ml of 4% yolk phosphatidyl ethanolamine solution are added and mixed 0.8 ml of 0.05 M Tris-hydrochloric acid buffer (pH 7.5) 0.3 ml of 1% Triton X-100, 0.3 ml of water and 0.2 ml of 10 mM aqueous solution of CaCI2 and the mixture is subjected to ultrasonic treatment for 10 minutes. To this are added 0.2 ml of ethyl ether and 0.1 ml of enzyme solution. The mixture is allowed to react at 37"C for 20 minutes and the reaction is suspended by adding 0.3 ml of 20% trichloroacetic acid.The reaction liquid is washed twice with 4 ml of ethyl ether, 0.5 ml of 0.2 N citrate buffer (pH 5.0) is added to 1 ml of the water layer, 0.1 ml of 2% aqueous solution of SnCI2 and 2 ml of 2% ninhydrin solution are added thereto, the mixture is heated at 1 00 C for 1 5 minutes to extricate ethanolamine, which is allowed to give forth color by the ninhydrin reaction and the absorbance is measured at OD 570 my. The activity of enzyme which extricates 1 /19 of ethanolamine per minute is defined as 1 unit (U).

(b) Method for the measurement of the activity of GK 0.2 M Tris-hydrochloric acid buffer (pH 9.0) 0.4 ml 0.1 M glycerol 0.05ml 10 mM ATP 0.1 ml 10 mM MgCI2 0.1 ml 0.25% Nitrotetrazolium Blue 0.1 ml 1% Bovine serum albumin 0. 14ml 10 mM NAD 0.1 ml 0.05% phenazine methosulfate 0.01 ml Glycerophosphate dehydrogenase (manufactured by Boehringer Sohn AG, 2 mg/ml, 65 U/mg) 5 ILl One mililiter of the reaction mixture each having a composition metnioned above is preincubated at 37"C for 5 minutes. To this is added 50 jul of a solution containing GK (10 mM phosphate buffer containing 10 mM of glycerol, appropriately diluted at pH 7.5). The mixture is allowed to react at 37"C for 10 minutes.Then, after the reaction is suspended by the addition of 0.1 N HCI, the color given forth is measured at a wave length of 550 nm to obtain the absorbance (A 550 nm). One unit of GK is defined as the activity to produce 1 ,umole of glycero-3-phosphate per minute. The equation for the calculation is as follows:: AA X dilution ratio 1 AA U/ml = X --=---- X dilution ratio O.05X10 4 2 (c) 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 (W/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 mixture having the composition mentioned above is charged in a small test tube and preincubated at 37"C for 3 minutes. To this is added 20 ILl of enzyme solution 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: AA 50 Activity of enzyme (U/ml) = ( ) x (-) 0.6 10 wherein AA shows the absorbance for 10 minutes at a wave length of 565 nm.

Hereafter the embodiment of the present invention will be mentioned with practical examples.

However, the present invention is never limited by them.

Example 1 [Preparation of calibration curve) A solution having the following composition was prepared.

(1) solution containing phosphatidyl glycerol: There was prepared a 1.0 ILmole/mi phosphatidyl glycerol solution containing 1.0% Triton X-100 (The concentration of phosphatidyl glycerol was determined in accordance with the method for the quantitative measurement of phosphate).

(2) Ca+ ±buffer reaction medium: 0.1 M Tris-HCI buffer (pH 8) 8.0 ml 1 M CaC12 0.2 ml Distilled water 1.8 ml Total 10 ml (3) Phospholipase D enzyme solution: One hundred mililiters of a solution consisting of 121.1 mg of Tris containing phospholipase D of 35 U/ml (0.6 mg/ml), 50.0 mg of cow serum albumin, 0.5 ml of 20% Triton C-100 and water.

(4) 60 mM EDTA (5) 40 mM MgCI2 solution containing 160 mM ATP: There were mixed 1.0 ml of 0.2 M ATP, 0.10 ml of 0.5 M MgCI2 and 0.15 ml of distilled water so that there was obtained 1.25 ml of the solution.

(6) GK enzyme solution: 10 mM Tris-HCI buffer containing 2 mg/ml of GK (pH 8).

(7) GPO enzyme solution: There was prepared 10 ml of GPO enzyme solution consisting of 10 mg of cow serum albumin containing 3 mg/ml of GPO, 560.75 mg of (NH4)2SO4, 1.0 ml of 0.1 M Tris-HCI buffer (pH 8) and 9 ml of distilled water.

(8) Indicator composition liquid containing GPO: 4-aminoantipyrine 0.24 ml aqueous solution of phenol (10 mg/ml) 0.16 ml Triton X-100 (1.25%) 0.8 ml Tris-HCI buffer (0.5 M, pH 8) 1.6 ml Peroxidase (90 U/ml distilled water) 0.8 ml GPO enzyme solution (3 mg/ml GPO) 0.8 ml Distilled water 15.6 ml Total 20 ml To each of O ml-0.10 ml of the solution containing phosphatidyl glycerol (PG) mentioned above was added 1% Triton X-1 00 so that the total amounted to 0.1 0 ml. To this mixture was further added 0.1 ml of Ca+ ±buffer. After the mixture was preheated at 37"C for 5 minutes, 0.05 ml of phospholipase D enzyme solution was added thereto and the mixture was allowed to react at 37"C for 10 minutes.After the reaction 0.04 ml of 60 mM EDTA was added and further 0.075 ml of 40 mM MgCI2 solution containing 160 mM ATP and 0.03 ml of GK enzyme solution were added thereto. The mixture was allowed to react at 37"C for 10 minutes.

After the reaction, 1.0 ml of the indicator composition liquid containing GPO was added to this and the mixture was allowed to react at 37"C for 20 minutes. Then, the absorbance due to the color given forth by the reaction was measured at a wave length of 500 nm (OD 500).

The results are as shown by Fig. 1. A satisfactory tendency of linear relation was obtained in a range from the point of 0.005 ml of PG-containing solution (PG content of 5 n moles) to that of 0.1 ml (PG content of 100 n moles).

Further, from this Fig. 1, the tendency of linear relation can be obtained when the PG content exceeded 2 n moles and the sensitivity was very sharp.

Example 2 In 1 8 ml of chloroform-methanol (2:1) was dissolved 5-6 ml of the amniotic fluid sampled.

The solution was treated by centrifugation at 2000 rpm. From three layers separated was taken out the chloroform layer, which was evaporated to dryness in nitrogen gas to obtain the total lipid. Then, this was dissolved in 0.1 ml of chloroform and the solution was charged into a silica gel column (0.3 g) wherein the neutral fat was removed by allowing 10 ml of chloroformmethanol (19:1) to flow and the phosphatidyl glycerol-containing fraction was recovered by allowing 10 ml of chloroform-methanol (2:1) to flow. Then, the solvent was removed and the residue was dissolved in 0.1 ml of 1% Triton X-100 to prepare the liquid to be inspected.

Further, 0.1 ml of Ca+ ±buffer was added to this liquid to be inspected and the mixture was preheated at 37"C for 5 minutes. Then, 0.05 ml of phospholipase D enzyme solution was added to this and the mixture was allowed to react at 37"C for 10 minutes. After the reaction, 0.04 ml of 60 mM EDTA was added to this followed by the addition of 0.075 ml of 40 mM MgCI2 solution containing 160 mM ATP and 0.03 ml of GK enzyme solution thereto and the mixture was allowed to react at 37"C for 10 minutes. Then, after the reaction, 1.0 ml of the indicator composition liquid containining GPO was added to this and the mixture was allowed to react at 37"C for 20 minutes.The absorbance due to the color given forth by the colored material produced by the reaction was measured at a wave length of 500 nm and the content of PG (mg/dl) in the amniotic fluid sampled was obtained from the calibration curve. The results were as shown in Fig. 2. In Fig. 2, X shows death case,

shows RDS occurrence case, A shows weak RDS occurrence case and

shows no RDS occurrence case.

Example 3 0.2 M Tris-HCI buffer (pH 7.5) 0.2 ml 10 mM CaCI2 0.2 ml 10mM MgCI2 0.2 ml 10% Triton X-100 0.05 ml 10 mM ATP 0.2 ml 20 U/ml phospholipase D, 5 U/ml GK and 50 U/ml GPO-containing liquid 0.1 ml 45 U/ml peroxidase 0.1 ml 0.3% 4-aminoantipyrine 0.2 ml 0.3% 3-methyl-N-ethyl-N-(fl- hydroxyethyl)aniline 0.2 ml Distilled water 0.55 ml Total 2.0 ml There was prepared 2.0 ml of the reaction mixture for the quantitative measurement of phosphatidyl glycerol having the composition mentioned above. To this was added 50 yl of the liquid to be inspected which is a liquid containing different amount of PG (PG content: 20 n moles-100 n moles). The mixture was allowed to react at 37"C for 15 minutes and then, the absorbance due to the color given forth by the colored material produced after the reaction was measured at a wave length of 550 nm (OD 550). The results were as shown in Fig. 3 and there was obtained a calibration curve which shows a satisfactory tendency of linear relation against the content of PG in the liquid to be inspected.

Claims

1. A method for quantitative measurement of phosphatidyl glycerol in a liquid to be inspected which comprises allowing an enzyme to act on the phosphatidyl glycerol to liberate glycerol and then quantitatively measuring glycerol produced.

2. A method for quantitative measurement according to Claim 1 wherein the enzyme is one which plays a role of catalyst in the reaction to produce glycerol and phosphatidic acid from phosphatidyl glycerol and water.

3. A method for the quantitative measurement according to Claim 2 wherein the enzyme which plays a role of the catalyst in the reaction to produce glycerol and phosphatidic acid from phosphatidyl glycerol and water is phospholipase D.

4. A method for the quantitative measurement according to Claim 1 wherein the liquid to be inspected is a body fluid.

5. A method for quantitative measurement of phosphatidyl glycerol in a liquid to be inspected which comprises the combination of following processes (a), (b), (c) and (d): (a) liberating glycerol by allowing phospholipase D to act on phosphatidyl glycerol, (b) producing glycero-3-phosphate by allowing glycerolkinase to act on glycerol in the presence of a phosphate donor, (c) allowing glycerophosphate oxidase to act on glycero-3-phosphate, and then (d) measuring the quantity of oxygen consumed or hydrogen peroxide produced in the reaction.

6. A method for the quantitative measurement according to Claim 5 wherein the phosphate donor is adenosine triphosphate.

7. A method for the quantitative measurement according to Claim 5 wherein, in the quantitative measurement of hydrogen peroxide, an indicator composition which reacts with hydrogen peroxide to produce a product which can be detected.

8. A method for the quantitative measurement according to Claim 7 wherein the indicator composition is a material containing a substance having a peroxidase action and a chromogen.

9. A method for the quantitative measurement according to Claim 8 wherein the chromogen is 4-aminoantipyrine and phenol.

1 0. A method for the quantitative measurement according to Claim 8 wherein the chromo gen is 4-aminoantipyrine and 3-methyl-N-ethyl-N-(ss-hydroxyethyl)aniline.

11. A method for the quantitative measurement according to Claim 9 wherein the measurement consists of an absorbance measurement at a wave length of about 500 nm.

1 2. A method for the quantitative measurement according to Claim 10 wherein the measurement consists of an absorbance measurement at a wave length of about 550 nm.

1 3. Method for quantitative measurement of phosphatidyl glycerol substantially as hereinbefore described with reference to the accompanying drawings.