FR2518117A1 - PROCESS FOR THE QUANTITATIVE MEASUREMENT OF PHOSPHATIDYLGLYCEROL - Google Patents

Abstract

METHOD OF QUANTITATIVE MEASUREMENT OF PHOSPHATIDYLGLYCEROL. THIS PROCESS CONSISTS OF REACTING AN ENZYME ON THE PHOSPHATIDYLGLYCEROL TO RELEASE GLYCEROL AND IN QUANTITATIVELY MEASURING THE GLYCEROL PRODUCED. PHARMACEUTICAL INDUSTRY.

Description

Method for the quantitative measurement of phosphatidyl

glycerol.

  The present invention relates to a

  new quantitative measurement of phosphatidyl-

  glycerol. Recently, the number of deaths among low birth weight newborns has been reduced due to

  improved treatment of newborns But the syndromes

  respiratory distress syndrome (RLS) in the perinatal period is still a serious problem and the deaths due to it represent a

  high percentage of all deaths.

  that the RMS occurs due to the default

  pulmonary surfactant This agent is important

  both to prevent contraction of the alveoli

  monaires that dilate immediately after the

  The defect of this pulmo- surfactant

  In consequence, if we can predict the onset of RLS early enough and if we can begin to treat the newborn soon enough, we can prevent the onset of RLS. appearance of the RMS, or it can be limited to a mild case. It is therefore necessary to measure this

pulmonary surfactant.

  The means available for this purpose can be

  can be broadly classified into the measurement method in which the physical property of the pulmonary surfactant is used, and the method in which the constituents of the pulmonary surfactant are biochemically determined. by measuring the ratio of lipids such as the ratio of phosphatidylcholine to sphingomyelin, that of phosphotidylglycerol to phosphatidylinositol or by the quantitative measurement of dipalmitoylphosphatidylcholine by means of the

  thin layer chromatography lAm J Obstet.

  Gynecol. 440: 109 (1971); Am J Obstet Gynecol, 613: 125 (1976); Am Obstet J Gynecol, 894: 133 (1979); Obstet & Gynecol, 295: 57 (1981);

  Am J Obstet Gynecol, 697: 138 (1980); etc. l.

  The results of these various examinations have recently made it possible

  to show that the existence of phosphatidylglycerol

  rol is an important factor for the emergence of

  SGR But the quantitative measurement of phosphatidylglycerol

  rol was very difficult, because it exists only in a quantity representing only about one-tenth

of that of phosphatidylcholine.

  So far, as a method of quantitative measurement

  phosphatidylglycerol, the process is known by thin layer chromatography in which,

  after eliminating the cellular component of the

  amniotic fluid by centrifugal separation,

  trait of the lipid constituents of the supernatant phase, the constituents of this extract are separated

  by thin-layer chromatography, we measure quantitatively

  At the titration rate of each phospholipid, the ratios of the various phospholipids are obtained and the amount of phosphatidylglycerol is obtained indirectly from the total phospholipid content obtained in advance by the quantitative measurement of phosphate by 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) 1, or the quantitative measurement method in which chromatography is used.

  high speed liquid graph lJournal of Chromato-

  graphy, 277: 223 (1981) l But this chromatic process

  thin layer mapping has disadvantages in that it takes two to three days to extract the lipid components of the amniotic fluid, in that the operations are complicated and, in addition, that it is necessary to heat to a temperature

  because of the detection of stains.

  also has the disadvantage of being a measuring procedure

  indirect reliance on ratios of quantities

  from stains and total phospholipid,

  and not to allow the treatment to be carried out

  multané many samples because we put

  thin layer chromatography is used.

  It has now been found that in the liquidation

  containing various lipid components to be examined

  such as amniotic fluid, one can quantitatively determine phosphatidylglycerol alone,

  simple, convenient and precise way, in a short time.

  We found, quite unexpectedly,

  that phospholipase D acts on phosphatidylglycerol

  by giving glycerol and phosphatidic acid

  that a satisfactory method of quantitative measurement of phosphatidylglycerol alone has been developed by quantitatively measuring that glycerol produced by the

reaction and released.

  Preferably, a process is used in which only phosphatidylglycerol can be

  measured in a satisfactory and quantitative way

  by acting phospholipase D on the test liquid to obtain glycerol, then by causing glycerol kinase to act on this glycerol in the presence of a phosphate donor such as adenosine triphosphate (ATP), then letting the glycerophosphate oxidase act on this product and then measuring the amount of oxygen consumed or

  of hydrogen peroxide produced by the reaction.

  The invention therefore relates to a method for the quantitative measurement of phosphatidylglycerol in

  the fluid to be studied which consists of releasing the gly-

  cerol by the action of an enzyme that plays the role of

  catalyst in the glycerol preparation reaction

  rol and phosphatidic acid from phospha-

  tidylglycerol and water and then measure quantitatively

  The glycerol produced is

  preferably a method for quantitatively measuring phosphorus

  phatidylglycerol in the test liquid which

  to perform in combination the following processes (a), (b), (c) and (d): (a) release of glycerol by the action of phospholipase D on phosphatidylglycerol, (b) production of glycerol-3-phosphate by action of glycerolkinam on glycerol in the presence of a phosphate donor, (c) action of glycerophosphate oxidase on glycero-3phosphate, and (d) measurement of the amount of oxygen consumed

  or hydrogen peroxide produced by the reaction.

  According to the invention, it is advantageous to

  prepare each reagent in a kit intended for quantitative measurement and, furthermore, this quantitative measurement can be carried out at ambient temperature of about 37 ° C., the operation being very simple and the time required for the reaction being very brief.

  Moreover, we can measure in a precise way the phos-

  phatidylglycerol to a very low concentration, and

  the phosphatidyl content can be measured directly

  glycerol, which proves to be very useful

  Measurement can be done in a simple and convenient way in a short time, it is possible to simultaneously measure several samples.

  a useful process for the quantitative measurement of phosphate

  dylglycérol. Figure 1 shows the calibration curve

  of Example 1 according to the invention, FIG.

  the results of the quantitative measurement of phospha-

  tidylglycerol when using various samples of amniotic fluid and Figure 3 shows the

  calibration curve of Example 3 of the invention.

  Phospholipase D is the enzyme that plays a role in

  role of catalyst in the reaction of phosphatidyl-

  glycerol and water to give glycerol and

  the phosphatidic acid according to the invention.

  This phospholipase D is known as a

  enzyme which is a catalyst for the reaction that pro-

  1 mole of phosphatidic acid and 1 mole of

  line from 1 mole lecithin and 1 mole

  As long as a substance can be a cat-

  lyser in the enzymatic reaction mentioned above.

  above, it can be used, and in particular for example that obtained by the extraction of cells containing phospholipase D or an enzymatic reagent found on the market, for example an enzyme from a microorganism obtained by culturing a bacterium producing phospholipase D which belongs to the genus Streptomyces lStreptomyces hachijoensis, strain A-1143 (FERM-P No. 1329); Japanese Patent Examined No. 39918/1977, Streptomyces

  chromofussus strain A-0848 (FERM-P No. 3519); disman-

  Japanese Patent Publication No. 130984/1977, etc., and enzymatic phospholipase D reagents.

sold on the market.

  The amount of phospholipase D to use

  must be modified in a suitable manner in

  the time required for the measurement and concentration of phosphatidylglycerol and no particular limitation is imposed on this quantity.

  for example, that for a test one can use

  Preferably, phospholipase D is used in an amount of not less than 0.1 units and preferably in an amount of about 1 to 10 units. Preferably, this phospholipase D is used after having dissolved in a buffer such as

  HCl-Tris buffer weakly acidic to weakly

  caline, a buffer of citric acid and a boric acid buffer, a PIPES-Na OH buffer or an imidazole buffer and, if necessary, a nonionic surfactant such as Triton X- 100 or serum albumin The enzymatic solution containing phospholipase D thus adjusted is then combined with the liquid to be examined, and glycerol L and phosphatidic acid are produced from the phosphatidylglycerol of the liquid to be examined with consumption of water. The mixing ratio of the two is not limited in a particular way and they

  can be mixed in a ratio such as

  From about 1 to about 10 units of phospholipase D are contained for a single test of liquid to be tested. The reaction temperature may be about 37 ° C. and the reaction time may be that which is sufficient for the release of glycerol.

  usually not less than 5 minutes and,

  not less than 10 minutes.

  The quantity of glycerol released by the reaction can be determined quantitatively. The phosphatidylglycerol content can be

the liquid to be examined.

  For the quantitative measurement of glycerol, various methods known for quantitative chemical measurement and those using enzymes can be used.

  Preferably, it is simple and convenient to

  to a quantitative enzymatic measurement process in which one or more species of enzymes whose substrate is

  glycerol and quantitatively measure the change

  detectable effect of enzymatic action in the reaction

  tion. Thus, for example, the GK-GPO method of quantitative measurement in which glycerol-3-phosphate is reacted with adenosine diphosphate (ADP) by the action on glycerol in the presence of a phosphate donor

  such as adenosine triphosphate (ATP) and glycerol

  rolkinase (GK), and then leave the glycerol

  Rophosphate oxidase (GPO) act on the glycero-3-

  phosphate so as to consume oxygen from the liquid

  of reaction to get oxygenated water is

  particularly simple and convenient.

  In this method of the GK-GPO type, the

  amount of oxygen consumed in the reaction liquid

  by an oxygen electrode or the quantity

  oxygenated water produced by an oxygenated water electrode.

  born according to the electric change the quantity

  oxygen or hydrogen peroxide can also be measured

  by an enzymatic electrode in which an oxygen electrode or a hydrogen peroxide electrode

  and an immobilized GPO are combined.

  may be in the form of membranes, fibers,

  pellets or tubes to facilitate installation

  tion of the enzyme to the detection part of the elec-

  In this embodiment, the electrode can be operated with a small amount of enzyme. The phospholipase D or GK used can also be immobilized in a similar manner or in another suitable manner. immobilization, one can implement any

  which process, such as, for example, the method of

  polymerization using, for example,

  acrylamide; an immobilization process

  a crosslinking agent for mixing with a protein such as albumin; a method

  inclusion using collagen or fibro-

  born; adsorption on an organic polymeric resin

  than porous or a covalent bond on such a resin; an inclusion method using a photocurable resin or a bonding method

  covalently using an aminated glass.

  In addition, other means of measuring

  of hydrogen peroxide, it is possible to carry out the

  quantitative measurement by spectrophotometric methods

  The indicators used are usually those in which a change can be quantitatively measured by spectrophotometric means, for example a colored reagent in which the turn occurs in the field. visible, a fluorescent reagent composition that

  is fluorescent, or a light reactive composition

  This is so, for example, that as a colored reagent composition a material containing a substance having a peroxidase action and a chromogen is used.

  the peroxidase action, it is often used

  peroxidase from horseradish and, as

  chromogen, the association is often used

  of an electron acceptor and a hydrogen donor As an electron acceptor, one can

  use, for example, 4-aminoantipyrine, 2-

  hydrazinobenizothiazole, with 3-methyl-2-benzothiazolinone hydrazine, 2-aminobenzothiazole, etc. As a hydrogen donor, for example phenol, 3-methyl-N-ethyl-N (-hydroxyethyl) aniline may be used. ,

  3,5-xylenol, N, N-dimethylaniline, N, N-di-

  ethylaniline, etc. As luminous substrates of a fluorescent reagent composition or a reagent composition

  photogenic, for example a bis (2,4,6-

  trichlorophenol) oxalate, phenylthiohydantoin,

  homovanillic acid, 4-hydroxyphenylacetic acid

  that vanillylamine, 3-methoxyethylamine,

  phloretic, hordenine, the luminol monoanion,

  lucigenine, etc. Each of them can be used

  read, if necessary, in association with an acceptor

  of electrons and / or a substance with a

  oxidase for the quantitative measurement of hydrogen peroxide.

  There is no particular limitation for the amount of enzyme reagent or chromogen

  This is how, for example, you can use

  for a test, usually not less than 0.01 units, preferably 0.05 to 100 units of GK and usually not less than 0.05 and preferably

  0.1 to 200 units of peroxidase It is also possible to use

  an adjusted solution such as concentration

  an electron acceptor or a hydrogen donor is not usually less than

  O, 1 m M and an adjusted solution such as the concentration

  of a phosphate donor such as ATP or cytosine triphosphate is not less than 0.1

  Preferably, to increase the enzymatic activity

  GK tick, a water-soluble salting salt is used

  magnesium ions, for example magnesium chloride,

  sium, and it can be dissolved in distilled water

  or in weakly acidic to weakly alkaline buffer

  These reagents can be used separately

  of the enzymatic solution of phospholipase D mention-

  above, or they may be mixed with that

  or, in addition, these reagents can be formed into an integrated laminate by applying them to papers

filters, film or other.

  The GK-GPO method of quantitative measurement

  tive has a high sensitivity to glycerol

  in the liquid to be examined and, since it is not affected by the impurities of the liquid to be examined, it is an excellent method for carrying out

a precise measurement.

  As GK used for this GK-type process

  GPO of quantitative measurement, it is possible to use

  What enzyme does it produce

  rol-3-phosphate and ADP, from glycerol and ATP, for example enzymes from rat liver or pigeon lJ Biol Chem, 211, 951 (1954) and Biochem Z, 329, 320 ( 1957) 1, those derived from microorganisms that are enzymes obtained

  from the culture medium of microorganisms pro-

  GK, such as Candida mycoderma or strepto-

  myces canus A-2408 (FERM-P No. 4977) Biochem Z, 333, 471 (1961), unexamined patent application in Japan published under No. 162987/1980, etc., and

  other enzymes sold on the market.

  In addition, as GPO any enzyme can be used as long as it plays a catalytic role

  in the dihydroxyacetone production reaction

  phosphate and hydrogen peroxide, from glycero-3-

  phosphate and oxygen, for example of the genus

  Streptococcus, of the genus Lactobacillus, of the genus Leuco-

  nostoc, bacteria producing glycerophospha-

  These enzymes belong to the genus Pediodoccus (Unexamined Japanese Patent Application Publication No. 72892/1978), an enzyme obtained by cultivation.

  of bacteria producing oxidized glycerophosphate

  which belong to the genus Aerococcus (Aerococcus

  viridans strain IFO 12219 and strain IFO 12317,

  unexamined patent and published in Japan under the

  No. 15746/1980) and enzymes sold on the market.

  Another enzymatic method for quantitatively measuring

  The glycerol method involves reacting glycerol kinase with glycerol in the presence of a phosphate donor such as ATP to obtain glycerol-3-phosphate and ADP, and then reacting the glycerol kinase with glycerol.

  glycerophosphate dehydrogenase on this glycero-3-

  phosphate in the presence of nicotine adenine

  cleotide (NAD) to produce dihydroxyacetone and reduced NAD, then quantitatively measure this reduced NAD to determine the amount of glycerol When carrying out the quantitative measurement of this reduced NAD, the absorbance measurement can usually be used at about 340 nm, or after having allowed it to give rise to a coloration using a water-soluble salt of tetrazolium such that

  3- (4,5-dimethyl) -2-thiazolyl-2H-tetrabromide bromide

  zolium, 2- (p-iodophenyl) -3- (p-nitro) chloride

  phenyl) -5-phenyl-2H-tetrazolium, 3,3 'chloride

  (3,3'-Dimethoxy-4,4'-biphenylene) -bis-1 2- (p-nitro-

  phenyl-5-phenyl-2H-tetrazoliuml (nitrotetrahydrate blue)

  zolium) or 2,6-dichlorophenol-indophenol in the presence of diaphorase or phenazine methosulphate,

  the color can be measured according to the absorp-

  bances using special wavelengths

absorption.

  As a process different from that mentioned above

  above it may be mentioned a means of quantitative measurement

  in which glycerol dehydrogenase is reacted with glycerol in the presence of NAD to produce dihydroxyacetone and reduced NAD

  and this reduced NAD is quantitatively measured.

  It is furthermore possible to mention a means which relies on a process in which glycerol kinase is made to act on glycerol in the presence of a phosphate donor such as ATP to obtain glycerophosphate and ADP, and reacts of

  pyruvate kinase on this ADP in the presence of phos-

  Phenolpyruvate to obtain pyruvic acid and ATP and this pyruvic acid is quantitatively measured. Methods for the quantitative measurement of this pyruvic acid are used such as that which consists in causing a hydrazine to act as

  2,4-dinitrophenylhydrazine on pyruvic acid

  that to give rise to a coloring and measuring

  color according to absorbance in use.

  reading a wavelength of about 440 nm, that

  according to which dehydrating lactate is reacted

  genase on pyruvic acid in the presence of reduced NAD to obtain oxidized NAD and lactic acid and measuring the decreased amount of NAD reduced in the reaction system by means such as an absorbance measurement at a wavelength of about 340 nm, that according to which pyruvate oxidase is made to act on pyruvic acid and the amount of oxygen consumed or that of oxygenated water of the reaction system is measured as a function of an electric variable and the one according to which the pyruvic acid is measured by spectrophotometric means using a water indicator composition.

oxygenated.

  As other processes than those mentioned above

  above, a quantitative measuring method can be used.

  glycerol according to which the

  glycerol oxidase on glycerol and measure the amount

  consumed oxygen or oxygenated water or

  of glyceraldehyde produced in the reaction system

  nel. As enzymes, reagents and others, it is

  simple and convenient to use those sold on the market.

  and the amount to be used can be appropriately determined. If necessary, a surfactant or

a stabilizing agent.

  As a liquid to be examined, mention may be made

  any sample, provided that it

  phosphatidylglycerol, for example fluids,

  of the body such as amniotic fluid.

* When the amniotic fluid is the liquid

  to consider, it is preferable to use the sample

  taken from the area containing phosphatidyl-

  glycerol; for example, a sample of 5 to 6 ml of amniotic fluid is extracted with 18 ml of the chloroform-methanol mixture (2: 1).

  the chloroform layer is collected by centrifugation

  at 2000 rpm and evaporate to dry

  in nitrogen gas to obtain the total lipid.

  Then, after having dissolved this total lipid in a defined amount of a 1% solution of Triton X-100, an enzymatic solution of phospholipase D is reacted and, for example, each enzyme on the

  basis of the quantitative measurement process of G-K-GPO

  In this case, there is no particular limitation to the ratio of use of the test liquid to the enzyme reagent and the like, and usually

  from about 0.1 to about 3 ml of the enzymatic reagent or

  0.01 ml to 1 ml of the test liquid As far as the reaction conditions are concerned, it is preferable to carry out the reaction at about 370 ° C. and for the reaction time, it is possible to choose any duration as long as the reaction is completed completely; habitually,

  the reaction is carried out for not less than 5 minutes.

  and, preferably, for not less than 10 minutes

  Similarly, with regard to the reaction medium,

  water or weakly acidic to weakly alkaline buffer is used as the solvent for each

reagent and others.

  So, by the quantitative measurement of the liquid

  to examine one can measure directly and quantitatively

  phosphatidylglycerol in a very short time and a phosphatidylglycerol content as small as 2.0 N moles, ie

  phosphatidylglycerol content of 0.03 mg / dl when used

  5 to 6 ml of amniotic fluid This means

  that the amount of phosphatidyl-

  glycerol in an extremely low concentration in comparison with the value of 0.2 mg / l of this which is the critical level for the appearance of the SGR In addition, no complicated operation is necessary and one can work to As a result, this is a good quantitative measurement process.

  Similarly, there is no particular limitation

  the method for measuring each phospholipase D, GK and GPO activity used in practical examples mentioned below according to the invention and these methods can be illustrated as follows:

  (a) Method for measuring the activity of phosphorus

  lipase D. AO, 1 ml of a solution of 4% phosphatidylethanolamine egg yolk, 0.8 ml of 0.05 M tris-hydrochloric acid buffer (p H 7.5) is added and mixed with 0.3 ml of 1% Triton X-100, 0.3 ml of water and 0.2 ml of a 10 mM aqueous solution of Ca C 12 and

  subject the mixture to ultrasonic treatment during

  After 10 minutes, 0.2 ml of ethyl ether and 0.1 ml of an enzymatic solution are added. The mixture is allowed to react at 37 ° C. for 20 minutes and the reaction is interrupted by the addition of 0.3 ml of acid. 20% trichloroacetic acid The reaction liquid is washed twice with 4 ml of ethyl ether, 0.5 ml of a 0.2 N citrate buffer (p H 5.0) is added to 1 ml of the aqueous layer, 0.1 ml of a 2% aqueous solution of Sn C 12 and 2 ml of 2% ninhydrin solution are added thereto, the mixture is heated at 100 ° C. for 15 minutes to release the ethanolamine, which gives rise to staining by the reaction of ninhydrin and measuring the absorbance at an OD of 570 mp. The activity of the enzyme which liberates 1 μg of ethanolamine per minute is defined as 1 unit (U).

  (b) Method for measuring GK activity.

  0.2 M Tris hydrochloric acid buffer (p H 9.0) 0.4 ml Glycerol 0.1 M 0.05 ml ATP 10 m M 0.1 ml Mg Cl 2 10 m M 0.1 ml Nitrotetrazolium blue 0.25% O, 1 ml Serum albumin of beef 1% 0.14 ml NAD 10 m M 0.1 ml Phenazine methosulfate 0.05% 0.01 ml Glycerophosphate dehydrogenase (manufactured by Boehringer Sohn AG,

2 mg / ml, 65 U / mg).

  One milliliter of the reaction mixture is pre-incubated.

  each containing the above-mentioned composition at 37 ° C. for 5 minutes. 50 μl of a solution containing GK (mM phosphate buffer containing 10 mM glycerol, suitably diluted to a pH of 7) are added thereto. , 5) The mixture is reacted

  at 37 ° C. for 10 minutes. After having interrupted the reaction by the addition of 0.1 N HCl, the color which

takes birth to a length

  550 nm waveform to obtain the absorbance (A 550 nm).

  A unit of GK is defined as the activity that pro-

  1 pmole of glycerol-3-phosphate per minute.

  The calculation equation is:

  U / ml = AA x Dilution ratio x 1 A x ratio 0.05 x 10 4 2 dilution (c) Method for measuring GPO activity Buffer with 0.2 M Trichlorohydric acid (p H 8 0.2 ml Peroxidase (0.5 mg / ml, 45 U / ml) 0.1 ml 4-aminoantipyridine 0.3 (w / v) 0.1 ml DL-glycero-3-phosphate 0, 1 M 0.1 ml N, N-Dimethylaniline 0.2% (V / V) 0.2 ml

Distilled water 0.3 ml.

  One milliliter of the reaction mixture is

  having the composition mentioned above in

  a small test tube and a pre-incubation

  at 37 ° C. for 3 minutes 20 μl of an enzymatic solution are added and the mixture is allowed to react for 10 minutes. The reaction is interrupted by the addition of 2.0 ml of 0.25% sodium laurylbenzene sulphonate ( P / V) and the absorbance of W rlo Ua qa 001-X uo: 7-Ti ao'uu q is evaluated, and is measured as follows: ## EQU1 ## 9, O) julil gc, ap uos-t,? X ni uuleu 9 zuoo 3 srud-13so-7 ap 3 ug (g Hd) UOT-4 nlos ZUDD ap ru :, z Tu 9 O n nc C, ozz - , a, dsoucl no 9: D qj-? Z 3

001 O'1 2 T O-Z

  -The Ds-Du N has a U-t? U O T O S O O UT-1 O S UOT ', Ua AUT, T: U 9 Sn aun

01 SUPO

  X (O'9) (It U / fl) 91 JLZ Ua j ap 9-4 TA Tq Y oq a: Ul PA Tn S UO Tq Pnba-julolas aul, zua T apouq al lnnolo UO - ulu 999 ap apuop 2 nan Buoi aun P q Tnpoad Li ZL L 8 L Sz

-To 2518117

(4) 60mM of EDTA.

  (5) 40 mM solution of Mg C 12 containing 160 mM ATP: 0.2 ml of 0.2 M ATP, 0.10 ml of Mg C 12 0.5 M and 0.15 ml are mixed together. ml of distilled water so as to obtain 1.25 ml of the solution.

(6) Enzymatic solution of GK.

  10 mM Tris-HCl buffer containing 2 mg / ml of

GK (p H 8).

(7) GPO enzymatic solution.

  10 ml of a GPO enzyme solution consisting of 10 mg of cow serum albumin containing 3 mg / ml of GPO, 560.75 mg of (NH 4) 2504, 1.0 ml of HCl-Tris buffer are prepared. 0.1 M (p H 8) and 9 ml of distilled water. (8) Liquid indicator composition containing GPO. ml 4aminoantipyridine 0.24 Phenol aqueous solution 0.16 (10 mg / ml) Triton X-100 (1.25%) 0.8 Tris-HCl buffer (0.5 M, p H 8) 1.6 Peroxidase (90 U / ml distilled water) 0.8 Enzyme solution of GPO 0.8 (3 mg / ml GPO) Distilled water 15.6 To each 0 ml sample at 0.10 ml of the solution containing phosphatidylglycerol (PG) Total 20 ml mentioned above, 1% Triton X 100% was added so that the total amount was 0.10 ml. To this mixture, 0.1 ml of the Ca ++ buffer was added after heating. the mixture at 37 ° C. for 5 minutes, 0.05 ml of an enzymatic solution of phospholipase D is added thereto and the mixture is allowed to react at 37 ° C. for 10 minutes. After the reaction, 0.04 ml of EDTA 60 is added. m M and in

  in addition to 0.075 ml of a solution of Mg C 12 40 m M containing

  160 mM of ATP and 0.03 ml of an enzymatic solution.

  The mixture is allowed to react at 37.degree.

  After the reaction, 1.0 ml of the GPO-containing indicator liquid composition is added and the mixture is allowed to react at 37 ° C. for 10 minutes.

  20 minutes Then we measure the absorbance due to the co-

  loration which originated as a result of the

  reaction at a wavelength of 500 nm (OD 500).

  The results are shown in FIG. 1. A trend is obtained for a satisfactory linear relationship in the range from 0.005 ml of solution containing PG (PG content of 5 N mol) to that of 0.1 ml ( PG content of

N moles).

  It also follows from this figure 1 that

  the tendency to a linear relationship can be obtained

  when the PG content exceeds 2 N moles and the

sensitivity is very big.

EXAMPLE 2

  In 18 ml of a mixture of chloroform and

  methanol (2: 1), dissolve 5 to 6 ml of a sample of

  The solution is centrifuged at 2000 rpm. The three layers which are separated are extracted by centrifugation and the chloroform layer is evaporated to dryness in nitrogen gas to obtain the total lipid. This total lipid is then dissolved. in 0.1 ml of chloroform and the solution is loaded into a column of silica gel (0.3 g), the neutral fat being removed by flowing ml of a mixture of chloroform and methanol (19: 1 ) and the fraction containing the phos-

  phatidylglycerol by flowing 10 ml of

  chloroform and methanol (2: 1) The solvent is then removed and the residue is dissolved in 0.1 ml of 1% Triton X-100 to prepare the liquid to be examined. Ca ++ buffer to the liquid to be examined and the mixture is heated at 370 ° C. for 5 minutes. 0.05 ml of an enzymatic solution of phospholipase D is then added and the mixture is allowed to react at 370 ° C. for 10 minutes. After the reaction 0.04 ml of 60 mM EDTA is added, followed by the addition of 0.075 ml of a solution of 40 mM MgCl 2 containing 160 mM ATP and 0.03 ml d an enzymatic solution of GK, and the mixture is allowed to react at 370 ° C for 10 minutes.

  the reaction, 1.0 ml of the liquid composition is added.

  of indicator containing GPO and the mixture is allowed to react at 370 ° C. for 20 minutes. The absorbance due to the coloration which originates under the effect of the colored material produced by the reaction at a wavelength of 500 is measured. and the PG content (mg / dl) of the amniotic fluid sample is obtained from the calibration curve.

  Results are as shown in Figure 2.

  In Figure 2, X represents fatal cases, O re-

  presents with cases of onset of respiratory distress syndrome, A represents cases of occurrence of mild respiratory distress syndrome and * represents

  cases where there is no evidence of

piratory.

EXAMPLE 3

  ml 0.2 M HCl-Tris Buffer (p H 7.5) 0.2 Ca C 12 10 m M 0.2 Mg C 12 10 m M 0.2 Triton X-100 at 10% 0.05 ATP 10 m M 0.2 Phospholipase D at 20 U / ml, GK at 5 U / ml and liquid containing 0.1 GPO at 50 U / ml Peroxidase at 45 U / ml 0.1 4-aminoantipyrin at 0.3 % 0.2 3-methyl-N-ethyl-N (-hydroxyethyl) 0.2 aniline 0.3% Distilled water 0.55 2.0 ml of the reaction mixture are prepared for the quantitative measurement of phosphatidylglycerol having the aforementioned composition To this mixture is added 50 μl of the test liquid which is a liquid containing different amounts of PG (PG content: 20 N moles to 100 N moles). The mixture is allowed to react at 37 ° C. for 15 minutes. , then measured at a wavelength of 550 nm (D 0550) the coloring developed by the colored material produced after the reaction The results obtained are recorded in Figure 3 and a calibration curve is obtained which shows a satisfactory linearity according to

  the PG content of the liquid to be examined.

Total 2.0 ml

Claims (13)

  1 Process for the quantitative measurement of phospha-   tidylglycerol of a liquid to be examined, characterized in that it consists in causing an enzyme to act on phosphatidylglycerol to release glycerol, and   quantitatively measure the glycerol produced.   Process according to claim 1, characterized   characterized in that the enzyme is an enzyme which acts as a catalyst in the preparation reaction of   glycerol and phosphatidic acid from phosphorus phatidylglycerol and water.   3. Process according to claim 2, characterized   in that the enzyme which plays a catalytic role   in the glycerol preparation reaction and   of phosphatidic acid from phosphatidylglycerol   rol and water is phospholipase D.   4 Process according to one of the claims   1 to 3, characterized in that the liquid to be examined is a fluid of the body.   5 Process for the quantitative measurement of phospha-   tidylglycerol of a liquid to be examined, characterized in that it comprises the combination of the following processes (a), (b), (c) and (d): (a) release of glycerol by   react phospholipase D with phosphatidyl   glycerol, (b) production of glycerol-3-phosphate by reacting glycerolase with glycerol in the presence of a phosphate donor, (c) action of glycerophosphate oxidase on glycerol-3-phosphate, and   (d) measuring the amount of oxygen con-   summed or hydrogen peroxide produced in the reaction.   Process according to claim 5, characterized   in that the phosphate donor is the appropriate nosine triphosphate.   Process according to Claim 5 or 6, characterized in that for the quantitative measurement of   hydrogen peroxide is used a composition to indi-   which reacts with hydrogen peroxide to give a product that can be detected.   The process of claim 7, wherein   characterized in that the indicator composition is a material containing a substance having an action peroxidase and a chromogen.   The method of claim 8, wherein   characterized in that the chromogen is 4-aminoantipyri- ne and phenol.   Process according to claim 8,   in that the chromogen is 4-aminoantipy-   and 3-methyl-N-ethyl-N- (e-hydroxyethyl) aniline.   The process of claim 9, wherein   in that the measurement consists of taking an absorbance measurement at a wavelength of about 500 nm.   The process of claim 10 wherein   characterized in that the measurement consists of taking an absorbance measurement at a wavelength of 550 nm approximately.