DE3246090C2 - - Google Patents

Description

The number of deaths of newborns with too little weight could lately thanks to the improved treatment lowered by newborns, however, represents the atomic not syndrome (RDS) is a major problem in the perinatal period, and the deaths caused by it make up a high percentage of total deaths. It is believed that the respiratory distress Syndrome due to a lack of lung wetting agent is. This remedy is important for preventing the Alveolar contractions of the lungs, occurring immediately after birth expand. The lack of this lung wetting agent causes respiratory atelectasis and the advent of RDS. Consequently, the occurrence of RDS could be prevented or prevented mild case, when the occurrence of RDS in one early period could be established and the treatment of the Newborns could be started early. Accordingly, the Determining this lung wetting agent a necessity. Roughly subdivided, there is a procedure for determining, in the physical properties of the lung wetting agent be used, and another, in which the ingredients of the lung wetting agent are biochemically examined.

The latter method was previously carried out so that the ratio  the lipids was determined as the ratio of lecithin to sphingomyelin or that of phosphatidyl-glycerol to phosphatidyl Inositol or that dipalmitoyl-phosphatidyl-choline by Thin-layer chromatography determined quantitatively was on. J. Obstet. Gynecol., 440, 109 (1971); 613, 125 (1976); 894, 133 (1979); Obstet. Gynecol., 295, 57 (1981); At the. J. Obstet. Gynecol., 697, 138 (1980)]. From the results These various investigations have recently recognized that the presence of phosphatidyl-glycerol is an important factor for the appearance of RDS. The quantitative determination However, phosphatidyl-glycerol was very difficult because the occurring amount is as low as about ¹ / ₁₀ the amount of lecithin. For the quantitative determination of phosphatidyl-glycerol previously known thin-layer chromatography, in the after Remove the cells from the amniotic fluid by centrifuging the lipids are extracted from the supernatant, the ingredients of this extract separated by thin layer chromatography Each phospholipid is quantified, the ratios the different phospholipids are calculated and the amount of phosphatidyl-glycerine indirectly from the value of Total phospholipids previously obtained by quantitative Determination of phosphoric acid according to the wet-burning method has been obtained [Am. J. Obstet. Gynecol., 613, 125 (1976); 1079, 135 (1979); 899, 133 (1979); 440, 109 (1971)]. Another Method for the quantitative determination of phosphatidyl-glycerol is the high-speed liquid chromatography [Journal of Chromatography, 277, 223 (1981)]. This method of However, thin-layer chromatography has the disadvantages that for the extraction of the lipid components from the amniotic fluid 2 to 3 days are needed, complicated measures and as well heating to high temperature is necessary for spot detection are. Other disadvantages are that they are an indirect method for determination based on the ratios of relative Stain levels and total phospholipid based, and that because the use of thin layer chromatography not simultaneously several samples can be examined.

The invention has carried out various investigations determine whether in the liquid, the various to be examined  Lipids, like fear water, contain only phosphatidyl Glycerol quantitative to simple, convenient and accurate Way in a short time can be determined.

The inventors have surprisingly found that Phospholipase D acts on phosphatidyl-glycerol and glycerol and phosphatidic acid forms. They found a satisfactory Method for the quantitative determination of phosphatidyl-glycerol by quantitative determination of the formed in the reaction and then glycerol extracted. Preferably, in this Process only phosphatidyl-glycerol quantitatively very satisfactory be determined by exposure to phospholipase D on the liquid to be examined for the formation of Glycerol, then exposing glycerol kinase to this Glycerol in the presence of a phosphate donor, such as adenosine triphosphate (ATP), finally, exposure to glycerol phosphate Oxidase on the product and determine the consumed Amount of oxygen or the hydrogen peroxide formed in the reaction.

The inventive method for quantitative determination of phosphatidyl-glycerol in the body fluid to be examined is therefore characterized in that the following Steps (a), (b), (c) and (d) are carried out:

• (a) exposing phospholipase D to a sample of Body fluid releasing glycerol from the phosphatidylglycerol contained in the sample;
• (b) forming glycerol-3-phosphate by exposure to Glycerol kinase on the released glycerol in the presence a phosphate donor;
• (c) exposing glycerol phosphate oxidase to the formed one Glycerol-3-phosphate and
• (d) determining the amount of consumed in the reaction (c) Oxygen or hydrogen peroxide formed in reaction (c).

The invention offers the advantages that each reagent for the quantitative determination in one package can, the quantitative determination at a temperature of about 37 ° C, they also easy and requires little time for the reaction.

In addition, the phosphatidyl-glycerol can be up to a very low Concentration accurately and directly determine what is in practice is of great benefit. Since the determination is easy and can be carried out expediently in a short time simultaneously several samples are examined, d. h., the invention provides a suitable method for quantitative determination of phosphatidyl-glycerin available.

There are various enzymatic methods in the literature described which are applied to glycerides and lipids but they are not suitable for quantitative determination of phosphatidyl-glycerol in body fluids, in particular Amniotic fluid. The underlying of the invention Task can therefore not be by these known methods to solve.

According to DE-OS 27 37 513 phospholipids, in particular Lecithin, sphingomyelin and lysolecithin, which combines occur in serum, enzymatically to form free choline degraded, which then quantified as an indicator substance becomes.

DE-OS 26 35 040 teaches Fettsäureglycerinester by certain Lipases (Rhizopus arrhizus lipase and Candida cylindrace- Lipase) hydrolytically in glycerol and the respective ones Split fatty acids. However, such fatty acid esters contain no phosphoric acid component.

Also, according to US-PS 42 41 178 only triglycerides by means a lipase-containing mixture to glycerol and the corresponding fatty acid components implemented.  

Furthermore, it is from the monograph "Methods of enzymatic Analysis "(H. U. Bergmeyer, Verlag Chemie), 3rd Edition, Volume I, S. 537/538, known that phospholipase D from white cabbage, the hydrolytic Cleavage of choline catalyzed by phosphatidylcholine, wherein phosphadidate is formed simultaneously.

The invention will be explained below with reference to the drawings.

Fig. 1 shows the calibration curve of Example 1;

Figure 2 shows the results of a quantitative determination of phosphatidyl-glycerol when examining various samples of amniotic fluid components.

Fig. 3 shows the calibration curve of Example 3.

As explained above, phospholipase D is itself an enzyme known that the reaction of 1 mole of phosphatidic acid and Choline catalyzed from 1 mole of lecithin and water. As part of Any type of phospholipase D can be used in the present invention be used, for. B. a preparation obtained by extraction of Phospholipase D containing cells has been obtained, or a commercially available enzyme preparation, e.g. B. one from microorganisms originating enzyme resulting from the culture of phospholipase D producing microorganisms has been obtained the too  of the genus Streptomyces, such as Streptomyces hachÿoensis A-1143 (FERM-P No. 1329, see JA-PS 39 918/1977), Streptomyces Chromofussus A-0848 (FERM-P No. 3519, unexamined JA patent application No. 1 30 984/1977). Also, a commercially available phospholipase D preparation is suitable.

The amount of phospholipase D to be used should be appropriate modified and to the time required for the determination as well adapted to the concentration of phosphatidyl-glycerol; it is not subject to any special limitation. For example, can one for a determination phospholipase D in an amount in general not less than 0.1 unit, preferably about 1 to 10 Use units. In addition, this phospholipase D is preferable used after being dissolved in a buffer is such as a weakly acidic to slightly alkaline tris Hydrochloric acid buffer, citric acid buffer, boric acid buffer, PIPES NaOH buffer or imidazole buffer and, if necessary, the Solution a non-ionic surfactant, such as alkylaryl polyether alcohols (Triton X-100) or serum albumin. The thus adjusted, phospholipase D containing enzyme solution and the liquid to be examined are mixed. In The liquid to be examined is formed by glycerol and phosphatidic acid from phosphatidyl-glycerol under consumption of water. The mixing ratio of the two is not especially limited, you can get them in such a relationship preferably, about 1 to 10 units of phospholipase D per sample of the liquid to be examined are included. The reaction temperature can be about 37 ° C, and the reaction time should be for the release of glycerin generally not less than 5 minutes, preferably not less than 10 minutes. The released by this reaction Glycerol is then quantified. Out the value of this quantitative determination is given the value for phosphatidyl-glycerol in the liquid to be tested.

For the quantitative determination of glycerol can various known methods for quantitative chemical  Determination or those that use enzymes. A preferred simple and convenient enzymatic Method of quantitative determination is that in which one or several enzymes whose substrate is glycerol and the detectable change in the enzyme action in the Reaction is determined quantitatively. For example, that is GK-GPO method for quantitative determination particularly simple and expediently, by glycerol-3-phosphate and adenosine diphosphate (ADP) from glycerol in the presence of a phosphate donor, such as adenosine triphosphate (ATP) and glycerol kinase (GK) then glycerol phosphate oxidase (GPO) on the glycerin 3-phosphate is allowed to act, so that the oxygen in the reaction liquid to form hydrogen peroxide is consumed.

The amount of consumed in the reaction liquid is Oxygen measured with an oxygen electrode, and the amount hydrogen peroxide formed is reacted with a hydrogen peroxide Electrode measured as electrical change or the amount of oxygen or hydrogen peroxide is with an enzyme electrode immobilized with glycerol phosphate immobilized Oxidase to an oxygen or hydrogen peroxide Electrode has been combined. The immobilized GPO may be in a membrane-like, fiber-like, granulate-like or tube-like shape to attach the Enzyme in the detector part of the oxygen or hydrogen peroxide To facilitate electrode. In this arrangement, the Working electrode with a small amount of enzyme. phospholipase D or the glycerol kinase used in the same way or immobilized accordingly. Immobilized enzymes can be prepared in various ways, eg. B. by Polymer inclusion in z. As acrylamide, by crosslinking Use of a crosslinking agent for a mixture with a Protein, such as albumin, by inclusion using Collagen or fibroin, by adsorption or by covalent Binding on a porous organic polymer or by Inclusion using a light-curing resin or  by covalent bonding using an aminated one Glass.

The quantitative determination of hydrogen peroxide can also using spectrophotometric methods, by using a Indicator formed by the reaction with hydrogen peroxide will be performed. As an indicator in the general such drugs used, their changes be determined quantitatively by spectroscopic means can, for. B. a color reagent mixture whose color is in Visible area changed, a fluorescent reagent that fluoresces, or one by irradiation with ultraviolet Light-generating reagent. For example, you can as Color reagent use a material containing a substance with peroxidase Contains activity and a chromogen. As substance with Peroxidase activity is generally made from horseradish originating peroxidase is used and as a combination chromogen an electron acceptor and a hydrogen donor. As electron acceptor such compounds are used such as 4-aminoantipyrine, 2-hydrazine-benzothiazole, 3-methyl- 2-benzothiazolone hydrazine or 2-aminobenzothiazole. As hydrogen Donor compounds are used such. Phenol, 3-methyl-N-ethyl-N- (β-hydroxyethyl) -aniline, 3,5-xylenol, N, N-dimethylaniline or N, N-diethylaniline.

As luminous substrates in a fluorescence reagent or a light-generating reagent mixture can be various known compounds are used, for. B. bis (2,4,6-trichlorophenol) - oxalate, phenylthiohydantoin, homovanillic acid, 4-hydroxyphenylacetic acid, vanillylamine, 3-methoxyethylamine, Phloretinsäure, Hordenin, Luminol monoanion or lucigenin. Each of these compounds can be combined if necessary with an electron acceptor and / or a substance with peroxidase Activity for the quantitative determination of hydrogen peroxide be used.

There is no particular limitation on the amount of enzyme or the chromogen used. So you should z. B. for a Examination generally not less than 0.01 units,  preferably from 0.05 to 100 units of glycerol kinase, not less than 0.05 units, preferably 0.1 to 200 units Glycerol phosphate oxidase and generally not less than 0.05 units, preferably 0.1 to 500 units of peroxidase use. Also, you can use a solution that is set up is that the concentration of an electron acceptor or a hydrogen donor, generally not less than 0.1 mmol is, and such a solution that the concentration a phosphate donor, such as ATP or cytosine triphosphate, not less than 0.1 mmol. Preferably, to increase the Glycerol kinase activity is a water-soluble, magnesium ion releasing salt used, for. As magnesium chloride, in distilled water or weakly acidic or weakly alkaline buffer can. These reagents can be separated from the phospholipase D Solution or mixed with it, further These reagents can be used as integrated composite preparations assembled by applying to filter paper or films become.

This GK-GPO method for quantitative determination shows a high sensitivity for that in the to be examined Liquid formed glycerin, and there it is due to the impurities not affected in the liquid to be examined It is an excellent method that one accurate determination allowed.

As glycerol kinase for this quantitative determination can Any enzyme can be used as long as it is glycerol-3-phosphate and ADP forms from glycerol and ATP, e.g. B. enzymes from the Liver of a rat or a pigeon [J. Biol. Chem., 211, P. 951 (1954), and Biochem. Z., 329, p. 320 (1957)], enzymes, derived from microorganisms, eg. B. from the culture medium of GK-producing microorganisms, such as Candida mycoderma or Streptomyces canus A-2408 (FERM-P 4977) [Biochem. Z., 333, p. 471 (1961), unexamined Japanese Patent Application No. 1 62 987 / 1980)] and commercially available preparations.  

The glycerophosphate used for this determination process Oxidase can be arbitrary as long as it plays the role of Catalyst in the reaction to form dihydroxyacetone phosphate and hydrogen peroxide from glycerol-3-phosphate and oxygen plays, and z. B. from the strains Streptococcus, Lactobacillus or Leuconostoc; it may also be glycerophosphate Oxidase be that of bacteria that cause the Stamm Pediococcus belong to (unaudited JA patent application No. 72 892/1978), an enzyme derived from the Culture of glycerol phosphate oxidase producing bacteria obtained from the strain Aerococcus (Aerococcus viridans IFO 12 219 and IFO 12 317, unexamined JA patent application No. 15 746/1980) and enzymes available on the market.

In another enzymatic method of quantitative Determination of glycerol will glycerol kinase on glycerol in Presence of a phosphate donor, such as ATP, allowed to act; glycerol-3-phosphate and ADP are obtained. Then glycerophosphate Dehydrogenase on this glycerol-3-phosphate allowed to act in the presence of nicotine adenine dinucleotide (NAD) to form dihydroxyacetone and NADH. This NADH is then quantified, and from it the amount of glycerin is calculated. In the quantitative Determination of NADH will generally increase absorption measured directly about 340 nm or after developing a color with 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 chloride, 3,3 '- (3,3'-dimethoxy- 4,4'-biphenylene) -bis [2- (p-nitrophenyl-5-phenyl-2H- tetrazolium chloride] (nitrotetrazolium blue) or 2,6-dichlorophenol indophenol in the presence of diaphorase or phenazine methosulfate, the color being measured at absorptions which correspond to the specific absorption wavelengths.

Another than the mentioned method for quantitative determination is characterized in that glycerol dehydrogenase act on glycerol in the presence of NAD becomes the formation of dihydroxyacetone and NADH and that this  NADH is determined quantitatively.

Another method is suitable in which glycerol kinase on glycerol in the presence of a phosphate donor, such as ATP, is allowed to form glycerol phosphate and ATP, then pyruvate kinase on this ATP in Presence of Phosphorenolpyruvat is allowed to act to the formation of pyruvic acid and ADP, said pyruvic acid is determined quantitatively. For quantitative determination this pyruvic acid you can use a method in which a hydrazine, such as 2,4-dinitrophenylhydrazine, on the pyruvic acid acts to form a color, measured their absorption at a wavelength of about 440 nm becomes. In another procedure is lactate dehydrogenase act on the pyruvic acid in the presence of NADH left to form NAD and lactic acid, which decreased Amount of NAD in the reaction system is determined by absorption measurement determined at a wavelength of about 340 nm. But you can also pyruvate oxidase on pyruvic acid let it act and the amount of oxygen consumed or hydrogen peroxide in the reaction system through the measure electrical change or the pyruvic acid spectrophotometrically using an indicator for Determine hydrogen peroxide.

Other than the above-mentioned quantitative methods Determination of glycerol is characterized in that Glycerol oxidase is allowed to act on glycerol and the Amount of oxygen consumed or of hydrogen peroxide or on glyceraldehyde formed in the reaction system.

As for the enzymes and reagents, it's easy and useful to use the commercially available preparations and to select the quantities to be used accordingly. You can also, if necessary, a surface active Use medium or a stabilizer.  

As a liquid, which is investigated in the method according to the invention Should be, every sample is suitable, as long as they are Phosphatidyl-glycerol contains, for. B. body fluids, such as Samples of amniotic fluid.

Will amniotic fluid as a fluid to be examined used, it is preferably made of a phosphatidyl taken from glycerol-containing area; for example 5 to 6 ml of amniotic fluid with 15 to 18 ml of chloroform / methanol in the ratio Extracted 2: 1; the chloroform phase is through Centrifuge at 2000 rpm and collect under nitrogen until evaporated to dryness. That's how you get that Total lipid that is in a certain amount of 1 percent Solution of Alkylarylpolyätheralkoholen (Triton X-100) solved becomes. Any enzyme solution, such as phospholipase, can be added to this solution D or glycerol phosphate oxidase or glycerol kinase for quantitative determination and other reagents act simultaneously or in succession. The ratio of fluid to enzyme solution is not particularly limited, in general will be about 0.1 to 3 ml of enzyme solution for 0.01 to 1 ml to be examined Liquid used. The determination is preferably at a temperature of about 37 ° C, the reaction time can be arbitrary, as long as the reaction is complete is; it is generally not less than 5 minutes, preferably not less than 10 minutes. As the reaction medium can Water or a weakly acidic or slightly alkaline buffer solution serve as a solvent for each reagent.

The inventive method for quantitative determination of phosphatidyl-glycerol in the liquid to be tested can be done directly in a very short time, man So low levels of phosphatidyl-glycerol as 2.0n moles, d. H. Phosphatidylglycerol in one Amount of 0.03 mg / dl, in an amniotic fluid sample of 5 to Determine 6 ml. This means that the amount of phosphatidyl glycerol is extremely low compared to a value of 0.2 mg / l, which is a critical value for the occurrence of RDS  is seen. Besides, no complicated measures necessary, the process can be carried out at normal temperature become.

The activity of phospholipase D and the activity of glycerophosphate Oxidase and glycerol kinase can be as follows be determined:

(a) Activity of phospholipase D

0.1 ml of a 4% solution of phosphatidyl-ethanolamine of egg yolk are mixed with 0.8 ml of 0.05M Tris-hydrochloric acid buffer, pH 7.5, 0.3 ml of a 1 percent solution of alkylaryl polyether alcohols (Triton X-100), 0.3 ml of water and 0.2 ml of one aqueous, 0.01m calcium chloride solution and mixed. The mixture is sonicated for 10 minutes, then with 0.2 ml Ethyl ether and 0.1 ml of enzyme solution. The mixture is Allowed to react for 20 minutes at 37 ° C, then with 0.3 ml 20prozentiger Trichloroacetic acid to interrupt the reaction. The reaction liquid is 2 times with 4 ml of ethyl ether washed. 1 ml of the aqueous phase is mixed with 0.5 ml 0.2n citrate buffer, pH 0.5, then 0.1 ml of a 2 percent aqueous stannous chloride solution and 2 ml of a 2 percent ninhydrin solution added. The mixture becomes 15 Heated at 100 ° C for a few minutes to extract the ethanolamine, its color reaction with ninhydrin at an absorption OD = 570 mμ is measured. The activity of the enzyme containing 1 μg Ethanolamine released per minute is defined as one unit.

(b) Glycerol kinase activity

From the following reagents, a reaction mixture is prepared:

0.2m Tris-hydrochloric acid buffer, pH 9.0 0.4 ml 0.1m glycerol 0.05 ml 10 mM ATP 0.1 ml 10 mmol MgCl₂ 0.1 ml Nitrotetrazolium blue (0.25%) 0.1 ml Bovine serum albumin (1%) 0.14 ml 10 mmol NAD 0.1 ml Phenazine methosulfate (0.05%) 0.01 ml Glycerol phosphate dehydrogenase (manufactured by Boehringer & Sohn AG, 2 mg / ml, 65 U / ml) 5 μl

1 ml of this reaction mixture is added during 5 minutes Preincubated at 37 ° C. with 50 μl of a glycerol kinase Solution (10 mM phosphate buffer containing 10 mM glycerol and diluted to a pH of 7.5, respectively). The mixture is allowed to react for 10 minutes at 37 ° C, then the reaction is stopped by adding 0.1N hydrochloric acid. The developed color becomes at a wavelength of 550 nm measured (A 550 nm). One unit of glycerol kinase is defined as the activity, which is 1 micromole of glycerol-3-phosphate per minute produced. The activity is calculated according to the following equation:

(c) Activity of glycerol phosphate oxidase

1 ml of the following reaction mixture is in a small Sample tube preincubated at 37 ° C for 3 minutes:

0.2m Tris-hydrochloric acid buffer, pH 8.0 | 0.2 ml Peroxidase (0.5 mg / ml, 45 U / ml) 0.1 ml 0.3 g / 100 ml 4-aminoantipyrine 0.1 ml 0.1m DL-glycerol-3-phosphate 0.1 ml 0.2vol% N, N-dimethylaniline 0.2 ml distilled water 0.3 ml

This mixture is mixed with 20 .mu.l of enzyme solution and 10 Allowed to react for a few minutes. The reaction is then by addition interrupted by 2.0 ml of 0.25 g / 100 ml sodium laurylbenzene sulphonate, the absorption of the product becomes at one wavelength measured at 565 nm.

The activity of the enzyme is calculated according to the following equation:

where ΔA is the absorption for 10 minutes at one wavelength of 565 nm.

The examples illustrate the invention.

Example 1 Create a calibration curve

The following solutions are produced:

(1) Phosphatidyl-glycerin-containing solution

A solution is prepared containing 1.0 μmol / ml phosphatidyl glycerol and 1.0% alkylaryl polyether alcohols (Triton X-100) contains. (The content of phosphatidyl-glycerol is determined by quantitative Determination of phosphate detected.)

(2) Ca⁺⁺ ion-containing buffer reaction medium 0.1 mol Tris-HCl buffer (pH 8) | 8.0 ml 1 mole of CaCl₂ 0.2 ml distilled water  1.8 ml 10 ml

(3) Phospholipase D-containing solution

100 ml of a solution (pH 8) consisting of 121.1 mg of tris- Buffer and 35 U / ml (0.6 mg / ml) phospholipase D, 50.0 mg Bovine serum albumin, 0.5 ml of a 20% solution of Alkylarylpolyätheralkoholen (Triton X-100) and water.

(4) 60 mM EDTA (5) A solution containing 40 mmol MgCl₂ and 160 mmol ATP

For this purpose, 1.0 ml of 0.2 mol of ATP, 0.10 ml of 0.5 mol MgCl₂ and 0.15 ml distilled water mixed so that 1.25 ml of the solution to be obtained.  

(6) Glycerol kinase solution

A solution containing 10 mM Tris-HCl buffer is used Contains 2 mg / ml glycerol kinase (pH 8).

(7) Glycerol phosphate oxidase solution

10 ml of a glycerol phosphate oxidase solution consisting of 10 mg Bovine serum albumin containing 3 mg / ml glycerol phosphate Oxidase, 560.75 mg of ammonium sulfate, 1.0 ml of 0.1 M Tris-hydrochloric acid buffer, pH 8, and containing 9 ml of water are prepared.

(8) Liquid glycerol phosphate oxidase-containing indicator mixture 4-aminoantipyrine | 0.24 ml aqueous phenol solution (10 mg / ml) 0.16 ml 1.25-percent alkylaryl polyether alcohol solution (Triton X-100) 0.8 ml 0.5m Tris-HCl buffer, pH 8 1.6 ml Peroxidase (90 U / ml in distilled water) 0.8 ml Glycerol phosphate oxidase solution (3 mg / ml) 0.8 ml distilled water 15.6 ml 20.0 ml

Each containing 0 to 0.10 ml of phosphatidyl-glycerol Solution becomes 1-percent Alkylarylpolyätheralkohol solution (Triton X-100) was added so that the total amount is 0.1 ml. This mixture is mixed with 0.1 ml Ca⁺⁺ buffer and heated to 37 ° C for 5 minutes. There are 0.05 ml of phospholipase D solution added, the mixture is at 37 ° C for 10 minutes react. After the reaction, 0.04 ml of 60 mmol EDTA and 0.075 ml of a 0.04m magnesium chloride Solution containing 160 mM ATP and 0.03 ml of the glycerol kinase solution contains, admitted. The mixture will react for 10 minutes at 37 ° C calmly. After the reaction, add 1.0 ml of the indicator mixture added, the mixture will react at 37 ° C for 20 minutes left, then the absorption at one wavelength measured at 500 nm (OD 500).  

The results are given in FIG . A satisfactory linear relationship is obtained in the range of 0.005 to 0.1 ml of phosphatidyl-glycerol-containing solution (phosphatidyl-glycerol content of 5 to 100 nmol).

A linear relationship is obtained when the phosphatidylglycerol Content is greater than 2 nmol, the sensitivity is pronounced.

example 2

5 to 6 ml of an amniotic fluid sample are dissolved in 18 ml of 2: 1 chloroform / methanol and centrifuged at 2000 rpm. There are 3 phases, of which the chloroform phase is evaporated to dryness under nitrogen protection. The total lipids are obtained, which are dissolved in 0.1 ml of chloroform and applied to a silica gel column (0.3 g). The neutral fat is obtained by elution with 10 ml of chloroform / methanol in the ratio 19: 1, the fraction containing phosphatidylglycerol is obtained by elution with 10 ml of chloroform / methanol in the ratio 2: 1. The solvent is removed, the residue is dissolved in 0.1 ml of a 1% Alkylarylpolyätheralkohol- solution (Triton X-100) and treated with 0.1 ml of Ca⁺⁺ buffer. The mixture is preheated to 37 ° C for 5 minutes, then treated with 0.05 ml of the phospholipase D solution and allowed to react for 10 minutes at 37 ° C. After the reaction, first 0.04 ml einr 60 mmol EDTA containing solution, then 0.075 ml of a 0.04m MgCl₂ solution containing 160 mmol of ATP and 0.03 ml of glycerol kinase solution was added. The mixture is allowed to react for 10 minutes at 37 ° C, then treated with 1.0 ml of the liquid indicator mixture. The mixture is allowed to react for 20 minutes at 37 ° C. The absorbance is measured at a wavelength of 500 nm, the content of phosphatidyl-glycerol (mg / dl) in the amniotic fluid samples is obtained from the calibration curve. The results are shown in Fig. 2, where × deaths, ○ the occurrence of RDS, Δ the occurrence of mild RDS cases, and ⚫ no RDS.

example 3

From the following ingredients, a reaction mixture for Quantitative determination of phosphatidyl-glycerol prepared:

0.2 mol Tris-HCl buffer, pH 7.5 | 0.2 ml 10 mmol CaCl₂ 0.2 ml 10 mmol MgCl₂ 0.2 ml 10% alkylaryl polyether alcohols (Triton X-100) 0.05 ml 10 mM ATP 0.2 ml Solution containing 20 U / ml phospholipase D, 5 U / ml glycerol kinase and 50 U / ml glycerol phosphate oxidase 0.1 ml 45 U / ml peroxidase 0.1 ml 0.3% 4-aminoantipyrine 0.2 ml 0.3% 3-methyl-N-ethyl-N- (β-hydroxyethyl) aniline 0.2 ml distilled water 0.55 ml 2,00 ml

2.0 ml of this mixture are mixed with 50 .mu.l of the liquid to be examined, which contains different amounts of phosphatidyl-glycerol (from 20 to 100 nmol). The mixture is allowed to react for 15 minutes at 37 ° C, then absorbance at a wavelength of 550 nm (OD₅₅₀) is measured. The results are shown in Fig. 3, and a calibration curve showing a satisfactory linear relationship with respect to the content of phosphatidyl-glycerol in the liquid to be examined is obtained.

Claims (9)

1. A method for the quantitative determination of phosphatidylglycerol in a body fluid to be examined, characterized in that the following steps are carried out:

• (a) exposing phospholipase D to a body fluid sample to release glycerol from phosphatidylglycerol contained in the sample;
• (b) forming glycerol-3-phosphate by exposing glycerol kinase to the released glycerol in the presence of a phosphate donor;
• (c) exposing glycerol phosphate oxidase to the glycerol-3-phosphate formed and then
• (d) determining the amount of oxygen consumed in the reaction (c) or hydrogen peroxide formed in the reaction (c).

2. Method for the quantitative determination according to claim 1, characterized in that the phosphate donor adenosine triphosphate is. 3. A method of quantitative determination according to claim 1, characterized in that the quantitative determination of Hydrogen peroxide using an indicator composition, the with hydrogen peroxide to a detectable Product reacts, is performed.   4. A method for the quantitative determination according to claim 3, characterized in that the indicator composition is a Contains substance with peroxidase activity and a chromogen. 5. A method of quantitative determination according to claim 4, characterized in that the chromogen is 4-aminoantipyrine and Phenol is. 6. Method for the quantitative determination according to claim 4, characterized in that the chromogen is 4-aminoantipyrine and 3-methyl-N-ethyl-N- (β-hydroxyethyl) -aniline. 7. Method for the quantitative determination according to claim 4, characterized in that the determination by measuring the Absorption performed at a wavelength of about 500 nm becomes. 8. A method for the quantitative determination according to claim 6, characterized in that the determination by measuring the Absorption performed at a wavelength of about 550 nm becomes. 9. The method according to claim 1 to 8, characterized that the body fluid is amniotic fluid.