DE3242652A1 - METHOD FOR THE QUANTITATIVE DETERMINATION OF PHOSPHATIDYL GLYCERINE - Google Patents

Description

Ohitocho-Mifuku, Tagata-gun, Shizuoka, Japan

"Method for the quantitative determination of phosphatidyl glycerine"

claimed priority:

November 20, 1981 _r Japan, No. 187486/81

In the treatment of newborns, respiratory distress syndrome (IBS) is a major problem in the perinatal period, and the resulting deaths make up a high percentage of total deaths. It is thought to be the dyspnoea syndrome is due to a lack of lung wetting agent. This remedy is important in preventing the Alveolar contractions of the lungs that expand immediately after birth. The lack of this lung wetting agent causes respiratory atelectasis and the emergence of IBS. Consequently, IBS could prevent or cause the occurrence of IBS mild case, when the occurrence of IBS could be detected in an early period and treatment of the newborn could be started early. Accordingly, the determination of this lung wetting agent is a necessity. There is roughly a method for determining the physical properties of the lung wetting agent and another in which the constituents of the lung wetting agent are biochemically examined will. The latter method has so far been carried out in such a way that the ratio of the lipids was determined as the ratio

or nis from lecithin to sphingomyelin, / that from phosphatidyl glycerol to phosphatidyl inositol, or / dipalmitoyl phosphatidyl choline was determined quantitatively by thin layer chromatography (Am. J. Obstet. Gynecol., 440, 109 (1971); 613, 125 (1976); 894, 133 (1979); Obstet. & Gynecol., 295, 57 (1981); Am. J. Obstet. Gynecol., 697, 138 (1980)). It has recently been recognized from the results of these various studies that the presence of phosphatidyl glycerin is an important factor in the occurrence of IBS. However, the quantitative determination of phosphatidyl glycerin was very difficult because the amount occurring is as little as about 1/10 the amount of lecithin. For the quantitative determination of phosphatidyl glycerol, thin-layer chromatography is known so far, in which, after removing the cells from the amniotic fluid by centrifugation _; the lipids are extracted from the supernatant, the components of this extract are separated by thin layer chromatography, each phospholipid is quantitatively determined, the ratios of the various phospholipids are calculated and the amount of phosphatidyl glycerol is obtained indirectly from the value of the total phospholipids, which is previously determined by quantitative Determination of phosphoric acid according to the wet combustion method (Am.J. Obstet. Gynecol. 613, 125 (1976); 1079, 135 (1979); 899, 133 (1979); 440, 109 (1971) J. Another method for the quantitative determination of phosphatidyl glycerol is high-speed liquid chromatography (Journal of Chromatography, 277, 223 (1981)). However, this method of thin-layer chromatography has the disadvantages that it takes 2 to 3 days to extract the lipid components from the amniotic fluid complicated measures and, in addition, heating to a high temperature for stain detection are necessary Further disadvantages are that it is an indirect method of determination which is based on the ratios of relative amounts of stains and total phospholipid and that several samples cannot be examined at the same time because of the use of thin layer chromatography.

The inventors made various studies in order to determine whether the liquid contains the various

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Seeking lipids, like amniotic fluid, only contains phosphatidyl glycerine can be determined quantitatively in a simple, functional and accurate manner in a short time.

The inventors have surprisingly found that phospholipase C / phospholipase C: 3.1.4.3 phosphatidyl choline phosphohydrolase /, which acts on lecithin and catalyzes the reaction to form 1,2-diglyceride and phosphatidylcholine, acts on phosphatidyl glycerin and forms glycerin-3-phosphate and diglyceride. They found a satisfactory one Method for the quantitative determination of phosphatidyl glycerol by quantitative determination of that formed in the reaction and then extracted glycerol-3-phosphate. Preferably only phosphatidyl glycerol can quantitatively very much in this process be determined satisfactorily by allowing the phospholipase C to act on the liquid to be examined for formation of glycerol-3-phosphate, glycerophosphate oxidase then acting on this glycerol-3-phosphate, and determining the amount of oxygen consumed or that formed in the reaction Hydrogen peroxide.

The present invention therefore relates to a method for the quantitative determination of phosphatidyl glycerol in the to investigating liquid, which is characterized in that glycerol-3-phosphate by the action of an enzyme that the The role of the catalyst in the reaction to form glycerol-3-phosphate and diglyceride from phosphatidyl glycerol and water plays, is released and then the released glycerol-3-phosphate is determined. The invention preferably relates to a method for the quantitative determination of phosphatidyl glycerol in the liquid to be examined, which is characterized in that the following steps (a), (b) and (c) are carried out will:

(a) Release of glycerol-3-phosphate by allowing phospholipase C to act on phosphatidyl glycerol,

(b) Exposing the. glycerol phosphate oxidase

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Glycerol-3-phosphate and

(c) Determining the amount of oxygen consumed or of hydrogen peroxide formed in the reaction.

The invention offers the advantages that each reagent can be prepared for the quantitative determination in a pack, the quantitative determination ^{can be carried out at a temperature of about 37 °} C., which is also easy to carry out and requires little time for the reaction.

In addition, the phosphatidyl glycerin can go down to a very low level Concentration can be precisely determined, the directly determined value for phosphatidyl glycerine turns out to be very expedient. As the determination is easy and expedient in a short time Time can be carried out, several samples can be examined at the same time. That is, the invention provides a suitable one Methods for the quantitative determination of phosphatidyl glycerol are available.

As an example of the enzyme that plays the role of the catalyst in the reaction to form glycerol-3-phosphate and diglyceride made up of phosphatidyl glycerine and water, phospholipase C can stand. This phospholipase C (E.C.3.1.4.3) is as Known enzyme that catalyzes the reaction of 1 mole each of diglyceride and phosphatidylcholine from 1 mole of lecithin and water. As the preparation serving as a catalyst in the aforementioned enzyme reaction, any preparation can be used, such as one obtained by extracting cells containing phospholipase C. or a commercially available one Enzyme preparation, e.g. an enzyme derived from microorganisms that is produced from the culture of phospholipase C. Microorganisms belonging to the genus Streptomyces such as Streptomyces hachijoensis A-T143 (FERM-P No. 1329), Streptomyces albireticuli IFO 12737, Streptomyces cinnamoneum IFO 12852 (Streptoverticillium cinnamoneum subsp. cinnamoneum IFO 12852), Streptomyces griseocarinensis IFO 12776 (Streptoverticillium griseocarneum IFO 12776) / under

the bacteria that belong to the Streptomyces strain / a distinction is made between those that form vortices in their aerobacteria and strep-

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called toverticillium (Baldacci, 1958) ^ 7 or Clostridium welchii and Bacillus cereus / tested and published JA-

Patent application No. 1356/1978, Lipid Metabolism P214 (1969JjT.

A commercially available phospholipase C preparation is also suitable.

The amount of phospholipase C to be used should be modified accordingly and adapted to the time required for the determination and to the concentration of phosphatidyl glycerol; it is not subject to any particular limitation. For example, phospholipase C can be used for a determination in an amount generally not less than 0.1 unit, preferably about 1 to 1 unit. In addition, this phospholipase C is preferably used after it has been dissolved in a buffer such as a weakly acidic to weakly alkaline tris-hydrochloric acid buffer, citric acid buffer, boric acid buffer, PIPES-NaOH buffer or imidazole buffer and, if necessary, a surfactant such as sodium deoxicholate or serum albumin is added to the solution. The enzyme solution containing phospholipase C and the liquid to be examined are mixed together. In the liquid to be examined, glycerine-3-phosphate is formed from phosphatidyl-glycerine with consumption of water. The mixing ratio of the two is not particularly limited; they can be mixed in such a ratio that about 1 to 100 units of phospholipase C are preferably contained per sample of the liquid to be examined. The reaction temperature can be about 37 ^° C. and the reaction time should be sufficient for the release of glycerol-3-phosphate, generally not less than 5 minutes, preferably not less than 10 minutes. The glycerol-3-phosphate released by this reaction is then determined quantitatively. The value for phosphatidyl glycerol in the liquid to be examined is obtained from the value of this quantitative determination.

For the quantitative determination of glycerol-3-phosphate you can various known methods for quantitative chemical

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Determination or those using enzymes can be used. A preferred, simple, and convenient enzymatic method for quantitative determination is the one in which one or more enzymes, the substrate of which is glycerol-3-phosphate, are used and quantifying the detectable change in enzyme action in the reaction. For example is the GPO process for quantitative determination particularly simple and expedient by adding glycerophosphate oxidase (GPO) to the released Glycerol-3-phosphate is allowed to act so that the oxygen in the reaction mixture is consumed to form hydrogen peroxide will.

The amount of oxygen consumed in the reaction liquid is measured with an oxygen electrode, the amount of Formed hydrogen peroxide is measured with a hydrogen peroxide electrode measured as an electrical change or the amount of oxygen or hydrogen peroxide is measured with an enzyme electrode which have been combined with an immobilized enzyme to form an oxygen or hydrogen peroxide electrode is. Immobilized enzymes can be produced in a number of ways, e.g. by entrapment, adsorption or binding, e.g. can GPO or GPO and PhosphoÜpase C in per se known Immobilized in a manner such as by having GPO or GPO and phospholipase C entrapped and immobilized in acrylamide will be, or GPO or GPO and Phospholipase C will be with one Protein, such as albumin, mixed, the proteins are cross-linked with each other and thus cause immobilization, or GPO or GPO and phospholipase C are included in collagen or fibroin or covalently bonded with it, or GPO or GPO and Phospholipase C are made by adsorption on a porous organic Polymer or immobilized by covalent bond with this, or GPO or GPO and phospholipase C are in a light-curing Resin trapped and immobilized. The immobilized enzyme can be in a membrane-like, fiber-like, granulate-like or. tube-like shape, which is preferably used for an enzyme electrode that is to a Oxygen electrode or hydrogen peroxide electrode are combined

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the should. The immobilized enzyme can be used as an enzyme electrode in which this immobilized enzyme is incorporated into the detector part of the electrode to be used, so that the amount of active enzyme used for quantitative determination by an electrical device is very small is.

The quantitative determination of hydrogen peroxide can also be carried out by using an indicator composition which forms a detectable material upon reaction with hydrogen peroxide. Preparations whose changes can be quantitatively determined by spectroscopic means are used as the indicator composition, e.g. a color reagent mixture whose color changes in the visible range, a fluorescent reagent which fluoresces when irradiated with ultraviolet light, or a light generating reagent. For example, a material containing a substance with peroxidase activity and a chromogen can be used as the color reagent composition. The peroxidase derived from horseradish is generally used as the substance with a peroxidase effect _and the combination of an electron acceptor and a phenolic compound is used as the chromogen. Compounds such as Γ. ·· ■ 4-aminoantipyrine, 2-hydrazine-benzothiazole, 3-methyl-2-benzothia- ^ are used as electron acceptors. zolone hydrazine or 2-aminobenzothiazole. Compounds such as phenol, 3-methyl-N-ethyl-N- (ß-hydroxyethyl) aniline, 3,5-xylenol, Ν, Ν-dimethylaniline or N, N, diethylaniline are used as phenolic components.

Various known compounds can be used as luminous substrates in a fluorescence reagent mixture or a light-generating reagent mixture, for example bis (2,4,6-trichloro-C-phenol) oxalate, pheny! Thiohydantoin, homovanillic acid, 4-hydro - J}, xyphenylacetic acid, vanillylamine, hi— »phloretin- %

. . 2,4, Sj-triphenvlimidazole ■ £

acidic, Hordenm, Lumxnol-Monoanion, Lucigenin or ^c / ■ * Each of these compounds can, if necessary, together with an electron acceptor and / or a substance with peroxidase effect for the quantitative determination of hydrogen peroxide

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be used.

There is no particular limitation on the amount of the enzyme or the choniogen used. So you can, for example, for an examination generally not less than 0.05 units, preferably 0.1 to 200 units of GPO, and generally not use less than 0.05 units, preferably 0.1 to 500 units, of peroxidase. You can also use a solution that with distilled water or weakly acidic or weakly alkaline buffer solution is adjusted so that the concentration of an electron acceptor or a phenolic compound is generally not less than 0.1 mmol. These reagents can can be used separately from the enzyme solution of phospholipase C or mixed with it, and these reagents in preparations for the. quantitative determination are packaged, such as freeze-dried preparations or integrated composite preparations, by applying them to filter paper or films.

This GPO quantitative method shows a high sensitivity to glycerol-3-phosphate in the liquid to be examined, and as it is caused by the impurities in the liquid being examined is not affected, it is an excellent method of making an accurate determination permitted.

The glycerol phosphate oxidase used for this determination method can be any, as long as it plays the role of a catalyst in the reaction for the formation of dihydroxyacetone phosphoric acid and hydrogen peroxide from glycerol-3-phosphate and oxygen, and for example from the strains Streptococcus, Lactobacillus or Lenconostoc originate; _It may also be glycerol phosphate oxidase produced by bacteria belonging to the Pediococcus strain (Japanese Unexamined Patent Application No. 72892/1978), an enzyme obtained from the culture of glycerol phosphate oxidase-producing bacteria which belong to the strain Aerococcus (Aerococcus viridans IFO 12219 and IFO 12317, unexamined Japanese patent application No. 15746/1980)

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and enzymes available on the market.

Another enzymatic method for quantitative determination Glycerol-3-phosphate becomes glycerol phosphate dihydrogenase, which takes the reaction to form dihydroxyacetone-phosphoric acid and NADH from glycerol-3-phosphate and nicotine-adenine-dinucleotide (NAD) catalyzed on the in the to be examined Solution released glycerol-3-phosphate left to act in the presence of NAD to form dihydroxyacetone-phosphoric acid and NADH. This NADH is then quantified, and from this the amount of glycerol-3-phosphate is calculated. This Methods employing quantitative determination of NADH are generally carried out by absorbance measurements at one wavelength performed from about 340 nm; 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 7 (Nitrotetrazolium blue) or 2,6-dichlorophenol-indophenol in Presence of diaphorase or phenazine methosulphate, the Color is measured at absorptions that correspond to the specific absorption wavelengths.

As for the enzymes and reagents, it is easy and convenient to use the commercially available preparations, the amount to be used can be selected accordingly. You can also use a surfactant if necessary Use agents or a stabilizer and incorporate them into various preparations.

As a liquid that is examined in the method according to the invention any sample is suitable as long as it contains phosphatidyl glycerine, e.g. body fluids such as samples of Amniotic fluid.

Used amniotic fluid as the fluid to be examined is used, the sample can be used as such or, if the content of phosphatidyl glycerol is too low, can

the sample can be concentrated. For example, 5 to 6 ml of amniotic fluid can be extracted with 15 to 18 ml of chloroform / methanol in a ratio of 2: 1; the chloroform phase is collected by centrifugation at 2000 rpm and evaporated to dryness under nitrogen protection. In this way the total lipid is obtained, which after dissolving in a certain amount of a 1 percent solution of alkylaryl polyether alcohols (Triton X-100) gives the solution which can be used as a sample of the amniotic fluid. In this way, any enzyme solution, such as that of phospholipase C or that of glycerol phosphate oxidase for quantitative determination, as well as other reagents, can act on the solution to be examined simultaneously or one after the other. The ratio of liquid to be examined to enzyme solution is not particularly critical, in general about 0.1 to 3 ml of enzyme solution are used for 0.01 to 1 ml of liquid to be examined. The determination is preferably carried out at a temperature of about 37 ^° C., the reaction time can be as long as the reaction is complete; it is generally not less than 5 minutes, preferably not less than 10 minutes. As the reaction medium, water or a weakly acidic or weakly alkaline buffer solution can be used as a solvent for each reagent.

The method according to the invention for quantitative determination of phosphatidylglycerin can be carried out directly to an extremely low concentration in a very short time. In addition, no complicated measures are necessary, the procedure can be carried out at normal temperature.

The activity of phospholipase C and the activity of glycerol phosphate oxidase can be determined as follows:

(a) Phospholipase C activity

0.1 ml of a 4 percent solution of phosphatidylcholine, the separated from egg yolk and purified, 0.3 ml 0.1 M tris-hydrochloric acid buffer (pH 8.5) and 0.1 ml of a 0.02 m

/ 14

aqueous magnesium chloride solution are mixed and 0.1 ml of the enzyme solution containing phospholipase C is added. The mixture is allowed to react at 37 ⁰ C for 15 minutes, the reaction is stopped by adding 0.8 ml of trichloroacetic acid and 0.1 ml 3,6prozentiger 5,5prozentigen serum albumin. The reaction mixture is allowed to stand in a water bath for 20 minutes, then filtered through Toyo filter paper (No. 5B). 0.5 are decomposed in a Kjeldahl flask with 0.55 ml perchloric acid 60prozentiger for 2 hours at 170 ⁰ C ml of the filtrate. The color developed by the released inorganic phosphorus by the addition of 0.5 ml of 2,4-aminophenol hydrochloride sulfide (amidosulfite), 0.25 ml of 3.3 percent ammonium molybdate and 3.2 ml of water is after standing at room temperature for 20 minutes at a Absorbance determined by 650 iv.ii. The activity of the enzyme, which releases one μΜοΙ inorganic phosphorus per minute, is defined as 1 unit (U).

(b) Activity of glycerol phosphate oxidase

0.2 ml of 0.2 M tris-hydrochloric acid buffer, pH 8.0, 0.1 ml of a peroxidase solution with 0.5 mg / ml (45 U / ml), 0.1 m 0.3 g / 100 ml 4-Aminoantipyrine, 0.1 ml of 0.1 M DL-glycerol-3-phosphate, 0.2 ml of 0.2% by volume of N, N-dimethylaniline and 0.3 ml of distilled water are placed in a small test tube for 3 minutes preheated to 37 ⁰ C and treated with 20 ul of a GPO-containing enzyme solution was added. The mixture is left to react for 10 minutes, then the reaction is stopped by adding 2.0 ml of 0.25 g of sodium laurylbenzene sulfonate / 100 ml. The absorbance of the product is measured at a wavelength of 565 nm.

The activity of the enzyme is calculated using the following equation:

Activity of the enzyme (U / ml) = (4 \) x ~

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

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The examples illustrate the invention.

example 1

A reaction mixture for the quantitative determination of phosphatidyl glycerine is prepared from the following reagents:

0.2 M dimethyl glutarate-NaOH buffer (pH 7.5) 0.2 ml

10 mmol CaCl ₂ 0.2 ml

TO% sodium deoxicholate 0.05 ml

1% bovine serum albumin 0.1 ml solution containing 40 U / ml phospholipase C.

"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-Methy1-N-äthy1-N- (ß-hydroxyethyl) aniline 0.2 ml

distilled water 0.85 ml

Total amount 2.0 ml

2.0 ml of this mixture are mixed with 50 μl of the solution to be examined, which contains various amounts of phosphatidylglycerol (from 20 to 100 nmoles). The mixture is allowed to ^{react for 25 minutes at 37 °} C., the color development of the pigment formed during the reaction is determined by absorption ) at a wavelength of 550 nm (OD 550).

The results are shown in FIG. 1, and a satisfactory one is obtained linear relationship with respect to the phosphatidyl glycerol content of the solution tested.

Example 2

A reaction mixture for the quantitative determination of phosphatidyl glycerine is prepared from the following reagents:

0.05 ml 0.1 ml 0.1 ml 0.2 ml 0.2 ml 0.1 ml 1, 05 ml

0.2 M dimethyl glutarate-NaOH buffer (pH 7.5) 0.2 ml

10% sodium deoxicholate

Solution containing 20 U / ml phospholipase C

and 50 U / ml GPO 45 U / ml peroxidase 0.3% 4-aminoantipyrine 0.3% phenol

1% bovine serum albumin distilled water

. Total amount _ 2.0 ml

^ 2.0 ml of the solution are mixed with 50 μΐ of the solution to be examined, which contains various amounts of phosphatidyl glycerol (from 10 to 100 η mol), added. The mixture becomes 15 Minutes at 37 ° C. reacted, the color developed by the pigment formed in the reaction is by absorption determined at a wavelength of 500 nm (OD 500).

The results are shown in FIG. 2, and a satisfactory one is obtained linear relationship to the content of phosphatidyl glycerine.

Claims (14)

Claims 1. A method for the quantitative determination of phosphatidyl glycerol in the liquid to be examined, characterized in that glycerol-3-phosphate by the action of an enzyme that acts as a catalyst in the reaction to form glycerol-3-pho ^ Sphat and diglyceride from phosphatidyl glycerin and water plays, is released and then the released Glycerol-3-phosphate is determined. 2. Method for quantitative determination according to claim 1, characterized in that the enzyme which plays the role of the catalyst in the reaction for the formation of glycerol-3-phosphate and diglyceride made from phosphatidyl glycerin and water, phospholipase C is. 3. A method for quantitative determination according to claim and 2, characterized in that the enzyme phospholipase C (EC3.1.4.3) is. 4. A method for quantitative determination according to claim 3, characterized in that the to be examined Fluid is a body fluid. 5. Method for the quantitative determination of phosphatidyl glycerol according to claim 1, characterized in that the following steps are carried out: (a) Release of glycerol-3-phosphate by allowing phospholipase C to act on phosphatidyl glycerol, (b) Allowing glycerol phosphate oxidase to act on glycerol-3-phosphate and subsequently (c) Determining the amount of oxygen consumed or at ■ -> - ■ hydrogen peroxide formed in the reaction. 6. A method for quantitative determination according to claim 5, characterized in that the quantitative determination of oxygen is carried out by an electrochemical method using an oxygen electrode. 7. A method for quantitative determination according to claim 5, characterized in that the quantitative determination of the Hydrogen peroxide by an electrochemical method using a hydrogen peroxide electrode will. 8. A method for quantitative determination according to claim 5, characterized in that the quantitative determination of the Hydrogen peroxide using an indicator composition, which turns into a detectable, through the reaction converts product formed with hydrogen peroxide will. 9. A method for quantitative determination according to claim 8, characterized in that the indicator composition a color developing, fluorescent, or light generating composition is. 10. A method for quantitative determination according to claim 9, characterized in that the color-developing composition a substance with peroxidase activity and a dye precursor contains. 11. A method for quantitative determination according to claim 10, characterized in that the dye precursor is 4-Aminoan.tipyrin and is phenol. 12. A method for quantitative determination according to claim 10, characterized in that the dye precursor is 4-aminoantipyrine and 3-methyl-N-ethyl-N- (ß-hydroxyethyl) aniline is. 13. A method for quantitative determination according to claim 11, characterized in that the determination is carried out by measuring the absorption at a wavelength of about 500 nm will. 14. Method for quantitative determination according to claim 12, characterized in that the determination is carried out by measuring the absorption at a wavelength of approximately 550 nm will.