CA1201047A - Method for quantitative measurement of phosphatidyl glycerol - Google Patents
Method for quantitative measurement of phosphatidyl glycerolInfo
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- CA1201047A CA1201047A CA000416607A CA416607A CA1201047A CA 1201047 A CA1201047 A CA 1201047A CA 000416607 A CA000416607 A CA 000416607A CA 416607 A CA416607 A CA 416607A CA 1201047 A CA1201047 A CA 1201047A
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- glycerol
- quantitative measurement
- phosphatidyl glycerol
- reaction
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2405/00—Assays, e.g. immunoassays or enzyme assays, involving lipids
- G01N2405/04—Phospholipids, i.e. phosphoglycerides
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Abstract
ABSTRACT
METHOD FOR QUANTITATIVE MEASUREMENT OF PHOSPHATIDYL GLYCEROL
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 phos-phatidyl 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 quantita-tively measured preferably by GK-GPO type method.
METHOD FOR QUANTITATIVE MEASUREMENT OF PHOSPHATIDYL GLYCEROL
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 phos-phatidyl 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 quantita-tively measured preferably by GK-GPO type method.
Description
~2~10~7 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 decreased owing to the im-proved 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 account for a high percentage of deaths.
It is regarded that RDS occurs owing to the lack of pulmo-nary 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. Conse~uently, 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 measure-ment of this pulmonary surfactant is necessary.
The means are roughly classified into the method for the measurement wherein the physical property of pulmo-nary surfactant is utilized and one wherein compositive ~omponents of pulmonary surfactant are biochemically 2S resolved. The latter method has been carried out by obtain-ing 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 30 chromatography IAm. J. Obstet. Gynecol., 440: 109 (1971);
Am. J. Obstet. Gynecol., 613: 125 (1976); Am. J. Obstet.
Gynecol., 894: 133 (1979); Obstet. & GynPcol., 295: 57 ~1981);
Am. J. Obstet. Gynecol., 697: 138 (1980); etc.].
~
j~!s 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 difficul~ 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 componen~s are extracted from the supernatant, the components of this extract are sepa-rated by the thin-layer chromatography, each phospholipid is quantitati~ely 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 lAm. J.
Obstet. Gynecol., 613: 125 tl976); Am. J. Obstet. Gynecol., 1079: 135 tl979); Am. J. Obstet. Gynecol., 899: 133 (1979);
Am. J. Obstet. Gynecol., 440: 109 (1971~]~ or the method for the ~uantitative measurement wherein the high speed liquid chromatography is used [Journal of Chromatography, 277: 223 (1981)]. However, this method of thin-layer chromatography had disddv~ Ps in that it takes tw~ 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 def~cts 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 trea~ment of many samples because of the thin-layer chromatography.
The present inventors carried out various studies ~ ., ,~'' ~2~
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 f~und quite unexpectedly that phospholipase D acts on phosphatidyl glycerol producing glycerol and phosphatidic acid and they achieved a satisfac-tory method for the ~uantitative measurement of only phospha-tidyl glycerol by quantitatively measuring this glycerolproduced by the reaction and extricated.
More preferably, there has been achieved a method whereby only phosphatidyl glycerol can be markedly satisfac-torily measured quantitatively by allowing phospholipase D~o 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 con-sumed or hydrogen peroxide produced in the reaction~
The present in~ention 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 ~lycerol by the action of an enzyme which plays a role of the cata-lyst in the reac~ion to produce glycerol and phosphatidic acid from phosphatidyl glycerol and water and then quantita-tively measuring glycerol produced. Preferably, it relatesto a method for the quantitative measurement of phosphatidyl glycerol in the liquid to be inspected whic`h comprises the combination of the following steps (a), (b), (c) and ~d):
(a) liberating glycerol by allowing phospholipase D
to act on phosphatidyl glycerol, :~Z~ '7 (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 37C 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 quantita-tive measurement of phosphatidyl glycerol.
In the drawings attached to this ~ecific~t;~ and illustrating embodiments of the present invention:
~ig. 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 0~
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 ex~raction of phospholipase D-con~a;n;ng cells or an enzyme reagent sold on the mar~et,for example, a microorganism-originated enzyme obtained from the culture of a phospholipase D--producing bacterium which belongs to a Streptomyces genus l[Streptomyces hachijoensis A-1143 strain ~FERM-P No. 1329); Japanese examined patent publication No. 39918J1977, Streptomyces chromofussus A-0848 strain (FERM-P No. 3519); Japanese un~m;ned patent publi-cation No. 130984/1977], etc. and enzyme reagents of phospho-lipase D sold on the market.
lS 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 phos-phatidyl 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 phospho-lipase D is preferably used after it is dissolved in a buffer such as weak acidic to weak alkali Tris-HCQ buffer, citric acid buffer, boric acid buffer, PIPES-NaOH buffer or imida-zole buffer and, if necessary, it may be adjusted by addingthereto a nonionic surfactant such asl'Triton X-100"or serum albumin. Then, the enzyme solution cont~ining phospholipase D thus adjusted and the liquid to be inspected are mixed and glycerol and phosphatidic acid are produced from phospha-tidyl glycerol in the liquid to be inspected with the consump-tion of water. The mixing ratio of the both is not particu~
larly 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 37C and the reaction time may be what is sufficient for the liberation of glycerol, usually * Tr~ .of Rohm and Haas Ca~ny for octyl~h~n~y polyethoxy ethanol.
., 12~10~7 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 obtainea 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 en2ymes.
Preferably, an enzymatic method for the quantitative measure-ment 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 quanti-tatively 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 ~he presence of a phosphate donor such as adeno-sine triphosphate (ATP) and glycerolkinase (GK), thenglycerophosphate oxidase (~PO) is allowed to act on glycero-3-phosphate so that the oxygen in the reaction liquid is consumed to produce hydrogen pèroxide is particularly simple and convenient.
In this GK~ type method, the quantity of oxygen consumed in the reaction liquid is measured with an oxyqen electrode or the quantity of hydrogen peroxide produced is measured wit~ a ~ydrogen 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 elec-trode or hydrogen peroxide electrode and an immobiliz~d 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 elec~rode. In this embodiment, , ., . .
o~
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, pclymerized 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; cova-lent-bonding method using colla~en or fibroin; ads~rption onto or covalent-bonding with a porous organic 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 measure-ment may be carried out by a spectrophotometric methodusing an indicator which is produced by the reaction with hydrogen peroxide. As the indicator, there are usually used ~hat wherein change can be quantitatively measured by a spectrophotometric means, for instance, a coloration reagent wherein color change occurs in the visible range, a fluores-cence reagen~ composition which fluoresces or a luminescent reagent composition which is luminescent by ultraviolet rays irradiation. For example, as à coloration reagent composition, there is used a material cont~; ni ng a substance having per-oxidase action and a chromogen. As the substance having theperoxidase 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, 2-hydrazinobenzothiazole, 3-methyl-2-benzothiazolone hydrazine, 2-aminobenzothiazole or the like. As a hydrogen donor, there is used, for example, phenol, 3-methy~-N-ethyl-N-(~-hydroxyethyl)aniline, 3,5-xylenol, N,N-dimethylaniline, N,N-diethylaniline or the like.
As luminous substrates in a ~luorescence reagent ~, 0~
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 measure-ment shows a h:igh sensitivity to the glycerol produced in the liquid to be inspected and, since it i5 not affected by the impurity in the liquid to be inspected, it is an excellent method whereby an accurate measurement can be carried out.
iZO10~
g 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 (1957)], 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 unP~m;ned 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-3-phosphate and oxygen, for example, Streptococcus genus, Lactobacillus genus, Leuconostoc genus, glycerophos-phate oxidase producing bacteria which belong to Pediococcus genus ~Japanese unPx~m;ne~ patent publication No. 72892/1978), an enzyme obtained from the culture of glycerophosphate oxidase-producing bacteria which belong to Aerococcus genus (Aerococcus viridans IFO 12219 strain and IFO 12317 strain, Japanese unexamined patent publication No. 15746/1980) and enzvmes 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 glycero-3-phosphate and ADP, then glycerophos-phate 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 12~1047 .
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-thia-zolyl-2H-tetrazolium bromide, 2-(p-iodophenyl~-3-~p-nitro-5 phenyl)-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 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 dihydroxy-acetone 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 lac~ate dehydrogenase is allowed to act on pyruvic acid in the presence of reduced NAD 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 . .
~2t~L0~7 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 c~mposition 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 inventiont there can be illustrated any sample as far as it contains phosphatidyl glycerol, for instance, bod~ 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 mQ of liquor amniotic fluid sampled is extracted with 15 - lg mQ
of chloroform-methanol (2:1), the chloroform layer is collected by centrifuging at 20~0 rpm and it is evaporated to dryness in nitrogen gas to obtain the to~al lipid. Then, a~ter 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 simultaneousl~. In this case, there is no particular * Trademark 0~1~
limitation on the use ratio of the liquid to be inspected to the enzyme reagent and the like and usually, about 0.1 -3 mQ of the enzyme reagent or the like is used for 0.01 mQ -1 mQ of the liquid to be inspected. As the reaction condi-tion, it is preferable to conduct the reaction at about 37Cand, 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/dQ when the liquor amnii of 5 - 6 mQ is used, can be measured. This means that the quantity of phosphatidyl glycerol in an extremely low con-centration compared to the value of 0.2 mg/Q thereof which isthe critical level for the occurrence of RDS can be measured.
In addition, no complicated operation is needed and the opera-tion can be carried out at the normal temperature. Consequently, this is a good method for the quantitative measurement. 5 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 0 methods:
(a) Method for the measurement of the activity of phospholipase D
l'o 0.1 mQ of 4% yolk phosphatidyl ethanolamine solution are added and mixed 0.8 mQ of 0.05 ~ Tris-hydro~
chloric acid buffer (pH 7.5), 0.3 mQ of 1% Triton X-100, 0.3 mQ of water and 0.2 mQ of 10 mM aqueous ~olution of CaCQ2 and the mixture is subjected to ultrasonic treatment for 10 minutes. To this are added 0.2 mQ of ethyl ether and O.1 mQ of enzyme solution. The mixture is allowed to react at 37C for 20 minutes and the reaction is suspended by add-ing 0.3 mQ of 20% trichloroacetic acid. The reaction liquid is washed twice with 4 mQ of ethyl ether, 0.5 mQ of 0.2 N
citrate buffer (pH 5.0) is added to l mQ of the water layer, 0.1 mQ of 2% aqueous solution of SnCQ2 and 2 mQ of 2% nin-hydrin solution are added thereto, the mixture is heated at100C for 15 minutes to extricate ethanolamine, which is allowed to give forth color by the ninhydrin reaction and the absorbance is measured at OD 570 m~. The activity of enzyme which extricates 1 ~g 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 mQ
0.1 M glycerol 0.05mQ
2Q 10 mM ATP 0.1 mQ
10 mM MgCQ2 0.1 mQ
0.25% Nitrotetrazolium Blue 0.1 mQ
1~ Bovine serum albumin 0.14mQ
10 mM NAD 0.1 mQ
0.05% phenazine methosulfate 0.01mQ
Glycerophosphate dehydrogenase (manufactured by Boehringer Sohn AG,
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 decreased owing to the im-proved 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 account for a high percentage of deaths.
It is regarded that RDS occurs owing to the lack of pulmo-nary 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. Conse~uently, 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 measure-ment of this pulmonary surfactant is necessary.
The means are roughly classified into the method for the measurement wherein the physical property of pulmo-nary surfactant is utilized and one wherein compositive ~omponents of pulmonary surfactant are biochemically 2S resolved. The latter method has been carried out by obtain-ing 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 30 chromatography IAm. J. Obstet. Gynecol., 440: 109 (1971);
Am. J. Obstet. Gynecol., 613: 125 (1976); Am. J. Obstet.
Gynecol., 894: 133 (1979); Obstet. & GynPcol., 295: 57 ~1981);
Am. J. Obstet. Gynecol., 697: 138 (1980); etc.].
~
j~!s 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 difficul~ 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 componen~s are extracted from the supernatant, the components of this extract are sepa-rated by the thin-layer chromatography, each phospholipid is quantitati~ely 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 lAm. J.
Obstet. Gynecol., 613: 125 tl976); Am. J. Obstet. Gynecol., 1079: 135 tl979); Am. J. Obstet. Gynecol., 899: 133 (1979);
Am. J. Obstet. Gynecol., 440: 109 (1971~]~ or the method for the ~uantitative measurement wherein the high speed liquid chromatography is used [Journal of Chromatography, 277: 223 (1981)]. However, this method of thin-layer chromatography had disddv~ Ps in that it takes tw~ 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 def~cts 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 trea~ment of many samples because of the thin-layer chromatography.
The present inventors carried out various studies ~ ., ,~'' ~2~
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 f~und quite unexpectedly that phospholipase D acts on phosphatidyl glycerol producing glycerol and phosphatidic acid and they achieved a satisfac-tory method for the ~uantitative measurement of only phospha-tidyl glycerol by quantitatively measuring this glycerolproduced by the reaction and extricated.
More preferably, there has been achieved a method whereby only phosphatidyl glycerol can be markedly satisfac-torily measured quantitatively by allowing phospholipase D~o 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 con-sumed or hydrogen peroxide produced in the reaction~
The present in~ention 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 ~lycerol by the action of an enzyme which plays a role of the cata-lyst in the reac~ion to produce glycerol and phosphatidic acid from phosphatidyl glycerol and water and then quantita-tively measuring glycerol produced. Preferably, it relatesto a method for the quantitative measurement of phosphatidyl glycerol in the liquid to be inspected whic`h comprises the combination of the following steps (a), (b), (c) and ~d):
(a) liberating glycerol by allowing phospholipase D
to act on phosphatidyl glycerol, :~Z~ '7 (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 37C 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 quantita-tive measurement of phosphatidyl glycerol.
In the drawings attached to this ~ecific~t;~ and illustrating embodiments of the present invention:
~ig. 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 0~
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 ex~raction of phospholipase D-con~a;n;ng cells or an enzyme reagent sold on the mar~et,for example, a microorganism-originated enzyme obtained from the culture of a phospholipase D--producing bacterium which belongs to a Streptomyces genus l[Streptomyces hachijoensis A-1143 strain ~FERM-P No. 1329); Japanese examined patent publication No. 39918J1977, Streptomyces chromofussus A-0848 strain (FERM-P No. 3519); Japanese un~m;ned patent publi-cation No. 130984/1977], etc. and enzyme reagents of phospho-lipase D sold on the market.
lS 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 phos-phatidyl 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 phospho-lipase D is preferably used after it is dissolved in a buffer such as weak acidic to weak alkali Tris-HCQ buffer, citric acid buffer, boric acid buffer, PIPES-NaOH buffer or imida-zole buffer and, if necessary, it may be adjusted by addingthereto a nonionic surfactant such asl'Triton X-100"or serum albumin. Then, the enzyme solution cont~ining phospholipase D thus adjusted and the liquid to be inspected are mixed and glycerol and phosphatidic acid are produced from phospha-tidyl glycerol in the liquid to be inspected with the consump-tion of water. The mixing ratio of the both is not particu~
larly 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 37C and the reaction time may be what is sufficient for the liberation of glycerol, usually * Tr~ .of Rohm and Haas Ca~ny for octyl~h~n~y polyethoxy ethanol.
., 12~10~7 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 obtainea 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 en2ymes.
Preferably, an enzymatic method for the quantitative measure-ment 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 quanti-tatively 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 ~he presence of a phosphate donor such as adeno-sine triphosphate (ATP) and glycerolkinase (GK), thenglycerophosphate oxidase (~PO) is allowed to act on glycero-3-phosphate so that the oxygen in the reaction liquid is consumed to produce hydrogen pèroxide is particularly simple and convenient.
In this GK~ type method, the quantity of oxygen consumed in the reaction liquid is measured with an oxyqen electrode or the quantity of hydrogen peroxide produced is measured wit~ a ~ydrogen 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 elec-trode or hydrogen peroxide electrode and an immobiliz~d 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 elec~rode. In this embodiment, , ., . .
o~
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, pclymerized 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; cova-lent-bonding method using colla~en or fibroin; ads~rption onto or covalent-bonding with a porous organic 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 measure-ment may be carried out by a spectrophotometric methodusing an indicator which is produced by the reaction with hydrogen peroxide. As the indicator, there are usually used ~hat wherein change can be quantitatively measured by a spectrophotometric means, for instance, a coloration reagent wherein color change occurs in the visible range, a fluores-cence reagen~ composition which fluoresces or a luminescent reagent composition which is luminescent by ultraviolet rays irradiation. For example, as à coloration reagent composition, there is used a material cont~; ni ng a substance having per-oxidase action and a chromogen. As the substance having theperoxidase 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, 2-hydrazinobenzothiazole, 3-methyl-2-benzothiazolone hydrazine, 2-aminobenzothiazole or the like. As a hydrogen donor, there is used, for example, phenol, 3-methy~-N-ethyl-N-(~-hydroxyethyl)aniline, 3,5-xylenol, N,N-dimethylaniline, N,N-diethylaniline or the like.
As luminous substrates in a ~luorescence reagent ~, 0~
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 measure-ment shows a h:igh sensitivity to the glycerol produced in the liquid to be inspected and, since it i5 not affected by the impurity in the liquid to be inspected, it is an excellent method whereby an accurate measurement can be carried out.
iZO10~
g 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 (1957)], 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 unP~m;ned 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-3-phosphate and oxygen, for example, Streptococcus genus, Lactobacillus genus, Leuconostoc genus, glycerophos-phate oxidase producing bacteria which belong to Pediococcus genus ~Japanese unPx~m;ne~ patent publication No. 72892/1978), an enzyme obtained from the culture of glycerophosphate oxidase-producing bacteria which belong to Aerococcus genus (Aerococcus viridans IFO 12219 strain and IFO 12317 strain, Japanese unexamined patent publication No. 15746/1980) and enzvmes 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 glycero-3-phosphate and ADP, then glycerophos-phate 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 12~1047 .
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-thia-zolyl-2H-tetrazolium bromide, 2-(p-iodophenyl~-3-~p-nitro-5 phenyl)-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 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 dihydroxy-acetone 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 lac~ate dehydrogenase is allowed to act on pyruvic acid in the presence of reduced NAD 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 . .
~2t~L0~7 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 c~mposition 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 inventiont there can be illustrated any sample as far as it contains phosphatidyl glycerol, for instance, bod~ 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 mQ of liquor amniotic fluid sampled is extracted with 15 - lg mQ
of chloroform-methanol (2:1), the chloroform layer is collected by centrifuging at 20~0 rpm and it is evaporated to dryness in nitrogen gas to obtain the to~al lipid. Then, a~ter 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 simultaneousl~. In this case, there is no particular * Trademark 0~1~
limitation on the use ratio of the liquid to be inspected to the enzyme reagent and the like and usually, about 0.1 -3 mQ of the enzyme reagent or the like is used for 0.01 mQ -1 mQ of the liquid to be inspected. As the reaction condi-tion, it is preferable to conduct the reaction at about 37Cand, 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/dQ when the liquor amnii of 5 - 6 mQ is used, can be measured. This means that the quantity of phosphatidyl glycerol in an extremely low con-centration compared to the value of 0.2 mg/Q thereof which isthe critical level for the occurrence of RDS can be measured.
In addition, no complicated operation is needed and the opera-tion can be carried out at the normal temperature. Consequently, this is a good method for the quantitative measurement. 5 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 0 methods:
(a) Method for the measurement of the activity of phospholipase D
l'o 0.1 mQ of 4% yolk phosphatidyl ethanolamine solution are added and mixed 0.8 mQ of 0.05 ~ Tris-hydro~
chloric acid buffer (pH 7.5), 0.3 mQ of 1% Triton X-100, 0.3 mQ of water and 0.2 mQ of 10 mM aqueous ~olution of CaCQ2 and the mixture is subjected to ultrasonic treatment for 10 minutes. To this are added 0.2 mQ of ethyl ether and O.1 mQ of enzyme solution. The mixture is allowed to react at 37C for 20 minutes and the reaction is suspended by add-ing 0.3 mQ of 20% trichloroacetic acid. The reaction liquid is washed twice with 4 mQ of ethyl ether, 0.5 mQ of 0.2 N
citrate buffer (pH 5.0) is added to l mQ of the water layer, 0.1 mQ of 2% aqueous solution of SnCQ2 and 2 mQ of 2% nin-hydrin solution are added thereto, the mixture is heated at100C for 15 minutes to extricate ethanolamine, which is allowed to give forth color by the ninhydrin reaction and the absorbance is measured at OD 570 m~. The activity of enzyme which extricates 1 ~g 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 mQ
0.1 M glycerol 0.05mQ
2Q 10 mM ATP 0.1 mQ
10 mM MgCQ2 0.1 mQ
0.25% Nitrotetrazolium Blue 0.1 mQ
1~ Bovine serum albumin 0.14mQ
10 mM NAD 0.1 mQ
0.05% phenazine methosulfate 0.01mQ
Glycerophosphate dehydrogenase (manufactured by Boehringer Sohn AG,
2 mg/mQ, 65 U/mg) 5 ~Q
One ~ ;ter of the reaction mixture each having a composition mentioned above is preincubated at 37C for 5 minutes. To this is added 50 ~Q of a solution contain;ng GK
(10 mM phosphate buffer cont~;~ing 10 mM of glycerol, appro-priately diluted at a pH 7.5). The mixture is allowed to react at 37C for 10 minutes. Then, after the reaction is suspended by the addition of 0.1N HcQ~ the color given forth :~L2C~10~
is measured at a wave length of 550 nm to obtain the absorbance (~ 550 nm). One unit of GK i5 defined as the activity to produce 1 ~mole of glycero-3-phosphate per minute. The equation for the calculation is as follows:
U/mQ = ~A x0di05utilon0 ratio x 1 = ~2 x dilution ratio (c) Method for the measurement of the activity of GPO
0.2 M Tris-hydrochloric acid buffer (pH 8.0) 0.2 mQ
peroxidase (0.5 mg/mQ, 45 U/mQ) 0.1 mQ
0.3 (W/V) 4-aminoantipyrine 0.1 mQ
0.1 M DL-glycero-3-phosphate 0.1 mQ
0.2% (V/V) N,N-dimethylaniline 0.2 mQ
Distilled water 0.3 mQ
One milili~er of the reaction mixture having the composition mentioned above is charged in a small test tube and preincubated at 37C for 3 minutes. To this is added 20 ~Q of enzyme solution and the mixture is allowed to react for 10 minutes. Then, the reaction is suspended by the addition of 2.0 mQ of 0.~5% (W/V) sodium laurylbenzene sulfonate and the absorbance of the product is measured at a wave length of 565 nm.
The activity of the enzyme is calculated in accordance with the following equation:
Activity of enzyme (U/mQ) = (~6 0) x (10) wherein ~A shows the absorbance for 10 minutes at a wave length o 565 nm.
~ exeafter the embodiment of the present invention will be mentioned with practical examples. However, the present invention is never limited by them.
. ~ . - .. . . :
---`" lZ6~0~7 Example 1 [Preparation of calibration curve]
A solution having the following composition was prepared.
(1) solution conta;n;ng phosphatidyl glycerol:
There was prepared a 1.0 ~mole/mQ phosphatidyl glycerol solution cont~;n;ng 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-HCQ buffer (pEI 8) 8.0 mQ
1 M CaCQ2 0.2 mQ
Distilled water 1.8 mQ
Total 10 mQ
One ~ ;ter of the reaction mixture each having a composition mentioned above is preincubated at 37C for 5 minutes. To this is added 50 ~Q of a solution contain;ng GK
(10 mM phosphate buffer cont~;~ing 10 mM of glycerol, appro-priately diluted at a pH 7.5). The mixture is allowed to react at 37C for 10 minutes. Then, after the reaction is suspended by the addition of 0.1N HcQ~ the color given forth :~L2C~10~
is measured at a wave length of 550 nm to obtain the absorbance (~ 550 nm). One unit of GK i5 defined as the activity to produce 1 ~mole of glycero-3-phosphate per minute. The equation for the calculation is as follows:
U/mQ = ~A x0di05utilon0 ratio x 1 = ~2 x dilution ratio (c) Method for the measurement of the activity of GPO
0.2 M Tris-hydrochloric acid buffer (pH 8.0) 0.2 mQ
peroxidase (0.5 mg/mQ, 45 U/mQ) 0.1 mQ
0.3 (W/V) 4-aminoantipyrine 0.1 mQ
0.1 M DL-glycero-3-phosphate 0.1 mQ
0.2% (V/V) N,N-dimethylaniline 0.2 mQ
Distilled water 0.3 mQ
One milili~er of the reaction mixture having the composition mentioned above is charged in a small test tube and preincubated at 37C for 3 minutes. To this is added 20 ~Q of enzyme solution and the mixture is allowed to react for 10 minutes. Then, the reaction is suspended by the addition of 2.0 mQ of 0.~5% (W/V) sodium laurylbenzene sulfonate and the absorbance of the product is measured at a wave length of 565 nm.
The activity of the enzyme is calculated in accordance with the following equation:
Activity of enzyme (U/mQ) = (~6 0) x (10) wherein ~A shows the absorbance for 10 minutes at a wave length o 565 nm.
~ exeafter the embodiment of the present invention will be mentioned with practical examples. However, the present invention is never limited by them.
. ~ . - .. . . :
---`" lZ6~0~7 Example 1 [Preparation of calibration curve]
A solution having the following composition was prepared.
(1) solution conta;n;ng phosphatidyl glycerol:
There was prepared a 1.0 ~mole/mQ phosphatidyl glycerol solution cont~;n;ng 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-HCQ buffer (pEI 8) 8.0 mQ
1 M CaCQ2 0.2 mQ
Distilled water 1.8 mQ
Total 10 mQ
(3) Phospholipase D enzYme solution: ~
One hundred mililiters of a solution (pH8) consisting of 121.1 mg of Tris con~A;n;ny phospholipase D of 35 U/mQ
(0.6 mg/mQ), 50.0 mg of cow serum albumin, 0.5 mQ of 20%
Triton X-100 and water.
One hundred mililiters of a solution (pH8) consisting of 121.1 mg of Tris con~A;n;ny phospholipase D of 35 U/mQ
(0.6 mg/mQ), 50.0 mg of cow serum albumin, 0.5 mQ of 20%
Triton X-100 and water.
(4) 60 mM EDTA
(5) 40 mM MgCQ2 solution cont~;n;ng 160 mM ATP:
There were mixed 1.0 mQ of 0.2 M ATP, 0.10 mQ of 0.5 M MgCQ2 ~nd 0.15 mQ of distilled water so that there was obtained 1.25 mQ of the solution.
There were mixed 1.0 mQ of 0.2 M ATP, 0.10 mQ of 0.5 M MgCQ2 ~nd 0.15 mQ of distilled water so that there was obtained 1.25 mQ of the solution.
(6) GK enzyme solution:
10 mM Tris-HCQ buffer cont~;n; ng 2 mg/mQ of GK
(p~ 8).
i2ai104~
10 mM Tris-HCQ buffer cont~;n; ng 2 mg/mQ of GK
(p~ 8).
i2ai104~
(7) GPO enzyme solution:
There was prepared 10 mQ of GPO enzyme solution consisting of 10 mg of cow serum albumin containing 3 mg/mQ
of GPO, 560.75 mg of (NH4)2SO4, 1.0 mQ of 0.1 M Tris-HCQ
buffer (pH 8) and 9 mQ of distilled water.
There was prepared 10 mQ of GPO enzyme solution consisting of 10 mg of cow serum albumin containing 3 mg/mQ
of GPO, 560.75 mg of (NH4)2SO4, 1.0 mQ of 0.1 M Tris-HCQ
buffer (pH 8) and 9 mQ of distilled water.
(8) Indicator composition liquid containing GPO:
4-aminoantipyrine 0.24 mQ
aqueous solution of phenol (10 mg/mQ) 0.16 mQ
Triton X-100 (1.25~) 0.8 mQ
Tris-HCQ buffer (0.5 M, pH 8)1.6 mQ
Peroxidase (90 U/mQ distilled water) 0.8 mQ
GPO enzyme solution (3 mg/mQ GPO) 0.8 mQ
Distilled water 15.6 mQ
Total 20 mQ
To each of 0 mQ - 0.10 mQ of the solution containing phosphatidyl glycerol (PG) mentioned above was added 1~ Triton X-100 so that the total amounted to 0.10 mQ. To this mixture was further added 0.1 mQ of Ca~+-buffer. After the mixture was preheated at 37C for 5 minutes, 0.05 mQ of phospholipase D enzyme solution was added thereto and the mixture was allowed to react at 37C for 10 minutes. After the reaction 0.04 mQ
of 60 mM EDTA was added and further 0.075 mQ of 40 mM MgCQ2 solution cont~;ning 160 mM ATP and 0.03 mQ of GK en~yme solu-tion were added thereto. The mixture was allowed to react at 37C for 10 minutes. After the reaction, 1.0 mQ of the indicator composition liquid cont~; ni ng GPO was added to this and the mixture was allowed to react at 37C 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 mQ of PG-containing solution (PG content of 5 nmoles) to that of 0.1 mQ (PG content of 100 n moles).
lZ~
Further, from this Fig. 1, the tendency of linear relation can be obtained when the PG con~ent exceeded 2 n moles and the sensitivity was very sharp.
Example 2 In 18 mQ of chloroform--methanol (2:1) was dissolved 5 - 6 mQ 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 mQ of chloroform and the solution was charged into a silica gel column (0.3 g) wherein the neutral fat was removed by allowing 10 mQ of chloroform-methanol (19:1) to flow and the phosphatidyl glycerol-containing fraction was recovered by allowing 10 mQ
of chloroform-methanol t2.1) to flow. Then, the solvent was removed and the residue was dissolved in 0.1 mQ of 1% Triton X-100 to prepare the liquid to be inspected. Further, 0.1 mQ
of Ca++-buffer was added to this liquid to be inspected and the mixture was preheated at 37C for 5 minutes. Then, 0.05 mQ of phospholipase D enzyme solution was added to this and the mixture was allowed to react at 37C for 10 minutes.
After the reaction, 0.04 mQ of 60 mM EDTA was added to this f~llowed by the addition of 0.075 mQ of 40 mM MgCQ2 solution containing 160 mM ATP and 0.03 mQ of GK enzyme solution thereto and the mixture was allowed to react at 37C for 10 minutes. Then, after the reaction, 1.0 mQ of the indicator composition liquid con~;n;n;ng GPO was added to this and the mixture was allowed to react at 37C 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/dQ) in the amniotic fluid sampled was obtained from the calibration curve. The results were as shown in ~ig. 2. In Fig. 2, X shows death case, O shows R~S occurrence case, ~ shows weak RDS occurrence case and ~ shows no RDS occurrence case.
10~
Example 3 0.2 M Tris-HCQ buffer (pH 7.5) 0.2 mQ
10 mM CaCQ2 0.2 mQ
10 mM MgCQ2 0.2 mQ
S 10% Triton X~100 0.05 mQ
10 mM ATP 0.2 mQ
20 U/mQ phospholipase D, 5 U/mQ GK
and 50 U/mQ GPO-containing liquid 0.1 mQ
45 U/mQ peroxidase 0.1 mQ
0.3~ 4-aminoantipyrine 0.2 mQ
0.3~ 3-methyl-N-ethyl-N-(~-hydroxyethyl)aniline 0.2 mQ
Distilled water 0.55 mQ
Total 2.C mQ
There was prepared 2.0 mQ of the reaction mixture for the quantitative measurement of phosphatidyl glycerol having the composition mentioned above. To this was added 50 ~Q of the liquid to be inspected which is a liquid contain-ing different amount of PG (PG content: 20 n moles - 100 n moles). The mixture was allowed to react at 37C 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.
4-aminoantipyrine 0.24 mQ
aqueous solution of phenol (10 mg/mQ) 0.16 mQ
Triton X-100 (1.25~) 0.8 mQ
Tris-HCQ buffer (0.5 M, pH 8)1.6 mQ
Peroxidase (90 U/mQ distilled water) 0.8 mQ
GPO enzyme solution (3 mg/mQ GPO) 0.8 mQ
Distilled water 15.6 mQ
Total 20 mQ
To each of 0 mQ - 0.10 mQ of the solution containing phosphatidyl glycerol (PG) mentioned above was added 1~ Triton X-100 so that the total amounted to 0.10 mQ. To this mixture was further added 0.1 mQ of Ca~+-buffer. After the mixture was preheated at 37C for 5 minutes, 0.05 mQ of phospholipase D enzyme solution was added thereto and the mixture was allowed to react at 37C for 10 minutes. After the reaction 0.04 mQ
of 60 mM EDTA was added and further 0.075 mQ of 40 mM MgCQ2 solution cont~;ning 160 mM ATP and 0.03 mQ of GK en~yme solu-tion were added thereto. The mixture was allowed to react at 37C for 10 minutes. After the reaction, 1.0 mQ of the indicator composition liquid cont~; ni ng GPO was added to this and the mixture was allowed to react at 37C 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 mQ of PG-containing solution (PG content of 5 nmoles) to that of 0.1 mQ (PG content of 100 n moles).
lZ~
Further, from this Fig. 1, the tendency of linear relation can be obtained when the PG con~ent exceeded 2 n moles and the sensitivity was very sharp.
Example 2 In 18 mQ of chloroform--methanol (2:1) was dissolved 5 - 6 mQ 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 mQ of chloroform and the solution was charged into a silica gel column (0.3 g) wherein the neutral fat was removed by allowing 10 mQ of chloroform-methanol (19:1) to flow and the phosphatidyl glycerol-containing fraction was recovered by allowing 10 mQ
of chloroform-methanol t2.1) to flow. Then, the solvent was removed and the residue was dissolved in 0.1 mQ of 1% Triton X-100 to prepare the liquid to be inspected. Further, 0.1 mQ
of Ca++-buffer was added to this liquid to be inspected and the mixture was preheated at 37C for 5 minutes. Then, 0.05 mQ of phospholipase D enzyme solution was added to this and the mixture was allowed to react at 37C for 10 minutes.
After the reaction, 0.04 mQ of 60 mM EDTA was added to this f~llowed by the addition of 0.075 mQ of 40 mM MgCQ2 solution containing 160 mM ATP and 0.03 mQ of GK enzyme solution thereto and the mixture was allowed to react at 37C for 10 minutes. Then, after the reaction, 1.0 mQ of the indicator composition liquid con~;n;n;ng GPO was added to this and the mixture was allowed to react at 37C 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/dQ) in the amniotic fluid sampled was obtained from the calibration curve. The results were as shown in ~ig. 2. In Fig. 2, X shows death case, O shows R~S occurrence case, ~ shows weak RDS occurrence case and ~ shows no RDS occurrence case.
10~
Example 3 0.2 M Tris-HCQ buffer (pH 7.5) 0.2 mQ
10 mM CaCQ2 0.2 mQ
10 mM MgCQ2 0.2 mQ
S 10% Triton X~100 0.05 mQ
10 mM ATP 0.2 mQ
20 U/mQ phospholipase D, 5 U/mQ GK
and 50 U/mQ GPO-containing liquid 0.1 mQ
45 U/mQ peroxidase 0.1 mQ
0.3~ 4-aminoantipyrine 0.2 mQ
0.3~ 3-methyl-N-ethyl-N-(~-hydroxyethyl)aniline 0.2 mQ
Distilled water 0.55 mQ
Total 2.C mQ
There was prepared 2.0 mQ of the reaction mixture for the quantitative measurement of phosphatidyl glycerol having the composition mentioned above. To this was added 50 ~Q of the liquid to be inspected which is a liquid contain-ing different amount of PG (PG content: 20 n moles - 100 n moles). The mixture was allowed to react at 37C 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 (12)
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 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 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 the following steps (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.
(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 is employed.
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.
10. A method for the quantitative measurement according to claim 8 wherein the chromogen is 4-aminoantipyrine and 3-methyl-N-ethyl-N-(.beta.-hydroxy-ehtyl)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.
12. 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.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP56203074A JPS58107199A (en) | 1981-12-16 | 1981-12-16 | Qualitative analysis of phosphatidyl glycerol |
| JP203074/81 | 1981-12-16 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1201047A true CA1201047A (en) | 1986-02-25 |
Family
ID=16467912
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000416607A Expired CA1201047A (en) | 1981-12-16 | 1982-11-29 | Method for quantitative measurement of phosphatidyl glycerol |
Country Status (6)
| Country | Link |
|---|---|
| JP (1) | JPS58107199A (en) |
| CA (1) | CA1201047A (en) |
| DE (1) | DE3246090A1 (en) |
| FR (1) | FR2518117B1 (en) |
| GB (1) | GB2111677B (en) |
| IT (1) | IT1155384B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3211347A1 (en) * | 1982-03-27 | 1983-09-29 | Boehringer Mannheim Gmbh, 6800 Mannheim | METHOD AND REAGENT FOR ENZYMATIC DETERMINATION OF PHOSPHATIDYLGLYCERIN |
| JPS59140900A (en) * | 1983-01-28 | 1984-08-13 | Toyo Jozo Co Ltd | Novel method for highly sensitive, enzymatic determination |
| JPS62215398A (en) * | 1986-01-06 | 1987-09-22 | アイソラブ，インク． | Method and kit for measuring quantity of phosphatidyl glycerol in amniotic fluid as indication element for medicaldiagnosis |
Family Cites Families (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1515195A (en) * | 1975-08-05 | 1978-06-21 | Hycel Inc | Triglyceride assay |
| JPS604716B2 (en) * | 1976-04-26 | 1985-02-06 | 東洋醸造株式会社 | Novel choline oxidase and its production method |
| JPS5324888A (en) * | 1976-08-19 | 1978-03-08 | Kyowa Hakko Kogyo Kk | Composition for quantitating fats containing phosphorus |
| US4241178A (en) * | 1978-01-06 | 1980-12-23 | Eastman Kodak Company | Process and composition for the quantification of glycerol ATP and triglycerides |
| DK401279A (en) * | 1978-09-26 | 1980-03-27 | Toyo Jozo Kk | MULTI-COMPONENT ANALYSIS METHOD AND APPARATUS FOR USE THEREOF |
| JPS55111800A (en) * | 1979-02-20 | 1980-08-28 | Toyo Jozo Co Ltd | Kit for analysis of lipid component |
| SU831129A1 (en) * | 1979-07-24 | 1981-05-23 | Дальневосточный Государственный Уни-Верситет | Method of obtaining phosphatidylglycerin |
| JPS5889199A (en) * | 1981-11-20 | 1983-05-27 | Toyo Jozo Co Ltd | Quantitative analysis of phosphatidylglycerol |
| JPS5898096A (en) * | 1981-12-07 | 1983-06-10 | Toyo Jozo Co Ltd | Determination of ethanolamine in solution containing the same |
-
1981
- 1981-12-16 JP JP56203074A patent/JPS58107199A/en active Pending
-
1982
- 1982-11-23 GB GB08233333A patent/GB2111677B/en not_active Expired
- 1982-11-29 CA CA000416607A patent/CA1201047A/en not_active Expired
- 1982-12-03 FR FR8220269A patent/FR2518117B1/en not_active Expired
- 1982-12-13 DE DE19823246090 patent/DE3246090A1/en active Granted
- 1982-12-15 IT IT24763/82A patent/IT1155384B/en active
Also Published As
| Publication number | Publication date |
|---|---|
| IT8224763A1 (en) | 1984-06-15 |
| IT1155384B (en) | 1987-01-28 |
| GB2111677B (en) | 1985-01-16 |
| JPS58107199A (en) | 1983-06-25 |
| DE3246090C2 (en) | 1991-10-10 |
| DE3246090A1 (en) | 1984-02-16 |
| FR2518117B1 (en) | 1986-10-10 |
| GB2111677A (en) | 1983-07-06 |
| FR2518117A1 (en) | 1983-06-17 |
| IT8224763A0 (en) | 1982-12-15 |
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